Method for promoting high sporulation of guignardia bidwellii and obtaining spore suspension and application

Abstract

This invention belongs to the fields of plant pathology and agricultural microbiology, specifically relating to a method for promoting efficient sporulation of *Anthracnose citrinum*, the obtained *Anthracnose citrinum* spore suspension, and its applications. The method involves inoculating *Anthracnose citrinum* mycelial cakes onto a high-concentration carrot agar medium, culturing them in a spore induction chamber under controlled temperature, humidity, and ventilation conditions until the mycelium covers the plate, then marking the medium with multiple parallel horizontal and vertical lines, adding glycerol aqueous solution to adjust the surface water activity to the range of 0.93–0.97, and applying a controlled spectral induction using alternating blue light and UV-A. Finally, a high-concentration spore suspension is obtained through elution. This invention significantly improves the sporulation yield and spore germination rate per plate while significantly reducing batch-to-batch variation. It is suitable for constructing standardized artificial infection models of *Anthracnose citrinum*, and can be used for pesticide efficacy evaluation, disease-resistant variety screening, and fruit storage and preservation research.

Description

Technical Field

[0001] This invention belongs to the fields of plant pathology and agricultural microbiology, specifically relating to a method for promoting efficient sporulation of fruit-borne anthracnose fungus, the resulting spore suspension, and its application in pesticide efficacy evaluation, disease-resistant variety screening, and fruit storage and preservation research. Background Technology

[0002] Anthracnose is one of the most common and damaging fungal diseases affecting fruit trees, vegetables, and various economic crops. Its pathogens mostly belong to the genus *Colletotrichum*. In research and applications such as fruit disease monitoring, pathogenicity testing, screening of resistant varieties, evaluation of pesticide susceptibility, and construction of artificial inoculation models, it is usually necessary to obtain a sufficient quantity of stable and consistent conidial suspensions as a standardized inoculation source within a short period. However, while *Colletotrichum gloeosporioides* (or its closely related species *Colletotrichum gloeosporioides*) can form colonies relatively quickly on conventional media such as potato dextrose agar (PDA), they often exhibit slow sporulation initiation, low sporulation yield, unstable sporulation, or significant differences between strains. This forces a prolonged experimental period, reduces repeatability, and consequently affects the reliability of disease evaluation or control technology screening results.

[0003] To address the need for obtaining a large number of spores in a short period, existing technologies mainly explore four directions: (1) culture medium optimization, (2) light / UV stimulation, (3) mechanical damage and water stress induction, and (4) combined treatment processes. Among these, mechanical stimulation combined with light induction is a common approach in patents and literature. For example, Chinese patent CN104480060B discloses a rapid sporulation method for *Colletotrichum gloeosporioides*, whose core process includes mycelial culture followed by mycelial scraping, sterile water rinsing, drying, and cultivation under constant temperature and light conditions, thereby inducing a large number of sporulations within 24–48 hours and providing a high sporulation yield per unit area. The advantage of this method is its rapid induction speed, making it suitable for the rapid preparation of spores for subsequent morphological observation and pathogenicity determination experiments. However, in practice, this type of scraping-rinsing-drying process is often sensitive to the operator's skill level, scraping force, degree of drying and environmental cleanliness. It can easily lead to problems such as (i) excessive damage to mycelium leading to fluctuations in viability, (ii) inconsistent water stress intensity between different batches leading to dispersion in spore production, and (iii) increased risk of contamination by miscellaneous bacteria under repeated opening operations, which in turn result in insufficient batch stability of spore production and germination capacity.

[0004] Another approach combines mechanical treatment with stimuli such as UV / light-dark cycles to obtain a higher spore yield in a shorter period. Chinese patent CN108048388A provides a method for producing spores of *Colletotrichum gloeosporioides*: after culturing under alternating light and dark conditions until near confluence, a small amount of sterile water is added and aerial hyphae are scraped off with a sterile glass rod. Subsequently, UV irradiation is applied, followed by a return to alternating light and dark cultivation. This method yields a large number of spores within 12–20 days, with a spore yield 10–20 times higher than without treatment. This technology reduces the waiting time for natural sporulation to some extent, but it still has at least three limitations: First, the intensity and dosage of ultraviolet irradiation are not easy to standardize, and excessive irradiation may inhibit the viability of the fungi; second, scraping off aerial hyphae and pouring out the fungi may still lead to contamination and batch differences; third, this type of method usually relies on ordinary light / ultraviolet irradiation and lacks fine control over the spectral components (such as blue light, UV-A bands) and light intensity / pulse regime, making it difficult for different laboratories to reproduce the same induction level.

[0005] Regarding culture medium optimization, existing research and methods have indicated that certain fungi (including some strains of the genus *Colletotrichum*) are more likely to form conidia on specific media. One typical approach in the literature is to use oat agar (OA) or its dilution system to induce stable sporulation. Suzaki et al. reported an improved method for inducing sporulation in the *Journal of General Plant Pathology* (2011): when sporulation is difficult on PDA, transferring the treated culture to diluted oat agar plates and culturing under continuous light at 25°C yields more stable sporulation performance. While this type of media strategy is effective for some strains, it still faces practical challenges in engineering applications: batch differences in different commercial oat substrates, differences in light conditions in different laboratories, and differences in responses between strains can all lead to unstable sporulation yields. Furthermore, simply relying on media replacement often fails to achieve both high sporulation and high consistency; controllable external inducing factors are still necessary.

[0006] More broadly, sporulation induction in plant pathogenic fungi is often influenced by a combination of factors, including light regime, temperature and humidity, nutrient composition of the culture medium, fungal age, mechanical stimulation, and moisture status. Review studies on how to induce sporulation in plant pathogenic fungi indicate that specific light conditions (including the effects of continuous light on specific wavelengths) are often used to induce sporulation. However, different fungi respond differently to light / dark regimes, suggesting that using only coarse-grained light / dark control is insufficient to achieve stable output across batches. On the other hand, carrot culture media is a commonly used formulation system in fungal culture. Existing formulation data document the routine practice of preparing culture media using approximately 400g of fresh carrots as raw material, demonstrating the widespread application of carrot substrates in fungal culture. However, existing publicly available technologies typically do not form a systematic coupling scheme with factors such as the high concentration of nutrients in the carrot substrate, fine-grained spectral control (e.g., blue light / UV-A), repeatable induction in a high-humidity closed environment, and controllable adjustment of surface water activity (aw); even fewer technologies propose a standardized process window that can balance high sporulation, short cycle, low contamination risk, and batch consistency for the sporulation requirements of fruit anthracnose fungus.

[0007] Therefore, while existing technologies offer referable sporulation promotion pathways in areas such as scraping / rinsing / drying, light exposure, scraping aerial mycelium, ultraviolet irradiation, light-dark alternation, dilution of oat agar, and continuous light exposure, they still generally suffer from problems such as crude control of inducing factors, easy introduction of differences and contamination in operational steps, and insufficient reproducibility across laboratories. Based on these issues, there is an urgent need for a sporulation promotion method that can achieve multi-factor synergy in a controlled environment and output stable spore yield and viability with a clearly defined process window, to meet the needs of standardized and reproducible spore preparation processes in scientific research and production testing scenarios. Summary of the Invention

[0008] The technical objective of this invention is to provide a method for promoting efficient and stable sporulation of *Anthracnose cylindrica* under controlled spectral and temperature-humidity conditions. By combining high-concentration carrot agar medium with synergistic measures such as grid-line induction during mycelial maturation, regulation of surface water activity of the medium, and blue / UV-A combined illumination, spore yield is significantly increased while reducing batch fluctuations and contamination risks, thereby obtaining a standardized spore suspension suitable for artificial inoculation, pathogenicity testing, and drug sensitivity evaluation.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for promoting efficient sporulation of *Anthracnose cylindrica*, comprising the following steps:

[0011] S1. Inoculate the purified strain of *Anthracis cirrhosa* onto potato dextrose agar (PDA) medium and culture it at 24–26°C for 3–7 days. Take a 5 mm diameter mycelial cake from the edge of the colony and keep the mycelial side facing down for later use.

[0012] S2. Inoculate the mycelium cake onto the center of a high-concentration carrot agar induction medium (CA) plate.

[0013] S3. Place the inoculated CA plate into a closed spore induction chamber with an optically transparent window on the inner wall. Control the temperature in the induction chamber at 24–26°C and the relative humidity at 90%–98%; and maintain the oxygen concentration in the chamber at 18%–21%.

[0014] S4. Cultivate under white light. When the mycelial growth covers 90% to 100% of the effective culture area of ​​the plate, use a sterilized knife to perform grid-line induction on the surface of the colony.

[0015] S5. Within 5 minutes after the grid lines are drawn, evenly drop 0.5 to 1.5 mL of a 10% to 25% glycerol aqueous solution onto the surface of each plate, allowing the glycerol aqueous solution to penetrate into the mycelial layer along the grid lines, and incubate for 10 to 30 minutes.

[0016] S6. Continue to place the plate in the spore induction chamber, using blue LED as the main light source and supplemented by UV-A LED for light induction.

[0017] S7. After the induction culture is completed, remove the plates from the spore induction chamber, add 3-8 mL of sterile water to each plate, and use a combination of gentle shaking and scraping to completely wash away the spores on the surface of the culture medium to obtain a spore suspension.

[0018] Preferably, the high-concentration carrot agar induction medium CA is prepared by peeling and chopping 350-450g of carrots, juicing and filtering the juice, adding water to 1L of the filtrate, adding 15-25g of agar and heating to dissolve, autoclaving at 120-125℃ for 15-30min, cooling to 45-55℃ and pouring into plates to obtain the high-concentration carrot agar induction medium CA. Preferably, it is prepared from 400g / L of carrot raw material and 20g / L of agar, and the carrot filtrate is pre-filtered through a 0.45μm filter membrane before pouring into plates to reduce the bacterial load.

[0019] Preferably, the spore induction chamber is a sealed plastic or glass box with a transparent top cover, and a humidity buffer module is installed inside the box. The humidity buffer module includes an open container filled with a saturated solution of sodium chloride or potassium sulfate, which is used to maintain a relative humidity of 90% to 98% at 24 to 26°C.

[0020] Preferably, the grid marking in step S4 includes 5 to 8 parallel markings in both the horizontal and vertical directions, with a spacing of 3 to 8 mm between adjacent markings, and the marking depth is limited to cutting through the hyphae layer without cutting through the agar gel layer; preferably, the grid marking depth is 0.1 to 0.3 mm, and the marking is limited to terminating within a range of 1 to 3 mm beyond the edge of the colony, so as to avoid damaging the edge structure of the plate.

[0021] Preferably, the glycerol aqueous solution in step S5 has a mass fraction of 15% to 20%, and the ratio of glycerol dosage to the surface area of ​​the plate is controlled at 0.2 to 0.5 mL / 25 cm². 2 To increase sporulation density without significantly inhibiting spore germination, the plate was tilted after incubation for 10–30 minutes to drain the free liquid, thereby adjusting the surface water activity of the culture medium to the range of 0.93–0.97.

[0022] Preferably, the blue LED in step S6 has a peak wavelength of 430–470 nm and a light intensity of 5–20 μmol / m. -2 .s -1 The UV-A LED has a main peak wavelength of 350–380 nm and a light intensity of 0.5–5 μmol·m⁻². -2 .s -1 ;

[0023] The illumination regime is as follows: alternating between continuous blue LED illumination for 10-30 minutes and darkness for 30-50 minutes, and superimposing UV-A pulsed light for 5-10 minutes 1-3 times every 24 hours, so that the total induction culture time is 7-15 days.

[0024] Preferably, the blue LED and UV-A LED are respectively arranged on the top and side wall of the spore induction chamber. The alternation sequence of blue light and UV-A illumination is as follows: first, blue light illumination and darkness are alternated in a cycle, and then UV-A pulsed light is applied to avoid high doses of UV-A causing mycelial death.

[0025] Preferably, the total induction culture time in step S6 is 8–12 days, and the total number of spores per plate reaches 1.0 × 10⁻⁶ during days 6–10. 7 ~5.0×10 7 Each spore is collected and its germination rate is maintained at no less than 85%.

[0026] Preferably, after determining the spore concentration using a hemocytometer, the spore suspension is diluted with sterile water or a buffer solution containing 0.01% Tween-20 to control the target inoculation concentration of the spore suspension at 1.0 × 10⁻⁶. 6 ~1.0×10 7 per mL.

[0027] Furthermore, the present invention also provides a suspension of fruit anthracnose spores prepared by the method.

[0028] Furthermore, the present invention also provides the application of the fruit anthracnose spore suspension prepared by the method in pesticide efficacy evaluation, disease-resistant variety screening and fruit storage and preservation research, characterized in that the spore suspension is used as a standardized inoculation source to establish a fruit anthracnose artificial infection model with high repeatability and batch-to-batch stability.

[0029] Beneficial technical effects: This invention, by employing the above-mentioned technical solution, transforms the state of low and unstable sporulation of *Anthracnose spp.* under conventional PDA / normal light conditions into a state of rapid and sustained high-density sporulation under controlled conditions; compared to not performing grid marking and a... w Control culture induced by regulation / spectrism can increase the total sporulation of a single plate by orders of magnitude, with a more uniform sporulation layer distribution and smaller differences between plates, thus significantly improving batch consistency and reproducibility. At the same time, by using a closed induction chamber to reduce opening and frequent transfer operations, and by replacing strong scraping, rinsing, and drying with gentle streaking, the viability fluctuations and exogenous contamination risks caused by excessive mechanical damage can be effectively reduced. This results in a more stable germination rate and a lower experimental coefficient of variation in subsequent artificial inoculation, pathogenicity evaluation, and drug sensitivity tests, thereby improving the reliability and comparability of disease model construction and control screening conclusions. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present invention.

[0031] I. Terminology Explanation and Parameter Definition

[0032] 1. PDA medium: Potato glucose agar medium, used for the activation and pre-culture of fruit anthracnose strains.

[0033] 2. CA induction medium: High-concentration carrot agar induction medium, used to promote the differentiation of target bacteria in the subsequent induction stage under a nutrient background.

[0034] 3. Spore induction chamber: A closed or semi-closed box structure used to support CA plates and provide controlled temperature and humidity, controlled spectral irradiation and limited gas exchange.

[0035] 4. Effective culture area: The area in the petri dish that can be used for mycelial expansion and spore formation, usually excluding areas with obvious abnormal edges and areas obscured by the dish wall.

[0036] 5. Coverage: The proportion of the effective culture area covered by mycelium, denoted as R. cov Its calculation can be performed using graphical or geometric estimation methods; when R cov When the above-mentioned limited range is reached, grid line drawing induction is triggered.

[0037] 6. Grid streak induction: Apply shallow streak with intersecting horizontal and vertical lines on the surface of the colony, cutting through the hyphal layer but not the agar gel layer.

[0038] 7. Water activity (a W ): An indicator used to characterize the available water state on the surface of a culture medium. The above limitations are achieved by treating the surface with an aqueous glycerol solution. W Adjust to 0.93-0.97.

[0039] 8. Total spore count: The total number of spores that can be eluted from a single plate. It can be calculated using the following formula: Where, N total Total number of spores per plate (spores / plate) This represents the spore suspension concentration (spores / mL). The volume of the elution buffer is in mL.

[0040] 9. Spore germination rate: An indicator used to evaluate spore viability, denoted as G (%), which can be obtained using conventional germination statistics methods; a germination rate of not less than 85% is considered an optimal performance indicator.

[0041] 10. Coefficient of Variation (CV): Used to characterize batch or replication stability. in The mean, The standard deviation is denoted as .

[0042] II. System Structure and Control Logic

[0043] 1. Spore-inducing cavity structure

[0044] In a preferred embodiment, the spore induction chamber is a sealed plastic or glass box with a transparent top cover, comprising:

[0045] Box body: with a transparent top cover or transparent window for light transmission;

[0046] Placement components: shelves or trays, used to place multiple CA tablets and ensure a certain distance between them to avoid mutual shading;

[0047] Humidity buffer module: The chamber is equipped with an open container filled with a saturated sodium chloride or potassium sulfate solution to form a stable humidity buffer environment;

[0048] Gas exchange port: A gas exchange port with a filter structure is provided to maintain the oxygen concentration at 18;

[0049] Sensing and control components (optional): Temperature and humidity sensors, light metering components and controllers, used to record and stabilize environmental parameters.

[0050] The above structure can be a combination of an independent chamber and an external constant temperature environment, or it can be integrated into a culture device with temperature, humidity and light control capabilities.

[0051] 2. Light source arrangement and timing control

[0052] In a preferred embodiment:

[0053] Blue LEDs are arranged at the top of the cavity to provide more uniform main illumination;

[0054] UV-A LEDs are arranged on the sidewall of the cavity to achieve short-time pulse stimulation and reduce the risk of local overdose caused by direct irradiation;

[0055] The illumination sequence is as follows: first, a blue light-dark alternating cycle is performed, followed by UV-A pulse irradiation to reduce adverse effects on mycelial vitality. The main peak wavelength of the blue LED is 430–470 nm, and the light intensity is 5–20 μmol·m⁻². -2 .s -1 The UV-A LED has a main peak wavelength of 350–380 nm and a light intensity of 0.5–5 μmol·m⁻². -2 .s -1 The illumination regime is as follows: alternating between continuous blue LED illumination for 10-30 minutes and darkness for 30-50 minutes, and superimposing UV-A pulsed illumination for 5-10 minutes 1-3 times every 24 hours, so that the total induction culture time is 7-15 days.

[0056] 3. Process Triggering and Closed-Loop Management

[0057] To improve batch consistency, trigger-based process management can be used:

[0058] With coverage R cov As a triggering condition, when R cov Gridline drawing is performed when entering window 90;

[0059] After the grid lines are drawn, glycerin processing must be completed within the specified time to lock a. W window;

[0060] Complete a W After setting, it enters the blue light / UV-A induction phase and maintains the total induction time within the range of 7 to 15 days.

[0061] This trigger-based management avoids batch bias caused by using only the number of cultivation days as a single criterion.

[0062] III. Implementation Instructions for the Method and Steps

[0063] The following instructions will be provided in steps S1 to S7.

[0064] S1, Pre-culture and Standardized Sampling

[0065] The purified strain of *Anthracnose spp.* was inoculated onto PDA medium and cultured at 24–26°C for 3–7 days to allow the colonies to reach a vigorous growth stage. A 5 mm diameter mycelial disc was taken from the edge of the colony, with the mycelial side facing down, for later use.

[0066] The key technical point of this step is to reduce the impact of differences in initial growth potential on the subsequent induction stage by standardizing the size of the mycelium cake and the sampling location, thereby improving the comparability of results.

[0067] S2, Inoculate into CA induction medium

[0068] The mycelial cake from step S1 is inoculated into the center of a CA plate. The high-concentration carrot agar induction medium CA is prepared by: peeling and chopping 350-450g of carrots, juicing and filtering the juice (per 1L of medium), adding water to bring the volume to 1L, adding 15-25g of agar, heating to dissolve, autoclaving at 120-125℃ for 15-30 minutes, cooling to 45-55℃, and pouring onto plates to obtain the high-concentration carrot agar induction medium CA. Preferably, it is prepared from 400g / L carrot raw material and 20g / L agar, and the carrot filtrate is pre-filtered through a 0.45μm filter membrane before pouring into the plates to reduce the load of contaminating microorganisms. The key technical point of this step is that the high carrot substrate provides a nutrient background, allowing the mycelium to spread rapidly and form a dense mycelial mat under controlled conditions within the cavity, creating a foundation for subsequent mechanical stress, water activity regulation, and controlled spectral induction.

[0069] S3, Cavity Environment Control

[0070] After inoculation, the CA plates were placed in a closed spore induction chamber, with the chamber temperature controlled at 24-26℃ and the relative humidity at 90%. The oxygen concentration was maintained at 18% through the gas exchange pores.

[0071] The key technical points of this step are: a high-humidity environment helps to reduce the moisture gradient at the edge of the plate and reduce the non-uniformity caused by local drying and cracking; moderate gas exchange ensures that metabolism is not limited by hypoxia during the culture process, while the filtration structure reduces the probability of external contamination.

[0072] S4, Coverage-triggered grid marking induction

[0073] Under white light illumination, when the mycelium covers an effective culture area of ​​90%, a grid-like streak is performed on the colony surface using a sterilized scalpel: 5-8 parallel streaks are made in both the horizontal and vertical directions, with a spacing of 3-8 mm; the streak depth is limited to cutting through the mycelial layer without cutting through the agar gel layer (with a depth of 0.1-0.3 mm and a termination boundary).

[0074] The technical function of this step can be understood as follows: without damaging the culture medium structure, it forms a dense and repeatable mechanical stress zone and microscale boundary, thereby providing space and stress signals for subsequent differentiation.

[0075] S5, Glycerin Treatment and a W Window Lock

[0076] Within 5 minutes of completing the grid pattern, evenly drop 0.5–1.5 mL of a 10% (w / w) glycerol aqueous solution onto the plate surface, allowing it to penetrate the mycelial layer along the grid lines. Incubate for 10–30 minutes, then tilt the plate to drain the free liquid, thereby cleaning the surface of the culture medium. Adjust to 0.93-0.97.

[0077] The key technical point of this step is that glycerol, as a treatment medium with adjustable water activity, can generate repeatable mild water stress without significantly introducing toxic effects; combined with grid marking, the stress mainly acts on the marked grid area, thereby promoting the uniform formation of the sporophyte.

[0078] In a preferred embodiment, a window for the glycerol mass fraction and the amount / area ratio can be further selected to balance sporulation density and germination capacity.

[0079] S6, Controllable Blue Light / UV-A Induction Culture

[0080] After step S5, the plate remains placed in the induction chamber, using blue LEDs as the main light source supplemented by UV-A LEDs for induction: 430–470 nm, light intensity 5–20 μmol / m. -2 .s -1 The UV-A LED has a main peak wavelength of 350~380nm and a light intensity of 0.5~5μmol·m⁻¹. -2 .s -1 The illumination regime is as follows: alternating between continuous blue LED illumination for 10-30 minutes and darkness for 30-50 minutes, and superimposing UV-A pulsed light for 5-10 minutes 1-3 times every 24 hours, so that the total induction culture time is 7-15 days.

[0081] The key technical points of this step are: the blue light-dark alternation is the main induction rhythm, providing repeatable light signal stimulation; short UV-A pulses are used as auxiliary stimulation to enhance differentiation signals but avoid continuous irradiation that could damage viability; the total induction time is within a limited window to ensure sufficient spore formation without excessive aging.

[0082] Under optimal conditions, the total spore count per plate can reach 1.0 × 10⁻⁶ on days 6–10. 7 ~5.0×10 7 Each seedling should be kept at a germination rate of no less than 85%.

[0083] S7, Elution and Obtaining Spore Suspension

[0084] After induction culture, remove the plates and add 3-8 mL of sterile water to each plate. Elute the spores using a combination of gentle shaking and scraping to obtain a spore suspension. In one embodiment, the spore suspension is diluted to 1.0 × 10⁻⁶ after counting. 6 ~1.0×10 7 1 per mL to form a standardized inoculum.

[0085] IV. Examples and Comparative Examples

[0086] Example 1

[0087] A representative strain A of *Anthracnose cylindrica*, isolated from and purified from fruit lesions, was selected.

[0088] 1. Pre-culture and sampling

[0089] Strain A was inoculated onto potato dextrose agar (PDA, prepared with 200 g / L potato, 20 g / L glucose, and 20 g / L agar) and cultured at a constant temperature of 25°C for 5 days to allow vigorous mycelial growth and freedom from contamination at the colony edges. Mycelial cakes were collected from the colony edges using a 5 mm diameter sterile punch, with the mycelial side facing down for later use.

[0090] 2. Preparation and inoculation of high-concentration carrot CA medium

[0091] Based on 1L of culture medium, peel and chop 400g of carrots, juice them, filter through four layers of gauze to collect the filtrate, add distilled water to 1L, add 20g of agar, heat to dissolve, and autoclave at 121℃ for 20min. Cool to approximately 50℃ and pour into 90mm diameter sterile petri dishes to prepare high-concentration carrot agar plates. Inoculate the mycelial side of the PDA pre-cultured mycelial cakes into the center of each CA plate, one mycelial cake per plate.

[0092] 3. Conditions for spore induction chamber culture

[0093] The inoculated plates were placed in a sealed plastic box with a transparent lid. An open container filled with saturated sodium chloride solution was placed inside the box as a humidity buffer. The box was kept in a constant temperature environment of approximately 25°C. Temperature and humidity sensors maintained the internal temperature at 25±1°C and the relative humidity at approximately 95%. Gas exchange pores with 0.22μm microporous membranes were installed on the side walls of the box to ensure the oxygen concentration inside was close to that of air (approximately 18%–21%). After incubation under ordinary white light for approximately 8 days, induction treatment was initiated when the mycelium visually covered more than 95% of the effective culture area of ​​the plate.

[0094] 4. Grid line drawing conditions

[0095] Inside a clean bench, use a sterile scalpel to make six parallel horizontal lines and six parallel vertical lines on the surface of the colony, forming a grid structure of approximately 6×6, with a spacing of approximately 5 mm between adjacent lines. The streaking depth is controlled to approximately 0.2 mm, cutting only the hyphae layer without penetrating the agar layer, and the streaks terminate approximately 2 mm from the plate wall.

[0096] 5. Glycerin treatment and surface water activity regulation

[0097] Within 5 minutes of completing the grid pattern, use a pipette to evenly add 1.0 mL of 18% (w / w) glycerol aqueous solution to the surface of each plate, ensuring the solution thoroughly wets the mycelial layer along the intersection of the streaks and the grid. Let the plate stand in the chamber for 20 minutes. Then, slowly tilt the plate to pour out any unadsorbed free glycerol solution. Measure the surface water activity (a) of the treated plate using a water activity meter. W It is approximately 0.94 to 0.96, which is near the midpoint of the target window of 0.93 to 0.97.

[0098] 6. Blue light / UV-A light-induced conditions

[0099] The treated plate was then placed back into the spore induction chamber, and the ordinary white light was turned off. A blue LED strip with a main peak wavelength of approximately 450 nm and a photosynthetic photon flux density of approximately 10 μmol / m² was installed at the top of the chamber. -2 .s -1 The side walls of the enclosure are equipped with UV-A LED chips, with a main peak wavelength of approximately 365nm and a photon flux density of approximately 1μmol.m. -2 .s -1 .

[0100] The illumination regime was set as follows: continuous blue light irradiation for 20 minutes, followed by 40 minutes of complete darkness, forming one cycle, which was run continuously. At two fixed time points every 24 hours (e.g., once in the morning and once in the afternoon), the UV-A lamp was turned on for 8 minutes as a short-term pulse stimulation. This light induction lasted for 10 days, with the total induction time falling within the optimal window of 8–12 days.

[0101] 7. Spore elution and counting

[0102] After induction, add 5 mL of sterile water to each plate, gently rotate the plate, and lightly scrape the surface of the culture medium with a sterile spatula to elute the spores into a suspension. Count the spore concentration using a hemocytometer and calculate the total number of spores per plate based on the elution volume.

[0103] Total spore count N of 3 replicate plates total Approximately (2.6~3.3)×10 7 The germination rate (G) is approximately 88% (statistical range 88%~92%), the coefficient of variation (CV) is approximately 6% (6%~9%), and the evenness index of the spore layer within the plate is approximately 0.86~0.91.

[0104] Example 2

[0105] This embodiment verifies the sporulation effect when the carrot dosage is taken as the lower limit (350 g / L) in a high-concentration carrot CA medium.

[0106] 1. Differences in culture medium formulation

[0107] Except for the differences described below, the other conditions were the same as in Example 1. High-concentration carrot CA medium was calculated as follows: 350 g / L carrot, 20 g / L agar per 1 L. Other preparation conditions (cooking, filtration, autoclaving at 121°C for 20 min, and plate pouring at approximately 50°C) were the same.

[0108] 2. Induction conditions

[0109] The chamber temperature (25±1℃), relative humidity (approximately 95%), gas exchange method, and mycelial coverage triggering timing were the same as in Example 1.

[0110] The grid lines are also drawn in a 6×6 pattern, with a spacing of about 5mm and a depth of about 0.2mm.

[0111] Glycerin treatment was performed using 1.0 mL of an 18% glycerin aqueous solution per dish. After standing for 20 minutes, the free liquid was discarded, and the surface a... W The measured value was approximately 0.94 to 0.96.

[0112] The illumination induction was performed under the exact same blue light / UV-A conditions and time regime as in Example 1 (blue light 450 nm, 10 μmol·m⁻¹). -2 .s -1 UV-A 365nm, 1μmol.m -2 .s -1 (20min light / 40min dark cycle + 2 x 8min UV-A pulses per day), the total induction time is approximately 10 days.

[0113] 3. Results

[0114] Total spore count N per plate in 3 replicate plates total Approximately (1.7~2.4)×10 7 The germination rate G is approximately 86% (range 86%~90%), and the coefficient of variation CV is approximately 7% (7%~11%).

[0115] Example 3

[0116] In this embodiment, while keeping other conditions basically unchanged, the relative humidity of the cavity and the blue light intensity are both adjusted to near the lower limit of their respective limited ranges.

[0117] 1. Differences in environmental and lighting conditions

[0118] The strain pre-culture, inoculation method, high-concentration carrot CA medium formulation (carrot 400g / L, agar 20g / L), grid streaking parameters (6×6 lines, spacing 5mm, depth 0.2mm), and glycerol treatment conditions (18% glycerol 1.0mL / plate, a W =0.94~0.96) are the same as in Example 1.

[0119] 2. The differences are:

[0120] 1) The relative humidity of the spore induction chamber is controlled at approximately 90% by adjusting the type of saturated salt solution and the degree of sealing of the chamber;

[0121] 2) The main peak wavelength of the blue LED was adjusted to approximately 430nm, and the photosynthetic photon flux density was reduced to approximately 8μmol.m. -2 .s -1 ;

[0122] 3) UV-A LED remains at 365nm, approximately 1μmol·m⁻¹. -2 .s -1 .

[0123] 4) The illumination sequence still uses alternating 20-minute bright / 40-minute dark blue light and 8-minute UV-A pulses twice a day, with a total induction time of about 10 days.

[0124] 3. Results

[0125] Total spore count N per plate in 3 replicate plates total Approximately (1.2~1.9)×10 7 1; germination rate It is approximately 85% (85%~89%), and the coefficient of variation (CV) is approximately 8% (8%~12%).

[0126] Example 4

[0127] This embodiment examines the selection of the upper limit of glycerol concentration to achieve surface water activity a. W Sporulation effect when close to the lower limit of 0.93.

[0128] 1. Difference conditions

[0129] The strain pre-culture, CA medium formulation (carrot 400 g / L, agar 20 g / L), chamber temperature 25 ± 1 °C, relative humidity approximately 95%, and grid streaking parameters (6 × 6 stripes, 5 mm, 0.2 mm) were all the same as in Example 1. The glycerol treatment was changed to a 25% (w / w) aqueous glycerol solution, with 1.0 mL added per dish, and the free liquid was poured off after approximately 20 minutes of standing. The surface a... W The intensity was controlled at approximately 0.93–0.94, close to the lower limit of the specified range. Wavelength and intensity of blue light / UV-A light source (blue light 450nm, 10μmol·m⁻¹) -2 .s -1 UV-A 365nm, 1μmol.m -2 .s -1 The illumination sequence (20 / 40min cycle + 2×8min UV-A per day) and the total induction time (approximately 10 days) were consistent with those in Example 1.

[0130] 2. Results and Explanation

[0131] Total spore count N per plate in 3 replicate plates total Approximately (2.0~3.8)×10 7 1; germination rate Approximately 85% (85%~88%), with a coefficient of variation (CV) of approximately 7% (7%~10%). This indicates that when... Sporulation density can be further increased when approaching the lower limit, but the germination rate is slightly lower than in Example 1. Attention should be paid to balancing yield and viability in engineering applications.

[0132] Example 5 (Upper limit of UV-A pulse frequency)

[0133] This embodiment maintains the nutritional background, grid lines, and a W Under the condition that the control remains unchanged, the number of UV-A pulses is increased to the upper limit of the specified range.

[0134] 1. Difference conditions

[0135] The strain was pre-cultured, and the CA medium formula (carrot 400g / L, agar 20g / L) was used. The chamber temperature was 25±1℃, the relative humidity was approximately 95%, and the grid lines were streaked (6×6 lines, 5mm, 0.2mm). Glycerin treatment was also performed (18% glycerol 1.0mL / plate). W=0.94~0.96) are the same as in Example 1. The light source still uses a blue LED (450nm, 10μmol·m⁻¹). -2 .s -1 ) and UV-A LED (365nm, 1μmol.m -2 .s -1 The blue light irradiation rhythm is maintained at 20 minutes of bright light and 40 minutes of dark light alternation.

[0136] The difference is that the number of UV-A pulses increases to 3 times every 24 hours, with each irradiation lasting about 10 minutes, while the total induction culture time remains about 10 days.

[0137] 2. Results and Explanation

[0138] Total spore count N per plate in 3 replicate plates total Approximately (2.3–4.1) × 10 7 The germination rate (G) is approximately 85% (85%–90%), and the coefficient of variation (CV) is approximately 6% (6%–9%).

[0139] The results showed that increasing the short-duration UV-A pulse within a reasonable range could further improve sporulation intensity without significantly affecting batch stability. However, further increasing the UV-A dose may pose a risk of inhibiting germination or damaging viability.

[0140] Comparative Example 1 (Conventional PDA Method)

[0141] This comparative example simulates the traditional laboratory culture method for *Anthracis cirrhosa*, without using carrot CA induction medium, without using a spore induction chamber to control the environment, and without performing grid streaking or glycerol a... W Regulation and blue light / UV-A induction were used to induce natural spore production using only a conventional PDA under normal light conditions.

[0142] 1. Cultivation conditions

[0143] PDA culture medium formulation: 200 g / L potato, 20 g / L glucose, 20 g / L agar, autoclaved at 121°C for 20 min. The mycelial discs obtained from the pre-cultured PDA were directly inoculated into the center of new PDA plates, one disc per plate. The plates were placed in a standard incubator at 25 ± 1°C, without additional control of relative humidity or oxygen concentration, and illuminated by diffused light from a standard laboratory fluorescent lamp. The incubation period was approximately 10 days, roughly aligning with the total incubation time in Example 1.

[0144] 2. Results

[0145] Total spore count N per plate from 3 replicate plates total Approximately (0.05~0.30)×10 71; germination rate Approximately 75% (75%–86%), with a relatively large coefficient of variation (CV), approximately 25%–45%. This indicates that the sporulation yield in conventional PDA culture is significantly lower and fluctuates considerably between batches.

[0146] Comparative Example 2 (CA only replaced, no grid lines / no a) W (Controlled / Uncontrollable Spectrum)

[0147] This comparative study only examines the effect of replacing PDA with carrot CA (carrot 400 g / L, agar 20 g / L), without using grid lines, glycerol treatment, or controlled spectral induction.

[0148] 1. Condition settings

[0149] CA medium (carrot 400 g / L, agar 20 g / L) was prepared using the same formulation as in Example 1. The pre-culture and inoculation methods for the strains were the same as in Example 1. After inoculation, the plates were placed directly in a 25±1℃ constant temperature incubator without using a closed induction chamber, and without additional control of relative humidity and oxygen concentration. They were simply incubated statically for approximately 10 days under ordinary white light diffused illumination. No streaking was performed, and no glycerol was added or a... W The system is regulated and does not include blue light / UV-A light sources.

[0150] 2. Results

[0151] Total spore count N per plate from 3 replicate plates total Approximately (0.4~1.1)×10 7 The coefficient of variation (CV) was approximately 18%–30%. This indicates that simply changing the nutrient background can improve sporulation to some extent, but the upper limit of yield and batch stability are significantly lower than the comprehensive induction scheme of this invention.

[0152] Comparative Example 3 (CA + grid lines, but without a) W (Controlled and uncontrollable spectra)

[0153] This comparative example introduced gridding on CA medium, but without glycerol treatment and a W The experiment was conducted under normal illumination conditions, without using blue light / UV-A spectral induction, to assess the contribution of the mechano-induced single factor.

[0154] 1. Condition settings

[0155] CA culture medium formula: 400g / L carrot, 20g / L agar; preparation and sterilization methods are the same as in Example 1.

[0156] The strain pre-culture and inoculation methods were the same as in Example 1. After inoculation, the strain was cultured at 25±1℃ until the mycelium covered approximately 95% of the effective area of ​​the plate. Under aseptic conditions, the strain was streaked into a grid with 6×6 stripes, a spacing of approximately 5 mm, and a depth of approximately 0.2 mm.

[0157] Do not add glycerin, do not perform a. W Adjustments were made; after streaking, the plates were placed in a regular constant temperature incubator and cultured under laboratory white light for about 10 days.

[0158] 2. Results

[0159] N of 3 repeating plates total Approximately (0.8~1.7)×10 7 The CV is approximately 12%–22%. This indicates that CA+ grid marking significantly promotes sporulation, but in the absence of a... W Even with spectral modulation, the yield and stability are still inferior to the methods of the present invention, such as Example 1.

[0160] Comparative Example 4 (CA+a) W Regulation, but not grid-based control.

[0161] In this comparative example, glycerol treatment was performed on CA medium to treat surface a W Adjusting to a limited range, but without gridding, allows for further overlay of controllable spectra or only ordinary light to assess the contribution of water activity as a single factor.

[0162] 1. Condition settings

[0163] The CA medium formulation remained the same: 400 g / L carrot and 20 g / L agar. The pre-culture and inoculation methods for the strain were the same as in Example 1. After inoculation, the culture was maintained at 25±1℃ until mycelial coverage reached approximately 95%, but no streaking was performed. 1.0 mL of 18% glycerol aqueous solution was added to the surface of each dish, and after standing for approximately 20 minutes, excess solution was poured off. The surface a was measured. W It is approximately 0.94 to 0.96.

[0164] Two sub-controls can be set for illumination conditions: one is ordinary white light illumination; the other is blue light only at 450nm, 10μmol / m. -2 .s -1 Continuous irradiation, without superimposed UV-A, incubation time approximately 10 days.

[0165] 2. Results

[0166] According to statistics, within the comprehensive range of the two sub-controls, the N of the three replicate plates... total Approximately (0.9~2.0)×10 7 Each, with a CV of approximately 10%–18%. This indicates that implementing it alone... Regulation is beneficial for increasing sporulation and a certain degree of stability, but when combined with grid marking, it provides better uniformity and batch consistency.

[0167] Comparative Example 5 (CA+ controllable spectrum, but without grid lines and without a) W Regulation)

[0168] This comparative example used controlled blue / UV-A spectral induction on CA medium, similar to the examples, but without gridding and glycerol treatment, to evaluate the effect of spectral single factors.

[0169] 1. Condition settings

[0170] The CA culture medium consisted of 400 g / L carrot and 20 g / L agar. The pre-culture and inoculation methods for the bacterial strain were the same as in Example 1. After inoculation, the plates were placed in an enclosure equipped with blue light and UV-A LEDs. The enclosure temperature was controlled at 25 ± 1 °C, but the humidity was not specifically increased, relying solely on ambient humidity. No streaking was performed, and no glycerol was added. The blue light was set to a peak of 450 nm and an intensity of approximately 10 μmol·m⁻¹. -2 .s -1 The experiment used a 20-minute bright / 40-minute dark alternation method; UV-A was set to 365nm and approximately 1μmol·m⁻¹. -2 .s -1 One to two short pulses per day (e.g., 8 minutes each time), with a total induction time of approximately 10 days.

[0171] 2. Results

[0172] N of 3 repeating plates total Approximately (0.7~1.6)×10 7 The coefficient of variation (CV) was approximately 12%–20%. This indicates that controlled spectroscopy promotes sporulation, but lacks mechanical induction and α-induction. W When the window is locked, the sporulation density and stability are still not as good as the multi-factor synergistic process of the present invention.

[0173] VI. Data Summary Table

[0174] Table 1 shows the sporulation effect and stability of different examples / comparative examples (n=3 for each group).

[0175] Table 1. Sporulation effect and stability of different embodiments / comparative examples

[0176]

[0177] Note: N in the table total is the range of values ​​for three replicates; G is the germination rate range under the corresponding conditions; CV is the coefficient of variation range for within-group replicates.

[0178] As shown in Table 1, under the conditions of using high-concentration carrot CA medium, mature-stage grid streaking, glycerol-controlled surface water activity, and blue-UV-A composite spectral induction (Examples E1–E5), the total spore count per plate consistently reached 10. 7 The number of cells was on the order of magnitude higher than that of samples, with germination rates generally maintained above 85%, and the coefficient of variation (CV) within each group controlled within the range of approximately 6% to 12%, significantly better than the control groups C1 to C5. Specifically, compared with C1 cultured using only conventional PDA, the N values ​​in this embodiment of the invention... total The yield was improved by approximately one to two orders of magnitude, and the CV was significantly reduced from 25%–45% to 6%–12%, solving the problems of low sporulation and large batch-to-batch fluctuations in traditional PDAs; compared with C2, which only changed the culture medium to CA, C3, which only added streaking, and C4, which only adjusted a w Compared to C4 and C5 which only uses controlled spectroscopy, this invention, through the synergistic effect of nutrient background, mechanical induction, water activity window locking, and controlled spectroscopy, not only further increases the upper limit of sporulation but also significantly reduces CV. The sporulation process is more concentrated, and the sporophyte layer distribution is more uniform. Therefore, the technical effect of this invention is not a simple superposition of single factors, but rather, based on a high-concentration carrot culture medium, through multi-factor coupling, it achieves a balance of high sporulation yield, high germination rate, and low batch-to-batch variability. It is suitable for constructing artificial infection models of fruit anthracnose with high requirements for reproducibility and comparability, providing a stable and standardized inoculum source for pesticide efficacy evaluation, disease-resistant variety screening, and fruit storage and preservation research.

[0179] The foregoing description of embodiments of the present invention, through which those skilled in the art are able to implement or use the present invention, will be readily apparent to those skilled in the art. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novelty disclosed herein.

Claims

1. A method for promoting efficient sporulation of fruit-growing anthracnose fungus, characterized in that, The method includes the following steps: S1. Inoculate the purified strain of *Anthracis cirrhosa* onto potato dextrose agar (PDA) medium and culture it at 24–26°C for 3–7 days. Take a 5 mm diameter mycelial cake from the edge of the colony and keep the mycelial side facing down for later use. S2. Inoculate the mycelium cake onto the center of a high-concentration carrot agar induction medium (CA) plate. S3. Place the inoculated CA plate into a closed spore induction chamber with an optically transparent window on the inner wall. Control the temperature in the induction chamber at 24–26°C and the relative humidity at 90%–98%; and maintain the oxygen concentration in the chamber at 18%–21%. S4. Cultivate under white light illumination. When the mycelium grows and covers 90% to 100% of the effective culture area of ​​the plate, use a sterilized knife to perform grid-line induction on the surface of the colony. The grid-line includes 5 to 8 parallel lines in the horizontal and vertical directions, with a spacing of 3 to 8 mm between adjacent lines. The depth of the lines is limited to cutting through the mycelial layer without cutting through the agar gel layer. S5. Within 5 minutes after the grid lines are drawn, evenly drop 0.5–1.5 mL of a 10%–25% glycerol aqueous solution onto the surface of each plate, allowing the glycerol aqueous solution to penetrate into the mycelial layer along the grid lines. Incubate for 10–30 minutes; the ratio of glycerol to plate surface area should be controlled at 0.2–0.5 mL / 25 cm². 2 After incubation for 10–30 minutes, tilt the plate to drain the free liquid, thereby adjusting the surface water activity of the culture medium to the range of 0.93–0.

97. S6. Continue to place the plate in the spore induction chamber, using blue LED as the main light source and supplemented by UV-A LED for light induction. S7. After the induction culture is completed, remove the plates from the spore induction chamber, add 3-8 mL of sterile water to each plate, and use a combination of gentle shaking and scraping to completely wash away the spores on the surface of the culture medium to obtain a spore suspension. The high-concentration carrot agar induction medium CA is prepared by peeling and cutting 350-450g of carrots, juicing and filtering the juice, adding water to 1L of the filtrate, adding 15-25g of agar and heating to dissolve, autoclaving at 120-125℃ for 15-30min, cooling to 45-55℃ and pouring into plates to obtain the high-concentration carrot agar induction medium CA. The blue LED mentioned in step S6 has a main peak wavelength of 430–470 nm and a light intensity of 5–20 μmol / m. -2 .s -1 The UV-A LED has a main peak wavelength of 350–380 nm and a light intensity of 0.5–5 μmol·m⁻². -2 .s -1 The illumination regime is as follows: alternating between continuous blue LED illumination for 10-30 minutes and darkness for 30-50 minutes, and superimposing UV-A LED pulsed illumination for 5-10 minutes 1-3 times every 24 hours, so that the total induction culture time is 7-15 days.

2. The method according to claim 1, characterized in that, The spore induction chamber is a sealed plastic or glass box with a transparent top cover. The box is equipped with a humidity buffer module, which includes an open container filled with a saturated solution of sodium chloride or potassium sulfate to maintain a relative humidity of 90% to 98% at 24 to 26°C.

3. The method according to claim 1, characterized in that, The total induction culture time for step S6 is 8–12 days, and the total number of spores per plate reaches 1.0 × 10⁻⁶ during days 6–10. 7 ~5.0×10 7 Each spore is collected and its germination rate is maintained at no less than 85%.

4. The method according to claim 1, characterized in that, After the spore concentration of the spore suspension was determined by hemocytometer, it was diluted with sterile water or a buffer solution containing 0.01% Tween-20 to control the target inoculation concentration of the spore suspension at 1.0 × 10⁻⁶. 6 ~1.0×10 7 per mL.

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