Liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites and its application

Abstract

The present invention discloses a liquid culture medium for inducing the production of secondary metabolites by twig-pathogenic fungi and its application. The liquid culture medium comprises the following components at mass concentrations: 25-300 g / L poplar bark powder, 2-6 g / L yeast extract, 5-10 g / L sucrose, and 1000-2000 g distilled water. Experimental results demonstrate that the liquid culture medium can induce the twig-pathogenic fungus, specifically Aspergillus chrysanthemi, to produce large quantities of toxic secondary metabolites, achieving efficient in vitro induction and stable cultivation of secondary metabolites. This is because the poplar bark powder in the liquid culture medium simulates the damaged host plant environment, providing a suitable simulated infection environment for Aspergillus chrysanthemi to secrete and produce secondary metabolites. The yeast extract serves as a source of amino acids, and the sucrose serves as a sugar source, providing basic nutrition for Aspergillus chrysanthemi and preventing excessive nutrients from interfering with the production of secondary metabolites. Furthermore, the liquid culture medium provided by the present invention features readily available raw materials, low cost, and simple preparation, thus possessing broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites and its application. Background Art

[0002] Branch and trunk pathogenic fungi, such as Cytospora chrysosperma (Pers.) Fr., are pathogenic fungi that cause ulcers or rot in plant branches and trunks. They belong to the phylum Ascomycota, orders Metaspermales, family Cytospora, and genus Cytospora. This genus of pathogenic fungi can infect a variety of woody economic tree species in my country. Among them, the poplar rot disease caused by Cytospora chrysosperma is a serious threat, seriously affecting the economic value of poplars and the stability of the ecological environment. At present, research reports on poplar rot diseases and pathogenic fungi mostly focus on the mechanism of rot disease, prevention and control methods, genetic diversity, etc. Therefore, the exploration of the molecular pathogenic mechanism of poplar rot pathogens is imminent.

[0003] Currently, no typical infection structure has been observed for Aspergillus chrysogenum. The germinating hyphae of the pathogen primarily invade through wounds on the host surface. Previous studies have shown that Aspergillus fungi produce toxic secondary metabolites during infection, thereby accelerating the pathogen's infection process. Therefore, understanding the synthesis mechanism and physicochemical properties of Aspergillus chrysogenum's secondary metabolites will accelerate the elucidation of its molecular pathogenic mechanism.

[0004] Currently, there are few reports on methods for culturing secondary metabolites of fungal pathogens of stem canker. Existing fungal culture techniques and liquid culture media are mainly targeted at agricultural pathogens such as Fusarium graminearum, Pyrenophora tritici-repentis, and Alternaria alternata, or at plant endophytes and marine endophytes with medicinal value, as well as engineered fungi. Among the existing forest pathogens, Huang Lili used apple bark broth (ABB) to culture the apple rot pathogen Valsa mali and extracted the crude toxin of the pathogen. However, since the main raw material of the apple bark broth (ABB) is one-year-old apple branches, there are limitations such as difficulty in obtaining the material and easy contamination. Therefore, the production of liquid culture medium that is not easily contaminated, can be cultured in large quantities and can induce the production of toxic secondary metabolites of Chrysospora chrysospora without being restricted by the seasonality of materials has become an urgent problem to be solved in the study of toxic secondary metabolites of Chrysospora chrysospora and even the pathogenic fungi of branch and trunk canker diseases.

[0005] The latent nature of the pathogen prevents it from becoming pathogenic in healthy poplar tissues. However, when the poplars weaken, the pathogen begins to infect and cause disease. Therefore, we believe that damaged host tissues facilitate the production of toxic secondary metabolites produced by the pathogen.

[0006] At present, the commonly used liquid culture media for fungal secondary metabolism are synthetic culture media without host tissue, such as the modified Czapek Dox Broth (CDB) commonly used for Fusarium spp. and the Fries3 liquid medium for culturing wheat yellow leaf blight pathogen. It is rare to find induction liquid culture media with added host tissue.

[0007] For weak parasites that rely on weakened trees for disease, damaged host tissue is a key factor in inducing and regulating the expression of genes that produce toxic secondary metabolites. Previous studies have attempted to prepare a poplar branch decoction medium based on the recipe for apple branch decoction medium (ABB). Although this achieved good induction results, the one-year-old poplar twigs required in the recipe are difficult to obtain, and the contamination rate during the culture process is relatively high. Furthermore, since subsequent structural analysis of toxic secondary metabolites requires a certain amount of crude secondary metabolite extract, there are certain requirements for the amount of basal culture fluid. Therefore, developing a liquid culture medium containing host tissue, capable of large-scale cultivation, and capable of stably inducing the production of secondary metabolites by branch pathogenic fungi is essential for exploring the molecular mechanisms of pathogenicity of these pathogens. Summary of the Invention

[0008] In response to the problems existing in the prior art, the purpose of the present invention is to provide a liquid culture medium and its application for inducing branch and trunk pathogenic fungi to produce secondary metabolites. By adding poplar bark powder, efficient in vitro induction and cultivation of secondary metabolites of branch and trunk pathogenic fungi are achieved.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites, which includes the following components in the following mass concentrations, based on a total volume of 1 L of the liquid culture medium: 25-300 g / L of poplar bark powder, 2-6 g / L of yeast extract, 5-10 g / L of sucrose, and 1000-2000 g of distilled water.

[0011] Preferably, based on the total volume of the liquid culture medium being 1 L, the following mass concentrations of components are included: poplar bark powder 25-150 g / L, yeast extract 2-4 g / L, sucrose 5-8 g / L, and distilled water 1000-1500 g.

[0012] The second aspect of the present invention provides a method for preparing the liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites, comprising the following steps:

[0013] A1. Add poplar bark powder to distilled water according to the recipe, boil for 15-20 minutes, and filter. The resulting filtrate is poplar bark juice.

[0014] A2. Add yeast extract and sucrose to poplar bark juice, mix well, then add distilled water to the volume and sterilize with high-pressure steam.

[0015] The third aspect of the present invention provides a method for inducing branch and trunk pathogenic fungi to produce secondary metabolites using the above-mentioned liquid culture medium, comprising the following steps:

[0016] S1. Inoculate the branch and trunk pathogenic fungi into the center of the PDA agar plate medium, and then place it in a 25°C incubator for bacterial activation culture;

[0017] S2. Use a hole puncher to punch out fresh growth on the edge of the activated cultured mycelium plate of the branch and trunk pathogenic fungus, inoculate it into the center of the PDA agar plate, and then place it in a constant temperature incubator at 25°C for 2-3 days;

[0018] S3. Using a borer, punch out fresh mycelial growth from an area on the twig pathogenic fungus plate after culturing for 2-3 days, inoculate the resulting mycelial mass into a liquid culture medium, and then culture in a constant temperature incubator at 25° C., 100-150 rpm, for 10-20 days to obtain a culture product comprising twig pathogenic mycelium and a bacterial solution;

[0019] S4. Use gauze to perform a first filtration on the culture product containing the branch and trunk pathogenic fungus mycelium and bacterial liquid to filter out the branch and trunk pathogenic fungus mycelium, and then use a microporous filter membrane to perform a second filtration to filter out spores, extract, and obtain the branch and trunk pathogenic fungus secondary metabolites.

[0020] Preferably, in step S1, the activation culture time of the bacterial strain is 4 to 5 days.

[0021] Beneficial effects:

[0022] 1. The present invention provides a liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites, wherein the liquid culture medium contains host tissue. Experimental results show that the liquid culture medium can culture in large quantities and stably induce branch and trunk pathogenic fungi, specifically Chrysospora chrysanthemi, to produce secondary metabolites. The poplar bark powder used in the raw materials can simulate the damaged host plant environment and provide a suitable simulated infection environment for inducing Chrysospora chrysanthemi to secrete toxic metabolites; yeast extract is used as an amino acid source, and sucrose is used as glycogen to provide basic nutrition for Chrysospora chrysanthemi, and avoid excessive nutrients interfering with the production of toxic secondary metabolites by Chrysospora chrysanthemi. In addition, culturing in the liquid culture medium of the present invention is more conducive to the subsequent preliminary extraction of secondary metabolites of branch and trunk pathogenic fungi.

[0023] 2. The liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites provided by the present invention has a wide range of application prospects because the various raw material components are easily available, are low in cost, and are easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Schematic diagram of the expression levels of the backbone genes of the secondary metabolism synthesis-related gene cluster in the liquid culture medium prepared with different bark powder concentrations in Examples 1-7;

[0026] Figure 2 Schematic diagram of the growth of Aspergillus niger in three culture media;

[0027] Figure 3 Schematic diagram of host leaf toxicity determination of three culture mediums of Aspergillus chrysogenum: (A) Inoculation diagram; (B) Schematic diagram of host leaf toxicity determination of three culture mediums;

[0028] Figure 4 Schematic diagram of the host leaf toxicity determination of the crude toxin of the secondary metabolism of the golden cyst: (A) inoculation diagram; (B) schematic diagram of the host leaf toxicity determination results of the crude toxin of the secondary metabolism. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Example 1

[0031] A liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites, comprising the following components in the following mass concentrations based on a total volume of 1 L of the liquid culture medium: 25 g / L of poplar bark powder, 2 g / L of yeast extract, 5 g / L of sucrose, and 1000 g of distilled water.

[0032] The preparation steps are as follows: weigh poplar bark powder according to the formula, add it to 1000g of distilled water, boil it for 20 minutes, filter it with 8 layers of gauze, and the filtrate is the poplar bark juice; add yeast extract and sucrose to the poplar bark juice, stir it evenly, put it into a 250ml triangular pyramidal flask, and sterilize it with high-pressure steam at 121°C for 20 minutes.

[0033] Example 2

[0034] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 50 g / L poplar bark powder, 2 g / L yeast extract, 5 g / L sucrose, and 1000 g distilled water. The preparation method is the same as in Example 1.

[0035] Example 3

[0036] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: poplar bark powder 75 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. The preparation method is the same as in Example 1.

[0037] Example 4

[0038] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 150 g / L poplar bark powder, 2 g / L yeast extract, 5 g / L sucrose, and 1000 g distilled water. The preparation method is the same as in Example 1.

[0039] Example 5

[0040] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 225 g / L poplar bark powder, 2 g / L yeast extract, 5 g / L sucrose, and 1000 g distilled water. The preparation method is the same as in Example 1.

[0041] Example 6

[0042] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 250 g / L poplar bark powder, 2 g / L yeast extract, 5 g / L sucrose, and 1000 g distilled water. The preparation method is the same as in Example 1.

[0043] Example 7

[0044] A liquid culture medium for inducing the production of secondary metabolites by branch-pathogenic fungi, comprising the following components at the following mass concentrations, based on a total volume of 1 L of the liquid culture medium: 300 g / L poplar bark powder, 2 g / L yeast extract, 5 g / L sucrose, and 1000 g of distilled water. The preparation method is the same as in Example 1.

[0045] Example 8

[0046] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 50 g / L poplar bark powder, 4 g / L yeast extract, 5 g / L sucrose, and 1500 g distilled water. The preparation method is the same as in Example 1.

[0047] Example 9

[0048] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 50 g / L poplar bark powder, 2 g / L yeast extract, 8 g / L sucrose, and 2000 g distilled water. The preparation method is the same as in Example 1.

[0049] Example 10

[0050] A liquid culture medium for inducing the production of secondary metabolites by branch-stem pathogenic fungi comprises the following components at the following mass concentrations, based on a total volume of 1 L: 50 g / L poplar bark powder, 6 g / L yeast extract, 5 g / L sucrose, and 1500 g distilled water. The preparation method is the same as in Example 1.

[0051] Application Example 1

[0052] Here, a method for inducing Cytospora chrysosperma to produce secondary metabolites using the liquid culture medium prepared in Examples 1-7 above is described using Cytospora chrysosperma cryopreserved at 4°C as an example. The Cytospora chrysosperma is classified as Cytospora chrysosperma, has a deposit number of CFCC 89981, and is deposited in the China Forestry Microorganism Collection Center. The method comprises the following steps:

[0053] (1) The cystospores of Chrysotrichum chrysanthemi stored at 4°C were inoculated into the center of a PDA agar plate, and then placed in a 25°C incubator for 4-5 days for bacterial activation culture.

[0054] (2) Use a 0.6 cm diameter puncher to punch out fresh growth on the edge of the plate of Mycelial spores of Chrysospora aurea after 4-5 days of activation culture, inoculate it into the center of the PDA agar plate again, and then place it in a constant temperature incubator at 25°C for 2-3 days;

[0055] (3) Using a 0.6 cm diameter puncher, three freshly grown mushroom blocks were punched out from the mycelial plate of the golden shell spore after culturing for 2 to 3 days, and the blocks were inoculated into 250 ml conical flasks respectively containing the poplar bark powder liquid culture medium prepared in Examples 1-7 and a blank liquid culture medium (except that the poplar bark powder was not added, the other components were the same as in Example 2). Then, the plates were placed in a constant temperature incubator at 25°C and 150 rpm for shaking culture for 10 days. At the same time, 200 ml of a control liquid culture medium of 50 g / L poplar bark powder (used to prove that the liquid culture medium itself is non-toxic) was prepared, and the control liquid culture medium was not inoculated with golden shell spores, and the plates were cultured together. After the culture was completed, a culture product containing golden shell spore mycelium and bacterial liquid was obtained. The culture product was filtered using 8 layers of gauze to remove the mycelium, and further filtered using a microporous filter membrane to remove the spores. The toxic secondary metabolites of golden shell spores were obtained by extraction and rotary evaporation.

[0056] Mycelial RNA was extracted and fluorescence quantitative PCR was used to detect the skeleton gene CcPpns1 of the gene cluster related to the secondary metabolism synthesis of golden cysts in each culture medium. acyltransferase The expression level of (under different bark powder concentration induction). Figure 1 .

[0057] Depend on Figure 1The results show that the expression level of the backbone genes of the secondary metabolism synthesis-related gene cluster was the highest when the liquid culture medium prepared in Example 2 (bark powder concentration of 50g / L) was used for cultivation. Therefore, it is believed that the optimal concentration for selecting the bark powder concentration in the culture medium is 50g / L. Therefore, in the following experiments, the liquid culture medium prepared in Example 2 is collectively referred to as 50g / L poplar bark powder culture medium. However, when the control liquid culture medium without poplar bark powder was used for cultivation under the same conditions, the expression level of the backbone genes of the secondary metabolism synthesis-related gene cluster was very low.

[0058] Comparative Application Example 1

[0059] 26g potato dextrose broth powder was added to 1000ml of distilled water and boiled until clarified. The mixture was poured into a 250ml conical flask and sterilized at 115°C for 20min. The mixture was cooled to make potato dextrose broth (PM0510), which was then inoculated with golden shell spores. The inoculation method was the same as in Application Example 1, and the culture was shaken at 150rpm at 25°C for 10 days. 200ml of potato dextrose broth was simultaneously made, but the culture was not inoculated with golden shell spores. The culture was co-cultivated to serve as a control medium (for demonstrating that the culture medium itself is nontoxic). After the culture was completed, the culture product of the culture medium was filtered using 8 layers of gauze to remove bacterial cells and further filtered using a microporous filter membrane to remove spores.

[0060] Comparative Application Example 2

[0061] 34.51g of modified Czapek medium powder was added to 1000ml of distilled water, boiled until clarified, poured into a 250ml conical flask, sterilized at 115°C with high pressure steam for 20min, cooled, and made into modified Czapek medium (MM1020). The spores were then inoculated with Chrysospora chrysanthemi. The inoculation method was the same as in Application Example 1, and the shaking culture was carried out at 25°C and 150rpm for 10 days. 200ml of modified Czapek medium was simultaneously made, which was not accessed with Chrysospora chrysanthemi, and co-cultivated as a culture medium control (for demonstrating that the culture medium itself is nontoxic). After the culture was completed, the culture product of the culture medium was filtered using 8 layers of gauze to remove bacteria, and further filtered using a microporous filter membrane to remove spores.

[0062] (a) Observe the growth and development of golden cyst spores in three different culture media as shown in Table 1 and Figure 2 shown.

[0063] Table 1 Effects of three culture media on the growth and development of mycelial balls

[0064]

[0065] From Table 1 and Figure 2The results showed that the growth of Cystella chrysogenum in the modified Czapek medium was the weakest among the three media, and the mycelial dry weight was the lightest. Cystella chrysogenum could grow well in potato dextrose broth and 50g / L poplar bark powder medium.

[0066] (b) Collect 1 L of culture medium of golden shell spores in 50 g / L poplar bark powder medium, potato glucose broth medium, and modified Czapek medium, and 200 ml of blank culture medium of 50 g / L poplar bark powder medium, blank potato glucose broth medium, and blank modified Czapek medium, respectively, and then use a rotary concentrator to concentrate them by rotary evaporation. After concentration, the toxicity test of poplar leaves was carried out, specifically: inoculation was performed on the poplar leaves with an inoculation needle, 5 μl of concentrated solution was added to the inoculation site, the treated poplar leaves were placed in a culture dish for moisturizing culture, the size of necrotic spots caused by the three culture concentrates on the poplar leaves was observed and recorded, and the induced toxin production effect of each culture medium was evaluated. The results are shown in Figure 3 .

[0067] Depend on Figure 3 The results showed that a concentrated solution of 50 g / L poplar bark powder culture medium of Aspergillus chrysogenum caused necrotic spots, while concentrated solutions of modified Czapek's medium and potato dextrose broth culture medium of Aspergillus chrysogenum did not cause obvious necrotic spots. Therefore, it is believed that the composition of the bark powder culture medium is superior to that of Czapek's medium and potato dextrose broth.

[0068] (c) Collect 5 L of culture solution of Chrysoporus chrysoporus in 50 g / L poplar bark powder culture medium and 200 ml of culture solution of corresponding blank 50 g / L poplar bark powder culture medium, extract the above culture solutions respectively with ethyl acetate, finally combine the organic phases, and concentrate the organic phases with a rotary concentrator to obtain the secondary metabolic crude toxin of Chrysoporus chrysoporus and the 50 g / L poplar bark powder culture medium extract, and dissolve the crude toxin in a mixture of dichloromethane and methanol. The biological activity of the secondary metabolic crude toxin of Chrysoporus chrysoporus was determined by leaf puncture method, specifically: inoculate the poplar leaves with an inoculation needle, add 5 μl of crude toxin and 50 g / L poplar bark powder culture medium extract to the inoculation site, place the treated poplar leaves in a culture dish for moisturizing culture, and observe and record the necrosis phenomenon caused by the secondary metabolic crude toxin of Chrysoporus chrysoporus on the poplar leaves. See the results. Figure 4 .

[0069] Depend on Figure 4 The results showed that 24 hours after inoculation, obvious necrotic spots appeared at the site of crude toxin inoculation, while the leaf structure was intact at the site of 50g / L poplar bark powder culture medium extract inoculation, and no necrotic spots appeared.

[0070] The experimental results above demonstrate that the liquid culture medium and corresponding culture method provided by the present invention can induce the production of large quantities of toxic metabolites in P. chrysotrichum, while the liquid culture medium itself has no toxic effects on poplar trees. The liquid culture medium provided by the present invention can also be applied to the induction culture of secondary metabolites of other branch and trunk pathogenic fungi (ulcer diseases), which will not be discussed here.

[0071] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A method for inducing golden shell cyst spores ( Cytospora chrysosperma ) a toxin-producing liquid culture medium, characterized in that The total volume of the culture medium is 1 L, and the following components are included in the culture medium at mass concentrations: poplar bark juice obtained by 25-300 g / L of poplar bark powder, 2-6 g / L of yeast extract, 5-10 g / L of sucrose, and 1000-2000 g of distilled water; The preparation method of the liquid culture medium comprises the following steps: A1. Add poplar bark powder to distilled water according to the recipe, boil, and filter. The resulting filtrate is poplar bark juice; A2. Add yeast extract and sucrose to poplar bark juice, mix well, then add distilled water to the volume and sterilize with high-pressure steam.

2. The method for inducing golden shell cyst spore according to claim 1 Cytospora chrysosperma ) a toxin-producing liquid culture medium, characterized in that The total volume of the culture medium is 1 L, and the following components are included in the culture medium at mass concentrations: poplar bark juice obtained by 25-150 g / L of poplar bark powder, 2-4 g / L of yeast extract, 5-8 g / L of sucrose, and 1000-1500 g of distilled water.

3. The induction of golden shell cyst spores by the liquid culture medium as described in claim 1 or 2 Cytospora chrysosperma ) A method for producing toxins, comprising the following steps: S1. Inoculate the cystospores of Chrysotrichum chrysanthemi into the center of the PDA agar plate medium, and then place it in a 25°C incubator for bacterial activation culture; S2. Use a punch to punch out fresh growth on the edge of the activated cultured Cystosporium chrysogenum mycelium plate, inoculate it into the center of the PDA agar plate, and then place it in a constant temperature incubator at 25°C for 2-3 days; S3. Using a cork puncher, punch out freshly grown bacterial masses from the plate of cystospora chrysanthemi after culturing for 2 to 3 days, inoculating the obtained bacterial masses into a liquid culture medium, and then culturing in a constant temperature incubator at 25° C. and 100-150 rpm for 10 to 20 days to obtain a culture product comprising cystospora chrysanthemi mycelia and a bacterial solution; S4. Using gauze to filter the culture product containing the mycelium of Chrysospora chrysanthemi and the bacterial liquid for the first time to remove the mycelium of Chrysospora chrysanthemi, and then using a microporous filter membrane to filter out the spores for the second time, extracting and obtaining the crude toxin.

4. The inducing golden shell cyst spore according to claim 3 ( Cytospora chrysosperma ) A method for producing toxins, characterized in that In step S1, the activation culture time of the bacterial strain is 4 to 5 days.

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