Application of barbadosweed endophytic fungus L2019 in barbadosweed-low-toxicity endophytic fungus symbiont

Abstract

The invention relates to the technical field of agricultural breeding, and particularly discloses application of a barbadosweed endophytic fungus L2019 in a barbadosweed-low-toxicity endophytic fungus symbiont. According to the method, oxytropis fallax seeds irradiated by heavy ion beams are soaked in a barbadosweed endophytic fungus L2019 bacterial suspension, and the barbadosweed-low-toxicity endophytic fungus symbiont is successfully constructed. According to the construction method, the transfection success rate is 61.1%-66.7%, swainsonine can be detected in the earlier stage, the flowering stage and the fruiting stage of the obtained symbiont whole plant, but the swainsonine content is reduced by 67.6%-87.9% compared with that of a wild control group, and a foundation is laid for further development of non-toxic / low-toxicity barbadosnut and field large-area planting. The construction method can be applied to construction of a plant-endophyte symbiont, and the constructed low-toxicity barbadosnut can be further applied to animal husbandry or ecological protection engineering.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural breeding, and specifically discloses an application of endophytic fungus L of locoweed 2019 in a locoweed-low toxicity endophytic fungus symbiont. Background Art

[0002] Locoweed is not a noun term for plant classification, but a general term for poisonous plants containing swainsonine in the genera Astragalus, Oxytropis and Swainsona internationally, which are mainly distributed in pastures in the western United States, Asia and South America. Long-term ingestion of locoweed can cause neurological dysfunction, reduced reproductive ability, growth inhibition and even death in livestock. Due to factors such as its wide distribution range, obvious interspecific competition advantages, strong seed germination ability, earlier greening season, higher content of toxic components after flowering, better palatability, susceptibility to almost all animals, and animal addiction to ingestion, locoweed causes serious harm to livestock breeding, and thus poses a huge threat to the development of the livestock industry. The main toxic substance of locoweed is swainsonine (SW), an indolizidine alkaloid produced by the metabolism of endophytic fungi of locoweed. SW can competitively inhibit the activity of mannosidase, causing enzyme dysfunction and the accumulation of complex oligosaccharides, thereby causing vacuolar degeneration in different cells, especially nerve cells.

[0003] Because of its drought and cold tolerance characteristics and strong vitality, locoweed has spread into a disaster in the western grasslands of China. The direct and indirect economic losses caused by animals being poisoned by ingesting locoweed reach tens of billions of yuan annually in China, seriously endangering the development of China's livestock industry. Therefore, it is urgent to construct low-toxic locoweed. In the prior art, first, it is necessary to select suitable endophytic strains, and some target strains cannot successfully construct the symbiont of the corresponding plants; when only soaking the host seeds in the target strain suspension to prepare the symbiont, transfection often fails; even if transfection is successful by means of a combination of soaking bacteria and injection treatment, or inoculating the target bacteria after making an incision in the hypocotyl of the seedling, the transfection success rate is generally low. Summary of the Invention

[0004] Aiming at the deficiencies in the complex inoculation process and generally low transfection success rate when constructing a plant-endophytic fungus symbiont in the prior art, the present invention provides an application of endophytic fungus L of locoweed 2019 in a locoweed-low toxicity endophytic fungus symbiont. In addition, the present invention also provides a method for constructing a low-toxic locoweed symbiont. By pre-treating Oxytropis ochrocephala Bunge seeds with heavy ion beam irradiation and then inoculating, the transfection success rate of the locoweed-low toxicity endophytic fungus symbiont is greatly improved. The locoweed-low toxicity endophytic fungus symbiont constructed by the present invention can be applied to ecological governance and the livestock industry, and has important social, environmental and economic benefits.

[0005] To achieve the above invention object, the present invention adopts the following technical solutions: First aspect, an application of an endophytic fungus L of locoweed 2019 in a locoweed-low-toxicity endophytic fungus symbiont, wherein the endophytic fungus L of locoweed 2019 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2019541.

[0006] The endophytic fungus L of locoweed selected in the present invention 2019 , is a mutant with stable low production of SW obtained by heavy ion irradiation mutagenesis of the endophytic fungus of locoweed( Alternaria Section Undifilum oxytropis ), and this strain was first publicly disclosed in the Chinese patent document with the application number ZL202310093034.9. This strain is the mutant strain 61 with low production of swainsonine recorded in the above patent document. On July 11, 2019, the inventor named this mutant strain 61 with low production of swainsonine as the endophytic fungus L of locoweed 2019 ( Undifilum oxytropis L 2019 ), and it is preserved in the China Center for Type Culture Collection, and its preservation number is CCTCC NO: M 2019541.

[0007] Through germplasm resource innovation and ecological function integration, the present invention successfully constructs a locoweed-low-toxicity endophytic fungus symbiont by using the endophytic fungus L of locoweed 2019 . This symbiont has multiple application values in ecological governance and the livestock industry, providing a new solution for the resource utilization of locoweed.

[0008] Preferably, the raw materials used to construct the locoweed-low-toxicity endophytic fungus symbiont include Oxytropis ochrocephala Bunge seeds irradiated by heavy ion beam.

[0009] Second aspect, the present invention provides a construction method of the above locoweed-low-toxicity endophytic fungus symbiont. The construction method includes the following steps: soaking the Oxytropis ochrocephala Bunge seeds irradiated by 12 C 6+ heavy ion beam in the bacterial suspension of the endophytic fungus L of locoweed 2019 , and culturing in the dark to obtain the locoweed-low-toxicity endophytic fungus symbiont; By irradiating Oxytropis ochrocephala Bunge seeds with heavy ion beam and soaking the irradiated seeds with bacteria, the present invention successfully constructs a locoweed-low-toxicity endophytic fungus symbiont, laying a foundation for cultivating low-toxic locoweed and large-scale field planting of low-toxic locoweed; at the same time, the present invention provides a new idea for the construction of plant-endophytic fungus symbiont. Compared with the conventional symbiont construction method, the transfection success rate of the present invention can be increased to 61.1% - 66.7%.

[0010] Preferably, the dose of the irradiation is 10 Gy - 20 Gy.

[0011] Preferably, the energy of the irradiation is 70 MeV / μ - 90 MeV / μ. In the present invention, the irradiation energy of 80 MeV / μ is taken as an example for illustration.

[0012] Preferably, the method for preparing the bacterial suspension comprises the following steps: taking the mycelium of the locoweed endophytic fungus L 2019 and suspending it in sterile water, culturing it with shaking to obtain the original bacterial suspension; mixing according to the volume ratio of the original bacterial suspension: sterile water of 5 - 15:95 - 85 to obtain the bacterial suspension.

[0013] In the present invention, the following method for preparing the original bacterial suspension is taken as an example for illustration: picking the mycelium of the mutant strain 61 that has grown for 20 d under dark conditions at 25°C on a PDA medium, breaking the mycelium with an inoculation loop under sterile conditions, grinding the mycelium with a tissue grinder, suspending it in 100 mL of sterile water, transferring it to a 250 mL conical flask, and culturing it in a shaker at 23°C - 27°C and 200 rpm - 280 rpm for 20 h - 28 h to separate the mycelium and prepare the original bacterial suspension.

[0014] Further preferably, the OD 600 of the original bacterial suspension is 1.0 - 1.5.

[0015] Preferably, the soaking time is 10 h - 14 h.

[0016] Preferably, the time for culturing in the darkroom is 6 days - 10 days and the temperature is 21°C - 25°C.

[0017] In the third aspect, the present invention provides a locoweed - low - toxicity endophytic fungus symbiont, which is prepared by the method for constructing the locoweed - low - toxicity endophytic fungus symbiont provided in the second aspect.

[0018] In the fourth aspect, the present invention provides the application of the above - mentioned locoweed - low - toxicity endophytic fungus symbiont in animal husbandry or ecological protection projects; wherein, the animal husbandry includes forage grasses in animal husbandry; the ecological protection projects include fields such as wind prevention and sand fixation, soil and water conservation, and desertification control.

[0019] The locoweed-low-toxic endophyte symbiont provided by the present invention can reduce the content of toxic components such as swainsonine in locoweed by regulating the metabolic level of endophytes, and can be further developed into a safe forage resource; if this symbiont is used to replace the conventional locoweed in the ecosystem, it can significantly reduce the economic losses of herdsmen caused by livestock poisoning and promote the sustainable development of grassland animal husbandry. Therefore, it has outstanding potential economic and social benefits. In addition, in view of the thick and developed roots of this symbiont and its extremely strong drought resistance and barren tolerance, it can be applied to the reconstruction of degraded grassland vegetation, the fixation of sandy land and the prevention and control of soil erosion. Its deep root network can effectively increase the content of soil organic matter, and its potential environmental benefits are prominent. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is the gel electrophoresis diagram of PCR products of different samples in the present invention, where lane 1 represents Marker, lane 2 represents the leaves of wild Oxytropis ochrocephala, and lane 3 represents the locoweed endophyte L 2019 , lane 4 represents the starting strain of the locoweed endophyte L 2019 , lane 5 represents the leaves of Oxytropis ochrocephala without endophytes, and lane 6 represents the negative control. Detailed Embodiments

[0022] In order to make the purpose, technical solutions and advantages of the present invention clearer, the following further details the present invention in combination with embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0023] The experimental materials, equipment and experimental methods used in the present invention are as follows: I. Experimental Materials and Main Instruments 1. The seeds of wild Oxytropis ochrocephala were obtained by the inventor after collection in Huangyuan County, Qinghai Province, and manually threshed after natural drying; Potato Dextrose Agar (PDA) and Potato Dextrose Broth powder medium were both purchased from Huankai Microorganisms; the plant genomic DNA extraction kit was purchased from Beijing Bioteke Corporation.

[0024] 2. Main instruments: The intelligent artificial climate chamber was purchased from Conviron Adapts, Canada; the QTRAP 5500 ultra-high performance liquid chromatography-mass spectrometry was purchased from AB SCIEX, USA; the PCR instrument was purchased from Biometra, Germany; the DYY-8C electrophoresis apparatus was purchased from Liuyi Instrument Factory, Beijing; the gel imaging system was purchased from Applied Biosystems, USA.

[0025] II. Experimental methods 1. Disinfection of seeds and obtaining of embryos The seeds of Oxytropis ochrocephala were surface-disinfected. The seeds were placed in a gauze piece and disinfected in 75% alcohol for 2 min, then in 1% sodium hypochlorite for 1 min, and finally rinsed 3 times with sterile water. After drying the surface moisture of the seeds with sterile filter paper, the seed coat was carefully scratched at the hilum with a scalpel without damaging the embryo, and then the seeds were soaked in sterile water for 12 h to absorb water, obtaining disinfected seeds.

[0026] Under a stereomicroscope in a laminar flow hood, the above-mentioned imbibed Oxytropis ochrocephala seeds were carefully peeled off the seed coat using tools such as micro-dissection forceps and dissection needles to obtain embryos.

[0027] 2. Obtaining of Oxytropis ochrocephala leaves in different groups (1) The above-separated embryos were placed on a petri dish containing double-layer sterile filter paper, 25 per dish. ① 5 mL of sterile water was added to each dish to moisten the filter paper, the embryos were placed on it, sealed with a sealing film, and cultured in the dark in a constant temperature incubator at 23 °C for one week to obtain germinated Oxytropis ochrocephala without endophytic fungi; ② According to the bacterial suspension prepared in different examples, 2 mL of sterile water was first added to each dish to moisten the filter paper, the embryos were placed on it, and then 3 mL of the corresponding bacterial suspension was added. After soaking at 20 °C - 25 °C for 10 h - 14 h, they were then cultured in the dark in a constant temperature incubator at 21 °C - 25 °C for 6 days - 10 days to obtain Oxytropis ochrocephala infected with endophytic fungus L 2019 of Oxytropis ochrocephala; (2) For the wild group, the seed coat was not separated, but instead, the seed coat was sanded until there were slight scratches and then placed in a petri dish containing double-layer filter paper for germination. There were 20 seeds per dish, and they were cultured in the dark in a constant temperature incubator at 23 °C for 1 week to obtain germinated wild Oxytropis ochrocephala.

[0028] (3) Mix the sterilized coconut bricks imported from India with Northeast China black soil and vermiculite, then divide them into disposable paper cups and compact them for later use. Use tweezers to carefully pick up the germinated endophytic fungus-free yellow flower thorn bean or the germinated wild yellow flower thorn bean and place it in the prepared soil. Transfer it to an intelligent artificial climate chamber for cultivation. The conditions are set to light intensity level 1, temperature 25℃, dark temperature 18℃, each for 12 hours, and humidity 0. Water the plants once a week with Meloko universal home gardening concentrated nutrient solution. The dilution method is to add 30mL of nutrient solution to 4L of distilled water. Water the plants normally during this period to obtain locoweed seedlings. Statistical analysis of the survival rate of the seeds after germination in the greenhouse and transplantation.

[0029] Survival rate = ×100% (4) Four weeks after transplanting (at this stage, the endophytes have essentially infected the locoweed seedlings), leaves from plants in different groups were collected. Leaves from endophyte-free Oxytropis flavescentis and wild Oxytropis flavescentis were used to determine whether the transfection was successful using PCR. The transplanted locoweed seedlings were cultured in an intelligent artificial climate chamber until flowering, and then moved outdoors for culture.

[0030] 3. In the present invention, specific primer PCR combined with agarose gel electrophoresis was used to detect endophytic fungi in transfected plants to verify whether the transfection was successful. The specific method is as follows: The primers used in the present invention are locoweed-specific primers OmtssuF / OmtssuR, and the primer sequences are shown in Table 1. Table 1 Tested primers

[0031] (1) The present invention uses wild Oxytropis lutea leaves and Oxytropis lutea leaves without endophytic fungi, and locoweed endophytic fungi L 2019 The starting strain (No. CICC2493), the endophytic fungus L 2019 PCR amplification of the DNA was performed using specific primers to ensure the specificity of the primers.

[0032] Plant DNA extraction: Take 100 mg of leaves from plants in different groups and grind them into fine powder in a mortar with liquid nitrogen. Transfer the fine powder to a 1.5 mL centrifuge tube, label it, and use a new rapid plant genomic DNA extraction kit according to the operating procedures to extract total DNA.

[0033] PCR amplification: The extracted DNA was subjected to PCR amplification using specific primers OmtssuF / OmtssuR. The PCR amplification reaction system (20 μL): 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of template DNA, 10 μL of Mixure enzyme, and 8 μL of water. For the PCR reaction, pre-denaturation was performed at 94 °C for 10 min, denaturation at 94 °C for 45 s, annealing at 52 °C for 45 s, extension at 72 °C for 45 s, for 30 cycles, and finally extension at 72 °C for 10 min.

[0034] The PCR products of the specific primers were detected by agarose gel electrophoresis using an electrophoresis apparatus. The gel electrophoresis diagrams of the PCR products of different samples are as Figure 1 shown, where Figure 1 lane 1 in 2019 represents Marker (DNA standard DL500), lane 2 represents the leaves of wild Oxytropis ochrocephala, lane 3 represents the endophytic fungus L of locoweed 2019 , lane 4 represents the starting strain of the endophytic fungus L of locoweed

[0035] From Figure 1 it can be seen that the wild Oxytropis ochrocephala, the endophytic fungus L of locoweed 2019 and the starting strain of the endophytic fungus L of locoweed 2019 (number: CICC2493) all showed target bands at the 164 bp length, while the leaves of Oxytropis ochrocephala without endophytic fungi and the negative control did not show target bands, indicating that the primers were specific and the detection method was feasible.

[0036] (2) The specific primer PCR method was used to detect whether the leaves of the transfected plants cultivated in different examples contained endophytic fungi, to judge whether the transfection was successful, and then to calculate the transfection success rate.

[0037] Example 1 The present invention first investigated the dose of irradiated Oxytropis ochrocephala seeds, and then constructed a locoweed - low - toxicity endophytic fungus symbiont using the preferred pretreated seeds. The specific content is as follows: I. Before constructing the locoweed - low - toxicity endophytic fungus symbiont of the present invention, the Oxytropis ochrocephala seeds need to be pretreated by heavy ion beam irradiation. In this example, the irradiation conditions were optimized to conduct subsequent research after obtaining seeds with a relatively high germination rate. The specific content is as follows: Randomly select 2400 brown Oxytropis ochrocephala Bunge seeds without lesions, wormholes, and plump under a stereomicroscope. Among them, 1800 seeds are randomly divided into 3 groups for irradiation, with 600 seeds per group. Place the seeds of these three groups into irradiation trays with a diameter of 35 mm, with 50 seeds added to each irradiation tray, 12 irradiation trays per group. After sealing with sealing film, place them on a rotating rack. Use the 12C6+ heavy ion beam provided by the shallow irradiation end (HIRFL) of the heavy ion accelerator in the State Key Laboratory of Heavy Ion Accelerator of the Institute of Modern Physics, Chinese Academy of Sciences, to irradiate the Oxytropis ochrocephala Bunge seeds with a carbon ion beam of 80 MeV / μ, LET of 40 Kev / μm. Three irradiation doses are set at 10 Gy, 20 Gy, and 40 Gy respectively, and three replicates are set for each dose. Test the germination rate of the three groups of seeds irradiated with different doses and the non-irradiated seeds (recorded as the control group). The results of the germination rate test are shown in Table 2.

[0038] Table 2

[0039] As can be seen from Table 1, the germination rate of the seeds in the 10 Gy dose group is 90.5 ± 4.20%, the germination rate of the 20 Gy dose group is 88.2 ± 2.83%, and the germination rate of the seeds in the 40 Gy dose group is 60.3 ± 5.61%. For the irradiated seeds, there is no significant difference in the germination rate between the first two groups (P > 0.05). Observing the embryo states of different treatments, it is found that the embryo state of the control group is the best, followed by the 20 Gy dose group, and the embryo state of the 40 Gy dose group is relatively poor. For the smooth progress of subsequent bacterium infection experiments, the Oxytropis ochrocephala Bunge seeds obtained from the 20 Gy dose group (recorded as pre-treated Oxytropis ochrocephala Bunge seeds) are selected for subsequent experiments in the present invention.

[0040] Second, this embodiment provides a method for constructing a locoweed - low-toxic endophytic fungus symbiont, and the construction method includes the following steps: S1. Preparation of bacterial suspension Pick the mycelia of the locoweed endophytic fungus L 2019 grown on PDA medium for 20 d under dark conditions at 25°C. Under sterile conditions, use an inoculation loop to break the mycelia, grind the mycelia with a tissue grinder, suspend them in 100 mL of sterile water, add 20 mL of sterilized PDA medium, transfer them to a 250 mL conical flask, and culture them in a shaker at 25°C and 250 rpm for 24 h to separate the mycelia, obtaining a stock solution of bacterial suspension with an OD 600 of 1.3; Mix the stock solution of bacterial suspension and sterile water according to the volume ratio of the stock solution of bacterial suspension: sterile water of 10:90 to prepare the bacterial suspension.

[0041] S2. Transfection treatment According to the method of "disinfection of seeds and obtaining of seed embryos" recorded in the present invention, disinfect 50 pre-treated Oxytropis ochrocephala Bunge seeds, and remove the seed coat to obtain the seed embryos; Place the separated embryos on a petri dish with double-layer sterile filter paper, 25 per dish. First, add 2 mL of sterile water to each dish to moisten the filter paper, place the embryos on it, then add 3 mL of the bacterial suspension, soak at 20 °C - 25 °C for 12 h, and then culture in the dark in an incubator at 23 °C for 7 days. During the dark culture period, add the bacterial suspension once every 3 days. After the dark culture ends, 44 germinated locoweed embryos are counted.

[0042] S3. Transplanting Mix the imported sterilized coconut brick from India with the black soil from Northeast China and vermiculite, then divide and pack them into disposable paper cups and compact for standby.

[0043] Carefully pick up the above-mentioned germinated embryo tissue with forceps and place it into the prepared soil, then transfer it to an intelligent artificial climate chamber for cultivation. The conditions are set as light intensity level 1, temperature 25 °C, temperature 18 °C in the dark, each for 12 h, and humidity 0. Water once a week with the Scotts All Purpose Flower & Vegetable Concentrate Nutrient Solution. The dilution method is to add 30 mL of nutrient solution to 4 L of distilled water. During this period, water normally to obtain locoweed seedlings. Two weeks after transplantation, it is counted that 38 of the seeds germinated and survived after transplantation and cultivation in the greenhouse. After calculation, the survival rate of locoweed seedlings is 86.4%.

[0044] Transplant the germinated Oxytropis ochrocephala Bunge obtained in the present invention under the same conditions. The survival rate of Oxytropis ochrocephala Bunge seedlings is 93.1%.

[0045] After soaking the seeds of Oxytropis ochrocephala Bunge with bacteria and germinating and growing the seedlings, the plants grow well after being transplanted into the intelligent artificial climate chamber.

[0046] S4. Endophytic fungus detection Four weeks after transplantation, collect the leaves of different plants, and use the specific primer PCR combined with agarose gel electrophoresis method to detect the endophytic fungi in the leaves of different plants. The PCR products of 24 plants show target bands in the electrophoresis diagram, and the corresponding plants are the locoweed - low-toxic endophytic fungus symbionts. After calculation, the transfection success rate of the locoweed - low-toxic endophytic fungus symbiont construction method provided in this example is 63.2%.

[0047] Remove the locoweed seedlings with unsuccessful transfection, culture the seedlings in the intelligent artificial climate chamber until they bloom, then transfer them to the outdoor for cultivation, and collect the seeds after the seeds mature.

[0048] Example 2 This example provides a method for constructing a locoweed - low-toxic endophytic fungus symbiont, and the construction method includes the following steps: S1. Preparation of bacterial suspension The method for preparing the bacterial suspension provided in this example is basically the same as that in Example 1, except that after transferring it into a 250 mL conical flask, it is cultured in a shaker at 27 °C and 200 rpm for 20 h to separate the hyphae, and a stock solution of the bacterial suspension with an OD 600 of 1.0 is obtained; the two are mixed according to the volume ratio of the stock solution of the bacterial suspension: sterile water of 15:85 to obtain the bacterial suspension.

[0049] S2. Transfection treatment According to the method of "disinfection of seeds and obtaining of seed embryos" recorded in the present invention, 50 Oxytropis ochrocephala seeds pretreated according to the method recorded in Example 1 are disinfected, and the seed coats are removed to obtain seed embryos; The separated seed embryos are placed on a culture dish with double-layer sterile filter paper, 25 in each dish. First, add 2 mL of sterile water to each dish to moisten the filter paper, place the seed embryos on it, then add 3 mL of the bacterial suspension, soak at 20 °C - 25 °C for 10 h, and then culture in the dark in a constant temperature incubator at 22 °C for 10 days. During the dark culture period, the bacterial suspension is replenished once every 3 days. After the dark culture is completed, 42 germinated locoweed seed embryos are counted.

[0050] S3. Transplanting The imported sterilized coconut coir from India is mixed with the black soil from Northeast China and vermiculite, and then divided and packed into disposable paper cups and compacted for standby.

[0051] Carefully pick up the above germinated seed embryo tissues with forceps and transfer them to the prepared soil, and then transfer them to an intelligent artificial climate chamber for cultivation. The conditions are set as light intensity level 1, temperature 25 °C, temperature 18 °C in the dark, each for 12 h, and humidity 0. Water once a week with the Scotts All Purpose Flower & Vegetable Plant Food concentrated nutrient solution. The dilution method is to add 30 mL of the nutrient solution to 4 L of distilled water. During this period, water normally to obtain locoweed seedlings. Two weeks after transplantation, it is counted that 27 plants survived after germination and transplantation in the greenhouse. After calculation, the survival rate of locoweed seedlings is 64.3%.

[0052] S4. Endophyte detection Four weeks after transplantation, collect the leaves of different plants, and use specific primer PCR combined with agarose gel electrophoresis to detect the endophytes in the leaves of different plants. The PCR products of 18 plants show target bands in the electrophoresis pattern, and the corresponding plants are the locoweed - low - toxicity endophyte symbionts. After calculation, the transfection success rate of the method for constructing the locoweed - low - toxicity endophyte symbiont provided in this example is 66.7%.

[0053] Remove the locoweed seedlings with unsuccessful transfection, culture the seedlings in an intelligent artificial climate chamber until they bloom, and then transfer them to the outdoor for cultivation. After the seeds are mature, collect the seeds.

[0054] Example 3 This embodiment provides a method for constructing a locoweed - low - toxicity endophytic fungus symbiont, and the construction method includes the following steps: S1. Preparation of bacterial suspension The method for preparing the bacterial suspension provided in this embodiment is basically the same as that in Example 1, except that after transferring it into a 250 mL conical flask, it is cultured in a shaker at 23 °C and 280 rpm for 28 h to separate the hyphae, and a stock solution of bacterial suspension with an OD 600 of 1.5 is obtained; the two are mixed according to the volume ratio of the stock solution of bacterial suspension: sterile water of 5:95, and the bacterial suspension is diluted with sterile water to obtain the bacterial suspension.

[0055] S2. Transfection treatment According to the method of "disinfection of seeds and obtaining of seed embryos" recorded in the present invention, 50 Oxytropis ochrocephala Bunge seeds pretreated according to the method recorded in Example 1 are disinfected, and the seed coats are removed to obtain seed embryos; The separated seed embryos are placed on a culture dish equipped with double - layer sterile filter paper, 25 per dish. First, 2 mL of sterile water is added to each dish to moisten the filter paper, the seed embryos are placed on it, then 3 mL of bacterial suspension is added, and they are soaked at 20 °C - 25 °C for 14 h, and then cultured in the dark in a constant - temperature incubator at 25 °C for 6 days. During the dark - room culture, the bacterial suspension is replenished once every 3 days. After the dark - room culture is completed, 43 germinated locoweed seed embryos are counted.

[0056] S3. Transplanting The imported Indian sterilized coconut coir is mixed with northeast black soil and vermiculite and then sub - packed into disposable paper cups and compacted for standby.

[0057] Use forceps to carefully pick up the above - mentioned germinated seed embryo tissues and place them into the prepared soil, and transfer them to an intelligent artificial climate chamber for cultivation. The conditions are set as light intensity level 1, temperature 25 °C, temperature 18 °C in the dark, each for 12 h, and humidity 0. Water once a week with Miracle - Gro general - purpose home gardening concentrated nutrient solution, and the dilution method is to add 30 mL of nutrient solution to 4 L of distilled water. During this period, water normally to obtain locoweed seedlings. Two weeks after transplanting, it is counted that 36 plants survived after germination and transplanting in the greenhouse.

[0058] After measurement, the survival rate of locoweed seedlings is 83.7%.

[0059] S4. Detection of endophytic fungi Four weeks after transplanting, collect the leaves of different plants, and use specific - primer PCR combined with agarose gel electrophoresis to detect the endophytic fungi in the leaves of different plants. The PCR products of 22 plants show target bands in the electrophoresis diagram, and the corresponding plants are the locoweed - low - toxicity endophytic fungus symbionts. After calculation, the transfection success rate of the method for constructing the locoweed - low - toxicity endophytic fungus symbiont provided in this embodiment is 61.1%.

[0060] Remove the locoweed seedlings that failed to be transfected. Cultivate the seedlings in an intelligent artificial climate chamber until they bloom, and then transfer them outdoors for cultivation. Collect the seeds after they mature.

[0061] Comparative Example 1 This comparative example provides a method for constructing a locoweed - low - toxicity endophytic fungus symbiont. The construction method is basically the same as that in Example 1, except that: the seeds of Oxytropis ochrocephala are not pretreated by irradiation, and the rest of the construction steps and process parameters are the same as those in Example 1.

[0062] When detecting the endophytic fungi in the leaves of different plants 4 weeks after transplantation in this comparative example, no target band appeared at 164 bp in the electrophoresis patterns of the 37 surviving transplanted plants, indicating that the transfection success rate was 0. Within the scope of this experiment, without irradiating the seeds of Oxytropis ochrocephala, it is impossible to successfully construct a locoweed - low - toxicity endophytic fungus symbiont. The reason may be that heavy - ion irradiation can damage the cell wall and cell membrane structures of plant cells, resulting in some small channels or pores, which improves the import efficiency of exogenous endophytes.

[0063] Effect Example 1 In the present invention, the wild Oxytropis ochrocephala seedlings described in Example 1 were also cultivated in an intelligent artificial climate chamber until they bloomed, then transferred outdoors for cultivation, and the seeds were collected after they matured, which were used as the wild control group.

[0064] In the present invention, after cultivating the locoweed - low - toxicity endophytic fungus symbionts constructed in Examples 1 - 3 and wild Oxytropis ochrocephala plants in an intelligent artificial climate chamber for 3 months, their plant height, above - ground dry weight, root dry weight and other indicators were measured. The measurement results are shown in Table 3.

[0065] Table 3

[0066] As can be seen from Table 1, there were no significant differences in plant height and biomass between the Oxytropis ochrocephala seed - infected plants and the wild control plants.

[0067] Effect Example 2 In the present invention, after harvesting the seeds of the locoweed - low - toxicity endophytic fungus symbionts constructed in Examples 1 - 3 and wild Oxytropis ochrocephala plants, the swainsonine content in different parts was measured. In addition, the swainsonine content in the whole grass of different plants during different growth periods was also measured. It should be noted that the present invention uses the UPLC - MS / MS method to measure the swainsonine content in plants, and the measurement results are shown in Table 4 below.

[0068] Table 4

[0069] Note: ND indicates not detected.

[0070] As can be seen from the data in Table 4, after the locoweed-low-toxic endophytic fungus symbionts constructed in Examples 1-3 grew in the greenhouse for 3 months, swainsonine was not detected in the plants, while swainsonine was detected during the fruiting period, but the content of swainsonine was significantly lower than that of the wild control group.

[0071] In summary, the present invention utilizes the locoweed endophytic fungus L 2019 ( Undifilum oxytropis L 2019 ) and the seeds of Oxytropis ochrocephala irradiated by 12 C 6+ heavy ion beams to successfully construct a locoweed-low-toxic endophytic fungus symbiont. The transfection success rate of this construction method is between 61.1% and 66.7%, which is greatly improved compared with the transfection success rate of the conventional construction method. At the same time, the symbiont constructed by the present invention grows well; the transfected endophytic fungus has little effect on the germination rate of Oxytropis ochrocephala seeds and the survival rate of seedlings, and has no significant effect on the plant height and biomass of Oxytropis ochrocephala plants growing for 3 months. Swainsonine was not detected in the symbiotic plant during this stage. Although swainsonine can be detected in the pre-flowering, flowering and fruiting periods, the content of swainsonine is significantly lower than that of the wild control group. Among them, the content of swainsonine in the whole herb at different growth stages is reduced by 67.6% - 87.9% compared with the wild control group, laying a foundation for the further development of non-toxic / low-toxic locoweed and large-scale field planting.

[0072] For the above reasons, the locoweed-low-toxic endophytic fungus symbiont provided by the present invention has great application potential in the livestock industry or ecological protection projects; among them, the livestock industry includes forage grass in the livestock industry; the ecological protection projects include fields such as wind prevention and sand fixation, soil and water conservation, and desertification control.

[0073] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of endophytic fungus L of locoweed 2019 in locoweed-low toxicity endophytic fungus symbiont, characterized in that The locoweed endophytic fungus L 2019 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M 2019541.

2. Application of the locoweed endophytic fungus L as described in claim 1 2019 in the locoweed-low toxicity endophytic fungus symbiont, characterized in that The raw materials used for constructing the locoweed - low - toxicity endophytic fungus symbiont include Oxytropis ochrocephala Bunge seeds irradiated by heavy ion beams.

3. A method for constructing a locoweed-low toxicity endophytic fungus symbiont as described in claim 1, characterized in that, The construction method includes the following steps: soaking the seeds of Oxytropis ochrocephala irradiated by heavy ion beams into the suspension of the endophytic fungus of locoweed L 2019 fungus suspension, and culturing in the dark to obtain a symbiont of locoweed and low-toxic endophytic fungus.

4. The method for constructing the locoweed-low toxicity endophytic fungus symbiont according to claim 3, characterized in that, The dose of the heavy ion beam irradiation is 10 Gy to 20 Gy; and / or The energy of the heavy ion beam irradiation is 70 MeV / μ to 90 MeV / μ.

5. The method for constructing a locoweed-low toxicity endophytic fungus symbiont according to claim 2, wherein The preparation method of the bacterial suspension includes the following steps: Take the mycelium of the endophytic fungus L of locoweed 2019 and suspend it in sterile water, and culture it by shaking to obtain the original stock solution of the bacterial suspension; mix it according to the volume ratio of the original stock solution of the bacterial suspension: sterile water of 5-15:95-85 to obtain the bacterial suspension.

6. The method for constructing a locoweed-low-toxic endophytic fungus symbiont according to claim 3 or 5, characterized in that, The OD of the original bacterial suspension 600 is 1.0 - 1.

5.

7. The method for constructing the locoweed-low toxicity endophytic fungus symbiont according to claim 3, characterized in that, The soaking time is 10 h to 14 h.

8. The method for constructing the locoweed-low toxicity endophytic fungus symbiont according to claim 3, characterized in that, The time for culturing in the darkroom is 6 days to 10 days; and / or The temperature for culturing in the darkroom is 21 °C to 25 °C.

9. A locoweed-low toxicity endophytic fungus symbiont, characterized in that, The symbiont is prepared by the construction method of the locoweed - low - toxicity endophytic fungus symbiont according to any one of claims 3 to 8.

10. The application of the locoweed - low - toxicity endophytic fungus symbiont according to claim 9 in animal husbandry or ecological protection projects.

Images