Rapid spore production method of alternaria alternata

Abstract

The invention belongs to the technical field of biology, and particularly discloses a rapid spore production method of ginseng alternaria alternata, which comprises the following steps: activating and culturing ginseng alternaria alternata in a PDA (potato dextrose agar) culture medium under the dark condition of 25 DEG C for 10 days, and punching a bacterial cake at the edge of a bacterial colony by using a puncher; inoculating a spore production culture medium with the fungus cake and carrying out sealed dark culture at 22 DEG C for 4 days; the method comprises the following steps of: performing #-shaped scratching at a position 1 cm away from the center of a bacterial colony, performing ultraviolet light treatment for 9 minutes, placing a culture dish at 22 DEG C in an unclosed manner, performing illumination culture for 12 hours in an illumination incubator with the relative humidity of 70%, then performing dark culture for 12 hours, and quickly producing spores at a wound. Compared with the prior art, the method has the advantages that the spore production period is greatly shortened (5 days), and the time cost is greatly saved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for rapid spore production of Alternaria ginseng. Background Art

[0002] Ginseng black spot disease is caused by Alternaria ginseng ( Alternaria panax ) is one of the most common diseases of ginseng, which usually leads to serious yield reduction of ginseng plants. Alternaria panax The problem of difficulty in spore production and the inability to obtain a large number of spores has greatly hindered the advancement of scientific research.

[0003] Alternaria panax The main reasons for sporulation difficulties include: 1. Environmental factors humidity: Alternaria panax The germination rate of conidia was the highest in a high humidity environment, while the germination rate decreased significantly in a dry environment. The soil had a strong inhibitory effect on spore germination, which may be due to the negative impact of microorganisms and other environmental factors in the soil on the germination and growth of spores.

[0004] Temperature: The starting temperature for conidia germination is 1°C, and the optimum temperature is 20-25°C. Too high or too low a temperature will affect the germination and spore production of conidia.

[0005] Lighting: Lighting conditions Alternaria panax Studies have shown that light intensity and light duration have a significant effect on spore germination and sporulation, but there is little specific research data.

[0006] 2. Nutritional factors Culture substrate: Different culture substrates have different Alternaria panax For example, PDA medium is often used to study the toxin production conditions of Alternaria fungi, but under different culture substrates, Alternaria panax There were significant differences in the sporulation ability.

[0007] Nutrient composition: the type and concentration of nutrients Alternaria panax The spore production capacity of the bacteria is also significantly affected. For example, the spore germination rate in 1% sugar water is higher, while the germination rate in distilled water and tap water is lower.

[0008] Therefore, it is necessary to develop a new rapid sporulation method for Alternaria ginseng. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for rapid spore production of Alternaria ginseng.

[0010] To achieve the above object, the present invention is implemented according to the following technical solutions: A method for rapid spore production of ginseng black spot pathogenic bacteria, comprising the following steps: S1. Activate and culture Alternaria ginseng in PDA medium at 25℃ in the dark for 10 days, and use a puncher to punch out the bacterial cake at the edge of the colony; S2, inoculate the bacterial cake into the spore-forming medium and culture it in a sealed dark place at 22℃ for 4 days; S3. Make a tic-tac-toe scratch at 1 cm from the center of the colony and then treat with ultraviolet light for 9 min. Place the culture dish at 22°C without sealing. Then, place it in a light incubator with a relative humidity of 70% for 12 h of light culture and then 12 h of dark culture to quickly produce spores at the wound.

[0011] Furthermore, each liter of the spore-forming medium consists of the following components: 100 mL of V8 juice agar medium, 2.4 g of CaCO3, 0.5 mg of CaCl2 and 20 g of agar powder.

[0012] Furthermore, the light source is a mixed light of red light: blue light: ultraviolet light = 40:9:1.

[0013] Preferably, the red light is emitted by a 40 W red light lamp, the blue light is emitted by a 40 W blue light lamp, and the ultraviolet light is emitted by an 8W ultraviolet lamp.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Increase spore production By precisely controlling the culture medium components (such as the precise selection of carbon source, nitrogen source, and inorganic salts), environmental factors (suitable temperature, humidity, light duration, and light quality), and culture methods (solid culture medium), Alternaria panax Create an ideal growth and reproduction environment. Under optimized conditions, the spore production can be greatly increased, and compared with traditional culture conditions, the spore production has increased by 6 times or more. This means that with the same resource input (such as culture medium raw materials, culture space and equipment use time, etc.), more spores can be harvested, providing a more sufficient material basis for subsequent related research or production applications.

[0015] 2. Shorten the training cycle The optimized sporulation conditions Alternaria panax The growth and development process of the plant is significantly accelerated. Precise condition setting avoids growth stagnation or slowness caused by environmental discomfort or nutrient deficiency, advances the time of spore formation, and shortens the entire culture cycle. For example, the culture process that originally required 7-11 days to achieve a considerable spore production can be shortened to 5-6 days under optimized conditions, and a large number of spores can be formed on the 6th day. This not only improves experimental or production efficiency, but also reduces the risk of bacterial contamination caused by long-term culture and the continuous investment of manpower and material resources, reducing costs and speeding up the advancement of scientific research or production.

[0016] 3. Enhance spore vitality and stability Appropriate spore production conditions help spores to better synthesize and accumulate various nutrients, bioactive ingredients and structural substances during the formation process. The spores produced in this way have stronger vitality and show higher stability during subsequent storage, transportation and inoculation applications. The spores produced under specific temperature and humidity conditions in this study have better cell membrane structural integrity and richer contents. They can maintain activity for a long time when stored at low or room temperature. When inoculated into a new culture medium or host environment, they can germinate and grow more quickly and efficiently. The germination rate is as high as 98.5% after only 4 hours of moisturizing culture, which is 2.45 times higher than that of previous studies, and the number of germ tubes formed has increased by 6.4 times, while improving the utilization efficiency and application effect of spores. In addition, this technology has also expanded its application scope and feasibility in related fields (such as biological control, fermentation industry, production of medicinal ingredients, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For different culture media Alternaria panax The effect of spore production.

[0018] Figure 2 UV treatment for different durations Alternaria panax The effect of spore production.

[0019] Figure 3 For different light intensity treatments Alternaria panax The effect of spore production.

[0020] Figure 4 Treating the Alternaria panax The effect of spore production.

[0021] Figure 5 Treat the Alternaria panax The effect of spore production.

[0022] Figure 6 For different light qualities and stimulation conditions Alternaria panax Colony status.

[0023] Figure 7 For the present invention, 4 pc·mL -1 of Alternaria panax Pathogenesis of ginseng 5 days after inoculation with spore suspension. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0025] The test materials used in the following examples are Test strains Alternaria ginseng strains isolated from ginseng producing areas in Jilin Province in 2018 Alternaria panax JY34, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 21, 2025, with the deposit number: CGMCC No.41814, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] Test materials The materials used in the experiment include: CaCO3, CaCl2, ZnSO4, yeast powder, peptone, V8 vegetable juice, ginseng leaves, ginseng stems, carrots, potatoes, glucose, sucrose and agar powder, etc.

[0027] The test culture medium is shown in Table 1.

[0028] Table 1 Test medium information

[0029] Experimental equipment Mold incubator, 40 W white light lamp, 40 W red light lamp, 40 W blue light lamp, two 8 W ultraviolet lamps, clean workbench, etc.

[0030] In order to optimize the spore production conditions of Alternaria ginseng strain JY34, the following experiments were performed: 1. Culture medium screening Will Alternaria panax The strain JY34 was activated and cultured in PDA medium at 25℃ in the dark for 10 days. A bacterial cake was punched at the edge of the colony with a borer with an inner diameter of 8 mm. The strain was inoculated on 9 preset spore-forming culture media and cultured in the dark at 22℃ for 4 days (the colony diameter was about 3 cm at this time). The dark culture was terminated and a sharp needle was used to scratch the culture dish 1 cm away from the center of the colony in a tic-tac-toe pattern. After the scratch, the culture dish was placed under an ultraviolet lamp at a distance of 20-30 cm for 9 minutes for stimulation. After the irradiation, it was placed in a light incubator for 12 hours of light culture and then 12 hours of dark culture. The spore production at the wound was observed, and the culture medium that could form spores was selected for subsequent experiments. Effects of different culture media on the growth of the culture dish Alternaria panax The effects of spore production are shown in Table 2 and Figure 1 shown.

[0031] Table 2 Effects of different culture media on Alternaria panax Effect of spore production

[0032] Note: The culture was carried out under the specific culture conditions of the pre-experiment, which were scratching and UV treatment followed by alternating light and dark culture for different periods of time. P <0.05 significant difference.

[0033] From Table 2 and Figure 1 It can be seen that the spore-producing medium with the best spore production is composed of the following components: 100 mL of V8 juice agar medium, 2.4 g of CaCO3, 0.5 mg of CaCl2 and 20 g of agar powder. Therefore, the subsequent examples use this spore-producing medium.

[0034] 2. Screening of light source conditions ① Under the optimal culture medium, set the ultraviolet light for 0, 3, 6, 9, 12, 15, and 30 min and the cold white light of 1500 Lux for irradiation. The other treatment methods and culture conditions are the same as those in the culture medium screening experiment; ② Under the optimal culture medium and ultraviolet conditions, set the cold white light treatment of 500, 750, 1000, 1500, 2000, 3000, and 5000 Lux, as well as the red light / blue light treatment of 40W, and the other treatment methods and culture conditions are the same as above; ③ After scratching, place the culture dish under a mixed light of red light: blue light: ultraviolet light = 40:9:1 (this light source condition simulates the composition ratio of sunlight) for 12 hours. After culturing under the above conditions for 24 hours, observe the spore production, spore morphology and germination rate. Effect of ultraviolet light treatment of different durations on the growth of the culture dish. A. panax The effects of spore production, spore morphology, germination rate and number of germ tubes formed are shown in Table 3 and Figure 2 As shown; different lighting conditions have an effect on A. panax The effects of spore production, spore morphology, germination rate and number of germ tubes formed are shown in Table 4. Figure 3 , Figure 4 and Figure 6 shown.

[0035] Table 3 Effect of different durations of UV treatment on Alternaria panax Effects of spore production, spore morphology, germination rate and number of germ tubes formed on

[0036] Note: The strain was cultured on V8-1 medium at 22℃ and 70% relative humidity for 5 days (sealed dark culture for 4 days + 12 h light culture followed by 12 h dark culture). Spore germination was determined by concave slide moisturizing culture method. Spore morphology was completed under a microscope. Different lowercase letters in the same column represent P <0.05 significant difference.

[0037] Table 4 Effects of different light conditions on Alternaria panax Effects of spore production, spore morphology, germination rate and number of germ tubes formed on

[0038] Note: The strain was cultured on V8-1 medium at 22℃ and 70% relative humidity for 5 days (sealed dark culture for 4 days + 12 h light culture followed by 12 h dark culture). Spore germination was determined by concave slide moisturizing culture method. Spore morphology was completed under a microscope. Different lowercase letters in the same column represent P <0.05 significant difference.

[0039] From Table 3, Table 4, Figure 2-Figure 4 and Figure 6 It can be seen that when the mixed light of red light: blue light: ultraviolet light = 40:9:1 is used as the light source Alternaria panax The spore production was the best, so this light source was used in subsequent experiments.

[0040] 3. Temperature and humidity screening The most suitable culture medium and light conditions were used to set up a single factor experiment, with temperature and humidity as independent variables. ① Temperature was the only variable: different temperature treatments were set at 20, 22, 25, 28, and 30°C. ② Humidity was the only variable: different relative humidity treatments were set at 50, 70, 80, 90, and 95%. After 24 hours of treatment under the above conditions, the spore production, spore morphology, and germination rate were observed. Alternaria panax The effects of spore production are shown in Table 5 and Figure 5 shown.

[0041] Table 5 Effect of different temperature and humidity conditions on Alternaria panax Effect of spore production

[0042] Note: The results of culturing the strains on V8-1 medium for 5 days under the same light and UV conditions (sealed dark culture for 4 days + 12 h light culture followed by 12 h dark culture) with temperature or humidity as the only variable. Different lowercase letters in the same column represent P <0.05 significant difference.

[0043] From Table 5 and Figure 5 It can be seen that when the temperature is 22℃ and the relative humidity is 70%, Alternaria panax The spore production was optimal, so this temperature and humidity were used in subsequent experiments.

[0044] 4. Evaluation of spore production and spore quality Use 10 4 Pieces / mL Alternaria panax The spore suspension was used for inoculation experiment. After 5 days of moisturizing culture (sealed dark culture for 4 days + 12 hours of light culture followed by 12 hours of dark culture), the disease situation was investigated. The ginseng roots were punctured and 20 μL of spore suspension was inoculated into each wound. The ginseng roots were moisturized at 22°C. CK was the water control group. Treatment 1 was ginseng roots with a main root length of 10-12 cm, treatment 2 was ginseng roots with a main root length of 5-10 cm, and treatment 3 was ginseng roots with a main root length of 3-5 cm. The results are shown in the figure. Figure 7 As shown by Figure 7 It is known that the present invention Alternaria panax The spores are highly pathogenic.

[0045] Therefore, Alternaria ginseng was activated and cultured in PDA medium at 25℃ in the dark for 10 days, and a bacterial cake was punched out at the edge of the colony with a cork hole; the bacterial cake was inoculated in a spore-forming medium (consisting of 100 mL of V8 juice agar medium, 2.4 g of CaCO3, 0.5 mg of CaCl2 and 20 g of agar powder) and sealed and cultured in the dark at 22℃ for 4 days; a well-shaped scratch was made 1 cm away from the center of the colony and then treated with ultraviolet light for 9 min. The culture dish was placed at 22℃ without sealing, and then cultured in a light incubator with a relative humidity of 70% (the light source was a mixed light of red light: blue light: ultraviolet light = 40:9:1) for 12 h and then in the dark for 12 h to quickly produce spores at the wound.

[0046] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for rapid spore production of Alternaria ginseng, characterized in that: The following steps are involved: S1. Activate and culture Alternaria ginseng in PDA medium at 25℃ in the dark for 10 days, and use a puncher to punch out the bacterial cake at the edge of the colony; S2, inoculate the bacterial cake into the spore-forming medium and culture it in a sealed dark place at 22℃ for 4 days; S3. Make a tic-tac-toe scratch at 1 cm from the center of the colony and then treat with UV light for 9 min. Place the culture dish at 22°C without sealing. Then, place it in a light incubator with a relative humidity of 70% for 12 h of light culture and then 12 h of dark culture to quickly produce spores at the wound.

2. The rapid conidia production method of Alternaria ginseng according to claim 1, characterized in that: The spore-forming medium is composed of the following components per liter: 100 mL of V8 juice agar medium, 2.4 g of CaCO3, 0.5 mg of CaCl2 and 20 g of agar powder.

3. The rapid conidia production method of Alternaria ginseng according to claim 1, characterized in that: The light source is a mixed light of red light: blue light: ultraviolet light = 40:9:

1.

4. The rapid conidia production method of Alternaria ginseng according to claim 3, characterized in that: The red light is emitted by a 40 W red light lamp, the blue light is emitted by a 40 W blue light lamp, and the ultraviolet light is emitted by an 8 W ultraviolet lamp.

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