Cultivation method for preventing and treating root rot and / or hemp mouth disease of angelica sinensis based on bacillus polymyxa fungicide

Abstract

The invention belongs to the technical field of biocontrol and angelica sinensis cultivation, and particularly relates to a cultivation method for preventing and treating angelica sinensis root rot and / or hemp mouth disease based on a bacillus polymyxa fungicide. The bacillus polymyxa fungicide is used for inhibiting the number and invasion of pathogenic bacteria in angelica sinensis rhizosphere soil, the disease index of angelica sinensis root rot and hemp mouth disease is reduced, meanwhile, the microbial diversity of the angelica sinensis rhizosphere soil is changed, and the microbial community structure of the angelica sinensis rhizosphere soil is optimized. By optimizing the fertilization mode, the chemical nitrogen fertilizer is reduced, the organic fertilizer is additionally applied, rich organic matters and trace elements are provided, and the angelica sinensis rhizosphere soil nutrition is improved, so that the angelica sinensis yield is improved, and the economic benefit of angelica sinensis planting is increased. Moreover, by intensively applying a green and environment-friendly pest killing technology in the angelica sinensis planting furrows, underground pests are effectively killed, the number of angelica sinensis rhizosphere and root explant-feeding nematodes is greatly reduced, a window for pathogenic bacteria to invade the roots of angelica sinensis is fundamentally closed, and the occurrence of angelica sinensis root rot and hemp mouth disease is effectively avoided.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol and angelica cultivation technology, specifically relating to a cultivation method for controlling root rot and / or root rot of angelica based on Bacillus polymyxa inoculant. Background Technology

[0002] Angelica sinensis ( Angelica sinensis (Oliv.) Diels Angelica sinensis, also known as Min Gui, Qin Gui, Xi Dang Gui, Chuan Gui, etc., is a perennial herbaceous plant of the Apiaceae family. It is mainly produced in Minxian County, Gansu Province, and is also cultivated in Yunnan, Sichuan, Shaanxi, Hubei and other places. Angelica sinensis has the effects of nourishing blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and promoting bowel movements. It is widely used in various departments of traditional Chinese medicine and as a food supplement.

[0003] With the development and application of new Angelica sinensis products, the increasing demand for healthy and natural products, and the expansion into overseas markets, the market demand for Angelica sinensis has been growing steadily in recent decades, and its market potential can be further explored, indicating a promising future. Therefore, farmers are highly motivated to cultivate it, leading to a continuous expansion of the planting area. However, due to the limited suitable planting areas for Angelica sinensis, the area of ​​continuous cropping has become increasingly large. Years of continuous cropping have resulted in a gradual increase in pests and diseases affecting Angelica sinensis, leading to quality degradation, declining yields, and seriously impacting the sustainable development of the Angelica sinensis industry.

[0004] Among the methods known to the inventors, soil-borne diseases are considered the most significant factor causing continuous cropping obstacles in Angelica sinensis. Root rot and root rot are the most severe, leading to a significant decline in both yield and quality, severely impacting the production and quality of authentic Angelica sinensis, significantly reducing planting efficiency, causing substantial losses to farmers, and severely dampening their enthusiasm. Furthermore, authentic Angelica sinensis producing areas are mostly located in high-altitude, remote mountainous regions with relatively backward production techniques. Farmers have weak awareness of disease prevention and control, and scientific planting techniques are outdated, resulting in widespread and frequent occurrences of root rot, root rot, and root rot, which are showing an increasing trend year by year and have become major limiting factors for Angelica sinensis production in these areas. Therefore, there is an urgent need to develop a cultivation method to prevent and control root rot and root rot in Angelica sinensis. Summary of the Invention

[0005] The purpose of this invention is to provide a cultivation method for controlling root rot and / or scab disease of Angelica sinensis based on Bacillus polymyxa inoculant. This cultivation method can effectively control root rot and scab disease of Angelica sinensis, thereby increasing the yield of Angelica sinensis and the economic benefits of Angelica sinensis cultivation.

[0006] This invention provides a cultivation method for preventing and controlling root rot and / or root rot in Angelica sinensis. The method involves applying a Bacillus polymyxa inoculant during the planting process. The application steps include: placing Angelica sinensis seedlings in seedling trenches, applying the Bacillus polymyxa inoculant, and then performing field management. The application rate of the Bacillus polymyxa inoculant is 15-225 kg / ha, and the effective viable count of the Bacillus polymyxa inoculant is 2-10 billion CFU / g.

[0007] Preferably, the cultivation method further includes fertilization and application of pesticides; the fertilization includes the application of base fertilizer and top dressing.

[0008] Preferably, the step of applying base fertilizer includes: applying base fertilizer before digging seedling trenches, wherein the base fertilizer includes nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer and organic fertilizer; the amount of nitrogen fertilizer applied is 180-210 kg / ha based on pure nitrogen, the amount of phosphorus fertilizer applied is 105-135 kg / ha based on phosphorus pentoxide, and the amount of potassium fertilizer applied is 60-75 kg / ha based on potassium oxide; the organic fertilizer includes commercial organic fertilizer, compost or manure, wherein the amount of commercial organic fertilizer applied is 2400-3000 kg / ha, the amount of compost applied is 3000-4500 kg / ha, and the amount of manure applied is 30000-45000 kg / ha.

[0009] The topdressing process includes: applying fertilizer after the first removal of the bolting seedlings of Angelica sinensis, applying the fertilizer to the Angelica sinensis planting rows, the fertilizer including nitrogen fertilizer, calculated as pure nitrogen, and the application rate of the nitrogen fertilizer is 30-45 kg / ha.

[0010] Preferably, the step of applying the insecticide includes: applying the insecticide after opening the seedling trench and before placing the Angelica dahurica seedlings; the insecticide includes high-fluorine chlorpyrifos-thiamethoxam granules or 4% diazinon granules, and the application rate of the insecticide is 15 kg / ha.

[0011] Preferably, the planting density of the Angelica seedlings is 150,000 to 170,000 plants per hectare, with a row spacing of 45 centimeters; The field management includes one or more of the following: weeding, removing bolting seedlings, and disease control.

[0012] Preferably, the disease control includes the control of brown spot disease and / or powdery mildew.

[0013] Preferably, the steps for controlling brown spot disease include: spraying with a 1500-fold dilution of 10% difenoconazole microemulsion at the initial stage of the disease, and spraying again in the same manner after 7 days; the steps for controlling powdery mildew include: spraying with a 4000-fold dilution of 40% flusilazole emulsifiable concentrate or a 2000-3000-fold dilution of 3% azoxystrobin at the initial stage of the disease, and spraying again with a 1500-fold dilution of 5% tebuconazole aqueous solution after 7 days.

[0014] Preferably, the cultivation method is to plant Angelica sinensis under full film covering; after applying the Bacillus polymyxa inoculant, cover with soil, and then cover with film.

[0015] Preferably, the Angelica seedlings are seedlings with a rhizome diameter greater than 2 mm; When selecting the Angelica sinensis seedlings, discard those with the following characteristics 1) and / or 2): 1) It has one or more of the following characteristics: seedling rot, mold, disease spots, insect damage, and breakage; 2) It has one or more of the following characteristics: rough skin, many branches, lateral roots growing backward, hard seedling texture, and lignified seedling heart.

[0016] This invention also provides the application of the cultivation method described in the above technical solution in improving the yield and / or quality of Angelica sinensis.

[0017] Beneficial effects: This invention provides a cultivation method for preventing and controlling root rot and / or root rot in Angelica sinensis. The method involves applying a Bacillus polymyxa inoculant during the planting process. The application steps include: placing Angelica sinensis seedlings in seedling trenches, applying the Bacillus polymyxa inoculant, and then performing field management. The application rate of the Bacillus polymyxa inoculant is 15-225 kg / ha, and the effective viable count of the Bacillus polymyxa inoculant is 2-10 billion CFU / g. During the cultivation of Angelica sinensis, applying the Bacillus polymyxa inoculant according to the method and dosage described in this invention can inhibit the number and invasion of pathogens in the rhizosphere soil of Angelica sinensis, showing a significant antibacterial effect, reducing the disease index of root rot and root rot, and simultaneously altering the microbial diversity of the rhizosphere soil, thus optimizing the microbial community structure of the rhizosphere soil.

[0018] Based on this, the cultivation method described in this invention includes scientific fertilization and green and environmentally friendly pest control technology. The optimized fertilization method reduces the amount of chemical nitrogen fertilizer used and increases the amount of organic fertilizer, reducing environmental pollution, providing abundant organic matter and trace elements, and improving the nutrition of the rhizosphere soil of Angelica sinensis, thereby increasing the yield of Angelica sinensis and enhancing the economic benefits of its cultivation. Furthermore, by applying green and environmentally friendly pest control technology intensively in the planting trenches of Angelica sinensis, underground pests are effectively eliminated and the number of phytophagous nematodes in the rhizosphere and ectophytic areas is significantly reduced, fundamentally closing the window for pathogens to invade the roots of Angelica sinensis and effectively preventing the occurrence of root rot and root rot. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This is a graph showing the effect of different microbial agents on the yield of Angelica sinensis in Example 2; Figure 2 This is a clustering result of OTU abundance of bacteria at the phylum level treated with different bacterial agents in Example 3; Figure 3 This is a clustering result of OTU abundance for fungi at the phylum level treated with different inoculants in Example 3; Figure 4 This is a graph showing the relative abundance of dominant bacterial genera in the rhizosphere soil of Angelica sinensis treated with different inoculants in Example 3. Figure 5 This is a graph showing the relative abundance of dominant fungal genera in the rhizosphere soil of Angelica sinensis treated with different inoculants in Example 3. Figure 6 The graph shows the effect of the cultivation method of controlling root rot and root rot of Angelica sinensis with Bacillus polymyxa inoculum in Example 4 on the yield of Angelica sinensis. Detailed Implementation

[0021] This invention provides a cultivation method for preventing and controlling root rot and / or root rot in Angelica sinensis. The method involves applying a Bacillus polymyxa inoculant during the planting process. The application steps include: placing Angelica sinensis seedlings in seedling trenches, applying the Bacillus polymyxa inoculant, and then performing field management. The application rate of the Bacillus polymyxa inoculant is 15-225 kg / ha, and the effective viable count of the Bacillus polymyxa inoculant is 2-10 billion CFU / g.

[0022] In one implementation method, the present invention selects plots of land with deep soil layers, convenient drainage, and rich organic matter content. In another implementation method, the present invention performs harrowing and tillage in mid-April; the harrowing and tillage method is not particularly limited, and conventional harrowing and tillage methods in the art can be used.

[0023] After harrowing and land preparation, as one embodiment, the present invention applies base fertilizer. As one embodiment, the base fertilizer includes nitrogen fertilizer, phosphate fertilizer, potassium fertilizer, and organic fertilizer. As one embodiment, the application rate of nitrogen fertilizer, calculated as pure nitrogen, is 180-210 kg / ha. As one embodiment, the application rate of phosphate fertilizer, calculated as phosphorus pentoxide, is 105-135 kg / ha. As one embodiment, the application rate of potassium fertilizer, calculated as potassium oxide, is 60-75 kg / ha. As one embodiment, the organic fertilizer includes commercial organic fertilizer, compost, or manure; the application rate of commercial organic fertilizer is 2400-3000 kg / ha; the application rate of compost is 3000-4500 kg / ha; the compost is high-temperature composting; and the application rate of manure is 30000-45000 kg / ha. The present invention does not specifically limit the source and preparation method of the commercial organic fertilizer, compost, and manure; they can be purchased from commercial sources or prepared according to conventional preparation methods in the art. The method of applying base fertilizer described in this invention can significantly reduce the amount of nitrogen fertilizer used and increase the amount of organic fertilizer used compared to conventional angelica planting fertilization. This provides angelica with abundant organic matter and trace elements, improves the nutrition of the rhizosphere soil, and thus increases the yield of angelica.

[0024] After applying the base fertilizer, the present invention performs trenching. The trenching method is not particularly limited; any conventional trenching method in the art can be used. As one embodiment, the row spacing is 45cm.

[0025] After trenching, as one implementation method, an insecticide is applied to the seedling trench. As one implementation method, the insecticide can be a low-toxicity, environmentally friendly granular insecticide for underground pests, or a high-fluorine chlorpyrifos-thiamethoxam granular formulation or a 4% diazinon granular formulation. As one implementation method, the application rate of the insecticide is 15 kg / ha. In this invention, the application method and selection of the insecticide are more efficient due to the triple action of contact, stomach poison, and systemic action, resulting in a longer effective period (60-90 days) and lower toxicity and environmental friendliness.

[0026] After applying the insecticide, as one implementation method, Angelica sinensis seedlings are placed in the seedling trench. As one implementation method, the Angelica sinensis seedlings of this invention are seedlings with a rhizome diameter greater than 2 mm. When selecting the Angelica sinensis seedlings, seedlings exhibiting the following characteristics 1) and / or 2) are discarded: 1) having one or more of the following characteristics: seedling rot, mold, disease spots, insect damage, and breakage; 2) having one or more of the following characteristics: rough epidermis, numerous branches, lateral root growth, hard seedling texture, and lignified seedling core. As one implementation method, the Angelica sinensis variety can be Mingui No. 1, Mingui No. 2, or Mingui No. 3, but these cannot be considered the entire scope of protection of this invention; the cultivation method of this invention can also be applied to other Angelica sinensis varieties. As one implementation method, the planting density of the Angelica sinensis seedlings is 150,000 to 170,000 plants / hectare. After placing the Angelica sinensis seedlings, Bacillus polymyxa inoculant is applied to the seedling trench. In one embodiment, when the effective viable count of the Bacillus polymyxa agent is 10 billion / gram, the application rate of the Bacillus polymyxa agent is 15-30 kg / ha; when the effective viable count of the Bacillus polymyxa agent is 2-1 billion / gram, the application rate of the Bacillus polymyxa agent is 150-225 kg / ha.

[0027] After applying the Bacillus polymyxa agent, the present invention performs soil covering and film covering.

[0028] After mulching, as one embodiment, the present invention applies topdressing after the first removal of the bolting seedlings of Angelica sinensis. As one embodiment, the topdressing is performed in early July, before rainfall. As one embodiment, the fertilizer is applied to the rows of Angelica sinensis plants. As one embodiment, the topdressing includes nitrogen fertilizer, and the application rate of the nitrogen fertilizer, calculated as pure nitrogen, is 30-45 kg / ha. In one embodiment, the cultivation method further includes field management. In one embodiment, field management includes one or more of weeding, removing bolting seedlings, and disease control. In one embodiment, disease control includes the control of brown spot and / or powdery mildew. In one embodiment, the steps for controlling brown spot include: spraying with a 1500-fold dilution of 10% difenoconazole microemulsion at the initial stage of disease, followed by a second spraying using the same method after 7 days. In one embodiment, the steps for controlling powdery mildew include: spraying with a 4000-fold dilution of 40% flusilazole EC or a 2000-3000-fold dilution of 3% azoxystrobin at the initial stage of disease, followed by a second spraying with a 1500-fold dilution of 5% tebuconazole Aqueous Solution after 7 days. During the control of brown spot and powdery mildew, those skilled in the art can select the dosage of the prepared pesticide according to the size of the Angelica sinensis seedlings, such as 600-1200 kg / ha, but this should not be considered as the entire scope of protection of this invention. In one implementation method, the bolting seedlings are removed between early July and early August.

[0029] In one implementation method, the angelica is harvested by removing the plastic film on a sunny day between mid-October and early November. The method of harvesting angelica is not particularly limited; any conventional method used in the field can be employed.

[0030] This invention also provides the application of the cultivation method described in the above technical solution in improving the yield and / or quality of Angelica sinensis. As one embodiment, improving the quality of Angelica sinensis can be achieved by increasing the content of one or more of the following: alcohol-soluble extracts, volatile oils, and ferulic acid.

[0031] The cultivation method described in this invention utilizes Bacillus polymyxa inoculant to inhibit pathogen invasion, improves the disease resistance of Angelica sinensis through rational and scientific fertilization and cultivation methods, and eliminates underground pests through green and environmentally friendly insecticides. By implementing these four steps, the occurrence or severity of Angelica sinensis root rot and root rot can be effectively prevented, thereby increasing the yield and quality of Angelica sinensis and improving the economic benefits of Angelica sinensis cultivation.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] In the following embodiments, unless otherwise specified, the field management methods are the same as those used for local Angelica sinensis cultivation.

[0034] Example 1 The effect of cultivation methods based on Bacillus polymyxa inoculants on the control of root rot and / or scab in Angelica sinensis. 1. Test materials The tested Angelica dahurica variety was Min Gui No. 1.

[0035] Test inoculants: Information on the five microbial inoculants selected for the experiment is shown in Table 1. All five inoculants are commercially available and are: Bacillus polymyxa inoculant (Product Registration Certificate No.: Microbial Fertilizer (2022) Approval No. (11443)), Bacillus colloidis inoculant (Product Registration Certificate No.: Microbial Fertilizer (2022) Approval No. (11440)), Paecilomyces lilacinus (Product Registration Certificate No.: Microbial Fertilizer (2018) Approval No. (3448)), Trichoderma harzianum (Product Registration Certificate No.: Microbial Fertilizer (2019) Approval No. (6988)), and Soil remediation inoculant (Product Registration Certificate No.: Microbial Fertilizer (2021) Approval No. (10587)). The dosage of each inoculant was based on the manufacturer's optimal recommended dosage.

[0036] Table 1 Information on the 5 tested microbial agents

[0037] Mulch film: Commercially available 35cm wide, 0.08mm thick black mulch film.

[0038] Physicochemical properties of the tested soil: organic matter 37.1 g / kg, total nitrogen 2.46 g / kg, total phosphorus 1.03 g / kg, total potassium 22.8 g / kg, available nitrogen 186 mg / kg, available phosphorus 48.9 mg / kg, available potassium 87 mg / kg, pH 6.10, EC 138.2 µs / cm.

[0039] 2. Overview of the Experimental Zone The experiment was conducted on farmland in Banyinpo Village, Huichuan Town, Weiyuan County, Dingxi City, Gansu Province. The experimental site was located at coordinates 35°02′39″N, 105°03′19″E, with an altitude of 2450 m. The average annual temperature was 5℃, the average temperature of the coldest month (January) was -12℃, the average temperature of the hottest month (July) was 15℃, the average annual precipitation was 566.4 mm, and the frost-free period was 131 days.

[0040] 3. Experimental Design This experiment employed a randomized block design with 6 treatments and 3 replicates: Treatment 1: No inoculation (CK); Treatment 2: Inoculated with Bacillus polymyxa (DN); Treatment 3: Inoculated with Bacillus colloidis (KC); Treatment 4: Inoculated with Paecilomyces lilacinus (DZ); Treatment 5: Inoculated with Trichoderma harzianum (HC); Treatment 6: Inoculated with soil remediation inoculants (TR). Each treatment received the same amount of chemical fertilizer, but no organic fertilizer was applied.

[0041] The experiment used film-side cultivation. The plot area was 13.5 m². 2Each area is planted with 6 rows of angelica, 25 plants per row, with a plant spacing of 20 cm and a row spacing of 45 cm. The previous crop was spring rapeseed. Fertilizer application: Base fertilizer: 150 kg / hm² of pure nitrogen. 2 P2O5 120 kg / hm 2 Topdressing: Apply 60 kg / hm² of pure nitrogen. 2 After applying base fertilizer and harrowing, the seedlings were transplanted. Topdressing was applied on July 7, 2023. After applying the microbial agent into the seedling furrows, the seedlings were placed, covered with soil, and then covered with mulch. The transplanting date for Angelica sinensis was April 16, 2023, and the harvest date was October 31, 2023.

[0042] 4. Measurement Method 4.1 Investigation of Angelica root rot disease After harvesting mature Angelica sinensis, the soil on the root surface was rinsed off with running water, and the incidence of root rot was investigated. Twenty plants were sampled from each plot. The evaluation grading of Angelica sinensis root rot was based on [Xin Zhongyao, Xu Hongxia, Cheng Xiurong. Bacillus subtilis (…]. Bacillus subtilis The disease prevention and growth promotion effects of strains B1 and B2 on Angelica sinensis and Astragalus membranaceus [J]. Plant Protection, 2008, 34(6):142-144. were evaluated using the method described in Table 2. The specific classification is shown in Table 2. Disease index of Angelica sinensis root rot % = 100 × ∑ (number of survey samples for each disease level × representative value of disease level) / (total number of survey samples × highest representative value of disease level). Control effect of Angelica sinensis root rot % = 100 × ((disease index of control - disease index of treatment) / disease index of control).

[0043] Table 2. Grading of Severity of Angelica Root Rot Disease

[0044] 4.2 Survey of the prevalence of oral canker disease in Angelica sinensis After the angelica root matures and is harvested, the soil on the root surface is rinsed off with running water, and the incidence of root rot is investigated. Twenty plants are sampled from each plot. The severity of root rot is graded into six levels based on the size of the lesion area relative to the total area of ​​a single root, as detailed in Table 3. The disease index and control efficacy for root rot are calculated using the same methods as for root rot.

[0045] Table 3. Severity classification of Angelica sinensis stomatitis disease.

[0046] 5. Results and Analysis Table 4. Effects of different microbial inoculants on the severity of root rot and scab disease in Angelica sinensis.

[0047] Note: Different letters in the same column indicate significant differences between treatments. P <0.05). The same applies below.

[0048] As shown in Table 4, different microbial inoculants exhibit varying degrees of effectiveness against root rot in Angelica sinensis. Treatment with Bacillus polymyxa and Bacillus collodion resulted in the lowest disease index and the best control efficacy against root rot, reaching 49.39% and 49.37% respectively. Trichoderma harzianum inoculant also showed a relatively low disease index and good control efficacy, at 34.25%. Finally, among soil remediation inoculants, Paecilomyces lilacinus showed the weakest control effect against root rot in Angelica sinensis.

[0049] Different microbial agents showed varying effects in preventing Angelica sinensis var. sarcodactylis disease. Treatments with Bacillus polymyxa, Trichoderma harzianum, and soil remediation agents resulted in the lowest disease index and the best prevention efficacy, with control efficacies of 17.42%, 16.51%, and 16.48%, respectively. Treatments with Bacillus collodion was the next best, showing a low disease index and good prevention efficacy of 11.80%. Paecilomyces lilacinus, however, showed the weakest prevention effect against Angelica sinensis var. sarcodactylis.

[0050] Therefore, it can be seen that the application of Bacillus polymyxa inoculant into the seed furrow during the sowing of Angelica sinensis, along with the supporting techniques, has a significant effect on the prevention and control of root rot and root rot in Angelica sinensis, and is better than other commonly used inoculants on the market.

[0051] Example 2 The effect of cultivation methods based on Bacillus polymyxa inoculants for controlling root rot and / or root rot in Angelica sinensis on increasing yield and efficacy of Angelica sinensis. The effects of Bacillus polymyxa inoculant on increasing the yield and efficacy of Angelica sinensis were studied using the same test materials, test areas and test designs as in steps 1-3 of Example 1.

[0052] Determination methods 1. Determination of Angelica sinensis yield Excluding the border rows, the roots of Angelica sinensis were harvested from the entire area, washed clean with high-pressure water, air-dried, weighed, and the yield of each plot was determined. The results are as follows: Figure 1 As shown in Table 5.

[0053] 2. The unit price of Angelica sinensis (Dang Gui) is calculated at 60 yuan / kg. The total production cost of Angelica sinensis is the sum of the following items: Seedling cost: 1125 kg / hm 2 ×12 yuan / kg = 13500 yuan / hm 2 Plastic film fee: 135 kg / hm 2 ×12 yuan / kg = 1620 yuan / hm 2 Pesticide cost: 900 yuan / hm² 2 Labor cost: 32,400 yuan / hm 2(Sowing + Harvesting + Land Preparation + Pesticide Application + Weeding = 7200 + 14400 + 2400 + 3600 + 4800 = 32400 yuan / hm²) 2 (Calculated at 80 yuan per workday); Fertilizer cost: 354 kg / hm² for urea. 2 ×2.65 yuan / kg = 938.1 yuan / hm 2 Diammonium phosphate 260.85 kg / hm 2 ×3.8 yuan / kg = 991.23 yuan / hm 2 Total: 50349.33 yuan / hm 2 Microbial agent cost: Calculated for each treatment based on the amount and price of microbial agent used.

[0054] Results and Analysis Applying Bacillus polymyxa inoculant to the seedbed during Angelica sinensis sowing can significantly increase the yield and economic benefits of Angelica sinensis, with its yield-increasing and efficiency-enhancing effects superior to other commercially available inoculants. Figure 1 It is evident that the treatments with Bacillus polymyxa and soil remediation inoculants resulted in the highest yield of Angelica sinensis, with increases of 32.71% and 32.48% respectively compared to the control. The treatment with Trichoderma harzianum resulted in the highest yield, with an increase of 17.36% compared to the control. The treatments with Bacillus colloidis and Paecilomyces lilacinus showed no significant yield increase compared to the control.

[0055] As shown in Table 5, the cost of applying soil remediation agents is the highest, followed by Trichoderma harzianum agents, which are also relatively expensive. Then comes Paecilomyces lilacinus, which is relatively expensive, while Bacillus polymyxa agents are relatively inexpensive. Based on production volume, the net profit and net profit increase, in descending order, are: Bacillus polymyxa agents > soil remediation agents > Trichoderma harzianum agents > Bacillus colloidis agents > control > Paecilomyces lilacinus agents. The net profit of Angelica sinensis treated with Paecilomyces lilacinus agents is lower than that of the control treatment.

[0056] Table 5. Economic Benefits of Inoculating Angelica Sinensis with Different Microbial Agents

[0057] Example 3 The effect of cultivation methods based on Bacillus polymyxa inoculants for controlling root rot and / or root rot in Angelica sinensis on improving the microecological environment of the rhizosphere soil of Angelica sinensis. The effects of Bacillus polymyxa inoculant on the improvement of the microecological environment of Angelica rhizosphere soil were studied using the same test materials, test areas and test designs as in steps 1-3 of Example 1.

[0058] 1. Measurement Method Soil DNA extraction and high-throughput sequencing Soil DNA extraction was performed using a soil total DNA extraction kit (EZNA). ® DNA was extracted using a Soil DNA Kit (Omega Bio-Tek, USA). The extracted DNA was then subjected to purity and concentration testing using a 1% agarose gel electrophoresis and a Nanodrop 2000 micro-spectrophotometer. Samples that passed quality control were sent to Shanghai Ling'en Biotechnology Co., Ltd. for 16S and ITS rDNA amplicon sequencing. Primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1) and 1492R (5'-GNTACCTTGTTACGACTT-3', SEQ ID NO:2) were used to amplify the V1-V9 regions of the 16S rDNA gene. The ITS region of the fungal ribosomal rDNA gene was amplified using primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO:3) and ITS4R (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:4), and third-generation high-throughput sequencing was performed using the PacBio platform.

[0059] Data processing Data from the PacBio platform was exported as BAM files. CCS (SMRT Link v7.0) was used to correct and filter the sequences, removing barcodes and primers. The resulting Reads were the final valid data (Clean Reads). Uparse software (Uparse v7.0.1001) was used to cluster all Clean Reads from all samples, classifying OTUs by 97% similarity and selecting representative sequences. Species annotation was performed on the representative sequences of the OTUs, and then abundance and diversity indices were analyzed for the OTUs.

[0060] Statistical analysis, graphing, and amplicon analysis of the experimental data were performed using Excel 2021, SPSS Statistics 26, Qiime 1.9.1 software (Version 1.9.1), and the Shanghai Ling'en Bioanalytical Platform (https: / / www.cloud.biomictoclass.com / ).

[0061] 2. Results and Analysis Effects of different microbial inoculants on the α-diversity of rhizosphere soil microorganisms in Angelica sinensis The results are shown in Table 6. Microbial inoculants significantly affected the α-diversity of the rhizosphere microbial community of *Angelica sinensis*. The number of observed species of bacteria and microorganisms in the rhizosphere of *Angelica sinensis* inoculated with *Bacillus polymyxa* and *Bacillus collodion* inoculations was significantly lower than the control treatment. Inoculation with *Trichoderma harzianum* significantly increased the number of observed species of bacteria and microorganisms in the rhizosphere of *Angelica sinensis*. The Chao1 index, reflecting the richness of the microbial community, showed that inoculation with *Bacillus polymyxa* and *Bacillus collodion* significantly reduced the richness of bacterial communities in the rhizosphere soil of *Angelica sinensis*, while inoculation with *Trichoderma harzianum* significantly reduced the richness of fungal communities in the rhizosphere soil of *Angelica sinensis*. The Shannon index, reflecting microbial community diversity, showed that inoculation with *Bacillus polymyxa*, *Bacillus collodion*, *Paecilomyces lilacinus*, *Trichoderma harzianum*, and soil remediation inoculants had no significant effect on the richness of bacterial and fungal communities in the rhizosphere of *Angelica sinensis*. The PD (Phylogenetic) index... Diversity reflects the diversity of the microbial community. Inoculation with Trichoderma harzianum and soil remediation agents significantly increased the diversity of the rhizosphere microbial community of Angelica sinensis. Inoculation with Bacillus polymyxa significantly increased the diversity of the rhizosphere bacterial community of Angelica sinensis. Inoculation with Bacillus collodioni significantly increased the diversity of the rhizosphere fungal community of Angelica sinensis. Inoculation with Paecilomyces lilacinus significantly decreased the diversity of the rhizosphere bacterial community of Angelica sinensis. The Simpson index reflects the evenness of the microbial community. Inoculation with Bacillus polymyxa significantly decreased the evenness of the rhizosphere bacterial community of Angelica sinensis.

[0062] Table 6. Effects of different microbial inoculants on α-diversity of microorganisms in rhizosphere soil of apple seedlings.

[0063] Effects of different microbial inoculants on the structure and composition of rhizosphere soil microorganisms of Angelica sinensis The results are as follows Figures 2-5 As shown, by Figure 2 It is evident that there are certain differences in the genetic distance between the rhizosphere bacterial communities of Angelica sinensis under different treatments. The treatments inoculated with Trichoderma harzianum and Paecilomyces lilacinus clustered together, while the control treatment was closer to them. The treatments inoculated with Bacillus polymyxa, soil remediation bacteria, and Bacillus colloidis all clustered separately. This indicates that inoculation with different bacteria can alter the structure of the rhizosphere soil bacterial community of Angelica sinensis.

[0064] Depend on Figure 3It is evident that there are certain differences in the genetic distance of the rhizosphere fungal community of Angelica sinensis under different treatments. The treatments inoculated with soil remediation inoculants and those inoculated with Bacillus polymyxa inoculants clustered together. The treatments inoculated with Trichoderma harzianum were relatively close to these treatments. The treatments inoculated with Paecilomyces lilacinus and Bacillus pectinus inoculants each clustered separately. The control treatment also clustered separately, and it was the furthest from the inoculated treatments. This indicates that the structure of the rhizosphere soil fungal community of Angelica sinensis differs depending on the inoculated inoculants.

[0065] Depend on Figure 4 It is evident that among the top 10 bacterial genera in terms of relative abundance, *Luteitalea* and *Pyrinomonas* were present in treatments inoculated with soil remediation microbial agents, *Trichoderma harzianum* agents, and *Paecilomyces lilacinus* agents, with their relative abundance increasing by 25.75%, 18.26%, and 10.22% and 33.94%, 35.38%, and 37.55%, respectively, compared to the control treatment. *Sphingomonas* was also present in treatments inoculated with soil remediation microbial agents and *Paecilomyces lilacinus* agents. The *Bacillus polymyxa*, *Bacillus colloides*, and *Bacillus colloides* inoculations were the most abundant, with relative abundances increasing by 31.17%, 21.75%, 16.88%, and 16.88%, respectively, compared to the control. *Chthoniobacter* was the most abundant in the *Pseudomonas lilacinus* and *Bacillus colloides* inoculations, with relative abundances increasing by 24.43% and 20.81%, respectively, compared to the control. *Gaiella* was less abundant in all five inoculations compared to the control.

[0066] Depend on Figure 5 It can be seen that among the top 10 fungal genera in terms of relative abundance, *Helicobacter brevipedus* (…) Trichocladium The proportion of *Bacillus colloides*, soil remediation agents, and *Trichoderma harzianum* inoculations was relatively high in these treatments, with their relative abundance increasing by 100.30%, 24.34%, and 16.26%, respectively, compared to the control treatment; *Fusarium* spp. ( Fusarium ) and Dow bacteria ( Tausonia The *Bacillus polymyxa* species accounted for a larger proportion in the treatments inoculated with the *Bacillus polymyxa* inoculant, with their relative abundance increasing by 30.05% and 35.72% respectively compared to the control treatment. However, they accounted for a smaller proportion in the treatments inoculated with the soil remediation inoculant and the *Trichoderma harzianum* inoculant, with their relative abundance decreasing by 28.90% and 15.30% and 9.58% and 26.53% respectively compared to the control treatment. *Morchella* species (… Mortierella ) were present in higher proportions in the treatments inoculated with soil remediation microbial agents and Trichoderma harzianum agents, with their relative abundance increasing by 28.68% and 29.18% respectively compared to the control treatment; *Clostridium* genus ( ThielaviaThe proportion of treatments inoculated with Bacillus polymyxa and soil remediation inoculants was relatively small, and their relative abundance decreased by 42.60% and 51.13% respectively compared with the control treatment. Tetracladium The proportion of Trichoderma species in all five treatments inoculated with different microbial agents was lower than that in the control; Trichoderma species ( Trichoderma In all five treatments inoculated with microbial agents, the proportion of Trichoderma was higher than that in the control. In particular, the treatments inoculated with Bacillus polymyxa, soil remediation agents, Trichoderma harzianum, and Bacillus colloidis agents showed that the relative abundance of Trichoderma increased by 7255.06%, 5101.47%, 2359.18%, and 1765.04%, respectively, compared with the control treatment.

[0067] The results above indicate that applying Bacillus polymyxa inoculant to the seed furrow during Angelica sinensis sowing can significantly alter the structure and diversity of the rhizosphere soil microbial community. In particular, it can recruit beneficial bacteria for the growth and development of Angelica sinensis, such as Trichoderma and Sphingomonas, thereby improving the microecological environment of the rhizosphere soil, reducing the severity of root rot and root rot, and promoting the growth of Angelica sinensis.

[0068] Example 4 The effect of a cultivation method based on Bacillus polymyxa inoculant for controlling root rot and root rot in Angelica sinensis on improving the quality and efficacy of Angelica sinensis. The steps are as follows: Control group: No Bacillus polymyxa inoculant was inoculated, no insecticides were applied to the seedbed, and no organic fertilizer was used. Other cultivation steps were the same as those for the treatment group treated with Bacillus polymyxa inoculant described below.

[0069] The cultivation site is located in Yangzhuang farmland, Huichuan Town, Weiyuan County, Dingxi City, Gansu Province. The experimental site coordinates are 35°05′19″N, 105°02′16″E, with an altitude of 2350 m. The average annual temperature is 5℃, the average temperature of the coldest month (January) is -12℃, the average temperature of the hottest month (July) is 15℃, the average annual precipitation is 566.4 mm, and the frost-free period is 131 days.

[0070] After harrowing and preparing the land in mid-April, apply base fertilizer: 180 kg / ha of pure nitrogen, 135 kg / ha of phosphorus pentoxide, 60 kg / ha of potassium oxide, and 2400 kg / ha of commercial organic fertilizer. The application method is to spread it over the entire plot.

[0071] After fertilization, harrow the land. Then, dig seedling furrows and apply insecticide in the furrows: 15 kg / ha of high-flufenicol·thiamethoxam granules. For transplanting Angelica sinensis: Select Min Gui No. 1 seedlings, discarding those that are rotten, moldy, have disease spots, insect damage, broken, or have rough skin, many branches, long lateral roots, hard texture, or lignified heartwood. Select robust seedlings with a rhizome diameter of approximately 0.5 cm. When transplanting Angelica sinensis, first place the seedlings, then apply Bacillus polymyxa inoculant to the seedling furrows. Planting density: 150,000-170,000 plants / ha, row spacing 45 cm. The application rate of the inoculant is: 15 kg / ha of Bacillus polymyxa inoculant with an effective viable count of 10 billion CFU / g. After applying the inoculant, cover with mulch film, using full mulch coverage.

[0072] In early July, after the first emergence of bolting seedlings, topdressing was applied with 45 kg / ha of pure nitrogen, and the fertilizer was applied to the rows of angelica plants.

[0073] Field management: Remove weeds in the field in a timely manner, and remove any angelica seedlings that bolt from early July to early August.

[0074] Prevention and treatment of brown spot disease in Angelica sinensis: In the early stage of the disease, spray with 10% difenoconazole microemulsion at a dilution of 1500 times, and spray again after 7 days to consolidate the effect.

[0075] Control of powdery mildew in Angelica sinensis: In the early stage of the disease, spray with 40% flusilazole EC at 4000 times dilution or 3% azoxystrobin at 2000-3000 times dilution. After 7 days, spray again with 5% tebuconazole WP at 1500 times dilution.

[0076] Harvesting: On October 10th, on a sunny day, remove the plastic film and harvest by hand or machine.

[0077] Determination of Angelica sinensis quality: The content of ferulic acid was determined by high performance liquid chromatography in Part I of the 2020 edition of the Chinese Pharmacopoeia; the content of alcohol-soluble extracts was determined by hot extraction in General Chapter 2201 of Part IV of the 2020 edition of the Chinese Pharmacopoeia; and the content of volatile oil was determined by method B in General Chapter 2204 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0078] Results and Analysis: The effects of cultivation methods on the control of root rot and root rot of Angelica sinensis by Bacillus polymyxa inoculum on the root growth of Angelica sinensis are shown in Table 7.

[0079] Table 7. Effects of cultivation methods on the control of root rot and root rot in Angelica sinensis by Bacillus polymyxa inoculant on the root growth of Angelica sinensis.

[0080] As shown in Table 7, the cultivation method of using Bacillus polymyxa inoculant to control root rot and root rot of Angelica sinensis has a significant promoting effect on the diameter of the rhizome, root length, fresh root weight and dry root weight of Angelica sinensis. Compared with the control treatment, the inoculation treatment with Bacillus polymyxa can increase the diameter of the rhizome, root length, fresh root weight and dry root weight of Angelica sinensis by 15.74%, 12.31%, 15.32% and 17.88%, respectively.

[0081] The impact of cultivation methods on the yield of Angelica sinensis, such as the control of root rot and root rot by Bacillus polymyxa inoculum. Figure 6 As shown, the cultivation method of using Bacillus polymyxa inoculant to control root rot and root rot of Angelica sinensis has a significant effect on increasing the yield of Angelica sinensis. The inoculation treatment with Bacillus polymyxa inoculant can increase the yield of Angelica sinensis by 43.08% compared with the control treatment.

[0082] The effects of cultivation methods on the control of root rot and root rot in Angelica sinensis by Bacillus polymyxa inoculum are shown in Table 8.

[0083] Table 8. Effects of Bacillus polymyxa inoculum on root rot and scab disease in Angelica sinensis.

[0084] It is evident that the cultivation method of using Bacillus polymyxa inoculants to control root rot and root rot in Angelica sinensis can significantly reduce the disease index of root rot, with a disease control effect of 59.39%. At the same time, the cultivation method of using Bacillus polymyxa inoculants to control root rot and root rot in Angelica sinensis can also significantly reduce the disease index of root rot, but the disease control effect is weaker than that for root rot. The control effect of Bacillus polymyxa inoculants on root rot can reach 29.34%.

[0085] The impact of cultivation methods for controlling root rot and root rot in Angelica sinensis with Bacillus polymyxa inoculant on the economic benefits of Angelica sinensis is shown in Table 9.

[0086] Table 9. Impact of cultivation methods for controlling root rot and root rot in Angelica sinensis with Bacillus polymyxa inoculants on the economic benefits of Angelica sinensis.

[0087] As can be seen from the economic benefit calculation in Table 9, although the cultivation method of using Bacillus polymyxa inoculant to control root rot and root rot of Angelica sinensis increased the total production cost, it significantly improved the economic benefits of Angelica sinensis cultivation by increasing the yield. Compared with the control, the total output value per hectare increased by RMB 45,411.8 and the net profit increased by RMB 44,736.18, which is very significant.

[0088] The effects of cultivation methods on the quality of Angelica sinensis, including the control of root rot and root rot by Bacillus polymyxa inoculum, are shown in Table 10.

[0089] Table 10. Effects of cultivation methods on the quality of Angelica sinensis in the control of root rot and root rot by Bacillus polymyxa inoculum.

[0090] As shown in Table 10, the cultivation method of using Bacillus polymyxa inoculant to control root rot and root rot in Angelica sinensis can significantly improve the quality of Angelica sinensis. Compared with the control, the cultivation method of using Bacillus polymyxa inoculant to control root rot and root rot in Angelica sinensis can increase the content of alcohol-soluble extract, volatile oil and ferulic acid in Angelica sinensis by 4.02%, 17.86% and 9.16%, respectively.

[0091] From the above embodiments, it can be concluded that the cultivation method described in this invention can effectively prevent and control root rot and root rot of Angelica sinensis, thereby improving the yield, quality, and economic benefits of Angelica sinensis cultivation.

[0092] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cultivation method for preventing and controlling root rot and / or root rot in Angelica sinensis, characterized in that, During the cultivation of Angelica sinensis, a Bacillus polymyxa agent is applied. The application steps include: placing Angelica sinensis seedlings in seedling trenches, applying the Bacillus polymyxa agent, and then carrying out field management; the application rate of the Bacillus polymyxa agent is 15-225 kg / ha, and the effective live bacteria count of the Bacillus polymyxa agent is 2-10 billion / gram.

2. The cultivation method according to claim 1, characterized in that, The cultivation method also includes fertilization and application of pesticides; the fertilization includes the application of base fertilizer and top dressing.

3. The cultivation method according to claim 2, characterized in that, The steps for applying base fertilizer include: applying base fertilizer before digging seedling trenches; the base fertilizer includes nitrogen fertilizer, phosphorus fertilizer, potassium fertilizer, and organic fertilizer; the application rate of nitrogen fertilizer is 180-210 kg / ha based on pure nitrogen, the application rate of phosphorus fertilizer is 105-135 kg / ha based on phosphorus pentoxide, and the application rate of potassium fertilizer is 60-75 kg / ha based on potassium oxide; the organic fertilizer includes commercial organic fertilizer, compost, or manure; the application rate of commercial organic fertilizer is 2400-3000 kg / ha, the application rate of compost is 3000-4500 kg / ha, and the application rate of manure is 30000-45000 kg / ha. The topdressing process includes: applying fertilizer after the first removal of the bolting seedlings of Angelica sinensis, applying the fertilizer to the Angelica sinensis planting rows, the fertilizer including nitrogen fertilizer, calculated as pure nitrogen, and the application rate of the nitrogen fertilizer is 30-45 kg / ha.

4. The cultivation method according to claim 2, characterized in that, The steps of applying the insecticide include: applying the insecticide after opening the seedling trench and before placing the Angelica dahurica seedlings; the insecticide includes high-fluorine chlorpyrifos-thiamethoxam granules or 4% diazinon granules, and the application rate of the insecticide is 15 kg / ha.

5. The cultivation method according to claim 1 or 2, characterized in that, The planting density of the Angelica sinensis seedlings is 150,000 to 170,000 plants per hectare, with a row spacing of 45 cm; the field management includes one or more of the following: weeding, removing bolting seedlings, and disease control.

6. The cultivation method according to claim 5, characterized in that, The disease control measures include the control of brown spot disease and / or powdery mildew.

7. The cultivation method according to claim 6, characterized in that, The steps for controlling brown spot disease include: spraying with a 1500-fold dilution of 10% difenoconazole microemulsion at the initial stage of the disease, and spraying again in the same manner after 7 days; the steps for controlling powdery mildew disease include: spraying with a 4000-fold dilution of 40% flusilazole emulsifiable concentrate or a 2000-3000-fold dilution of 3% azoxystrobin at the initial stage of the disease, and spraying again with a 1500-fold dilution of 5% tebuconazole aqueous solution after 7 days.

8. The cultivation method according to claim 1 or 2, characterized in that, The cultivation method involves planting Angelica sinensis under full film mulch; after applying the Bacillus polymyxa inoculant, the soil is covered, and then the film is covered again.

9. The cultivation method according to claim 1 or 2, characterized in that, The Angelica seedlings are those with a rhizome diameter greater than 2 mm; When selecting the Angelica sinensis seedlings, discard those with the following characteristics 1) and / or 2): 1) It has one or more of the following characteristics: seedling rot, mold, disease spots, insect damage, and breakage; 2) It has one or more of the following characteristics: rough skin, many branches, lateral roots growing backward, hard seedling texture, and lignified seedling heart.

10. The application of the cultivation method according to any one of claims 1 to 9 in improving the yield and / or quality of Angelica sinensis.

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