Cultivation method of pseudomonas

Abstract

The invention discloses a method for cultivating pseudomonas. The method comprises the following steps: step 1, planting tomatoes in soil; 2, after the tomatoes are mature, tomato rhizosphere soil is extracted; 3, preparing bacterial suspensions with multiple concentrations by adopting a gradient dilution method, and coating the bacterial suspensions on the NA solid culture medium; and 4, after single colonies grow from the bacterial suspensions with various concentrations at the temperature of 25-29 DEG C, selecting the single colonies, carrying out streak purification on a new NA solid culture medium, and naming the purified pseudomonas strain as the pseudomonas stutzeri strain X54. According to the method, the due characteristics of the strain are kept in the culture process, and the defects of a strain culture method are overcome.

Description

Technical Field

[0001] The invention belongs to the general field of plant technology, and specifically relates to a method for cultivating pseudomonads and a method for dissolving phosphorus and improving disease resistance of tomatoes. Background Art

[0002] Tomato is the crop with the largest area of ​​greenhouse vegetable cultivation in my country and the world. It has high nutritional value and economic value and is one of the most popular vegetable types on the public table. Due to the rapid development of the scale and intensiveness of the greenhouse vegetable industry, excessive application of fertilizers and pesticides, and long-term monoculture of vegetable crops, the continuous cropping obstacles in the greenhouse soil have become more serious year by year, and soil-borne diseases such as tomato wilt have occurred frequently, seriously restricting the sustainable development of greenhouse tomatoes. Tomato wilt is caused by infection with tomato-specific Fusarium oxysporum f.sp.Lycopersici. It is a fungal soil-borne disease belonging to the subphylum Ascomycota. It has strong pathogenicity and a wide range of incidence. The incidence rate is generally 20%-30%, and in severe cases, it may be completely eliminated. The main ways to repair the soil with continuous cropping obstacles in the facility are crop rotation, selection of disease-resistant varieties, soil disinfection and biological control. Among them, chemical disinfection has the best effect, but it will cause pathogens to increase drug resistance, pollute the environment and product quality (El-Sheekhet al 2022, Pei D et al 2023). Due to geographical and time constraints, crop rotation and disease-resistant varieties cannot achieve good results. Therefore, biological control methods have become a research hotspot for green control of soil-borne diseases (Im SM et al 2020). There are many beneficial microorganisms (biocontrol bacteria) in the rhizosphere and soil of crops. At present, many bacteria with the above-mentioned effects have been isolated from the soil, mainly Bacillus, Streptomyces, etc.

[0003] Phosphorus is an essential nutrient for all organisms and plays a vital role in maintaining ecosystem functions and crop growth. In pursuit of high efficiency, farmers have applied excessive amounts of chemical fertilizers, resulting in serious nutrient enrichment in the soil of greenhouse vegetables. Most of the greenhouse soils that have been planted for more than five years have mild or moderate salt damage, especially the effective phosphorus content in the greenhouse soil has reached 345 mg / kg, resulting in a phosphorus utilization rate of only 10%-26% in the greenhouse soil in the season, causing a large amount of phosphorus to remain in the greenhouse soil, which not only causes a waste of phosphorus fertilizer resources, but also increases the risk of soil phosphorus leaching and environmental pollution.

[0004] Because biocontrol bacteria have many types, fast reproduction rate, wide parasitic range, and easy cultivation, bacteria have better survival and competitive advantages. At present, the biocontrol bacteria for tomato diseases are mainly Bacillus and Pseudomonas, both of which have good antagonistic effects on tomato diseases. Biocontrol actinomycetes are widely present in different natural ecological environments such as soil and ocean, and have the characteristics of a wide variety and different metabolic functions. The antibiotics and enzymes produced by actinomycetes have great application value in the biological control of crop diseases. In the screening process of antagonistic bacteria, it was found that some actinomycetes have good antagonistic effects on tomato early blight, tomato bacterial wilt, etc. Streptomyces among actinomycetes are widely used in industrial production of antibiotics.

[0005] In the past, research on biocontrol bacteria mainly focused on separation, purification and disease prevention mechanisms, but most of the strains obtained in this way had unstable biocontrol effects in field trials, and lacked a method for cultivating the strains. The present invention discloses a method for cultivating Pseudomonas and dissolving phosphorus and improving tomato disease resistance. The present invention maintains the proper characteristics of the strain during the cultivation process, and provides a new method for strain cultivation. Summary of the invention

[0006] In view of the problems existing in the background technology, the present invention provides a method for cultivating Pseudomonas, dissolving phosphate and improving disease resistance of tomatoes, and the technical scheme includes:

[0007] Step 1: Plant tomatoes in soil;

[0008] Step 2: After the tomatoes are ripe, extract the tomato rhizosphere soil;

[0009] Step 3: Prepare bacterial suspensions of multiple concentrations by gradient dilution method and spread them on NA solid culture medium;

[0010] Step 4: After a single colony grows out of the bacterial suspension at each concentration at a temperature of 25°C-29°C, pick the single colony and streak it onto a new NA solid medium for purification. The purified Pseudomonas strain is named Pseudomonas stutzeri strain X54.

[0011] The beneficial effects of the present invention are:

[0012] 1. The proper characteristics of the strain are maintained during the cultivation process, which makes up for the shortcomings of the strain cultivation method, enables the characteristics of the strain to be stably inherited, and provides a new method for strain cultivation.

[0013] 2. The surface of the cultivated and collected single colonies is smooth, and the bacteria are slightly wrinkled. As phosphate-solubilizing bacteria, they can dissolve inorganic phosphorus such as calcium phosphate very well, and the phosphate-solubilizing capacity can reach 4.87, which is beneficial to the absorption of phosphorus by crops, improves and improves soil fertility, and alleviates the obstacle of soil salt accumulation.

[0014] 3. The cultivated and collected single colonies have a significant inhibitory effect on tomato wilt, with an inhibition rate of 57%, and can be used as a biocontrol bacterium to prevent and control tomato wilt caused by Fusarium oxysporum. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The present invention is a schematic diagram of a method for cultivating Pseudomonas and a process for dissolving phosphate.

[0016] Figure 2 This is a schematic diagram of the results of the treatment group in the method for improving disease resistance of tomatoes according to an embodiment of the present invention.

[0017] Figure 3 This is a schematic diagram of the results of the control group in the method for improving disease resistance of tomatoes in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0019] like Figure 1 In the embodiment of the present invention shown, the cultivation method includes the following steps:

[0020] Step 1: Plant tomatoes in soil;

[0021] Step 2: After the tomatoes mature (3 to 6 months), extract the tomato rhizosphere soil;

[0022] Step 3: Prepare bacterial suspensions of various concentrations by gradient dilution method, and spread them on NA solid medium (5 g beef extract powder, 10 g peptone, 15 g agar, 10 g NaCl, 1000 mL water);

[0023] Step 4: After a single colony grows out of the bacterial suspension at each concentration at a temperature of 25°C-29°C, pick a single colony and streak it onto a new NA solid culture medium for purification. The purified Pseudomonas strain is named Pseudomonas stutzeri strain X54 and stored at -80°C for future use.

[0024] In this embodiment, before coating (inoculation) in step 3, the NA solid culture medium needs to be sterilized at 121° C. for 20 min.

[0025] In this embodiment, the purification process of step 4 needs to be repeated 2-3 times.

[0026] After step 5, step 6 is performed, a method for improving disease resistance of tomatoes, comprising:

[0027] Step 61, prepare the fermentation liquid of the strain: activate the Pseudomonas stutzeri X54 strain stored at -80°C, pick the activated colonies and culture them in NA solid medium at 28°C for 48h. Then pick a single colony to NA liquid medium, culture it at 28°C, 180rpm for 24h to obtain seed liquid. Take 200uL of the seed liquid and add it to the NA liquid medium, and shake and culture it at 28°C, 180rpm for 24h to obtain the fermentation liquid of Pseudomonas stutzeri X54 strain, the concentration of which is about 1×10 8 ~6×10 8 cfu / mL;

[0028] Step 62, using the four-point standoff method, the fermentation liquid of the Pseudomonas stutzeri strain X54 strain prepared in step 61 is used for determination, the indicator bacteria is (the crop pathogen preserved in the laboratory: Fusarium oxysporum f.sp. lycopersici), the control group is dripped with an equal amount of NA liquid culture medium, and three replicates are set; cultured at 28° C. for 7 days, and the antibacterial status and the size of the antibacterial circle are observed. The colony diameter is recorded, and the antibacterial rate is calculated. The calculation formula of the antibacterial rate is as follows:

[0029] Inhibition rate = (control colony diameter × treated colony diameter) / control colony diameter × 100%;

[0030] Step 63, mixing the cultured Pseudomonas stutzeri strain X54 with NA liquid culture medium (5 g beef extract powder, 10 g peptone, 10 g NaCl, 1000 mL water), and pouring the mixture into the soil to serve as tomato planting soil;

[0031] The process of planting tomatoes in the soil is carried out before or after watering in step 63 .

[0032] After step 62, it is observed whether a transparent circle is produced. The results show that the Pseudomonas stutzeri strain X54 can produce a transparent circle on the PKO medium, indicating that the X54 strain has the ability to solubilize phosphate, and the phosphate solubilization capacity can reach 4.87, which is beneficial to the absorption of phosphorus by crops, increases and improves soil fertility, and alleviates the obstacle of soil salt accumulation.

[0033] The calculation result of the inhibition rate in step 62 shows that the strain has a good inhibition effect on it, and the inhibition rate is 57%, which can be used as a biocontrol bacteria for the prevention and treatment of crop diseases.

[0034] After step 63, a pot test of the effect of Pseudomonas stutzeri strain X54 on tomato wilt is performed.

[0035] The soil for the potted experiment was the rhizosphere soil of tomato plants grown in Shenyang Agricultural University's research base for 38 years. Two days after the tomatoes were transplanted, Figure 2 The control group shown in the figure was added with an equal amount of sterile water (fermentation broth of Pseudomonas stutzeri strain X54); after 2 days, the pathogen of tomato wilt was inoculated. The base of the tomato stem was damaged with a sterile needle and then the pathogen was inoculated by root irrigation. The inoculation volume of the pathogen was 50 mL (the concentration of the pathogen spore solution was 1×108 cfu / mL). After 14 days, the control effect of Pseudomonas stutzeri strain X54 on tomato wilt was determined, which was the same as that of Figure 2 Compared with the control group shown in the figure, the prevention efficiency is 90%, which can effectively prevent and control the occurrence of tomato wilt.

[0036] The present invention provides a method for cultivating Pseudomonas and a method for solubilizing phosphate and improving disease resistance of tomatoes. The proper characteristics of the strain are maintained during the cultivation process, the phosphate solubilizing capacity is 4.87, the antibacterial rate of tomato wilt is 57%, the potted control effect is 90%, and the Pseudomonas can be used as a biocontrol bacterium to prevent and control tomato wilt, providing a new microbial strain and control method strategy for the control of crop pathogenic fungi.

Claims

1. A method for cultivating Pseudomonas, characterized in that: include: Step 1: Plant tomatoes in soil; Step 2: After the tomatoes are ripe, extract the tomato rhizosphere soil; Step 3: Prepare bacterial suspensions of multiple concentrations by gradient dilution method and spread them on NA solid culture medium; Step 4: After a single colony grows out of the bacterial suspension at each concentration at a temperature of 25°C-29°C, pick the single colony and streak it onto a new NA solid culture medium for purification. The purified Pseudomonas strain is named Pseudomonas stutzeri strain X54.

2. The method for cultivating Pseudomonas according to claim 1, characterized in that: The components of the NA solid culture medium are: 5 g beef extract powder, 10 g peptone, 15 g agar, 10 g NaCl, and 1000 mL water.

3. The method for cultivating Pseudomonas according to claim 1 or 2, characterized in that: Before coating in step 3, the NA solid culture medium needs to be sterilized at 121°C for 20 minutes.

4. The method for cultivating Pseudomonas according to claim 1, characterized in that: The purification process in step 4 needs to be repeated 2-3 times.

5. The method for cultivating Pseudomonas according to claim 1 or 4, characterized in that: The purified Pseudomonas stutzeri strain X54 was stored at -80°C for future use.

6. The method for cultivating Pseudomonas according to claim 5, characterized in that: After step 4, step 5 is performed, which is a method for improving disease resistance of tomatoes, comprising: Step 51, prepare the fermentation liquid of the strain: activate the Pseudomonas stutzeri X54 strain stored at -80°C, pick the activated colonies and culture them in NA solid medium at 28°C for 48h. Then pick a single colony to NA liquid medium, culture it at 28°C, 180rpm for 24h to obtain seed liquid. Take 200uL of the seed liquid and add it to the NA liquid medium, and shake and culture it at 28°C, 180rpm for 24h to obtain the fermentation liquid of Pseudomonas stutzeri X54 strain, the concentration of which is about 1×10 8 ~6×10 8 cfu / mL; Step 52, culturing at 28°C for 7 days; Step 53: Mix the cultured Pseudomonas stutzeri strain X54 with the NA liquid culture medium, and then irrigate the mixture into the soil to serve as tomato planting soil.

7. The method for cultivating Pseudomonas according to claim 6, characterized in that: The components of the NA liquid culture medium are: 5 g beef extract powder, 10 g peptone, 10 g NaCl, and 1000 mL water.

8. The method for cultivating Pseudomonas according to claim 6, characterized in that: The process of planting tomatoes in the soil is carried out before or after watering in step 53 .

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