Streptomyces murinus JKTJ-3 fungicide as well as preparation method and application thereof

Abstract

The invention discloses a streptomyces murinus JKTJ-3 microbial inoculum and a preparation method and application thereof.The streptomyces murinus JKTJ-3 is inoculated into a solid fermentation culture medium for fermentation culture, the streptomyces murinus JKTJ-3 microbial inoculum with the streptomyces spore number reaching 1 * 10 < 9 > cfu / g-1 * 10 < 12 > cfu / g is obtained, the obtained streptomyces murinus JKTJ-3 microbial inoculum is applied to strawberry seedling raising, and the yield of strawberry seedlings is increased. The streptomyces murinus JKTJ-3 microbial inoculum can remarkably increase the plant height, stem diameter, root length, leaf number and the like of strawberry stock plant seedlings, and in the later strawberry stock plant transplanting and propagation expanding period, the stock plant cultivated by using the streptomyces murinus JKTJ-3 microbial inoculum has the advantages that the stolon growing time is advanced, and the growing number and the seedling number are remarkably increased. Therefore, the streptomyces murinus JKTJ-3 has a very good application prospect in the aspects of strawberry mother plant seedling cultivation and strawberry mother seedling breeding efficiency improvement.

Description

Technical Field

[0001] The invention belongs to the field of microecological preparations, and specifically relates to a Streptomyces muridarum JKTJ-3 bacterial agent and a preparation method and application thereof. Background Art

[0002] Probiotic preparations, also known as live bacterial preparations, probiotics or stimulants, refer to live bacterial preparations made by special processes using the theory of microecology, through microorganisms or their growth-promoting substances that are beneficial to the host. These preparations have been widely used in agriculture, forestry, animal husbandry, fishery, food processing and water treatment. As a type of higher actinomycetes, Streptomyces is suitable for preparing various forms of live bacterial preparations, including spore powder, seed coating and mycelium culture, due to its high spore production and long spore survival time. In addition, Streptomyces can not only secrete factors that promote plant growth, but also has the ability to produce antibiotics. By screening strains from Streptomyces that have a probiotic effect on plant and soil microecology, efficient probiotic preparations can be developed. These preparations can significantly improve the biocontrol and growth-promoting effects during the growth and development of plants. Therefore, the research and development of Streptomyces probiotic agents has broad application prospects and commercial value.

[0003] In order to achieve the efficient application of microecological preparations, obtaining high-quality products is a necessary prerequisite. In recent years, solid fermentation has become the preferred fermentation technology due to its advantages in production cost, secondary metabolite production and high-quality spore concentration. Solid fermentation refers to the use of wet materials that are insoluble in deionized water as physical support and nutrient sources to simulate the natural living environment of microorganisms and promote their growth and synthesis of metabolites (Bhargav S et al., 2008; Thomas L et al., 2013). In solid fermentation, industrial and agricultural wastes and by-products such as bran, rice husks and straw are widely used as raw materials, which not only solves the problem of the treatment of these wastes, but also adds economic value to them, while reducing production costs, meeting the needs of sustainable development.

[0004] Solid fermentation is affected by many factors, including substrate type, carbon source, nitrogen source, initial water content, inoculation amount and fermentation temperature (Nigam PSN et al., 2009). Different strains also have different tolerance and adaptability to different factors. In addition, the fermentation equipment required for solid fermentation is relatively simple and economical, and the solid fermentation products have the advantages of strong activity, long survival time, easy storage and transportation (Ting S et al., 2016). At present, there have been reports on solid fermentation technology involving Streptomyces. For example, Chinese patent CN106011022A discloses the fermentation of rose yellow Streptomyces using solid raw materials such as sand, corn flour and rice husks, while Chinese patent CN108277177A describes the solid fermentation of fine yellow Streptomyces using solid raw materials such as bran and rice husks.

[0005] In terms of strawberry seedling cultivation, the current mainstream model still relies on methods such as fertilizers, humic acid and plant growth regulators to promote the growth of strawberry mother plant seedlings and increase the number of runners and seedlings. However, the research and application of using microbial agents to promote the propagation of strawberry runners is still limited. Therefore, the development of a new microbial agent is expected to fill the gap in this field and provide an innovative solution for improving strawberry seedling cultivation technology. Summary of the invention

[0006] Based on the above-mentioned prior art, the present invention provides a Streptomyces muridarum JKTJ-3 bacterial agent and a preparation method and application thereof. The present invention uses a bacterial agent based on the solid fermentation product of Streptomyces muridarum JKTJ-3 for strawberry seedling cultivation, which can effectively promote the expansion and propagation of strawberry runners and improve the reproduction coefficient of strawberry mother seedlings.

[0007] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0008] A solid fermentation medium for Streptomyces muridarum JKTJ-3 comprises a solid matrix, glycerol and deionized water, wherein the material-water ratio of the solid matrix to the deionized water is 1:0.6-1.8, preferably the material-water ratio is 1:1.1-1.6, the mass of glycerol is 0-5% of the mass of the solid matrix, preferably the mass of glycerol is 1-4% of the mass of the solid matrix, wherein the solid matrix comprises, by mass percentage, 10-90% of peat, 10-90% of bran, preferably 30-70% of coconut bran and 30-70% of bran.

[0009] A method for preparing a solid fermentation medium of Streptomyces muridarum JKTJ-3 comprises the following steps:

[0010] S1. Mix peat, bran and deionized water and stir evenly;

[0011] S2, adding glycerol to the mixture obtained in step S1 and stirring evenly;

[0012] S3, sterilizing the mixture obtained in step S2 to obtain the solid fermentation medium of Streptomyces muridarum JKTJ-3.

[0013] A Streptomyces muridarum JKTJ-3 bacterial agent is a fermentation product obtained by inoculating Streptomyces muridarum JKTJ-3 into a solid fermentation medium.

[0014] A method for preparing a Streptomyces muridarum JKTJ-3 bacterial agent comprises the following steps:

[0015] P1, activating and culturing Streptomyces muridarum JKTJ-3 until the spores are full, thereby obtaining an activated strain;

[0016] P2. Use a bacterial block puncher to punch holes on the activated strain plate to obtain activated bacterial blocks;

[0017] P3. Inoculate 1 to 5 activated bacterial blocks into the solid fermentation medium of Streptomyces muridarum JKTJ-3 according to claim 2, and carry out fermentation culture for 5 to 15 days to obtain the Streptomyces muridarum JKTJ-3 bacterial agent.

[0018] Further, the activation culture method is:

[0019] The Streptomyces muridarum JKTJ-3 bacterial agent was inoculated on ISP-2 medium, and then placed in a 28-34°C incubator for activation culture for 3-5 days.

[0020] Further, the fermentation culture method is:

[0021] The solid fermentation medium inoculated with Streptomyces muridarum JKTJ-3 is placed in a constant temperature incubator at 24-40° C. (preferably 28-35° C.) and stirred every 20-28 hours.

[0022] The invention discloses an application of a Streptomyces muridarum JKTJ-3 bacterial agent in strawberry seedling cultivation (promoting the propagation of strawberry runners).

[0023] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0024] In the present invention, Streptomyces muridarum JKTJ-3 is inoculated into a solid fermentation medium for fermentation culture, and the number of Streptomyces spores obtained reaches 1×10 9 cfu / g~1×10 12 cfu / g of Streptomyces muridarum JKTJ-3 agent, the obtained Streptomyces muridarum JKTJ-3 agent is applied to strawberry seedling cultivation, which can significantly increase the plant height, stem thickness, root length and leaf number of strawberry mother plant seedlings, and in the subsequent strawberry mother plant transplanting and expansion period, the mother plant cultivated with Streptomyces muridarum JKTJ-3 agent has an early generation time of runners, and the number of generation and the number of seedlings are significantly increased. It can be seen that the Streptomyces muridarum JKTJ-3 agent of the present invention has a very good application prospect in cultivating strawberry mother plant seedlings and improving the reproduction efficiency of strawberry mother seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a comparison chart of the spore production of Streptomyces muridarum JKTJ-3 strain fermented in different raw material fermentation media.

[0026] Figure 2 This is a comparison chart of the spore production of Streptomyces muridarum JKTJ-3 strain fermented with solid fermentation medium with different raw material ratios.

[0027] Figure 3This is a comparison chart of the spore production of Streptomyces cinerea JKTJ-3 strain fermented with different bottled amounts of solid fermentation medium.

[0028] Figure 4 This is a comparison chart of the spore production of Streptomyces muridarum JKTJ-3 strain fermented in solid fermentation medium with different material-water ratios.

[0029] Figure 5 This is a comparison chart of the spore production of the fermentation culture of Streptomyces muridarum JKTJ-3 strain with different inoculation amounts.

[0030] Figure 6 This is a comparison chart of the spore production of Streptomyces muridarum JKTJ-3 strain under different fermentation culture temperatures.

[0031] Figure 7 This is a comparison chart of the effects of different carbon sources on the spore production of the fermentation culture of Streptomyces muridarum JKTJ-3 strain.

[0032] Figure 8 This is a comparison chart of the spore production of Streptomyces muridarum JKTJ-3 strain fermentation culture under different glycerol concentrations.

[0033] Fig. 9 Actual photos of strawberry mother plant seedlings cultivated in different seedling media.

[0034] Fig.10 These are actual photos of strawberry mother plants propagating seedlings using different seedling media when the runners begin to develop. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to specific embodiments.

[0036] Unless otherwise specified, the sources of the raw materials used in the following examples are as follows:

[0037] Species: Streptomyces murinus JKTJ-3 (Mihong Ge, Xiang Cai, Dehuan Wang, et al. Efficacy of Streptomyces murinus JKTJ-3 in Suppression of Pythium Damping-Off of Watermelon. Microorganisms, 2023, 11, 1360.)

[0038] Fermentation raw materials: peat and bran are purchased from the market.

[0039] Example 1

[0040] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0041] 2. Put peat and bran into a 250mL Erlenmeyer flask in a mass ratio of 4:6. The total mass of peat and bran (solid matrix) is 20g. Add 24g of water (material-water ratio is 1:1.2) to the Erlenmeyer flask and stir evenly.

[0042] 3. Add 0.9 g of carbon source glycerol (the mass of glycerol is 3% of the total mass of the solid matrix) to the conical flask, stir evenly, and then sterilize at 121°C for 40 min to obtain the solid fermentation medium of Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3).

[0043] 4. Inoculate three activated bacterial blocks on the culture medium JKTJ-3, then stir every 24 hours in a constant temperature incubator at 30°C. After 7 days of fermentation, obtain the JKTJ-3 bacterial agent of Streptomyces muridarum (referred to as bacterial agent JKTJ-3). Determine the number of spores produced by the JKTJ-3 strain of Streptomyces muridarum (referred to as strain JKTJ-3) in the bacterial agent JKTJ-3 by the dilution plate method. Repeat the experiment 3 times, and calculate the average number of spores produced by strain JKTJ-3 in the bacterial agent JKTJ-3. After measurement and calculation, the average number of spores of strain JKTJ-3 is 7.73×10 11 cfu·g -1 .

[0044] Experiment 1: Solid fermentation inoculation method experiment

[0045] Experimental methods:

[0046] 1. Inoculate S.murinus JKTJ-3 on a plate containing ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full, thereby obtaining an activated strain plate.

[0047] 2. Use a bacterial block puncher to punch holes on the activated strain plate to obtain activated bacterial blocks of strain JKTJ-3 with a diameter of 8 mm and set aside.

[0048] 3. Use sterile water to scrape the spores on the activated strain plate, and then use sterile water to prepare a 10 8 CFU / mL of strain JKTJ-3 spore liquid is set aside.

[0049] 4. Pipette 1 mL of strain JKTJ-3 spore liquid and inoculate it into 100 mL of ISP-2 liquid culture medium. Cultivate it at 28°C and 220 rpm for 24 hours to prepare strain JKTJ-3 shaking culture liquid for later use.

[0050] 5. Weigh 30 g of bran and put it into a 250 mL Erlenmeyer flask. Add 30 g of deionized water into the flask and stir evenly. Then sterilize the material at 121°C for 40 min to obtain a sterilized matrix.

[0051] 6. Inoculate strain JKTJ-3 into the sterilized matrix in the form of activated bacterial blocks, spore liquid and shaking culture liquid, respectively. The inoculation amount is 1 bacterial block, 1 mL spore liquid and 1 mL shaking culture liquid, respectively. After uniform stirring, place it in a constant temperature incubator at 28°C and turn it over every 24 hours. After 5 days of fermentation and culture, the fermentation products of the three inoculation methods were obtained. The number of spores produced by the three inoculation methods was determined by the dilution plate method, and the experiment was repeated 3 times to calculate the average number of spores of strain JKTJ-3 under the three inoculation methods.

[0052] Experimental results:

[0053] S.murinus JKTJ-3 was cultured in sterilized bran materials using three different inoculation methods. The average number of spores produced by strain JKTJ-3 is shown in Table 1. The number of spores produced by strain JKTJ-3 using the activated bacterial block inoculation method was significantly higher than that using the other two inoculation methods.

[0054] Table 1 Effects of three inoculation methods on spore production of strain JKTJ-3

[0055]

[0056] Experiment 2: Screening experiment of raw materials of solid matrix in solid fermentation medium

[0057] Experimental methods:

[0058] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0059] 2. Select crushed mushroom residue, peat, rice husk, corn stalk, coconut bran, sawdust, rape straw, distiller's grains, and earthworm castings as candidate matrix raw materials. Each candidate raw material is placed in a 250mL triangular flask with bran in a mass ratio of 1:1. The total mass of the solid matrix in the triangular flask is 30g. Add 36g of deionized water (material-water ratio is 1:1.2) to the triangular flask, stir evenly, and obtain a mixed material. Then sterilize the mixed material at 121°C for 40min to obtain a solid fermentation medium for Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). After the above operation, 9 different mediums JKTJ-3 are obtained, and the medium JKTJ-3 with only bran as the solid matrix is ​​used as the control medium JKTJ-3.

[0060] 3. Treat 10 different culture media JKTJ-3 as follows:

[0061] Two activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then stirred every 24 hours in a constant temperature incubator at 28°C. After fermentation and culture for 7 days, the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) was obtained. The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0062] Experimental results:

[0063] S.murinus JKTJ-3 was fermented in 10 different culture media. The average number of spores produced by strain JKTJ-3 was as follows: Figure 1 As shown by Figure 1 It can be seen that the strain JKTJ-3 can grow and produce spores in the other 9 culture media except the culture medium JKTJ-3 prepared with wine grains and bran as solid matrix.

[0064] In the medium JKTJ-3 prepared with peat and bran as solid matrix, the strain JKTJ-3 produced the highest amount of spores, reaching 1.71×10 9 cfu·g -1 Compared with other medium JKTJ-3, the spore production of strain JKTJ-3 was significantly different. Therefore, peat and bran were selected as solid matrix to prepare medium JKTJ-3.

[0065] Experiment 3: Optimization experiment of solid matrix ratio in solid fermentation medium

[0066] Experimental methods:

[0067] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0068] 2. Peat and bran were placed in 11 250 mL triangular flasks at a mass ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 9:1, and 0:10, respectively, and the total mass of the solid matrix in each triangular flask was 30 g. 36 g of deionized water (material-water ratio of 1:1.2) was added to each triangular flask, stirred evenly, and a mixed material was obtained, and then the mixed material was sterilized at 121°C for 40 min to obtain a solid fermentation medium for Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). After the above operation, 11 different mediums JKTJ-3 were obtained.

[0069] 3. 11 different culture media JKTJ-3 were treated as follows:

[0070] Two activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then stirred every 24 hours in a constant temperature incubator at 28°C. After fermentation and culture for 7 days, the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) was obtained. The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0071] Experimental results:

[0072] S.murinus JKTJ-3 was fermented in 11 different culture media. The average number of spores produced by strain JKTJ-3 was as follows: Figure 2 As shown by Figure 2 It can be seen that when the mass ratio of raw materials peat to bran is 4:6, the amount of spores produced by strain JKTJ-3 is the largest, reaching 2.33×10 9 cfu·g -1 Compared with the solid matrix with other raw material ratios, the strain JKTJ-3 produced significantly different spores. Therefore, the mass ratio of peat to bran in the solid fermentation medium of Streptomyces muridarum JKTJ-3 was 4:6.

[0073] Experiment 4: Optimization of bottle filling volume in solid fermentation medium

[0074] Experimental methods:

[0075] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0076] 2. Peat and bran were respectively placed in 5 250 mL triangular flasks at a mass ratio of 4:6. The total mass of peat and bran (solid material) was 20 g, 25 g, 30 g, 35 g and 40 g, respectively. 24 g, 30 g, 36 g, 42 g and 48 g of deionized water (material-water ratio was 1:1.2) were added to each triangular flask, respectively, and stirred evenly to obtain a mixed material. The mixed material was then sterilized at 121 ° C for 40 min to obtain a solid fermentation medium of Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). After the above operation, 5 different mediums JKTJ-3 were obtained.

[0077] 3. Five different culture media JKTJ-3 were treated as follows:

[0078] Two activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then stirred every 24 hours in a constant temperature incubator at 28°C. After fermentation and culture for 7 days, the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) was obtained. The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0079] Experimental results:

[0080] S.murinus JKTJ-3 was fermented in five different culture media. The average number of spores produced by strain JKTJ-3 was as follows: Figure 3 As shown by Figure 3 It can be seen that with the increase of bottle filling volume, the amount of spores produced by strain JKTJ-3 gradually decreased. When the bottle filling volume was 20g, the amount of spores produced by strain JKTJ-3 was the largest, which was 3.20×10 9 cfu·g -1 .

[0081] Experiment 5: Optimization experiment of material-water ratio in solid fermentation medium

[0082] Experimental methods:

[0083] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0084] 2. Peat and bran were respectively placed in 7 250 mL triangular flasks at a mass ratio of 4:6. The total mass of peat and bran (solid material) was 30 g. Deionized water was added to the 7 triangular flasks. The material-water ratios in the 7 triangular flasks were 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, and 1:1.8, respectively. They were stirred evenly to obtain 7 portions of mixed materials. Each portion of the mixed material was then sterilized at 121°C for 40 min to obtain a solid fermentation medium for Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). After the above operation, 7 different mediums JKTJ-3 were obtained.

[0085] 3. Seven different culture media JKTJ-3 were treated as follows:

[0086] Two activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then stirred every 24 hours in a constant temperature incubator at 28°C. After 7 days of fermentation, the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) was obtained. The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0087] Experimental results:

[0088] S.murinus JKTJ-3 was fermented in seven different culture media. The average number of spores produced by strain JKTJ-3 was as follows: Figure 4 As shown by Figure 4 It can be seen that with the increase of water content in the culture medium JKTJ-3, the amount of spores produced by strain JKTJ-3 gradually increased. When the material-water ratio was 1:1.2, the amount of spores produced by strain JKTJ-3 reached a maximum of 2.29×10 9 cfu·g-1, then, with the increase of feed-water ratio, the spore production of strain JKTJ-3 decreased.

[0089] Experiment 6: Optimization of the inoculum size of Streptomyces muridarum JKTJ-3

[0090] Experimental methods:

[0091] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0092] 2. Peat and bran were placed in a 250 mL triangular flask at a mass ratio of 4:6. The total mass of peat and bran (solid material) was 30 g. 36 g of deionized water (material-water ratio was 1:1.2) was added to each triangular flask, stirred evenly, and a mixture was obtained. The mixture was then sterilized at 121°C for 40 min to obtain a solid fermentation medium for Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). Five portions of medium JKTJ-3 were prepared according to the above method.

[0093] 3. The activated strain was inoculated on 5 portions of culture medium JKTJ-3, with the inoculation amount being 1, 2, 3, 4, and 5 bacterial blocks (diameter of bacterial blocks being 8.0 mm), respectively. Subsequently, each portion of culture medium JKTJ-3 inoculated with Streptomyces muridarum JKTJ-3 was placed in a constant temperature incubator at 28°C, and stirred every 24 hours. After 7 days of fermentation and culture, Streptomyces muridarum JKTJ-3 bacterial agent (referred to as bacterial agent JKTJ-3) was obtained. The number of spores produced by Streptomyces muridarum JKTJ-3 strain (referred to as strain JKTJ-3) in bacterial agent JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in bacterial agent JKTJ-3 was calculated.

[0094] Experimental results:

[0095] Different inoculum amounts of Streptomyces murinus (S.murinus) JKTJ- were fermented in medium JKTJ-3. The average number of spores produced by strain JKTJ-3 was as follows: Figure 5 As shown by Figure 5 It can be seen that when the inoculation amount of strain JKTJ-3 was 3 bacterial blocks, the spore amount produced by strain JKTJ-3 was the largest, which was 4.35×10 9 cfu·g -1 , which is significantly different from other inoculation amounts. Experiment 7: Optimization experiment of fermentation culture temperature

[0096] Experimental methods:

[0097] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0098] 2. Peat and bran were placed in a 250 mL triangular flask at a mass ratio of 4:6. The total mass of peat and bran (solid matrix) was 30 g. 36 g of deionized water (material-water ratio was 1:1.2) was added to the triangular flask and stirred evenly to obtain a mixture. The mixture was then sterilized at 121° C. for 40 min to obtain a solid fermentation medium for Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). Five portions of medium JKTJ-3 were prepared according to the above method.

[0099] 3. Inoculate 2 activated bacterial blocks on 5 culture media JKTJ-3, and then place the five culture media JKTJ-3 inoculated with Streptomyces muridarum JKTJ-3 in constant temperature incubators at 24°C, 28°C, 30°C, 35°C, and 38°C, respectively, and stir once every 24 hours. After fermentation and culture for 7 days, obtain the Streptomyces muridarum JKTJ-3 bacterial agent (referred to as bacterial agent JKTJ-3), and determine the number of spores produced by the Streptomyces muridarum JKTJ-3 strain (referred to as strain JKTJ-3) in the bacterial agent JKTJ-3 by the dilution plate method. Repeat the experiment 3 times, and calculate the average number of spores produced by strain JKTJ-3 in the bacterial agent JKTJ-3.

[0100] Experimental results:

[0101] The average number of spores produced by Streptomyces murinus JKTJ-3 at different fermentation temperatures is shown in Figure 2. Figure 6 As shown by Figure 6 It can be seen that when the fermentation culture temperature is between 24℃ and 30℃, the amount of spores produced by strain JKTJ-3 gradually increases with the increase of temperature. When the temperature reaches 30℃, the amount of spores produced by strain JKTJ-3 reaches the maximum value of 5.26×10 9 cfu·g -1 When the temperature was above 30℃, the amount of spores produced by strain JKTJ-3 decreased.

[0102] Experiment 8: Screening experiment of carbon source in solid fermentation medium

[0103] Experimental methods:

[0104] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0105] 2. Put peat and bran into 6 250mL Erlenmeyer flasks in a mass ratio of 4:6. The total mass of peat and bran (solid material) is 30g. Add 36g of deionized water (material-water ratio is 1:1.2) to each Erlenmeyer flask, stir well to obtain a mixed material.

[0106] 3. Add 0.6 g of soluble starch, glycerol, glucose, sucrose, maltose, and mannitol (the mass of the carbon source is 2% of the total mass of the solid matrix) as carbon sources to 6 triangular flasks, respectively, stir evenly, and then sterilize the 6 triangular flasks at 121° C. for 40 min to obtain 6 solid fermentation media of Streptomyces muridarum JKTJ-3 (referred to as medium JKTJ-3). At the same time, medium JKTJ-3 without adding carbon source was used as control medium JKTJ-3.

[0107] 4. The seven different culture media JKTJ-3 were treated as follows:

[0108] Two activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then placed in a constant temperature incubator at 28°C, turned over every 24 hours, and fermented for 7 days to obtain the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3). The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0109] Experimental results:

[0110] S.murinus JKTJ-3 was fermented in seven different culture media. The average number of spores produced by strain JKTJ-3 was as follows: Figure 7 As shown by Figure 7 It can be seen that when glycerol was used as the carbon source, the strain JKTJ-3 produced the largest amount of spores, which was 4.1×10 9 cfu·g -1 , while the other five carbon sources were not conducive to the growth of strain JKTJ-3, and the amount of spores produced was lower than that of the control.

[0111] Experiment 9: Optimization of the amount of glycerol added to solid fermentation medium

[0112] Experimental methods:

[0113] 1. Inoculate S.murinus JKTJ-3 on a plate with ISP-2 medium, streak evenly, and culture in a 28°C incubator for 5 days until the spores are full to obtain an activated strain plate. Use a bacterial block puncher to punch holes in the activated strain plate to obtain an activated bacterial block with a diameter of 8 mm.

[0114] 2. Put peat and bran into 6 250mL Erlenmeyer flasks in a mass ratio of 4:6. The total mass of peat and bran (solid material) is 20g. Add 24g of deionized water (material-water ratio is 1:1.2) to each Erlenmeyer flask, stir well to obtain a mixed material.

[0115] 3. Add 0%, 1%, 2%, 3%, 4%, and 5% (W / W) glycerol to 6 Erlenmeyer flasks, respectively, and stir evenly. Then sterilize the 6 Erlenmeyer flasks at 121°C for 40 min to obtain 6 solid fermentation culture media of Streptomyces muridarum JKTJ-3 (referred to as culture medium JKTJ-3).

[0116] 4. The six medium JKTJ-3 with different glycerol concentrations were treated as follows:

[0117] The three activated bacterial blocks were inoculated on the culture medium JKTJ-3, and then placed in a constant temperature incubator at 30°C, turned over every 24 hours, and fermented for 7 days to obtain the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3). The number of spores produced by the strain of Streptomyces muridarum JKTJ-3 (referred to as strain JKTJ-3) in the strain JKTJ-3 was determined by the dilution plate method. The experiment was repeated 3 times, and the average number of spores produced by the strain JKTJ-3 in the strain JKTJ-3 was calculated.

[0118] Experimental results:

[0119] S.murinus JKTJ-3 was fermented in 6 different glycerol concentrations. The average number of spores produced by strain JKTJ-3 was as follows: Figure 8 As shown by Figure 8 It can be seen that when the glycerol concentration in the solid fermentation matrix is ​​between 0 and 3%, as the glycerol concentration increases, the spore amount of strain JKTJ-3 in the matrix gradually increases. When the glycerol concentration is 3%, the spore production of strain JKTJ-3 is the largest, which is 4.32×10 11 cfu·g-1, but when the glycerol concentration was between 3% and 5%, the spore production of strain JKTJ-3 gradually decreased with the increase of glycerol concentration.

[0120] Example 2

[0121] 1. Common matrix (matrix CK, mixed by peat and perlite in a volume ratio of 3:1) is used as a control group strawberry mother plant seedlings seedlings matrix, and then the bacterial agent JKTJ-3 prepared by Example 1 is added to the common matrix (the ratio of bacterial agent JKTJ-3 to the common matrix is ​​3g / L) and mixed to make a seedling matrix JKTJ-3 (referred to as matrix JKTJ-3) as an experimental group strawberry mother plant seedlings seedlings matrix. Matrix JKTJ-3 and matrix CK are respectively loaded into disposable seedling pots, strawberry runner seedlings are cut, and cut into seedling pots, and managed according to conventional mother seedlings seedling method.

[0122] 2. After 70 days of cultivation, the following measurements were performed on the strawberry mother plant seedlings in the experimental group and the control group:

[0123] 1) Take photos of strawberry mother plants grown on different seedling substrates. Fig. 9 shown.

[0124] 2) The biomass indices such as plant height, stem thickness, root length and leaf number of strawberry mother plant seedlings cultivated in different seedling cultivation media were measured. The biomass indices of strawberry mother plant seedlings cultivated in different seedling cultivation media are shown in Table 2 below:

[0125] Table 2 Effects of different seedling culture media on the biomass of strawberry mother plant seedlings

[0126]

[0127]

[0128] From Table 2 and Fig. 9 It can be seen that compared with the control group, the biomass indicators such as plant height, stem thickness, root length and leaf number of the strawberry mother plant seedlings in the experimental group were significantly increased, which shows that the bacterial agent JKTJ-3 of the present invention has a good growth-promoting effect on strawberry mother plants.

[0129] 3) The nutrient contents of P, K, Ca and Mg in strawberry mother plant seedlings cultivated with different seedling cultivation media were measured. The nutrient contents of the elements in strawberry mother plant seedlings cultivated with different seedling cultivation media are shown in Table 3 below:

[0130] Table 3 Effects of different seedling culture media on the nutrient content of strawberry mother plant seedlings

[0131]

[0132] As shown in Table 3, the matrix JKTJ-3 can promote the absorption and accumulation of nutrients in strawberry mother plant seedlings. The P, K, Ca and Mg nutrient contents of the strawberry mother plant seedlings in the experimental group were significantly higher than those in the control group, especially the P content increased by 63.1% compared with the control.

[0133] 4) The plant hormone contents of IAA, GA, ABA, and ETH and the activities of SOD, POD, and CAT resistance-related enzymes in strawberry mother plant seedlings cultivated with different seedling cultivation substrates were measured. The plant hormone contents and resistance-related enzyme activities in strawberry mother plant seedlings cultivated with different seedling cultivation substrates are shown in Table 4 below:

[0134] Table 4 Effects of different seedling culture media on hormones and resistance-related enzyme activities in strawberry mother plant seedlings

[0135]

[0136] As shown in Table 4, compared with the control group, the IAA, GA, ABA, ETH plant hormone contents and the activities of SOD, POD, CAT resistance-related enzymes in the strawberry seedlings of the experimental group (cultivated with JKTJ-3 seedling medium) were significantly increased.

[0137] 3. Place common substrate (mixed with peat and perlite in a volume ratio of 3:1) into two groups of planting bags respectively, set the strawberry mother seedlings of the experimental group obtained in step 2 as the strawberry mother plants of the experimental group in one group of planting bags, and set the strawberry mother plant seedlings of the control group obtained in step 2 as the strawberry mother plants of the control group in another group of planting bags. When the runners of the strawberry mother plants of the control group begin to occur and end, perform the following measurements:

[0138] 1) When 25% of the strawberry mother plants in the control group began to form runners, the strawberry mother plants cultivated with different seedling media were photographed. The results are as follows: Fig.10 shown.

[0139] 2) When 25% of the strawberry mother plants in the control group began to grow runners, the reproduction indicators such as the number of runners and the number of seedlings of the strawberry mother plants cultivated with different seedling cultivation substrates were measured. The reproduction indicators of the strawberry mother plants cultivated with different breeding substrates are shown in Table 5 below:

[0140] 3) When the runners of 75% of the strawberry mother plants in the control group were completed, the reproduction indicators of the strawberry mother plants cultivated with different seedling substrates were statistically shown in Table 5 below:

[0141] Table 5 Effects of different seedling medium on the propagation of strawberry mother plants

[0142]

[0143] Depend on Fig.10 As shown in Table 5, the strawberry mother plants cultivated with the seedling medium JKTJ-3 showed early occurrence of runners and fast seedling formation. At the same time, the number of runners and seedlings at different stages were significantly higher than those of the control group, indicating that the seedling medium has a significant promoting effect on the reproduction of strawberry seedlings.

Claims

1. A solid fermentation medium for Streptomyces muridarum JKTJ-3, characterized in that: The invention comprises a solid matrix, glycerol and deionized water, wherein the material-water ratio of the solid matrix to the deionized water is 1:0.6-1.8, the mass of the glycerol is 0-5% of the mass of the solid matrix, and the solid matrix, calculated by mass percentage, comprises: 10-90% of peat and 10-90% of bran.

2. A method for preparing the solid fermentation medium of Streptomyces muridarum JKTJ-3 according to claim 1, characterized in that The steps include: S1. Mix peat, bran and deionized water and stir evenly; S2, adding glycerol to the mixture obtained in step S1 and stirring evenly; S3, sterilizing the mixture obtained in step S2 to obtain the solid fermentation medium of Streptomyces muridarum JKTJ-3.

3. A Streptomyces muridarum JKTJ-3 bacterial agent, characterized in that: The bacterial agent is a fermentation product obtained by inoculating the Streptomyces muridarum JKTJ-3 described in claim 1 into the solid fermentation medium described in claim 2.

4. A method for preparing the Streptomyces muridarum JKTJ-3 bacterial agent according to claim 3, characterized in that The steps include: P1, activating and culturing Streptomyces muridarum JKTJ-3 until the spores are full, thereby obtaining an activated strain; P2. Use a bacterial block puncher to punch holes on the activated strain plate to obtain activated bacterial blocks; P3. Inoculate 1 to 5 activated bacterial blocks into the solid fermentation medium of Streptomyces muridarum JKTJ-3 according to claim 2, and carry out fermentation culture for 5 to 15 days to obtain the Streptomyces muridarum JKTJ-3 bacterial agent.

5. The preparation method of the Streptomyces muridarum JKTJ-3 bacterial agent according to claim 4, characterized in that The method of activation culture is: The Streptomyces muridarum JKTJ-3 bacterial agent was inoculated on ISP-2 medium, and then placed in a 28-34°C incubator for activation culture for 3-5 days.

6. The preparation method of the Streptomyces muridarum JKTJ-3 bacterial agent according to claim 4, characterized in that The fermentation culture method is: The solid fermentation medium inoculated with Streptomyces muridarum JKTJ-3 was placed in a constant temperature incubator at 24-40°C and stirred every 20-28 hours.

7. The preparation method of the Streptomyces muridarum JKTJ-3 bacterial agent according to claim 4, characterized in that: The diameter of the activated bacterial block is 6-10 mm.

8. Use of the Streptomyces muridarum JKTJ-3 agent according to claim 4 in strawberry seedling cultivation.

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