Construction method of oat-endophytic fungus symbiont

Abstract

The invention discloses an oat-endophytic fungus symbiont construction method, which comprises: 1, disinfecting oat seeds, and carrying out surface sterilization by using ethanol and sodium hypochlorite so as to remove external microbial contamination; step 2, culturing aseptic seedlings of the oats, namely culturing the seeds treated in the step 1 on a culture medium; step 3, carrying out enlarged culture, and adopting a secondary culture strategy to ensure that a bacterial source with high activity and enough concentration is available during inoculation; 4, artificial inoculation, wherein artificial inoculation is conducted on the epicotyl parts of the oat aseptic seedlings to obtain oat seedlings; 5, transplanting and culturing, wherein the oat seedlings in the step 4 are transplanted into a growth matrix to be cultured until the seeds are harvested; according to the method, the grass endophytic fungi are inoculated on the epicotyl of the oat, a new oat-endophytic fungi symbiont is constructed, a new oat germplasm resource is created, the good disease-resistant effect is achieved, the stability and heredity of the symbiont are optimized, and stable inheritance of disease-resistant characters is achieved through vertical propagation.

Description

Technical Field

[0001] This invention relates to the field of agricultural breeding technology, and in particular to a method for constructing an oat-endophytic fungus symbiosis. Background Technology

[0002] Endophytic fungi can widely colonize multiple species in the Pooideae subfamily. Their symbiotic relationship with grasses can enhance the host's resistance to various biotic and abiotic stresses. However, they can also produce toxic alkaloids, causing livestock poisoning and seriously jeopardizing the healthy development of grassland animal husbandry. Due to these dual characteristics, grass-endophytic fungal symbionts have become one of the hottest research areas in the past 20 years. Currently, they have been found in more than 300 species of grasses from 80 genera worldwide. These are endophytic fungi belonging to the gramineous phylloxera. The primary mode of dispersal for gramineous endophytic fungi is vertical dispersal through infection of host seeds, preventing widespread interspecific and individual proliferation. Furthermore, desirable traits mediated by endophytic fungi can be stably inherited through vertical dispersal. Therefore, artificially constructing new symbiotic relationships between endophytic fungi and gramineous plants for gramineous germplasm innovation, particularly in resistance breeding, holds significant application potential and is hailed as "next-generation molecular breeding."

[0003] Oats are a high-quality dual-purpose crop for both food and forage. As a globally cultivated crop, oats rank sixth in the world in terms of both planting area and yield. Compared with other crops, oats have higher nutritional value and stronger tolerance to harsh environments, giving them broad planting prospects and extremely high production value. However, many factors still restrict oat production in practice, among which diseases are the main factor affecting oat production and are prevalent in oat-growing regions worldwide. Breeding and planting disease-resistant varieties is an effective way to control oat diseases. Endophytic fungi in grasses can enhance host disease resistance through niche competition, secretion of antimicrobial compounds, and activation of host immune responses, and their traits are stable with a wide range of resistance. Therefore, utilizing endophytic fungi in grasses for oat germplasm innovation can better address the impact of diseases, shorten the breeding cycle, and has good application prospects.

[0004] In recent years, plant-microbe interactions have received widespread attention for enhancing crop stress resistance and promoting growth. Among these, endophytic fungi, a class of microorganisms capable of colonizing plant tissues and forming symbiotic relationships with their hosts, have been shown to enhance plant stress resistance, increase growth rates, promote nutrient absorption, and reduce pathogen invasion. Numerous studies have demonstrated that endophytic fungi can help plants cope with environmental stress through various mechanisms. For example, some endophytic fungi can produce growth hormones, promoting root development and improving the absorption of water and nutrients; others can reduce the accumulation of reactive oxygen species by secreting antioxidant enzymes (such as peroxidase and superoxide dismutase), thereby mitigating stress-induced damage to plant cells. Furthermore, some endophytic fungi can induce systemic resistance in plants, enhancing their defense against pathogens and pests.

[0005] Therefore, this invention creates an oat-endophytic fungus symbiosis, utilizing the biological functions of endophytic fungi to improve the growth ability and disease resistance of oats under adverse environmental conditions such as drought, salinity, and low temperature. This has significant agricultural application value and ecological benefits. Therefore, this invention proposes a method for constructing an oat-endophytic fungus symbiosis to solve the problems existing in the prior art. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to propose a method for constructing an oat-endophytic fungus symbiosis. This method involves inoculating oat hypocotyls with grass endophytic fungi to create a new oat-endophytic fungus symbiosis, establishing a new oat germplasm resource with good disease resistance. The method optimizes the stability and heritability of the symbiosis, and the endophytic fungi achieve stable inheritance of disease resistance traits through vertical transmission, shortening the breeding cycle. By providing an efficient and sustainable solution in agricultural applications, this method improves the yield stability of oats under disease pressure and reduces dependence on chemical pesticides, thus offering significant ecological and economic benefits.

[0007] To achieve the objectives of this invention, the invention is implemented through the following technical solution: a method for constructing an oat-endophytic fungus symbiosis, comprising the following steps:

[0008] Step 1: Disinfect oat seeds by surface sterilization with 75% ethanol and 10% sodium hypochlorite to remove external microbial contamination.

[0009] Step 2: Oat sterile seedling culture. Place the seeds treated in Step 1 on a culture medium for culture to obtain oat sterile seedlings.

[0010] Step 3: Expand the culture. Use a secondary culture strategy to periodically select newly formed endophytic fungal mycelia from the outermost edge of the colony and transfer them to fresh culture medium to simulate natural growth conditions.

[0011] Step 4: Artificial inoculation. Oat seedlings are obtained by artificially inoculating the hypocotyl of sterile oat seedlings.

[0012] Step 5: Transplanting and Cultivation. After washing the roots of the oat seedlings from Step 4, transplant them into the growth substrate for cultivation until the seeds are harvested.

[0013] Step 6: Endophytic fungal monitoring. After transplanting, the inoculation status of endophytic fungi is tested. Seedlings that have not been successfully inoculated are removed, and the seeds of the remaining seedlings are collected after they mature.

[0014] A further improvement is made in the following steps: In step one, the seeds are first disinfected with a 75% ethanol solution for 5 minutes, rinsed with sterile water 3 to 5 times, then disinfected with a 10% sodium hypochlorite solution for 5 minutes, rinsed with sterile water 3 to 5 times, and then the treated seeds are placed on sterile filter paper until the seeds are dry.

[0015] A further improvement is made in the following steps: the culture medium preparation method in step two is to add 15g of agar to 1L of water, then perform high-temperature and moist heat sterilization, and then incubate in the dark for 3-5 days at 25±1℃ in an intelligent light incubator.

[0016] A further improvement is that the endophytic fungal mycelium in step three is... When expanding the culture of bromicola, select the outermost mycelium and place it in a new culture medium to ensure the viability of the mycelium.

[0017] A further improvement is made in step four: the culture medium with mycelia on the periphery of the colony is cut into small pieces and stuffed into the cut of the oat to ensure the viability and concentration of the inoculated mycelia.

[0018] A further improvement is made in that the specific steps of artificial inoculation in step four include:

[0019] S1. Make a 2-3 mm transverse cut at the hypocotyl of the oat sterile seedling and stuff the mycelium into the cut.

[0020] S2. Place the inoculated seedlings in the dark under intelligent light culture and constant temperature conditions for 2-3 days, and control the temperature at 25±1℃.

[0021] S3. Cultivate the inoculated seedlings under constant temperature and light conditions for 5 to 7 days, controlling the temperature at 25±1℃, the light intensity at 25000LUX, and the light duration at 16h·d-1 to obtain oat seedlings.

[0022] A further improvement is that the growth substrate in step five is composed of vermiculite and nutrient soil, and the volume ratio of vermiculite to nutrient soil is 1:1.

[0023] The further improvement lies in the following: the cultivation conditions in step five are 25±2℃, light intensity of 2000~3000lx, and light duration of 16h·d.-1 .

[0024] The beneficial effects of this invention are as follows: By inoculating oat hypocotyls with grass endophytic fungi, this invention constructs a new oat-endophytic fungal symbiosis, creating a new oat germplasm resource with good disease resistance, optimizing the stability and heritability of the symbiosis, and achieving stable inheritance of disease resistance traits through vertical transmission of endophytic fungi, thus shortening the breeding cycle. By providing an efficient and sustainable solution in agricultural applications, this invention improves the yield stability of oats under disease pressure and reduces dependence on chemical pesticides, thus having significant ecological and economic benefits. Attached Figure Description

[0025] Figure 1 This is a flowchart of the steps of the present invention;

[0026] Figure 2 This is a flowchart of the oat sterile seedling inoculation process of the present invention;

[0027] Figure 3 Microscopic image of the leaf sheath of the inoculation material of this invention;

[0028] Figure 4 This is a molecular bacterial detection gel electrophoresis image of the inoculation material of the present invention;

[0029] Figure 5 This describes the greenhouse cultivation of the inoculum material of the present invention.

[0030] Figure 6 This is a comparison diagram showing the disease resistance identification of the novel oat-endophytic fungus symbiotic material of the present invention. Detailed Implementation

[0031] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0032] Endophytic fungi are fungi that survive within plant tissues at a certain stage of their life cycle without causing obvious diseases. Endophytic fungi are defined as fungal groups that exist within healthy plant tissues at a specific stage of their life cycle, obtaining nutrients through mutualistic symbiosis without causing obvious pathological symptoms in the host. Based on functional characteristics, they can be divided into growth-promoting types: secreting indoleacetic acid to promote plant growth, such as the apple endophytic pink broom fungus; defensive types: producing antimicrobial substances to resist pathogens, such as the Sophora flavescens strain BS001 inhibiting 16 pathogenic bacteria; and metabolic types: synthesizing special secondary metabolites, such as the Humulus scandens strain producing novel alkaloids.

[0033] Based on this, according to Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown, this embodiment provides a method for constructing an oat-endophytic fungus symbiont, including the following steps:

[0034] Step 1: Disinfect oat seeds by surface sterilization with 75% ethanol and 10% sodium hypochlorite to remove external microbial contamination.

[0035] Select mature oat seeds with uniform color and size, remove the lemma, disinfect with 75% ethanol solution for 5 minutes, rinse with sterile water 3-5 times, then disinfect with 10% sodium hypochlorite solution for 5 minutes, rinse with sterile water 3-5 times. Place the treated seeds on sterile filter paper until the seeds are dry. This effectively kills bacteria, fungi and other contaminants on the seed surface. The oat seeds used are of the Dafuweng variety, originating from Canada. Their uniform color and size make them easier to standardize, improving disinfection efficiency and success rate. The main function is to remove external microbial contamination from the surface of the oat seeds, ensuring that subsequent operations are carried out in a sterile environment, thus laying the foundation for sterile seedling cultivation.

[0036] Step 2: Oat sterile seedling culture. The seeds treated in Step 1 are placed on a culture medium for culture to obtain oat sterile seedlings. The sterilized seeds are cultivated into healthy oat sterile seedlings under sterile conditions, providing an ideal host for endophytic fungal inoculation, effectively preventing contamination by other microorganisms, and ensuring the sterility of the seedlings.

[0037] The culture medium was prepared by adding 15g of agar to 1L of water and then sterilizing it by high temperature and humidity for 20min to prepare water agar medium. The medium was then poured onto plates for later use. During cultivation, the medium was cultured in the dark at 25±1℃ in an intelligent light incubator for 3-5 days. The agar concentration of the medium optimized the water and nutrient supply, and the dark culture simulated the natural germination conditions of the seeds, thus improving the survival rate of seedlings.

[0038] Step 3: Expand the culture. Use a secondary culture strategy to periodically select newly formed endophytic fungal mycelia from the outermost edge of the colony and transfer them to fresh culture medium to simulate natural growth conditions. The outermost mycelia have the strongest vitality and the most vigorous growth, which can avoid inoculation failure caused by aging mycelia. By proliferating and activating the endophytic fungal mycelia, ensure that there is a high vitality and sufficient concentration of inoculum source at the time of inoculation.

[0039] Endophytic fungal hyphae are When expanding the culture of bromicola, select the outermost mycelium and place it in a new culture medium to ensure the viability of the mycelium.

[0040] Step 4: Artificial inoculation. Oat seedlings are obtained by artificially inoculating the hypocotyl of sterile oat seedlings.

[0041] Cut off a piece of culture medium with mycelium attached to the periphery of the colony, cut it into small pieces and stuff it into the cut of the oat to ensure the viability and concentration of the inoculated mycelium. The scratching of the epicotyl is an optimized design for oats, which is conducive to the rapid colonization of fungi.

[0042] The specific steps for artificial inoculation include:

[0043] S1. Make a 2-3 mm transverse cut at the hypocotyl of the oat sterile seedling and stuff the mycelium into the cut.

[0044] S2. Place the inoculated seedlings in the dark under intelligent light culture and constant temperature conditions for 2-3 days, and control the temperature at 25±1℃.

[0045] S3. Incubate the seedlings under constant temperature and light conditions for 5–7 days, controlling the temperature at 25±1℃, the light intensity at 25000LUX, and the photoperiod at 16h·d. -1 Oat seedlings were obtained.

[0046] Step 5: Transplanting and Cultivation. After washing the roots of the oat seedlings from Step 4, transplant them into the growth substrate for cultivation until seed harvest. The purpose is to transfer the inoculated oat seedlings to the growth substrate to simulate the natural environment, support the plants to grow from the seedling stage to maturity and harvest seeds. At the same time, the light conditions are similar to the natural day length, which promotes photosynthesis and seed formation, ensuring that the symbiont grows stably in the "field-like" environment, laying the foundation for the final seed harvest.

[0047] The growth substrate consisted of vermiculite and nutrient soil, with a volume ratio of vermiculite to nutrient soil of 1:1. The cultivation conditions were 25±2℃, light intensity of 2000~3000lx, and light duration of 16h·d-1.

[0048] Step 6: Endophytic fungal monitoring. After transplanting, the inoculation status of endophytic fungi is tested. Seedlings that have not been successfully inoculated are removed, and the seeds of the remaining seedlings are collected after they mature.

[0049] Four weeks after transplanting, basal leaf sheaths were collected for monitoring. Leaf sheath sections were treated with aniline blue staining solution, and endophytic fungal mycelia were examined under a microscope. Plant DNA was extracted, and tefA was used as a specific primer for detection; successful inoculation resulted in the observation of bright bands. Cultivation continued until successful plants were established, and seeds were harvested.

[0050] Resistance to *Desmodium styracifolium* leaf spot pathogen in oats was assessed using successfully inoculated oat-endophytic fungal symbiotic materials, with oat materials of the same variety but without endophytic fungi serving as a control. Both materials were cultured and separated under identical conditions: a) control group (oat-endophytic fungal symbiotic material); b) inoculated group (oat-endophytic fungal symbiotic material); c) control group (oat material without endophytic fungi); d) inoculated group (oat material without endophytic fungi). Four weeks later, healthy plants with uniform growth were selected and inoculated with the pathogen using the filter paper method. Both materials had filter paper treated with sterile water as a blank treatment group. Characteristics were observed one week after pathogen inoculation. The experiment showed that both the oat-endophytic fungal symbiotic material and the blank treatment group (without endophytic fungi) developed the disease. One week after pathogen inoculation, the oat-endophytic fungal symbiotic material showed little or no disease, while the blank treatment group (without endophytic fungi) exhibited overall yellowing of leaves and numerous leaf spots. The results showed that endophytic fungal symbiosis could significantly improve oat resistance to Delcytosis leaf spot.

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing an oat-endophytic fungus symbiosis, comprising the following steps: Step 1: Disinfect oat seeds by surface sterilization with 75% ethanol and 10% sodium hypochlorite to remove external microbial contamination. Step 2: Oat sterile seedling culture. Place the seeds treated in Step 1 on a culture medium for culture to obtain oat sterile seedlings. Step 3: Expand the culture. Use a secondary culture strategy to periodically select newly formed endophytic fungal mycelia from the outermost edge of the colony and transfer them to fresh culture medium to simulate natural growth conditions. Step 4: Artificial inoculation. Oat seedlings are obtained by artificially inoculating the hypocotyl of sterile oat seedlings. Step 5: Transplanting and Cultivation. After washing the roots of the oat seedlings from Step 4, transplant them into the growth substrate for cultivation until the seeds are harvested. Step 6: Endophytic fungal monitoring. After transplanting, the inoculation status of endophytic fungi is tested. Seedlings that have not been successfully inoculated are removed, and the seeds of the remaining seedlings are collected after they mature.

2. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: In step one, the seeds are first disinfected with 75% ethanol solution for 5 minutes, then rinsed with sterile water 3-5 times, and then disinfected with 10% sodium hypochlorite solution for 5 minutes, then rinsed with sterile water 3-5 times. After that, the treated seeds are placed on sterile filter paper until the seeds are dry.

3. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: The culture medium preparation method in step two is to add 15g of agar to 1L of water, then perform high-temperature moist heat sterilization, and then incubate in the dark at 25±1℃ in an intelligent light incubator for 3-5 days.

4. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: The endophytic fungal mycelium in step three is When expanding the culture of bromicola, select the outermost mycelium and place it in a new culture medium to ensure the viability of the mycelium.

5. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: In step four, the culture medium with mycelia around the colony is cut into small pieces and inserted into the cut of the oat to ensure the viability and concentration of the inoculated mycelia.

6. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: The specific steps for artificial inoculation in step four include: S1. Make a 2-3 mm transverse cut at the hypocotyl of the oat sterile seedling and stuff the mycelium into the cut. S2. Place the inoculated seedlings in the dark under intelligent light culture and constant temperature conditions for 2-3 days, and control the temperature at 25±1℃. S3. Cultivate the inoculated seedlings under constant temperature and light conditions for 5 to 7 days, controlling the temperature at 25±1℃, the light intensity at 25000LUX, and the light duration at 16h·d-1 to obtain oat seedlings.

7. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: In step five, the growth substrate consists of vermiculite and nutrient soil, with a volume ratio of vermiculite to nutrient soil of 1:

1.

8. The method for constructing an oat-endophytic fungus symbiosis according to claim 1, characterized in that: The cultivation conditions in step five are 25±2℃, light intensity of 2000~3000lx, and light duration of 16h·d. -1 .

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