A method for efficiently inducing the production of ascospores by kanamycini monospora and a culture medium used in the method

Abstract

This invention belongs to the field of microbial culture technology, specifically relating to a culture medium formulation and corresponding culture method for efficiently inducing Cannomonas to produce perithecia. This invention provides a method for inducing Cannomonas (… Monosporascus cannonballus The culture medium for producing ascocarps: Add water to a mixture of carbon source, nitrogen source, inorganic salts, and plant tissue to a final volume of 1 L, adjust the pH to 7±0.1, add 20.0 g of agar powder, and then sterilize. This invention also provides a method for inducing ascocarp production in *Cannomonas*: Inoculate *Cannomonas* onto a culture medium plate, place the inoculated plate at 32±0.5℃, and culture using a light-dark cycle of 16 hours of light and 8 hours of darkness per day for 14-18 days. The method of this invention has technical advantages such as high sporulation efficiency and a short cycle.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture technology, specifically relating to a method for efficiently inducing Cannodomonas aeruginosa (… Monosporascus cannonballus The culture medium formula and corresponding culture method for producing perithecia. Background Technology

[0002] Cannolymph ( Monosporascus cannonballus *Ascophyllum hexandrum* (AHH) is a devastating soil-borne pathogen causing black spot root rot in cucurbits such as watermelon and melon. During its sexual reproduction stage, it produces ascophylls containing numerous ascospores. Its strong resistance and long lifespan are key to the disease's long-term survival in the soil and completion of its disease cycle. Field symptoms manifest as large areas of leaves turning yellow and dying during the fruit enlargement and ripening stage of cucurbits such as watermelon and melon, along with rapid yellowing, wilting, and drying of vines, preventing normal fruit ripening. A distinguishing feature is the appearance of black, spherical granules embedded in the root epidermis, which detach and fall off as the root epidermis rots upon maturity, leaving sunken marks on the root. This disease causes severe economic losses in cucurbit producing regions worldwide. Therefore, achieving efficient, stable, and reproducible large-scale production of its ascophylls in the laboratory is a fundamental prerequisite for conducting downstream research on the pathogen's population genetics, pathogenic mechanisms, drug resistance, and host interactions.

[0003] To achieve this goal, existing technologies have provided some basic solutions. Chinese patent CN112725198B discloses a synthetic culture medium (Cz-RT) using lactose as the sole carbon source and potassium nitrate as the inorganic nitrogen source, which induces perithecia maturation by static incubation for 20-30 days under constant temperature (23-25℃) and darkness. This method establishes preliminary laboratory standards for the sexual culture of this bacterium.

[0004] However, this approach has the following significant limitations: First, a single carbon and nitrogen source combination may not be able to fully meet all the nutritional requirements for efficient ascocarp formation, thus limiting further improvement in sporulation potential. Second, the constant temperature and darkness static culture mode used fails to actively utilize temperature changes and specific photoperiods—two key environmental factors proven by various ascomycetes to effectively regulate sexual development. Finally, the traditional single-point inoculation method fails to introduce potential biological stress or quorum sensing signals generated by mycelial interactions, which are often important factors triggering fungal sexual reproduction in nature. These shortcomings collectively lead to problems such as limited sporulation yield, long production cycles, and insufficient batch-to-batch stability in existing methods.

[0005] In addition, researchers in this field often use nutrient-rich universal culture media (such as potato dextrose agar PDA, oat medium OMA, etc.) to culture Cannodomonas in their routine work. Although these media can support its mycelial growth well, they are generally difficult to effectively induce or can only weakly induce its sexual sporulation process, resulting in difficulties in obtaining perithecia and a heavy reliance on the time-consuming natural isolation of soil disease samples, which is inefficient and uncontrollable.

[0006] Therefore, there is an urgent need in this field for a systematic approach that can integrate complex nutrition, environmental factors, and biological interaction induction to fundamentally overcome the current bottlenecks in the production efficiency and quality of Cannospora perithecia. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for inducing Cannodomonas aeruginosa to produce perithecia with high sporulation efficiency and short cycle, and the culture medium used therein.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for inducing Cannosum (… Monosporascus cannonballus Culture medium for producing perithecia: Add water to the carbon source, nitrogen source, inorganic salts, and plant tissue to a final volume of 1 L, adjust the pH to 7±0.1, then add 20.0 g of agar powder (until the agar is completely dissolved) and sterilize.

[0009] Note: The sterilization process is conventional 121℃ high-pressure steam sterilization for 20 minutes.

[0010] As an improvement to the culture medium of the present invention: The carbon source consisted of 10±0.1 g of glucose and 15±0.1 g of malt extract; The nitrogen source consisted of 2 ± 0.1 g of peptone, 1 ± 0.1 g of yeast extract, and 1 ± 0.1 g of casein amino acids. The inorganic salt consists of 0.5±0.05 g sodium nitrate, 0.5±0.05 g potassium chloride, 0.5±0.05 g magnesium sulfate heptahydrate, and 1.5±0.05 g potassium dihydrogen phosphate. The plant tissue was 5 ± 0.1 g of dried and pulverized host plant tissue (moisture content ≤ 10%, w / w).

[0011] As a further improvement to the culture medium of the present invention: the host plant tissue is the stem or root of watermelon or melon.

[0012] The present invention also provides a method for inducing Cannolymphoidea to produce perithecia: using any of the culture media described above, comprising the following steps: Inoculate Cannodomonas on a culture medium plate; After inoculation, the plates were placed at 32±0.5℃ and cultured with a light-dark cycle of 16 hours of light and 8 hours of darkness per day for 14 to 18 days.

[0013] As an improvement to the method of the present invention: the inoculation adopts the mycelium cake inoculation method, and the mycelium cake has a diameter of 5 mm.

[0014] As a further improvement to the method of the present invention: during the culture process, the perithecia form as early as day 14-15.

[0015] This invention has the following technical advantages: 1. The combination of a complex carbon source of "glucose-malt extract" and a complex organic nitrogen source of "peptone-yeast extract-casein amino acids" provides a more balanced and easily utilized nutritional basis; the specially added dried pulverized host plant tissue introduces potential host-specific chemical signals, which can more effectively simulate the natural infection environment, thereby directionally inducing the sexual reproduction process of pathogens.

[0016] 2. The optimized composite nutrient matrix, combined with host signals, provides a sufficient material and signal basis for the large-scale formation of perithecia.

[0017] 3. By setting the culture temperature at 32℃ (the optimal temperature for pathogens) and using a long-day (16h / 8h) light-dark cycle for the entire induction process, an efficient and standardized culture process was established, which can stably obtain a large number of mature ascocarps within 14-21 days.

[0018] 4. All ingredients in the formula are precisely quantified chemical reagents or standard extracts, supplemented by clear culture parameters (temperature, time, photoperiod), which effectively avoids batch differences of traditional natural raw materials, ensuring high reproducibility and stability of sporulation results between different laboratories and different batches, and providing a reliable guarantee for subsequent scientific research. Attached Figure Description

[0019] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Figure 1 Comparative photographs of Cannocystis cannoli after 21 days of cultivation using a single-point inoculation method on eight different culture media (including the culture medium specifically for this invention and seven control culture media). From left to right and top to bottom: the culture medium of this invention, the synthetic culture medium (Cz-RT) of Chinese patent CN112725198B, OMA, PDA, PSA, WA, MEA, and 1 / 2 MS culture medium. Figure 1 This clearly demonstrates that only on the medium of this invention can black perithecia be induced earliest and most densely, while sterile sporulation structures are absent on most conventional media.

[0021] Figure 2 Comparison of the microscopic morphology of ascospores produced by Cannosum on the special medium of this invention, synthetic medium (Cz-RT) of Chinese patent CN112725198B and oat medium (OMA) (×400x). like Figure 2 As shown, all three culture media can produce ascospores, but there are differences in spore morphology, size and maturity, which further confirms the influence of different culture media on the quality of sporulation. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Except for dried and pulverized watermelon or melon seedling tissue (stem or root), all other components of the culture medium of the present invention can be obtained commercially.

[0023] Malt extract, for example, is available from Thermo Fisher Scientific (LP0039B). Yeast extract, for example, is available from Sangon Biotech (Shanghai) Co., Ltd. (A515245-0500).

[0024] Cannolymph ( Monosporascus cannonballus ZJUP0990-2) in "First Report of Monosporascus cannonballus Causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China" (Diseases Caused by Fungi and Fungus-LikeOrganisms) has clearly informed.

[0025] Example 1: Preparation of Culture Medium Weigh the raw materials according to the following formula: 10.0 g glucose, 15.0 g malt extract, 2.0 g peptone, 1.0 g yeast extract, 1 g casein amino acid, 0.5 g sodium nitrate (NaNO3), 0.5 g potassium chloride (KCl), 0.5 g magnesium sulfate heptahydrate (MgSO4·7H2O), 1.5 g KH2PO4, 5.0 g dried and pulverized watermelon seedling tissue (stem and root), and 20.0 g agar powder (for solid culture medium).

[0026] The dried and pulverized tissue of watermelon seedlings (stems and roots) refers to: the stems and roots of watermelon seedlings are mixed in any ratio, dried to a moisture content of ≤10% (w / w), and then pulverized to pass through a 60-mesh sieve; the watermelon seedlings refer to the watermelon seedlings from seed emergence to the growth of 2-4 true leaves.

[0027] Add all components except agar powder to approximately 900 mL of distilled water and stir until fully dissolved or evenly dispersed. Then, add distilled water to bring the volume to 1 L. Adjust the pH to 7.0 using 1 M NaOH or HCl solution. Add agar powder, heat to a gentle boil, and stir continuously until the agar is completely dissolved. Dispense the culture medium into Erlenmeyer flasks and autoclave at 121°C for 20 minutes using standard autoclaving. After sterilization, allow the culture medium to cool to approximately 50-55°C, then pour approximately 20 mL into sterile petri dishes (90 mm in diameter) in a laminar flow hood. After allowing to solidify, the solid agar plate culture medium specific to this invention is obtained and used for subsequent sporulation induction culture.

[0028] Experiment 1: Comparison of the effects of different culture media on sporulation of Cannosum. To verify the effectiveness of the special culture medium of the present invention (Example 1) in inducing Cannodomonas aeruginosa (… Monosporascus cannonballus The superiority of ZJUP0990-2 in perithecia production was demonstrated through parallel comparative experiments with various commonly used or reported bacterial culture media. The specific procedures are as follows: 1. Test culture medium A total of eight culture media were used in the experiment. Except for the culture medium of this invention, the others were commonly used basic or synthetic culture media in the field: The special culture medium of this invention is prepared according to the formula of Example 1.

[0029] The synthetic culture medium (Cz-RT) of Chinese patent CN112725198B has the following formula (1 L): KNO3 2 g, KCl 0.5 g, K2HPO4 1 g, MgSO4·7H2O 0.5 g, FeSO4 0.01 g, lactose 30 g, agar 17 g; Potato Gluconate Agar Medium (PDA): The formula includes 200 g of peeled potatoes, 20 g of glucose, 20 g of agar, and distilled water to a final volume of 1 L. The method is as follows: Wash and peel the potatoes, weigh out 200 g of potatoes, cut them into small pieces, add water and boil until tender (boil for 20-30 minutes, until easily pierced with a glass rod). Filter through eight layers of gauze, heat, and add 20 g of agar as needed for the experiment. Continue heating and stirring until the agar is completely dissolved. Add glucose, stir well, cool slightly, and then add water to a final volume of 1 L. Dispense into test tubes or Erlenmeyer flasks, stopper and seal, sterilize (1.1 atmospheres, 121 °C for 20 min), cool, and store for later use.

[0030] Potato Sucrose Agar (PSA): The formula consists of 200 g potatoes (peeled), 20 g sucrose, 20 g agar, and distilled water to a final volume of 1 L; the preparation method is the same as that of the PDA medium described above.

[0031] Oat culture medium (OMA) has the following formula (1L): 30 g oats, 20 g agar, and distilled water to a final volume of 1 L; Malt extract culture medium (MEA): 34 g malt extract, 20 g agar, distilled water to a final volume of 1 L; Glucose beef extract peptone medium (WA): 5 g peptone, 10 g glucose, 3 g beef extract powder, 5 g sodium chloride, 15 g agar, distilled water to a final volume of 1 L, pH adjusted to 7.2. 1 / 2MS medium (MS): MS salt 2.2 g, sucrose 5.0 g, MES 0.5 g, agar 10 g, distilled water to a final volume of 1 L, pH adjusted to 5.7.

[0032] All culture media were autoclaved at 121°C for 20 minutes, and then equal volumes were poured into plates for later use.

[0033] 2. Inoculation and Culture Take Cannolysia var. cannoli stored in an ultra-low temperature freezer Monosporascus cannonballus ZJUP0990-2 was revived on PDA medium at 32°C for 3 days. After colonies grew, it was transferred to new PDA medium and cultured at 32°C for 6 to 8 days before use.

[0034] Take the strain Monosporascus cannonballusFrom the edge of fresh PDA colonies of ZJUP0990-2, use a sterile punch (5 mm diameter) to cut out mycelial discs and inoculate them into the center of each of the above-mentioned culture medium plates (90 mm diameter, approximately 20 mL), with the mycelial side facing down. Five replicates were set up for each treatment. All inoculated plates were placed in a 32°C incubator for alternating light and dark incubation (16 hours of light per day, 7500 lux light intensity / 8 hours of darkness).

[0035] Count of blastocysts Observe perithecia formation daily and record the time of initial appearance. On days 18 and 25 of culture, use a stereomicroscope to directly count three 1cm samples from the edge and center of each plate colony. 2 The field of view was determined, the number of daughter shells was counted, and the mean and standard deviation were calculated.

[0036] 4. Results and Analysis As shown in Table 1, different culture media showed significant differences in mycelial growth and sporulation induction ability of strain ZJUP0990-2.

[0037] Table 1. Effects of different culture media on the growth and sporulation of Cannosum coli.

[0038] Experimental results show that: The culture medium of this invention has comprehensive and significant advantages in inducing sexual sporulation of Cannomonas. Ascocarps form as early as day 14–15 of culture; by day 18, the ascocarp density reaches 71 ± 2 cells / cm²; and by day 25, the number of ascocarps further increases to 105 ± 8 cells / cm², with fully mature, dark brown, and abundant spores. Compared with the culture medium of the comparative patent (CN112725198B, Cz-RT) which is also intended to induce the sexual generation of this fungus, the culture medium of this invention advances the onset time of ascocarp formation by about 5–6 days, and the final (day 25) ascocarp yield is about 4.2 times that of the latter, with significantly better sporulation efficiency and maturity.

[0039] Although oat culture medium (OMA) has a certain induction ability, its sporulation effect is still lower than that of the culture medium of this invention and Cz-RT culture medium.

[0040] Nutrient-rich media such as PDA, PSA, and WA, suitable for the growth of a wide range of fungi, failed to induce perithecia production under the conditions of this experiment, indicating that simply providing abundant nutrients is not the key to inducing sexual reproduction in Cannomonas. 1 / 2 MS medium did not support the growth of this fungus at all.

[0041] Example 2: The "dried and pulverized watermelon seedling tissue (stem and root)" was replaced with "dried and pulverized melon seedling tissue (stem and root)," while the rest remained the same as the formula in Example 1. This formula was then cultured according to the method described in Experiment 1.

[0042] The results obtained from the formulation in Example 2 were not significantly different from those obtained from the formulation in Example 1.

[0043] Comparative Example 1-1: In Example 1, “10.0 g of glucose and 15.0 g of malt extract” was changed to “8.5 g of glucose and 16.5 g of malt extract”, and the rest was the same as in Example 1.

[0044] Comparative Examples 1-2: In Example 1, “10.0 g of glucose and 15.0 g of malt extract” were changed to “5 g of glucose and 20 g of malt extract”, and the rest were the same as in Example 1.

[0045] Comparative Example 2-1: The ingredients in Example 1, "0.5 g sodium nitrate, 0.5 g potassium chloride, 0.5 g magnesium sulfate heptahydrate, 1.5 g potassium dihydrogen phosphate", were changed to "1.5 g sodium nitrate, 0.4 g potassium chloride, 0.4 g magnesium sulfate heptahydrate, 0.7 g potassium dihydrogen phosphate", while the rest remained the same as in Example 1.

[0046] Comparative Example 2-2: The ingredients in Example 1, "0.5 g sodium nitrate, 0.5 g potassium chloride, 0.5 g magnesium sulfate heptahydrate, 1.5 g potassium dihydrogen phosphate", were changed to "2 g sodium nitrate, 0.5 g potassium chloride, 0.5 g magnesium sulfate heptahydrate, 1.5 g potassium dihydrogen phosphate", while the rest remained the same as in Example 1.

[0047] Comparative Example 3: The use of “5.0 g of dried and pulverized watermelon seedling tissue (stem, root)” in Example 1 was omitted, and the rest was the same as in Example 1.

[0048] The culture media obtained from the above 5 comparative examples were operated according to the above experimental method, and the results were compared with those of Example 1 in Table 2 below.

[0049] Table 2

[0050] In Comparative Experiment 1, the formula of Example 1 was used, but the "alternating light and dark culture (16 hours of light / 8 hours of darkness per day)" in Experiment 1 was changed to complete darkness culture, while the temperature remained unchanged at 32℃. The results are shown in Table 3 above.

[0051] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A culture medium for inducing Cannodomonas cannoli to produce perithecia, characterized in that: Add water to the carbon source, nitrogen source, inorganic salts, and plant tissue to a final volume of 1 L, adjust the pH to 7±0.1, add 20.0 g of agar powder, and then sterilize.

2. The culture medium according to claim 1, characterized in that: The carbon source consisted of 10±0.1 g of glucose and 15±0.1 g of malt extract; The nitrogen source consisted of 2 ± 0.1 g of peptone, 1 ± 0.1 g of yeast extract, and 1 ± 0.1 g of casein amino acids. The inorganic salt consists of 0.5±0.05 g sodium nitrate, 0.5±0.05 g potassium chloride, 0.5±0.05 g magnesium sulfate heptahydrate, and 1.5±0.05 g potassium dihydrogen phosphate. The plant tissue was 5 ± 0.1 g of dried and pulverized host plant tissue.

3. The culture medium according to claim 2, characterized in that: The host plant tissue is the stem or root of a watermelon or cantaloupe.

4. A method for inducing Cannodomonas to produce perithecia, characterized in that, Using the culture medium as described in any one of claims 1 to 3, the method includes the following steps: Inoculate Cannodomonas on a culture medium plate; After inoculation, the plates were placed at 32±0.5℃ and cultured with a light-dark cycle of 16 hours of light and 8 hours of darkness per day for 14 to 18 days.

5. The method according to claim 4, characterized in that: The inoculation method used was the mycelium cake inoculation method, with a mycelium cake diameter of 5 mm.

6. The method according to claim 4 or 5, characterized in that: During the culture process, the perithecia formed as early as day 14-15.

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