Novel method for strengthening colonization of growth-promoting bacteria in root system

Abstract

The invention relates to the technical field of plant root system colonization, and discloses a novel method for strengthening colonization of growth-promoting bacteria in a root system, which comprises the following steps: S1, seed coating: inoculating rhizobium into a culture medium for culturing, sterilizing, dissolving a fungicide suspension into a solution containing sodium alginate, starch, CaCl, ferrous sulfate and zinc sulfate to form microcapsules, and drying to obtain a seed coating solution; disinfecting the seeds and then wrapping the seeds with microcapsules; s2, planting associated plants; s3, mycorrhizal fungi inoculation and allogenic material treatment; and S4, monitoring the plant height of the plant and mycorrhiza infection related indexes. By means of the novel method for strengthening the colonization of the growth-promoting bacteria in the root system, the problem that the colonization efficiency is low in an existing growth-promoting bacteria inoculation method is effectively solved, the colonization ability of the growth-promoting bacteria in the root system is improved, the stress resistance and nutrition supply of plants are enhanced, infection and propagation of rhizobium and arbuscular mycorrhizal fungi are promoted, and the yield of the plants is increased. Powerful support is provided for green and sustainable development of agricultural production.

Description

Technical Field

[0001] This invention relates to the field of plant root colonization technology, specifically a new method for enhancing the colonization of growth-promoting bacteria in roots. Background Technology

[0002] In agricultural production, plant growth-promoting bacteria, as a green and environmentally friendly biological resource, play multiple important roles, including promoting plant growth, enhancing plant resistance to stress, and improving soil fertility. They provide nutrients to plants through nitrogen fixation, phosphorus solubilization, and potassium solubilization, and can also secrete plant hormones and antibiotics to regulate plant growth and resist pests and diseases. For example, some nitrogen-fixing bacteria can convert atmospheric nitrogen into nitrogen that plants can utilize, significantly improving the nitrogen nutrition level of crops; AM fungi can promote nutrient absorption, increase crop yield and quality, and reduce the application of pesticides and fertilizers, thus having broad application prospects in agricultural production. Furthermore, growth-promoting bacteria can also enhance plant tolerance to stresses such as drought, salinity, and diseases by inducing systemic resistance, thereby improving crop yield and quality.

[0003] However, despite the numerous advantages of plant growth-promoting bacteria, their colonization effect on plant roots is often unsatisfactory in practical applications. Currently, commonly used inoculation methods include seed dressing, soil irrigation, and rhizosphere application, but these methods share some common problems. Firstly, growth-promoting bacteria have weak survival capabilities in soil and are easily affected by soil environmental factors (such as temperature, humidity, pH, and competition from soil microorganisms), leading to a rapid decrease in their numbers and difficulty in effectively colonizing the root system. Secondly, existing inoculation methods often fail to ensure sufficient contact and interaction between the growth-promoting bacteria and plant roots, resulting in low colonization efficiency and failing to fully utilize the biological functions of the bacteria. Therefore, to fully leverage the role of plant growth-promoting bacteria in agricultural production and improve their colonization ability in the root system, there is an urgent need to develop a new and efficient colonization method for growth-promoting bacteria. Summary of the Invention

[0004] This invention provides a novel method for enhancing the colonization of growth-promoting bacteria in plant roots. It addresses the problem of low colonization efficiency caused by existing inoculation methods, which often fail to ensure sufficient contact and interaction between the bacteria and plant roots. The method involves seed coating with beneficial growth-promoting bacteria, followed by inoculation with mycorrhizal fungi (AM), and the addition of flavonoids and fatty acids to jointly promote the efficient propagation of arbuscular mycorrhizal fungi. Simultaneously, clover is planted as a companion plant to further promote plant growth. By combining effective growth-promoting bacteria with seed coating technology, the stress resistance and nutrient supply of seeds are enhanced, thereby promoting the infection and propagation of rhizobia and arbuscular mycorrhizal fungi.

[0005] To achieve the above objectives, the present invention provides a novel method for enhancing the colonization of growth-promoting bacteria in roots, comprising the following steps: S1. Seed coating: Rhizobium is inoculated into a culture medium and cultured. After sterilization, the bacterial suspension is dissolved in a solution containing sodium alginate, starch, CaCl, ferrous sulfate and zinc sulfate to form microcapsules. After seed sterilization, the microcapsules are coated. S2. Planting of companion plants: After the seeds germinate, sterilized clover is planted as a companion plant, and the planting density of clover is a certain proportion of the target plant. S3. Mycorrhizal fungi inoculation and exogenous substance treatment: Inoculate the soil with a certain mass ratio of Moses tuberculosis mycorrhizal fungi agent. After the plants emerge, water them with a solution containing specific exogenous substances at a certain time. Determine the watering volume according to the soil moisture content and water regularly. S4. Monitor plant height and indicators related to mycorrhizal infection.

[0006] Preferably, the culture medium is LB medium, the culture temperature is 25-30℃, the culture speed is 150-200 rpm, and the culture time is 15-20 hours; During the seed coating process, the mass ratio of microcapsules to seeds is (1:5)-(1:10).

[0007] Preferably, the sodium alginate concentration is 1-2%, the starch concentration is 2-4%, and the CaCl concentration is 1-3%.

[0008] Preferably, the concentration range of ferrous sulfate is 0.1-0.4%, and the concentration range of zinc sulfate is 0.03-0.15%.

[0009] Preferably, the disinfectant used for seed disinfection is 8-12% H2O2, and the disinfection time is 25-35 minutes.

[0010] Preferably, the planting density of the clover is 30-50% of the target plant density; The target plant is corn, alfalfa, or other grass or legume plants; When the target plant is maize, the concentration of hesperidin is 1000-2000 nmol / L and the concentration of myristic acid is 0.5-1.5 mmol / L; When the target plant is alfalfa, the concentration of quercetin is 30-70 μmol / L, the concentration of hesperidin is 1000-2000 nmol / L, the concentration of myristic acid is 1-3 mmol / L, and the concentration of palmitoleic acid is 80-120 μmol / L.

[0011] Preferably, the mass ratio of the *M. mossioides* mycorrhizal fungal agent is 2-5%, and each gram of the agent contains 100-200 spores.

[0012] Preferably, the exogenous substance applied 3-4 weeks after the plant seedlings emerge includes one or more combinations of hesperidin, myristic acid, quercetin, and palmitoleic acid.

[0013] Preferably, the soil moisture content is maintained at 75-85% of field capacity, and watering is performed once a week; The mycorrhizal infection-related indicators include one or more of the following: spore density, hyphal density, mycorrhizal infection rate, and arbuscular abundance.

[0014] Preferably, the monitoring of plant height and mycorrhizal infection-related indicators is conducted once every 1-2 weeks; the mycorrhizal fungi inoculation is carried out by strip application, hole application, or broadcast application.

[0015] This invention provides a novel method for enhancing the colonization of growth-promoting bacteria in roots. It offers the following beneficial effects: 1. The novel method for enhancing the colonization of growth-promoting bacteria in the root system, as described in this invention, effectively solves the problem of low colonization efficiency in existing growth-promoting bacteria inoculation methods. It not only improves the colonization ability of growth-promoting bacteria in the root system, but also enhances the plant's stress resistance and nutrient supply, promotes the infection and propagation of rhizobia and arbuscular mycorrhizal fungi, and provides strong support for the green and sustainable development of agricultural production.

[0016] 2. The present invention adopts a technical solution of inoculating rhizobia into a culture medium and then making microcapsules for seed coating. This achieves the technical effect of protecting the growth-promoting bacteria in the complex soil environment and enabling them to better combine with the seeds and slowly release the growth-promoting bacteria. Compared with the simple seed mixing method in the prior art, this invention solves the problem that the growth-promoting bacteria are easily affected by the soil environment and have difficulty in effectively colonizing the root system.

[0017] 3. This invention employs a technical solution that involves planting sterilized clover as a companion plant after seed germination, with a planting density of 30-50% of the target plant. This achieves the technical effect of improving the micro-ecological environment around the plant roots, promoting plant growth, and providing a more suitable living environment for growth-promoting bacteria. Compared with the existing technology that does not focus on the planting of companion plants and only inoculates growth-promoting bacteria, this invention solves the shortcomings of the poor root micro-ecological environment, which is not conducive to the colonization and function of growth-promoting bacteria.

[0018] 4. This invention employs a technical solution that involves inoculating the soil with a certain mass ratio of *Mallotus fasciatus* mycorrhizal fungi agent, and then irrigating the soil 3-4 weeks after plant emergence with a solution containing exogenous substances such as hesperidin and myristic acid. The irrigation volume is determined based on the soil moisture content, and irrigation is carried out weekly to maintain the soil moisture content at 75-85% of field capacity. This achieves the technical effect of effectively promoting the efficient propagation of arbuscular mycorrhizal fungi and enhancing the colonization effect of growth-promoting fungi on the root system. Compared with existing technologies that lack precise control over the inoculation amount of mycorrhizal fungi and the use of exogenous substances, this invention solves the shortcomings of low colonization efficiency and the inability to fully utilize the biological functions of growth-promoting fungi.

[0019] 5. This invention employs a technical solution that monitors plant height and mycorrhizal infection-related indicators, including spore density and mycelial density, with a monitoring frequency of once every 1-2 weeks. This achieves real-time monitoring of plant growth and mycorrhizal colonization, allowing for timely adjustments to planting strategies and ensuring optimal colonization results. Compared to existing technologies that lack systematic monitoring or are not timely, this invention addresses the shortcomings of existing technologies that cannot flexibly adjust planting measures according to actual conditions to guarantee the colonization effect of mycorrhizal colonization.

[0020] 6. This invention adopts a technical solution that sets different concentrations of exogenous substances for different target plants, such as corn and alfalfa, achieving the technical effect of accurately adapting to the needs of different plants and maximizing the role of growth-promoting bacteria. Compared with the existing technology that adopts a uniform inoculation scheme for growth-promoting bacteria and the use of exogenous substances without distinguishing plant species, this invention solves the problem that it cannot meet the differentiated needs of different plants for growth-promoting bacteria and exogenous substances. Attached Figure Description

[0021] Figure 1 The effects of different combinations of exogenous substances on spore density, hyphal density and maize mycorrhizal infection rate of AM fungi were investigated. Figure 2 The effects of different combinations of exogenous substances on spore density, hyphal density and alfalfa mycorrhizal infection rate of AM fungi were investigated. In the figure, M% represents the mycorrhizal infection intensity of the entire root system, and A% represents the arbuscular abundance of the entire root system. Data are presented as mean ± standard error, n=5. Different lowercase letters indicate significant differences in Duncan's multiple comparisons among different exogenous material treatments (P<0.05). Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] In agricultural production, plant growth-promoting bacteria have become a key biological resource for the development of green and environmentally friendly agriculture due to their advantages in promoting plant growth, enhancing stress resistance, and improving soil fertility. For example, nitrogen-fixing bacteria can convert atmospheric nitrogen into nitrogen that plants can utilize, while agronomical fungi (AM) can promote nutrient absorption by plants, reducing the use of pesticides and fertilizers. However, currently used methods for inoculating growth-promoting bacteria, such as seed dressing, soil irrigation, and rhizosphere application, have many problems. On the one hand, growth-promoting bacteria are easily affected by environmental factors such as temperature, humidity, pH, and competition from soil microorganisms in the soil, resulting in weak survival ability and difficulty in effectively colonizing the root system. On the other hand, these methods cannot allow growth-promoting bacteria to have sufficient contact and interaction with plant roots, leading to low colonization efficiency and failing to fully utilize the biological functions of growth-promoting bacteria.

[0024] To address the aforementioned technical problems, this application provides a novel method for enhancing the colonization of growth-promoting bacteria in roots. Specifically, it includes the following steps: Seed coating: Rhizobium is inoculated into a culture medium and cultured. After sterilization, the inoculum suspension is dissolved in a solution containing sodium alginate, starch, CaCl₂, ferrous sulfate, and zinc sulfate to form microcapsules. After seed sterilization, the microcapsules are coated. This coating method not only protects the growth-promoting bacteria in complex soil environments but also allows for the slow release of the bacteria, enabling them to better bind with the seeds.

[0025] Companion plant cultivation: After seed germination, sterilized clover is planted as a companion plant, with the clover planting density being a certain proportion of the target plant. By cultivating companion plants, the micro-ecological environment around the plant roots can be improved, promoting plant growth and providing a more suitable living environment for growth-promoting bacteria.

[0026] Mycorrhizal fungi inoculation and exogenous substance treatment: A specific mass ratio of *M. mossioides* mycorrhizal fungi agent was inoculated into the soil. After the plants emerged, a solution containing a specific exogenous substance was applied at a certain time. The volume of application was determined based on the soil moisture content, and application was carried out regularly. By precisely controlling the inoculation amount of mycorrhizal fungi and the application of exogenous substances, the efficient propagation of arbuscular mycorrhizal fungi was effectively promoted, and the colonization effect of growth-promoting fungi on the root system was enhanced.

[0027] Monitor plant height and indicators related to mycorrhizal infection. Real-time monitoring of these indicators helps to adjust planting strategies in a timely manner, ensuring optimal colonization of growth-promoting fungi.

[0028] This invention effectively solves the problem of low colonization efficiency in existing methods for inoculating growth-promoting bacteria. It not only improves the colonization ability of growth-promoting bacteria in the root system but also enhances plant stress resistance and nutrient supply, promotes the infection and propagation of rhizobia and arbuscular mycorrhizal fungi, and provides strong support for the green and sustainable development of agricultural production.

[0029] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0030] Please see the appendix Figure 1 -Appendix Figure 2 This invention provides a novel method for enhancing the colonization of growth-promoting bacteria in roots, comprising the following steps: I. Seed coating Microbial culture: Rhizobium was inoculated into LB medium. The inoculated medium was placed in a shaker and the temperature was set to 28°C and the rotation speed to 180 rpm for 18 hours.

[0031] Microcapsule preparation: After the culture is completed, the inoculum is sterilized.

[0032] Prepare a solution of 1.5% sodium alginate + 3% starch + 2% CaCl + 0.2%-0.3% ferrous sulfate + 0.05-0.1% zinc sulfate.

[0033] The sterilized bacterial agent suspension is dissolved in the above solution to form microcapsules.

[0034] Seed disinfection and coating: Select the target plant seeds and disinfect them with a 10% H2O2 solution for 30 minutes.

[0035] After disinfection, the seeds are coated with prepared microcapsules containing a microbial agent.

[0036] II. Cultivation of Associated Plants Clover seed treatment: Two weeks after the seeds germinate, prepare to plant clover as a companion plant.

[0037] Clover seeds are also disinfected with a 10% H2O2 solution before planting.

[0038] Planting density and operation: The planting density of clover should be controlled at 30-50% of the target plant. Clover seeds should be sown around the target plant according to this ratio.

[0039] III. Mycorrhizal fungi inoculation and exogenous substance irrigation Mycorrhizal fungal inoculation: Inoculate the soil with 2-5% by weight of Moses tuberculosis mycorrhizal fungicide (each gram of fungicide contains 100-200 spores).

[0040] External material irrigation: Irrigate with exogenous substances 3-4 weeks after the target plants emerge.

[0041] Hesperidin 1500 nmol / L + myristic acid 1 mmol / L was selected as the exogenous substance.

[0042] The irrigation volume is determined based on the soil moisture content, maintaining the soil moisture content at 80-85% of field capacity, and irrigating once a week.

[0043] IV. Monitoring plant growth and mycorrhizal infection Monitoring indicators and methods: corn: Spore density: Soil samples were collected periodically, and the number of AM fungal spores in the soil was determined by specific separation and counting methods. The changes in spore density before and after propagation of different treatment groups were compared. For example, T1 was the spore density before propagation, and the spore density after propagation of treatments such as T2, T3, T4, and T5 were compared.

[0044] Mycelial density: Mixed root and soil samples were collected, and after a series of treatments, the length of mycelia per unit mass of soil was observed and calculated using a microscope to obtain the mycelial density. The differences in mycelial density between different treatment groups and the control T1 were compared.

[0045] Mycorrhizal infection rate: Corn root samples were collected, stained, and then the infection of the roots by AM fungi was observed under a microscope. The mycorrhizal infection rate and arbuscular abundance were calculated, and the infection status of different treatment groups was compared with that of control T1.

[0046] Alfalfa: Spore density: Similar to corn, soil samples were collected periodically to determine the number of AM fungal spores in alfalfa-grown soil and to compare the changes in spore density between different treatment groups and control T1.

[0047] Mycelial density: Mixed samples of alfalfa roots and soil were collected, treated, and observed under a microscope to calculate the length of mycelia per unit mass of soil. The differences in mycelial density between different treatment groups and control T1 were compared.

[0048] Mycorrhizal infection rate: Alfalfa root samples were collected, stained, and observed under a microscope to determine the infection status of the roots by AM fungi. The mycorrhizal infection rate and arbuscular abundance were calculated, and the infection status of different treatment groups was compared with that of control T1.

[0049] Data recording and analysis: Each monitoring session requires detailed recording of various data, including spore density, mycelial density, mycorrhizal infection rate, and arbuscular abundance for different treatment groups.

[0050] Statistical analysis was performed on the recorded data, such as by using analysis of variance, to determine the significant differences between different treatment groups, thereby determining the effects of different treatments on the sporulation, mycelial density, and mycorrhizal infection degree of AM fungi.

[0051] Example 1: refer to Figure 1 This embodiment provides a method for enhancing the colonization of growth-promoting bacteria in maize roots, including the following steps: I. Experimental Preparation (a) Material preparation Plant materials: Select plump, disease-free corn seeds.

[0052] Microbial materials: Prepare a fungal inoculum of *M. mossioides*, ensuring that each gram of inoculum contains 100-200 spores; obtain rhizobia for seed coating.

[0053] Chemical reagents: Purchase LB medium, 10% H2O2, 1.5% sodium alginate, 3% starch, 2% CaCl, ferrous sulfate, zinc sulfate, hesperidin, myristic acid, quercetin, palmitoleic acid, etc., and ensure that the purity of the reagents meets the experimental requirements.

[0054] Other materials: Prepare sterilized flower pots, soil and other planting materials to reduce interference from miscellaneous bacteria in the experiment.

[0055] (II) Instrument Preparation Prepare experimental instruments such as shakers, sterilizers, microscopes, centrifuges, petri dishes, and pipettes, ensuring that the instruments are in good working order and have been calibrated and cleaned.

[0056] II. Seed Coating Rhizobium was inoculated into LB medium and cultured for 18 hours at 28°C and 180 rpm in a shaker.

[0057] After cultivation, the bacterial agent is sterilized. Then, the bacterial agent suspension is dissolved in a solution composed of 1.5% sodium alginate, 3% starch, 2% CaCl, 0.2%-0.3% ferrous sulfate, and 0.05%-0.1% zinc sulfate, and stirred thoroughly to form microcapsules.

[0058] Disinfect corn seeds with 10% H2O2 for 30 minutes, then coat the disinfected seeds with the aforementioned microcapsules.

[0059] III. Planting and Treatment Plant the coated corn seeds in prepared flower pots and provide suitable temperature, light and water conditions to promote seed germination.

[0060] Two weeks after seed germination, clover sterilized with 10% H2O2 was planted as a companion plant. The planting density of clover was determined according to 30-50% of the number of corn plants, and the clover was planted evenly around the corn plants.

[0061] Inoculate the soil with a 2-5% (w / w) concentration of *Tetranychus mossioides* mycorrhizal fungicide. Three to four weeks after corn emergence, irrigate the soil with the following treatment groups: Group T1: As a control, no additional exogenous substances such as hesperidin and myristic acid were added.

[0062] Group T2: Irrigated with quercetin 1500 nmol / L + myristic acid 1 mmol / L.

[0063] Group T3: Irrigated with quercetin 1500 nmol / L + myristic acid 1 mmol / L + palmitoleic acid 50 μmol / L.

[0064] Group T4: Irrigated with hesperidin 1500 nmol / L + myristic acid 1 mmol / L.

[0065] Group T5: Irrigated with hesperidin 1500 nmol / L + myristic acid 1 mmol / L + palmitoleic acid 100 μmol / L.

[0066] Determine the irrigation volume based on the soil moisture content, maintain the soil moisture content at 80-85% of field capacity, and irrigate once a week.

[0067] IV. Monitoring Indicators and Methods (a) Monitoring plant height Use a ruler to measure the height of the corn plant from the base to the top growing point each week, and record the plant height data regularly.

[0068] (II) Monitoring of mycorrhizal infection-related indicators Spore density determination: Soil samples were collected before propagation (labeled as T1 spore density before propagation) and after propagation in each treatment group. Spores in the soil were separated by wet sieving decanting-sucrose centrifugation, counted under a microscope, and the spore density of different treatment groups was statistically analyzed.

[0069] Mycelial density determination: After the appropriate treatment time, a mixed sample of roots and soil was collected. After washing the sample, it was stained with acid fuchsin, and the length of mycelia per unit mass of soil was calculated under a microscope using the streak method to obtain the mycelial density.

[0070] Mycorrhizal infection rate and wisteria abundance determination: Maize root samples were collected, washed, and stained with alkaline fuchsin. The infection status of the roots by AM fungi was observed under a microscope. One hundred root segments were randomly selected, and the mycorrhizal infection rate (number of infected root segments / total number of observed root segments × 100%) and wisteria abundance (number of root segments containing wisteria / total number of observed root segments × 100%) were calculated.

[0071] V. Experimental Results Spore density: The spore density after propagation was significantly higher in all treatments than before propagation. The order of spore density was: T4 > T3 > T2 > T1 > T5. The T4 treatment (hesperidin 1500 nmol / L + myristic acid 1 mmol / L) had the highest spore density, significantly higher than other treatments; there was no significant difference in spore production among T1, T2, and T5.

[0072] Mycelial density: Compared with the control T1, the T3, T4 and T5 treatments all significantly increased the mycelial density of the system. Among them, the mycelial density of the T4 treatment reached 3.9 m / g, which was significantly higher than that of the T1 control treatment (1.1 m / g).

[0073] Mycorrhizal infection rate and wisteria abundance: The mycorrhizal infection intensity and wisteria abundance of the applied treatments were both higher than those of the control treatment. The highest root infection rate of maize plants was T5 (64.1%), while the root infection rate of the control T1 was 28.7%; the highest wisteria abundance was T2 treatment (22.7%), while the control was 8.4%.

[0074] Overall, the infection intensity and arbor abundance of the T4 treatment were significantly higher than those of other treatments.

[0075] Comparing T4 and T2, the flavonoid components are different, and it can be seen that hesperidin is more effective than quercetin on corn. Comparing T3 and T2, T3 was applied with a certain concentration of palmitoleic acid, which had a better effect, indicating that palmitoleic acid, combined with quercetin and myristic acid, has a promoting effect on the infection and colonization of Funneliformismosseae.

[0076] Example 2: refer to Figure 2 This embodiment provides a method for enhancing the colonization of growth-promoting bacteria in alfalfa roots, including the following steps: I. Experimental Preparation (a) Material preparation Plant materials: Select plump alfalfa seeds that are free from pests and diseases.

[0077] Microbial materials: Prepare *M. mossioides* root fungicide (each gram of fungicide contains 100-200 spores) and rhizobia.

[0078] Chemical reagents: Prepare LB medium, 10% H2O2, 1.5% sodium alginate, 3% starch, 2% CaCl, ferrous sulfate, zinc sulfate, hesperidin, myristic acid, quercetin, palmitoleic acid and other chemical reagents, and ensure the quality of the reagents.

[0079] Other materials: Prepare sterilized flower pots and soil, etc.

[0080] (II) Instrument Preparation Prepare instruments such as shakers, autoclaves, microscopes, centrifuges, petri dishes, and pipettes, and ensure they are in good working order.

[0081] II. Seed Coating Rhizobium was inoculated into LB medium and cultured on a shaker at 28°C and 180 rpm for 18 hours.

[0082] After sterilizing the cultured bacterial agent, the bacterial agent suspension is dissolved in a solution composed of 1.5% sodium alginate, 3% starch, 2% CaCl, 0.2%-0.3% ferrous sulfate, and 0.05%-0.1% zinc sulfate to form microcapsules.

[0083] Sterilize alfalfa seeds with 10% H2O2 for 30 minutes, then encapsulate them in microcapsules.

[0084] III. Planting and Treatment Plant the coated alfalfa seeds in flowerpots and provide a suitable growing environment to promote germination.

[0085] Two weeks after the seeds germinate, clover sterilized with 10% H2O2 is planted as a companion plant. The planting density of clover is determined to be 30-50% of the number of alfalfa plants and they are planted evenly.

[0086] Inoculate the soil with a 2-5% (w / w) dose of *Tetranychus mossioides* mycorrhizal fungicide. Three to four weeks after alfalfa emergence, irrigate the following treatment groups with the exogenous substance: Group T1: Control group, with no additional exogenous substances added.

[0087] Group T2: Irrigated with quercetin 1500 nmol / L + myristic acid 1 mmol / L.

[0088] Group T3: Irrigated with quercetin 1500 nmol / L + myristic acid 1 mmol / L + palmitoleic acid 50 μmol / L.

[0089] Group T4: Irrigated with hesperidin 1500 nmol / L + myristic acid 1 mmol / L.

[0090] Group T5: Irrigated with quercetin 50 μmol / L + hesperidin 1500 nmol / L + myristic acid 2 mmol / L + palmitoleic acid 100 μmol / L.

[0091] Determine the irrigation volume based on the soil moisture content, maintain the soil moisture content at 80-85% of field capacity, and irrigate once a week.

[0092] IV. Monitoring Indicators and Methods (a) Monitoring plant height Measure the height of the alfalfa plants weekly using a ruler, from the base to the top growing point, and record the data.

[0093] (II) Monitoring of mycorrhizal infection-related indicators Spore density determination: Soil samples were collected before propagation (spore density before propagation in T1) and after propagation in each treatment group. Spores were separated by wet sieving decanting-sucrose centrifugation and counted under a microscope.

[0094] Mycelial density determination: After the corresponding treatment time, a mixed sample of roots and soil was collected, washed, stained with acid fuchsin, and the length of mycelium per unit mass of soil was calculated under a microscope using the streak method.

[0095] Mycorrhizal infection rate and wisteria abundance determination: Alfalfa root samples were collected, washed, stained with alkaline fuchsin, and observed under a microscope. 100 root segments were randomly selected, and the mycorrhizal infection rate (number of infected root segments / total number of observed root segments × 100%) and wisteria abundance (number of root segments containing wisteria / total number of observed root segments × 100%) were calculated.

[0096] V. Experimental Results Spore density: Compared with the control, all treatments increased the spore density of AM fungi. The spore density ranking was T5 > T2 > T4 > T3 > T1. The T5 treatment had the highest spore density, followed by T2, both of which were significantly higher than the other treatments; there was no significant difference in spore production among T1, T3, and T4.

[0097] Mycelial density: Compared with the control, all treatments significantly increased mycelial density. The mycelial density was ranked as T4 > T5 > T3 > T2 > T1, with the mycelial density of treatment T4 reaching 3.8 m / g, significantly higher than that of the control T1 (1.7 m / g).

[0098] Mycorrhizal infection rate and wilting abundance: There was no significant difference in mycorrhizal infection intensity between the treatments and the control, but the wilting abundance of each treatment was higher than that of the control. The highest wilting abundance was in treatment T2 (35.3%), compared to 15.7% in the control.

[0099] Overall, treatment T2 showed better infection rates. Comparing T4 and T2, both treatments promoted mycorrhizal colonization on alfalfa. However, compared to T3 and T2, T3 with added palmitoleic acid was less effective, indicating that higher concentrations of palmitoleic acid do not necessarily promote mycorrhizal colonization on alfalfa.

[0100] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A novel method for enhancing the colonization of growth-promoting bacteria in roots, characterized in that, Includes the following steps: S1. Seed coating: Rhizobium is inoculated into a culture medium and cultured. After sterilization, the bacterial suspension is dissolved in a solution containing sodium alginate, starch, CaCl, ferrous sulfate and zinc sulfate to form microcapsules. After seed sterilization, the microcapsules are coated. S2. Planting of companion plants: After the seeds germinate, sterilized clover is planted as a companion plant, and the planting density of clover is a certain proportion of the target plant. S3. Mycorrhizal fungi inoculation and exogenous substance treatment: Inoculate the soil with a certain mass ratio of Moses tuberculosis mycorrhizal fungi agent. After the plants emerge, water them with a solution containing specific exogenous substances at a certain time. Determine the watering volume according to the soil moisture content and water regularly. S4. Monitor plant height and indicators related to mycorrhizal infection.

2. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The culture medium is LB medium, the culture temperature is 25-30℃, the culture speed is 150-200 rpm, and the culture time is 15-20 hours; During the seed coating process, the mass ratio of microcapsules to seeds is (1:5)-(1:10).

3. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The concentration of sodium alginate is 1-2%, the concentration of starch is 2-4%, and the concentration of CaCl is 1-3%.

4. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The concentration range of ferrous sulfate is 0.1-0.4%, and the concentration range of zinc sulfate is 0.03-0.15%.

5. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The disinfectant used for seed disinfection is 8-12% H2O2, and the disinfection time is 25-35 minutes.

6. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The planting density of the clover is 30-50% of the target plant. The target plant is corn, alfalfa, or other grass or legume plants; When the target plant is maize, the concentration of hesperidin is 1000-2000 nmol / L and the concentration of myristic acid is 0.5-1.5 mmol / L; When the target plant is alfalfa, the concentration of quercetin is 30-70 μmol / L, the concentration of hesperidin is 1000-2000 nmol / L, the concentration of myristic acid is 1-3 mmol / L, and the concentration of palmitoleic acid is 80-120 μmol / L.

7. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The mass ratio of the Moses tuberculosis mycorrhizal fungicide is 2-5%, and each gram of fungicide contains 100-200 spores.

8. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The exogenous substances applied 3-4 weeks after the plants emerge include one or more combinations of hesperidin, myristic acid, quercetin, and palmitoleic acid.

9. The novel method for enhancing root colonization of growth-promoting bacteria according to claim 1, characterized in that, The soil moisture content should be maintained at 75-85% of field capacity, and watering should be done once a week. The mycorrhizal infection-related indicators include one or more of the following: spore density, hyphal density, mycorrhizal infection rate, and arbuscular abundance.

10. The novel method for promoting root colonization of growth-enhancing bacteria according to any one of claims 1-9, characterized in that, The monitoring of plant height and mycorrhizal infection-related indicators is conducted every 1-2 weeks; the mycorrhizal fungi are inoculated by strip application, hole application, or broadcast application.

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