Method for inducing artificial sporulation of bionectria ochroleuca

Abstract

The present application relates to the technical field of plant pathology, and particularly relates to a method for artificially inducing large sporulation of Ascochyta aspidii, comprising the following steps: S1, strain activation: inoculating Ascochyta aspidii into PDA culture medium, and culturing under light at 20 DEG C for 7 days; S2, sporulation induction: inoculating the activated Ascochyta aspidii into an optimized sporulation culture medium, culturing under near-ultraviolet light at 20 DEG C for 14 days, and obtaining a large amount of spores, wherein the components of the optimized sporulation culture medium include 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15-20 g / L agar. The present application establishes a rapid, efficient and simple in-vitro sporulation method of Ascochyta aspidii, which can effectively solve the technical problem of difficult sporulation under conventional culture conditions in a laboratory, and provides sufficient experimental materials for in-depth analysis of the pathogenic mechanism.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant pathology, more particularly to a method for artificially inducing Mycocentrospora acerina to produce spores in large quantities. BACKGROUND

[0002] Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. is a perennial herbaceous medicinal plant of Aristolochiaceae Asarum, mainly distributed in China, Japan and Russia, and is a native medicinal material with great economic and medicinal value in China. Its dried roots and rhizomes are important raw materials for traditional Chinese medicine prescriptions and modern Chinese medicine preparations. Modern pharmacological studies have confirmed that it is rich in bioactive components such as volatile oils, lignans and organic acids, and has anti-inflammatory, antibacterial and analgesic pharmacological activities, and is widely used in modern Chinese clinical research. In recent years, the cultivation area of Asarum has been expanding, however, the long production cycle, lack of disease-resistant varieties, and backward cultivation mode have led to frequent diseases of Asarum. Among them, leaf blight caused by Mycocentrospora acerina (R. Hartig) Deighton is the most common and serious leaf disease due to its multiple cycle infection characteristics, with a field incidence rate of 15-20%, and a disease prevalence rate of 50-100% in diseased plants in the field, which greatly damages the yield and quality of Asarum. At present, the research on Asarum leaf blight is focused on occurrence regularity and chemical control, but there is a bottleneck of low efficiency of conidium induction in vitro, and the in-depth exploration of the key links such as the transmission characteristics, epidemic regularity and pathogenesis of the pathogen is still insufficient, which seriously restricts the in-depth study of infection cycle, pathogenesis and pathogen-host interaction mechanism, and brings significant challenges to the scientific prevention and control of Asarum leaf blight.

[0003] Efficient preparation of conidia is the basis for the study of pathogenesis of Mycocentrospora acerina and the development of fungicides. As the main source of infection of Mycocentrospora acerina, conidia are an important basis for studying pathogenesis and screening control agents. Their yield and activity directly affect the accuracy of pathogenicity assessment and pesticide virulence testing. At present, the main methods for inducing large-scale production of spores include in vitro leaf cutting, mycelium cutting, inverted medium, carrot substrate and soil liquid induction. In the prior art, patent application CN106085945A uses soil liquid to induce spore production, but has problems such as complex operation and impure spore suspension; patent application CN112322573A induces the pathogen to produce 1×10 3 conidia / mL by mycelium cutting combined with light and dark alternation, but has problems such as low spore yield and long spore production cycle, which is difficult to break through the research bottleneck of rapid and large-scale production of spores.

[0004] Current induction methods focus on improving spore production, ignoring the verification of the correlation between spore biological activity and field infection efficiency. Traditional culture medium is difficult to produce conidia, and although leaf inoculation can produce spores, it is difficult to meet the requirements of morphological observation, and the spore production is small and the method is not reproducible, which is difficult to meet the research of fungicide indoor toxicity data and field control efficiency. In addition, the existing patent technology (such as patent application CN105695389A) produces spores through in vitro leaf inoculation of cercospora sooty mould, which increases the spore production to 1.0×10 4 spores / mL, but does not establish a spore activity evaluation system, which cannot guarantee the reliability of its application in bioassay. SUMMARY

[0005] In order to make up for the shortcomings of the above-mentioned prior art, the purpose of the present application is to provide a method for artificially inducing large amounts of spores of aspidium leaf blight fungus, which uses near-ultraviolet induction method to obtain a large amount of conidia, and the obtained conidia have high infection activity and can be used for pathogenic fungus biological research and fungicide bioassay.

[0006] In order to achieve the above-mentioned purpose, the present application realizes the technical scheme as follows:

[0007] A method for artificially inducing large amounts of spores of aspidium leaf blight fungus, comprising the following steps:

[0008] S1, strain activation: inoculate aspidium leaf blight fungus on PDA medium, and culture at 20℃ under light for 7d;

[0009] S2, spore induction: inoculate the activated aspidium leaf blight fungus on the optimized spore production medium, and culture at 20℃ under near-ultraviolet light with initial pH value of 7 for 14d to obtain a large amount of spores.

[0010] The components of the optimized spore production medium include 200mL / L V8 juice, 0.3g / L CaCO3, 1g / L glucose, 1g / L potassium nitrate and 15-20g / L agar.

[0011] Under the preferred scheme, the light intensity of the near-ultraviolet light is 0.04-0.14μmol·m -2 ·s -1 , and the wavelength is 283-365nm.

[0012] Under the preferred scheme, the light intensity of the near-ultraviolet light is 0.09μmol·m -2 ·s -1 , and the wavelength is 365nm.

[0013] Under the preferred scheme, the diameter of the aspidium leaf blight fungus cake is 5mm.

[0014] Under the preferred scheme, the components of the optimized sporulation medium include 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15 g / L agar.

[0015] Under the preferred scheme, the light intensity in step S1 is 92 μmol·m -2 ·s -1 .

[0016] Under the preferred scheme, white light is used for the activation of the strain in step S1, and the light cycle in steps S1-S2 is 24 h / d.

[0017] In a more specific embodiment, a method for artificially inducing a large amount of sporulation of Ascochyta taxicola includes the following steps:

[0018] (1) Isolation of high-purity strain

[0019] A 3×3 mm healthy tissue at the diseased-healthy interface is cut from a diseased leaf of Ascochyta taxicola, and the tissue surface is sterilized (75% ethanol for 30 s, 1% sodium hypochlorite for 3 min, and sterile water for 3 times of rinsing); the sterilized tissue is inoculated on PDA medium and cultured at 20°C under light for 7 days to obtain primary colonies; the pure culture strain is obtained by successive transfer for 3 times by tip mycelium purification method.

[0020] (2) Preparation of optimized sporulation medium

[0021] V8 juice 200 mL / L, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, agar 15 g / L, and constant volume to 1000 mL; after high-pressure sterilization at 120°C, the initial pH of the medium is adjusted to 7.0 to obtain the optimized medium.

[0022] (3) Cultivation

[0023] a. Pre-cultivation of strain

[0024] The purified strain is inoculated on PDA medium and cultured at 20°C under light for 7 days (light intensity 92 μmol·m -2 ·s -1 , 24 h / d).

[0025] b. Induced sporulation of pathogenic fungus

[0026] The activated strain is inoculated on the optimized medium and placed in a near-ultraviolet light (light intensity 0.09 μmol·m -2 ·s -1 ) incubator for continuous irradiation and incubation at 20°C for 14 days.

[0027] In the present application, the sporulation amount determination method includes eluting the conidia on the culture dish with 2% Tween 20 to prepare a conidium suspension, and counting with a hemocytometer.

[0028] In the present application, the method for determining the biological activity of conidia comprises diluting the spore suspension described above to a uniform spray on healthy leaves of Asarum, keeping the culture moist, and observing after 72 hours.

[0029] The present application has the following beneficial effects:

[0030] (1) The culture medium component materials used in the method for artificially inducing mass sporulation of Asarum leaf blight fungus provided by the present application are all common consumables on the market, the culture method is simple, and the cost of inducing sporulation is significantly reduced.

[0031] (2) The method for artificially inducing mass sporulation of Asarum leaf blight fungus provided by the present application has a sporulation time of 14 days, and a high success rate (> 95%), thereby improving the sporulation efficiency of Asarum leaf blight fungus.

[0032] (3) The Asarum leaf blight fungus spores obtained by the method for artificially inducing mass sporulation of Asarum leaf blight fungus provided by the present application have high infection force, and can be used for biological activity determination of control agents. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a colony morphology diagram of Asarum leaf blight fungus on the optimized sporulation culture medium for 14 days;

[0034] Figure 2 is a microscopic observation diagram of conidia produced by Asarum leaf blight fungus cultured on the optimized sporulation culture medium for 14 days. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0036] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The experimental methods in the embodiments, unless otherwise specified, are all conventional methods. The specific conditions not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specified by the manufacturer, are all conventional products that can be obtained by purchase.

[0037] The V8 juice used in the present application is purchased from the Campbell Soup Company in the United States; the Asarum leaf blight fungus is a high-purity strain obtained by extracting the fungus from the diseased leaves of Asarum and purifying the extracted strain by laboratory methods.

[0038] Collection and processing of disease samples

[0039] Source of disease samples: The tested Asarum leaf blight samples were collected from Xinbin County, Fushun City, Liaoning Province, and typical disease-healthy junction tissue (3mm×3mm) was selected.

[0040] Treatment of diseased samples: The surface of the diseased-healthy junction tissue is disinfected by immersing the diseased-healthy junction tissue in 75% ethanol for 30 seconds, immersing it in 1% sodium hypochlorite for 3 minutes, rinsing it three times with sterile water, and then blotting off excess water with sterile filter paper.

[0041] The method for isolating and purifying Asarum leaf blight pathogens includes the following steps:

[0042] (1) Primary culture of the strain: The sterilized disease-healthy junction tissue was inoculated into PDA medium and incubated in a constant temperature incubator at 20℃ under continuous white light illumination at a light intensity of 92 μmol·m⁻¹. -2 ·s -1 After culturing for 7 days, primary colonies were obtained.

[0043] PDA medium: 200 g / L potato, 15 ± 5 g / L glucose, 15 g / L agar, and distilled water to a final volume of 1000 mL.

[0044] (2) Strain purification: The strain was continuously transferred for 3 generations using the advanced mycelial purification method. The purified strain was identified by morphological and molecular identification as Mycocentrospora acerina (R.Hartig) Deighton, and was continuously preserved in an incubator.

[0045] The environmental conditions set for the constant temperature incubator are: temperature 20±0.5℃, light cycle L:D=24h:0h.

[0046] Specifically, a method for artificially inducing the massive sporulation of Asarum leaf blight pathogen includes the following steps:

[0047] S1. Activation of the strain: The Asarum leaf blight pathogen was inoculated into PDA medium and cultured at 20℃ under light for 7 days;

[0048] S2. Induction of sporulation: The activated Asarum leaf blight pathogen was inoculated into an optimized sporulation medium, and cultured at 20°C with an initial pH of 7 under near-ultraviolet light for 14 days to obtain a large number of spores.

[0049] The optimized sporulation medium consists of 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15–20 g / L agar.

[0050] In a further preferred embodiment, the near-ultraviolet light intensity is 0.04–0.14 μmol·m⁻². -2 ·s -1, and the wavelength is 283-365 nm. More preferably, the intensity of the near-ultraviolet light is 0.09 μmol·m -2 ·s -1 , and the wavelength is 365 nm.

[0051] In a further preferred embodiment, the components of the optimized sporulation medium include 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15 g / L agar.

[0052] In a further preferred embodiment, the intensity of the light in step S1 is 92 μmol·m -2 ·s -1 .

[0053] Example 1 Conidial preparation

[0054] The optimized sporulation medium was configured as follows: 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15 g / L agar, and the volume was made up to 1 L. The medium was autoclaved at 121°C, and the initial pH of the medium was adjusted to 7 using 1 mol / L NaOH in a clean bench to obtain the optimized sporulation medium.

[0055] A method for artificially inducing Ascochyta taxicola to produce a large number of spores includes the following steps:

[0056] S1. Strain activation: Ascochyta taxicola was inoculated into PDA medium and cultured under light at 20°C for 7 days;

[0057] S2. Spore induction: An activated Ascochyta taxicola colony with a diameter of 5 mm was transferred to the center of the optimized sporulation medium, and was irradiated with near-ultraviolet light at a wavelength of 365 nm at 20°C for 24 h / d for 14 days. As shown in Figures 1-2 , a large number of spores were produced by Ascochyta taxicola after 14 days of spore production culture.

[0058] Spore collection and counting: 10 mL of eluent was added to each dish to gently scrape the mycelium on the surface of the colony, and double-layered lens paper was used to filter out the mycelium. The blood cell counting plate was used to count 10 fields of the same area for three times, and the calculation formula of the spore concentration was as follows:

[0059] Spore concentration (pieces / mL) = count average × 10 3 × dilution factor

[0060] Example 2 Spore bioactivity assay

[0061] 1. Test material

[0062] Healthy Asarum plants were cultivated in a cold shed with a temperature of 20±2℃.

[0063] 2. Inoculation experiment

[0064] The spores prepared in Example 1 were respectively configured into spore suspensions (containing 2% Tween 20) with concentrations of 1.0×10 3 / mL, 1.0×10 4 / mL and 1.0×10 5 / mL. A handheld sprayer was used to uniformly spray the leaf surface (1 mL / plant). After 72 h of inoculation, the lesion area was measured under the condition of 20℃, light and moisture. SPSS 22.0 was used to analyze the difference significance by the least difference method (P<0.05).

[0065] Comparative Example 1

[0066] In this comparative example, the spore production of Asarum leaf blight fungus was induced by cutting the mycelial block, including the following steps:

[0067] S1. Strain activation: Asarum leaf blight fungus was inoculated in PDA medium and cultured under light for 7 days at 20℃.

[0068] S2. Spore induction: The activated Asarum leaf blight fungus was cut into small blocks with a diameter of 1-2 mm. The small blocks were placed on a sterile glass slide containing water droplets, and the glass slide was placed in a culture dish with moistened filter paper for moisture culture. The culture was carried out under the condition of 20℃ and white light until the Asarum leaf blight fungus produced spores.

[0069] The produced spores were collected and counted by the same method as in Example 1.

[0070] Inoculation experiment: The spores produced by cutting the mycelial block were configured into a spore suspension (containing 2% Tween 20) with a concentration of 1.0×10 5 / mL. Healthy Asarum plants with consistent growth vigor were selected, and a handheld sprayer was used to uniformly spray the leaf surface (1 mL / plant). After 72 h of inoculation, the lesion area was measured under the condition of 20℃, light and moisture. SPSS 22.0 was used to analyze the difference significance by the least difference method (P<0.05).

[0071] Table 1 Spore production results of Asarum leaf blight fungus by different culture methods

[0072]

[0073] Table 1 shows the spore production results of the culture methods in Example 1 and Comparative Example 1. The experimental results show that the spore production method provided in Example 1 increases the spore production by 86.32 times (P<0.01) compared to the traditional mycelial block cutting method.

[0074] Table 2 Pathogenicity results of the spores produced by different culture methods

[0075]

[0076] Note: Different letters represent significant differences (P < 0.05).

[0077] As shown in Table 2, compared with the traditional mycelium cutting method, the present application can increase the spore production of Ascochyta morindae by more than 86 times through special light induction and optimization of the culture medium components, and the produced spores have stronger invasiveness, and the spore concentration is 1.0 x 10 4 ~ 1.0 x 10 5 Under the spore concentration of 1.0 x 10

[0078] Comparative Example 2

[0079] In this comparative example, the culture process of Example 1 was used to culture Ascochyta morindae, except that the culture medium used for spore production induction in step S2 was PDA medium, and a plurality of different light sources (see Table 3) were used for parallel experiments. The activated Ascochyta morindae in step S1 was subjected to spore production induction, and the Ascochyta morindae was cultured until spores were produced.

[0080] Table 3 Light quality information table

[0081] Light quality White light Red light Blue light Green light Near ultraviolet light Darkness Wavelength Full wavelength 630 nm 475 nm 520 nm 365 nm —

[0082] The produced spores were collected and counted by the same method as in Example 1, and the results are shown in Table 3.

[0083] Table 4 Spore production results of Ascochyta morindae under different light quality conditions

[0084]

[0085] Table 4 shows the spore production results of Ascochyta morindae under different light quality conditions. The experimental results show that Ascochyta morindae can produce a large number of conidia only under the induction of near-ultraviolet light, and cannot produce spores under white light, red light, blue light, green light, and darkness.

[0086] Comparative Example 3

[0087] The Aspidium leaf blight fungus was cultured according to the culture process of Example 1, except that the culture medium used in step S2 was different. A plurality of culture media (see Table 5) were used in parallel experiments to induce spore production of the Aspidium leaf blight fungus after activation in step S1, and compared with the optimized spore production culture medium of Example 1.

[0088] Table 5: Culture medium and its formula information table

[0089]

[0090] Note: Solution A in Table 2 is calcium nitrate tetrahydrate 10 g, potassium dihydrogen phosphate 2 g, sodium chloride 1.5 g, magnesium sulfate heptahydrate 2.5 g, and the volume is made up to 100 mL.

[0091] The spores produced were collected and counted using the same method as in Example 1, and the results are shown in Table 6.

[0092] Table 6: Spore production results of Aspidium leaf blight fungus under different culture medium conditions

[0093] Culture medium Spore yield (x 10 3 spores / mL) MM medium 0 OA medium 2.88 CM medium 0 Czapek medium 0 WA medium 0 PDA medium 1.37 V8 medium 4.60

[0094] Table 6 shows the spore production results of Aspidium leaf blight fungus under different culture medium conditions. Compared with the spore production results of Example 1 and Comparative Example 3, the Aspidium leaf blight fungus can produce a large amount of spores only in the optimized spore production culture medium provided by the present application compared with other culture media.

[0095] Comparative Example 4

[0096] The Aspidium leaf blight fungus was cultured according to the culture process of Example 1, except that the initial pH of the culture used in step S2 was different. The initial pH values of 5, 6, 8, and 9 were used in parallel experiments to induce spore production of the Aspidium leaf blight fungus after activation in step S1, and compared with the culture initial pH of Example 1.

[0097] The spores produced were collected and counted using the same method as in Example 1, and the results are shown in Table 7.

[0098] Table 7: Spore production results of Aspidium leaf blight fungus under different initial pH conditions

[0099] Initial pH value Spore yield (x 10 3 spores / mL) 5 0 6 17.44 7 101.55 8 5.95 9 0

[0100] Table 7 shows the spore production results of Aspidium leaf blight fungus under different initial pH conditions. Compared with the spore production results of Example 1 and Comparative Example 4, the spore production amount of the Aspidium leaf blight fungus in the optimized spore production culture medium with an initial pH of 7 is significantly higher than that of other initial pH values.

[0101] Comparative Example 5

[0102] The Ascalophyllum kawakamii was cultured according to the culture process of Example 1, except that the culture temperature in step S2 was different, and 15℃ and 25℃ were used as the culture temperature for parallel experiments, respectively, to compare with the culture temperature of Example 1. The Ascalophyllum kawakamii activated in step S1 was used for spore induction.

[0103] Table 8 Spore production results of Ascalophyllum kawakamii under different culture temperature conditions

[0104] Culture temperature (°C) Spore yield (x 10 3 spores / mL) 15 0 20 101.55 25 0

[0105] Table 8 is the spore production results of Ascalophyllum kawakamii under different culture temperature conditions. By comparing the spore production results of Example 1 and Comparative Example 5, the spore production amount of Ascalophyllum kawakamii cultured at 20℃ is significantly higher than that cultured at 15℃ and 25℃.

[0106] The above description is only the preferred specific implementation of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can make equivalent replacements or changes according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A method for artificially inducing mass sporulation of Asarum leaf blight pathogen, characterized in that, Includes the following steps: S1. Activation of the strain: The Asarum leaf blight pathogen was inoculated into PDA medium and cultured at 20℃ under light for 7 days; S2. Sporulation induction: The activated Asarum leaf blight pathogen was inoculated into the optimized sporulation medium and cultured under near-ultraviolet light at 20℃ with an initial pH of 7 for 14 days to obtain a large number of spores. The optimized sporulation medium consists of 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15–20 g / L agar. The pathogen causing leaf blight in Asarum is... Mycocentrospora acerina (R.Hartig) Deighton.

2. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 1, characterized in that, The intensity of near-ultraviolet light is 0.04~0.14 μmol·m. -2 ·s -1 The wavelength is 283~365nm.

3. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 2, characterized in that, The intensity of near-ultraviolet light was 0.09 μmol·m. -2 ·s -1 The wavelength is 365nm.

4. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 1, characterized in that, The diameter of the fungal cake of Asarum leaf blight is 5 mm.

5. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 1, characterized in that, The optimized sporulation medium consisted of 200 mL / L V8 juice, 0.3 g / L CaCO3, 1 g / L glucose, 1 g / L potassium nitrate, and 15 g / L agar.

6. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 1, characterized in that, The light intensity in step S1 is 92 μmol·m -2 ·s -1 .

7. The method for artificially inducing mass sporulation of Asarum leaf blight pathogen according to claim 1, characterized in that, The strain was activated by white light irradiation in step S1, and the light cycle for steps S1 to S2 was 24 h / d.

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