Method for promoting efficient spore production of colletotrichum gloeosporioides, obtained spore suspension and application

Abstract

The invention belongs to the technical field of phytopathology and agricultural microorganisms, and particularly relates to a method for promoting efficient spore production of colletotrichum gloeosporioides, an obtained colletotrichum gloeosporioides spore suspension and application of the colletotrichum gloeosporioides spore suspension. The method comprises the following steps: inoculating a high-concentration carrot agar culture medium with a colletotrichum gloeosporioides cake, culturing in a spore induction cavity with controllable temperature, humidity and ventilation conditions until hyphae cover a flat plate, carrying out grid scribing according to a plurality of horizontal and vertical parallel lines, dropwise adding a glycerol aqueous solution to adjust the surface water activity of the culture medium to be 0.93-0.97, and culturing at the temperature of 20-30 DEG C to obtain the colletotrichum gloeosporioides. Controllable spectrum induction with blue light and UV-A alternating combination is applied, and finally high-concentration spore suspension is obtained through elution. According to the method, the single-plate sporulation quantity and the spore germination rate are remarkably improved, meanwhile, the batch-to-batch variation is remarkably reduced, and the method is suitable for constructing a standardized artificial infection model of the fruit anthracnose and is used for pesticide effect evaluation, disease-resistant variety screening and related research of fruit storage and preservation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant pathology and agricultural microbiology technology, and specifically relates to a method for promoting high-efficiency sporulation of Glomerella cingulata, a spore suspension obtained therefrom, and application thereof in pesticide efficacy evaluation, disease-resistant variety screening, and fruit storage and preservation research. BACKGROUND

[0002] Anthracnose is one of the most common and significant fungal diseases of fruit trees, vegetables, and various economic crops, and its pathogen belongs to the genus Colletotrichum. In the research and application scenarios of fruit disease monitoring, pathogenicity determination, disease-resistant variety screening, pesticide sensitivity evaluation, and artificial inoculation model construction, it is often necessary to obtain a sufficient number of stable and batch-consistent conidial suspensions as a standardized inoculum source in a short period of time. However, although Glomerella cingulata (or its close relative, G. acutatum, etc.) can form colonies relatively quickly on general culture media such as potato dextrose agar (PDA), it often has problems such as slow sporulation initiation, low sporulation yield, unstable sporulation, or large differences between different strains, which prolongs the experimental period, reduces repeatability, and thus affects the reliability of disease evaluation or control technology screening results.

[0003] To solve the problem of obtaining a large number of spores in a short period of time, existing technologies mainly explore four directions: (1) culture medium optimization, (2) light / ultraviolet stimulation, (3) mechanical damage and water stress induction, and (4) combined treatment process. Among them, 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 G. acutatum, which includes scraping mycelium, washing with sterile water, drying, and culturing under constant temperature and light conditions after mycelium culture, thereby inducing a large number of sporulation in 24-48 hours, and giving a relatively high sporulation yield per unit area. The advantage of this scheme is that the induction speed is fast, which is suitable for rapid preparation of spores for subsequent morphological observation and pathogenicity determination experiments. However, in actual operation, such scraping-washing-drying strong stimulation process is often sensitive to the proficiency of the operator, the scraping intensity, the drying degree, and the environmental cleanliness, which may introduce problems such as (i) excessive damage to mycelium leading to fluctuation in activity, (ii) inconsistent water stress intensity in different batches leading to sporulation yield dispersion, and (iii) increased risk of contamination by other microorganisms under repeated opening of the cover, thereby causing insufficient batch stability of spore yield and germination force.

[0004] Another type of solution combines mechanical treatment with stimuli such as UV / alternating light and dark to obtain higher spore yield in a shorter period. Chinese patent CN108048388A provides a method for Colletotrichum gloeosporioides to produce spores: after culturing under alternating light and dark conditions until nearly full, a small amount of sterile water is added and the aerial mycelium is scraped off with a sterile glass rod, followed by UV irradiation, and then back to alternating light and dark culture, thereby obtaining a large amount of spores in 12-20 days, and the spore yield can be increased by 10-20 times compared to no treatment. This technology reduces the waiting time for natural spore production to some extent, but still has at least three limitations: first, the intensity and dose of UV irradiation are not easy to standardize, and excessive irradiation may inhibit the vitality of the fungus; second, scraping off the aerial mycelium and pouring out the operation may still cause contamination and batch differences; third, this type of method usually uses ordinary light / UV irradiation as the main method, lacks fine control of spectral composition (such as blue light, UV-A, etc. waveband) and light intensity / pulse system, making it difficult to reproduce the same induction level in different laboratories.

[0005] In terms of medium optimization, some research and methods have pointed out that certain fungi (including some strains of Colletotrichum) are more likely to form conidia on specific media. One of the more typical approaches in the literature is to use oat agar (OA) or its dilution system to induce stable spore production. Suzaki et al. reported an improved method for inducing spore production in Journal of General Plant Pathology (2011): when spore production is difficult on PDA, the treated culture is transferred to a diluted oat agar plate and incubated under continuous light at 25°C, which can obtain a more stable spore production performance. This type of medium strategy is effective for some strains, but still faces practical pain points in engineering applications: differences in different batches of commercial oat substrates, differences in light conditions in different laboratories, and differences in responses among strains, all of which can lead to unstable spore production. In addition, simply relying on medium replacement often cannot balance high spore production and high consistency, and still needs to add controllable external induction factors.

[0006] More broadly, sporulation induction of plant pathogenic fungi is often affected by multi-factor coupling, including light regime, temperature and humidity, medium nutrient composition, mycelial age, mechanical stimulation, water status, etc. The review on how to induce sporulation of plant pathogenic fungi pointed out that specific light conditions (including continuous light and specific wavelength-related effects) are often used to induce spore formation, and different fungi respond differently to light / dark regime, suggesting that only using coarse-grained light / dark control cannot obtain stable output across batches. On the other hand, carrot agar and other medium systems are commonly used in fungal culture, and the existing formula data records the conventional practice of preparing medium with about 400g of fresh carrots as raw material, showing the universal application of carrot substrate in the field of fungal culture. However, existing public technologies usually do not form a system coupling scheme of high concentration of carrot substrate, fine-grained spectrum control (such as blue light / UV-A), repeatable induction in 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 yield, short cycle, low pollution risk, and batch consistency for the sporulation of Guignardia bidwellii.

[0007] Therefore, although the existing technology provides a reference for sporulation promotion path in the direction of scraping / rinsing / drying, light, scraping aerial mycelium, ultraviolet irradiation, light / dark alternation, and diluting oat agar, continuous light, etc., there are still problems such as rough control of induction factors, easy introduction of differences and pollution in operation links, and insufficient reproducibility across laboratories. Based on the above status, there is an urgent need for a sporulation promotion method that can realize multi-factor synergy in a controllable environment and output stable spore yield and activity with a clear process window, to meet the demand for standardized and repeatable spore preparation process in scientific research and production detection scenarios. SUMMARY

[0008] The technical purpose of the present application is to provide a method for promoting high-efficiency and stable sporulation of Guignardia bidwellii under controllable spectrum and controllable temperature and humidity environment. By combining grid line induction during mycelial maturation, medium surface water activity regulation, and blue light / UV-A composite light regime on high-concentration carrot agar medium, the method realizes significant improvement in spore yield and reduces batch fluctuation and pollution risk, thereby obtaining standardized spore suspension suitable for artificial inoculation, pathogenicity determination, and drug sensitivity evaluation.

[0009] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0010] A method for promoting high-efficiency sporulation of Guignardia bidwellii, the method comprising the following steps:

[0011] S1, inoculate the Guosheng Guanqiao bacteria purified strain on potato dextrose agar medium PDA, cultivate at 24-26℃ for 3-7 days, take the mycelium cake with a diameter of 5mm at the edge of the colony, and make the mycelium face consistent downward for standby;

[0012] S2, inoculate the mycelium cake in the central part of the high-concentration carrot agar induction medium CA plate;

[0013] S3, place the inoculated CA plate in a closed spore induction cavity with an optically transparent window on the inner wall, control the temperature in the induction cavity at 24-26℃, control the relative humidity at 90%-98%, and control the oxygen concentration in the cavity at 18%-21%;

[0014] S4, cultivate under white light illumination, and when the mycelium growth covers 90%-100% of the effective cultivation area of the plate, use sterilized knives to perform grid line induction on the surface of the colony;

[0015] S5, within 5 minutes after the completion of the grid line, evenly add 0.5-1.5mL of 10%-25% glycerol aqueous solution to the surface of each plate, so that the glycerol aqueous solution penetrates into the mycelium layer along the grid line, and incubate for 10-30 minutes;

[0016] S6, continue to place the plate in the spore induction cavity, and use blue light LED as the main light source and UV-A LED as the auxiliary light source for illumination induction;

[0017] S7, after the end of the induction cultivation, take out the plate from the spore induction cavity, add 3-8mL of sterile water to each plate, and completely elute the spores on the surface of the medium by combining gentle shaking and scraping washing, to obtain a spore suspension.

[0018] As preferred, the high-concentration carrot agar induction medium CA is prepared by filtering the juice of 350-450g of peeled and chopped carrots, taking the filtrate to 1L, adding 15-25g of agar, dissolving by heating, high-pressure wet heat sterilizing at 120-125℃ for 15-30min, and then cooling to 45-55℃ to pour the plate, to obtain the high-concentration carrot agar induction medium CA; preferably, it is prepared from 400g / L of carrot raw materials and 20g / L of agar, and the carrot filtrate is pre-filtered through a 0.45μm filter membrane before pouring the plate, to reduce the bacterial load.

[0019] As preferred, the spore induction cavity is a closed plastic or glass box with a transparent top cover, a humidity buffer module is arranged inside the box, and the humidity buffer module includes an open container filled with saturated sodium chloride or potassium sulfate solution, which is used to maintain a relative humidity of 90%-98% at 24-26℃.

[0020] As preferred, the grid lines in step S4 include 5-8 parallel lines in horizontal and vertical directions, with a distance of 3-8 mm between adjacent lines, and the depth of the lines is limited to cutting the mycelium layer without cutting the agar gel layer; preferably, the depth of the grid lines is 0.1-0.3 mm, and the lines are terminated within the range of 1-3 mm outside the colony edge to avoid damaging the structure of the plate edge.

[0021] As preferred, the mass fraction of the glycerol aqueous solution in step S5 is preferably 15%-20%, and the ratio of the glycerol amount to the surface area of the plate is controlled to be 0.2-0.5 mL / 25 cm 2 , so as to improve the sporulation density without significantly inhibiting the spore germination force; after incubation for 10-30 minutes, the plate is tilted to drain the free liquid, thereby adjusting the water activity on the surface of the culture medium to the range of 0.93-0.97.

[0022] As preferred, the main peak wavelength of the blue light LED in step S6 is 430-470 nm, and the illumination intensity is 5-20 μmol.m -2 .s -1 , the main peak wavelength of the UV-A LED is 350-380 nm, and the illumination intensity is 0.5-5 μmol.m -2 .s -1 ;

[0023] The illumination regime is: alternating cycles of 10-30 min continuous illumination with the blue light LED and 30-50 min darkness, and superimposed 1-3 times per 24 h, each time for 5-10 min of UV-A pulsed illumination, so that the total induction culture time is 7-15 days.

[0024] As preferred, the blue light LED and the UV-A LED are arranged at the top and the side wall of the spore induction cavity, respectively, and the illumination alternating sequence of the blue light and the UV-A is: first performing the alternating cycles of the blue light illumination and darkness, and then performing the UV-A pulsed illumination, so as to avoid mycelium death caused by high-dose UV-A.

[0025] As preferred, the total induction culture time of step S6 is preferably 8-12 days, and during the period of 6-10 days, the total amount of spores in a single plate reaches 1.0×10 7 -5.0×10 7 , and the spore germination rate is maintained to be no less than 85%.

[0026] As preferred, after the spore concentration is determined by a hemocytometer, the spore suspension is diluted using sterile water or a buffer containing 0.01% Tween-20, so that the target inoculation concentration of the spore suspension is controlled to be 1.0×10 6 -1.0×10 7 / mL.

[0027] Further, the present application also provides the fruit anthracnose spore suspension prepared by the method.

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

[0029] Beneficial technical effects: the fruit anthracnose is changed from the state of less and unstable sporulation under conventional PDA / ordinary light to the state of rapid start and continuous high-density sporulation under controlled conditions by adopting the technical scheme, and the sporulation amount of the grid-drawn and a w Compared with the non-grid-drawn and non-spectrum-induced control culture, the total sporulation amount of the single plate can be improved by an order of magnitude, the spore layer distribution is more uniform, the difference between plates is smaller, the batch consistency and repeatability are significantly improved, and at the same time, the closed induction cavity reduces the opening and frequent transfer operations, and the mild streaking replaces the rough stimulation such as strong scraping, washing and drying, so that the activity fluctuation caused by mechanical overdamage and the risk of exogenous pollution are effectively reduced, the obtained spore suspension shows more stable germination rate and lower experimental coefficient of variation in subsequent artificial inoculation, pathogenicity evaluation and pesticide sensitivity test, and the reliability and comparability of the disease model construction and prevention and control screening conclusion are improved. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0031] I. Explanation of terms and definition of parameters

[0032] 1. PDA medium: potato dextrose agar medium, used for activation and preculture 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 the nutrition background.

[0034] 3. Spore induction cavity: a closed or quasi-closed box structure for carrying the CA plate and providing controlled temperature and humidity, controlled spectrum illumination and limited gas exchange.

[0035] 4. Effective culture area: the area of the Petri dish available for mycelial expansion and sporulation, usually excluding the area of the distinct marginal anomaly and the area blocked by the dish wall.

[0036] 5. Coverage: the proportion of the effective culture area covered by mycelium, denoted as R cov , which can be calculated by image method or geometric estimation method; when R cov reaches the above defined interval, grid line induction is triggered.

[0037] 6. Grid line induction: shallow lines crossing horizontally and vertically are made on the surface of the colony, which is required to cut through the mycelial layer without cutting through the agar gel layer.

[0038] 7. Water activity (a W ): an index used to characterize the state of available water on the surface of the culture medium. The above defined adjustment of the surface a W to 0.93~0.97 is achieved by treating the surface with a glycerol aqueous solution.

[0039] 8. Total amount of spores: the total number of spores obtained by elution from a single plate. It can be calculated as follows: where N total is the total number of spores per plate (number / plate), is the concentration of the spore suspension (number / mL), and is the volume of the eluent (mL).

[0040] 9. Spore germination rate: an index used to evaluate the viability of spores, denoted as G (%), which can be obtained by conventional germination statistics method; a germination rate of no less than 85% is used as a preferred effect index.

[0041] 10. Coefficient of variation (CV): an index used to characterize the stability between batches or repetitions: where is the mean value, and is the standard deviation.

[0042] II. System structure and control logic

[0043] 1. Spore induction cavity structure

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

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

[0046] Placing assembly: shelves or trays for placing multiple CA plates and ensuring a certain distance to avoid mutual light blocking;

[0047] Humidity buffer module: an open container is provided in the box, filled with saturated sodium chloride or potassium sulfate solution, to form a stable humidity buffer environment;

[0048] Gas exchange holes: Gas exchange holes with filter structure are set to maintain the oxygen concentration at 18%;

[0049] Sensing and control components (optional): Temperature and humidity sensors, light metering components and controllers for recording and stabilizing environmental parameters.

[0050] The above structure can be a combination of an independent box, an external constant temperature environment, or 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 light LEDs are arranged at the top of the cavity to provide more uniform main light;

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

[0055] The light sequence is to first perform blue light-dark alternating cycle, and then perform UV-A pulse irradiation to reduce the adverse effects on mycelial activity. The main peak wavelength of the blue light LED is 430-470 nm, and the light intensity is 5-20 μmol.m -2 .s -1 The main peak wavelength of the UV-A LED is 350-380 nm, and the light intensity is 0.5-5 μmol.m -2 .s -1 ; The light regime is: alternating cycle of 10-30 min continuous light irradiation with blue light LED and 30-50 min darkness, and superimposed 1-3 times of 5-10 min UV-A pulse light irradiation within every 24 h, 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] The coverage rate R cov is used as a trigger condition, and when R cov enters the 90 window, grid lines are drawn;

[0059] After grid lines are drawn, glycerol processing is completed within a specified time to lock the a W window;

[0060] After a W is set, the blue light / UV-A induction phase is entered, and the total induction time is maintained within the range of 7-15 days.

[0061] The trigger management can avoid batch deviation caused by taking culture days as the only criterion.

[0062] III. Implementation of method steps

[0063] The following is implemented according to steps S1-S7.

[0064] S1, pre-culture and standardized sampling

[0065] A purified strain of Guignardia bidwellii is inoculated on PDA culture medium and cultured at 24-26°C for 3-7 days to make the colony in the vigorous growth stage. A 5mm-diameter fungus cake is taken from the edge of the colony, with the mycelium surface kept downward for standby.

[0066] The technical key point of this step is to reduce the influence of initial growth potential difference on the subsequent induction stage by unifying the fungus cake size and sampling position, and to improve the comparability of the results.

[0067] S2, inoculation into CA induction medium

[0068] The fungus cake of step S1 is inoculated in the center of the CA plate. The high-concentration carrot agar induction medium CA is prepared as follows: 350-450g of peeled and chopped carrots are juiced, the filtrate is filtered, water is added to 1L, 15-25g of agar is added and dissolved by heating, and the mixture is autoclaved at 120-125°C for 15-30min, then cooled to 45-55°C and poured into a plate to obtain the high-concentration carrot agar induction medium CA; preferably, 400g / L of carrot raw material and 20g / L of agar are used, and the carrot filtrate is pre-filtered through a 0.45μm filter membrane before pouring the plate to reduce the load of foreign bacteria. The technical key point of this step is to provide a nutrient background by using a higher carrot substrate, so that the mycelium can rapidly spread and form a dense fungal carpet in a controlled environment, creating a foundation for subsequent mechanical stress, water activity regulation, and controllable spectrum induction.

[0069] S3, cavity environment control

[0070] The inoculated CA plate is placed in a closed spore induction cavity, the temperature of the cavity is controlled at 24-26°C, the relative humidity is controlled at 90, and the oxygen concentration is maintained at 18 through gas exchange holes.

[0071] The technical key point of this step is that a high-humidity environment is conducive to reducing the water gradient at the edge of the plate and reducing non-uniformity caused by local dry cracking; moderate gas exchange ensures that metabolism is not limited by hypoxia during cultivation, and the filtering structure reduces the probability of external contamination.

[0072] S4, grid line induction triggered by coverage

[0073] When the mycelium covers 90% of the effective culture area under white light illumination, grid streaking is performed on the colony surface using sterilized knives: 5-8 parallel streaks in the horizontal and vertical directions, with a spacing of 3-8 mm; the depth of the streaks is limited to cutting through the mycelium layer without cutting through the agar gel layer (and the depth can be 0.1-0.3 mm and the boundary can be terminated).

[0074] The technical role of this step can be understood as follows: under the premise of not destroying the culture medium structure, dense, repeatable mechanical stress zones and micro-scale boundaries are formed, thereby providing space and stress signals for subsequent differentiation.

[0075] S5, glycerol treatment and a W Window locking

[0076] Within 5 minutes after the completion of grid streaking, 0.5-1.5 mL of a 10% glycerol aqueous solution is uniformly added to the surface of the plate, so that it penetrates into the mycelium layer along the grid of streaks; after incubation for 10-30 minutes, the free liquid is tilted and drained, thereby exposing the surface of the culture medium to the air. Adjust to 0.93-0.97.

[0077] The technical points of this step are as follows: glycerol, as a water activity-adjustable treatment medium, can produce repeatable mild water stress without significantly introducing toxic effects; in combination with grid streaking, the stress mainly acts on the grid-streaked area, thereby promoting the uniform formation of spore layers.

[0078] In preferred embodiments, the window of the glycerol mass fraction and the amount / area ratio can be further selected to balance the spore production density and germination force.

[0079] S6, controllable blue light / UV-A induced culture

[0080] After step S5, the plate is continuously placed in the induction cavity, and blue light LEDs are used as the main light source and UV-A LEDs are used as auxiliary light sources for induction: 430-470 nm, light intensity 5-20 μmol.m -2 .s -1 The main peak wavelength of the UV-A LED is 350-380 nm, and the light intensity is 0.5-5 μmol.m -2 .s -1 The light regimen is as follows: continuous illumination with blue light LEDs for 10-30 min and alternating cycles of darkness for 30-50 min, with 1-3 times of 5-10 min UV-A pulse illumination superimposed within every 24 h, so that the total induction culture time is 7-15 days.

[0081] The technical points of this step are: blue light-dark alternation is the main induction rhythm, providing repeatable light signal stimulation; UV-A short pulse as an auxiliary stimulus, used to strengthen the differentiation signal but avoid continuous irradiation leading to viability damage; the total induction time is within a limited window, so that spore formation is sufficient but not over-aging.

[0082] Under the preferred conditions, the total amount of single plate spores can reach 1.0×10 7 ~ 5.0×10 7 per plate on the 6th to 10th day, and the germination rate is maintained at no less than 85%.

[0083] S7, elution of spore suspension from the plate

[0084] After the induction culture is completed, the plate is removed, 3-8 mL of sterile water is added to each plate, and the spores are eluted by gentle shaking combined with scraping washing to obtain a spore suspension. In an embodiment, the spore suspension is diluted to 1.0×10 6 ~ 1.0×10 7 per mL after counting to form a standardized inoculum source.

[0085] IV. Examples and Comparative Examples

[0086] Example 1

[0087] A representative strain A of G. cingulata was selected, which was isolated from fruit lesions and purified.

[0088] 1. Pre-culture and sampling

[0089] Strain A was inoculated on potato dextrose agar medium (PDA, prepared according to 200 g / L potato, 20 g / L glucose, 20 g / L agar) and cultured at 25°C for 5 days to make the colony edge mycelium grow vigorously and free of contamination. A sterile puncher with a diameter of 5 mm was used to take a bacterial cake at the edge of the colony, with the mycelium face facing down for standby.

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

[0091] According to 1 L of medium, 400 g of peeled and chopped carrots were juiced, the filtrate was collected by filtering through 4 layers of gauze, distilled water was added to 1 L, 20 g of agar was added and dissolved by heating, and then autoclaved at 121°C for 20 min. When cooled to about 50°C, pour into a sterile petri dish with a diameter of 90 mm to prepare a high-concentration carrot agar plate. The mycelium face of the bacterial cake obtained by PDA pre-culture was inoculated in the center of the CA plate, with 1 cake per dish.

[0092] 3. Spore induction cavity culture conditions

[0093] The inoculated plates were placed in a sealed plastic box with a transparent top cover. An open container filled with saturated sodium chloride solution was placed in the box as a humidity buffer module, and the box was placed in a constant temperature environment at about 25°C. The temperature in the cavity was maintained at 25±1°C and the relative humidity was about 95% as monitored by a temperature and humidity sensor. The side wall of the box was provided with a gas exchange hole with a 0.22 μm microporous filter membrane, so that the oxygen concentration in the cavity was close to the air level (about 18% to 21%). The culture was carried out under ordinary white light illumination for about 8 days, and when the mycelium covered more than 95% of the effective culture area of the plate under visual observation, the induction treatment was carried out.

[0094] 4. Grid line conditions

[0095] In the clean bench, 6 parallel lines were drawn on the surface of the colony in the horizontal direction and 6 parallel lines were drawn in the vertical direction with a sterilized scalpel to form a grid structure of about 6x6, with a spacing of about 5 mm between adjacent lines. The depth of the line was controlled at about 0.2 mm, only cutting the mycelium layer without cutting through the agar layer, and the line ended at about 2 mm from the wall of the plate.

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

[0097] Within 5 minutes after the grid lines were drawn, 1.0 mL of 18% glycerol aqueous solution was uniformly added to the surface of each plate using a pipette, so that the solution fully wetted the mycelium layer along the grid intersection. After 20 minutes in the cavity, the plate was slowly tilted and the free glycerol solution was poured out. The water activity meter was used to measure that the water activity a of the plate surface after treatment was about 0.94-0.96, close to the median value of the target window of 0.93-0.97. W

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

[0099] The treated plate was continuously placed in the spore induction cavity, and the ordinary white light was turned off. A blue light LED lamp strip was installed on the top of the box, with a main peak wavelength of about 450 nm and a photosynthetic photon flux density of about 10 μmol.m -2 .s -1 A UV-A LED lamp bead was installed on the side wall of the box, with a main peak wavelength of about 365 nm and a photon flux density of about 1 μmol.m -2 .s -1 .

[0100] The light regime was set as follows: blue light was continuously irradiated for 20 minutes, followed by 40 minutes of complete darkness, which was one cycle, and continuous operation; at two fixed time points (such as once in the morning and once in the afternoon) within 24 hours, the UV-A lamp bead was turned on for 8 minutes as a short pulse stimulation. The above light induction lasted for 10 days, and the total induction time fell near the median value of the preferred window of 8-12 days. ​

[0101] 7. Spore elution and counting

[0102] After the induction was completed, 5 mL of sterile water was added to each plate, the plates were gently rotated and the surface of the medium was lightly scraped with a sterile spatula to elute the spores into a suspension. The concentration of spores was counted using a hemocytometer and the total number of spores per plate was calculated by combining the elution volume.

[0103] 3. Total number of spores per plate N total about (2.6-3.3) x 10 7 about 88% (statistical range 88%-92%) and a coefficient of variation CV of about 6% (6%-9%) and a uniformity index U of about 0.86-0.91.

[0104] Example 2

[0105] This example verifies the sporulation effect of the high concentration carrot CA medium when the lower limit (350 g / L) of the carrot dosage is used.

[0106] 1. Medium formulation differences

[0107] The remaining conditions were the same as in Example 1, except for the following differences. The high concentration carrot CA medium was prepared in 1 L: carrot 350 g / L, agar 20 g / L. The other preparation conditions (cooking, filtering, autoclaving at 121 °C for 20 min, pouring the plates at about 50 °C) were the same.

[0108] 2. Induction conditions

[0109] The cavity temperature (25 ± 1 °C), relative humidity (about 95%), gas exchange mode, and timing of mycelium coverage triggering were the same as in Example 1.

[0110] The grid lines were also drawn in 6 x 6 lines with a spacing of about 5 mm and a depth of about 0.2 mm.

[0111] The glycerol treatment used 18% glycerol solution 1.0 mL / plate, and after standing for 20 min, the free liquid was poured off, and the surface of the medium was covered with a sterile glass plate. W about 0.94-0.96 was measured.

[0112] The light induction used exactly the same blue light / UV-A conditions and time regime as in Example 1 (blue light 450 nm, 10 μmol.m -2 .s -1 UV-A 365 nm, 1 μmol.m -2 .s -1 20 min light / 40 min dark cycle + 2 x 8 min UV-A pulses per day) for a total induction time of about 10 days.

[0113] 3. Results

[0114] Total amount of single plate spores N of 3 repeated plates total about (1.7~2.4)×10 7 ; germination rate G about 86% (range 86%~90%), coefficient of variation CV about 7% (7%~11%).

[0115] Example 3

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

[0117] 1. Differences in environmental and lighting conditions

[0118] The strain pre-culture, inoculation method, high-concentration carrot CA medium formula (carrot 400 g / L, agar 20 g / L), grid streaking parameters (6x6 lines, 5 mm spacing, 0.2 mm depth), and glycerol treatment conditions (18% glycerol 1.0 mL / dish, a W =0.94~0.96) are the same as in Example 1.

[0119] 2. Differences are:

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

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

[0122] 3) The UV-A LED is still 365 nm, about 1 μmol.m -2 .s -1 .

[0123] 4) The lighting timing still uses blue light 20 min bright / 40 min dark alternation and 2 times per day, 8 min UV-A pulse each time, with a total induction time of about 10 days.

[0124] 3. Results

[0125] Total amount of single plate spores N of 3 repeated plates total about (1.2~1.9)×10 7 ; germination rate about 85% (85%~89%), coefficient of variation CV about 8% (8%~12%).

[0126] Example 4

[0127] The present example investigates the upper limit of glycerol concentration, to make the surface water activity a W The sporulation effect near the lower limit of 0.93.

[0128] 1. Difference conditions

[0129] The strain pre-culture, CA medium formula (carrot 400 g / L, agar 20 g / L), cavity temperature 25±1℃, relative humidity about 95%, grid streaking parameters (6×6, 5mm, 0.2mm) are the same as Example 1. The glycerol treatment is changed to use a glycerol aqueous solution with a mass fraction of 25%, and the amount of glycerol added per dish is still 1.0mL. After standing for about 20min, the free liquid is poured out. The surface a W is measured by the water activity meter. The control is about 0.93~0.94, close to the lower limit of the defined range. The wavelength and intensity of the blue light / UV-A light source (blue light 450nm, 10μmol.m -2 .s -1 ; UV-A 365nm, 1μmol.m -2 .s -1 ), light timing (20 / 40min cycle + 2×8min UV-A per day) and total induction time (about 10d) are consistent with Example 1.

[0130] 2. Results and explanations

[0131] The total number of spores N of single plate of 3 repeated plates total is about (2.0~3.8)×10 7 ; the germination rate is about 85% (85%~88%), and the coefficient of variation CV is about 7% (7%~10%). It is shown that when the sporulation density can be further improved near the lower limit, but the germination rate is slightly lower than Example 1, and attention should be paid to balance the yield and activity in engineering applications.

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

[0133] In this example, the number of UV-A pulses is increased to the upper limit of the defined range while keeping the nutritional background, grid streaking and a W control conditions unchanged.

[0134] 1. Difference conditions

[0135] The strain pre-culture, CA medium formula (carrot 400 g / L, agar 20 g / L), cavity temperature 25±1℃, relative humidity about 95%, grid streaking (6×6, 5mm, 0.2mm) and glycerol treatment (18% glycerol 1.0mL / dish, a W=0.94~0.96) are the same as Example 1. The light source part still uses blue LED (450 nm, 10 μmol.m -2 .s -1 ) and UV-A LED (365 nm, 1 μmol.m -2 .s -1 ), the blue light illumination rhythm maintains 20 min light / 40 min dark alternation.

[0136] The difference is that the number of UV-A pulses is increased to 3 times per 24 h, and each illumination time is about 10 min, and the total induction culture time is still about 10 d.

[0137] 2. Results and explanations

[0138] 3. Total spore amount N of single plate of 3 repeated plates total about (2.3~4.1) × 10 7 ; the germination rate G is about 85% (85%~90%), and the coefficient of variation CV is about 6% (6%~9%).

[0139] The results show that increasing short-time UV-A pulses within a reasonable range can further improve the spore production intensity and has no obvious adverse effect on batch stability, but if the UV-A dose is further increased, there may be a risk of inhibiting germination or damaging viability.

[0140] Comparative Example 1 (conventional PDA method)

[0141] This comparative example simulates the fruiting body culture method commonly used in traditional laboratories, does not use carrot CA induction medium, does not use spore induction cavity to control the environment, does not carry out grid streaking, glycerol a W regulation and blue light / UV-A induction, and only uses conventional PDA under ordinary illumination conditions for natural spore production.

[0142] 1. Culture conditions

[0143] PDA medium formula: 200 g / L potato, 20 g / L glucose, 20 g / L agar, high pressure sterilization 121℃ 20 min. The mycelium cake obtained by pre-culturing PDA is directly inoculated into the center of a new PDA plate, 1 mycelium cake per dish. The plate is placed in a normal constant temperature incubator, the temperature is 25±1℃, the relative humidity and oxygen concentration are not additionally controlled, and the illumination is ordinary laboratory fluorescent light scattering light. The culture time is selected to be about 10 d to roughly align with the total culture time of Example 1.

[0144] 2. Results

[0145] 3. Total spore amount N of single plate of 3 repeated plates total about (0.05~0.30) × 10 7Spores per plate about 75% (75%-86%), with a large coefficient of variation CV of about 25%-45%. This shows that the spore yield in conventional PDA culture is significantly low and has large batch fluctuations.

[0146] Comparative Example 2 (only CA is replaced, without grid lines / a W Control / without controllable spectrum

[0147] This comparative example only investigates the effect of replacing PDA with carrot CA (carrot 400 g / L, agar 20 g / L), without grid lines, glycerol treatment, and controllable spectrum induction.

[0148] 1. Condition setting

[0149] CA medium (carrot 400 g / L, agar 20 g / L) was prepared with the same formula as in Example 1, and the strain pre-culture and inoculation methods were the same as in Example 1. After inoculation, the plate was directly placed in a 25±1°C constant temperature incubator, without using a sealed induction cavity, without additional control of relative humidity and oxygen concentration, and only under the illumination of ordinary white light lamp scattering. The plate was incubated for about 10 days. No grid lines were drawn, no glycerol was added, and no a W control, without configuring a blue light / UV-A light source.

[0150] 2. Results

[0151] Total spore amount N of a single plate of 3 repeated plates total about (0.4-1.1)×10 7 individual, with a coefficient of variation CV of about 18%-30%. This shows that the spore yield can be increased to some extent by only changing the nutritional background, but the upper limit of the yield and the batch stability are both significantly lower than the comprehensive induction scheme of the present application.

[0152] Comparative Example 3 (CA+grid lines, but without a W control and without controllable spectrum

[0153] This comparative example introduces grid lines on the CA medium, but does not perform glycerol treatment and a W adjustment, nor does it use blue light / UV-A spectrum induction, but only under ordinary light conditions to evaluate the contribution of the mechanical induction single factor.

[0154] 1. Condition setting

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

[0156] Strains were pre-cultured and inoculated as in Example 1. After inoculation, the plates were incubated at 25±1°C until mycelium covered about 95% of the effective area of the plate. Grid streaking was performed under sterile conditions with 6x6 lines, spacing about 5 mm, and depth about 0.2 mm.

[0157] No glycerol was added, and no a W Control; after streaking, the plates were kept in a normal incubator and incubated under laboratory white light illumination for about 10 days.

[0158] 2. Results

[0159] N of 3 replicate plates total about (0.8-1.7)xlO 7 , CV about 12%-22%. It can be seen that CA+grid streaking had obvious promoting effect on sporulation, but in the absence of a W and spectral regulation, the yield and stability were still inferior to the methods of Example 1 and the like.

[0160] Comparative Example 4 (CA+a W regulation, but no grid streaking)

[0161] This comparative example was implemented on CA medium with glycerol treatment, and the surface a W regulation to a limited range, but no grid streaking, can further superimpose controllable spectrum or only normal light illumination, to evaluate the contribution of the single factor of water activity.

[0162] 1. Condition setting

[0163] The CA medium formula was still carrot 400 g / L, agar 20 g / L. The strains were pre-cultured and inoculated as in Example 1. After inoculation, the plates were incubated at 25±1°C until mycelium covered about 95%, but no grid streaking was implemented. 1.0 mL of 18% glycerol aqueous solution was added to the surface of each dish, and after standing for about 20 min, the excess solution was poured out, and the surface a W about 0.94-0.96.

[0164] The illumination conditions can be set to two kinds of sub-contrasts: one is normal white light illumination; the other is only blue light 450 nm, 10 μmol.m -2 .s -1 Continuous illumination, no superimposition of UV-A, incubation time about 10 days.

[0165] 2. Results

[0166] According to statistics, within the comprehensive range of the two sub-contrasts, N of 3 replicate plates total about (0.9-2.0)xlO 7 , CV about 10%-18%. It shows that the implementation of The regulation is beneficial to increase the sporulation amount and stability to some extent, but the uniformity and batch consistency are better when combined with grid marking.

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

[0168] This comparative example uses a similar blue light / UV-A controllable spectrum induction on CA medium as the examples, but without grid marking and glycerol treatment, to evaluate the effect of the single factor of spectrum.

[0169] 1. Condition setting

[0170] The CA medium formula is carrot 400 g / L and agar 20 g / L, and the strain pre-culture and inoculation method are the same as Example 1. After inoculation, the plate is placed in a box equipped with blue light and UV-A LED, and the box temperature is controlled at 25±1℃, but the cavity humidity is not specially increased, and only relies on the environmental humidity. No grid marking is performed, and no glycerol is added. The blue light is set to a main peak of 450 nm, with an intensity of about 10 μmol.m -2 .s -1 , with 20 min light / 40 min dark alternation; the UV-A is set to 365 nm, about 1 μmol.m -2 .s -1 , with 1-2 times of short pulses per day (such as 8 min each time), and the total induction time is about 10 d.

[0171] 2. Results

[0172] The N total of the 3 repeated plates is about (0.7-1.6)×10 7 , and the CV is about 12%-20%. It is shown that the controllable spectrum has a promoting effect on sporulation, but lacks mechanical induction and a W window locking, and the sporulation density and stability are still not as good as the multi-factor synergistic process of the present application.

[0173] Six, data summary table

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

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

[0176]

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

[0178] As can be seen from Table 1, under the conditions of using high-concentration carrot CA medium, grid streaking at the maturation stage, glycerol regulating surface water activity, and blue light-UV-A composite spectrum induction (Examples E1-E5), the total amount of single-plate spores stably reached 10 7 orders of magnitude, the germination rate was generally maintained at more than 85%, and the coefficient of variation CV was controlled within about 6%-12%, which was significantly better than each of the comparative examples C1-C5. Specifically, compared with C1 which only used conventional PDA culture, the N total of the examples of the present application increased by about 1-2 orders of magnitude, and the CV was significantly reduced from 25%-45% to 6%-12%, solving the problems of low sporulation yield and large batch fluctuation of traditional PDA; compared with C2 which only replaced the culture medium with CA, C3 which only increased streaking, C4 which only regulated a w of the present application, under the synergistic effect of nutritional background, mechanical induction, water activity window locking, and controllable spectrum, not only further improved the upper limit of sporulation, but also significantly reduced the CV, and the sporulation process was more concentrated and the spore layer distribution was more uniform. It can be seen that the technical effect of the present application is not a simple superposition of single factors, but through the coupling of multiple factors in the present application, high sporulation yield, high germination rate, and low batch variation are achieved, which is suitable for constructing an artificial inoculation model of fruit anthracnose with high repeatability and comparability, and provides a stable standardized inoculum for pesticide efficacy evaluation, disease-resistant variety screening, and fruit storage and preservation research.

[0179] The above describes the embodiments of the present application, and through the above description of the disclosed embodiments, those skilled in the art can implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting high sporulation efficiency of Guignardia bidwellii, characterized by, The method comprises the following steps: S1, inoculate the purified Guignardia bidwellii strain on potato dextrose agar medium PDA, and culture at 24-26℃ for 3-7 days, take a 5mm-diameter mycelium cake at the edge of the colony, and make the mycelium face consistent downward for standby; S2, inoculate the mycelium cake in the center of high-concentration carrot agar induction medium CA plate; S3, place the inoculated CA plate in a closed spore induction cavity with an optically transparent window on the inner wall, control the temperature in the induction cavity at 24-26℃, and control the relative humidity at 90%-98%; and control the oxygen concentration in the cavity to maintain at 18%-21%; S4, culture under white light illumination, when the mycelium growth covers 90%-100% of the effective culture area of the plate, use sterilized knives to perform grid line induction on the surface of the colony; S5, within 5 minutes after the completion of grid line induction, evenly add 0.5-1.5mL of 10%-25% glycerol aqueous solution to the surface of each plate, so that the glycerol aqueous solution penetrates into the mycelium layer along the grid line, and incubate for 10-30 minutes; S6, continue to place the plate in the spore induction cavity, and use blue light LED as the main light source and UV-A LED as the auxiliary light source for illumination induction; S7, after the completion of induction culture, take out the plate from the spore induction cavity, add 3-8mL of sterile water to each plate, and completely elute the spores on the surface of the culture medium by combining gentle shaking and scraping washing, to obtain a spore suspension.

2. The method of claim 1, wherein, The high-concentration carrot agar induction medium CA is prepared as follows: peel and chop 350-450g of carrots, filter the juice, take the filtrate, add water to 1L, add 15-25g of agar, heat and dissolve, high-pressure wet heat sterilize at 120-125℃ for 15-30min, and then cool to 45-55℃, pour the plate, and obtain the high-concentration carrot agar induction medium CA.

3. The method of claim 1, wherein, The spore induction cavity is a closed plastic or glass box with a transparent top cover, and a humidity buffer module is arranged in the box, which comprises an open container filled with saturated sodium chloride or potassium sulfate solution, and is used to maintain a relative humidity of 90%-98% at 24-26℃.

4. The method of claim 1, wherein, The grid line induction in step S4 comprises 5-8 parallel lines in the horizontal direction and the vertical direction, the distance between adjacent lines is 3-8mm, and the depth of the line is limited to cut off the mycelium layer without cutting off the agar gel layer.

5. The method of claim 1, wherein, The mass fraction of the glycerol aqueous solution in step S5 is 15% to 20%, and the ratio of the amount of glycerol to the surface area of the plate is controlled to be 0.2 to 0.5 mL / 25 cm 2 After incubation for 10 to 30 minutes, the plate is tilted to drain the free liquid, thereby adjusting the water activity of the medium surface to the range of 0.93 to 0.

97.

6. The method of claim 1, wherein, The main peak wavelength of the blue LED in step S6 is 430-470 nm, and the light intensity is 5-20 μmol.m -2 .s -1 The main peak wavelength of the UV-A LED is 350-380 nm, and the light intensity is 0.5-5 μmol.m -2 .s -1 ; The illumination system is as follows: continuously illuminate with blue light LED for 10-30min, and alternately cycle with darkness for 30-50min, and superimpose 1-3 times of 5-10min UV-A pulse illumination every 24h, so that the total induction culture time is 7-15 days.

7. The method of claim 1, wherein, The total induction culture time of step S6 is 8-12 days, and the total amount of single plate spores reaches 1.0×10 7 ~5.0×10 7 and the spore germination rate is not less than 85%.

8. The method of claim 1, wherein, The spore suspension was diluted with sterile water or buffer containing 0.01% Tween-20 after the spore concentration was determined by a hemocytometer, so that the target inoculum concentration of the spore suspension was controlled at 1.0 x 10 6 ~ 1.0 x 10 7 spores / mL.

9. The Guignardia bidwellii spore suspension prepared by the method according to any one of claims 1-8.

10. The use of the spore suspension of G. fructigenum prepared according to the method of any one of claims 1 to 8 in the evaluation of the efficacy of pesticides, the screening of disease-resistant varieties and the study of fruit storage and preservation, characterized in that, The spore suspension is used as a standardized inoculum to establish a Guignardia bidwellii artificial inoculation model with high repeatability and batch stability.

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