Intercropping method for transmitting disease-resistant signal through AMF mycelium bridge

Abstract

The invention provides an intercropping method for transmitting a disease-resistant signal through an AMF hypha bridge. The intercropping method comprises the following steps that crops suitable for symbiosis with arbuscular mycorrhizal fungi are selected as intercropping plants; inoculating arbuscular mycorrhizal fungi strains to rhizospheres of the two crops to form a hypha bridge; inoculating pathogenic bacteria to the rhizosphere of one crop, and simulating a disease invasion process; disease-resistant signals caused by diseases are transmitted to another crop from the infected crop through the AMF hypha bridge, so that the disease-resistant capability of the other crop is improved; by detecting physiological and biochemical indexes, agronomic characters and disease-resistant reactions of crops in different time periods, the transmission effect of the AMF hypha bridge and the improvement effect of the AMF hypha bridge on indexes such as disease resistance and disease-resistant enzyme activity are evaluated. The disease resistance of intercropping crops can be effectively improved. According to the method, the AMF hypha bridge establishes a network at the rhizosphere of the crop, so that when one crop is attacked by diseases, the other crop obtains a disease-resistant signal, and the immune response and disease resistance of the crop are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to an intercropping method for transmitting disease resistance signals through AMF hyphae bridges. Background Art

[0002] Currently, plant disease resistance is one of the important topics in agricultural production. Arbuscular mycorrhizal fungi (AMF), as a microorganism widely present in the soil, connect the roots of different plants through hyphae bridges, forming a complex rhizosphere interaction network. It has been proven that it can transmit disease resistance signals between plants and enhance the disease resistance of crops. However, the mechanism and application of AMF hyphae bridges in transmitting disease resistance signals between crops have not been fully explored. Through research, it was found that AMF hyphae bridges can transmit disease resistance signals between Aconitum carmichaelii and corn, enhancing Aconitum carmichaelii's resistance to diseases such as small leaf spot. Therefore, a new agricultural production method has been developed that can use AMF hyphae bridges to transmit disease resistance signals between crops and enhance the overall disease resistance of crops. Summary of the Invention

[0003] The purpose of the present invention is to provide an intercropping method that transmits disease resistance signals through AMF hyphae bridges, which can effectively improve the disease resistance of intercropped crops and enable one crop to receive disease resistance signals when the other crop is attacked by a disease, thereby enhancing its immune response and disease resistance.

[0004] According to one object of the present invention, the present invention provides an intercropping method for transmitting disease resistance signals through AMF hyphae bridges, comprising the following steps: S1. Select crops: Select crops that are suitable for symbiosis with arbuscular mycorrhizal fungi as intercrops; S2. Inoculation of AMF strains: Arbuscular mycorrhizal fungal strains were inoculated into the rhizosphere of the two crops to form hyphal bridges; S3. Pathogen inoculation: Inoculate the rhizosphere of one of the crops with pathogens to simulate the disease invasion process; S4. Mycelial bridges transmit disease resistance signals: Through the AMF hyphae bridge, disease-induced resistance signals are transmitted from the infected crop to another crop, thereby improving the disease resistance of the other crop; S5. Observe and evaluate the effects: By testing the physiological and biochemical indicators, agronomic traits and disease resistance responses of crops in different time periods, the transmission effect of AMF mycelial bridge and its role in improving indicators such as disease resistance and disease-resistant enzyme activity were evaluated.

[0005] Furthermore, the crops are Aconitum carmichaelii and corn.

[0006] Furthermore, the AMF strain is Glomus mosseae.

[0007] Furthermore, the inoculated pathogen is the pathogen of corn leaf blight.

[0008] Furthermore, the hyphae bridge is established by separating with a nylon mesh.

[0009] Furthermore, the hyphae bridge is established by thin film separation.

[0010] Furthermore, disease resistance was evaluated by measuring the content of photosynthetic pigments.

[0011] Furthermore, the disease resistance was evaluated by measuring the superoxide dismutase (SOD) activity.

[0012] Furthermore, it is characterized in that disease resistance is evaluated by measuring catalase (CAT) activity.

[0013] Furthermore, disease resistance was evaluated by measuring amino acid content.

[0014] Beneficial effects The technical solution of the present invention can effectively improve the disease resistance of intercropped crops, especially the disease resistance of Aconitum kusnezoffii. Through this method, the AMF hyphae bridge establishes a network in the crop rhizosphere, enabling one crop to receive disease resistance signals when attacked by a disease, thereby enhancing its immune response and disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the effects of different treatments on catalase (CAT) activity in Aconitum kusnezoffii according to the present invention; Figure 2 This is a graph showing the effects of different treatments on the peroxidase (POD) activity of Aconitum carmichaelii in different embodiments of the present invention; Figure 3 This is a graph showing the effects of different treatments on the superoxide dismutase (SOD) activity of Aconitum carmichaelii in accordance with the present invention; Figure 4 This is a graph showing the effects of different treatments on the polyphenol oxidase (PPO) activity of Aconitum carmichaelii in different embodiments of the present invention; Figure 5 This is a graph showing the effects of different treatments on the amino acid content of Aconitum carmichaelii in the examples of the present invention. DETAILED DESCRIPTION

[0016] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like to indicate directions or positional relationships are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as limiting the present invention.

[0018] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the said features. In the description of the present invention, "multiple" means two or more, unless otherwise clearly and specifically defined. In addition, the terms "installed", "connected", and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a communication between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances.

[0019] Example 1 An intercropping method for transmitting disease resistance signals through AMF hyphae bridges, comprising the following steps: S1. Select crops: Select crops suitable for symbiosis with arbuscular mycorrhizal fungi (AMF) as intercropping plants, especially Aconitum carmichaelii and corn.

[0020] S2. Inoculation of AMF strains: Arbuscular mycorrhizal fungi (AMF) strains (e.g., Glomus mosseae) were inoculated into the rhizosphere of the two crops to allow for the formation of hyphal bridges. The hyphal bridges were established by separating them with nylon mesh or by separating them with film. S3. Pathogen inoculation: Pathogens (such as corn leaf blight) are inoculated into the rhizosphere of one of the crops to simulate the disease invasion process.

[0021] S4. Mycelial bridges transmit disease resistance signals: Through the AMF hyphal bridge, disease-induced resistance signals are transmitted from the infected crop to another crop, thereby improving the disease resistance of the other crop.

[0022] S5. Observe and evaluate the effects: The effectiveness of AMF hyphal bridges and their impact on improving disease resistance and anti-disease activity were evaluated by measuring crop physiological and biochemical indicators, agronomic traits, and disease resistance responses over different time periods. Specifically, disease resistance was assessed by measuring photosynthetic pigment content, superoxide dismutase (SOD) activity, catalase (CAT) activity, and amino acid content.

[0023] Example 2 Intercropping system of Aconitum kusnezoffii and corn Crop selection and processing: Huangwu and corn were selected as intercrops.

[0024] The Glomus mosseae strain was inoculated into the roots of Aconitum carmichaelii and corn respectively through rhizosphere inoculation.

[0025] The roots of the two crops were separated by nylon mesh, but a channel was left between the rhizospheres to promote the formation of hyphal bridges.

[0026] Pathogen inoculation: Corn leaves were inoculated with the pathogen of southern leaf spot.

[0027] Observe and record the changes in disease resistance of Aconiti kusnezoffii at 0 hours, 6 hours, 12 hours, and 24 hours after pathogen inoculation.

[0028] Effect observation and evaluation: Disease resistance of Aconitum carmichaelii was evaluated by measuring growth parameters (such as plant height, stem diameter, leaf area) and enzyme activities (such as superoxide dismutase SOD, catalase CAT, and peroxidase POD) of Aconitum carmichaelii.

[0029] The results showed that the disease resistance of Aconitum carmichaelii was significantly improved after inoculation with AMF mycelial bridge, as manifested by increased disease resistance enzyme activity and alleviated disease symptoms.

[0030] Example 3 The rhizosphere separation system between Aconitum kusnezoffii and maize Crop selection and processing: Huangwu and corn were selected as intercrops.

[0031] Film separators were used to separate the rhizosphere of Aconiti kusnezoffii and maize to maintain the formation of AMF hyphae bridges.

[0032] Pathogen inoculation: Corn leaves were inoculated with the pathogen of southern leaf spot.

[0033] Observe and record the changes in disease resistance of Aconiti kusnezoffii at 0 hours, 6 hours, 12 hours, and 24 hours after pathogen inoculation.

[0034] Effect observation and evaluation: Determine the photosynthetic pigment content of Aconitum carmichaelii (such as chlorophyll a, chlorophyll b and total chlorophyll content).

[0035] The results showed that the establishment of AMF hyphae bridge increased the photosynthetic pigment content of Aconitum kusnezoffii, thereby improving its disease resistance.

[0036] Example 4 Control experiment under sterile silk bridge system Crop selection and processing: Huangkuiwu and corn were selected as the control group.

[0037] No AMF strain was inoculated between Aconiti kusnezoffii and corn to maintain a sterile silk bridge system.

[0038] Pathogen inoculation: Corn leaves were inoculated with the pathogen of southern leaf spot.

[0039] Effect observation and evaluation: To determine the agronomic traits and disease resistance indexes of Aconitum carmichaelii.

[0040] The results showed that under the sterile silk bridge system, the disease resistance of Aconitum kusnezoffii was poor and its enzyme activity level was low.

[0041] Example 5 Experimental design This experiment used Aconiti kusnezoffii intercropped with corn as the research subject. A single-factor field potting method was used. Four treatments (Table 1) were used: Aconiti kusnezoffii intercropped with corn intercropped with AMF strains separated by nylon mesh in the rhizosphere and inoculated with Southern Blight (creating a hyphal bridge and transmitting corn susceptibility signals); B, Aconiti kusnezoffii intercropped with corn intercropped with AMF strains, was used as the rhizosphere without AMF inoculation (CK, no hyphal bridge); C, Aconiti kusnezoffii intercropped with corn intercropped with AMF strains separated by film inoculation and inoculated with Southern Blight (no hyphal bridge and no corn susceptibility signals); and D, Aconiti kusnezoffii intercropped with AMF strains (creating a hyphal bridge and no corn susceptibility signals). Each pot represented one treatment, with nine replicates for each treatment, for a total of 36 pots. Treatments A, B, and C were all inoculated with Southern Blight before the early flowering stage; treatment D was left uninoculated.

[0042] The experimental flowerpots were 930 cm long × 380 cm wide × 280 cm high. During planting, the chamber partition method was used, and the flowerpots were divided into three parts in the length direction. Each part of the flowerpots treated with A, B, and D was separated by nylon mesh (to prevent roots from contacting each other, but microorganisms and mycorrhizal hyphae can transmit signals between the underground rhizosphere), and each part of the flowerpot treated with C was separated by plastic film (roots, hyphae, water, small molecules, and root secretions cannot pass through). Corn and Aconitum carmichaelii were planted in the two side chambers, respectively, and no crops were planted in the middle to prevent the direct diffusion of water and nutrients.

[0043] Planting techniques: Huangcaowu was raised using seedlings, and corn was sown on-demand. Transplanting and sowing were carried out on May 27, 2023. Post-planting field management procedures, including tillage, weeding, and watering, were the same for all treatments.

[0044] Table 1 Different treatments of intercropping Aconitum carmichaelii and corn Note: Y stands for maize; H stands for Aconitum caryophyllus; Gm stands for Glomus mosseae; B stands for Microbacterium maydis; the symbol + before Y / H stands for the accessed strain, and - stands for the unaccessed strain.

[0045] Inoculation time of corn leaf blight pathogen: On August 11, 2023, corn leaves in treatments A, B, and C were inoculated with corn leaf blight pathogen.

[0046] Measurement indicators and methods Measurement time On August 11, 2023, corn leaves were inoculated with southern leaf spot disease to measure agronomic traits. Three pots of Aconitum kusnezoffii plants were randomly selected from each of the four treatments, each located on a uniform contour line. Leaf and root samples were collected from each treatment three times. Labeled, sealed bags were placed in a portable ice box, and these samples, representing the 0-hour inoculation period, were brought back to the school for testing. (All equipment used for sampling was disinfected and sterilized.)

[0047] Determination method of AMF fixed value rate 1 cm lateral root segments were fixed with a FAA solution prepared with 130 ml formaldehyde, 50 ml glacial acetic acid, and 2000 ml 15% ethanol for 24 hours. The segments were then rinsed with pure water, immersed in a 10% KOH solution, and heated in a 90°C waterbath for 60 minutes. After rinsing with pure water, the segments were acidified in a 2% hydrochloric acid solution for 10 minutes and stained with a 0.05% trypan blue glycerol solution (lactic acid, glycerol, and water in a volume ratio of 3:1:1). The prepared maize root segments were placed in a 1.5 mL centrifuge tube, 1 / 3 volume of glycerol was added, and the tubes were refrigerated for later use. AMF infection and colonization structures were observed under a microscope, and the colonization rate was calculated.

[0048] Calculation formula: AMF colonization rate = ∑ m × n / N = ∑ (0% × number of root segments + 10% × number of segments + 20% Determination of growth physiological indicators Plant height: Use a steel tape measure to measure the distance from the base of the plant to the tip of the top leaf.

[0049] Stem diameter: Measure the stem diameter at the first node above the ground using a vernier caliper.

[0050] Leaf area index: Select a plant, unfold its leaves and measure its length and width. Leaf area = leaf length × leaf width × 0.75.

[0051] Determination of photosynthetic indexes and chlorophyll content The photosynthetic parameters were determined by light response curve measurement using a portable photosynthetic meter. The net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were measured at noon when the sunlight intensity was maximum. The measurement of these four photosynthetic indices began at 0 hours after inoculation. Three plants with the same growth were selected for each treatment. Three leaves were randomly selected from each plant of Aconitum carmichaelii. The four photosynthetic parameters of Aconitum carmichaelii leaves were measured in the field using the Li-6400 portable photosynthetic meter.

[0052] The leaves used to measure the chlorophyll content should be freshly collected from Aconitum carmichaelii. They should be wrapped in tin foil, labeled, and immediately immersed in liquid nitrogen. Return to the laboratory to determine the chlorophyll content. If the experiment cannot be conducted immediately, transfer them to a -80°C refrigerator for storage. Use the ethanol extraction method. According to the Lambert-Beer law A=kCL, the different pigments in the extract have different absorption of the visible spectrum. Observe and record the absorbance A of each pigment at the maximum absorption wavelength on the photosynthetic chromatograph. Then use the formula to calculate the concentration of each pigment in the extract. The empirical formula is: Chlorophyll a concentration: Ca=13.95A665-6.88A649 Chlorophyll b concentration: Cb = 24.96A649-7.32A665 Carotenoid concentration: Cx = (1000A470-2.05Ca-114.8Cb) / 245 Determination of leaf enzyme activity Aconitum kusnezoffii samples were collected at 0, 6, 12, and 24 hours after inoculation with southern corn leaf spot to test physiological parameters. The effects of the mycelial network system on various physiological parameters at different time periods after inoculation were investigated. The collected Aconitum kusnezoffii leaves were immediately frozen in liquid nitrogen, transported back to the laboratory, and stored in a -80°C refrigerator. These samples were then used to measure key enzymes of the reactive oxygen species system (SOD and POD) in the leaves. POD activity was determined using the guaiacol method, following the method of Yuan Qinghua et al. Furthermore, the activity of superoxide dismutase (SOD), a key enzyme in the defense system (PPO), was determined using the enzyme activity assay method described by Sui Li et al.

[0053] Determination of amino acid content As described above, the amino acid content in the leaves of Aconiti Kusnezoffii was measured using samples collected 0 hours, 6 hours, 12 hours, and 24 hours after the first inoculation with southern corn leaf blight, and the ninhydrin colorimetric method was selected for determination.

[0054] Results and Analysis AMF determination rate under different treatments From the experimental data results in Table 2, it can be seen that only group B was not inoculated with Rhizobacterium Moses, and the colonization rate in the later stage was 0, while the colonization rate of group C was the most significant.

[0055] Table 2 Colonization rate of Aconitum kusnezoffii Note: Data are mean ± standard deviation. Different lowercase letters indicate statistical significance between groups (P < 0.05) (SPSS data analysis, univariate analysis). The following tables are the same.

[0056] Effects of different mycelial network treatments on agronomic traits of Aconitum kusnezoffii Table 3, treatments C and D inoculated with Glomus mosseae significantly increased the plant height, stem diameter and leaf area of Aconitum kusnezoffii, and had a superior effect on the agronomic traits of Aconitum kusnezoffii. The hyphae network system of treatment A showed more obvious advantages in agronomic traits.

[0057] Table 3 Effects of mycelial network on agronomic traits of Aconitum kusnezoffii Effects of different treatments on the content of photosynthetic pigments in Aconitum kusnezoffii As shown in Table 4, the photosynthetic pigment contents in treatments A, C, and D, which established mycelial networks, were significantly higher than those in treatment B (CK). Furthermore, the Chla, Chlb, and Chl (a + b) contents in treatment D were all lower than those in the other two treatments. During plant growth, light is a crucial factor influencing plant growth. Light energy is absorbed by plants, generating photosynthesis. Within the plant, the tricarboxylic acid cycle is carried out, converting primary substances into energy required for growth and development. Plants are categorized as sun-loving, shade-loving, and neutral based on their light requirements. Sun-loving plants require stronger light and can grow and develop even under very high saturating light intensities. Shade-loving plants are more shade-tolerant and require appropriate shade, growing and developing better under low light conditions. Both excessively high and low light intensities can reduce the content of photosynthetic pigments in plants, inhibiting photosynthesis and even damaging chloroplast and mitochondrial structures. Chlorophyll provides the material basis for photosynthesis and influences its rate. It is distributed throughout leaves and stems. Increased chlorophyll content facilitates the capture and absorption of light energy.

[0058] Table 4 Effects of different treatments on the content of photosynthetic pigments in Aconitum kusnezoffii Effects of different treatments on photosynthetic parameters of Aconitum kusnezoffii As shown in Table 5, Pn and Tr in the experimental groups A, C, and D that established mycelial networks were significantly higher than those in group B (CK), and Ci in group B was higher than that in the other three treatment groups. The data show that Ci of the leaves of Aconitum carmichaelii was positively correlated with Pn, Tr, and Gs, indicating that the photosynthesis of Aconitum carmichaelii was affected by stomatal factors. The reduction of Gs hindered CO2 from entering the mesophyll cells to participate in the reaction, resulting in lower photosynthesis of Aconitum carmichaelii.

[0059] Table 5 Effects of different treatments on photosynthetic parameters of Aconitum kusnezoffii Effects of different treatments on enzyme activities of Aconitum carmichaelii Depend on Figure 1 As can be seen from the test results, catalase (CAT) activity in all four treatments showed a downward trend from 0 to 6 hours. Treatments A, C, and D showed an increase after 6 hours. Treatment A showed a decrease to a certain value after 6 hours before recovering and returning to the original enzyme activity level within 12 hours. The fluctuation in enzyme activity was also minimal. Treatment B continued to decline after 6 hours, and only showed an upward trend after 12 hours. Catalase is present in all plant tissues, and its activity is correlated with a plant's metabolic intensity and resistance to cold and disease.

[0060] Depend on Figure 2 It can be seen that the peroxidase (POD) activity of treatment B decreased significantly in the 0-12h stage, and began to recover when it reached an extremely low value. The POD activity of groups A, C, and D fluctuated slightly. The enzyme activity of treatments B and C at 24h was lower than that at 0h, and could not return to the original enzyme activity level within 24h. Treatment A recovered to above the original enzyme activity level within 24h after infection with pathogens and tended to be stable.

[0061] Depend on Figure 3 As can be seen, the superoxide dismutase activity test results showed that the SOD activity of all four treatments showed an upward trend from 0 to 6 hours. Treatment A had higher enzyme activity than the other three treatments, and at 12 hours, it tended to the SOD activity level before infection. The enzyme activity of treatments B and C remained at a relatively low level. The enzyme activity level of treatment B was the most affected by the disease stress and was significantly lower than that of the other three treatments. SOD is an important antioxidant, widely present in animals, plants, and microorganisms, and has the specific function of scavenging free radicals.

[0062] Depend on Figure 4As can be seen, the impact of key enzymes in the defense enzyme system, polyphenol oxidase activity test results showed that PPO activity tended to a constant value in the 6-hour period. When subjected to stress, the PPO activity of treatment A dropped to a certain value in the 0-6-hour period, then began to rise again, returning to the original enzyme activity level in the 24-hour period. The PPO value of group B dropped sharply in the 0-6-hour period, and the PPO activity fluctuated the most when subjected to stress, and could not return to the original enzyme activity level within 24 hours. In treatment D, which was not exposed to corn leaf blight, PPO showed an upward trend in the 0-6-hour period, reached a certain value in 6 hours, began to decline, and returned to the original PPO activity level.

[0063] Effects of different treatments on the amino acid content of Aconitum kusnezoffii Depend on Figure 5 As can be seen, the amino acid content of all four treatments increased from 0 to 12 hours and then decreased after 12 hours. However, the amino acid content of treatments A, C, and D was significantly higher than that of treatment B at all time periods, with the amino acid content ranking A > D > C > B. Amino acids are important components of plant tissues and are essential for many structural components. Their content reflects plant quality and also has certain effects on plant immunity and stress resistance.

[0064] Changes in defense enzyme activity are one of the mechanisms by which plants activate their own immune defense responses. Numerous researchers have conducted in-depth research on the relationship between various physiological and biochemical metabolic reactions in plants after pathogen infection and plant disease resistance. They believe that superoxide dismutase (SOD) and peroxidase (POD) play a crucial role in defending against damage to cell membranes by reactive oxygen species and oxygen free radicals. Polyphenol oxidase (PPO) promotes the production of various secondary metabolites in plants, hindering the invasion and proliferation of pathogens. Literature has reported a positive correlation between the activities of these enzymes and disease resistance. In recent years, numerous studies have reported that plant growth-promoting bacteria promote plant growth by influencing the activity of defense enzymes such as PPO and POD. In summary, establishing a mycelial network between grain and medicine can increase the activity of key enzymes in the reactive oxygen system (SOD and POD) and the defense system (PPO), thereby enhancing the stress resistance of Aconitum kusnezoffii.

[0065] In our study, when infected with pathogens, Aconitum kusnezoffii, which establishes a fungal hyphal bridge with maize, induces the expression of key defense signaling enzymes (POD, SOD, CAT, and PPO) in the roots of the donor Aconitum kusnezoffii through interplant communication, stimulating the plant's disease defense system and synergizing mycorrhizal resistance. Although Treatment B meets the requirements for intercropping Aconitum kusnezoffii, the agronomic performance of both maize and Aconitum kusnezoffii during the same period is poor compared to the other three treatments, with significantly lower defense efficacy after disease infection. Treatment C, which only inoculated with G. mosseae without establishing a fungal hyphal bridge with maize, while exhibiting better defense efficacy than Treatment B, remains inferior to Treatment A. Numerous studies have shown that mycorrhizae themselves absorb soil nutrients and promote plant growth through symbiotic interactions with plant roots. Compared to aboveground plant communication mediated by volatiles, these mycorrhizal bridges are more stable and reliable, less susceptible to factors, and less susceptible to weather. Using underground mycorrhizal hyphal bridges as channels for interplant communication can transmit signals over greater distances.

[0066] The interspecific rhizosphere of plants embodies the "plant-plant, plant-microbe, and plant-soil" interactions, and the mechanisms promoting these interspecific rhizosphere interactions remain largely unknown. Therefore, this study used the arbuscular mycorrhizal fungus (AMF) Glomus mosseae intercropped with Aconiti kusnezoffii to establish a hyphal bridge between the two crops and investigate the signaling effects of rhizosphere interactions. Four treatments were set: A) nylon mesh-separated rhizospheres of Aconiti kusnezoffii intercropped with maize and inoculated with AMF strains, followed by inoculation of corn leaves with Southern Blight (establishing hyphal bridges and transmitting maize susceptibility signals); B) no AMF strains were inoculated in the rhizospheres of Aconiti kusnezoffii intercropped with maize (CK, no hyphal bridges); C) film-separated rhizospheres of Aconiti kusnezoffii intercropped with AMF strains and inoculated with Southern Blight (no hyphal bridges and no maize susceptibility signals); and D) inoculated maize rhizospheres of Aconiti kusnezoffii intercropped with AMF strains (establishing hyphal bridges and no maize susceptibility signals). Physiological, biochemical, and resistance indicators of Aconiti kusnezoffii plants under different treatments were observed at 0, 6, 12, and 24 hours after inoculation with Southern Blight. The results were as follows: (1) Compared with B (CK), the height, stem diameter and leaf area of A. aconitifolia inoculated with AMF strains A, C and D increased. Among them, the height, stem diameter and leaf area of A. aconitifolia inoculated with AMF strains A, C and D increased by about 1.5 times compared with B. (2) The enzyme activities of A. aconitifolia inoculated with AMF strains A, C and D, including CAT, POD, SOD, PPO, photosynthetic index and chlorophyll content, were higher than those in B. At 24 h, the POD and SOD enzyme activities of A were higher than those of B and C. The amino acid content of the four treatments was A>D>C>B, and A was significantly higher than B. This indicates that the hyphal bridge formed by arbuscular mycorrhizal fungi can transmit maize disease signals between the rhizosphere of A. aconitifolia intercropped with maize, stimulate the growth of A. aconitifolia plants, increase the activity of resistance enzymes and increase the total amount of free amino acids, thereby improving the disease resistance of A. aconitifolia. This provides a certain basis for further revealing the effect of intercropping of two crops on reducing pests and diseases.

[0067] This invention describes a method for transmitting disease resistance signals between different crops (Aconiti kusnezoffii and corn) using hyphal bridges formed by arbuscular mycorrhizal fungi (AMF). Research results show that by inoculating AMF strains, such as Glomus mosseae, to establish a hyphal bridge between Aconiti kusnezoffii and the corn rhizosphere, the hyphal bridge effectively transmits disease resistance signals generated by the corn after pathogen invasion, significantly improving Aconiti kusnezoffii's disease resistance. Four treatment groups were set up in the experiment: Group A, inoculated the Aconiti kusnezoffii and corn rhizospheres with AMF strains, established a hyphal bridge, and then inoculated with southern leaf spot; Group B, inoculated neither the Aconiti kusnezoffii nor the corn rhizospheres with AMF strains, served as a control; Group C, separated the Aconiti kusnezoffii and corn rhizospheres by a film and inoculated with AMF strains; and Group D, inoculated the Aconiti kusnezoffii and corn rhizospheres with AMF strains but without pathogen inoculation. In the experiment, the AMF hyphal bridge significantly improved the agronomic traits, photosynthetic pigment content, and defense enzyme activity of Aconiti kusnezoffii, demonstrating strong disease resistance. This method can effectively enhance crop disease resistance in agricultural production and has important practical applications.

[0068] This invention utilizes hyphal bridges formed by arbuscular mycorrhizal fungi (AMF) to transmit disease resistance signals between different crops (Aconiti kusnezoffii and corn), thereby improving the crop's disease resistance, particularly when intercropping Aconiti kusnezoffii with corn. Studies have shown that AMF hyphal bridges can effectively enhance Aconiti kusnezoffii's resistance to diseases such as leaf spot.

[0069] Specifically: Establishment of mycelial network: By establishing an AMF mycelial bridge between Aconitum carmichaelii and the rhizosphere of corn, the study found that AMF inoculation significantly improved the colonization rate, agronomic traits (such as plant height, stem diameter, leaf area) and photosynthetic pigment content (such as chlorophyll a, chlorophyll b, etc.) of Aconitum carmichaelii, which had obvious advantages over the control group (CK) that was not inoculated with AMF.

[0070] Transmission of disease resistance signals: Pathogen inoculation experiments revealed that AMF hyphae bridges effectively transmit disease resistance signals triggered by pathogen attack in corn. After receiving these signals, the A. aconitifolia plants demonstrated strong disease resistance. For example, enzyme activities (such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)) varied significantly across treatments, with particularly rapid recovery and maintenance of high levels in treatments A, C, and D.

[0071] Physiological response: By measuring photosynthetic parameters (such as net photosynthetic rate, stomatal conductance, etc.) and amino acid content, the study found that the AMF hyphae bridge enhanced the resistance of Aconitum carmichaelii. The disease resistance of treatment groups A, C, and D was significantly higher than that of treatment group B without the hyphae bridge.

[0072] In summary, the present invention improves the growth, disease resistance and physiological state of crops through the establishment of AMF mycelial bridges, showing good agricultural application potential, especially in improving the disease resistance of crops such as Aconitum carmichaelii.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An intercropping method for transmitting disease resistance signals through AMF hyphae bridges, characterized in that: The following steps are involved: S1. Select crops: Select crops that are suitable for symbiosis with arbuscular mycorrhizal fungi as intercrops; S2. Inoculation of AMF strains: Arbuscular mycorrhizal fungal strains were inoculated into the rhizosphere of the two crops to form hyphal bridges; S3. Pathogen inoculation: Inoculate the rhizosphere of one of the crops with pathogens to simulate the disease invasion process; S4. Mycelial bridges transmit disease resistance signals: Through the AMF hyphae bridge, disease-induced resistance signals are transmitted from the infected crop to another crop, thereby improving the disease resistance of the other crop; S5. Observe and evaluate the effects: By testing the physiological and biochemical indicators, agronomic traits and disease resistance responses of crops in different time periods, the transmission effect of AMF mycelial bridge and its role in improving indicators such as disease resistance and disease-resistant enzyme activity were evaluated.

2. The method according to claim 1, characterized in that The crops are Aconitum carmichaelii and corn.

3. The method according to claim 1, characterized in that The AMF strain is Glomus mosseae.

4. The method according to claim 1, wherein The inoculated pathogen is the pathogen of corn leaf blight.

5. The method according to claim 1, wherein The hyphae bridges were separated by nylon mesh.

6. The method according to claim 1, characterized in that The hyphal bridges are established by thin membrane separations.

7. The method according to claim 1, characterized in that Disease resistance was assessed by measuring the content of photosynthetic pigments.

8. The method according to claim 1, characterized in that Disease resistance was assessed by measuring superoxide dismutase (SOD) activity.

9. The method according to claim 1, characterized in that Disease resistance was assessed by measuring catalase (CAT) activity.

10. The method according to claim 1, characterized in that Disease resistance was assessed by measuring amino acid content.

Images