Complex microbial inoculant for efficiently degrading p-hydroxybenzoic acid in soil and application of complex microbial inoculant

Abstract

The invention provides a complex microbial inoculant for efficiently degrading p-hydroxybenzoic acid in soil, the complex microbial inoculant comprises a compost bacillus Sys-Gs-008 strain and a bacillus licheniformis Sys-Gs-011 strain, the preservation number of the compost bacillus Sys-Gs-008 strain is CCTCC (China Center for Type Culture Collection) NO: M20252817, and the preservation number of the bacillus licheniformis Sys-Gs-011 strain is CCTCC NO: M20252820. The complex microbial inoculant provided by the invention can efficiently degrade the main phenolic acid allelochemical-p-hydroxybenzoic acid produced by ginseng, and the two strains in the complex microbial inoculant are non-ginseng pathogenic strains and have no antagonism between each other, so that the complex microbial inoculant can be applied to preparation of a ginseng allelochemical-p-hydroxybenzoic acid degradation microbial inoculant, and has a wide application prospect. The continuous cropping obstacle of the ginseng is greatly improved, and the method has a good application prospect in the field of ginseng planting.

Description

Technical Field

[0001] This invention relates to a compound microbial agent and its uses, including Bacillus compostii (Bacillus subtilis). Bacillus stercoris Sys-Gs-008 strain and Bacillus licheniformis ( Bacillus licheniformis. The Sys-Gs-011 strain, a compound microbial agent, can efficiently degrade p-hydroxybenzoic acid in soil and belongs to the field of microbial technology. Background Technology

[0002] Ginseng ( Pannax gxinseng Ginseng (CA Meyer), also known as Dijing, Shencao, or Huangshen, is a perennial herbaceous medicinal plant belonging to the genus Panax in the family Araliaceae. The medicinal parts are its roots and rhizomes, which possess remarkable effects such as greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, promoting body fluid production and nourishing blood, and calming the mind and improving intelligence. Ginseng has been cultivated for over 400 years, and in recent years, with the further increase in market demand, artificial cultivation has become the only way to meet that demand.

[0003] As a perennial herb, ginseng has a long cultivation cycle and a unique growing environment, which can lead to severe continuous cropping obstacles, significantly impacting the yield of artificially cultivated ginseng. Continuous cropping obstacles in ginseng are a classic agroecological problem, rooted in a highly specialized and fragile balance between ginseng and the soil environment, which is difficult to restore once broken.

[0004] The main reasons for ginseng continuous cropping obstacles are: 1. Accumulation of soil pathogenic microorganisms: During the growth process, ginseng roots secrete specific organic substances (such as saponins and phenolic acids). These secretions selectively stimulate the growth of certain pathogenic fungi (such as Fusarium, Phytophthora, and rust fungi) and nematodes in the soil, leading to a year-on-year increase in soil-borne diseases. During continuous cropping, the pathogenic fungal community forms a "dominant flora," directly infecting the roots of newly planted ginseng. 2. Allelopathic autotoxicity: Certain compounds (such as phenolic acids and saponin derivatives) produced by ginseng root secretions and the decomposition of plant residues can be toxic to themselves, inhibiting root development and seedling growth, damaging cell membrane structure, and interfering with the activity of beneficial microorganisms in the soil. 3. Decreased soil microbial diversity: Long-term continuous cropping leads to a shift in soil from a "bacterial" to a "fungal" type, reducing the proportion of beneficial bacteria (such as actinomycetes and Trichoderma), weakening the soil ecosystem's regulatory capacity, and making pathogens more prone to outbreaks.

[0005] Therefore, degrading specific allelochemicals like benzoic acid is a crucial step in addressing continuous cropping obstacles. Benzoic acid autotoxic substances are interconnected with soil microecological imbalance and deterioration of physicochemical properties, collectively leading to continuous cropping problems. Degrading benzoic acid can help break the vicious cycle: directly removing toxic substances and mitigating direct damage to crops; and indirectly regulating the microbial community: by removing this "false signal," it helps suppress pathogens and creates conditions for the recovery of beneficial microorganisms.

[0006] p-Hydroxybenzoic acid (p-hydroxybenzoic acid) belongs to the benzoic acid class of substances. It is a hydroxy-substituted derivative of benzoic acid and is one of the most common and widely studied members of the phenolic allelochemicals secreted by plants. Numerous academic papers have confirmed that p-hydroxybenzoic acid is one of the main phenolic acids accumulated in the soil of old ginseng cultivation sites. Its concentration is significantly positively correlated with the incidence of ginseng root diseases (such as rust rot). Therefore, finding microorganisms with p-hydroxybenzoic acid degradation capabilities is an urgent need in this field. Summary of the Invention

[0007] This invention solves the problems in the prior art by providing a compound microbial agent for the efficient degradation of p-hydroxybenzoic acid. This compound microbial agent can efficiently degrade p-hydroxybenzoic acid, the main phenolic allelochemical produced by ginseng, with a degradation rate of up to 81.5%. Furthermore, it has been proven to degrade p-hydroxybenzoic acid in the soil of ginseng-continuously cropped fields.

[0008] Two novel strains were screened in this application, namely Bacillus compostii (Bacillus compostii). Bacillus stercoris Sys-Gs-008 strain and Bacillus licheniformis ( Bacillus licheniformis. Both strains, namely Sys-Gs-011 and Sys-Gs-008, are deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, with a deposit date of December 10, 2025. The accession number for Bacillus compostii Sys-Gs-008 is CCTCC NO: M20252817, and its 16S rDNA sequence is shown in SEQ ID NO.1. The accession number for Bacillus licheniformis Sys-Gs-011 is CCTCC NO: M20252820, and its 16S rDNA sequence is shown in SEQ ID NO.2.

[0009] The above-mentioned compound microbial agent is cultured by inoculating Bacillus compostii strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 into a culture medium to obtain a culture of the compound microbial agent. Specifically, *Bacillus compostii* strain Sys-Gs-008 and *Bacillus licheniformis* strain Sys-Gs-011 were first inoculated separately into LB liquid medium and activated by incubation at 37°C with a shaker at 150 rpm for 24 hours to obtain seed culture. Then, the seed culture was re-inoculated into MSM medium at a 3% (v / v) inoculation rate and incubated at 28°C with a shaker at 180 rpm for 2 days to obtain separate bacterial suspensions of *Bacillus compostii* strain Sys-Gs-008 and *Bacillus licheniformis* strain Sys-Gs-011. The bacterial suspensions of *Bacillus compostii* strain Sys-Gs-008 and *Bacillus licheniformis* strain Sys-Gs-011 were then mixed at a volume ratio of 2:1 to obtain the culture of the composite bacterial agent. The LB liquid medium contained the following components: 10.0 g / L tryptone, 5.0 g / L yeast extract, and NaCl. 10.0 g / L; The MSM medium comprises the following components: (NH4)2SO4 2.0 g / L, CaCl2 0.1 g / L, K2HPO4·3H2O 1.0 g / L, NaH2PO4 1.0 g / L, MgSO4·7H2O 0.35 g / L.

[0010] This application further provides a microbial agent, the effective components of which include Bacillus compostii strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011, or a culture of a compound microbial agent.

[0011] This application also provides a method for alleviating the obstacle of continuous cropping of ginseng, comprising the following steps: treating the soil of the ginseng continuous cropping site with a compound microbial agent, a culture of the compound microbial agent, or the above-mentioned microbial agent.

[0012] As a preferred method, the culture of the compound microbial agent is diluted and sprayed onto the ginseng-continuously-cropped land.

[0013] Furthermore, the culture of the compound microbial agent was diluted with water at a volume ratio of 1:40 to obtain a diluted microbial solution. The diluted microbial solution was sprayed onto the ginseng-co-cropped land at a dosage of 40L / mu, and then deep plowing was carried out.

[0014] Compared with the prior art, the present invention has the following advantages: the compound microbial agent provided by the present invention can efficiently degrade p-hydroxybenzoic acid, the main phenolic allelochemical produced by ginseng, and the two strains in the compound microbial agent are both non-ginseng pathogenic strains and have no antagonistic effect on each other. It can be used to prepare a microbial agent for degrading p-hydroxybenzoic acid, a ginseng allelochemical, which greatly improves the continuous cropping obstacles of ginseng and has good application prospects in the field of ginseng cultivation. Attached Figure Description

[0015] Figure 1 This is an HPLC chromatogram of p-hydroxybenzoic acid detection in the blank control culture medium; Figure 2 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation by Bacillus composting strain Sys-Gs-008. Figure 3 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation of Bacillus licheniformis Sys-Gs-011 strain. Figure 4 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation of bacterial group A. Figure 5 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation of bacterial group B. Figure 6 The image shows the HPLC detection of p-hydroxybenzoic acid in the culture medium after fermentation of bacterial group C. Figure 7 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation of bacterial group D. Figure 8 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation of bacterial group E. Figure 9 A comparison of the degradation effects of p-hydroxybenzoic acid in different groups of culture media; Figure 10 A comparative graph showing the degradation effects of different groups of cultures on p-hydroxybenzoic acid in soil; Figure 11 A comparison of soil in ginseng-continuous cropping fields before and after treatment with diluted bacterial solution. Detailed Implementation

[0016] The present invention will now be described in detail with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0017] culture medium

[0018] The composition of the culture medium described in this embodiment is as follows (based on 1L of culture medium): Beef extract peptone medium (Nutrient Agar, NA): 3.0g beef extract, 10.0g peptone, 5.0g NaCl, 15.0g agar; Luria-Bertani (LB) liquid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl; Minimal salt medium (MSM): (NH4)2SO4 2.0g, CaCl2 0.1g, K2HPO4·3H2O 1.0g, NaH2PO4 1.0g, MgSO4·7H2O 0.35g.

[0019] Isolation and culture of strains

[0020] In this embodiment, 5g of rhizosphere soil sample from wild ginseng collected in Benxi, Liaoning Province, was placed in a 250mL Erlenmeyer flask, and 100mL of basal inorganic salt medium containing 50mg / L p-hydroxybenzoic acid was added. The mixture was incubated at 28℃ and 180r / min for 7 days. After incubation, 10mL of the culture solution was transferred to 90mL of basal inorganic salt medium containing 100mg / L p-hydroxybenzoic acid, and incubated at 28℃ and 180r / min for 7 days. After incubation, 10mL of the culture solution was transferred to 90mL of basal inorganic salt medium containing 200mg / L p-hydroxybenzoic acid, and incubated at 28℃ and 180r / min for 7 days. The final culture solution was diluted with sterile water to three concentrations (10...). -5 10 -6 10 -7 The colonies were plated separately on NA medium containing 200 mg / L p-hydroxybenzoic acid and incubated at 28°C for 3 days. Then, single colonies with different morphological characteristics were picked and streaked onto new NA plates containing 200 mg / L p-hydroxybenzoic acid for repeated purification three times.

[0021] The purified and stably growing strains on the plate were picked and transferred to 1.5 mL EP tubes containing 600 μL LB liquid medium. The tubes were sealed with sealing film and then cultured in a shaker at 37°C and 180 r / min for 1 day. An equal volume of 40% glycerol was added, and the mixture was mixed and stored in an ultra-low temperature freezer at -80°C. At least 3 copies of each strain were stored as candidate strains capable of degrading p-hydroxybenzoic acid.

[0022] 10 μL of the preserved bacterial strain was inoculated into 5 mL of LB liquid medium and activated by incubation at 37°C and 150 rpm for 24 h to obtain the seed culture. 2 mL of the seed culture was fermented in 100 mL of basal inorganic salt medium containing 200 mg / L p-hydroxybenzoic acid as the experimental group. A basal inorganic salt medium containing 200 mg / L p-hydroxybenzoic acid without bacterial culture served as the control group (CK). Both cultures were pre-cultured at 28°C and 180 rpm for 3 days. After the above fermentation culture, the fermentation broth was collected.

[0023] Determination of the strain's ability to degrade p-hydroxybenzoic acid

[0024] By performing HPLC analysis on the fermentation broth samples after filtration of each strain, and comparing the results with the data in the blank control medium (CK), it can be preliminarily determined whether the strain has the ability to degrade p-hydroxybenzoic acid.

[0025] The culture medium was extracted three times with equal volumes of ethyl acetate. The upper layer of ethyl acetate was extracted, evaporated to dryness by rotary evaporation, and reconstituted in acetonitrile. The solution was filtered through a 0.22 µm organic filter membrane and used as the HPLC sample solution for HPLC detection.

[0026] After the above tests, two strains capable of degrading p-hydroxybenzoic acid were screened and identified: *Bacillus compostii*, named strain Sys-Gs-008; and *Bacillus licheniformis*, named strain Sys-Gs-011. HPLC detection is as follows: Figure 1-3 As shown: Figure 1 This is an HPLC chromatogram of p-hydroxybenzoic acid detection in the blank control culture medium; Figure 2 The HPLC chromatogram shows the detection of p-hydroxybenzoic acid in the culture medium after fermentation by Bacillus composting strain Sys-Gs-008. Figure 3 The image shows the HPLC detection chromatogram of p-hydroxybenzoic acid in the culture medium after fermentation of Bacillus licheniformis strain Sys-Gs-011. The HPLC detection conditions were as follows: an Agilent 1290 chromatographic system, an ODS-C18 reversed-phase column (4.6 mm × 250 mm, 5 μm, Agilent), a mobile phase of acetonitrile:0.1% formic acid water = 50:50, a flow rate of 0.4 mL / min, a UV wavelength of 230 nm, and a column temperature of 30℃.

[0027] By Figure 1 and Figure 2 , Figure 3 Comparative analysis clearly shows that the p-hydroxybenzoic acid (p-hydroxybenzoic acid) content in the fermentation medium of *Bacillus compostii* strain Sys-Gs-008 and *Bacillus licheniformis* strain Sys-Gs-011 was reduced to varying degrees compared to the blank control medium. *Bacillus compostii* strain Sys-Gs-008 exhibited stronger p-hydroxybenzoic acid degradation ability than *Bacillus licheniformis* strain Sys-Gs-011. Further calculations showed that *Bacillus compostii* strain Sys-Gs-008 achieved a degradation rate of 46.8%-48.3% for 200 mg / L p-hydroxybenzoic acid in the culture medium, while *Bacillus licheniformis* strain Sys-Gs-011 achieved a degradation rate of 12.8%-18.1%. Both strains showed only moderate p-hydroxybenzoic acid degradation abilities.

[0028] Sequencing and sequence alignment and analysis

[0029] Two strains were sent to a biotechnology company for sequencing. The 16S rDNA sequences of the two strains are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The sequencing results were analyzed using BLAST in the NCBI nucleic acid database. After BLAST sequence alignment and phylogenetic analysis, the 16S rDNA sequence of strain Sys-Gs-008 was found to be similar to... Bacillus stercoris (GeneBank ID: NR_181952.1) With a similarity of 99%, it was identified as Bacillus compostii ( Bacillus stercoris The bacteria. The 16S rDNA sequence of strain Sys-Gs-011 is similar to... Bacillus licheniformis (GeneBank ID: NR_118996.1) With a similarity of 99%, it was identified as Bacillus licheniformis. Bacillus licheniformis .) bacteria.

[0030] Preservation of strains

[0031] Two strains were deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, on December 10, 2025. The accession number for Bacillus compostii Sys-Gs-008 is CCTCC NO: M20252817, and the accession number for Bacillus licheniformis Sys-Gs-011 is CCTCC NO: M20252820.

[0032] Determination of the optimal ratio of compound microbial agents

[0033] Preliminary experiments showed no antagonistic effect between the two strains. 10 μL of each of the two strains capable of degrading p-hydroxybenzoic acid was inoculated into 5 mL of LB liquid medium and cultured at 37℃ with a shaker at 150 rpm for 24 h to obtain the seed culture. Subsequently, 3 mL of the seed culture was inoculated into 150 mL of MSM medium and cultured at 28℃ with a shaker at 180 rpm for 2 days to obtain the compound bacterial culture. Five bacterial groups (AE) were set up with a compound ratio of *Bacillus compostii* Sys-Gs-008 : *Bacillus licheniformis* Sys-Gs-011 = 3:1, 2:1, 1:1, 1:2, and 1:3. P-hydroxybenzoic acid was then added to achieve a final concentration of 200 mg / L. A sterile MSM medium containing 200 mg / L p-hydroxybenzoic acid was set up as the control group (CK). The cultures were pre-cultured at 28℃ with a shaker at 180 rpm for 3 days. After the above fermentation culture, the fermentation broth was collected.

[0034] HPLC analysis was performed on the fermentation broth samples after filtration from each bacterial group to detect p-hydroxybenzoic acid. The results were compared with those of the control group to preliminarily determine the optimal ratio of the compound bacterial agent. HPLC detection is as follows: Figure 4-8 As shown in the figure, the degradation effect of p-hydroxybenzoic acid in different groups of culture media is compared. Figure 9 As shown, from Figure 9 As can be seen, after compounding, the degradation rate of p-hydroxybenzoic acid in bacterial group B was significantly improved, far exceeding the degradation rate of individual strains, reaching a degradation rate of 80.0%-81.5% for p-hydroxybenzoic acid.

[0035] Determination of the degradation ability of compound microbial agents on p-hydroxybenzoic acid in soil 10 μL of each of the two strains was inoculated into 5 mL of LB liquid medium and activated by incubation at 37℃ and 150 rpm for 24 h as seed culture. Then, 3 mL of the seed culture was inoculated into 150 mL of MSM medium and incubated at 28℃ and 180 rpm for 2 days to obtain the compound bacterial solution. This solution was then mixed according to the optimal ratio of Bacillus compostii Sys-Gs-008: Bacillus licheniformis Sys-Gs-011 = 2:1. Soil from wild ginseng cultivation in Benxi, Liaoning Province was mixed and divided into a control group and a bacterial treatment group, with three replicates in each group. Each replicate sample was placed in pots, and 2% exogenous p-hydroxybenzoic acid (p-hydroxybenzoic acid) was added to the soil volume of each pot. The bacterial treatment group was inoculated with 10 mL of bacterial solution, while the control group was inoculated with 10 mL of sterile MSM medium. After mixing, the samples were incubated at room temperature for 60 days. Soil samples were collected every 15 days for HPLC analysis, with six soil samples collected from each replicate.

[0036] 10 g of the obtained soil sample was weighed through a 100-mesh sieve and placed in a 250 mL Erlenmeyer flask. 50 mL of 2 mol / L NaOH solution was added, and the mixture was shaken at 25 ℃ and 200 r / min for 12 h, followed by standing at room temperature for 2 h. The sample was then centrifuged at 4000 r / min for 15 min, and the supernatant was collected. The pH of the supernatant was adjusted to 2.5 with 12 mol / L HCl, sonicated for 10 min, and allowed to stand for 2 h. The supernatant was filtered and extracted five times with ethyl acetate. The upper ethyl acetate layer was evaporated to dryness and reconstituted in acetonitrile. The solution was filtered through a 0.22 µm organic filter membrane and used as the HPLC sample solution for HPLC detection. The detection results are as follows: Figure 10 As shown, compared with the control group, the degradation rate of p-hydroxybenzoic acid in the treatment group increased significantly with the cultivation of microorganisms, confirming that the compound microbial agent provided in this application has the function of efficiently degrading p-hydroxybenzoic acid in soil.

[0037] Field application of compound microbial agents 10 μL of each of the two strains was inoculated into 5 mL of LB liquid medium and activated by incubation at 37℃ and 150 rpm for 24 h as seed culture. A 3% (v / v) inoculum was then added to LB medium and incubated at 28℃ and 180 rpm for 2 days to prepare the compound bacterial solution. This solution was mixed according to the optimal ratio of Bacillus compostii Sys-Gs-008: Bacillus licheniformis Sys-Gs-011 = 2:1 and set aside. The bacterial solution was diluted with water at a volume ratio of 1 L (before dilution) per acre and sprayed onto the field, followed by deep plowing. A comparison of the soil in a ginseng-co-cropping field before and after treatment with the diluted bacterial solution is shown in the figure below. Figure 11 As shown, after treatment, p-hydroxybenzoic acid in the soil of ginseng-continuously cropped fields was efficiently degraded.

Claims

1. A highly efficient compound microbial agent for degrading p-hydroxybenzoic acid, comprising Bacillus compostii (… Bacillus stercoris Sys-Gs-008 strain and Bacillus licheniformis ( Bacillus licheniformis. The Sys-Gs-011 strain, of which the preservation number of Bacillus compostii Sys-Gs-008 is: CCTCC NO: M20252817, and the preservation number of Bacillus licheniformis Sys-Gs-011 strain is: CCTCC NO: M20252820.

2. The compound microbial agent according to claim 1, characterized in that: The 16S rDNA sequence of Bacillus compostii strain Sys-Gs-008 is shown in SEQ ID NO.1, and the 16S rDNA sequence of Bacillus licheniformis strain Sys-Gs-011 is shown in SEQ ID NO.

2.

3. The compound microbial agent according to claim 1, characterized in that: The ratio of Bacillus compostii strain Sys-Gs-008 to Bacillus licheniformis strain Sys-Gs-011 in the compound microbial agent is 2:

1.

4. The application of the compound microbial agent according to claim 1 in alleviating the obstacle of continuous ginseng cropping, wherein the obstacle of continuous ginseng cropping is specifically achieved by degrading p-hydroxybenzoic acid, an autotoxic substance produced by ginseng in the soil.

5. The method for cultivating the compound microbial agent according to claim 1, characterized in that: Bacillus compostii strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 were inoculated into a culture medium and cultured to obtain a culture of the compound microbial agent.

6. The cultivation method according to claim 5, characterized in that: First, Bacillus composting strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 were separately inoculated into LB liquid medium and activated by incubation at 37℃ and 150 r / min for 24 h as seed culture. Then, the seed culture was re-inoculated into MSM medium at a volume fraction of 3% and incubated at 28℃ and 180 r / min for 2 days to obtain the bacterial cultures of Bacillus composting strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011. Then, the bacterial cultures of Bacillus composting strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 were mixed at a volume ratio of 2:1 to obtain the culture of the compound bacterial agent. The LB liquid culture medium comprises the following components: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L NaCl; The MSM culture medium comprises the following components: (NH4)2SO4 2.0 g / L, CaCl2 0.1 g / L, K2HPO4·3H2O 1.0 g / L, NaH2PO4 1.0 g / L, and MgSO4·7H2O 0.35 g / L.

7. A microbial inoculant, characterized in that: The effective components of the microbial agent include the Bacillus composting strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 as described in claim 1, or the culture of the compound agent obtained by the culture method described in claim 5.

8. A method for alleviating ginseng continuous cropping obstacles, characterized in that: Includes the following steps: The soil of ginseng-continuous cropping land was treated with the Bacillus composting strain Sys-Gs-008 and Bacillus licheniformis strain Sys-Gs-011 as described in claim 1, or the culture of the compound microbial agent obtained by the culture method described in claim 5, or the microbial agent described in claim 7.

9. The method according to claim 8, characterized in that: The culture of the compound microbial agent was diluted and sprayed onto the ginseng-continuously-cropped land.

10. The method according to claim 9, characterized in that: The culture of the compound microbial agent was diluted with water at a volume ratio of 1:40 to obtain a diluted microbial solution. The diluted microbial solution was sprayed onto the ginseng-co-cropped land at a dosage of 40L / mu, and then deep plowing was carried out.

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