Planting method for increasing chromone content in radix saposhnikoviae

Abstract

The invention provides a planting method for increasing chromone content in radix saposhnikoviae, a medium trace element composition and a microbial preparation are jointly applied to planting of radix saposhnikoviae, and the composition is CaCl2, MgSO4, FeCl3, MnCl2, CuCl2 and ZnCl2. The strain in the microbial preparation is bacillus subtilis. The medium trace element composition has significant influence on the contents of cimicifugin, cimicifugin glycoside and 5-O-methylvisammioside in the radix saposhnikoviae.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fertilizers, and particularly relates to the application of a microbial preparation and medium and trace elements in the planting of Saposhnikovia divaricata. Background Art

[0002] Saposhnikovia divaricata is the dried root of the plant Saposhnikovia divaricata (Turcz.) Schischk. of the Umbelliferae family. The roots of plants that have not bolted are dug in spring and autumn, the fibrous roots and sediment are removed, and then dried in the sun. Saposhnikovia divaricata has the effects of expelling wind and relieving exterior syndrome, eliminating dampness and relieving pain, and stopping convulsions. It is used for cold headache, rheumatic arthralgia, rubella pruritus, and tetanus.

[0003] Saposhnikovia divaricata contains components such as volatile oils, coumarins, and chromones. Among them, chromone compounds are the main components for Saposhnikovia divaricata to exert its medicinal effects. Prim-O-glucosylcimifugin and 5-O-methylvisammioside are often used as the index components in the Chinese Pharmacopoeia to evaluate the quality of Saposhnikovia divaricata, and the total amount of the two is required to be not less than 0.24%. The latest research found that 3 ’ The changes in the two components of 3-O-angeloylhyperin and prim-O-glucosylcimifugin contribute greatly to distinguishing Saposhnikovia divaricata with different growth modes and years, and they can be considered as quality markers for controlling the growth process of Saposhnikovia divaricata. 3 ’ 3-O-angeloylhyperin can be used as a substrate for peroxidase, and through the action of the enzyme, it can significantly improve the antioxidant capacity and eliminate excessive hydrogen peroxide.

[0004] Wang Yingfan et al. (Journal of Jilin Agricultural University, 2006, 28(3): 289-291) compared and analyzed the active ingredients of cultivated Saposhnikovia divaricata in the first and second years with the contents of 4 chromones and polysaccharides as indicators. The results showed that the sum of the contents of prim-O-glucosylcimifugin and 5-O-methylvisammioside in Saposhnikovia divaricata in the first and second years were 0.481% and 0.506% respectively, both far exceeding the specified limits in the Chinese Pharmacopoeia. However, the contents of prim-O-glucosylcimifugin, prim-O-glucosylcimifugin, and sec-O-glucosylhamaudol in Saposhnikovia divaricata in the second year were significantly higher than those in the first year. Only the content of 5-O-methylvisammioside in the first-year Saposhnikovia divaricata was significantly higher than that in the second-year Saposhnikovia divaricata.

[0005] Extending the planting years of Saposhnikovia divaricata can increase the content of most chromones, but at the same time, it increases the planting time cost and may lead to a decrease in the content of 5-O-methylvisammioside. Due to the limited cultivated land area in the genuine producing area, in order to pursue the economic benefits of planting Saposhnikovia divaricata, it is necessary not to extend the planting years as much as possible. This requires research on how to increase the content of chromones in the first-year Saposhnikovia divaricata.

[0006] Guo Xu et al. (Journal of South China Agricultural University, 2020, 41(4): 30-37) studied the differences in the quality of radix fangfengi cultivated from different origins and the relationship between soil factors and the quality of the medicinal materials. The results showed that the total amount of radix fangfengi chromones was significantly negatively correlated with soil electrical conductivity, available phosphorus content and available calcium content, and significantly positively correlated with available manganese content. Available phosphorus, available manganese and total phosphorus content can explain 71.8% of the total chromone information of radix fangfengi.

[0007] Plant growth requires balanced nutrition. In the growth, development and metabolism of medicinal plants, in addition to the role played by macroelements such as nitrogen, phosphorus and potassium, trace elements such as calcium, magnesium, iron, manganese and zinc are also indispensable elements. Although plants do not require large amounts of trace elements, they are crucial to plant yield and quality. Lack or excess of these elements will affect the root nutrition and physiological functions of plants, thereby affecting the accumulation of their effective chemical components, leading to reduced yield or quality of medicinal plants. Summary of the invention

[0008] The invention provides a planting method for increasing the content of chromones in siler. A medium and trace element composition and a microbial preparation are jointly applied to siler planting. The strain in the microbial preparation is Bacillus subtilis. The chromones capable of increasing the content are cimicifuga glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, chelidonol glycoside and 3'-O-angeloylchelidonol.

[0009] The trace element composition is CaCl 2 MgSO 4 、FeCl 3 、MnCl 2 , CuCl 2 、ZnCl 2 .

[0010] CaCl in the trace element composition 2 300-1200 parts by weight, MgSO 4 60-180 parts by weight, FeCl 3 15-120 parts by weight, MnCl 2 10-80 parts by weight, CuCl 2 10-80 parts by weight, ZnCl 2 5-40 parts by weight. Preferably CaCl 2 300-900 parts by weight, MgSO 4 60-120 parts by weight, FeCl 3 15-60 parts by weight, MnCl 2 10-40 parts by weight, CuCl 2 10-40 parts by weight, ZnCl 2 5-20 parts by weight. More preferably CaCl2 is 600 parts by weight, MgSO 4 is 90 parts by weight, FeCl 3 is 30 parts by weight, MnCl 2 is 20 parts by weight, CuCl 2 is 20 parts by weight, ZnCl 2 is 10 parts by weight.

[0011] The trace and minor element composition has a significant effect on the contents of prim-O-glucosylcimifugin, cimifugin and 5-O-methylvisammioside in Saposhnikovia divaricata.

[0012] The present invention unexpectedly finds that, compared with the single use of trace and minor elements, the combined application of the trace and minor element composition and the microbial agent can significantly increase the content of chromone in Saposhnikovia divaricata.

[0013] The strain in the microbial agent is selected from Bacillus Subtilis tkm-1, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No. 14950, and has been disclosed in Patent CN108865949A.

[0014] Inoculate Bacillus Subtilis tkm-1 into LB medium and culture it at 30 - 32 °C for 24 - 48 h to obtain the culture solution of Bacillus Subtilis tkm-1.

[0015] Among them, the LB medium is composed of 5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of sodium chloride, 20 g / L of glucose, and 20 g / L of agar powder.

[0016] The microbial agent also includes shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, compound fertilizers. The weight ratio of the strain culture solution to shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, compound fertilizers is 1:(1 - 10):(1 - 10):(0.1 - 1):(10 - 50):(1 - 20):(1 - 10), preferably 1:4:3:0.3:20:10:5.

[0017] The shrimp and crab shells are derived from the shells of any chitin-rich aquatic animals in rivers, lakes and seas, and the dried products are fine particle powders passed through a 20 - 100 mesh sieve. Preferably, they are shrimp shell powder, crab shell powder or shrimp and crab powder.

[0018] The collagen is selected from one or more of porcine skin collagen, fish skin collagen or bovine skin collagen.

[0019] The main components of the compound amino acids are glycine, alanine, leucine, isoleucine, valine, cystine, cysteine, methionine, threonine, serine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, glutamic acid, and aspartic acid.

[0020] The carbohydrate is selected from one or more of glucose, fructose, and sucrose.

[0021] The weight ratio of nitrogen, phosphorus, and potassium in the compound fertilizer is (10 - 20):(10 - 20):(10 - 20), preferably 15:15:15.

[0022] The preparation method of the microbial agent includes: (1) Add the Bacillus subtilis culture solution, shrimp and crab shells, collagen, and sodium chloride to water, mix, ferment at a temperature of 25 - 35°C for 3 - 5 days, and bubble and ventilate to obtain a fermentation broth; (2) Add the compound amino acids, carbohydrate, and compound fertilizer to the fermentation broth obtained in step (1), ferment at a temperature of 25 - 35°C for 3 - 5 days, and bubble and ventilate to obtain the microbial agent.

[0023] The viable count in the microbial agent obtained in step (2) is not less than 1×10 9 ~2×10 10 cfu / ml, preferably the viable count is not less than 1×10 10 cfu / ml.

[0024] The application method of the medium and trace elements and the microbial agent is to dilute the microbial agent 1 - 10 times with water to obtain a diluted solution of the microbial agent, add the above medium and trace element composition to the diluted solution of the microbial agent, mix evenly, and irrigate the roots of Saposhnikovia divaricata. The usage amount of the diluted solution of the microbial agent is 100L - 1000L per mu.

[0025] Compared with the clear water group, when the medium and trace elements and the microbial agent of the present invention are jointly applied to the cultivation of Saposhnikovia divaricata, the content of 3'-O-angeloyl-hamaudol can be increased by more than 50%, the contents of prim-O-glucosylcimifugin and 5-O-methylvisamminol can be increased by more than 80%, and the contents of cimifugin and sec-O-glucosylhamaudol can be increased by more than 150%. Description of the Drawings

[0026] Figure 1 It is the HPLC chart of the reference solution for Test Example 1 (Peak 1 is prim-O-glucosylcimifugin, Peak 2 is cimifugin, Peak 3 is 5-O-methylvisamminol, Peak 4 is sec-O-glucosylhamaudol, and Peak 5 is 3'-O-angeloyl-hamaudol).

[0027] Figure 2HPLC chromatogram of the test sample solution for Test Example 1 (Peak 1 is cimifugin, Peak 2 is cimifugin, Peak 3 is 5-O-methylvisammioside, Peak 4 is sec-O-glucosylhamaudol, Peak 5 is 3'-O-angeloylsec-O-glucosylhamaudol). Detailed implementation method

[0028] Example 1

[0029] Effect of medium and trace elements on chromone content of Saposhnikovia divaricata

[0030] In this study, the orthogonal experiment method was adopted, and the L 25 (5 6 ) orthogonal experiment table was selected. Each factor had 5 levels, with a total of 25 experiments, 3 replicates for each experiment, a total of 75 bags. The factor levels are shown in Table 1, and the experimental design is shown in Table 2.

[0031] Table 1 Orthogonal experiment factor level table (mg / kg) Factor level <![CDATA[CaCl 2 (A)]]> <![CDATA[MgSO 4 (B)]]> <![CDATA[FeCl 3 (C)]]> <![CDATA[MnCl 2 (D)]]> <![CDATA[CuCl 2 (E)]]> <![CDATA[ZnCl 2 (F)]]> 1 0 0 0 0 0 0 2 300 60 15 10 10 5 3 600 90 30 20 20 10 4 900 120 60 40 40 20 5 1200 180 120 80 80 40

[0032] Table 2 Experimental design Experiment A (mg / kg) B (mg / kg) C (mg / kg) D (mg / kg) E (mg / kg) F (mg / kg) 1 0 0 0 0 0 0 2 0 60 15 10 10 5 3 0 90 30 20 20 10 4 0 120 60 40 40 20 5 0 180 120 80 80 40 6 300 0 15 20 40 40 7 300 60 30 40 80 0 8 300 90 60 80 0 5 9 300 120 120 0 10 10 10 300 180 0 10 20 20 11 600 0 30 80 10 20 12 600 60 60 0 20 40 13 600 90 120 10 40 0 14 600 120 0 20 80 5 15 600 180 15 40 0 10 16 900 0 60 10 80 10 17 900 60 120 20 0 20 18 900 90 0 40 10 40 19 900 120 15 80 20 0 20 900 180 30 0 40 5 21 1200 0 120 40 20 5 22 1200 60 0 80 40 10 23 1200 90 15 0 80 20 24 1200 120 30 10 0 40 25 1200 180 60 20 10 0

[0033] Select plump and uniform Saposhnikovia divaricata seeds, soak them in 35°C water for 24 hours and then fish them out, sow them in a petri dish lined with two layers of moist filter paper, and place them at room temperature for germination. After the seed germ grows to about 1 cm, select seedlings with similar growth vigor and plant them into mushroom bags filled with substrate soil. Add the corresponding content of medium and trace elements to the substrate soil according to Table 2, mix evenly, divide each experiment into 3 bags, a total of 75 bags, plant 5 plants in each mushroom bag, place them in the greenhouse to grow, and cultivate them under the conditions of a daytime temperature of 25°C, a nighttime temperature of 20°C, a humidity of 50%, ventilation for 8 - 12 hours, and a light of 16 hours / darkness of 8 hours. After two true leaves grow, thin the seedlings to 3 plants.

[0034] Samples were sampled after four months of growth, the roots were rinsed clean, dried and crushed, and the chromone content was measured.

[0035] Example 2

[0036] Preparation of microbial agent dilution

[0037] 1 Kg of Bacillus subtilis tkm-1 culture solution, 4 Kg of crab powder, 3 kg of porcine skin collagen, 0.3 kg of sodium chloride, 20 kg of compound amino acids, 10 kg of fructose, 5 kg of compound fertilizer (nitrogen:phosphorus:potassium ratio 15:15:15).

[0038] Bacillus subtilis tkm-1 is from Beijing Techmax High-Tech Co., Ltd.

[0039] Preparation method of microbial agent: (1) Add 1000 L of water, Bacillus subtilis culture solution, crab powder, collagen, sodium chloride, and conduct cultivation with bubbling aeration. Control the cultivation temperature at 28 °C and the cultivation time at 4 days to obtain a fermentation broth. (2) Add compound amino acids, fructose, and compound fertilizer to the fermentation broth in step (1), continue cultivation with bubbling aeration, control the cultivation temperature at 32 °C, and the cultivation time at 3 days to obtain a microbial preparation for standby.

[0040] Conduct a detection on the bacterial density of the obtained microbial preparation, and the bacterial density is 2.2×10 11 cfu / ml.

[0041] Dilute the obtained microbial preparation 10 times with water to obtain a diluted solution of the microbial preparation.

[0042] Example 3

[0043] Influence of medium and trace elements and microbial preparation on chromone in Saposhnikovia divaricata

[0044] Select plump and uniform Saposhnikovia divaricata seeds, soak them in 35 °C water for 24 hours and then take them out. After taking them out, place them on a wet cloth, then place them in a room temperature environment, cover them with a moistened hemp sheet to achieve the purpose of germination. Then stir the seeds with sand, and then sow them on the ridge surface. Gently sweep them with a broom to cover the seeds and then slightly compact them. After the seedlings emerge evenly, in addition to conventional fertilization and watering, select 100 seedlings in each group and conduct root irrigation with the liquid according to the method in Table 3, once a month for a total of 3 times.

[0045] Table 3 Root irrigation liquid and volume Group Root irrigation solution Volume of root irrigation solution Water group Water 50 ml / plant Group 1 Water + Medium and trace element composition 50 ml / plant Group 2 Diluent of Example 2 50 ml / plant Group 3 Diluent of Example 2 + Medium and trace element composition 50 ml / plant

[0046] The composition ratio of the medium and trace element composition in Group 1 and Group 3 is the same as that of Level 3 in Example 1. Add the medium and trace elements to the diluted solution for root irrigation together. The ratio of the diluted solution to the medium and trace element composition is as follows: The medium and trace elements added to each milliliter of the diluted solution are CaCl 2 72 mg, MgSO 4 10.8 mg, FeCl 3 3.6 mg, MnCl 2 2.4 mg, CuCl 2 2.4 mg, ZnCl 2 1.2 mg.

[0047] Test Example 1

[0048] Determination of chromone content in Saposhnikovia divaricata

[0049] Determination method

[0050] Preparation of reference substance solution: Weigh appropriate amounts of prim-O-glucosylcimifugin reference substance, cimifugin reference substance, 5-O-methylvisammioside reference substance, sec-O-glucosylhamaudol reference substance, and 3'-O-angeloylsec-O-glucosylhamaudol reference substance accurately, and prepare a mixed reference substance solution with methanol such that each 1 mL contains 295.08 μg of prim-O-glucosylcimifugin, 76.51 μg of cimifugin, 356.13 μg of 5-O-methylvisammioside, 444.40 μg of sec-O-glucosylhamaudol, and 499.19 μg of 3'-O-angeloylsec-O-glucosylhamaudol.

[0051] Preparation of test solution: Take about 1.25 g of the fine powder of this product, weigh accurately, place it in a stoppered conical flask, accurately add 50 mL of methanol, weigh, reflux in a water bath for 2 h, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0052] Detection conditions: Use a GL Sciences-C18 column (4.6 mm × 250 mm, 5 μm) as the chromatographic column, use acetonitrile as mobile phase A, use 0.1% phosphoric acid solution as mobile phase B, elute according to the gradient (0 - 11 min, 15% - 21% A; 11 - 20 min, 21% - 31% A; 20 - 40 min, 31% - 83% A; 40 - 48 min, 83% - 90% A; 48 - 55 min, 90% - 100% A); the detection wavelength is 254 nm, the flow rate is 1.0 mL·min -1 , and the injection volume is 10 μL.

[0053] The HPLC chromatograms of the reference substance solution and the test solution are respectively as Figure 1 and Figure 2 shown. Peak 1 is prim-O-glucosylcimifugin, peak 2 is cimifugin, peak 3 is 5-O-methylvisammioside, peak 4 is sec-O-glucosylhamaudol, peak 5 is 3'-O-angeloylsec-O-glucosylhamaudol. Calculate the content of chromone in the test sample based on the ratio of the peak area of the test sample to that of the reference substance. The larger the ratio of the peak areas, the higher the content of this chromone.

[0054] Determination results of chromone content in the sample of Example 1

[0055] Table 4 Determination results of chromone content in Saposhnikovia divaricata (mg / g) Experiment Cimifugin Cimifugin 5-O-Methylvisammioside Sec-O-glucosylhamaudol 3’-O-Angeloylsec-O-glucosylhamaudol 1 2.04±0.07 0.22±0.03 2.41±0.12 0.21±0.03 0.15±0.04 2 1.95±0.12 0.37±0.05 2.55±0.23 0.30±0.04 0.20±0.04 3 3.46±0.21 0.27±0.05 3.12±0.15 0.52±0.09 0.35±0.04 4 4.25±0.09 0.70±0.06 3.53±0.22 0.49±0.06 0.23±0.05 5 3.41±0.26 0.68±0.04 3.52±0.18 0.42±0.06 0.21±0.03 6 3.47±0.16 0.38±0.04 3.19±0.17 0.24±0.06 0.23±0.06 7 3.42±0.22 0.53±0.06 3.15±0.20 0.34±0.06 0.23±0.05 8 2.63±0.20 0.65±0.04 2.73±0.15 0.35±0.06 0.28±0.04 9 3.21±0.13 0.29±0.05 2.53±0.19 0.37±0.04 0.28±0.05 10 3.65±0.17 0.42±0.04 3.18±0.16 0.33±0.05 0.23±0.06 11 3.19±0.11 0.62±0.07 2.95±0.11 0.36±0.03 0.20±0.05 12 3.75±0.13 0.46±0.07 2.68±0.13 0.29±0.04 0.30±0.03 13 3.04±0.10 0.31±0.03 3.37±0.38 0.39±0.06 0.29±0.05 14 3.46±0.11 0.45±0.06 3.15±0.25 0.38±0.06 0.25±0.06 15 2.99±0.12 0.38±0.03 3.30±0.33 0.48±0.07 0.19±0.03 16 4.03±0.12 0.59±0.05 2.99±0.15 0.38±0.04 0.24±0.05 17 3.68±0.15 0.48±0.05 3.67±0.31 0.39±0.04 0.31±0.02 18 2.79±0.24 0.36±0.04 3.35±0.24 0.34±0.04 0.26±0.04 19 3.23±0.06 0.35±0.07 2.94±0.14 0.45±0.07 0.35±0.07 20 4.13±0.16 0.52±0.05 3.27±0.27 0.43±0.06 0.34±0.06 21 2.78±0.25 0.58±0.07 3.41±0.24 0.29±0.03 0.37±0.05 22 2.78±0.22 0.23±0.05 2.99±0.12 0.32±0.06 0.31±0.04 23 3.06±0.28 0.74±0.08 3.15±0.19 0.40±0.06 0.26±0.05 24 3.52±0.20 0.22±0.04 3.05±0.24 0.40±0.05 0.30±0.07 25 2.90±0.23 0.19±0.03 2.94±0.18 0.38±0.03 0.28±0.07

[0056] Perform an analysis of variance on the above results, and the results are shown in Table 5

[0057] The influence of trace elements on the chromone content in Table 5 Element Cimifugin Cimifugin 5-O-Methylvisammioside Sec-O-glucosylhamaudol 3’-O-Angeloylsec-O-glucosylhamaudol <![CDATA[CaCl 2 (A)]]> P＜0.01 P＜0.01 P＜0.05 P＜0.01 P＜0.01 <![CDATA[MgSO 4 (B)]]> P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＜0.05 <![CDATA[FeCl 3 (C)]]> P＜0.01 P＜0.01 P＜0.01 P＜0.01 P＞0.05 <![CDATA[MnCl 2 (D)]]> P＜0.01 P＜0.01 P＜0.01 P＞0.05 P＞0.05 <![CDATA[CuCl 2 (E)]]> P＜0.01 P＜0.01 P＜0.05 P＞0.05 P＜0.01 <![CDATA[ZnCl 2 (F)]]> P＜0.01 P＜0.01 P＜0.01 P＜0.05 P＞0.05

[0058] The results showed that medium and trace elements had significant effects on the contents of cimifugin, cimifugin glycoside and 5-O-methylvisammioside. MnCl 2 (D) and CuCl 2 (E) had no significant effect on the content of sec-O-glucosylhamaudol. FeCl 3 (C), MnCl 2 (D), ZnCl 2 (F) had no significant effect on the content of 3'-O-angeloylsec-O-glucosylhamaudol.

[0059] Determination results of chromone content in the sample of Example 3

[0060] Table 6 Determination results of chromone content in Saposhnikovia divaricata (mg / g) Group Cimifugin Cimifugin 5-O-Methylvisammioside Sec-O-glucosylhamaudol 3’-O-Angeloylsec-O-glucosylhamaudol Water group 2.66 0.37 2.23 0.22 0.33 Group 1 3.7 0.52 2.98 0.30 0.42 Group 2 3.36 0.68 3.34 0.41 0.40 Group 3 5.07 0.95 4.12 0.63 0.52

[0061] Compared with the water group, the percentage increase of the active ingredients is shown in Table 7:

[0062] Table 7 Percentage increase of active ingredients compared with the water group (%) Group Cimifugin Cimifugin 5-O-Methylvisammioside Sec-O-glucosylhamaudol 3’-O-Angeloylsec-O-glucosylhamaudol (Group 1 - Water group) / Water group 39.10 40.54 33.63 36.36 27.27 (Group 2 - Water group) / Water group 26.32 83.78 49.78 86.36 21.21 (Group 3 - Water group) / Water group 90.60 156.76 84.75 186.36 57.58

[0063] As can be seen from Table 7, compared with the water group, groups 1-3 can all significantly increase the content of active ingredients in Saposhnikovia divaricata. Compared with groups 1 and 2, group 3 can significantly increase the content of active ingredients in Saposhnikovia divaricata.

[0064] The above content describes the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications all belong to the protection scope of the present invention.

[0065] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any way without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0066] Furthermore, any combination can be made between different embodiments of the present invention, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for increasing the content of chromone in siler, characterized in that: The medium and trace element composition and the microbial preparation are jointly applied to the planting of windproof. The strain in the microbial preparation is Bacillus subtilis, and the chromones are cimicifuga glycoside, cimicifuga glycoside, 5-O-methylvisaminol glycoside, chelidonol glycoside and 3'-O-angeloylchelidonol.

2. The planting method according to claim 1, characterized in that: The medium and trace element composition is CaCl2, MgSO4, FeCl3, MnCl2, CuCl2 and ZnCl2.

3. The planting method according to claim 2, characterized in that: The trace element composition comprises 300-1200 parts by weight of CaCl2, 60-180 parts by weight of MgSO4, 15-120 parts by weight of FeCl3, 10-80 parts by weight of MnCl2, 10-80 parts by weight of CuCl2 and 5-40 parts by weight of ZnCl2.

4. The planting method according to claim 3, characterized in that: The trace element composition comprises 300-900 parts by weight of CaCl2, 60-120 parts by weight of MgSO4, 15-60 parts by weight of FeCl3, 10-40 parts by weight of MnCl2, 10-40 parts by weight of CuCl2 and 5-20 parts by weight of ZnCl2.

5. The planting method according to claim 4, characterized in that: The trace element composition comprises 600 parts by weight of CaCl2, 90 parts by weight of MgSO4, 30 parts by weight of FeCl3, 20 parts by weight of MnCl2, 20 parts by weight of CuCl2 and 10 parts by weight of ZnCl2.

6. The planting method according to claim 1, characterized in that: The Bacillus subtilis is tkm-1, and its preservation number is CGMCC No.14950.

7. The planting method according to claim 6, characterized in that: The Bacillus subtilis is inoculated into LB culture medium and cultured at 30-32° C. for 24-48 hours to obtain a Bacillus subtilis culture solution.

8. The planting method according to claim 7, characterized in that: The microbial preparation also includes shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates and compound fertilizers.

9. The planting method according to claim 8, characterized in that: The weight ratio of the Bacillus subtilis culture solution to the shrimp and crab shells, collagen, sodium chloride, compound amino acids, carbohydrates, and compound fertilizer is 1: (1-10): (1-10): (0.1-1): (10-50): (1-20): (1-10).

10. The planting method according to claim 9, characterized in that: The microbial preparation is prepared by the following steps: (1) Adding Bacillus subtilis culture solution, shrimp and crab shells, collagen, and sodium chloride into water, mixing, fermenting at a temperature of 25 to 35° C. for 3 to 5 days, bubbling and ventilating, and obtaining a fermentation solution; (2) Adding compound amino acids, carbohydrates and compound fertilizers to the fermentation liquid of step (1), fermenting at a temperature of 25-35° C. for 3-5 days, and bubbling and aerating to obtain the product.

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