Composite microcapsule of bacillus subtilis and trichoderma asperellum and application thereof

Abstract

The invention discloses a composite microcapsule of bacillus subtilis and trichoderma asperellum, which is prepared by the following steps: embedding bacillus subtilis Pn1 and trichoderma asperellum Ta1 by adopting sodium alginate, and carrying out microencapsulation treatment, so that the controllable release of the bacillus subtilis Pn1 and the trichoderma asperellum Ta1 is realized; therefore, the stability of the composite biocontrol bacterium under adverse environmental factors is improved, meanwhile, the inhibition effect on root rot bacteria is enhanced, and the sodium alginate embedded bacillus subtilis Pn1 and trichoderma asperellum Ta1 have important application value in prevention and treatment of panax notoginseng biological root rot.

Description

Technical Field

[0001] This invention belongs to the field of plant biological control, specifically involving a composite microcapsule of Bacillus subtilis and Trichoderma hydathodes and its application in the biological control of root rot in Panax notoginseng. Background Technology

[0002] Sanqi [ Panax notoginseng (Burk.) FH Chen] (also known as Tianqi, Xueshen, or Renshen Sanqi) belongs to the Araliaceae family ( Araliaceae ) Ginseng ( Panax Panax notoginseng is a perennial herb and a very important traditional and precious medicinal plant in my country. Because of its long growth cycle and preference for warm, humid environments, it is highly susceptible to diseases, especially fungal diseases, when cultivated under shade netting (Su LL, Li WY, Chen XH, et al. Proline-rich protein PRPL1 enhances...). Panax notoginseng defense against Fusarium solani By regulating reactive oxygen species balance and strengthening the cell wall barrier. Plant cell and environment, 2024, 47(7): 2377-2395). Among them, root rot is an important disease of Panax notoginseng. The large-scale occurrence of root rot not only leads to a significant decrease in the yield of Panax notoginseng, but also seriously affects the quality of the medicinal material. Studies have shown that root rot of Panax notoginseng is caused by a complex infection of multiple pathogens, including bacteria, fungi and nematodes (Wu ZX, Hao ZP, Zeng Y, et al. Molecular characterization of microbial communities in the rhizosphere soils and roots of diseased and healthy). Panax notoginseng Antonie van Leeuwenhoek, 2015, 108(5): 1059-1074), including Fusarium solani ( Fusarium solani (Zhao Q, Qiu BL, Li S, et al. Osmotin-like protein gene from...) is the main pathogen of root rot in Panax notoginseng. Panax notoginseng is regulated by jasmonic acid and involved in defense responses to Fusarium solani Phytopathology, 2020, 110(8): 1419-1427. Currently, due to the lack of efficient and environmentally friendly control methods, spraying chemical pesticides is still the main means of controlling root rot. However, long-term and large-scale use not only pollutes the environment and threatens human health, but also leads to an increase in pesticide residues in Panax notoginseng, posing a hidden danger to the safe use of the medicinal material. Therefore, it is urgent to establish environmentally friendly and sustainable methods for controlling root rot.

[0003] Microbial biological control agents (MBCAs) offer an environmentally friendly, safe, and practical solution for controlling plant pathogens. Biological control effectively addresses many problems arising from chemical control, such as reducing environmental pollution and pesticide residues, while overcoming the limitations of physical control, providing a new approach for controlling root rot in Panax notoginseng. MBCAs protect crops from disease damage through multiple mechanisms of action. For example, during colonization, MBCAs can directly inhibit pathogen growth by competing for niches and nutrients, producing antimicrobial substances such as antibiotics and volatile organic compounds, and secreting hydrolytic enzymes such as cellulase, protease, and β-1,3-glucanase. Furthermore, MBCAs can induce systemic resistance (ISR) in plants, enhancing their own defense mechanisms. Therefore, MBCAs can play a positive role in disease control and plant growth and development through one or more mechanisms.

[0004] Common MBCAs mainly include two categories: fungi and bacteria. Among fungi, the most common is the genus Trichoderma ( Trichoderma Among bacteria, the most common are Bacillus spp. (spp.). Bacillus In addition, there are also Pseudomonas (spp.). Pseudomonas Bacillus subtilis (spp.), etc. Bacillus subtilis Hashem A. Tabassum B., Abd Allah E F. are members of the genus Bacillus and are common MBCAs. Their biocontrol activities against plant pathogens include biofilm formation, promoting plant growth, competing for nutrients and niches, inducing cell lysis, and activating systemic resistance in plants (Hashem A. Tabassum B, Abd Allah E F). Bacillus subtilis : A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences, 2019, 26(6):1291-1297). Trichoderma acanthosporium ( Trichoderma asperellum(Trichoderma) is a common type of MBCA (Mycorrhizae) that exerts its biocontrol function mainly through parasitism, competition for nutrients and space, secretion of secondary metabolites such as antibiotics, and induction of ISR (Induced Seed Rational Response). Loc et al. found that... T. asperellum The chitinase produced by PQ34 can inhibit mango ( Mango indicate ) and anthracnose bacteria in peppers ( Colletotrichum The growth of peanuts (spp.) is improved. Peanut underground Align the whole sclerotium ( Sclerotium rolfsii ) defense ability (Loc NH, Huy ND, Quang HT, et al. Characterization and antifungal activity of extracellular chitinase from a biocontrol fungus, Trichoderma asperellum PQ34. Mycology, 2019, 11(1):38-48). T. asperellum The xylanase secreted by ACCC30536 can activate poplar ( Populus davidiana × P. albavar. pyramidalis In addition, it can stimulate the auxin and jasmonic acid signaling pathways, promote plant growth and enhance resistance to pathogens, and also induce catalase activity to enhance its antioxidant capacity (Guo RT, Ji SD, Wang ZY, et al.). Trichoderma asperellum Xylanases promote growth and induce resistance in poplars. (Microbiological Research, 2021, 248: 126767). These studies indicate that these two biocontrol bacteria have broad application potential in plant disease control and plant growth promotion.

[0005] Microcapsules are microbial biocontrol agents that encapsulate microbial biochemical agents (MBCAs) within a protective coating or matrix using encapsulation technology. Encapsulation is a process of physically incorporating active substances (core materials) into the matrix structure of another material (wall material), followed by chemical stabilization (Mariello M, Kim K, Wu KL, et al. Recent advances in encapsulation of flexible bioelectronic implants: materials, technologies, and characterization methods. Advanced Materials, 2022, 34(34):2201129). Encapsulation of MBCAs provides a more suitable microenvironment and, combined with physical protection, safeguards them from abiotic stresses to prevent rapid decline in MBCA populations, while maintaining their metabolic activity for extended periods during storage and after application.

[0006] In the group's previous research, Liang Tingting isolated and screened a strain of Bacillus subtilis Pn1 ( Bacillus subtle Pn1 can secrete extracellular lysins such as protease, β-1,3-glucanase, and cellulase, exhibiting broad-spectrum antibacterial activity. It can effectively inhibit the growth of Fusarium solani and reduce the occurrence of root rot in Panax notoginseng. In addition, the fermentation supernatant, extracellular protein, and crude lipopeptide extract of Bacillus subtilis Pn1 can significantly inhibit the growth of Fusarium solani (Liang Tingting. Screening and Mechanism of Action of Antagonistic Bacteria for Root Rot in Panax notoginseng [D]. Yunnan: Master's Thesis of Kunming University of Science and Technology, 2023.). Li Youyu isolated and screened a Trichoderma echinococcus Ta1 strain ( Trichoderma asperellum Ta1 was found to have significant inhibitory activity against Fusarium solani, reducing the severity of root rot in Panax notoginseng and promoting its growth (Li Youyu. Mechanism of Echinosporium spp.'s effect on growth promotion and disease prevention in Panax notoginseng [D]. Yunnan: Master's thesis, Kunming University of Science and Technology, 2024). Therefore, Bacillus subtilis Pn1 and Trichoderma spp. Ta1, as MBCAs, can effectively control root rot in Panax notoginseng. However, due to the influence of environmental conditions and microbial cell vitality, their application alone may not be effective. B. subtilis Pn1 or T. asperellum Ta1 limits its full potential for biological control of root rot in Panax notoginseng. Summary of the Invention

[0007] This invention provides a composite microcapsule of Bacillus subtilis and Trichoderma echinococcosis, which uses sodium alginate to encapsulate Bacillus subtilis (… Bacillus subtilis Pn1 and Trichoderma acicularis ( Trichoderma asperellumTa1 was microencapsulated to achieve controlled release of Bacillus subtilis Pn1 and Trichoderma hydathodes Ta1, which were then applied to the prevention and control of root rot in Panax notoginseng, effectively reducing the occurrence of root rot and promoting the growth of Panax notoginseng.

[0008] The composite microcapsules of Bacillus subtilis and Trichoderma hydathodes of this invention specifically contain Bacillus subtilis (… Bacillus subtilis Pn1 bacterial suspension, Trichoderma acicularis ( Trichoderma asperellum Ta1 spore suspension and sodium alginate solution (2-3% by volume) were mixed and then added dropwise to a calcium chloride solution (1-3% by volume). The mixture was cross-linked and solidified at 100-200 rpm for 15-20 minutes. After solid-liquid separation, the solid was washed with sterile water and filtered to remove excess water. The resulting product contained ≥1×10⁻⁶ viable Bacillus subtilis cells. 11 CFU / g and Trichoderma spore content ≥ 1×10 9 The composite microcapsules are in the form of 1 g of Bacillus subtilis Pn1 bacterial suspension and Trichoderma echinosporum Ta1 spore suspension, with a volume ratio of 1:1 and a volume ratio of Bacillus subtilis Pn1 bacterial suspension to sodium alginate solution of 1:3-5.

[0009] When using the compound microcapsules of Bacillus subtilis and Trichoderma echinococcosis of this invention, the compound microcapsules are applied to the roots of Panax notoginseng during the growth process of two-year-old Panax notoginseng. The application is repeated every 2 days for a total of 7 times. Finally, the biocontrol effect of the compound microcapsules of Bacillus subtilis Pn1 and Trichoderma echinococcosis Ta1 on root rot of Panax notoginseng is evaluated.

[0010] The beneficial effects of this invention are as follows: The composite biocontrol bacteria prepared by encapsulating two antagonistic bacteria, Bacillus subtilis Pn1 and Trichoderma echinocandi Ta1, with sodium alginate enhances the inhibitory effect on the root rot pathogen of Panax notoginseng. Furthermore, microencapsulation allows for the controlled release of Bacillus subtilis Pn1 and Trichoderma echinocandi Ta1, improving their stability under adverse environmental conditions. Applying the composite microcapsules during Panax notoginseng cultivation not only reduces the occurrence of root rot but also promotes the growth of Panax notoginseng. The preparation method of this capsule is simple and effective, providing a new solution for the biological control of root rot in Panax notoginseng, and is of great value for ensuring the quality of medicinal materials and promoting the sustainable development of the industry. Attached Figure Description

[0011] Figure 1 The images show the morphology of the composite microcapsule of Bacillus subtilis Pn1 and Trichoderma hydathodes Ta1. Figures A, B, and C are schematic diagrams of the morphology under different conditions. Figure 2This is a stability diagram of Bacillus subtilis and Trichoderma echinococcus in composite microcapsules under abiotic stress; the upper figure shows the statistical results of Bacillus subtilis in composite microcapsules and unencapsulated Bacillus subtilis, and the lower figure shows the statistical results of Trichoderma echinococcus in composite microcapsules and unencapsulated Trichoderma echinococcus Ta1 spores; in the figure, Alg-BsTa represents the composite microcapsule, Bs represents unencapsulated Bacillus subtilis Pn1, and Ta represents unencapsulated Trichoderma echinococcus Ta1; Figure 3 This is a schematic diagram of the antibacterial activity test results of the composite microcapsules; in the figure, CK is the control group, Alg-BsTa is the composite microcapsule, Bs is the unencapsulated Bacillus subtilis Pn1, and Ta is the unencapsulated Trichoderma hydatopsima Ta1. Figure 4 This is a schematic diagram of the statistical results of the antibacterial activity detection of the composite microcapsules; the upper figure shows the statistical results of the colony diameter, and the lower figure shows the statistical results of the antibacterial rate. In the figure, CK is the control group, Alg-BsTa is the composite microcapsule, Bs is the unencapsulated Bacillus subtilis Pn1, and Ta is the unencapsulated Trichoderma hydatopsima Ta1. Figure 5 This is a diagram showing the biocontrol effect of Bacillus subtilis Pn1 and Trichoderma echinosporum Ta1 composite microcapsules on root rot of Panax notoginseng; Figure 6 This is a graph showing the growth-promoting effect of Bacillus subtilis Pn1 and Trichoderma echinococcosis Ta1 composite microcapsules on Panax notoginseng. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, culture media, methods, and equipment used in the present invention are conventional reagents, culture media, methods, and equipment in this technical field.

[0013] Example 1: Preparation of composite microcapsules of Bacillus subtilis Pn1 and Trichoderma echinococcosis Ta1 Bacillus subtilis Pn1 was inoculated into LB medium and cultured at 28°C with shaking at 150 rpm for 2 days. The bacterial cells were collected by centrifugation at 4°C and 10,000 rpm for 15 min, and resuspended in physiological saline to a final concentration of 1×10⁻⁶. 12 CFU / mL; *Trichoderma echinococcus* Ta1 was inoculated onto PDA plates and incubated upside down at 28°C for 4 days. After *Trichoderma echinococcus* Ta1 had completely covered the PDA plates, the plates were washed with physiological saline, the liquid was collected, and the mycelium was removed by filtration. This was the spore suspension of *Trichoderma echinococcus* Ta1, which was then adjusted to a concentration of 5 × 10⁻⁶ CFU / mL. 10Weigh 2g of sodium alginate, add 100mL of distilled water, and dissolve in a 45℃ water bath with stirring to prepare a 2% sodium alginate solution. Mix the Bacillus subtilis Pn1 bacterial suspension, Trichoderma echinococcus Ta1 spore suspension, and sodium alginate solution in a volume ratio of 1:1:4, stir well, and then slowly inject the mixture into a 1mL disposable syringe into a 2% calcium chloride solution (g:mL). Immobilize the mixture in a shaker at 150rpm for 15min to obtain microcapsules. Rinse the microcapsules three times with sterile water to remove excess calcium ions and unencapsulated Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1 from the surface of the microcapsules. Filter to obtain sodium alginate-encapsulated Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1 composite microcapsules (Alg-BsTa) (the concentration of Bacillus subtilis Pn1 after encapsulation is 1.466×10⁻⁶). 11 CFP / mL, the concentration of Trichoderma echinocandes Ta1 after embedding was 7.8 × 10⁻⁶. 9 The collected composite microcapsules (number / mL) were stored at 4℃ for later use. The morphology of the composite microcapsules is as follows: Figure 1 As shown, the composite microcapsules are approximately spherical individual solid particles with a particle size mainly ranging from 1.8 to 2.1 mm. Furthermore, the composite microcapsules exhibit no adhesion, are light green in color, and have no odor.

[0014] Example 2: Stability analysis of Bacillus subtilis and Trichoderma hydatopsica in composite microcapsules under abiotic stress The protective effects of composite microcapsules against Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1 under abiotic stresses, including high temperature (55℃), ultraviolet (UV), strong acid (HCl, pH=4), strong alkali (NaOH, pH=10), high salt (0.2 mol / L NaCl), and hydrogen peroxide (0.05 mol / L H2O2) stress, were investigated. 2 g of composite microcapsules were weighed and placed in 10 mL centrifuge tubes, 3 mL of physiological saline was added, and the tubes were incubated at 55℃ for 1 h under UV light, respectively. 2 g of composite microcapsules were also weighed and placed in 10 mL centrifuge tubes containing 3 mL of HCl solution (pH=4), NaOH solution (pH=10), H2O2 solution, and NaCl (0.2 mol / L) solution, respectively, and incubated at room temperature for 24 h. After treatment, 3 mL of lysis buffer (0.06 mol / L sodium citrate, 0.2 mol / L sodium bicarbonate) was added and the mixture was shaken to form a gel solution. A mixture of equal volumes of unencapsulated *Bacillus subtilis* Pn1 and *Trichoderma echinococcus* Ta1 spore suspensions was used as a control. The viable conidia of *Bacillus subtilis* Pn1 and *Trichoderma echinococcus* Ta1 were counted using the plate dilution method. 100 μL of the treated gel solution and bacterial suspension were serially diluted and then evenly spread on LB and PDA plates. After incubation at 28°C for 24 h, colony counting was performed. LB plates were used to count viable *Bacillus subtilis* Pn1 spores; PDA plates were used to count viable *Trichoderma echinococcus* Ta1 conidia.

[0015] The results are as follows Figure 2 As shown, after treatment with abiotic stresses such as high temperature, ultraviolet light, strong acid, strong alkali, high salt, and hydrogen peroxide, the number of surviving Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1 in the composite microcapsules was significantly higher than that in the free Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1. This indicates that under abiotic stress, the composite microcapsules can effectively improve the survival rate of Bacillus subtilis Pn1 and Trichoderma echinococcus Ta1, thus ensuring their viability.

[0016] Example 3: Analysis of the broad-spectrum antibacterial activity of the composite microcapsules First, a suspension of Bacillus subtilis Pn1 bacteria (1.466 × 10⁻⁶) was prepared. 11 CFP / mL) and Trichoderma acicularis Ta1 spore suspension (concentration 7.8×10⁻⁶) 9 (Spores / mL). 400 μL of Bacillus subtilis Pn1 was inoculated into LB medium and cultured at 37℃ and 200 rpm for 24 h. The culture was then aliquoted into 1.5 mL centrifuge tubes for later use. Trichoderma echinocandes Ta1 was inoculated into PDA plates and cultured upside down at 28℃ for 4 days. After the plates were fully colonized, the plates were rinsed with sterile water, and the liquid was collected. The mycelium was removed by filtration, yielding a suspension of Trichoderma echinocandes Ta1 spores, which was then aliquoted into 1.5 mL centrifuge tubes for later use.

[0017] The broad-spectrum antibacterial activity of the composite microcapsules was detected using the plate confrontation method. Pathogens included *Fusarium solani* (…). F. nightshade Fusarium oxysporum ( F. oxysporum ), Fusarium truncatum ( F. chlamydosporum Fusarium graminearum ( ), F. grasses The pathogen was inoculated into the center of a PDA plate. Then, Bacillus subtilis Pn1 suspension, Trichoderma echinococcus Ta1 spore suspension, and composite microcapsules were inoculated in a cross-hatching pattern at a distance of 2 cm. The plate was divided into four groups: a control group inoculated only with the corresponding pathogen; a Bs group inoculated only with Bacillus subtilis Pn1 suspension; a Ta group inoculated only with Trichoderma echinococcus Ta1 spore suspension; and an Alg-BsTa group inoculated with composite microcapsules. After incubation at 28℃ for 5 days, the colony diameter was measured, and the inhibition rate was calculated using the following formula: Inhibition rate (%) = (D0 - D1) ÷ D0 × 100% Where: D0 is the pathogen colony diameter in the control group (cm); D1 is the pathogen colony diameter on the PDA plates inoculated with Bacillus subtilis Pn1 suspension, Trichoderma echinococcus Ta1 spore suspension, and composite microcapsules (cm).

[0018] The results are as follows Figure 3 , 4 As shown, compared with the control group (CK), the composite microcapsules significantly inhibited the growth of *Fusarium solani* (a type of bacteria). F. solani Fusarium oxysporum ( F. oxysporum ), Fusarium truncatum ( F. chlamydosporum ) and Fusarium graminearum ( F. graminearum The growth of multiple pathogens, including *Fusarium solani*, was inhibited. Colony growth was significantly inhibited in the Bs, Ta, and Alg-BsTa groups, but the colony diameter of pathogens in the Alg-BsTa group was significantly smaller than that in the Bs and Ta groups, while in the control group, pathogen colonies almost filled the entire plate. The colony diameter of pathogens in the Alg-BsTa group was significantly smaller than that in the Bs, Ta, and control groups, indicating that the composite microcapsules significantly inhibited the growth of pathogens. The inhibition rate of the composite microcapsules against pathogens reached over 74%, with an inhibition rate of 80.67 ± 1.36% against *Fusarium solani*. In conclusion, compared with the two unencapsulated antagonistic bacteria, the composite microcapsules exhibited stronger antibacterial activity.

[0019] Example 4: Pot experiment to evaluate the biocontrol effect of composite microcapsules The efficacy of compound microcapsules against root rot in Panax notoginseng was evaluated through a pot experiment. Two-year-old Panax notoginseng plants of uniform growth were transplanted into flowerpots and divided into five groups: CK group, Fs group, Bs-Fs group, Ta-Fs group, and Alg-BsTa-Fs group. The CK group served as the control group, receiving only sterile water; the Fs group was treated with Fusarium solani, receiving only 15 mL (1×10⁻⁶) of Fusarium solani spore suspension. 6 Spores / mL); The Bs-Fs group was a co-treatment group of Bacillus subtilis and Fusarium solani. First, 15 mL of Bacillus subtilis Pn1 suspension was applied (once every 2 days, for a total of 7 applications), followed by irrigation with 100 mL of Fusarium solani spore suspension (1×10⁻⁶). 6 Spores / mL; applied once every 2 days, for 3 applications); Ta-Fs group, i.e., the co-treatment group of Trichoderma echinococcus and Fusarium solani, was first treated with a suspension of Trichoderma echinococcus Ta1 spores (concentration 7.8 × 10⁻⁶). 9 After applying the compound microcapsules (15g / time, once every 2 days, for a total of 7 times), the plants were then irrigated with a Fusarium solani spore suspension (as above). The Alg-BsTa-Fs group, which is the group treated with both compound microcapsules and Fusarium solani, was first treated with compound microcapsules (15g / time, once every 2 days, for a total of 7 times), and then irrigated with a Fusarium solani spore suspension (as above). The potted plants were placed in a greenhouse for cultivation, and watered once every 3 days. After 50 days of cultivation, the occurrence of root rot in Panax notoginseng was observed.

[0020] The results are as follows Figure 4 As shown, the CK group showed good growth and no obvious symptoms of root rot. The Fs group, however, exhibited complete root rot symptoms, with yellowing leaves that began to wither and even the entire plant dying. Observation of the roots revealed that all the roots were rotten, with the rotten area covering more than three-quarters of the root system in most cases. Bs- Fs Group, Ta- Fs The group of Panax notoginseng exhibited more obvious symptoms of root rot, with some leaves turning yellow and beginning to wither, and the rotten area of ​​the roots reaching one-fifth. In contrast, the Alg-BsTa-Fs group showed mild root rot symptoms, with a few leaves showing localized yellowing, but most remaining green; some roots showed localized rot, but the rotten area did not exceed one-tenth of the root. This indicates that the composite microcapsules had a better biocontrol effect than the two unencapsulated antagonistic bacteria, effectively controlling root rot in Panax notoginseng caused by Fusarium solani.

[0021] Example 5: Field trial to evaluate the growth-promoting effect of compound microcapsules A field trial was conducted in A'e Village, Shupi Township, Qiubei County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province (N 23° 54′ 28″, E 104°08′ 09″) to evaluate the growth-promoting effect of compound microcapsules on Panax notoginseng. A control group (CK group) and three treatment groups (Bs, Ta, Alg-BsTa) were set up. The control group was irrigated only with sterile water, while the three treatment groups were treated only with Bacillus subtilis suspension (Bs) (1.466 × 10⁻⁶). 11 CFP / mL, applied once a week, 15mL each time), Trichoderma acicularis conidia suspension (Ta) (concentration 7.8×10⁻⁶). 9 The study employed a randomized block design, following local Panax notoginseng management strategies. The growth of Panax notoginseng was observed until harvest season. Fifteen plants were randomly selected from each group, and agronomic parameters were measured, including plant height, plant weight, root length, fresh root weight, dry root weight, stem length, stem diameter, stem scape height, leaf length, and leaf width. (The text also mentions spores / mL, applied once weekly, 15 mL each, and compound microcapsules (Alg-BsTa) 15 g / time, applied weekly.)

[0022] The results are as follows Figure 5 As shown, the results indicate that the composite microcapsules significantly promoted the growth of Panax notoginseng, specifically manifested in a significant increase in multiple agronomic parameters. Compared with the Bs and Ta groups, the agronomic parameters of Panax notoginseng treated with composite microcapsules increased to varying degrees. Plant weight increased by 49.49% and 29.99%, respectively; root fresh weight increased by 41.59% and 20.72%, respectively; stem length increased by 37.67% and 8.36%, respectively; root length increased by 23.25% and 3.52%, respectively; stem diameter increased by 27.68% and 8.13%, respectively; plant height increased by 11.76% and 6.08%, respectively; and stem height increased by 10.32% and 8.96%, respectively.

Claims

1. A composite microcapsule of Bacillus subtilis and Trichoderma echinococcosis, characterized in that: Sodium alginate was used to embed Bacillus subtilis ( Bacillus subtilis Pn1 and Trichoderma acicularis ( Trichoderma asperellum Ta1 was microencapsulated to achieve controlled release of Bacillus subtilis Pn1 and Trichoderma acicularis Ta1.

2. The composite microcapsule of Bacillus subtilis and Trichoderma echinococcosis according to claim 1, characterized in that: Bacillus subtilis ( Bacillus subtilis Pn1 bacterial suspension, Trichoderma acicularis ( Trichoderma asperellum Ta1 spore suspension and sodium alginate solution were mixed and then added dropwise to a 1-3% (w / v) calcium chloride solution. The mixture was cross-linked and solidified at 100-200 rpm for 15-20 min. Solid-liquid separation was performed, and the solid was washed with sterile water and filtered to obtain a Bacillus subtilis viable count ≥1×10⁻⁶. 11 CFU / g and Trichoderma spore content ≥ 1×10 9 1 / g composite microcapsule 3. The composite microcapsule of Bacillus subtilis and Trichoderma echinococcosis according to claim 2, characterized in that: The sodium alginate solution has a mass-volume concentration of 2-3%, the volume ratio of Bacillus subtilis Pn1 bacterial suspension to Trichoderma echinosporum Ta1 spore suspension is 1:1, and the volume ratio of Bacillus subtilis Pn1 bacterial suspension to sodium alginate solution is 1:3-5.

4. The application of the composite microcapsules of Bacillus subtilis and Trichoderma hydathodes as described in claim 1 in the prevention and control of root rot and the promotion of Panax notoginseng growth.

5. The application according to claim 4, characterized in that: The pathogen causing root rot is Fusarium solani (Solanum lycopersicum). Fusarium solani Fusarium oxysporum ( Fusarium oxysporum ), Fusarium truncatum ( Fusarium chlamydosporum ) and Fusarium graminearum ( Fusarium graminearum ).

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