A method for rapid sporulation of Alternaria ginseng

Abstract

This invention belongs to the field of biotechnology, specifically disclosing a rapid sporulation method for *Alternaria ginseng*. The method involves activating and culturing *Alternaria ginseng* in PDA medium at 25°C in the dark for 10 days. Mycelial cakes are then collected from the colony edges using a punch. These mycelial cakes are inoculated onto a sporulation medium and cultured in a sealed dark environment at 22°C for 4 days. After making crisscross cuts 1 cm from the center of the colony, the cells are treated with ultraviolet light for 9 minutes. The culture dish is then left uncovered and placed at 22°C. Finally, the cells are cultured in a light incubator at 70% relative humidity for 12 hours followed by 12 hours in the dark, resulting in rapid sporulation at the cuts. Compared to existing technologies, this invention significantly shortens the sporulation cycle (5 days), greatly saving time and costs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for rapid sporulation of Alternaria ginseng. Background Technology

[0002] Ginseng black spot disease is caused by Alternaria ginseng ( Alternaria panax Caused by [unclear], it is one of the most common diseases of ginseng, usually leading to severe yield reduction in ginseng plants. However, Alternaria panax The difficulty in spore production and the inability to obtain a large number of spores have greatly hindered the progress of scientific research.

[0003] Alternaria panax The main reasons for difficulty in sporulation include:

[0004] 1. Environmental factors

[0005] humidity: Alternaria panax The conidia of the plant germinate at the highest rate under high humidity conditions, while the germination rate decreases significantly in dry environments. Soil has a strong inhibitory effect on spore germination, which may be due to the negative impact of soil microorganisms and other environmental factors on spore germination and growth.

[0006] Temperature: The starting temperature for conidial germination is 1℃, and the optimal temperature is 20-25℃. Temperatures that are too high or too low will affect spore germination and sporulation capacity.

[0007] Light: Lighting conditions affect Alternaria panax Light intensity and duration of illumination also have some impact on sporulation. Studies have shown that light intensity and duration of illumination have a significant effect on spore germination and sporulation, but specific research data are limited.

[0008] 2. Nutritional factors

[0009] Culture substrate: Different culture substrates have different effects on... Alternaria panax The sporulation capacity is significantly affected. For example, PDA medium is often used to study the toxin-producing conditions of Alternaria species, but under different culture media, Alternaria panax There are significant differences in sporulation capacity among them.

[0010] Nutritional components: The types and concentrations of nutrients affect Alternaria panax The spore-producing ability is also significantly affected. For example, spores in 1% sugar water have a higher germination rate, while those in distilled water and tap water have lower germination rates.

[0011] Therefore, it is necessary to develop a new method for rapid sporulation of Alternaria ginseng. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention provides a method for rapid sporulation of Alternaria ginseng.

[0013] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0014] A rapid sporulation method for the pathogen causing ginseng black spot disease includes the following steps:

[0015] S1. Activate and culture *Alternaria ginseng* in PDA medium at 25°C in the dark for 10 days, and then use a punch to collect mycelial cakes from the edge of the colony.

[0016] S2. Inoculate the mycelium cakes onto the sporulation medium and incubate in a sealed dark environment at 22°C for 4 days;

[0017] S3. After making a crisscross pattern at 1 cm from the center of the colony, treat with ultraviolet light for 9 min. Place the petri dish unsealed at 22℃, and then incubate in a light incubator with a relative humidity of 70% for 12 h of light culture followed by 12 h of dark culture to rapidly produce spores at the wound site.

[0018] Furthermore, each liter of the sporulation 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.

[0019] Furthermore, the light source is a mixture of red light: blue light: ultraviolet light = 40:9:1.

[0020] Preferably, the red light is emitted using a 40 W red light lamp, the blue light is emitted using a 40 W blue light lamp, and the ultraviolet light is emitted using an 8 W ultraviolet light lamp.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] I. Increase sporulation

[0023] By precisely controlling the composition of the culture medium (such as the precise selection of carbon sources, nitrogen sources, and inorganic salts), environmental factors (suitable temperature, humidity, light duration, and light quality), and the culture method (solid culture medium culture), it is possible to achieve [the desired results]. Alternaria panax It creates an extremely ideal growth and reproduction environment. Under optimized conditions, sporulation can be significantly increased, by 6 times or more compared to traditional culture conditions. This means that with the same resource input (such as culture medium raw materials, culture space, and equipment usage time), more spores can be harvested, providing a more sufficient material basis for subsequent related research or production applications.

[0024] II. Shorten the cultivation cycle

[0025] Optimized sporulation conditions enable Alternaria panaxThe growth and development process is significantly accelerated. Precise condition settings avoid growth stagnation or slowness caused by unsuitable environment or nutrient deficiency, thus advancing spore formation and shortening the entire culture cycle. For example, the culture process that originally required 7-11 days to achieve a considerable amount of spores can be shortened to 5-6 days under optimized conditions, with a large number of spores forming as early as day 6. This not only improves experimental or production efficiency but also reduces the risk of contamination by other microorganisms that may occur during long-term culture, as well as the continuous investment of human and material resources, thereby reducing costs and accelerating the progress of scientific research or production.

[0026] III. Enhancing spore viability and stability

[0027] Suitable sporulation conditions help spores better synthesize and accumulate various nutrients, bioactive components, and structural substances during formation. Spores produced in this way have stronger viability and exhibit greater stability during subsequent preservation, transportation, and inoculation applications. The spores produced in this study under specific temperature and humidity conditions have better cell membrane integrity and richer contents. They maintain activity for a longer period when stored at low or room temperature, and germinate and grow more rapidly and efficiently when inoculated into new culture media or host environments. The germination rate reached 98.5% after only 4 hours of moist culture, a 2.45-fold increase compared to previous studies, and the number of germ tubes formed increased by 6.4 times, simultaneously improving spore utilization efficiency and application effects. Furthermore, this technology expands its application scope and feasibility in related fields (such as biological control, fermentation industry, and pharmaceutical ingredient production). Attached Figure Description

[0028] Figure 1 For different culture media to Alternaria panax The effect of sporulation rate.

[0029] Figure 2 For different durations of ultraviolet light treatment Alternaria panax The effect of sporulation rate.

[0030] Figure 3 Processing for different light intensities Alternaria panax The effect of sporulation rate.

[0031] Figure 4 Processing under different lighting conditions Alternaria panax The effect of sporulation rate.

[0032] Figure 5 Treatment for different temperature and humidity conditions Alternaria panax The effect of sporulation rate.

[0033] Figure 6 For different light qualities and stimulation conditions Alternaria panax Colony state.

[0034] Figure 7 For the present invention, 10 4 pcs·mL -1 of Alternaria panax Disease incidence in ginseng 5 days after inoculation with spore suspension. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0036] The test materials used in the following examples

[0037] Test strains

[0038] Alternaria ginseng strains were collected and isolated from ginseng-producing areas in Jilin Province in 2018. Alternaria panax JY34 was deposited on February 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41814, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] Test materials

[0040] The materials used in the experiment included: CaCO3, CaCl2, ZnSO4, yeast powder, peptone, V8 vegetable juice, ginseng leaves, ginseng stems, carrots, potatoes, glucose, sucrose, and agar powder.

[0041] The test culture media are shown in Table 1.

[0042] Table 1 Information on test culture media

[0043]

[0044] Experimental equipment

[0045] Mold incubator, 40W white light, 40W red light, 40W blue light, two 8W ultraviolet lamps, clean bench, etc.

[0046] To optimize the sporulation conditions of Alternaria ginseng strain JY34, the following experiments were conducted:

[0047] 1. Culture medium screening

[0048] Will Alternaria panaxStrain JY34 was activated and cultured in PDA medium at 25℃ in the dark for 10 days. Mycelial cakes were collected from the colony edges using an 8 mm inner diameter punch. The strain was inoculated onto nine pre-designed sporulation media and cultured in the dark at 22℃ for 4 days (at which point the colony diameter was approximately 3 cm). After the dark culture was completed, a crisscross pattern was made 1 cm from the center of the colony using a sharp needle. The culture dish was then placed 20-30 cm away from a UV lamp for 9 minutes to stimulate sporulation. After irradiation, the dish was placed in a light incubator for 12 hours of light culture followed by 12 hours of dark culture. Sporulation at the wound sites was then observed, and a suitable culture medium for sporulation was selected for subsequent experiments. Different culture media showed varying effects on sporulation. Alternaria panax The effects on sporulation yield are shown in Table 2 and Figure 1 As shown.

[0049] Table 2 Effects of different culture media on Alternaria panax Effect of sporulation

[0050]

[0051] Note: Cultivation was conducted under specific conditions in the preliminary experiment, namely, scratching followed by UV treatment and alternating light and dark incubation for different durations. Different lowercase letters in the same column indicate... P <0.05 Significant difference.

[0052] From Table 2 and Figure 1 It is known that the optimal sporulation 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. Therefore, this sporulation medium was used in subsequent examples.

[0053] 2. Selection of light source conditions

[0054] ① Under optimal culture medium, UV light was applied for 0, 3, 6, 9, 12, 15, and 30 min, and a fixed 1500 Lux cool white light was applied. All other treatments and culture conditions were the same as in the culture medium screening experiment. ② Under optimal culture medium and UV conditions, cool white light treatments of 500, 750, 1000, 1500, 2000, 3000, and 5000 Lux were applied, along with a fixed 40W red / blue light treatment. All other treatments and culture conditions were the same. ③ After scratching, the culture dishes were placed under a mixed light source of red:blue:UV light = 40:9:1 (simulating the composition ratio of sunlight) for 12 h. After 24 h of culture under these conditions, sporulation, spore morphology, and germination rate were observed. The effects of different durations of UV light treatment on... A. panax The effects of sporulation yield, spore morphology, germination rate, and germ tube formation are shown in Table 3 and Figure 2 As shown; treatment under different lighting conditions A. panaxThe effects of sporulation yield, spore morphology, germination rate, and number of germ tubes formed are shown in Table 4. Figure 3 , Figure 4 and Figure 6 As shown.

[0055] Table 3 Effects of UV treatment for different durations Alternaria panax Effects of sporulation yield, sporulation morphology, germination rate and number of germ tubes formed

[0056]

[0057] Note: Results are from strain cultured on V8-1 medium at 22°C and 70% relative humidity for 5 days (4 days of sealed dark culture + 12 hours of light culture followed by 12 hours of dark culture). Spore germination was determined using the concave slide moist culture method. Spore morphology was determined under a microscope. Different lowercase letters in the same column indicate different morphological characteristics. P <0.05 Significant difference.

[0058] Table 4 Effects of treatments under different lighting conditions Alternaria panax Effects of sporulation yield, sporulation morphology, germination rate and number of germ tubes formed

[0059]

[0060] Note: Results are from strain cultured on V8-1 medium at 22°C and 70% relative humidity for 5 days (4 days of sealed dark culture + 12 hours of light culture followed by 12 hours of dark culture). Spore germination was determined using the concave slide moist culture method. Spore morphology was determined under a microscope. Different lowercase letters in the same column indicate different morphological characteristics. P <0.05 Significant difference.

[0061] From Table 3, Table 4, Figures 2-4 and Figure 6 It can be seen that when a mixture of red light: blue light: ultraviolet light = 40:9:1 is used as a light source... Alternaria panax The light source produced the best sporulation, so it was used in subsequent experiments.

[0062] 3. Temperature and humidity screening

[0063] Single-factor experiments were conducted using the most suitable culture medium and light conditions after screening, with temperature and humidity as independent variables. ① Temperature was the sole variable: different temperature treatments of 20, 22, 25, 28, and 30℃ were set. ② Humidity was the sole variable: different relative humidity treatments of 50%, 70%, 80%, 90%, and 95% were set. Sporulation, spore morphology, and germination rate were observed 24 hours after treatment. The effects of different temperature and humidity treatments on... Alternaria panax The effects on sporulation yield are shown in Table 5 and Figure 5 As shown.

[0064] Table 5 Effects of treatment under different temperature and humidity conditions Alternaria panax Effect of sporulation

[0065]

[0066] Note: Results of strains cultured on V8-1 medium under the same light and UV conditions for 5 days (4 days of sealed dark culture + 12 hours of light culture followed by 12 hours of dark culture) with temperature or humidity as the only variable are represented by different lowercase letters in the same column. P <0.05 Significant difference.

[0067] From Table 5 and Figure 5 It can be seen that at a temperature of 22℃ and a relative humidity of 70%, Alternaria panax The optimal sporulation rate was achieved, so this temperature and humidity were used in subsequent experiments.

[0068] 4. Evaluation of sporulation yield and sporulation quality

[0069] Use 10 4 cells / mL Alternaria panax Inoculation experiments were conducted using spore suspension. After 5 days of moist incubation (4 days of sealed dark incubation followed by 12 hours of light followed by 12 hours of dark incubation), disease incidence was investigated. Ginseng roots were treated with puncture wounds, and each wound was inoculated with 20 μL of spore suspension and incubated at 22℃. The control group (CK) was water. Treatment 1 used ginseng roots with a main root length of 10-12 cm, treatment 2 used ginseng roots with a main root length of 5-10 cm, and treatment 3 used ginseng roots with a main root length of 3-5 cm. Results are as follows: Figure 7 As shown, by Figure 7 It can be seen that the present invention Alternaria panax The spores are of high pathogenicity.

[0070] Therefore, *Alternaria ginseng* was activated and cultured in PDA medium at 25°C in the dark for 10 days. Mycelial cakes were collected from the edge of the colonies using a punch. The mycelial cakes were inoculated into a sporulation medium (composed of 100 mL V8 juice agar, 2.4 g CaCO3, 0.5 mg CaCl2, and 20 g agar powder) and sealed in the dark at 22°C for 4 days. After making a crisscross pattern at 1 cm from the center of the colony, the cells were treated with ultraviolet light for 9 min. The culture dish was placed open at 22°C and then incubated 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 of light incubation followed by 12 h of dark incubation to rapidly produce sporulation at the wound site.

[0071] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for rapid sporulation of *Alternaria ginseng*, characterized in that, Includes the following steps: S1. Activate and culture Alternaria ginseng in PDA medium at 25°C in the dark for 10 days, and use a punch to collect the mycelial cake at the edge of the colony. S2. Inoculate the mycelium cakes onto the sporulation medium and incubate in a sealed dark environment at 22°C for 4 days. S3. After making a crisscross pattern at 1 cm from the center of the colony, treat with ultraviolet light for 9 min. Place the petri dish open at 22℃, then incubate in a light incubator with 70% relative humidity for 12 h of light incubation followed by 12 h of dark incubation to rapidly produce sporulation at the wound site. The light source is a mixed light of red light: blue light: ultraviolet light = 40:9:

1. The red light is emitted by a 40W red light lamp, the blue light by a 40W blue light lamp, and the ultraviolet light by an 8W ultraviolet light lamp. Each liter of the sporulation 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; The *Alternaria ginseng* strain is *Alternaria ginseng*. Alternari apanax JY34 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41814 and deposit date of February 21, 2025.

Images