A method for cultivating symbiotic bacteria for promoting the growth of Psoralea corylifolia and its application

Abstract

The present invention provides a method for cultivating symbiotic bacteria that promote the growth of Pseudomonas aeruginosa and its application. The cultivation method comprises the following steps: (1) sterilizing the symbiotic bacteria culture medium at 120-125° C. for 10-20 minutes; (2) adding Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria albus BB-8 to the symbiotic bacteria culture medium respectively; maintaining the temperature of the symbiotic bacteria culture medium at 29-30° C., first aerobically fermenting and culturing for 6-7 hours, then anaerobic fermenting and culturing for 3-4 hours, and repeating this process for 50-80 hours to obtain a symbiotic culture product. The present invention uses DSEs of Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria albus BB-8 to establish a mutualistic symbiotic relationship with the host plant Pseudomonas aeruginosa 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] Eulaliopsis binata (Retz.) CE Hubb., a member of the Poaceae family, has smooth, glabrous culms 30–80 cm tall, often branched at the top, with longitudinal grooves on one side, and 3–5 nodes. The leaf sheaths, except for the lower ones, are shorter than the internodes. The basal leaf sheaths are densely covered with white hairs, forming a thick base. The ligule is a circle of short ciliate hairs, and the leaves are narrowly linear. The inflorescence is densely covered with light yellow-brown hairs, 2–4.5 cm long, arranged in a finger-like pattern. The anthers are approximately 2.5 mm long, with a broom-like stigma, yellow-brown or purple-black. This species is widely distributed and highly variable, but its variations often converge, making it difficult to forcibly separate species or varieties. It grows in grassy areas on sunny hillsides. Eulaliopsis binata is an excellent fiber plant, a good raw material for papermaking, rayon, and artificial silk. The young rhizome is flat and slightly bitter in nature, can promote qi and break blood, and is mainly used to treat female dry diseases and prolapse of the anus.

[0003] Because the dominant fungal genus is a pathogen, previous studies have reported that dark septate endophytes (DSEs) are widely colonized in ecosystems worldwide, particularly in plants growing in extreme environments, where DSE colonization rates are high. Lugo et al. conducted a survey of endophytic fungi in arid regions of Argentina and found that endophytic fungi colonized the roots of 42 plant species, and that DSE colonization was associated with plant functional traits and phylogeny. DSEs are also prevalent in arid grasslands of Hungary. Other studies have found that endophytic fungi isolated from pioneer plants growing in saline soils have a certain degree of adaptability to high-salt environments. Summary of the Invention

[0004] The present invention aims to provide a method for cultivating symbiotic bacteria for promoting the growth of Pseudomonas aeruginosa and application thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for culturing symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa, the method comprising the following steps:

[0007] (1) Sterilize the symbiotic bacteria culture medium at 120-125°C for 10-20 minutes;

[0008] (2) Separately, Alternaria alternata ( Alternaria alternata )BB-1, Alternaria brassicae ( Alternaria cabbages) BB-6, and Alternaria alstroemeriae BB-8 are added to the symbiotic bacteria culture medium; the temperature of the symbiotic bacteria culture medium is maintained at 29-30° C., and aerobic fermentation culture is first carried out for 6-7 hours, and then anaerobically fermented for 3-4 hours, and this process is repeated for 50-80 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215. Furthermore, the symbiotic bacteria culture medium includes 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 phellinus igniarius 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.

[0009] Furthermore, the preparation method of the apple juice is: adding 3 times the mass of water to Aksu Fuji apples, beating into juice, adding cellulase in an amount of 500 mg / L to the juice, enzymolyzing at 45° C. for 2 hours, and filtering to obtain apple juice.

[0010] Furthermore, the preparation method of the Phellinus linteus extract is:

[0011] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0012] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0013] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

[0014] Furthermore, the powder was ultrafinely pulverized and passed through an 80-mesh sieve.

[0015] Furthermore, the pore size of the ultra-microfiltration membrane is 0.45 μm.

[0016] Furthermore, the vitamin complex comprises the following components by weight: 11-15 parts vitamin B1, 10-12 parts vitamin D, 1-3 parts riboflavin, 3-5 parts pantothenic acid, and 9-11 parts vitamin C. Furthermore, the aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at an aeration ratio of 0.35-0.45 and a rotation speed of 110-120 rpm.

[0017] 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.

[0018] The present invention provides application of the symbiotic bacteria cultivation method in promoting the growth of Pseudomonas aeruginosa.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0020] The present invention uses DSEs (Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8) to establish a mutually beneficial symbiotic relationship with the host plant, Pseudomonas aeruginosa, through a long-term ecological adaptation process. Pseudomonas aeruginosa provides the DSEs with essential living space and nutrients, while the DSEs may produce compounds that alter plant root morphology, maintain cell wall rigidity, regulate stomatal opening and closing, and enhance plant antioxidant activity, thereby enhancing the drought resistance of the host plant, Pseudomonas aeruginosa. The present invention utilizes a specific culture medium supplemented with multiple components, including apple juice, mulberry linterae extract, and astragalus root residue, while simultaneously maintaining aerobic and anaerobic culture conditions. The two act synergistically to promote the growth of all three bacteria. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] Example 1: This example provides a method for cultivating symbiotic bacteria that promote the growth of Pseudomonas aeruginosa, the cultivation method comprising the following steps:

[0023] (1) Sterilize the symbiotic bacteria culture medium at 123°C for 15 minutes;

[0024] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic culture medium respectively; the temperature of the symbiotic culture medium was maintained at 30°C, and aerobic fermentation culture was first performed for 6.5 hours, and then anaerobic fermentation culture was performed for 3.5 hours, and this process was repeated for 70 hours to obtain a symbiotic culture product; wherein: the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213, the preservation unit is: China Center for Type Culture Collection, and the recommended classification name is: Alternaria alternata) BB-1, the culture was received by the China Center for Type Culture Collection on November 13, 2023, and the deposit address is Wuhan University, Wuhan, China, and it has been registered. The viability of the culture was tested by this collection on November 20, 2023, and the result was viable. The deposit number of Alternaria brassicae BB-6 is: CCTCCNO: M20232214, and the depository is: China Center for Type Culture Collection. The recommended classification name is: Alternaria brassicae brassicae) BB-6, the culture was received by the China Center for Type Culture Collection on November 13, 2023, and the deposit address is Wuhan University, Wuhan, China, and it has been registered; the viability of this culture was tested by this collection on November 20, 2023, and the result was viable; the deposit number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215, the depositor is: China Center for Type Culture Collection, and the suggested taxonomic name is Alternaria alstroemeriae BB-8. The culture was received by the China Center for Type Culture Collection on November 13, 2023, and the deposit address is Wuhan University, Wuhan, China, and it has been registered; the viability of this culture was tested by this collection on November 20, 2023, and the result was viable;

[0025] 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 root residue, 4 parts of yeast extract, 3 parts of apple juice, 2 parts of phellinus igniarius extract, 0.4 parts of multivitamins, 0.25 parts of sodium hydrogen phosphate, 1 part of wolfberry and 2 parts of artemisia annua.

[0026] The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0027] The preparation method of the Phellinus linteus extract is as follows:

[0028] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0029] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0030] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

[0031] After ultrafine grinding, pass through 80 mesh sieve.

[0032] The pore size of the ultramicrofiltration membrane is 0.45 μm.

[0033] The vitamin complex consists 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.

[0034] The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0035] The anaerobic fermentation culture conditions are as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0036] Example 2: This example provides a method for cultivating symbiotic bacteria that promote the growth of Pseudomonas aeruginosa, the cultivation method comprising the following steps:

[0037] (1) Sterilize the symbiotic bacteria culture medium at 120°C for 10 minutes;

[0038] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic bacteria culture medium respectively; the temperature of the symbiotic bacteria culture medium was maintained at 29°C, and aerobic fermentation culture was first carried out for 6 hours, and then anaerobically fermented for 3 hours, and this process was repeated for 68 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215.

[0039] 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 root residue, 2 parts of yeast extract, 2 parts of apple juice, 1 part of phellinus igniarius extract, 0.2 parts of multivitamins, 0.2 parts of sodium hydrogen phosphate, 0.5 parts of wolfberry and 1 part of artemisia annua.

[0040] The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0041] The preparation method of the Phellinus linteus extract is as follows:

[0042] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0043] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0044] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

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

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

[0047] The vitamin complex consists 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.

[0048] 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 and a rotation speed of 110 rpm.

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

[0050] Example 3: This example provides a method for cultivating symbiotic bacteria that promote the growth of Pseudomonas aeruginosa, the cultivation method comprising the following steps:

[0051] (1) Sterilize the symbiotic bacteria culture medium at 125°C for 20 minutes;

[0052] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic bacteria culture medium respectively; the temperature of the symbiotic bacteria culture medium was maintained at 30°C, and aerobic fermentation culture was first carried out for 7 hours, and then anaerobically fermented for 4 hours, and this process was repeated for 75 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215.

[0053] 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 root residue, 5 parts of yeast extract, 5 parts of apple juice, 3 parts of phellinus igniarius extract, 0.6 parts of multivitamins, 0.3 parts of sodium hydrogen phosphate, 1.5 parts of wolfberry and 3 parts of artemisia annua.

[0054] The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0055] The preparation method of the Phellinus linteus extract is as follows:

[0056] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0057] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0058] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

[0059] After ultrafine grinding, pass through 80 mesh sieve.

[0060] The pore size of the ultramicrofiltration membrane is 0.45 μm.

[0061] The vitamin complex consists of the following components in parts by mass: 15 parts of vitamin B, 12 parts of vitamin D, 3 parts of riboflavin, 5 parts of pantothenic acid and 11 parts of vitamin C.

[0062] 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.

[0063] The anaerobic fermentation culture conditions are as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0064] Example 4: This example provides a method for culturing symbiotic bacteria that promotes the growth of Psoralea corylifolia, the culturing method comprising the following steps:

[0065] (1) Sterilize the symbiotic bacteria culture medium at 123°C for 15 minutes;

[0066] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic bacteria culture medium respectively; the temperature of the symbiotic bacteria culture medium was maintained at 30°C, and aerobic fermentation culture was first carried out for 6.5 hours, and then anaerobically fermented for 3.5 hours, and this process was repeated for 70 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215.

[0067] 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 root residue, 2 parts of yeast extract, 5 parts of apple juice, 1 part of phellinus linteus extract, 0.6 parts of multivitamins, 0.2 parts of sodium hydrogen phosphate, 1.5 parts of wolfberry and 3 parts of artemisia annua.

[0068] The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0069] The preparation method of the Phellinus linteus extract is as follows:

[0070] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0071] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0072] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

[0073] After ultrafine grinding, pass through 80 mesh sieve.

[0074] The pore size of the ultramicrofiltration membrane is 0.45 μm.

[0075] The vitamin complex consists 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.

[0076] The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0077] The anaerobic fermentation culture conditions are as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0078] Comparative Example 1

[0079] The difference between this comparative example and Example 1 is that the symbiotic culture medium is different.

[0080] A method for culturing symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa, the method comprising the following steps:

[0081] (1) Sterilize the symbiotic bacteria culture medium at 123°C for 15 minutes;

[0082] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic bacteria culture medium respectively; the temperature of the symbiotic bacteria culture medium was maintained at 30°C, and aerobic fermentation culture was first carried out for 6.5 hours, and then anaerobically fermented for 3.5 hours, and this process was repeated for 70 hours to obtain a symbiotic culture product; wherein the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215.

[0083] 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.

[0084] The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0085] The anaerobic fermentation culture conditions are as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

[0086] Comparative Example 2

[0087] The difference between this comparative example and Example 1 is that the fermentation conditions are different.

[0088] A method for culturing symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa, the method comprising the following steps:

[0089] (1) Sterilize the symbiotic bacteria culture medium at 123°C for 15 minutes;

[0090] (2) Alternaria alternata BB-1, Alternaria brassicae BB-6, and Alternaria alstroemeriae BB-8 were added to the symbiotic bacteria culture medium respectively; the temperature of the symbiotic bacteria culture medium was maintained at 30°C, and aerobic fermentation culture was carried out for 70 hours to obtain a symbiotic culture product; wherein, the preservation number of Alternaria alternata BB-1 is: CCTCC NO: M20232213; the preservation number of Alternaria brassicae BB-6 is: CCTCC NO: M20232214; the preservation number of Alternaria alstroemeriae BB-8 is: CCTCC NO: M20232215.

[0091] 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 root residue, 4 parts of yeast extract, 3 parts of apple juice, 2 parts of phellinus igniarius extract, 0.4 parts of multivitamins, 0.25 parts of sodium hydrogen phosphate, 1 part of wolfberry and 2 parts of artemisia annua.

[0092] The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

[0093] The preparation method of the Phellinus linteus extract is as follows:

[0094] (1) Ultrafine grinding of Phellinus igniarius to obtain Phellinus igniarius powder;

[0095] (2) Add 11 times the mass of water to the mulberry ignia powder, sonicate for 150 min, and centrifuge to obtain the extract;

[0096] (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, the precipitate was retained, and freeze-dried until free of 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 free of moisture to obtain the mulberry ignia extract.

[0097] After ultrafine grinding, pass through 80 mesh sieve.

[0098] The pore size of the ultramicrofiltration membrane is 0.45 μm.

[0099] The vitamin complex consists 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.

[0100] The aerobic fermentation culture conditions are as follows: sterile air is intermittently introduced into the symbiotic bacteria culture medium at a ventilation ratio of 0.4 and a rotation speed of 115 rpm.

[0101] Performance testing:

[0102] 1. The colony counts of the symbiotic bacteria obtained by the cultivation methods of the examples of the present invention and the comparative examples were performed, as shown in Table 1.

[0103] Table 1 (Unit 10 7 cfu / mL)

[0104]

[0105] 2. Experimental group (28 days): Select mature and plump spikelets of Agropyron chinense. 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 then dry them for later use.

[0106] Use a sterilized toothpick to peel the inner and outer lemma 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 culture medium was inoculated at the center of the culture dish with the symbiotic bacteria of Example 1. The sealed culture dish was placed in a light incubator at 25° C. with a 12-hour light and 12-hour dark period.

[0107] 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 Pseudomonas aeruginosa were used in each of the experimental and control groups.

[0108] 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℃ high temperature and high pressure for 20min, and set aside.

[0109] The germination rate was calculated using the formula: number of germinated Psoralea corylifolia seeds / 50*100%.

[0110] Table 2 Performance test results

[0111]

[0112] As shown in Example 1, when the symbiotic bacteria of Example 1 and the seeds of Psoralea corylifolia were symbiotically germinated, the germination rate was as high as 88%. Without symbiotic germination, the germination rate was only 14%.

[0113] 3. Two groups of Pseudocrocyprosciaenae with similar growth conditions (growth cycle, height, etc.) were selected and replicated five times. One group was treated with the symbiotic bacteria described in Example 1, while the other served as a blank control. After three weeks of incubation, the plants were kept under water deprivation. After three weeks, growth observation revealed that the Pseudocrocyprosciaenae with the symbiotic bacteria exhibited superior growth, demonstrating that the symbiotic bacteria can enhance the drought resistance of the host plant.

[0114] 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 within the scope of protection of the present invention.

Claims

1. A method for cultivating symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa, characterized in that: The culture method comprises the following steps: (1) Sterilize the symbiotic bacteria culture medium at 120-125°C for 10-20 minutes; (2) Separately, Alternaria alternata ( Alternaria alternata )BB-1, Alternaria brassicae ( Alternaria brassicae )BB-6, Alternaria alternata ( 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 process is repeated for 50-80 hours to obtain a symbiotic culture product; wherein, Alternaria alternata ( Alternaria alternata )BB-1 has a deposit number of CCTCC NO: M20232213; Alternaria brassicae ( Alternaria brassicae )BB-6 is deposited with CCTCC NO: M20232214; Alternaria alstroemeria ( Alternaria alstroemeriae ) The deposit number of BB-8 is: CCTCC NO: M20232215; the symbiotic bacteria culture medium comprises the following raw materials in percentage by weight: 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 phellinus linteus 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.

2. The method for cultivating symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa according to claim 1, characterized in that: The preparation method of the apple juice comprises the following steps: adding 3 times the mass of water to Aksu Fuji apples to make juice, adding 500 mg / L of cellulase to the juice, performing enzymatic hydrolysis at 45° C. for 2 hours, and filtering to obtain the apple juice.

3. The method for cultivating symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa 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.

4. The method for cultivating symbiotic bacteria that promotes the growth of Pseudomonas aeruginosa according to claim 1, characterized in that: The anaerobic fermentation culture conditions are as follows: the stirring of the symbiotic bacteria culture medium is stopped, and the introduction of air is stopped.

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