Complex microbial inoculant capable of overcoming continuous cropping obstacles of pinellia ternate and application of complex microbial inoculant
By screening and culturing Sphingomonas sphingosine monocytogenes and cold-resistant Bacillus subtilis from Kyung Hee University to prepare a compound bacterial agent, the problem of allelopathic autotoxic substances accumulation in Pinellia ternata under continuous cropping obstacles was solved, thereby improving soil ecological function and Pinellia ternata growth.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NORTHWEST A & F UNIV
- Filing Date
- 2026-01-22
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies have failed to effectively utilize the interactions between strains and have overlooked the potential of compound microbial agents in alleviating the obstacles of continuous cropping of Pinellia ternata, especially in the degradation of allelopathic autotoxic substances, leading to an imbalance in the soil microbial community and a deterioration of soil physicochemical properties.
Sphingomonas kyungheensis JN21 and Peribacillus frigoritolerans F16, strains from Kyung Hee University, were screened and cultured to prepare a compound microbial agent. This compound microbial agent was applied to soils continuously cropped with Pinellia ternata to degrade the allelopathic autotoxic substance p-hydroxybenzoic acid, promote carbon, nitrogen, and sulfur cycles, and increase the abundance of genes related to the hydrogen sulfide production pathway.
It significantly reduces the accumulation of allelopathic autotoxic substances in soils where Pinellia ternata is continuously cropped, improves soil ecological function, promotes the growth of Pinellia ternata, and provides a biological control solution to alleviate the obstacles of continuous cropping.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology, specifically relating to a compound microbial agent for overcoming the obstacle of continuous cropping of Pinellia ternata and its application. Background Technology
[0002] The main reasons for the obstacles to continuous cropping of Pinellia ternata include imbalance of soil microbial flora, accumulation of allelopathic autotoxic substances, and deterioration of soil physicochemical properties.
[0003] Plant rhizosphere microorganisms are a group of microorganisms that are closely attached to the surface of plant roots or inhabit the soil surrounding the roots. They exist in a specific micro-ecological environment influenced by root exudates and shed material, and can utilize allelochemicals and autotoxins as a carbon source for growth. However, current utilization of rhizosphere microorganisms is largely limited to artificial inoculation of single strains, often neglecting the interactions and synergistic effects between strains. Compound microbial agents utilize strains derived from key functional species symbiotic with dominant plants, especially plant rhizosphere microorganisms. Therefore, the strains in compound microbial agents have several advantages over natural microbial communities and single strains. First, the strains in compound microbial agents can exert a weighted effect, enabling simpler and more efficient utilization of the microbiome. Second, compound microbial agents can optimize metabolic pathways, performing multiple functions simultaneously, reducing the metabolic burden on cells, and exhibiting better degradation of allelochemicals and autotoxins.
[0004] Currently, there are no reports on compound microbial agents for degrading allelopathic autotoxins in Pinellia ternata. Therefore, it is of great significance to explore strains that can degrade allelopathic autotoxins in soil and construct compound microbial agents to alleviate the obstacles of continuous cropping of Pinellia ternata and to enhance the artificial cultivation and sustainable development of Pinellia ternata. Summary of the Invention
[0005] To solve the above-mentioned technical problems, this invention screened and obtained *Sphingosine Monoclonalella* from the rhizosphere soil of *Pinellia ternata*. (Kyung Hee University) Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16, obtained from Kyung Hee University's Sphingosine Monoclonal strain. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans A bacterial suspension of F16 was prepared by mixing it to obtain a compound bacterial agent. Application of this compound bacterial agent to soil continuously cropped with Pinellia ternata significantly reduced the content of p-hydroxybenzoic acid, an allelochemical autotoxic substance in Pinellia ternata, thus alleviating the obstacles of continuous cropping. Furthermore, absolute quantitative analysis of the metagenomics of rhizosphere soil inoculated with the compound bacterial agent revealed that it promoted carbon, nitrogen, and sulfur cycling in the soil, particularly increasing the abundance of genes involved in hydrogen sulfide production pathways, thus promoting the growth of Pinellia ternata in in-situ soil after continuous cropping. This invention provides a new solution for preventing and alleviating the obstacles of continuous cropping of Pinellia ternata.
[0006] On one hand, the present invention provides a compound microbial agent, wherein the active ingredient of the microbial agent is *Sphingosine Monoclonalella* from Kyung Hee University. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16, the Kyung Hee University Sphingosine Monoclonal strain Sphingomonas kyungheensis JN21 has the accession number CGMCC No. 36292, and the described cold-resistant *Pseudomonas aeruginosa*... Peribacillus frigoritolerans F16 has the accession number CGMCC No. 36291.
[0007] Secondly, the invention also provides strains for preparing the compound bacterial agent described in this invention, the strains including *Sphingosaminomonas* from Kyung Hee University. Sphingomonas kyungheensis JN21, the Kyung Hee University Sphingosaminomonas Sphingomonas kyungheensis JN21 was obtained by screening from the rhizosphere soil of Pinellia ternata, and its preservation information is as follows: Strain name: JN21; Classification name: Kyung Hee University Sphingosine Monoclonalella Sphingomonas kyungheensis ; The collection was received by the preservation center on October 22, 2025. Date of issuance of preservation certificate: November 5, 2025; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Accession number: CGMCC No.36292.
[0008] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0009] Furthermore, the Kyung Hee University Sphingosine Monoclonal strain... Sphingomonas kyungheensis JN21 is degradable by p-hydroxybenzoic acid.
[0010] Thirdly, another bacterial strain for preparing the compound bacterial agent described in this invention is also provided, the strain comprising *Bacillus subtilis*. Peribacillus frigoritolerans F16, the cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans F16 was obtained by screening from the rhizosphere soil of Pinellia ternata, and its preservation information is as follows: Strain name: F16; Classification name: Cold-resistant Bacillus subtilis Peribacillus frigoritolerans ; The collection was received by the preservation center on October 22, 2025. Date of issuance of preservation certificate: November 5, 2025; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Accession number: CGMCC No.36291.
[0011] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0012] Furthermore, the cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans F16 can degrade p-hydroxybenzoic acid.
[0013] Fourthly, the present invention also provides the application of the compound microbial agent described herein in the degradation of p-hydroxybenzoic acid.
[0014] Fifthly, the invention also provides the application of the compound microbial agent described herein in alleviating or preventing the obstacle of continuous cropping of Pinellia ternata.
[0015] Furthermore, in the application of the compound microbial agent in alleviating or preventing the obstacles of continuous cropping of Pinellia ternata, the alleviation or prevention is achieved by applying the compound microbial agent to the soil in which Pinellia ternata is continuously cropped to degrade the autotoxic substances in the soil, wherein the autotoxic substances are p-hydroxybenzoic acid.
[0016] Furthermore, in the application of the compound microbial agent in alleviating or preventing the obstacles of continuous cropping of Pinellia ternata, the alleviation or prevention is achieved by applying the compound microbial agent to the soil of continuous cropping of Pinellia ternata to promote the carbon, nitrogen and sulfur cycle in the soil of continuous cropping of Pinellia ternata. The promotion of carbon, nitrogen and sulfur cycle includes increasing the abundance of genes related to the hydrogen sulfide production pathway in the rhizosphere soil of continuous cropping of Pinellia ternata.
[0017] Finally, a method for preparing the compound microbial agent described in this invention is also provided, characterized by comprising the following steps: S1. Sphingosine monocytogenes from Kyung Hee University were cultured in LB medium. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16 was obtained from Kyung Hee University's Sphingosine Monoclonal antibody. Sphingomonas kyungheensis JN21 bacterial suspension and cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans F16 bacterial suspension; S2, the *Sphingosomalomonas* strain obtained from the mixing step S1 (Kyung Hee University) Sphingomonas kyungheensis JN21 bacterial suspension and cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans The bacterial suspension of F16 was used to obtain a compound bacterial agent.
[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention provides Sphingosine Monoclonalella from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritoleransThe compound microbial agent composed of F16 can effectively degrade p-hydroxybenzoic acid, a key allelopathic autotoxic substance that causes continuous cropping obstacles in Pinellia ternata, thus reducing autotoxicity at its source.
[0019] (2) This compound microbial agent can not only degrade specific harmful substances, but also improve the overall soil ecological function. Metagenomic analysis shows that it can promote the carbon, nitrogen and sulfur cycle in the rhizosphere soil, especially significantly increase the abundance of genes related to the hydrogen sulfide production pathway, which may create a more favorable growth environment for Pinellia ternata by producing beneficial signaling molecules such as H2S or improving soil nutrient cycling.
[0020] (3) The two functional bacteria used were Kyung Hee University's Sphingosine Monoclonal strain. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16 strains were all screened from the rhizosphere of Pinellia ternata, exhibiting greater stability and adaptability. Applying a compound microbial agent prepared from these two strains to the in-situ soil after continuous cropping of Pinellia ternata significantly reduced the accumulation of p-hydroxybenzoic acid in the rhizosphere soil and improved the growth of Pinellia ternata plants. This invention provides an effective biological control solution for overcoming the obstacles of continuous cropping of Pinellia ternata. Attached Figure Description
[0021] Figure 1 Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans A morphological diagram of F16 on an LB solid plate.
[0022] Figure 2 Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans Phylogenetic tree of F16. Where A represents *Sphingosine monocytogenes* from Kyung Hee University. Sphingomonas kyungheensis JN21; B is a cold-resistant *Bacillus subtilis*. Peribacillus frigoritolerans Phylogenetic tree of F16.
[0023] Figure 3 Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans Morphological image of F16 under a scanning electron microscope.
[0024] Figure 4 High-performance liquid chromatography (HPLC) chromatograms of p-hydroxybenzoic acid under different treatments are shown. A represents the positive control, i.e., the HPLC chromatogram of 20 mg / L p-hydroxybenzoic acid; B represents *Sphingosomalmonella* from Kyung Hee University. Sphingomonas kyungheensis High-performance liquid chromatography (HPLC) chromatogram of JN21 degradation of p-hydroxybenzoic acid; C represents cold-resistant *Bacillus subtilis*. Peribacillus frigoritolerans High-performance liquid chromatography (HPLC) chromatogram of F16 degradation of p-hydroxybenzoic acid.
[0025] Figure 5 The graph shows the results of p-hydroxybenzoic acid content determination in different treatments.
[0026] Figure 6 Agronomical phenotypes of Pinellia ternata growth in in-situ soil under different treatments for continuous cropping. A represents the positive control, i.e., Pinellia ternata seedlings in in-situ soil treated with sterile phosphate buffer; B represents *Sphingomonas* from Kyung Hee University. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16 compound microbial agent was used to treat Pinellia ternata seedlings in in-situ soil after continuous cropping.
[0027] Figure 7 Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans Figure 1 shows the results of determining the p-hydroxybenzoic acid content in the in-situ soil after continuous cropping of Pinellia ternata following treatment with F16 compound microbial agent.
[0028] Figure 8 Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans The results of soil nutrient cycling determination for Pinellia ternata treated with F16 compound microbial agent are shown in the figure. A represents the carbon cycle; B represents the nitrogen cycle; and C represents the sulfur cycle. Detailed Implementation
[0029] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.
[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.
[0031] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0032] Example 1 This example demonstrates the isolation and acquisition of Sphingomonas JN21 and frost-resistant Pseudomonas aeruginosa F16 from Kyung Hee University.
[0033] Using healthy and vigorous Pinellia ternata collected from fields in Qingshui County, Tianshui City, Gansu Province, after a 10-year crop rotation with wheat, 10g of rhizosphere soil was suspended in 90mL of sterile PBS (0.1M, pH 7.4) and shaken at 200rpm for 30min in the dark to obtain a soil suspension. The soil suspension was then continuously diluted (10... -3 Up to 10 -6100 μL aliquots were spread onto Luria Bertani (LB, 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, pH 7.4) agar (1.5% agar). The plates were incubated upside down at 30°C for 2–5 days, with daily monitoring of colony development. When colonies appeared on the plates, the bacterial strain was isolated and purified based on the size and morphology of the colonies. Each colony was picked using a sterile inoculating loop and streaked onto a fresh LB agar plate for further purification until distinct, morphologically varied single colonies were formed. Single colonies were then inoculated onto LB liquid medium and incubated at 30°C and 180 rpm / min for 24 hours. The bacterial culture was then thoroughly mixed with 50% glycerol at a 1:1 volume ratio and transferred to sterile cryovials for storage at -80°C.
[0034] The growth morphology of *Sphingomonas* JN21 and *Ligusticum striatum* F16 strains from Kyung Hee University on LB plates is as follows: Figure 1 As shown, strain JN21 has round, golden-yellow colonies with a smooth surface; strain F16 has round, white colonies with a smooth surface.
[0035] Example 2 This example is for the identification of Sphingomonas JN21 and frost-resistant Leptospira F16 from Kyung Hee University.
[0036] Genomic DNA was extracted from *Sphingomonas JN21* and *Ligustrum lucidum* F16 from Kyung Hee University using the FastPure Bacteria DNA Isolation Mini Kit, and 16S rRNA gene amplification was performed. The PCR products were purified from 1% agarose gels and sent to a biotechnology company for bidirectional Sanger sequencing. The 16S rRNA gene sequence of strain *Sphingomonas JN21* from Kyung Hee University is shown in SEQ ID NO:1, and the 16S rRNA gene sequence of *Ligustrum lucidum* F16 is shown in SEQ ID NO:2.
[0037] The 16S rRNA gene sequence of Sphingosine Monoclonalella JN21 from Kyung Hee University is as follows:
[0038] The 16S rRNA gene sequence of Bacillus paraibonensis F16 that is cold-tolerant is as follows: CTTTGTCACCTTAGGCGGCTGGCTCCATGAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAATGGCTTTATGGGATTCGCTTACCTTCGCAGGTTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCAC CTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGC TGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATC TCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCC GAAGGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGT AAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC GGGCCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCA GGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCT AACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCC TGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGAAAG TCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTAC ACTTGGAATTCCACTTTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAAT GACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGGAACCAC CTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGT ATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTAC CGTCAAGGTACCAGCAGTTACTCTGGTACTTGTTCTTCCCTAACAACAGA ACTTTACGACCCGAAGGCCTTCTTCGTTCACGCGGCGTTGCTCCGTCAGA CTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTG GGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACG CATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATGCGCCGCG GGCCCATCTATAAGTGACAGCGTAAAACCGTCTTTCCATCTTCTCTCATGC GAGAAAAGAACGTATCCGGTATTAGCTCCGGTTTCCCGAAGTTATCCCAG TCTTATAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGCCGCTAATCTC AGGGAGCAAGCTCCCGTCGATTCGCTCGACTGCATGATTAGGCACCC.
[0039] The 16S rRNA gene sequences of *Sphingomonas* JN21 and *Ligusticum striatum* F16 from Kyung Hee University were compared with the NCBI database, and a phylogenetic tree was constructed using MEGA (v11.0.13). The phylogenetic tree is shown below. Figure 2 As shown, they were identified as *Sphingosine monocytogenes* and *Ligusticum striatum* from Kyung Hee University, and were named *Sphingosine monocytogenes* from Kyung Hee University. Sphingomonas kyungheensisJN21 (abbreviated as JN21) and cold-resistant Bacillus subtilis Peribacillus frigoritolerans F16 (abbreviated as F16).
[0040] Example 3 This example demonstrates the observation of *Sphingosaminomonas* from Kyung Hee University using a scanning electron microscope. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans The form of F16.
[0041] Kyung Hee University Sphingosine Monoclonal Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16 cells were cultured in LB liquid medium at 30°C and 37°C for 24 hours, respectively. The cells were washed three times with PBS, and then mixed with an equal volume of PBS. 1 mL of the bacterial culture was added to 300 μL of 2.5% glutaraldehyde, mixed, and incubated overnight at 4°C for fixation. The cells were washed three times with PBS to remove the fixative, and then dehydrated using an ethanol gradient (30%, 50%, 70%, 80%, 95%, 100%). After each addition of ethanol, the cells were incubated at 4°C for 15 min, followed by centrifugation at 8000 rpm. Finally, 100% anhydrous ethanol was added, followed by centrifugation and washing twice (5000 rpm, 10 min). The cells were then resuspended in 200 μL of anhydrous ethanol for later use.
[0042] Take 10 μL of Kyung Hee University Sphingosine Monoclonal bacteria respectively Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16 ethanol suspension was dropped onto the silicon wafer surface and dried with CO2. The dried sample was then separated into front and back sides, adhered to conductive adhesive, and further sputter-coated with gold before observation. (Kyung Hee University, Sphingosine Monoclonalella) Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans The morphology of F16 under a scanning electron microscope is as follows: Figure 3 As shown. Sphingosine monocytogenes from Kyung Hee University Sphingomonas kyungheensis JN21 exhibits a dense, wrinkled aggregate morphology under a scanning electron microscope; it is a cold-resistant, near-spore-forming bacterium. Peribacillus frigoritolerans F16 exhibits a clear rod-shaped single-cell morphology under a scanning electron microscope.
[0043] Example 4 This example demonstrates the in vitro evaluation of *Sphingosomalmonella* from Kyung Hee University. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans Degradation efficiency of F16 p-hydroxybenzoic acid.
[0044] Kyung Hee University Sphingosine Monoclonal Sphingomonas kyungheensis JN21 (JN21) and cold-resistant Bacillus subtilis Peribacillus frigoritoleransF16 cells were cultured in LB liquid medium at 30°C and 37°C for 24 hours at 180 rpm. After fermentation, the cells were collected by centrifugation at 4°C and 8000 rpm for 10 minutes, and then resuspended in PBS to a concentration of 1×10⁻⁶. 8 CFU / mL. The bacterial suspension (1%, v / v) was inoculated into MSM medium containing 20 mg / L p-hydroxybenzoic acid. After incubation at 30°C and 180 rpm for 48 hours, centrifugation was performed at 4°C and 10,000 rpm for 10 min. The supernatant was acidified to pH 2.3 with 6 M HCl, and extracted three times with equal volumes of ethyl acetate. The extracts were combined. The combined extract was evaporated to dryness under nitrogen (40°C, 0.5 L / min), redissolved in 3 mL of 50% methanol and 0.1% formic acid, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane into a high-performance liquid chromatography (HPLC) vial. HPLC analysis was performed on an Agligent 1260 system equipped with a 246 nm UV detector. The results are shown below. Figure 4 and Figure 5 As shown. The formulation of the MSM medium is as follows: 1 g / L KH2PO4, 1.5 g / L NaH2PO4·12H2O, 0.5 g / L NaNO3, 0.5 g / L (NH4)2SO4, 0.2 g / L MgSO4·7H2O, 0.02 g / L CaCl2, 0.005 g / L FeSO4·7H2O, pH 6.5.
[0045] Depend on Figure 4 It can be seen that the peak value of p-hydroxybenzoic acid was detected at 13.06667 min. Figure 5 It is known that Kyung Hee University's Sphingosine Monoclonal strain Sphingomonas kyungheensis JN21 (JN21) and cold-resistant Bacillus subtilis Peribacillus frigoritolerans F16 (F16) can degrade p-hydroxybenzoic acid by 16.33% and 20.17% respectively in vitro.
[0046] Example 5 This example is for evaluating *Sphingosomalmonella* from Kyung Hee University. Sphingomonas kyungheensis JN21 (JN21) and cold-resistant Bacillus subtilis Peribacillus frigoritolerans The effect of F16 (F16) compound microbial agent on the growth of Pinellia ternata in in-situ soil after continuous cropping.
[0047] Kyung Hee University Sphingosine Monoclonal Sphingomonas kyungheensis JN21 (JN21) and cold-resistant Bacillus subtilis Peribacillus frigoritoleransF16 was inoculated into LB liquid medium and cultured in the dark with shaking (180 rpm) at 30°C and 37°C for 24 hours, respectively. The fermentation product was collected by centrifugation at 12000 rpm for 5 minutes, and the cells were resuspended in 50 mL of sterile PBS to obtain Kyung Hee University's Sphingosine Monoclonal strain. Sphingomonas kyungheensis JN21 bacterial suspension and cold-resistant *Lisobacterium* Peribacillus frigoritolerans F16 bacterial suspension, 2.5 mL (10 8 Sphingosomalidobacterium sphingosine mononucleosis (CFU / mL) from Kyung Hee University Sphingomonas kyungheensis JN21 bacterial suspension and 2.5 mL (10 8 Cold-resistant *Ligusticum striatum* (CFU / mL) Peribacillus frigoritolerans The compound bacterial agent was prepared by uniformly mixing F16 bacterial suspension.
[0048] The test group (SynCom) was inoculated with 5 mL of compound microbial agent in the in-situ soil of Pinellia ternata after continuous cropping, while the control group (Control) was inoculated with 5 mL of PBS in the same soil. The soil was watered with 10 mL of sterile deionized water every three days. After 60 days, the growth of Pinellia ternata in the in-situ soil was observed, and the results are as follows: Figure 6 As shown, the content of p-hydroxybenzoic acid in the soil was determined using high performance liquid chromatography, and the results are as follows. Figure 7 As shown. By Figure 6 It can be seen that the growth of Pinellia ternata was better 60 days after inoculation with the compound microbial agent, and the biomass was significantly higher than that of the control, indicating that inoculation with the compound microbial agent in the in-situ soil after continuous cropping significantly alleviated the continuous cropping obstacles of Pinellia ternata. Figure 7 It can be seen that the p-hydroxybenzoic acid content in the soil inoculated with compound microbial agent was 0.031 mg / g soil, which was significantly lower than the p-hydroxybenzoic acid content in the control group soil (0.041 mg / g soil). This indicates that the compound microbial agent alleviates the obstacle of continuous cropping of Pinellia ternata by degrading the p-hydroxybenzoic acid content in the soil in situ after continuous cropping.
[0049] Example 6 This example presents a metagenomic absolute quantitative analysis of the rhizosphere response of Pinellia ternata to inoculation with a compound microbial agent.
[0050] Rhizosphere soil samples from the control and test groups (compound microbial agent) of *Pinellia ternata* were collected after 60 days of treatment in Example 5 and sent to Shanghai Tianhao Biotechnology Co., Ltd. for absolute metagenomic quantification of the rhizosphere soil. Results are as follows: Figure 8 As shown, the absolute abundance of carbon, nitrogen, and sulfur cycling-related pathways and genes in the rhizosphere soil of Pinellia ternata treated with the compound microbial agent for 60 days was significantly higher than that in the control group. In the sulfur cycle, the absolute abundance of genes involved in the hydrogen sulfide production pathway was the highest, indicating that hydrogen sulfide may act as a signaling molecule to help Pinellia ternata grow in in-situ soil after continuous cropping. Therefore, metagenomic analysis suggests that the compound microbial agent alleviates the continuous cropping obstacle of Pinellia ternata by promoting carbon, nitrogen, and sulfur cycling in in-situ soil after continuous cropping.
[0051] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.
Claims
1. A compound microbial agent, characterized in that, The active ingredient of the bacterial agent is *Sphingosine Monoclonalella* from Kyung Hee University. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16, the Kyung Hee University Sphingosine Monoclonal strain Sphingomonas kyungheensis JN21 has the accession number CGMCC No. 36292, and the described cold-resistant *Pseudomonas aeruginosa* is... Peribacillus frigoritolerans F16 has the accession number CGMCCNo.36291.
2. The bacterial strain for preparing the compound bacterial agent according to claim 1, characterized in that, The strains included *Sphingosaminomonas* from Kyung Hee University. Sphingomonas kyungheensis JN21, the Kyung Hee University Sphingosaminomonas Sphingomonas kyungheensis JN21 has the accession number CGMCC No.36292.
3. The strain according to claim 2, characterized in that, The Kyung Hee University Sphingosine Monoclonal Sphingomonas kyungheensis JN21 is degradable by p-hydroxybenzoic acid.
4. The bacterial strain for preparing the compound bacterial agent according to claim 1, characterized in that, The strains include cold-resistant *Bacillus subtilis*. Peribacillus frigoritolerans F16, the cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans F16 has the accession number CGMCC No. 36291.
5. The strain according to claim 4, characterized in that, The cold-resistant Bacillus subtilis Peribacillus frigoritolerans F16 can degrade p-hydroxybenzoic acid.
6. The application of the compound microbial agent according to claim 1 in the degradation of p-hydroxybenzoic acid.
7. The application of the compound microbial agent according to claim 1 in alleviating or preventing the obstacle of continuous cropping of Pinellia ternata.
8. The application according to claim 7, characterized in that, The mitigation or prevention is achieved by applying the compound microbial agent to the soil where Pinellia ternata is continuously cropped, thereby degrading the autotoxic substances in the soil where Pinellia ternata is continuously cropped, wherein the autotoxic substances are p-hydroxybenzoic acid.
9. The application according to claim 7, characterized in that, The mitigation or prevention is achieved by applying the compound microbial agent to the soil where Pinellia ternata is continuously cropped, thereby promoting the carbon, nitrogen, and sulfur cycle in the soil. This promotion of the carbon, nitrogen, and sulfur cycle includes increasing the abundance of genes related to the hydrogen sulfide production pathway in the rhizosphere soil of Pinellia ternata continuously cropped.
10. A method for preparing the compound microbial agent according to claim 1, characterized in that, Includes the following steps: S1. Sphingosine monocytogenes from Kyung Hee University were cultured in LB medium. Sphingomonas kyungheensis JN21 and cold-resistant *Ligusticum striatum* Peribacillus frigoritolerans F16, obtained from Kyung Hee University's Sphingosine Monoclonal strain. Sphingomonas kyungheensis JN21 bacterial suspension and cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans F16 bacterial suspension; S2, the *Sphingosomalomonas* strain obtained from the mixing step S1 (Kyung Hee University) Sphingomonas kyungheensis JN21 bacterial suspension and cold-resistant *Bacillus subtilis* Peribacillus frigoritolerans The bacterial suspension of F16 was used to obtain a compound bacterial agent.