CN114907981B - Preservation method of solid microbial inoculum of Pantoea ananatis - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a method for preserving a solid microbial inoculum of pantoea ananatis. The preservation method specifically comprises the following steps: mixing the pantoea ananatis solid microbial inoculum with a solid carrier, uniformly stirring, standing and culturing for 24-48 h at room temperature, vacuum sealing and packaging, and storing at a drying place at 20-25 ℃; wherein the solid carrier consists of 40 parts by weight of bran, 25 parts by weight of corncob, 15 parts by weight of coconut husk 15 and 20 parts by weight of diatomite. The preservation method of the invention has simple operation and easy implementation, can effectively preserve the activity of the solid bacterial agent of the pantoea ananatis for a long time, and ensures that the effective viable count reaches 2.8x10 after 12 months of the preservation ¹² cfu/g. The pineapple pantoea fungicide prepared by the preservation method has obvious growth promoting effect on tripterygium wilfordii, can obviously improve the chlorophyll content, the alpha-chlorophyll content and the biomass of the tripterygium wilfordii, and is worth popularizing.

Description

A kind of preservation method of Pantoea pineapple solid inoculant

Technical field

The present invention relates to the technical field of microorganisms, and specifically relates to a method for preserving Pantoea pineapple solid inoculant.

Background technique

Due to its various pharmacological activities and being a natural biological raw material, Tripterygium wilfordii has become one of the most popular traditional Chinese medicinal materials in research at home and abroad. However, the sharp increase in drug demand has also resulted in the predatory development of natural resources of Tripterygium wilfordii, which is facing depletion of Tripterygium wilfordii resources. Due to the extremely complex chemical structures of plant secondary metabolites, no effective or economical synthesis method has been found yet. Therefore, artificial cultivation of Tripterygium wilfordii has become an important means to meet current market demand and protect natural resources. However, most of the plant raw materials of Tripterygium wilfordii come from retail cultivation. Due to the lack of scientific technical guidance and the excessive abuse of pesticides and fertilizers, both the yield and quality have been affected, and pollutants have appeared in varying degrees. Problems such as excessive standards, variety degradation, and serious decline in resistance to diseases, insect pests, and weeds have resulted in low economic benefits for the raw materials of Tripterygium wilfordii produced. Therefore, it is necessary to strengthen the standardization of Tripterygium wilfordii planting and develop efficient and safe green microbial preparations with practical application value to increase the yield of Tripterygium wilfordii in our province, improve its quality, and transform resource advantages into economic advantages. The rapid development of the Tripterygium wilfordii industry is particularly necessary.

Plant endophytes refer to those bacteria that live in plant tissues during some or all periods of their life history and do not cause obvious disease symptoms to plant tissues. Existing studies have shown that endophytic fungi live in special environments within plants for a long time and co-evolve with their hosts, which can have an important impact on the growth, physiology and biochemistry of host plants. At present, some progress has been made in the application research of endophytes in promoting crop growth, disease control, insect pest control, nematode control and resistance to abiotic stress. By studying the interaction between endophytes and crops, endophytes The biological functions of bacteria on crops have been confirmed. In terms of medicinal plants, in addition to having probiotic functions, some endophytes can also produce or promote the accumulation of medicinal active substances in the host. If they are transferred into plants, they can not only increase the yield of traditional Chinese medicines, but also Increase the content of its active ingredients and enhance its "authentic" quality. However, since endophytic bacteria are all selected from plants and are relatively sensitive to external environmental conditions, how to preserve them for a long time and maintain a certain level of bacterial activity is an important factor affecting their application.

Contents of the invention

The object of the present invention is to provide a method for preserving the solid inoculant of Pantoea pineapple.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The present invention first provides a method for preserving the Pantoea pineapple solid inoculant. The method is to mix the Pantoea pineapple solid inoculant with a solid carrier, stir evenly, and then let it stand for 24 to 48 hours at room temperature, then vacuum seal the package and place it. Store in a dry place at 20-25°C; wherein, the solid carrier is composed of 40 parts by weight of bran, 25 parts by weight of corn cobs, 15 parts by weight of coconut shell 15 and 20 parts by weight of diatomaceous earth.

Further, the weight ratio of the above-mentioned Pantoea pineapple solid inoculant to the solid carrier is 1:20.

Further, the preparation method of the above-mentioned Pantoea pineapple solid inoculant includes the following steps:

1) Inoculate the cryopreserved Pantoea pineapple onto LB solid medium and incubate it upside down at 30°C for 36 hours. Pick a single colony and inoculate it into the LB liquid medium and culture it at 30°C and 180 rpm for 24 hours. Obtain seed liquid;

2) Inoculate the seed liquid into LB liquid medium at an inoculation amount of 1vol%, ferment and culture for 36 hours at 30°C and 180 rpm, then centrifuge the fermentation liquid at 4°C and 4000g for 20 minutes to collect the cells. Precipitate, resuspend the bacterial cell sediment with sterile water to prepare a bacterial suspension with a concentration of 1.0×10 ⁹ cfu/mL;

3) Take 1L of bacterial suspension, add 320g of freeze-drying protective agent to it, stir and mix before pre-freezing; after pre-freezing, vacuum freeze-drying is completed to obtain strain freeze-dried powder, which is Pantoea pineapple LY-3 solid bacteria agent.

Furthermore, the above-mentioned Pantoea ananatis is Pantoea ananatis LY-3, which has been deposited in the General Microbiology Center of China Microbial Culture Collection Committee on March 27, 2013, with the deposit number CGMCC No. 7371.

Further, the above-mentioned freeze-drying protective agent is composed of 100 parts by weight of skimmed milk powder, 120 parts by weight of sodium alginate and 100 parts by weight of glycerin.

Further, the above-mentioned specific method of pre-freezing is: set the cooling rate to 0.3°C/min, cool to -15~-20°C, and pre-freeze at a constant temperature for 12 hours.

Further, the specific method of the above-mentioned vacuum freeze-drying is: after the pre-freezing is completed, the vacuum pump is turned on to evacuate. When the vacuum degree reaches 20 Pa, the vacuum degree is maintained and the temperature is raised at a rate of 1°C/30min, and the sample is heated to 5°C. , keep for 24 hours to end the freeze-drying process.

The present invention also provides the application of the above-mentioned preservation method in preparing a microbial inoculant that increases the chlorophyll content of Tripterygium wilfordii.

The present invention also provides the application of the above-mentioned preservation method in the preparation of microbial inoculants that increase the content of triptolide.

The present invention also provides the application of the above-mentioned preservation method in preparing microbial inoculant for increasing the biomass of Tripterygium wilfordii.

The significant advantages of the present invention are:

The preservation method of the present invention is simple to operate and easy to implement, and can effectively preserve the vitality of the Pantoea pineapple solid bacterial agent for a long time. After 12 months of storage, the effective number of viable bacteria reaches 2.8×10 ¹² cfu/g. The Pantoea pineapple inoculant prepared by the preservation method of the present invention has an obvious growth-promoting effect on Tripterygium wilfordii, can significantly increase the chlorophyll content, A content and biomass of Tripterygium wilfordii, and is worthy of vigorous promotion.

Description of the drawings

Figure 1 shows the Pantoea pineapple LY-3 solid inoculant mixed with a solid carrier.

Figure 2 shows the effect of fungicides on Tripterygium wilfordii plants.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. . The specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Various changes can readily be made to the precise description of the invention by those skilled in the art, based on the information contained in this application, without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the processes, properties, or components described, as these embodiments and other descriptions are merely illustrative of certain aspects of the invention. In fact, it is obvious that various changes that can be made to the embodiments of the present invention by those skilled in the art or related fields are covered by the scope of the appended claims.

The strain material Pantoea ananatis used in the present invention is classified and named Pantoea ananatis . It was registered and deposited at the General Microorganism Center of the China Microbial Culture Collection Committee on March 27, 2013. The deposit number is It is: CGMCC No. 7371, and its address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Pantoea pineapple LY-3 is isolated and purified from the tissue of Tripterygium wilfordii. It is yellow on the front and yellowish brown on the back on a flat culture medium, with a single round shape; peritrichous flagella; and the colonies often appear round and moist. , smooth and even texture. The strain was amplified and sequenced by Bao Bioengineering (Dalian) Co., Ltd. for 16s rDNA, and was identified as Pantoea ananatis through comparison in NCBI.

Example 1 Preparation of Pantoea pineapple LY-3 solid inoculant

(1) Preparation of culture medium:

1) LB solid medium: 10 g tryptone, 5 g yeast extract, 5 g NaCl, 1000 mL sterile water, pH7.0-7.2.

2) LB liquid medium: 10 g tryptone, 5 g yeast extract, 5 g NaCl, 15 g agar, 1000 mL sterile water, pH 7.0-7.2.

(2) Bacteria activation:

Inoculate the cryopreserved Pantoea pineapple LY-3 onto the LB solid medium and incubate it upside down at 30°C for 36 hours. Pick a single colony and inoculate it into the LB liquid medium and culture it at 30°C and 180 rpm for 24 hours. Used as seed liquid.

(3) Preparation of bacterial suspension:

The seed liquid was inoculated into LB liquid culture medium at an inoculation amount of 1 vol%, and fermented and cultured for 36 hours at 30°C and 180 rpm. The fermentation liquid was then centrifuged at 4°C and 4000 g for 20 minutes to collect the bacterial sediment. ; Resuspend the bacterial pellet in sterile water to prepare a bacterial suspension with a concentration of 1.0×10 ⁹ cfu/mL.

(4) Freeze-drying of strains:

Take 1L bacterial suspension, add a freeze-drying protective agent composed of 100 g skimmed milk powder, 120 g sodium alginate and 100 g glycerol, stir and mix, then pre-freeze, set the cooling rate to 0.3°C/min, and cool to -20℃, constant temperature pre-freezing for 12 hours; after the pre-freezing is completed, turn on the vacuum pump to evacuate. When the vacuum degree reaches 20 Pa, maintain this vacuum degree and heat it at a rate of 1℃/30min. When the sample heats up to 5℃, keep After 24 hours, the freeze-drying process is completed, and the bacterial strain freeze-dried powder is obtained, which is Pantoea pineapple LY-3 solid bacterial agent. The number of viable bacteria in this bacterial agent is 1.3×10 ¹⁰ cfu/g.

Example 2 Preservation method of Pantoea pineapple LY-3 solid inoculant

(1) Preparation of solid carrier:

The solid carrier is composed of 40 parts by weight of bran, 25 parts by weight of corn cobs, 15 parts by weight of coconut shell, and 20 parts by weight of diatomaceous earth. These raw materials are mixed and crushed through a 60-80 mesh sieve, and then autoclaved at 121°C. Sterilize for 30 minutes. After sterilization, put it in a drying box at 40°C for 40 hours and set aside.

(2) Preservation of Pantoea pineapple LY-3 solid inoculant

Mix the Pantoea pineapple LY-3 solid inoculant and the solid carrier at a mass ratio of 1:20, stir evenly, incubate at room temperature for 48 hours, vacuum package, and seal and store in a dry place at 20 to 25°C.

Figure 1 shows the Pantoea pineapple LY-3 solid inoculant mixed with a solid carrier.

After storage for 1, 2, 3, 6, and 12 months, samples were taken to detect the number of viable bacteria. The results are shown in the table below. After 12 months, the viable bacterial count of the solid microbial agent was as high as 2.8×10 ¹² cfu/g. When not mixed with the solid carrier, the viable bacterial count was only 2.2×10 ⁷ . This shows that the preservation method of the present invention can effectively protect pineapples. Pantoea LY-3 solid inoculant has low bacterial mortality, which is helpful for the promotion and application of Pantoea pineapple LY-3 solid inoculant.

Table 1: Changes in the number of bacteria over time

After 12 months of storage, samples were taken to test its growth-promoting effect on Tripterygium wilfordii. Take 20g of the preserved inoculant, mix it with 1000mL of sterile water, stir evenly, and apply 50mL/plant into the rhizosphere of Tripterygium wilfordii seedlings. After three months of natural growth, take samples to measure the biomass of the whole plant of Tripterygium wilfordii seedlings, leaf chlorophyll content and Root A content. Sterile water was used as a control. The results are shown in Figure 2 and Table 2. After three months of growth, it can be found that the growth of Tripterygium wilfordii seedlings treated with fungicides was significantly better than that of the control group, with more developed root systems, biomass, leaf chlorophyll content, root The A contents were significantly increased; specifically, compared with the control seedlings, the biomass of Tripterygium wilfordii seedlings treated with fungicides increased by 22.53%, the chlorophyll content of leaves increased by 33.33%, and the triptolide content of roots increased by 35.54%. .

Table 2: Growth-promoting effects of fungi on Tripterygium wilfordii

Claims (1)

1. A method for preparing and preserving the Pantoea pineapple solid inoculant, which is characterized in that: the Pantoea pineapple solid inoculant is mixed with a solid carrier, stirred evenly, and then left to be cultured at room temperature for 24 to 48 hours, then vacuum sealed and packaged. Store in a dry place at 20~25°C; wherein, the solid carrier is composed of 40 parts by weight of bran, 25 parts by weight of corn cobs, 15 parts by weight of coconut shell and 20 parts by weight of diatomaceous earth; the solid state of Pantoea pineapple The weight ratio of bacterial agent to solid carrier is 1:20; The preparation method of the solid inoculant of Pantoea pineapple includes the following steps: 1) Inoculate the cryopreserved Pantoea pineapple onto LB solid medium, invert it for 36 hours at 30°C, pick a single colony and inoculate it into the LB liquid medium, and cultivate it for 24 hours at 30°C and 180rpm to obtain the seed liquid. ; 2) Inoculate the seed liquid into the LB liquid medium at an inoculation amount of 1vol%, and ferment and culture it at 30°C and 180rpm for 36 hours. Then, centrifuge the fermentation liquid at 4°C and 4000g for 20 minutes to collect the bacterial sediment. Resuspend the bacterial pellet in sterile water to prepare a bacterial suspension with a concentration of not less than 1.0×10 ⁸ cfu/mL; 3) Take 1L of bacterial suspension, add 320g of freeze-drying protective agent to it, stir and mix before pre-freezing; after pre-freezing, vacuum freeze-drying is completed to obtain strain freeze-dried powder, which is Pantoea pineapple LY-3 solid bacteria agent; The Pantoea ananatis is Pantoea ananatis LY-3, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee on March 27, 2013, with the preservation number of CGMCC No. 7371; The freeze-drying protective agent is composed of 100 parts by weight of skimmed milk powder, 120 parts by weight of sodium alginate and 100 parts by weight of glycerin; The specific method of pre-freezing is: set the cooling rate to 0.3°C/min, cool to -20°C, and pre-freeze at a constant temperature for 12 hours; The specific method of vacuum freeze-drying is: after the pre-freezing is completed, the vacuum pump is turned on to evacuate. When the vacuum degree reaches 20pa, the vacuum degree is maintained and the temperature is raised at a rate of 1°C/30min. When the sample is heated to 5°C, the temperature is maintained After 24 hours, the freeze-drying process ends.