Culture medium for inducing Stemphylium solani to produce spores as well as preparation method and application of culture medium

Abstract

The invention relates to the technical field of fungus culture, and discloses a culture medium for inducing Stemphylium solani to produce spores, a preparation method and application, the preparation raw materials of the culture medium comprise a tomato extract, a carrot extract and agar, the total mass of the tomato extract and the carrot extract is 50g / L to 200g / L, and the content of the tomato extract is not less than 50g / L. According to the culture medium for inducing the Stemphylium solani to produce the spores, the preparation method and the application, the tomato extract and the carrot extract in a specific ratio are combined, so that optimal nutrition and environmental conditions are provided for the Stemphylium solani to produce the spores. Experimental data show that the sporulation quantity of the culture medium is remarkably higher than that of a conventional culture medium (such as CA, V8, PDA and the like), the technical problem that an existing solid culture medium is unstable in sporulation or does not produce sporulation is solved, the selected core raw materials tomato and carrot are common fruits and vegetables, low in cost and easy to obtain, expensive or special chemical reagents are avoided, and the production cost is reduced. And the research and development and application cost is greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fungus culture, in particular to a culture medium for inducing sporulation of tomato botryospaeria dothidea, a preparation method and application thereof. BACKGROUND

[0002] Tomato botryospaeria dothidea is a widespread and serious destructive disease that seriously restricts the healthy development of the tomato industry. The key to studying the epidemic pattern and control technology of this disease lies in obtaining stable and sufficient pathogen inoculum (conidia) through artificial inoculation. Currently, the conventional method for obtaining conidia mainly has two major bottlenecks: one is to use PDA, V8, oat and other solid culture media for cultivation and induction, but these culture media generally have the problems of no sporulation, low sporulation yield and poor stability; the other is to use fresh tomato leaves and other host materials for induction, which can obtain spores, but the operation process is complicated, the cycle is long, and it is difficult to scale up, which cannot meet the needs of large-scale resistance identification and pathogenic biology research.

[0003] The defects of the prior art seriously hinder the in-depth study and effective control of tomato botryospaeria dothidea. Therefore, there is an urgent need in the art to develop a new culture medium that can stably, efficiently and conveniently induce tomato botryospaeria dothidea to produce a large amount of conidia, in order to solve the core technical problem of difficulty in obtaining artificial inoculum. SUMMARY

[0004] To solve the problems presented in the background, the present application provides the following technical solutions: a culture medium for inducing sporulation of tomato botryospaeria dothidea, a preparation method and application thereof. The preparation raw materials of the culture medium include tomato extract, carrot extract and agar, and the total mass of the tomato extract and the carrot extract is 50g / L to 200g / L, wherein the content of the tomato extract is not less than 50g / L.

[0005] Preferably, the mass ratio of the tomato extract to the carrot extract is 1:1 to 3:1.

[0006] Preferably, the mass ratio of the tomato extract to the carrot extract is 1:1.

[0007] The preparation method of the culture medium comprises the following steps:

[0008] S1, weighing and cutting tomato and carrot according to the proportion, boiling after adding water, and then filtering to obtain a mixed extract;

[0009] S2, adding agar to the mixed extract, stirring uniformly and constant volume;

[0010] S3, sterilizing the constant-volume culture medium solution, and obtaining the sporulation culture medium after cooling.

[0011] Preferably, the boiling time in step S1 is 20-40 minutes.

[0012] Preferably, the sterilization condition in step S3 is 121℃, 15-25 minutes.

[0013] A method for inducing tomato Sclerotinia sclerotiorum to produce conidia, comprising the following steps:

[0014] Inoculating the activated tomato Sclerotinia sclerotiorum on the culture medium, and culturing at 20-28℃ for 7-14 days in dark condition to induce the production of conidia.

[0015] Preferably, the culture temperature is 25℃, and the culture time is 10 days.

[0016] Preferably, the culture medium is used for preparing inoculum for artificial inoculation of tomato gray leaf spot.

[0017] Preferably, the culture medium is used for biological property research of tomato Sclerotinia sclerotiorum or disease resistance identification of tomato varieties.

[0018] Compared with the prior art, the present application provides a culture medium for inducing tomato Sclerotinia sclerotiorum to produce conidia, a preparation method and application, which has the following beneficial effects:

[0019] 1. The culture medium for inducing tomato Sclerotinia sclerotiorum to produce conidia, the preparation method and application, by combining tomato and carrot extracts in a specific ratio, provide optimal nutritional and environmental conditions for tomato Sclerotinia sclerotiorum conidial production. Experimental data show that the conidial production of this culture medium is significantly higher than that of conventional culture media (such as CA, V8, PDA, etc.), solving the technical problems of unstable or no conidial production of existing solid culture media.

[0020] 2. The culture medium for inducing tomato Sclerotinia sclerotiorum to produce conidia, the preparation method and application, use common fruits and vegetables as core raw materials, which are low in cost and easy to obtain, avoiding the use of expensive or special chemical reagents, greatly reducing the research and application cost.

[0021] 3. The culture medium for inducing tomato Sclerotinia sclerotiorum to produce conidia, the preparation method and application, the preparation method of the culture medium is simple, without complex equipment and special process. Compared with the fresh host induction method, the present application directly produces conidia on the culture medium, eliminating multiple cumbersome steps such as cultivating host plants, inoculation, and moisture retention, significantly shortening the period of obtaining a large number of spores and greatly improving the efficiency.

[0022] 4. The culture medium, preparation method, and application of this method for inducing sporulation of *Strombus stolonifer* in tomato: This culture medium can stably and rapidly produce a large number of conidial suspensions, which can fully meet the large demand for high-quality artificial inoculum in tomato variety disease resistance identification, pathogen pathogenicity research, disease epidemic pattern analysis, and fungicide efficacy testing. It has extremely high practical application and promotion value. Attached Figure Description

[0023] Figure 1 This is a schematic diagram showing the results of conidia production by the tomato creeping mold on different culture media according to the present invention;

[0024] Figure 2 A schematic diagram of sporulation on a tomato and carrot culture medium according to the present invention;

[0025] Figure 3 A schematic diagram illustrating sporulation on a rye culture medium for invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Please see Figures 1-3 The present invention provides a technical solution:

[0028] Example 1: Isolation and activation of *Pseudomonas stolonifera*

[0029] Diseased leaves were collected from tomato plants exhibiting typical symptoms of gray leaf spot in the field in Nanchang City, Jiangxi Province. The pathogen was isolated using tissue isolation. Tissue blocks from the boundary between diseased and healthy tissue were cut, surface-sterilized with 75% ethanol for 30 seconds, then with 1% sodium hypochlorite solution for 90 seconds, rinsed three times with sterile water, and placed in the center of a PDA plate for incubation in the dark at 25°C for 5-7 days. After colonies grew, mycelia from the colony edges were selected for purification. Genomic DNA was extracted from the strain, and PCR amplification and sequence alignment of the ITS and GAPDH genes identified the pathogen as *Stemphylium lycopersici*. The purified strain was inoculated onto PDA slant agar and stored at 13°C for later use. Before use, the strain was inoculated from the slant onto a fresh PDA plate for activation and incubated in the dark at 25°C for 5-7 days.

[0030] Example 2: Preparation of the culture medium (tomato-carrot culture medium) of the present invention

[0031] Weigh out 100g of fresh tomatoes and 100g of fresh carrots, and wash them with deionized water. Cut the tomatoes and carrots into pieces about 1cm long. 3 Place the small pieces of the tomato and carrot mixture into a beaker, add 1000 mL of deionized water, and heat on an electric stove until boiling, maintaining a gentle boil for 30 minutes. Filter through two layers of gauze, collect the filtrate, and discard the residue. Add 15 g of agar powder to the filtrate and stir with a glass rod to ensure thorough dispersion. Then, bring the filtrate to a final volume of 1000 mL with deionized water and stir well. Dispense the prepared culture medium solution into Erlenmeyer flasks and autoclave at 121°C for 20 minutes. After sterilization, allow the culture medium to cool to 50-60°C, and under aseptic conditions, pour it into sterilized 9 cm Petri dishes, approximately 15-20 mL per dish, and allow to stand and cool to solidify, thus obtaining the tomato and carrot culture medium of this invention.

[0032] Example 3: Preparation of control culture medium

[0033] To verify the superiority of the culture medium of the present invention, several commonly used culture media were also prepared as comparative examples:

[0034] Carrot culture medium (CA): Weigh 200g of carrots, boil and filter them as in Example 2, add 15g of agar powder, make up to volume, sterilize, and pour into plates.

[0035] V8 medium: Take 100mL of commercially available V8 vegetable juice, add 3g of CaCO3, stir well, let stand for 30 minutes, and filter through two layers of gauze. Take the filtrate, add 15g of agar powder, and make up to 1000mL. Sterilize and pour into plates.

[0036] Rye medium (RA): Weigh 50g of rye seeds, soak them in 1000mL of deionized water for 24 hours, sterilize at 121℃ for 30 minutes, filter, add 15g of agar powder to the filtrate, make up to 1000mL, sterilize, and pour into plates.

[0037] Oat medium (OA): Weigh 30g of oat flakes, add 1000mL of deionized water, incubate at 60℃ for 1 hour, filter, add 15g of agar powder to the filtrate, bring the volume to 1000mL, sterilize, and pour into plates.

[0038] Potato glucose agar (PDA) medium: Weigh 200g of peeled potatoes, boil and filter them as in Example 2, add 20g of glucose and 15g of agar powder to the filtrate, bring the volume to 1000mL, sterilize, and pour into plates.

[0039] Corn flour culture medium (CMA): Weigh 30g of corn flour, boil and filter it as in Example 2, add 15g of agar powder to the filtrate, make up to 1000mL, sterilize and pour into plates.

[0040] Tomato and carrot culture media with different ratios:

[0041] Ingredients A: 50g tomatoes, 150g carrots.

[0042] Formula B: 150g tomatoes, 50g carrots.

[0043] The preparation method is the same as in Example 2.

[0044] Example 4: Conidia induction and sporulation yield determination

[0045] Using a sterile punch with a diameter of 5 mm, mycelial cakes were collected from the edge of the activated *Stemona japonica* colony (Example 1). Using a sterile inoculation needle, the mycelial cakes were inoculated into the center of various culture medium plates prepared in Examples 2 and 3, with three replicates for each treatment. All inoculated plates were placed in a 25°C incubator and incubated in the dark for 10 days.

[0046] After cultivation, conidia were counted. 3 mL of sterile distilled water was added to each petri dish, and the mycelia and spores on the surface of the medium were gently brushed with a sterile brush to ensure the conidia were fully suspended in the water. The spore suspension was filtered through double-layered sterile gauze to remove mycelial debris. A small amount of filtrate was added to a hemocytometer, and the number of conidia was observed and counted under an optical microscope (10×40x). Each plate was counted three times, and the average value was taken as the conidia yield of that plate. The average value of the three replicate plates on the same medium was then calculated, and the results were expressed as "conidia / plate". Duncan's multiple range test was used for significance analysis (p<0.05).

[0047] Example 5: Results and Analysis

[0048] The sporulation results of *Stemona japonica* on different culture media are as follows: Figure 1 As shown.

[0049] Depend on Figure 1 It is evident that the tomato-carrot medium (100g tomato + 100g carrot) of this invention exhibits the best sporulation induction effect, with a significantly higher sporulation yield than all other control media (p<0.05). Among different ratios of tomato-carrot media, the sporulation yield was as follows: 100g / L tomato > 50g / L tomato > 150g / L tomato, indicating that a 1:1 mass ratio of tomato and carrot provides the best synergistic induction effect for sporulation of *Stenophyllus tomatous*. In contrast, traditional PDA and CMA media showed virtually no sporulation under these experimental conditions, and the sporulation yields of other media were significantly lower than those of the medium of this invention. This demonstrates that the medium provided by this invention has unexpectedly significant advantages in inducing sporulation of *Stenophyllus tomatous*, effectively solving the problems of unstable sporulation and low sporulation yield in existing technologies.

[0050] Example 6: Application of the culture medium of the present invention in disease resistance identification

[0051] The culture medium prepared in Example 2 was used for disease resistance identification of tomato varieties. The method of Example 4 was used to induce *Stenophyllus tomatous* to produce a large number of conidia, with a concentration of 1×10⁻⁶. 5 A spore suspension at spore density (mL) was prepared. Four-leaf stage seedlings of five different tomato varieties (AE) were selected and sprayed evenly with the spore suspension until both sides of the leaves were moistened. Plants sprayed with sterile water served as a control. After inoculation, the plants were placed in a humidifier at 25℃ and >95% relative humidity for 24 hours in the dark, and then transferred to a greenhouse under normal temperature and light conditions. Seven days after inoculation, the disease incidence of each variety was investigated, and the disease index was calculated. The results showed that varieties A and B were highly susceptible, with disease indices of 85.6 and 78.9, respectively; variety C was moderately resistant, with a disease index of 35.2; and varieties D and E were resistant, with disease indices of 15.8 and 10.4, respectively. This application example demonstrates that the culture medium of this invention can rapidly and stably obtain high-quality artificial inoculum, which has been successfully used for the identification of resistance to gray leaf spot in tomato varieties, and has important application value.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A culture medium for inducing sporulation of *Pseudomonas stolonifera*, its preparation method, and its application, characterized in that, The raw materials for preparing the culture medium include tomato extract, carrot extract and agar, wherein the total mass of the tomato extract and carrot extract is 50 g / L to 200 g / L, and the content of tomato extract is not less than 50 g / L.

2. The culture medium according to claim 1, characterized in that, The mass ratio of the tomato extract to the carrot extract is 1:1 to 3:

1.

3. The culture medium according to claim 1, characterized in that, The mass ratio of the tomato extract to the carrot extract is 1:

1.

4. The method for preparing the culture medium according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Weigh and chop tomatoes and carrots according to the proportion, add water, boil, and then filter to obtain a mixed extract. S2. Add agar to the mixed extract, stir well and make up to volume; S3. Sterilize the culture medium solution after it has been brought to a fixed volume, and then cool it to obtain the sporulation culture medium.

5. The culture medium, preparation method, and application for inducing sporulation of *Stemona japonica* as described in claim 4, characterized in that... In step S1, the boiling time is 20-40 minutes.

6. The culture medium, preparation method, and application for inducing sporulation of *Pseudomonas stolonifera* according to claim 4, characterized in that, In step S3, the sterilization conditions are 121°C for 15-25 minutes.

7. A method for inducing *Stemona japonica* to produce conidia using the culture medium according to any one of claims 1-3, characterized in that, Includes the following steps: The activated *Pseudomonas tomatoensis* was inoculated onto the culture medium and cultured in the dark at a constant temperature of 20-28°C for 7-14 days to induce the production of conidia.

8. The method according to claim 7, characterized in that, The culture temperature was 25℃, and the culture time was 10 days.

9. The use of the culture medium according to any one of claims 1-3 in the preparation of inoculum for artificial inoculation of tomato gray leaf spot.

10. The application of the culture medium according to any one of claims 1-3 in the study of the biological characteristics of *Stenophyllus scutellarioides* or in the identification of disease resistance in tomato varieties.

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