CN105039214B - Separation and purification method of peony vegetative organ endophytic bacteria, culture medium and preparation method thereof - Google Patents
Separation and purification method of peony vegetative organ endophytic bacteria, culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a separation and purification method of peony vegetative organ endophytic bacteria, a culture medium and a preparation method thereof, and belongs to the technical field of separation and culture of plant endophytic bacteria. The separation and purification culture medium for peony vegetative organ endophytic bacteria comprises the following components in every 1000mL culture medium: 1.5-2.5 g of dried cicada powder, 4-6 g of yeast powder, 1.5-2.5 g of gellan gum, 0.2-1 g of trehalose, 0.1-1 g of inulin, 8-10 g of NaCl, 150-250 mL of peony rhizosphere soil leaching liquor, 14-18 g of agar powder and the balance of distilled water. The culture medium is fine and appropriate in material matching on the whole, is suitable for the separation culture of the peony root, stem and leaf vegetative organ endophytic bacteria, can also be used for streak purification, and has the advantages of richer types of strains which can be separated and cultured, obvious diversity advantages, simple preparation method, convenience in operation and good popularization and application values compared with the traditional culture medium.
Description
Technical Field
The invention relates to a separation and culture technology of plant endophytes, in particular to a separation and purification method of peony vegetative organ endophyte, a culture medium used in the separation and purification operation and a preparation method thereof.
Background
The endophyte generally refers to a kind of microorganism which lives in plant tissues at a certain stage of life history and does not cause obvious disease symptoms to the growth and development of plants, such as fungi, bacteria, actinomycetes and the like. The endophyte is often a stable symbiotic or parasitic relationship formed by long-term co-evolution of different types of microorganisms of plants in a specific habitat, and the physiological activity characteristics of the endophyte are different from the indigenous microorganism groups, so that the endophyte is a good material source for screening specific strains, strains producing functional enzymes or agricultural biocontrol strains. In recent years, there have been increasing reports on endophytes.
At present, the most classical and common bacteria separation medium is beef extract peptone medium, and the formula of the beef extract peptone medium is as follows: 3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, 15-20 g/L of agar powder and distilled water with constant volume of 1000mL, wherein the natural pH value is 7.0-7.2, and the beef extract is sterilized by high-pressure steam at 121 ℃ for 20 min. The culture medium is extremely widely applied, and makes an important contribution to the isolated culture of various materials of bacteria. However, with the continuous expansion and deepening of the research field of microbial science, the traditional bacteria culture medium formula and the preparation method thereof also face certain embarrassment, and the prominent problems are mainly reflected in the following aspects: (1) the culture medium formula is lack of continuous innovation, and the single formula is repeatedly used for a long time, so that the diversity of the strains is severely restricted during the bacterial screening; (2) when special materials are used for screening bacteria, the traditional bacteria culture medium formula has no good pertinence, and often cannot achieve ideal test effects, such as insect endophytic bacteria, plant endophytic bacteria and the like; the waste of assay consumables and assay time due to repeated screening is also an immense problem due to lack of medium innovation.
In some literature reports, the formulation of a bacterial culture medium is sometimes adjusted, but the fundamental adjustment is not much, so that repeated screening of common bacteria in microbial research becomes a bottleneck problem limiting relevant experimental research. The data show that the diversity of microbial resources is far from being fully developed and utilized, and the types of microorganisms which can be isolated and cultured by people only account for 1-2% of the total amount, so that the necessity and important practical significance of continuously innovating and improving the culture medium are met.
Peony Paeonia suffruticosa belongs to Ranunculaceae, Paeonia suffruticosa is luxurious in taste and vigor, and is known as the king of flowers. The peony industry develops rapidly, and has been developed greatly in the aspects of cultivation, appreciation, medicine, porcelain art, essential oil, cosmetics, diet and the like, and the situation is happy. However, researches on peony endophytes are rarely reported, and researches on nutrient organ endophytes are blank, so that a culture medium formula for peony endophyte separation is innovated, a peony endophyte separation culture medium formula and a preparation technology thereof are determined, a microorganism resource screening approach can be expanded, technical support is provided for downstream researches, diversity of screened strains of peony endophytes can be improved, a peony endophyte development and utilization theory is enriched, unnecessary repeated screening is reduced to the greatest extent, more possibility is provided for screening out rare species, and the method is significant.
Disclosure of Invention
The invention aims to provide a separation and purification culture medium specially aiming at peony vegetative organ endophytic bacteria.
Meanwhile, the invention also provides a preparation method of the separation and purification culture medium.
Finally, the invention also provides a method for separating and purifying the peony vegetative organ endophytic bacteria.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the separation and purification culture medium for peony vegetative organ endophytic bacteria comprises the following components in every 1000mL culture medium: 1.5-2.5 g of dried cicada powder, 4-6 g of yeast powder, 1.5-2.5 g of gellan gum, 0.2-1 g of trehalose, 0.1-1 g of inulin, 8-10 g of NaCl, 150-250 mL of peony rhizosphere soil leaching liquor, 14-18 g of agar powder and the balance of distilled water.
The dried powder of the golden cicada is obtained by sterilizing, drying and crushing the aged nymph of the golden cicada. Wherein the sterilization, drying and crushing treatment can be carried out by conventional operation in the field, such as drying, baking or freeze-drying. Specifically, the cicada dry powder can be prepared according to the following steps: taking a plurality of old nymphs of the golden cicadas, sterilizing the nymphs for 20min by high-pressure steam at the temperature of 121 ℃, putting the nymphs in a constant-temperature drying oven for overnight drying at the temperature of 90 ℃, taking out the nymphs, cutting off the forepaws, grinding the nymphs by using a mortar until no obvious particles exist, and sieving the nymphs by using a sieve of 160-180 meshes to obtain the dried powder of the golden cicadas.
The peony rhizosphere soil leaching liquor is an extracting liquor obtained after water extraction of peony rhizosphere soil, for example, 200-400 g of peony rhizosphere fine soil is added into every 500mL of water, and the extraction operation is a conventional technology in the field. Specifically, the peony rhizosphere soil leaching liquor can be prepared by referring to the following steps: taking a plurality of peony rhizosphere soil, manually removing impurities, sieving with a 40-60-mesh sieve to obtain rhizosphere fine soil, putting 300g of the rhizosphere fine soil into a 1000mL triangular flask, adding 500mL of distilled water, shaking for 2 hours at room temperature at 220r/min, standing for 30 minutes, pouring the supernatant into a sand core filter, and filtering to obtain filtrate, namely the peony rhizosphere soil leaching liquor.
Preferably, the culture medium for separating and purifying the peony vegetative organ endophytic bacteria comprises the following components in every 1000mL of culture medium: 2g of cicada dry powder, 5g of yeast powder, 2g of gellan gum, 0.5g of trehalose, 0.3g of inulin, 9g of NaCl, 200mL of peony rhizosphere soil leaching liquor, 16g of agar powder and the balance of distilled water.
The preparation method of the culture medium for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) adding 0.2-1 g of trehalose and 0.1-1 g of inulin into distilled water, sterilizing for 20-30 min by high-pressure steam at the temperature of 98-105 ℃, standing, and sterilizing for 20-30 min by high-pressure steam at the temperature of 98-105 ℃ to obtain a solution A for later use;
2) adding 1.5-2.5 g of cicada dry powder, 4-6 g of yeast powder, 1.5-2.5 g of gellan gum, 8-10 g of NaCl, 14-18 g of agar powder and 150-250 mL of peony rhizosphere soil leaching liquor into distilled water, and sterilizing to obtain a solution B for later use;
3) and mixing the solution A and the solution B, and then metering to 1000mL to obtain the product.
The standing in the step 1) can be performed at a constant temperature of 37 ℃, the standing time is 18-30 h (preferably 24h), the standing time is used for recovering and growing the high-temperature resistant spores which are not killed after the first sterilization, the spores have strong high-temperature resistance, the spores can recover and grow after the first thermal stimulation, and the bacteria in the growing state can be easily killed after the second sterilization treatment.
The distilled water in the steps 1) and 2) can be properly used, and the sum of the two dosages does not exceed 1000 mL.
And 2) preparing the dried powder of the golden cicada and the leaching liquor of the peony rhizosphere soil in the same way.
The pH value of the mixed solution can be adjusted to be neutral before sterilization in the step 2).
The sterilization in the step 2) is a conventional sterilization operation, namely high-pressure steam sterilization at 121 ℃ for 20-30 min.
And 3) mixing and constant volume operation in the step 3) are carried out under aseptic conditions, and the distilled water for constant volume is aseptic.
The method for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) cleaning annual healthy nutrient organs (such as roots, stems, leaves and rootstocks can be cut into small segments) of peony, sealing the abnormal exposed parts with melted paraffin (such as two ends of roots and stems and petiole cuts of leaves), and washing with sterile water;
2) transferring the peony root into 65-75% ethanol solution for surface disinfection (3-10 min), then disinfecting with 0.05-0.15 mol/L mercuric chloride (3-5 min), shearing off sealed paraffin under aseptic operation conditions, crushing the rest part, standing for precipitation, taking supernatant, inoculating into a separation and purification culture medium of endophytic bacteria of a peony vegetative organ, and culturing at constant temperature of 30-35 ℃ (2-3 days);
3) picking single colonies (with obvious requirements and typical characteristics), and performing streak purification (each single colony is subjected to streak purification for more than 3 times continuously) to obtain living pure cultures of different single colonies.
In the step 2), ethanol solution with the concentration of 70 percent and 0.1mol/L mercuric chloride are adopted.
And 3) preliminarily identifying the obtained typical strain by adopting a method combining the colony characteristics, conventional physiological and biochemical index determination and 16S rDNA sequence analysis.
The invention has the beneficial effects that:
different culture medium formulas are suitable for growth of different microorganism varieties, the number and types of screened strains are different due to the difference of material components and even the adjustment of trace components, and no culture medium can meet the growth requirements of all microorganisms at the same time. The invention discloses a culture medium formula specially for the separation culture of peony vegetative organ endophyte through continuous exploration and optimization, and tests prove that the culture medium formula has good separation and culture effects on the endophyte.
In the invention, the dried powder of the aged nymphs of the golden cicadas is introduced into a culture medium, and the aged nymphs of the golden cicadas are taken as food for sucking plant root juice during the growth and development time of the golden cicada nymphs under the ground for 2-3 years and even as long as 12-13 years, and finally become the aged nymphs as an energy storage body after long-time nutrition accumulation. The data show that every 100g of cicada nymphs are rich in 72g of protein, 15g of fat and 1.8g of ash, and also contain calcium, phosphorus, iron, various vitamins, amino acids and the like, so the cicadas are good materials for preparing bacterial microorganism culture media. The nutrient substances of the dried cicada powder in the culture medium and the yeast powder in the formula are mutually supplemented, and the aim is to meet the requirement of the endogenous bacteria on nutrition.
The addition of inulin in a microbial culture medium is beneficial to maintaining the diversity of background microorganisms of a test sample, and besides the structural specificity, the inulin also has good moisture retention and thermal stability, which have direct influence on improving the quality of the culture medium, so the inulin is a good material for a bacterial culture medium.
The gellan gum and trehalose added in the culture medium formula have the functions of providing energy and stimulating cell growth and also have the nonspecific protection function, and the main function principle is that the gellan gum and the trehalose have the unique functions of preventing and treating bacterial cells from drying and dehydrating and stabilizing metabolism. Therefore, the gellan gum and the trehalose have positive significance in the aspects of protecting weak strains, reducing the death rate of thalli and enhancing the adaptability, and particularly have more obvious effect on screening endophytes of plants.
The peony rhizosphere soil leaching liquor is matched and used in the culture medium based on the consideration of balanced nutrition, particularly the balance of trace elements. The peony rhizosphere soil leaching liquor is a natural nutrient solution which is closer to the growth of peony endophytes, and the addition of the peony rhizosphere soil leaching liquor plays an auxiliary role in preserving and improving the physiological activity and survival probability of peony endophyte metabolism.
In the separation and culture processes of peony vegetative organ endophytic bacteria, the change of the osmotic pressure of the endophytic bacteria habitat is difficult to avoid, and in order to buffer the influence of drastic change of the osmotic pressure on the growth of thalli, the sodium chloride component in the culture medium is specially controlled to be about 9% of the ion concentration of normal saline, so that the growth of more endophytic bacteria is protected.
The culture medium is precise and appropriate in material matching on the whole, has a unique point in the selection of core components, is suitable for the separation culture of bacteria, can also be used for streak purification, and has good actual use effect. Compared with the traditional culture medium, the identified strains are rich in types and obvious in diversity advantage. In addition, the method has good reference significance for isolated culture of other types of plant endophytic bacteria.
The preparation method of the culture medium is simple, is convenient to operate, and has good popularization and application values. In the preparation process of the culture medium, trehalose and inulin are sterilized by high-pressure steam for 2 times at the temperature of 98-105 ℃, because the structure of the saccharides is usually damaged at the conventional sterilization temperature of 121 ℃, and most microorganisms can be killed after the trehalose and the inulin are sterilized by high-pressure steam for 2 times at the temperature of 98-105 ℃. The bacteria capable of forming spores are revived and grow after being stressed by heat at 98-105 ℃, the spore state is relieved, and the bacteria can be killed in the second sterilization.
Detailed Description
The following examples are intended to illustrate the invention in further detail, but are not to be construed as limiting the invention in any way.
Example 1
In this example, the composition of each 1000mL of the culture medium for separating and purifying the peony vegetative organ endogenous bacteria is as follows: 2g of cicada dry powder, 5g of yeast powder, 2g of gellan gum, 0.5g of trehalose, 0.3g of inulin, 9g of NaCl, 200mL of peony rhizosphere soil leaching liquor, 16g of agar powder and the balance of distilled water, wherein the pH value is 7.0.
The preparation method of the culture medium for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) preparing cicada dry powder:
weighing a proper amount of aged nymphs of the golden cicadas, sterilizing the nymphs for 20min by high-pressure steam at the temperature of 121 ℃, placing the nymphs in a constant-temperature drying oven at the temperature of 90 ℃ for overnight drying, cutting off the forelegs, grinding the nymphs to be free of obvious particles by using a mortar, and sieving the nymphs with a 160-mesh sieve to obtain dried golden cicada powder;
2) preparing a peony rhizosphere soil leaching solution:
taking a plurality of peony rhizosphere soil, manually removing impurities, sieving with a 40-mesh sieve to obtain 300g of rhizosphere fine soil, placing the fine soil in a 1000mL triangular flask, adding 500mL of distilled water, shaking for 2h in a shaking table at room temperature and 220r/min, standing for 30min, pouring the supernatant into a sand core filter, and filtering to obtain filtrate, namely peony rhizosphere soil leaching liquor;
3) weighing 0.5g of trehalose and 0.3g of inulin, slowly adding the trehalose into a beaker filled with 500mL of distilled water in sequence for dissolving, stirring uniformly, transferring the mixture into a triangular flask, sterilizing the mixture for 20min by high-pressure steam at 105 ℃, standing the mixture in a constant temperature box at 37 ℃ for 48h, sterilizing the mixture for 30min by high-pressure steam at 105 ℃, and finally placing the mixture in a constant temperature box at 65 ℃ for later use;
4) respectively weighing 2g of gellan gum, 2g of dried cicada powder, 5g of yeast powder, 9g of NaCl and 16g of agar powder, sequentially adding 200mL of mixed solution of distilled water and 200mL of peony rhizosphere soil leaching liquor, adjusting the pH value to 7.0, fixing the volume of the distilled water to 500mL, transferring into a triangular flask, sterilizing with high-pressure steam at 121 ℃ for 20min, cooling to 65 ℃ (both about), mixing with the standby solution obtained in the step 3) under the aseptic operation condition, shaking uniformly, and turning over, wherein the obtained flat plate is a peony vegetative organ endophytic bacteria separation flat plate.
The method for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) respectively taking roots, stems and leaves of current-year healthy nutritive organs of peony Paeonia suffruticosa, firstly washing the roots, the stems and the leaves with tap water, cutting the roots and the stems into small sections of 5cm, sealing two ends with melted paraffin, sealing a petiole incision of the leaves with the paraffin, and then respectively washing the roots, the stems and the leaves with sterile water for three times;
2) sterilizing with 70% ethanol solution for 5min, sterilizing with 0.1mol/L mercuric chloride for 3min, cutting off sealing paraffin under aseptic condition, grinding and mixing the rest part in mortar, standing for precipitation, coating appropriate amount of supernatant on solid separation culture medium, and culturing at constant temperature of 30 deg.C for 48 hr;
meanwhile, taking a traditional beef extract peptone bacterial culture medium (formula: 3g of beef extract, 10g of peptone, 5g of sodium chloride, 20g of agar powder and 1000mL of distilled water) as a reference, and coating 15 plates respectively;
3) and (3) selecting different single colonies with obvious differences and typical characteristics by using an inoculating loop, carrying out streaking purification respectively, and continuously streaking each single colony for 3 times to obtain living pure cultures of different single colonies.
The obtained typical strain is preliminarily identified by a method combining the colony characteristics, the conventional physiological and biochemical index determination and the 16S rDNA sequence analysis. The results show that the strains grow well on the separation culture medium, the number is large, the repeatability is good, the colony characteristics are various, the identified species are rich, the main species list is shown in the following table 1, and compared with the species of peony endophytic bacteria obtained by separation of the traditional formula, the peony endophytic bacteria have obvious advantages in diversity.
TABLE 1 peony endophytic bacterial species isolated, purified and identified using different media
| Strains separated by the culture medium formula of the invention | Strain separated by traditional formula (beef extract peptone culture medium) |
| Bacillus subtilis | Bacillus subtilis |
| Pseudomonas aeruginosa | Pseudomonas aeruginosa |
| Bacillus licheniformis | LichenBacillus licheniformis |
| Acinetobacter Acinetobacter sp. | Acinetobacter Acinetobacter sp. |
| Serratia nematophila Serratia nematodiphila | Serratia nematophila Serratia nematodiphila |
| Enterobacter sp. | / |
| Novel Sphingobacterium Novosphingobium sp. | / |
| West Davisae Cedecena davisae | / |
| Pantoea agglomerans | / |
| Terribacillus sp. | / |
| Burkholderia phytofmans | / |
| Bacillus cereus | Bacillus cereus |
| Pseudomonas hibiscicola (Pseudomonas hibiscicola) | / |
| / | Bacillus megaterium |
| / | Klebsiella pneumoniae Pneumoniae |
Example 2
In this example, the composition of each 1000mL of the culture medium for separating and purifying the peony vegetative organ endogenous bacteria is as follows: 1.5g of cicada dry powder, 6g of yeast powder, 2.5g of gellan gum, 1g of trehalose, 0.1g of inulin, 8g of NaCl, 250mL of peony rhizosphere soil leaching liquor, 18g of agar powder and the balance of distilled water, wherein the pH value is 7.0.
The preparation method of the culture medium for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) preparing cicada dry powder:
weighing a proper amount of aged nymphs of the golden cicadas, sterilizing the nymphs for 20min by high-pressure steam at the temperature of 121 ℃, placing the nymphs in a constant-temperature drying oven at the temperature of 90 ℃ for overnight drying, cutting off the forelegs, grinding the nymphs to be free of obvious particles by using a mortar, and sieving the nymphs with a 160-mesh sieve to obtain dried golden cicada powder;
2) preparing a peony rhizosphere soil leaching solution:
taking a plurality of peony rhizosphere soil, manually removing impurities, sieving with a 40-mesh sieve to obtain 200g of rhizosphere fine soil, placing the fine soil in a 1000mL triangular flask, adding 500mL of distilled water, shaking for 2h in a shaking table at room temperature and 220r/min, standing for 30min, pouring the supernatant into a sand core filter, and filtering to obtain filtrate, namely peony rhizosphere soil leaching liquor;
3) weighing 1g of trehalose and 0.1g of inulin, slowly adding the trehalose and the inulin into a beaker filled with 300mL of distilled water in sequence for dissolving, stirring uniformly, transferring the mixture into a triangular flask, sterilizing the mixture for 30min by high-pressure steam at 98 ℃, standing the mixture in a constant temperature box at 37 ℃ for 48h, sterilizing the mixture for 25min by high-pressure steam at 98 ℃, and finally placing the mixture in a constant temperature box at 65 ℃ for later use;
4) weighing 2.5g of gellan gum, 1.5g of dried cicada powder, 6g of yeast powder, 8g of NaCl and 18g of agar powder, sequentially adding into a mixed solution of 300mL of distilled water and 250mL of peony rhizosphere soil leaching liquor, adjusting the pH value to 7.0, transferring into a triangular flask, sterilizing with high-pressure steam at 121 ℃ for 20min, cooling to 65 ℃ (about both), mixing with the standby solution in the step 3) under the aseptic operation condition, fixing the volume to 1000mL, shaking uniformly, and pouring the plate, wherein the obtained plate is the peony vegetative organ endophytic bacteria separation plate.
The method for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) respectively taking roots, stems and leaves of current-year healthy nutritive organs of peony Paeonia suffruticosa, firstly washing the roots, the stems and the leaves with tap water, cutting the roots and the stems into small sections of 5cm, sealing two ends with melted paraffin, sealing a petiole incision of the leaves with the paraffin, and then respectively washing the roots, the stems and the leaves with sterile water for three times;
2) sterilizing the surface of the mixture by using 65% ethanol solution for 8min, sterilizing the surface of the mixture by using 0.05mol/L mercuric chloride for 5min, shearing off sealing paraffin under the aseptic operation condition, fully grinding and uniformly mixing the rest part of the mixture in a mortar, standing and precipitating, taking a proper amount of supernatant, coating the supernatant on a solid separation culture medium, and culturing at the constant temperature of 33 ℃ for 54 h;
3) and (3) selecting different single colonies with obvious differences and typical characteristics by using an inoculating loop, carrying out streaking purification respectively, and continuously streaking each single colony for 3 times to obtain living pure cultures of different single colonies.
Example 3
In this example, the composition of each 1000mL of the culture medium for separating and purifying the peony vegetative organ endogenous bacteria is as follows: 2.5g of cicada dry powder, 4g of yeast powder, 1.5g of gellan gum, 0.2g of trehalose, 1g of inulin, 10g of NaCl, 150mL of peony rhizosphere soil leaching liquor, 14g of agar powder and the balance of distilled water, wherein the pH value is 7.0.
The preparation method of the culture medium for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) preparing cicada dry powder:
weighing a proper amount of aged nymphs of the golden cicadas, sterilizing the nymphs for 20min by high-pressure steam at the temperature of 121 ℃, placing the nymphs in a constant-temperature drying oven at the temperature of 90 ℃ for overnight drying, cutting off the forelegs, grinding the nymphs to be free of obvious particles by using a mortar, and sieving the nymphs with a 180-mesh sieve to obtain dried golden cicada powder;
2) preparing a peony rhizosphere soil leaching solution:
taking a plurality of peony rhizosphere soil, manually removing impurities, sieving with a 60-mesh sieve to obtain 400g of rhizosphere fine soil, placing the fine soil in a 1000mL triangular flask, adding 500mL of distilled water, shaking for 2h in a shaking table at room temperature and 220r/min, standing for 30min, pouring the supernatant into a sand core filter, and filtering to obtain filtrate, namely peony rhizosphere soil leaching liquor;
3) weighing 0.2g of trehalose and 1g of inulin, slowly adding the trehalose into a beaker filled with 500mL of distilled water in sequence for dissolving, stirring uniformly, transferring the mixture into a triangular flask, carrying out steam sterilization at the temperature of 101 ℃ for 25min, standing the mixture in a constant temperature box at the temperature of 37 ℃ for 48h, carrying out steam sterilization at the temperature of 101 ℃ for 30min, and finally placing the mixture in a constant temperature box at the temperature of 65 ℃ for later use;
4) weighing 1.5g of gellan gum, 2.5g of dried cicada powder, 4g of yeast powder, 10g of NaCl and 14g of agar powder respectively, sequentially adding 300mL of mixed solution of distilled water and 150mL of peony rhizosphere soil leaching liquor, adjusting the pH value to 7.0, fixing the volume of the distilled water to 500mL, transferring the mixed solution into a triangular flask, sterilizing the mixed solution for 20min by high-pressure steam at the temperature of 121 ℃, cooling the mixed solution to 65 ℃ (the mixed solution can be cooled to about 65 ℃), mixing the mixed solution with the standby solution obtained in the step 3) under the aseptic operation condition, shaking uniformly, and then pouring the mixed solution, wherein the obtained flat plate is a peony vegetative organ endophytic bacteria separation flat plate.
The method for separating and purifying the peony vegetative organ endophytic bacteria comprises the following steps:
1) respectively taking roots, stems and leaves of current-year healthy nutritive organs of peony Paeonia suffruticosa, firstly washing the roots, the stems and the leaves with tap water, cutting the roots and the stems into small sections of 5cm, sealing two ends with melted paraffin, sealing a petiole incision of the leaves with the paraffin, and then respectively washing the roots, the stems and the leaves with sterile water for three times;
2) sterilizing the surface of the mixture for 3min by transferring the mixture into 75% ethanol solution, sterilizing the mixture for 3min by using 0.15mol/L mercuric chloride, shearing off sealing paraffin under the aseptic operation condition, fully grinding and uniformly mixing the rest part in a mortar, standing and precipitating, taking a proper amount of supernatant, coating the supernatant on a solid separation culture medium, and culturing at the constant temperature of 35 ℃ for 48 h;
3) and (3) selecting different single colonies with obvious differences and typical characteristics by using an inoculating loop, carrying out streaking purification respectively, and continuously streaking each single colony for 3 times to obtain living pure cultures of different single colonies.
Claims (6)
1. The separation and purification culture medium for peony vegetative organ endophytic bacteria is characterized in that: the composition per 1000mL of medium was: 1.5-2.5 g of dried cicada powder, 4-6 g of yeast powder, 1.5-2.5 g of gellan gum, 0.2-1 g of trehalose, 0.1-1 g of inulin, 8-10 g of NaCl, 150-250 mL of peony rhizosphere soil leaching liquor, 14-18 g of agar powder and the balance of distilled water; the dried powder of the golden cicada is obtained by sterilizing, drying and crushing the aged nymph of the golden cicada.
2. The culture medium for separating and purifying peony vegetative organ endophytic bacteria according to claim 1, wherein: the composition per 1000mL of medium was: 2g of cicada dry powder, 5g of yeast powder, 2g of gellan gum, 0.5g of trehalose, 0.3g of inulin, 9g of NaCl, 200mL of peony rhizosphere soil leaching liquor, 16g of agar powder and the balance of distilled water.
3. The isolated and purified culture medium of peony vegetative organ endophytic bacteria according to claim 1 or 2, wherein: the peony rhizosphere soil leaching liquor is prepared by adding 200-400 g of peony rhizosphere fine soil into every 500mL of water and leaching.
4. The preparation method of the culture medium for separating and purifying the peony vegetative organ endophytic bacteria is characterized by comprising the following steps: the method comprises the following steps:
1) adding 0.2-1 g of trehalose and 0.1-1 g of inulin into distilled water, sterilizing for 20-30 min by high-pressure steam at the temperature of 98-105 ℃, standing, and sterilizing for 20-30 min by high-pressure steam at the temperature of 98-105 ℃ to obtain a solution A for later use;
2) adding 1.5-2.5 g of cicada dry powder, 4-6 g of yeast powder, 1.5-2.5 g of gellan gum, 8-10 g of NaCl, 14-18 g of agar powder and 150-250 mL of peony rhizosphere soil leaching liquor into distilled water, and sterilizing to obtain a solution B for later use; the dried powder of the golden cicada is obtained by sterilizing, drying and crushing the aged nymph of the golden cicada;
3) and mixing the solution A and the solution B, and then metering to 1000mL to obtain the product.
5. The method for preparing culture medium for separating and purifying peony vegetative organ endophytic bacteria according to claim 4, wherein the culture medium comprises: the peony rhizosphere soil leaching liquor is prepared by adding 200-400 g of peony rhizosphere fine soil into every 500mL of water and leaching.
6. The method for separating and purifying the peony vegetative organ endophytic bacteria is characterized by comprising the following steps: the method comprises the following steps:
1) cleaning the current-year healthy nutritive organs of peony, sealing the abnormal exposed parts with melted paraffin, and washing with sterile water;
2) transferring the mixture into 65-75% ethanol solution for surface disinfection for 3-10 min, then disinfecting for 3-5 min by 0.05-0.15 mol/L mercuric chloride, shearing off sealed paraffin under the aseptic operation condition, crushing the rest part, standing for precipitation, taking supernatant, inoculating the supernatant into the separation and purification culture medium according to any one of claims 1-3, and culturing for 2-3 days at constant temperature of 30-35 ℃;
3) and (3) selecting single colonies with obvious differences and typical characteristics, carrying out streaking purification respectively, and continuously streaking and purifying each single colony for more than 3 times to obtain living pure cultures of different single colonies.
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| 食用昆虫的开发利用与产业化;魏永平;《西北农业大学学报》;19981231;第26卷(第6期);摘要，第89页第1段，表1 * |
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