A comprehensive prevention and treatment method for iron deficiency in *Polygonatum sibiricum* grown in forests.

Abstract

This invention relates to the field of ecological cultivation technology of Chinese medicinal materials, specifically to a comprehensive prevention and control method for iron deficiency in *Polygonatum sibiricum* under forest cover. This invention addresses the symptoms of iron deficiency in *Polygonatum sibiricum* under forest cover by adjusting soil pH, providing effective iron supply, and combining a compound microbial agent of *Bacillus subtilis*, *Bacillus pumilus*, *Bacillus tekirae*, *Bacillus mycoides*, and *Bacillus amyloliquefaciens* with *Pseudomonas fluorescens* to release iron carriers. This fundamentally solves the problems of chlorosis and yellowish-white discoloration in young leaves, oily yellowish-brown spots on leaves, wilting of young shoots, elongated internodes, and easy lodging. Using *Polygonatum sibiricum* as the material, this invention proposes a three-step comprehensive approach to eliminate deficiency symptoms caused by iron deficiency during its ecological cultivation in forests. This allows the chloroplast thylakoid membrane and chlorophyll synthesis in *Polygonatum sibiricum* plants under forest cover to recover, photosynthesis to return to normal, and chlorophyll fluorescence kinetic parameters to a healthy state.

Description

Technical Field

[0001] This invention relates to the field of ecological cultivation technology of Chinese medicinal materials, specifically a comprehensive prevention and control method for iron deficiency in the rhizosphere soil microenvironment of *Polygonatum sibiricum* planted under forest cover. Background Technology

[0002] Iron is an essential micronutrient for plant growth and development, participating in many metabolic reactions, including photosynthesis. As a major factor influencing the activity of various enzymes in the cytochrome structure-porphyrin ring synthesis pathway, it plays an important role in respiration and photosynthesis, the two main sources of cellular energy, and affects many physiological processes such as nitrogen metabolism, organic acid metabolism, carbohydrate metabolism, and protoplasmic traits.

[0003] Iron deficiency hinders photosynthetic electron transport, thus impairing photosynthesis and chloroplast synthesis, resulting in typical iron-deficiency chlorosis in young leaves. Iron plays a crucial role in the synthesis and functional development of chlorophyll precursors. Most of the iron in plant cells is evenly distributed on the thylakoid membrane and is strongly correlated with chlorophyll content. Therefore, when plants are iron deficient, thylakoids easily disintegrate, leading to a decrease in chlorophyll content.

[0004] The available iron content in soil is mainly affected by various environmental factors such as parent material, redox potential, pH, organic matter, and microorganisms. Among these, pH has the greatest impact on iron content. Due to the high oxygen content at the Earth's surface, iron gradually transforms into hydroxyl oxide polymers with extremely low solubility. Soils with low pH values ​​have a relatively high concentration of soluble iron, and the minimum solubility of iron oxide is only 10 in the pH range of 7.5-8.5. -10 mol / L is far from meeting the needs of normal growth and development of plants, microorganisms, etc.

[0005] In the distribution areas of *Polygonatum sibiricum*, there are many plots of soil with neutral or slightly alkaline conditions. As *Polygonatum sibiricum* is a medicinal and edible plant with multiple physiological and health benefits and rich nutrients, its shade tolerance and perennial nature endow it with the natural endowment of forest granaries and land for storing grain. Therefore, the understory ecological cultivation of *Polygonatum sibiricum* has been widely carried out in Northeast and North China, as well as in Shandong, Henan, Shaanxi, and Hubei provinces. In some plain ecological forests, iron deficiency often occurs during the vigorous growth season, with particularly severe damage to young leaves and apical meristems. Simple fertilization can only partially alleviate the symptoms and cannot fundamentally solve the problem of negative impacts on the growth and development of *Polygonatum sibiricum* under forest ecology. Therefore, there is an urgent need to provide a comprehensive prevention and control method for iron deficiency in understory *Polygonatum sibiricum*. Summary of the Invention

[0006] This invention provides a comprehensive prevention and control method for iron deficiency in Solomon's seal under forest cover, including the following steps: S1, adjusting the pH of the rhizosphere soil: forest tending residue-based biochar is applied to the rhizosphere soil after being acidified with wood vinegar.

[0007] S11. The tree waste obtained from forest tending is crushed into particles with a diameter of 2 mm and placed in a pyrolysis carbonization furnace (4) for anaerobic pyrolysis at 520℃ for 2 h under nitrogen conditions to obtain garden biochar C0.

[0008] S12, Immerse biochar CO in a solution containing 0.5 mol·L⁻¹ -1 FeCl3 and 0.5 mol·L -1 In the FeSO4 solution, the Fe:C mass ratio is 0.56, and the biochar CO:solution mass-volume ratio is 1:8. After stirring for 30 min until homogeneous, the solution is placed in a pyrolysis carbonization furnace (4) and calcined at 430℃ for 2 h to obtain modified biochar C1.

[0009] S13. Dilute the wood vinegar stock solution 10 times. Mix the modified biochar C1 with the diluted wood vinegar stock solution at a ratio of 1g:10ml. After stirring thoroughly, let it stand and acidify for 2 days to obtain acidified modified biochar. Apply the acidified modified biochar to the root zone of the Polygonatum sibiricum under the forest in early spring by opening shallow trenches with a diameter of 10cm. Irrigate 5L of the mixed solution per square meter. Use 1500L per acre of Polygonatum sibiricum under the forest.

[0010] S2. Effective supply of iron: Apply chelated iron organic fertilizer to the rhizosphere soil of Polygonatum sibiricum under the forest after step S1, and at the same time spray the leaves with diluted chelated iron organic fertilizer multiple times to quickly alleviate the iron deficiency symptoms of Polygonatum sibiricum plants.

[0011] S21. Crush the horse hoof and foot slices, pass them through a 40-mesh sieve, take 12 kg and put them into a corrosion-resistant container, add 4.6 kg of water (pH 7.2), and mix in 48 g of Actinomadura keratinilytica (EU637009, strain WCC-2265T) bacterial agent, with a viable bacteria content of 2×10⁻⁶. 10 CFU / g, stir evenly, spray water once every 12 hours at 42-45℃ to keep it moist, and treat stubborn keratin by biodegradation for 5 days; then add 30L of 1.0mol / L sulfuric acid, stir evenly with a glass rod, and react for 12 hours; evaporate and deacidify in a water bath (≤90℃) to concentrate to 10L, slowly pour into another corrosion-resistant container containing 20L of water, evaporate and deacidify again, and repeat this operation 3 times to obtain a water chestnut hydrolyzed amino acid solution.

[0012] S22. Crush 260 kg of sesame cake into pieces less than 1 cm in diameter. Add 60 kg of millet bran to a composting tank 300 cm long, 200 cm wide, and 90 cm deep. Add 240 L of water and stir well. Slowly pour in a hydrolyzed amino acid solution from water chestnuts and stir well. Add a compound microbial agent and mix thoroughly until the total bacterial count of the mixed material is not less than 700,000 CFU / g. Record the temperature at the center of the compost pile and the ambient temperature regularly. Turn the pile every 2 days and add water to maintain a moisture content of 62% throughout the composting process. The composting time is 30 days. After the composting is completed, accurately weigh and uniformly sample at least 5 kg of the compost to determine the moisture content, protein nitrogen, total nitrogen, and mineral nitrogen (NH4+). + +NO3 - The total amino acid and organic C content were calculated, and the composting degree and protein N degradation rate of cake fertilizer under different microbial agent treatments were calculated. The compound microbial agents included one or more of yeast, Bacillus subtilis, black mold, and Pseudomonas.

[0013] S23. Add 22 kg of ferrous sulfate, add a large amount of water to completely dissolve it, and finally the liquid level reaches 50 cm. Stir well and allow the chelation reaction to continue for one week to obtain chelated iron organic fertilizer.

[0014] S24. From late April to early May, take 100L of the prepared fertilizer solution and dilute it 10 times. Spray it on the leaves 4 times, with an interval of 7-15 days between the two applications. Take 900L of the prepared fertilizer solution and dilute it 5 times. Drip irrigate the roots of the Solomon's seal rhizome under the forest with water. Each batch of prepared fertilizer can meet the needs of 10,000 square meters, or 1 hectare.

[0015] S3, high-yield siderophore-producing bacterial agent secretes siderophores;

[0016] S31, Preparation of Bacillus compound inoculant

[0017] S311. Preparation of cow dung biochar: Fresh cow dung is collected and cleaned of stones and other debris. The cow dung is spread evenly on a clean core board with a flat cement floor underneath. It is air-dried under full light and natural ventilation until the moisture content is below 18%. The dried cow dung is then preheated by drying at 105℃ for 3 hours. The preheated cow dung is then loaded into a pyrolysis carbonization furnace 4, and the heating rate is controlled at 100℃·h⁻′. When the pyrolysis temperature reaches 380℃, nitrogen source 1 is injected into the pyrolysis carbonization furnace 4 through the air inlet 3. The pyrolysis is carried out for 3 hours under oxygen-deficient conditions. After the pyrolysis carbonization furnace 4 is turned off, it is cooled to below 80℃. The pyrolyzed cow dung is cooled and stored for later use. The cow dung biochar prepared by pyrolysis is crushed in a pulverizer and passed through a 40-mesh sieve to obtain cow dung biochar.

[0018] S312. Fermentation broth preparation: Single colonies of Bacillus tekirae, Bacillus mycosis fungoides, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus pumilus were picked and inoculated into Erlenmeyer flasks containing LB medium as seed culture. The culture was incubated at 30℃ and 180 r / min for 12 h. The culture was then inoculated into a fermenter containing LB medium at a 2% inoculum and incubated for another 24 h at 30℃ and 180 r / min to obtain the fermentation broth.

[0019] S313. Preparation of Bacterial Powder: The fermentation broth cultured in the fermenter was removed, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. Maltodextrin, a preservative, was added at a ratio of 20:3, and the mixture was shaken for 30 s. It was then placed on a shaker at 150 rpm and incubated at 30°C for 30 min, followed by spray drying to obtain the bacterial powder. The bacterial powder was then uniformly mixed with cow dung biochar at a volume ratio of 23:77 to obtain a Bacillus compound inoculant. The spore content of the inoculant prepared by this formula was 3.9 × 10⁻⁶. 8 CFU·g -1 The moisture content (w) is ≤0.9%, the fineness is ≥99%, the wetting time is 33.0s, and the suspension rate reaches 81.0%. All indicators meet the national standards.

[0020] S32, Preparation of fluorescent Pseudomonas agent

[0021] S321. Activate *Pseudomonas fluorescens* on KB plates at 28°C for 24 hours. After activation, pick a single colony with an inoculation loop and inoculate it into 5 mL of liquid seed KB medium. Incubate at 28°C and 150 rpm for 24 hours. Then inoculate it into solid seed KB medium and incubate at 28°C for 24 hours. Pick a loopful of the cultured primary seed and inoculate it into 50 mL of liquid seed KB medium. Incubate at 28°C and 150 rpm for 24 hours to obtain secondary seed.

[0022] S322. The fermentation medium was first treated with 1g of 8-hydroxyquinoline to remove trace amounts of iron. The fermentation medium composition was: 1.5g glycerol, 1.0g hydrolyzed casein, 2.5g nitric acid sulfate, 2.5g dipotassium hydrogen phosphate, and 7.0g glucose, with a pH of 6.5. The culture conditions were: 200 / 500 mL flasks (200 mL of medium in a 500 mL flask), a secondary seed inoculation of 2%, and an optimal culture time of 48 hours. 3+ The concentration was 0.8 mg / L, and the final viable count was 5.0 × 10⁻⁶. 9 CFU·g -1 .

[0023] S323. Under aseptic conditions, collect *Pseudomonas fluorescens* cells in the logarithmic growth phase by centrifugation, and resuspend them in fresh liquid seed KB medium to achieve a viable count of 5.0 × 10⁻⁶.9 CFU·g -1 Add sodium carboxymethyl cellulose according to the volume ratio of bacterial solution to stabilizer = 10:1. After thorough mixing, mix with wetting agent, dispersant, protectant and carrier. The wetting agent is sodium dodecyl sulfate (9%), the dispersant is sodium tripolyphosphate (9%), the protectant is humic acid (1.2%), and the carrier is diatomaceous earth with a particle size of 4 mm (52.8%). After standing at room temperature for 48 hours, package and store at room temperature. When using, use the plate count method to detect the number of viable bacteria in the fluorescent Pseudomonas agent.

[0024] S33. Detection of the siderophore production capacity of the strain: The strain (Bacillus compound agent and fluorescent Pseudomonas agent) was activated and cultured on NB plates for 24 h. Single colonies were picked with a toothpick and inoculated onto CAS plates. After culturing at 28℃ for 2 days, single colonies were picked and inoculated into MSA medium. The culture was carried out at 28℃ and 150 r / min for 2 days. The bacterial suspension was centrifuged at 10000 r / min for 10 min. An equal volume of bacterial culture supernatant was mixed with the CAS detection solution and reacted in the dark for 1 h. The absorbance As was measured at 680 nm wavelength. At the same time, the absorbance Ar of the reaction between the blank medium and an equal volume of CAS detection solution was measured. The siderophore production capacity of the strain was analyzed by As / Ar. The As / Ar ratio was between 0 and 1. Bacteria with high siderophore production capacity had an As / Ar ratio below 0.5. The siderophore production of the strain was expressed as siderophore activity units (SU), calculated by the formula: SU = [(Ar-As) / Ar] × 100.

[0025] S4. Apply the microbial agent in early July to mid-August during the high-temperature season, and observe the disappearance of yellowing symptoms and the content of ferrous iron in the surface soil below 0.01 μmol·L⁻¹. -1 At this time, apply a high-side-carrier bacterial agent, and apply a fluorescent Pseudomonas agent near the drip irrigation line, in the soil at a depth of 5-10 cm in the rhizosphere, at a concentration of 10 g / m². 2 Apply Bacillus compound inoculant to the side of the drip irrigation line, at a depth of 5-10 cm in the soil around the roots, at a concentration of 30 g / m². 2 .

[0026] In one possible implementation, the compound microbial agent added in step S22 is Bacillus subtilis and Aspergillus niger, with a bacterial count ratio of Bacillus subtilis to Aspergillus niger of 60:2.

[0027] In one possible implementation, the degree of decomposition in step S22 is measured by the humic acid E4 / E6 ratio. The specific determination method is as follows: the sample is extracted with distilled water (the mass ratio of distilled water to sample is 20:1), filtered, and the absorbance of the filtrate is measured at wavelengths of 465 nm and 665 nm using a spectrophotometer. The ratio is then calculated as E4 / E6. The decomposition requirements are: C / N less than 6.50, protein nitrogen degradation rate greater than 22%, humic acid E4 / E6 value less than 3.50, and total amino acid content greater than 27.3 mg / g.

[0028] In one possible implementation, the LB medium in step S312 is prepared by sterilizing 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1L of distilled water at 121°C for 20min.

[0029] In one possible implementation, the liquid seed KB medium (1L) in step S321 consists of: 15mL glycerol, 20g peptone, 0.3g K2HPO4, 1.5g MgSO4·7H2O, and pH 7.0; the solid seed KB medium is Kings' B medium with 20g agar added.

[0030] In one possible implementation, the NB medium (1L) in step S33 consists of: 3.0g beef extract powder, 10.0g peptone, 5.0g NaCl, and 18.0g agar; the MSA medium (1L) consists of: 20.0g sucrose, 2.0g L-asparagine, 1.0g K2HPO4, and 0.5g MgSO4·7H2O.

[0031] In one possible implementation, the CAS medium in step S33 consists of: 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, 100 μL of 1 mmol / L CaCl2, 2 mL of 1 mmol / L MgSO4, and 1.8 g of agar per 100 mL. 5 mL each of phosphate buffer and CAS staining solution are slowly added at approximately 60°C to obtain CAS blue medium.

[0032] The composition of the CAS detection solution is as follows: Solution A: 500 mL of 1 mmol / L CAS (chromium azure), 100 mL of 0.1 mmol / L FeCl3·6H2O (prepared with 12 mmol / L hydrochloric acid); Solution B: 400 mL of 2 mmol / L HDTMA (hexadecyltrimethylammonium bromide); Slowly pour Solution A into Solution B to prepare 1 L of blue CAS staining solution.

[0033] Beneficial Effects: The comprehensive prevention and control method for iron deficiency in *Polygonatum sibiricum* under forest cover provided by this invention addresses the symptoms of iron deficiency in *Polygonatum sibiricum* under forest cover by adjusting soil pH, providing effective iron supply, and combining a compound microbial agent of *Bacillus subtilis*, *Bacillus pumilus*, *Bacillus tekirae*, *Bacillus mycoides*, and *Bacillus amyloliquefaciens* with *Pseudomonas fluorescens* agent to release iron carriers. This fundamentally solves the problems of chlorosis and yellowish-white discoloration of young leaves, oily yellowish-brown spots on leaves, wilting of young shoots, elongated internodes, and easy lodging. For physiological disorders caused by iron deficiency in *Polygonatum sibiricum* under forest cover, a three-step comprehensive approach is proposed: pH control, effective iron supply, and application of iron carrier microbial agents. This eliminates the deficiency symptoms, allowing the chloroplast thylakoid membrane and chlorophyll synthesis of *Polygonatum sibiricum* plants under forest cover to recover, photosynthesis to return to normal, and chlorophyll fluorescence kinetic parameters to show a healthy state. Attached Figure Description

[0034] Figure 1 A schematic diagram of the structure for preparing biochar from cow dung pyrolysis.

[0035] Figure 2 A diagram showing the application location of rhizosphere fungicide for Polygonatum sibiricum under forest cover.

[0036] Figure 3 The yellowing symptoms are caused by iron deficiency in *Polygonatum sibiricum* grown under forest cover.

[0037] Figure 4 The yellowing symptoms of leaves in the Polygonatum sibiricum plant in Case 1.

[0038] Figure 5 In Case 1, the iron deficiency in *Polygonatum sibiricum* manifested as severe yellowing of young leaves, interveinal chlorosis in semi-mature leaves, and milder yellowing in older leaves.

[0039] Figure 6 This is a detailed observation of the interpulsal chlorosis symptoms caused by iron deficiency in the chicken-head polygonatum in Case 1.

[0040] Figure 7 The image shows the growth status of the Polygonatum sibiricum in Case 1 after being treated by the system of this invention.

[0041] Figure 8 This refers to the mild iron deficiency symptoms of the Solomon's seal rhizome in Case 2.

[0042] Figure 9 This is a growth diagram of the Polygonatum sibiricum in Case 2 after treatment group 2.

[0043] Figure 10 This is a growth diagram of the Polygonatum sibiricum in Case 2 after treatment group 3.

[0044] Figure 11 The symptoms of iron deficiency in Polygonatum sibiricum in Case 3.

[0045] Figure 12 The iron deficiency symptoms were observed in Case 3 when Polygonatum divaricata and Polygonatum esculentum were mixed.

[0046] Figure 13 This is a growth diagram of Polygonatum sibiricum and Polygonatum tigrinum in Case 3 after treatment group 2.

[0047] In the diagram: 1. Nitrogen source; 2. Gas flow meter; 3. Gas inlet; 4. Pyrolysis carbonization furnace; 5. Gas outlet; 6. Alkali solution; 7. Operating table. Detailed Implementation

[0048] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described below, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0049] A comprehensive prevention and treatment method for iron deficiency in *Polygonatum sibiricum* grown in forests includes the following steps:

[0050] S0, Iron Deficiency Assessment;

[0051] S01, Chlorophyll Content Determination

[0052] Accurately weigh 100 mg of Polygonatum sibiricum leaves, transfer them to a mortar, add a pre-prepared mixture of anhydrous ethanol, acetone and distilled water in a ratio of 4.5:4.5:1 and a small amount of quartz sand, and grind until homogeneous. After dark treatment for 2 hours, using the extract as a control, measure the absorbance of the sample at 645 nm and 663 nm using a UV spectrophotometer, and calculate the chlorophyll content in the leaves using the Arnon formula.

[0053] S02, chlorophyll fluorescence measurement

[0054] Fluorescence parameters were measured from 8:00 to 12:00 using a MINI-PAM (ultra-portable modulated chlorophyll fluorometer) from WALZ GmbH, Germany. After a 20-minute dark reaction, saturated light intensity was applied to measure the initial fluorescence F0, maximum fluorescence Fm, and PS II maximum photon conversion efficiency Fv / Fm.

[0055] S03, Leaf Color Measurement

[0056] Leaf color parameters were measured using a fully automatic colorimeter CR-400 (Konica Minolta, Japan). The values ​​of L*, a*, and b* were recorded, with each index repeated three times. Among them, L* represents gloss brightness, with a higher value indicating higher brightness; a* represents the red-green saturation of the sample, with positive values ​​indicating red and negative values ​​indicating green. The higher the a* value, the deeper the red, and vice versa; b* represents the yellow-blue saturation of the sample, with positive values ​​indicating yellow and negative values ​​indicating blue. The higher the b* value, the deeper the yellow, and vice versa.

[0057] S04, Determination of available iron content in leaves

[0058] The leaves were washed with a 0.1% sodium dodecyl sulfate solution, then rinsed thoroughly with tap water, and rinsed three times with deionized water. The total Fe content in the leaves was determined by inductively coupled plasma atomic emission spectrometry. 2+ The content was determined by dilute hydrochloric acid extraction and o-phenanthroline colorimetric method.

[0059] S05, available iron content in soil

[0060] Available iron was determined using the diethylenetriaminepentaacetic acid (DTPA) extraction method. DTPA-CaCl2-TEA buffer solution at pH 7.3 was used as the extraction solvent to extract available iron from the soil. The iron content in the extract was determined using an atomic absorption spectrophotometer.

[0061] When the soil pH is higher than 7.2 and the available iron content is lower than 1.93-4.38 mg / kg, iron deficiency is likely to occur in *Polygonatum sibiricum* under forest cover. Symptom identification is shown in Table 1.

[0062] Table 1. Symptoms and characteristics of iron deficiency in *Polygonatum sibiricum* plants grown in forests.

[0063]

[0064] The higher the disease index, the more severe the iron deficiency in the leaves.

[0065] S1, regulation of rhizosphere soil pH;

[0066] S11. The tree waste obtained from forest tending is crushed into particles with a particle size of 2mm and placed in a pyrolysis carbonization furnace 4 for anaerobic pyrolysis at 520℃ under nitrogen conditions for 2h to obtain garden biochar C0.

[0067] S12, Immerse biochar CO in a solution containing 0.5 mol·L⁻¹ -1 FeCl3 and 0.5 mol·L -1 In a FeSO4 solution, the Fe:C mass ratio is 0.56, and the biochar CO:solution mass-volume ratio is 1:8. After stirring for 30 minutes until homogeneous, the solution is placed in a furnace and calcined at 430℃ for 2 hours to obtain modified biochar C1.

[0068] S13. The wood vinegar solution was provided by the Wood Laboratory of Beijing Forestry University. It had a pH of 3.1, an EC (electrical conductivity) of 3.48 mS / cm, and an organic carbon content of 32.87 g / L. The wood vinegar solution was diluted 10 times. Modified biochar C1 was mixed with the diluted wood vinegar solution at a ratio of 1 g: 10 ml. After thorough stirring, the mixture was allowed to stand for 2 days to acidify, resulting in acidified modified biochar with a pH of approximately 4.51, a carbon content of over 75%, and an iron content of over 40 mg / kg. In early spring, the mixture was applied to the root zone of the Polygonatum sibiricum under the forest by digging shallow trenches 10 cm deep. 5 L of the mixed solution was applied per square meter, and 1500 L was used per acre of Polygonatum sibiricum under the forest.

[0069] S2. Effective supply of iron: Apply chelated iron organic fertilizer to the rhizosphere soil of *Polygonatum odoratum* under the forest after step S1, and spray diluted chelated iron organic fertilizer on the leaves multiple times.

[0070] S21. Crush the horse hoof slices, pass them through a 40-mesh sieve, take 12 kg and put them into a corrosion-resistant container, add 4.6 kg of water with a pH of 7.2, and mix in Actinomadura keratinilytica (EU637009, strain WCC-2265). T 48g of bacterial agent, containing 2×10⁻⁶ live bacteria. 10 CFU / g, stir evenly, spray water once every 12 hours at 42-45℃ to keep it moist, and treat stubborn keratin by biodegradation for 5 days; then add 30L of 1.0mol / L sulfuric acid, stir evenly with a glass rod, and react for 12 hours; evaporate and deacidify in a water bath (≤90℃) to concentrate to 10L, slowly pour into another corrosion-resistant container containing 20L of water, evaporate and deacidify again, and repeat this operation 3 times to obtain a water chestnut hydrolyzed amino acid solution.

[0071] S22. Purchase sesame cake meal with a crude protein content of over 40% and an iron content greater than 900 mg / kg. Crush 260 kg of sesame cake meal into pieces less than 1 cm in diameter. Add 60 kg of millet bran to a composting tank with a length of 300 cm, a width of 200 cm, and a depth of 90 cm. Add 240 L of water and stir evenly. Slowly pour in a hydrolyzed amino acid solution from water chestnuts and stir evenly. Add a compound microbial agent and mix thoroughly to ensure that the total bacterial count of the mixed material is not less than 700,000 CFU / g. The compound microbial agent consists of Bacillus subtilis and Aspergillus niger, with a bacterial count ratio of 60:2. Record the temperature at the center of the compost pile and the ambient temperature at 10:00 AM daily. Turn the pile over every two days and add water to maintain a moisture content of 62% throughout the composting process. The composting time is 30 days.

[0072] After the fertilizer composting is completed, accurately weigh and evenly sample at least 5 kg of the sample to determine the moisture content, protein nitrogen, total nitrogen, and mineral nitrogen (NH4+).+ +NO3 - The total amino acid and organic C content were calculated, and the composting degree and protein N degradation rate of cake fertilizer under different microbial agent treatments were calculated.

[0073] The degree of decomposition is measured by the humic acid E4 / E6 ratio. The determination method is as follows: the sample is extracted with distilled water, filtered, and the absorbance of the filtrate is measured by spectrophotometer at wavelengths of 465 nm and 665 nm. The ratio of the absorbance values ​​at the two wavelengths is calculated, i.e., E4 / E6. The decomposition requirements are C / N less than 6.50, protein N degradation rate greater than 22%, humic acid E4 / E6 ratio less than 3.50, and total amino acid content greater than 27.3 mg / g. The mass ratio of distilled water to sample is 20:1.

[0074] S23. Add 22 kg of ferrous sulfate, add a large amount of water to completely dissolve it, and finally the liquid level reaches 50 cm. Stir evenly and allow the chelation reaction to continue for one week to obtain chelated iron organic fertilizer.

[0075] S24. From late April to early May, take 100L of the prepared fertilizer solution and dilute it 10 times, then spray it on the leaves 4 times, with an interval of 7-15 days between the two applications; take 900L of the prepared fertilizer solution and dilute it 5 times, then drip irrigate the roots of the Solomon's seal rhizome under the forest with water.

[0076] S3, high-yield iron carrier-secreting bacteria

[0077] S31, Preparation of Bacillus compound bacterial agent;

[0078] S311. Preparation of cow dung biochar: Fresh cow dung is collected, and stones and other debris are removed. The cow dung is spread evenly on a clean core board, with a flat cement floor underneath. It is air-dried under full light and natural ventilation until the moisture content is below 18%. The dried cow dung is then preheated by drying at 105℃ for 3 hours. The preheated cow dung is then loaded into pyrolysis carbonization furnace 4, and the heating rate is controlled at 100℃·h⁻¹. When the pyrolysis temperature reaches 380℃, nitrogen source 1 is introduced through the inlet. Nitrogen gas is blown into the pyrolysis carbonization furnace 4. The amount of nitrogen gas introduced can be observed by the gas flow meter 2. The pyrolysis time is 3 hours under oxygen-deficient conditions. After the pyrolysis carbonization furnace 4 is turned off, it is cooled to below 80°C. The pyrolyzed cow dung is cooled and stored for later use. The cow dung biochar prepared by pyrolysis is crushed in a pulverizer and passed through a 40-mesh sieve to obtain cow dung biochar. The nitrogen gas is finally discharged from the gas outlet 5 and absorbed by the alkali solution 6. The nitrogen source 1, gas flow meter 2, gas inlet 3, pyrolysis carbonization furnace 4, gas outlet 5, and alkali solution 6 are all located on the operating table 7.

[0079] The biochar sample was degassed at room temperature. After degassed, it was cooled and transferred to a physical adsorption analyzer for analysis to obtain the specific surface area of ​​the biochar. The value was required to be ≥130 m². 2 / g.

[0080] S312. Fermentation broth preparation: Single colonies of Bacillus tekirae, Bacillus mycosis fungoides, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus pumilus were picked and inoculated into Erlenmeyer flasks containing LB medium as seed culture. The culture was incubated at 30℃ and 180 r / min for 12 h. The culture was then inoculated into a fermenter containing LB medium at a 2% inoculum and incubated at 30℃ and 180 r / min for another 24 h to obtain the fermentation broth. The LB medium was prepared by sterilizing 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, and 1 L of distilled water at 121℃ for 20 min.

[0081] S313. Preparation of Bacterial Powder: The fermentation broth cultured in the fermenter was removed, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. Maltodextrin, a preservative, was added at a ratio of 20:3, and the mixture was shaken for 30 s. It was then placed on a shaker at 150 rpm and incubated at 30°C for 30 min, followed by spray drying to obtain the bacterial powder. The bacterial powder was then uniformly mixed with cow dung biochar at a volume ratio of 23:77 to obtain a Bacillus compound inoculant. The spore content of the inoculant prepared by this formula was 3.9 × 10⁻⁶. 8 CFU·g -1 The moisture content (w) is ≤0.9%, the fineness is ≥99%, the wetting time is 33.0s, and the suspension rate reaches 81.0%. All indicators meet the national standards.

[0082] S32, Preparation of fluorescent Pseudomonas agent

[0083] S321. Activate *Pseudomonas fluorescens* on KB plates at 28°C for 24 hours. After activation, pick a single colony with an inoculation loop and inoculate it into 5 mL of liquid seed KB medium. Incubate at 28°C and 150 rpm for 24 hours. Then inoculate it into solid seed KB medium and incubate at 28°C for 24 hours. Pick a loopful of the cultured primary seed and inoculate it into 50 mL of liquid seed KB medium. Incubate at 28°C and 150 rpm for 24 hours to obtain secondary seed. The composition of liquid seed KB medium (1 L) is: 15 mL glycerol, 20 g peptone, 0.3 g K2HPO4, 1.5 g MgSO4·7H2O, pH 7.0. Solid seed KB medium: Kings' B medium with 20 g agar added.

[0084] S322. The fermentation medium was first treated with 1g of 8-hydroxyquinoline to remove trace amounts of iron. The composition of the fermentation medium was: 1.5g glycerol, 1.0g hydrolyzed casein, 2.5g magnesium sulfate, 2.5g dipotassium hydrogen phosphate, and 7.0g glucose, with a pH of 6.5. The pH of the fermentation medium was controlled at 6.5 using ammonia and phosphoric acid. The culture conditions were: 200 / 500 mL flask volume, 2% inoculum, and an optimal culture time of 48 h. 3+ The concentration was 0.8 mg / L, and the final viable count was 5.0 × 10⁻⁶. 9 CFU·g -1 .

[0085] S323. Under aseptic conditions, collect *Pseudomonas fluorescens* cells in the logarithmic growth phase by centrifugation, and resuspend them in fresh liquid seed KB medium to achieve a viable count of 5.0 × 10⁻⁶. 9 CFU·g -1 The above steps are followed by adding sodium carboxymethyl cellulose at a volume ratio of bacterial solution to stabilizer of 10:1. After thorough mixing, the mixture is combined with a wetting agent, dispersant, protectant, and carrier. The wetting agent is sodium dodecyl sulfate (9%), the dispersant is sodium tripolyphosphate (9%), the protectant is humic acid (1.2%), and the carrier is diatomaceous earth with a particle size of 4 mm (52.8%). After being placed at room temperature for 48 hours, the mixture is packaged and stored at room temperature to obtain the fluorescent Pseudomonas aeruginosa agent.

[0086] S33. Detection of the siderophore production capacity of the strain: The strain (Bacillus compound inoculum and Pseudomonas fluorescens inoculum) was activated and cultured on NB plates for 24 h. Single colonies were picked with a toothpick and inoculated onto CAS plates. After culturing at 28℃ for 2 days, single colonies were picked and inoculated into MSA medium. The culture was carried out at 28℃ and 150 r / min for 2 days. The bacterial suspension was centrifuged at 10000 r / min for 10 min. An equal volume of bacterial culture supernatant was mixed with the CAS detection solution and reacted in the dark for 1 h. The absorbance As was measured at 680 nm. At the same time, the absorbance Ar of the reaction between the blank medium and an equal volume of CAS detection solution was measured. The siderophore production capacity of the strain was analyzed by As / Ar. The As / Ar ratio was between 0 and 1. Bacteria with high siderophore production capacity had an As / Ar ratio below 0.5. The siderophore production of the strain was expressed as siderophore activity units (SU), calculated by the formula: SU = [(Ar-As) / Ar] × 100.

[0087] The composition of NB medium (1L) is: 3.0g beef extract powder, 10.0g peptone, 5.0g NaCl, and 18.0g agar; the composition of MSA medium (1L) is: 20.0g sucrose, 2.0g L-asparagine, 1.0g K2HPO4, and 0.5g MgSO4·7H2O.

[0088] The composition of CAS medium is as follows: per 100 mL, there is 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolyzed casein, 100 μL of 1 mmol / L CaCl2, 2 mL of 1 mmol / L MgSO4, and 1.8 g of agar. At about 60 °C, 5 mL each of phosphate buffer and CAS staining solution are slowly added to obtain CAS blue medium.

[0089] The composition of the CAS detection solution is as follows: Solution A: 500 mL of 1 mmol / L CAS (chrome azure) and 100 mL of 0.1 mmol / L FeCl3·6H2O; Solution B: 400 mL of 2 mmol / L HDTMA (hexadecyltrimethylammonium bromide); Slowly pour Solution A into Solution B to prepare 1 L of blue CAS staining solution.

[0090] S4. Apply the microbial agent during the hot and humid season from early July to mid-August, and observe until the yellowing symptoms disappear and the content of ferrous iron in the surface soil is below 0.01 μmol·L⁻¹. -1 At this time, apply a high-side-carrier bacterial agent, and apply a fluorescent Pseudomonas agent near the drip irrigation line, in the soil at a depth of 5-10 cm in the rhizosphere, at a concentration of 10 g / m². 2 Apply Bacillus compound inoculant to the side of the drip irrigation line, at a depth of 5-10 cm in the soil around the roots, at a concentration of 30 g / m². 2 .

[0091] Case 1: Iron deficiency chlorosis of Polygonatum sibiricum under Sophora japonica trees in Tongzhou District, Beijing

[0092] Two-year-old Solomon's seal rhizome under a Chinese scholar tree forest in Tongzhou District, Beijing. The soil is brown alluvial soil with a pH of 8.05, available iron content of less than 2.17 mg / kg, and bulk density of 1.372 g / cm3, which is classified as slightly alkaline and iron-deficient soil. The forest stand is a plain ecological forest with the dominant tree species, the Chinese scholar tree, being 12 years old, and a canopy closure of 0.7.

[0093] Comparative experiments revealed that *Polygonatum sibiricum* planted under *Sophora japonica* forests without the implementation of the integrated technology of pH control, effective iron supply, and application of iron-carrier microbial agents began to show iron deficiency symptoms in mid-May, which became more pronounced by early June. Specifically, as follows... Figure 4 , Figure 5 and Figure 6 .

[0094] In the experimental plots treated with pH regulation, effective iron supply, and application of iron-carrier microbial agents (a ternary system treatment method), the growth of Polygonatum sibiricum was normal.

[0095] Data was collected in mid-July. Plant samples were randomly selected using a grid method, with 50 plants selected from each of the control and treatment groups. The average value was calculated after measurement. The measurement methods were as follows: Urease in the rhizosphere soil at 10 cm depth was measured using a UV spectrophotometer according to the sodium phenoxide-sodium hypochlorite colorimetric method; chlorophyll content was calculated by measuring the absorbance of the samples at 645 nm and 663 nm using a UV spectrophotometer; the chlorophyll fluorescence parameter Fv / Fm (PSII maximum photon conversion efficiency) was measured using a WALZ MINI-PAM (Germany); the leaf color saturation parameter a* was measured using a CR-400 fully automatic colorimeter (Konica Minolta, Japan); and the available iron content in leaves was determined using the dilute hydrochloric acid extraction method and the o-phenanthroline colorimetric method. The results are shown in Table 2.

[0096] Table 2. Effects of the ternary system treatment method on the prevention and treatment of iron deficiency in *Polygonatum sibiricum* under forest cover.

[0097]

[0098] As shown in Table 2, no iron deficiency was observed in the *Polygonatum sibiricum* plants under the forest canopy treated with the method of this invention. The Fv / Fm ratio in the chlorophyll fluorescence kinetics parameter represents the plant's health status; the value obtained in this experiment was greater than 0.8, indicating that the *Polygonatum sibiricum* plants treated with the ternary system were in a healthy growth state. Furthermore, the rhizosphere soil urease activity was 23% higher than the control, indicating higher soil activity and more effective supply of mineral nutrients. The application effect of this invention in Case 1 is shown in [the table]. Figure 7 .

[0099] Case 2: Ecological Cultivation of Polygonatum odoratum under Chestnut Forests in Chengde City, Hebei Province

[0100] The soil in the ecological planting area of ​​Polygonatum cyrtonema under chestnut forests in Chengde City, Hebei Province, is mountain brown soil with a pH of 7.4 and a topsoil bulk density of 1.302 g / cm³. 3 The soil has an available iron content of 4.61 mg / kg and is considered near-neutral. The canopy closure of the chestnut economic forest is 0.5.

[0101] The experiment was designed with three treatment groups: treatment group 1 was a blank control, treatment group 2 was iron-carrying biochar acidified with wood vinegar + foliar spraying of ferrous sulfate, and treatment group 3 was iron-carrying biochar acidified with wood vinegar + foliar spraying of chelated iron organic fertilizer + high-yield iron carrier bacterial agent.

[0102] like Figure 8 As shown, the iron deficiency symptoms of Polygonatum sibiricum in the blank control plot were yellowish-white at the top, lighter color between the veins of the middle leaves, and basically normal color of the lower leaves.

[0103] like Figure 9As shown, the leaf characteristics of Polygonatum sibiricum in the plot of iron-carrying biochar acidified with wood vinegar and foliar sprayed with ferrous sulfate showed that although the iron deficiency symptoms were alleviated, some of the sprayed ferrous sulfate remained on the leaf surface in the form of ferric iron, which was ineffective iron. The tender leaves at the top still showed intervein chlorosis.

[0104] like Figure 10 As shown, the *Polygonatum sibiricum* bred in treatment group three did not show obvious iron deficiency symptoms during the vigorous growth season. The specific breeding process is as follows:

[0105] a. Before the buds of Poria cocos emerge in early spring, apply iron-loaded biochar acidified with wood vinegar in shallow trenches around the roots. After one week, measure the pH value of the soil around the roots. The value will decrease by 0.3-0.5. The available iron in the soil can increase by 30-80% from the original background of 2.0-6.0 mg / kg, and the effect will last for about 6-7 weeks.

[0106] b. By May, when the stem segments of Polygonatum sibiricum have about 6-9 nodes, the pH value of the rhizosphere soil has basically recovered to the original 7.0-7.5, and the available iron in the soil around the roots of Polygonatum sibiricum has decreased to a level that is not significantly different from the content in the basal layer, that is, an average of about 4.0 mg / kg.

[0107] c. Before the Solomon's seal faces iron deficiency stress again, timely foliar spraying with chelated iron organic fertilizer can increase the iron content of the three whorls of leaves below the top from 46.13 mg / kg to 52.49 mg / kg after one week, an average increase of 13.78%.

[0108] d. Spray once every 10 days. By mid-June, the available iron content in the leaves will be maintained at a normal level of about 50 mg / kg, the soil pH value will decrease slightly by 0.1-0.2, and the average available iron content in the soil will be about 4.2 mg / kg.

[0109] e. With the arrival of the rainy season, stop applying foliar fertilizers. The soil pH value rises slowly, while the soil available iron content gradually decreases. At this time, apply fluorescent Pseudomonas aeruginosa agent and high-side-carrier Bacillus compound agent according to the water gradient. This can maintain the available iron chelated by side carriers in the roots of Polygonatum sibiricum for a long time, ensuring the full absorption of available iron.

[0110] Case 3: Ecological Cultivation of Polygonatum odoratum under Forest Trees in Jizhou District, Tianjin

[0111] The experiment was designed with two treatment groups: treatment group one was a blank control, and treatment group two was the same as treatment group three in Case 2.

[0112] Mixed sowing of Polygonatum sibiricum and Polygonatum cyrtonema under the forest canopy, such as... Figure 11 As shown, three-year-old Polygonatum cyrtonema exhibited interveinal chlorosis in the blank control experiment, such as... Figure 12As shown, *Polygonatum sibiricum* exhibited interveinal chlorosis in the blank control experiment, while *Polygonatum sibiricum* and *Polygonatum bisporum* cultured in treatment group II did not show iron deficiency symptoms. Figure 13 .

[0113] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made based on the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A comprehensive prevention and treatment method for iron deficiency of under-forest chicken head polygonatum, characterized by: Comprising the following steps, S1, adjusting the pH of the rhizosphere soil; S11, crushing the forest tending tree waste into particles with a particle size of 2 mm, placing it in a pyrolysis carbonization furnace (4) for anaerobic pyrolysis at 520°C for 2 hours under nitrogen to obtain garden biochar C0; S12. Immerse biochar CO in a solution containing 0.5 mol·L⁻¹ -1 FeCl3 and 0.5 mol·L -1 In the FeSO4 solution, the Fe:C mass ratio is 0.56, and the biochar CO:solution mass-volume ratio is 1:

8. After stirring for 30 min until homogeneous, the solution is placed in a pyrolysis carbonization furnace (4) and calcined at 430℃ for 2 h to obtain modified biochar C1. S13, diluting the wood vinegar stock solution 10 times, mixing the modified biochar C1 with the diluted wood vinegar stock solution at a ratio of 1g:10ml, stirring thoroughly, and then standing for 2 days to obtain acidified modified biochar, which is applied to the rhizosphere of the forest chicken head yellow root in early spring by digging a 10cm deep trench and applying 5L of the biochar per square meter; S2, effective supply of iron element, applying chelated iron organic fertilizer to the rhizosphere soil of the forest chicken head yellow root improved by step S1, and spraying dilute chelated iron organic fertilizer on the leaves multiple times; S3, high-yield siderophore-producing bacterial agent secretes siderophores, and the high-yield siderophore-producing bacterial agent includes fluorescent pseudomonas agent and bacillus complex bacterial agent; S32, preparation of fluorescent pseudomonas agent S321, activating the fluorescent pseudomonas on a KB plate at 28°C for 24 hours, after activation, using a inoculation loop to pick single colonies, inoculating into 5mL liquid seed KB medium, culturing at 28°C, 150 r / min in a shaker for 24 hours, then inoculating into solid seed KB medium, culturing at 28°C for 24 hours, using an inoculation loop to pick a ring of the well-cultured first-level seed, inoculating into 50mL liquid seed KB medium, culturing at 28°C, 150 r / min in a shaker for 24 hours to obtain a second-level seed; S322, the fermentation medium is first treated with 1 g of 8-hydroxyquinoline to remove trace iron, the fermentation medium is composed of glycerol 1.5 g, hydrolyzed casein 1.0 g, magnesium sulfate 2.5 g, dipotassium hydrogen phosphate 2.5 g, and glucose 7.0 g, pH is 6.5, the culture condition is that 200 mL of the fermentation medium is loaded in a 500 mL culture bottle, the secondary seed inoculation amount is 2%, and the optimal culture time is 48 h, Fe 3+ concentration is 0.8 mg / L, and the final viable cell count is 5.0×10 9 CFU·g –1 ; S323、in sterile conditions, centrifugal collection of Pseudomonas fluorescens cells in logarithmic growth phase, suspended with fresh liquid seed KB medium, so that the viable bacterial count reaches 5.0×10 9 CFU·g –1 The above, according to the volume ratio of bacteria liquid: stabilizer = 10:1, add sodium carboxymethyl cellulose, mix thoroughly, and then mix with wetting agent, dispersing agent, protective agent and carrier, wherein the wetting agent is 9% sodium dodecyl sulfate, the dispersing agent is 9% sodium tripolyphosphate, the protective agent is 1.2% humic acid, and the carrier is 52.8% diatomite, the particle size of diatomite is 4mm, after placing at room temperature for 48h, packaging, storing at room temperature, to obtain Pseudomonas fluorescens agent; S4, inoculation of microbial agent, in the high temperature and high humidity season from early July to mid-August, observe the disappearance of yellowing symptoms, the content of divalent iron element in the surface soil is less than 0.01 μmol·L -1 If the content of divalent iron element in the surface soil is more than 0.01 μmol·L, then inoculate high-yield siderophore microbial agent, inoculate Pseudomonas fluorescens microbial agent near the drip irrigation pipe line, 10 g / m 2 , inoculate Bacillus complex microbial agent far from the drip irrigation pipe line, 30 g / m 2 .

2. The method according to claim 1, characterized in that: The step S2 comprises: S21, crushing the palm piece of horse hoof, passing through 40 mesh sieve, taking 12 kg into an anti-corrosion container, adding water 4.6 kg, mixing Actinomadura keratinilytica bacterial agent 48 g, wherein the viable bacterial content is 2×10 10 CFU / g, stirring uniformly, spraying water once every 12 h at 42-45℃, keeping the wet state, treating the stubborn keratin by biodegradation for 5 days; then adding 1.0 mol / L sulfuric acid 30 L, stirring uniformly with a glass rod, reacting for 12 h; deacidifying and concentrating to 10 L, slowly injecting into another anti-corrosion container containing 20 L water, deacidifying again, a total of 3 times, obtaining horse hoof palm hydrolyzed amino acid solution; S22, crushing 260kg of sesame cake into pieces with a diameter of less than 1cm, adding 60kg of millet bran to a retting pool, adding water 240L, and stirring uniformly; slowly injecting horse hoof palm hydrolyzed amino acid solution, stirring uniformly, adding a compound bacterial agent, and thoroughly mixing and stirring uniformly; recording the center temperature of the fertilizer pile and the ambient temperature at regular intervals, turning the pile every 2 days, and supplementing water to maintain the moisture content at 62% throughout the pile preparation process, and the pile preparation time is 30 days; S23, adding 22kg of ferrous sulfate, completely dissolving by adding a large amount of water, finally the liquid level reaches 50cm, stirring uniformly, and chelating for one week to obtain chelated iron organic fertilizer; S24, in late April to early May, taking 100L of retted fertilizer liquid and diluting it 10 times, spraying it on the leaves in 4 times, with an interval of 7-15 days; taking 900L of retted fertilizer liquid and diluting it 5 times, and dripping it into the roots of the forest chicken head yellow root with water.

3. The method according to claim 2, characterized in that: The compound bacterial agent added in step S22 is Bacillus subtilis and Aspergillus niger, and the ratio of the number of Bacillus subtilis and Aspergillus niger is 60:

2.

4. The method according to claim 2, characterized in that: After the end of the fertilizer pile preparation in step S22, accurately weigh and uniformly sample more than 5kg, measure the moisture content, protein N, total N, mineral nitrogen, total amino acid, and organic C content, and calculate the composting degree and protein N degradation rate of the cake fertilizer under different bacterial agent treatments; The humus maturity is measured by humic acid E4 / E6 value, and the determination method is as follows: the sample is immersed in distilled water, filtered, and then the filtrate is measured for absorbance at 465 nm and 665 nm by a spectrophotometer, and the ratio of the absorbance at the two wavelengths, i.e., E4 / E6, is calculated; the C / N is less than 6.50, the protein N degradation rate is higher than 22%, the humic acid E4 / E6 value is less than 3.50, and the total amino acid content is greater than 27.3 mg / g, wherein the mass ratio of the distilled water to the sample is 20:

1.

5. The method according to claim 1, characterized in that: The step S3 comprises: S31, preparation of bacillus complex inoculant; S311, preparation of cow dung biochar: after collecting fresh cow dung, remove the sundries therein, lay the cow dung on a clean large core board, the lower layer is a flat cement floor, dry in the air under full light and natural ventilation conditions until the moisture content is below 18%, lay the dried cow dung on a preheating treatment for 3h at 105℃, load the preheating treated cow dung into a pyrolysis carbonization furnace (4), control the heating rate to be 100℃·h -1 -380℃, pyrolyze for 3h under anoxic conditions; after the pyrolysis carbonization furnace (4) is turned off, cool it to below 80℃, cool the pyrolyzed cow dung for standby use, put the pyrolyzed cow dung biochar into a crusher to break it, pass it through a 40-mesh sieve, and obtain the cow dung biochar; S312, preparation of fermentation liquor: single colonies of Bacillus tequilensis, Bacillus mycoides, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus pumilus are inoculated into a conical flask containing LB culture medium, and are cultured at 30 DEG C and 180 r / min for 12 h as seed liquid, and then are inoculated into a fermentation tank containing LB culture medium at a 2% inoculation amount, and are further cultured at 30 DEG C and 180 r / min for 24 h to obtain fermentation liquor; S313、Bacterial powder preparation: the fermentation broth cultured in the fermentor was taken out, centrifuged at 5000 rpm for 10 min, the supernatant was removed, the protective agent malt dextrin was added according to the ratio of 20:3, oscillated for 30 s, and placed in a shaking bed with a rotation speed of 150 rpm for 30 min at 30℃, then spray dried to obtain bacterial powder; the bacterial powder was uniformly mixed with the cow manure biochar according to the volume ratio of 23:77 to obtain bacillus complex inoculant; the bacillus content of the inoculant prepared by the formula is 3.9 x 10 8 CFU·g –1 , the moisture content is ≤0.9%, the fineness is ≥99%, the wetting time is 33.0 s, and the suspension rate reaches 81.0%.

6. The method according to claim 5, wherein the method is characterized by: The step S3 further comprises: S33, detection of iron carrier production capacity of the strain, the strain is activated and cultured on NB plate for 24 h, single colonies are picked up with a toothpick and are spotted on CAS plate, after 2 d of culture at 28 DEG C, single colonies are inoculated into MSA culture medium, and are cultured at 28 DEG C and 150 r / min for 2 d, bacterial suspension is centrifuged at 10 000 r / min for 10 min, and equal volume of bacterial culture supernatant and CAS detection solution are mixed, and are reacted for 1 h in the dark, and the absorbance As is detected at 680 nm, and the absorbance Ar of the reaction of equal volume of blank culture medium and CAS detection solution is also detected, the iron carrier production capacity of the strain is analyzed through As / Ar, As / Ar is between 0 and 1, the bacteria with high iron carrier production capacity have As / Ar lower than 0.5, and the yield of iron carrier of the strain is expressed by iron carrier activity unit SU, and the calculation formula is as follows: SU = [(Ar-As) / Ar] x 100.

7. The method according to claim 5, characterized in that: The LB culture medium in the step S312 is prepared by sterilizing 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water at 121 DEG C for 20 min.

8. The method according to claim 1, characterized in that: The composition of the liquid seed KB culture medium in the step S321 is as follows: 15 mL of glycerol, 20 g of proteose peptone, 0.3 g of K2HPO4 and 1.5 g of MgSO4·7H2O, and the pH is 7.0; the composition of the solid seed KB culture medium is as follows: 20 g of agar is added to Kings'B culture medium.

9. The method according to claim 6, characterized in that: The composition of the NB culture medium in the step S33 is as follows: 3.0 g of beef extract powder, 10.0 g of proteose peptone, 5.0 g of NaCl and 18.0 g of agar; and the composition of the MSA culture medium is as follows: 20.0 g of sucrose, 2.0 g of L-asparagine, 1.0 g of K2HPO4 and 0.5 g of MgSO4·7H2O. The composition of the CAS culture medium in the step S33 is: 1 mL of 20% sucrose solution, 3 mL of 10% acid hydrolysis casein, 100 μL of 1 mmol / L CaCl2, 2 mL of 1 mmol / L MgSO4, 1.8 g of agar, 5 mL of phosphate buffer and 5 mL of CAS dyeing solution added slowly at 60°C, to make 100 mL of CAS blue culture medium. The composition of the CAS detection solution is: A liquid: 500 mL of 1 mmol / L CAS and 100 mL of 0.1 mmol / L FeCl3·6H2O; B liquid: 400 mL of 2 mmol / L HDTMA; A liquid is slowly poured into B liquid to prepare 1 L of blue CAS dyeing solution.

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