Culture method and application of symbiotic bacteria for promoting growth of Arabidopsis arundinacea

Abstract

The invention provides a culture method of symbiotic bacteria for promoting growth of Arabidopsis arundinacea and application of the symbiotic bacteria. The culture method comprises the following steps: (1) sterilizing a symbiotic bacteria culture medium at 120-125 DEG C for 10-20 minutes; (2) respectively adding the alternaria alternata BB-1, the alternaria brassicae BB-6 and the alternaria hexaphoria BB-8 into the symbiotic bacterium culture medium; and keeping the temperature of the symbiotic bacterium culture medium at 29-30 DEG C, firstly carrying out aerobic fermentation culture for 6-7 hours, then carrying out anaerobic fermentation culture for 3-4 hours, and repeating the steps for 50-80 hours to obtain a symbiotic culture product. According to the invention, DSE of alternaria alternata BB-1, DSE of alternaria brassicae BB-6 and DSE of alternaria hexangularis BB-8 are used to establish a reciprocal symbiotic relationship with host plant Arabidopsis arundinacea in a long-term ecological adaptation process.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a cultivation method and application of symbiotic bacteria for promoting the growth of Pseudomonas aeruginosa. Background Art

[0002] Eulagopsis binata (Retz.) CE Hubb. is a plant of the genus Eulagopsis in the Poaceae family. The culm is 30-80 cm high, smooth and hairless, often branched at the top, with longitudinal grooves on one side and 3-5 nodes. The leaf sheaths are shorter than the internodes except for the lower ones. The basal leaf sheaths are densely covered with white hairs to form a thick base; the ligule is a circle of short cilia, and the leaves are narrow and linear. The racemes are densely covered with light yellow-brown hairs, 2-4 arranged in a finger-like manner, 2-4.5 cm long; the anthers are about 2.5 mm long, the stigma is broom-like, yellow-brown or purple-black. This species is widely distributed and has many variations, but its variant traits are often connected, and it is not appropriate to force it to be separated into species or variants. It grows in the grass on sunny hillsides. Eulagopsis binata is an excellent fiber plant and a good raw material for papermaking, artificial cotton and artificial silk. The young rhizome is mild and slightly bitter in nature, can promote qi and relieve blood, and is mainly used to treat women's dry diseases and prolapse of the anus.

[0003] Because the dominant fungal genus is a pathogen, previous studies have also reported that dark septate endophytes (DSE) are widely colonized in global ecosystems, especially plants growing in extreme environments, and their DSE colonization rate is high. Lugo et al. conducted a survey on endophytic fungi in arid areas of Argentina. The roots of 42 plant species were colonized by endophytic fungi, and DSE colonization was related to plant functional traits and phylogeny. DSE is also common in arid grasslands in Hungary. Other studies have found that endophytic fungi isolated from pioneer plants growing in salinized soils have a certain ability to adapt to high-salt environments. Summary of the invention

[0004] The invention aims to provide a method for culturing symbiotic bacteria for promoting the growth of Pseudocalli chinensis and application thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 120-125° C. for 10-20 min; (2) Alternaria BB-1 ( Alternaria alternata BB-1), Alternaria brassicae BB-6 ( Alternaria brassicae BB-6)、Alternaria alternata BB-8( Alstroemeria AlternariaBB-8) is added to the symbiotic bacteria culture medium; the temperature of the symbiotic bacteria culture medium is maintained at 29-30°C, and aerobic fermentation is first carried out for 6-7 hours, and then anaerobic fermentation is carried out for 3-4 hours, and this is repeated for 50-80 hours to obtain a symbiotic culture product; wherein, Alternaria BB-1 ( Alternaria alternata BB-1) is deposited as: CCTCCNO: M20232213; Alternaria brassicae BB-6 ( Alternaria brassicae BB-6) is deposited as: CCTCCNO:M20232214; Alternaria spp. BB-8 ( Alstroemeria Alternaria BB-8) is deposited as CCTCCNO: M20232215. Further, the symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 4-6 parts of corn flour, 3-5 parts of soybean meal, 2-4 parts of magnesium ammonium phosphate, 1-2 parts of astragalus residue, 2-5 parts of yeast extract, 2-5 parts of apple juice, 1-3 parts of mulberry linterus extract, 0.2-0.6 parts of multivitamins, 0.2-0.3 parts of sodium hydrogen phosphate, 0.5-1.5 parts of wolfberry and 1-3 parts of artemisia annua.

[0006] Furthermore, the preparation method of the apple juice is: add 3 times the mass of water to Aksu Fuji apples, beat into juice, add cellulase in an amount of 500 mg / L to the juice, enzymolyze for 2 hours at 45° C., and filter to obtain apple juice.

[0007] Furthermore, the preparation method of the Phellinus linteus extract is: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0008] Furthermore, the ultrafine powder is sieved through an 80-mesh sieve.

[0009] Furthermore, the pore size of the ultrafiltration membrane is 0.45 μm.

[0010] Further, the composite vitamin is composed of the following components by mass: 11-15 parts of vitamin B1, 10-12 parts of vitamin D, 1-3 parts of riboflavin, 3-5 parts of pantothenic acid and 9-11 parts of vitamin C. Further, the aerobic fermentation culture conditions are: in the symbiotic bacteria culture medium, sterile air is intermittently introduced at a ventilation ratio of 0.35-0.45, and the rotation speed is 110-120 rpm.

[0011] Furthermore, the anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0012] The invention provides application of the symbiotic bacteria culture method in promoting the growth of Pseudocrophyllales chinensis.

[0013] Compared with the prior art, the advantages and beneficial effects of the present invention are: The present invention uses DSE of Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to establish a mutualistic symbiotic relationship with the host plant Pseudo-chrysanthemum in the long-term ecological adaptation process. Pseudo-chrysanthemum provides necessary living space and nutrients for DSE, and DSE may produce some compounds to change the morphological structure of plant roots, maintain the rigidity structure of cell walls, regulate the opening and closing of stomata, and improve the antioxidant activity of plants, thereby enhancing the drought resistance of the host Pseudo-chrysanthemum. The present invention uses a specific culture medium, adds apple juice, mulberry linterus extract, astragalus residues and other components, and simultaneously uses aerobic and anaerobic cycles for culture conditions. The two work synergistically to promote the growth of the three bacteria at the same time. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0015] This embodiment provides a method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 123° C. for 15 min; (2) Add Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; keep the temperature of the symbiotic bacteria culture medium at 30°C, first perform aerobic fermentation culture for 6.5 hours, then perform anaerobic fermentation culture for 3.5 hours, and repeat this process for 70 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCCNO: M20232213, the preservation unit is China Center for Type Culture Collection, and the recommended classification name is: Alternaria BB-1 ( Alternaria alternata BB-1), the culture was received and registered by China Center for Type Culture Collection on November 13, 2023; the viability of the culture was tested by this collection on November 20, 2023, and the result was viable; the preservation number of Alternariabrassicae BB-6 is: CCTCCNO:M20232214, the preservation unit is: China Center for Type Culture Collection, the recommended classification name: Alternaria brassicae BB-6, the culture was received and registered by China Center for Type Culture Collection on November 13, 2023; the viability of the culture was tested by this collection on November 20, 2023, and the result was viable; Alternariaalstroemeriae BB-8 The deposit number of the culture is: CCTCCNO: M20232215, and the depository is: China Center for Type Culture Collection. The suggested classification name is: Alternaria alstroemeriae BB-8. The culture was received by China Center for Type Culture Collection on November 13, 2023 and registered. The viability of the culture was tested by this collection on November 20, 2023, and the result was viable. The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 5 parts of corn flour, 4 parts of soybean meal, 3 parts of magnesium ammonium phosphate, 1.5 parts of astragalus residues, 4 parts of yeast extract, 3 parts of apple juice, 2 parts of mulberry linterus extract, 0.4 parts of multivitamins, 0.25 parts of sodium hydrogen phosphate, 1 part of wolfberry and 2 parts of artemisia annua.

[0016] The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0017] The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0018] After ultrafine grinding, pass through 80 mesh sieve.

[0019] The pore size of the ultrafiltration membrane is 0.45 μm.

[0020] The complex vitamin is composed of the following components in parts by mass: 12 parts of vitamin B, 11 parts of vitamin D, 2 parts of riboflavin, 4 parts of pantothenic acid and 10 parts of vitamin C.

[0021] The aerobic fermentation culture condition is as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0022] The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped. Example

[0023] This embodiment provides a method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 120° C. for 10 min; (2) Adding Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; maintaining the temperature of the symbiotic bacteria culture medium at 29° C., aerobically fermenting the culture medium for 6 h, then anaerobic fermenting the culture medium for 3 h, and repeating the process for 68 h to obtain a symbiotic culture product; wherein the deposit number of Alternaria alternata BB-1 is: CCTCCNO: M20232213; the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the deposit number of Alternaria alstroemeriae BB-8 is: The deposit number of BB-8 is: CCTCCNO:M20232215.

[0024] The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 4 parts of corn flour, 3 parts of soybean meal, 2 parts of magnesium ammonium phosphate, 1 part of astragalus residue, 2 parts of yeast extract, 2 parts of apple juice, 1 part of mulberry linterus extract, 0.2 parts of multivitamins, 0.2 parts of sodium hydrogen phosphate, 0.5 parts of wolfberry and 1 part of artemisia annua.

[0025] The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0026] The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0027] After ultrafine grinding, pass through 80 mesh sieve.

[0028] The pore size of the ultrafiltration membrane is 0.45 μm.

[0029] The complex vitamin is composed of the following components in parts by mass: 11 parts of vitamin B1, 10 parts of vitamin D, 1 part of riboflavin, 3 parts of pantothenic acid and 9 parts of vitamin C.

[0030] The aerobic fermentation culture condition is as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.35 and a rotation speed of 110 rpm.

[0031] The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped. Example

[0032] This embodiment provides a method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 125° C. for 20 min; (2) Adding Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; maintaining the temperature of the symbiotic bacteria culture medium at 30°C, aerobically fermenting the culture medium for 7 hours, then anaerobic fermenting the culture medium for 4 hours, and repeating the process for 75 hours to obtain a symbiotic culture product; wherein the deposit number of Alternaria alternata BB-1 is: CCTCCNO: M20232213; the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the deposit number of Alternaria alstroemeriae BB-8 is: BB-8) is deposited in: CCTCCNO:M20232215.

[0033] The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 6 parts of corn flour, 5 parts of soybean meal, 4 parts of magnesium ammonium phosphate, 2 parts of astragalus residues, 5 parts of yeast extract, 5 parts of apple juice, 3 parts of mulberry linterus extract, 0.6 parts of multivitamins, 0.3 parts of sodium hydrogen phosphate, 1.5 parts of wolfberry and 3 parts of artemisia annua.

[0034] The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0035] The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0036] After ultrafine grinding, pass through 80 mesh sieve.

[0037] The pore size of the ultrafiltration membrane is 0.45 μm.

[0038] The complex vitamin is composed of the following components in parts by mass: 5 parts of vitamin B1, 12 parts of vitamin D, 3 parts of riboflavin, 5 parts of pantothenic acid and 11 parts of vitamin C.

[0039] The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.45 and a rotation speed of 120 rpm.

[0040] The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped. Example

[0041] This embodiment provides a method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 123° C. for 15 min; (2) Adding Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; maintaining the temperature of the symbiotic bacteria culture medium at 30°C, aerobically fermenting the culture medium for 6.5 hours, then anaerobic fermenting the culture medium for 3.5 hours, and repeating the process for 70 hours to obtain a symbiotic culture product; wherein the deposit number of Alternaria alternata BB-1 is: CCTCCNO: M20232213; the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the deposit number of Alternaria alstroemeriae BB-8 is: BB-8) is deposited in: CCTCCNO:M20232215.

[0042] The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 4 parts of corn flour, 5 parts of soybean meal, 2 parts of magnesium ammonium phosphate, 2 parts of astragalus residues, 2 parts of yeast extract, 5 parts of apple juice, 1 part of mulberry linterus extract, 0.6 parts of multivitamins, 0.2 parts of sodium hydrogen phosphate, 1.5 parts of wolfberry and 3 parts of artemisia annua.

[0043] The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0044] The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0045] After ultrafine grinding, pass through 80 mesh sieve.

[0046] The pore size of the ultrafiltration membrane is 0.45 μm.

[0047] The complex vitamin is composed of the following components in parts by mass: 12 parts of vitamin B, 11 parts of vitamin D, 2 parts of riboflavin, 4 parts of pantothenic acid and 10 parts of vitamin C.

[0048] The aerobic fermentation culture condition is as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0049] The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0050] Comparative Example 1 The difference between this comparative example and Example 1 is that the symbiotic culture medium is different.

[0051] A method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 123° C. for 15 min; (2) Adding Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; maintaining the temperature of the symbiotic bacteria culture medium at 30°C, aerobically fermenting the culture medium for 6.5 hours, then anaerobic fermenting the culture medium for 3.5 hours, and repeating the process for 70 hours to obtain a symbiotic culture product; wherein the deposit number of Alternaria alternata BB-1 is: CCTCCNO: M20232213; the deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214; the deposit number of Alternaria alstroemeriae BB-8 is: BB-8) is deposited in: CCTCCNO:M20232215.

[0052] The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 5 parts of corn flour, 4 parts of soybean meal, 3 parts of magnesium ammonium phosphate, 4 parts of yeast extract, 0.25 parts of sodium hydrogen phosphate, 1 part of wolfberry and 2 parts of artemisia annua.

[0053] The aerobic fermentation culture condition is as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0054] The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0055] Comparative Example 2 The difference between this comparative example and Example 1 is that the fermentation conditions are different.

[0056] A method for culturing symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, the culturing method comprising the following steps: (1) sterilizing the symbiotic bacteria culture medium at 123° C. for 15 min; (2) Add Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 to the symbiotic bacteria culture medium respectively; maintain the temperature of the symbiotic bacteria culture medium at 30°C, and perform aerobic fermentation culture for 70 hours to obtain a symbiotic culture product; wherein the deposition number of Alternaria alternata BB-1 is CCTCCNO: M20232213; the deposition number of Alternaria brassicae BB-6 is CCTCCNO: M20232214; and the deposition number of Alternaria alstroemeriae BB-8 is CCTCCNO: M20232215.

[0057] The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 5 parts of corn flour, 4 parts of soybean meal, 3 parts of magnesium ammonium phosphate, 1.5 parts of astragalus residues, 4 parts of yeast extract, 3 parts of apple juice, 2 parts of mulberry linterus extract, 0.4 parts of multivitamins, 0.25 parts of sodium hydrogen phosphate, 1 part of wolfberry and 2 parts of artemisia annua.

[0058] The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0059] The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

[0060] After ultrafine grinding, pass through 80 mesh sieve.

[0061] The pore size of the ultrafiltration membrane is 0.45 μm.

[0062] The complex vitamin is composed of the following components in parts by mass: 12 parts of vitamin B, 11 parts of vitamin D, 2 parts of riboflavin, 4 parts of pantothenic acid and 10 parts of vitamin C.

[0063] The aerobic fermentation culture condition is as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0064] 1. The colony counts of the symbiotic bacteria obtained by the cultivation methods of the embodiments of the present invention and the comparative examples were performed, as shown in Table 1.

[0065] Table 1 (Unit 10 7 cfu / mL)

[0066] 2. Experimental group (28 days): Select mature and full spikelets of Ampelopsis chinensis, and soak the whole spikelets in distilled water, 75% alcohol, 100% alcohol, 75% alcohol, and sterile water in a clean bench for one minute each, and dry them for later use.

[0067] Use a sterilized toothpick to peel the inner and outer lemmas and scatter the seeds, mix the seeds evenly, and then use a small spoon to sow the seeds evenly on 5g·L -1 The oatmeal medium was inoculated with the symbiotic bacteria of Example 1 in the center of the culture dish. The sealed culture dish was placed in a light incubator at 25° C. with 12 h light and 12 h dark.

[0068] Control group: The sterilized seeds were directly sown in 5g·L -1 The oatmeal medium was not inoculated with bacteria, and other conditions were the same as those of the experimental group. 50 seeds of Pseudocallum chinense were used in each of the experimental and control groups.

[0069] The preparation method of oat culture medium is as follows: take 5g of oats, add 550ml of distilled water and boil for 1h, filter through 8 layers of gauze, take the filtrate and make the volume to 1L, then add 15g of agar powder, sterilize at 121℃ and high temperature and high pressure for 20min, and set aside.

[0070] The germination rate was calculated using the formula: number of germinated Pseudocallum chinense seeds / 50*100%.

[0071] Table 2 Performance test results

[0072] It can be seen from Example 1 that when the symbiotic bacteria of Example 1 and the seeds of Pseudomonas aeruginosa are symbiotically germinated, the germination rate is as high as 88%. Without symbiotic germination, the germination rate is only 14%.

[0073] 3. Select two groups of Pseudocrocodactyla chinensis with similar growth conditions (growth cycle, height, etc.), set 5 repetitions, one group added with the symbiotic bacteria of Example 1, and the other group as a blank control, and keep the water shortage state after culturing for 3 weeks. After 3 weeks, the growth condition was observed and it was found that the Pseudocrocodactyla chinensis with the symbiotic bacteria grew better, indicating that the symbiotic bacteria can enhance the drought resistance of the host Pseudocrocodactyla chinensis.

[0074] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis, characterized in that: The culture method comprises the following steps: (1) sterilizing the symbiotic bacteria culture medium at 120-125° C. for 10-20 min; (2) Alternaria BB-1 ( Alternaria alternata BB-1), Alternaria brassicae BB-6 ( Alternaria brassicae BB-6)、Alternaria alternata BB-8( Alternaria alstroemeriae BB-8) is added to the symbiotic bacteria culture medium; the temperature of the symbiotic bacteria culture medium is maintained at 29-30°C, and aerobic fermentation is first carried out for 6-7 hours, and then anaerobic fermentation is carried out for 3-4 hours, and this is repeated for 50-80 hours to obtain a symbiotic culture product; wherein, Alternaria BB-1 ( Alternaria alternata BB-1) is deposited as: CCTCCNO: M20232213; Alternaria brassicae BB-6 ( Alternaria brassicae BB-6) is deposited as: CCTCCNO:M20232214; Alternaria spp. BB-8 ( Alternaria alstroemeriae The deposit number of BB-8 is: CCTCCNO:M20232215.

2. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The symbiotic bacteria culture medium comprises the following raw materials in percentage by mass: 4-6 parts of corn flour, 3-5 parts of soybean meal, 2-4 parts of magnesium ammonium phosphate, 1-2 parts of astragalus residues, 2-5 parts of yeast extract, 2-5 parts of apple juice, 1-3 parts of mulberry linterus extract, 0.2-0.6 parts of complex vitamins, 0.2-0.3 parts of sodium hydrogen phosphate, 0.5-1.5 parts of wolfberry and 1-3 parts of artemisia annua.

3. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The preparation method of the apple juice is as follows: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

4. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The preparation method of the Phellinus linteus extract is as follows: (1) ultrafine grinding Phellinus linteus to obtain Phellinus linteus powder; (2) adding water 11 times the mass of the mulberry linterus powder to the mulberry linterus powder, ultrasonicating for 150 min, and centrifuging to obtain an extract; (3) The extract was rotary evaporated to 1 / 5 of its original volume to obtain a concentrated solution; 2 / 3 of the volume of anhydrous ethanol was added to the concentrated solution, stirred evenly, and allowed to stand at 6°C for 27 h. The solution was filtered, and the precipitate was retained and freeze-dried until there was no moisture. Water was added to fully dissolve the solution, and the solution was filtered through an ultra-microfiltration membrane. The filtrate was freeze-dried until there was no moisture to obtain the mulberry ignia extract.

5. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 4, characterized in that: After ultrafine grinding, pass through 80 mesh sieve.

6. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 4, characterized in that: The pore size of the ultrafiltration membrane is 0.45 μm.

7. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The complex vitamin is composed of the following components in parts by mass: 11-15 parts of vitamin B1, 10-12 parts of vitamin D, 1-3 parts of riboflavin, 3-5 parts of pantothenic acid and 9-11 parts of vitamin C.

8. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.35-0.45 and a rotation speed of 110-120 rpm.

9. The method for cultivating symbiotic bacteria that promotes the growth of Pseudocypsum chinensis according to claim 1, characterized in that: The anaerobic fermentation culture condition is as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

10. Use of the symbiotic bacteria culture method according to any one of claims 1 to 9 in promoting the growth of Pseudocalli chinensis.

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