Liquid culture medium for inducing branch pathogenic fungi to generate secondary metabolites and application of liquid culture medium

Abstract

The invention discloses a liquid culture medium for inducing branch pathogenic fungi to generate secondary metabolites and application. The liquid culture medium comprises the following components in mass concentration: 25-300g / L of poplar bark powder, 2-6g / L of yeast extract, 5-10g / L of sucrose and 1000-2000g of distilled water. Experimental results show that the liquid culture medium can induce branch and trunk pathogenic fungi, particularly aureocystis aureocystis, to produce toxic secondary metabolites in large quantities, and efficient in-vitro induction and stable culture of the secondary metabolites are realized. In the liquid culture medium, the poplar bark powder can simulate a damaged host plant environment, so that a suitable simulated infection environment is provided for inducing the aureocystis aureocystis to secrete and generate secondary metabolites; the yeast extract can be used as an amino acid source, cane sugar is used as a sugar source, basic nutrition is provided for the aureocystis aureocystis, and the situation that excessive nutrient substances interfere with the aureocystis aureocystis to generate secondary metabolites is avoided. In addition, the liquid culture medium provided by the invention has the advantages of easily available raw materials, low cost and simplicity and convenience in preparation, thereby having a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically, relates to a liquid medium for inducing secondary metabolites of pathogenic fungi of branches and trunks and its application. Background Art

[0002] Pathogenic fungi of branches and trunks, such as Cytospora chrysosperma (Pers.) Fr., are pathogenic fungi that cause plant branch ulcers or rots, belonging to Ascomycota, Diaporthales, Cytosporaceae, and Cytospora. Pathogenic fungi of this genus can infect various woody economic tree species in China. Among them, poplar canker caused by Cytospora chrysosperma is seriously harmful, seriously affecting the economic value of poplar and the stability of the ecological environment. At present, research reports on poplar canker and pathogenic fungi mainly focus on aspects such as the occurrence mechanism of canker, control methods, and genetic diversity. Therefore, it is urgent to explore the molecular pathogenic mechanism of poplar canker pathogens.

[0003] Currently, no typical infection structures have been observed in Cytospora chrysosperma. The hyphae germinated from pathogenic fungi mainly invade through wounds on the host surface. Existing research has shown that fungi of the genus Cytospora will accelerate the infection process of pathogenic fungi by producing toxic secondary metabolites during the infection process. Therefore, clarifying the synthesis mechanism and physicochemical properties of the secondary metabolites of Cytospora chrysosperma will accelerate the analysis of its molecular pathogenic mechanism.

[0004] Currently, there are few reports on the culture methods of secondary metabolites of pathogenic fungi of branch canker diseases. Existing research on fungal culture techniques and liquid media mainly focuses on pathogenic fungi in agriculture such as Fusarium graminearum, Pyrenophora tritici-repentis, and Alternaria alternata, or on plant endophytic fungi, marine endophytic fungi, and engineering fungi with medicinal value. Among existing forest tree pathogenic fungi, Huang Lili used the apple bark broth medium ABB (Apple bark broth) to culture Valsa mali and extract the crude toxin of this pathogen. However, due to the main raw material of the apple bark broth medium ABB being one-year-old apple branches, there are limitations in material acquisition and easy contamination. Therefore, making a liquid medium that is not easily contaminated, can be cultured in large quantities, and can induce Cytospora chrysosperma to produce toxic secondary metabolites without being restricted by material seasonality has become an urgent problem to be solved in the study of the toxic secondary metabolites of Cytospora chrysosperma and even pathogenic fungi of branch canker diseases.

[0005] The latent infection characteristic of Cytospora chrysosperma enables this pathogen not to show pathogenicity in healthy poplar tissues. However, when the poplar tree vigor weakens, Cytospora chrysosperma begins to infect and cause diseases. Therefore, we believe that under the induction of damaged host tissues, the toxic secondary metabolites of Cytospora chrysosperma are more likely to be produced.

[0006] Currently, the commonly used liquid media for fungal secondary metabolism are synthetic media without host tissues. For example, the commonly used modified Czapek Dox Broth (CDB) in Fusarium, and the Fries3 liquid medium for culturing Mycosphaerella graminicola on wheat. There are few induced liquid media with added host tissues.

[0007] For facultative parasites that develop diseases under the condition of weakened tree vigor, damaged host tissues are the key factors inducing and regulating the gene expression of toxic secondary metabolites. Existing studies have attempted to prepare a poplar branch decoction medium according to the formula of the apple branch decoction medium ABB. Although good induction effects have also been achieved, the required one-year-old poplar twig materials in the formula are not easily obtained, and the contamination rate is relatively high during the culture process. In addition, since a certain amount of crude secondary metabolites is required for the subsequent analysis of the structure of toxic secondary metabolites, there are certain requirements for the amount of the basic culture solution. Therefore, it is very necessary to develop a liquid medium with host tissues that can be cultured in large quantities and can stably induce the production of secondary metabolites by trunk and branch pathogenic fungi for exploring the pathogenic molecular mechanism of such pathogens. Summary of the Invention

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

[0009] To achieve the above purpose, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a liquid medium for inducing the production of secondary metabolites by trunk and branch pathogenic fungi. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: 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, calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: 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.

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks, comprising the following steps:

[0013] A1. Add poplar bark powder to distilled water according to the formula, boil for 15 - 20 min, filter, and the obtained filtrate is the poplar bark powder juice;

[0014] A2. Mix yeast extract and sucrose into the poplar bark powder juice, then add distilled water and make up the volume, and sterilize by high-pressure steam.

[0015] The third aspect of the present invention provides a method for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks using the above-mentioned liquid medium, comprising the following steps:

[0016] S1. Inoculate the pathogenic fungi of branches and trunks into the center of a PDA agar plate medium, and then place it in an incubator at 25°C for strain activation culture;

[0017] S2. Use a punch to cut out fresh-growing edge fungal blocks on the mycelial plate of the pathogenic fungi of branches and trunks after activation culture, inoculate them into the center of a PDA agar plate, and then place it in a constant-temperature incubator at 25°C for 2 - 3 days of culture;

[0018] S3. Use a punch to cut out fungal blocks from the area with fresh mycelial growth on the plate of the pathogenic fungi of branches and trunks after 2 - 3 days of culture, inoculate the obtained fungal blocks into the liquid medium, and then place it in an incubator at 25°C, 100 - 150 rpm for 10 - 20 days of culture to obtain a culture product containing the mycelia and broth of the pathogenic fungi of branches and trunks;

[0019] S4. First filter the culture product containing the mycelia and broth of the pathogenic fungi of branches and trunks with gauze to filter out the mycelia of the pathogenic fungi of branches and trunks, then use a microporous filter membrane for a second filtration to filter out spores, and extract to obtain the secondary metabolites of the pathogenic fungi of branches and trunks.

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

[0021] Beneficial effects:

[0022] 1. The present invention provides a liquid medium for inducing secondary metabolites in pathogenic fungi of branches and trunks, which contains host tissues. Experimental results show that this liquid medium can be used to culture a large amount of pathogenic fungi of branches and trunks, specifically Cytospora chrysosperma, and stably induce the production of secondary metabolites. Among them, the poplar bark powder used in the raw materials can simulate the environment of damaged host plants, providing a suitable simulated infection environment for inducing Cytospora chrysosperma to secrete toxic metabolites; yeast extract serves as a source of amino acids, and sucrose is used as glycogen, providing basic nutrients for Cytospora chrysosperma and avoiding excessive nutrients from interfering with the production of toxic secondary metabolites by Cytospora chrysosperma. In addition, culturing in the liquid medium of the present invention is more conducive to the preliminary extraction of secondary metabolites of pathogenic fungi of branches and trunks in the subsequent process.

[0023] 2. In the formula of the liquid medium provided by the present invention for inducing secondary metabolites in pathogenic fungi of branches and trunks, the sources of various raw material components are easy to obtain, the cost is low, and the preparation is simple. Therefore, it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

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

[0026] Figure 2 It is a schematic diagram of the growth of Cytospora chrysosperma in three media;

[0027] Figure 3 It is a schematic diagram of the toxicity determination of the host leaves of the culture solutions of three media of Cytospora chrysosperma: (A) inoculation schematic diagram; (B) schematic diagram of the toxicity determination of the host leaves of the culture solutions of three media;

[0028] Figure 4 It is a schematic diagram of the toxicity determination of the host leaves of the secondary metabolite crude toxin of Cytospora chrysosperma: (A) inoculation schematic diagram; (B) schematic diagram of the toxicity determination results of the host leaves of the secondary metabolite crude toxin. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Based on a total volume of 1 L of the liquid medium, it includes the following components in mass concentration: poplar bark powder 25 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g.

[0032] Its preparation steps: Weigh the poplar bark powder according to the formula, add it to 1000 g of distilled water, boil for 20 min, filter with 8 layers of gauze, and the filtrate is the poplar bark powder juice; add yeast extract and sucrose to the poplar bark powder juice, stir evenly, fill it into a 250 ml Erlenmeyer flask, and sterilize it at 121 °C under high-pressure steam for 20 min.

[0033] Example 2

[0034] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Based on a total volume of 1 L of the liquid medium, it includes the following components in mass concentration: poplar bark powder 50 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0035] Example 3

[0036] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Based on a total volume of 1 L of the liquid medium, it includes the following components in mass concentration: poplar bark powder 75 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0037] Example 4

[0038] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Based on a total volume of 1 L of the liquid medium, it includes the following components in mass concentration: poplar bark powder 150 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0039] Example 5

[0040] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 225 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0041] Example 6

[0042] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 250 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0043] Example 7

[0044] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 300 g / L, yeast extract 2 g / L, sucrose 5 g / L, and distilled water 1000 g. Its preparation method is the same as that of Example 1.

[0045] Example 8

[0046] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 50 g / L, yeast extract 4 g / L, sucrose 5 g / L, and distilled water 1500 g. Its preparation method is the same as that of Example 1.

[0047] Example 9

[0048] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 50 g / L, yeast extract 2 g / L, sucrose 8 g / L, and distilled water 2000 g. Its preparation method is the same as that of Example 1.

[0049] Example 10

[0050] A liquid medium for inducing the production of secondary metabolites by pathogenic fungi of branches and trunks. Calculated based on the total volume of the liquid medium being 1 L, it includes the following components with the following mass concentrations: poplar bark powder 50 g / L, yeast extract 6 g / L, sucrose 5 g / L, and distilled water 1500 g. Its preparation method is the same as that of Example 1.

[0051] Application Example 1

[0052] Taking Cytospora chrysosperma preserved at 4°C as an example, the taxonomic name of this Cytospora chrysosperma is Cytospora chrysosperma, and the preservation number is CFCC 89981. It is preserved in the China Forestry Microorganism Preservation Center. The method for inducing Cytospora chrysosperma to produce secondary metabolites using the liquid medium prepared in the above Examples 1-7 is described as follows:

[0053] (1) Inoculate Cytospora chrysosperma preserved at 4°C into the center of a PDA agar plate medium, and then place it in an incubator at 25°C for strain activation culture for 4-5 days;

[0054] (2) Use a punch with a diameter of 0.6 cm to punch out fresh-growing edge fungal blocks on the Cytospora chrysosperma mycelium plate after 4-5 days of activation culture, and inoculate them again into the center of the PDA agar plate, and then place it in a constant-temperature incubator at 25°C for culture for 2-3 days;

[0055] (3) Use a punch with a diameter of 0.6 cm to punch out 3 fresh-growing edge fungal blocks on the Cytospora chrysosperma mycelium plate after 2-3 days of culture, and inoculate them into 250 ml conical flasks containing the poplar bark powder liquid media and blank liquid media (except that poplar bark powder is not added, and other components are the same as in Example 2) prepared in Examples 1-7, and then place them in a constant-temperature incubator at 25°C and 150 rpm for shaking culture for 10 days. At the same time, prepare a control liquid medium of 200 ml of 50 g / L poplar bark powder (to prove that the liquid medium itself is non-toxic), and it is not inoculated with Cytospora chrysosperma and co-cultured. After the culture is completed, a culture product containing Cytospora chrysosperma mycelium and broth is obtained. Filter the culture product with 8 layers of gauze to filter out the mycelium, and further filter it with a microporous filter membrane to filter out the spores, extract, and rotary evaporate to obtain the toxic secondary metabolites of Cytospora chrysosperma.

[0056] Extract the mycelium RNA, and detect the expression level of the skeleton gene CcPpns1 of the secondary metabolite synthesis-related gene cluster of Cytospora chrysosperma in each medium by fluorescence quantitative PCR technology acyltransferase (under the induction of different bark powder concentrations). The results are shown in Figure 1 .

[0057] From Figure 1The results show that when cultivating with the liquid medium prepared in Example 2 (the bark powder concentration is 50 g / L), the expression level of the skeleton genes of the gene cluster related to secondary metabolism synthesis is the highest. It is thus considered that for the selection of the bark powder concentration in the medium, the optimal concentration is 50 g / L. Therefore, in the following experiments, the liquid medium prepared in Example 2 will be collectively referred to as the 50 g / L poplar bark powder medium; while when cultivating under the same conditions in the control liquid medium without adding poplar bark powder, the expression level of the skeleton genes of the gene cluster related to secondary metabolism synthesis is very low.

[0058] Comparative Application Example 1

[0059] Add 26 g of potato dextrose broth medium powder to 1000 ml of distilled water, boil until clear, pour it into a 250 ml conical flask, sterilize it at 115 °C under high-pressure steam for 20 min, and cool to prepare potato dextrose broth medium (PM0510). Then inoculate Cytospora chrysosperma, and the inoculation method is the same as that in Application Example 1. Incubate it on a constant temperature shaker at 25 °C with an oscillation speed of 150 rpm for 10 days. At the same time, prepare 200 ml of potato dextrose broth medium without inoculating Cytospora chrysosperma, and co-culture it as a medium control (used to prove that the medium itself is non-toxic). After the cultivation is completed, filter the cultivation product of the medium with 8 layers of gauze to filter out the bacteria, and further filter it with a microporous filter membrane to filter out the spores.

[0060] Comparative Application Example 2

[0061] Add 34.51 g of modified Czapek's medium powder to 1000 ml of distilled water, boil until clear, pour it into a 250 ml conical flask, sterilize it at 115 °C under high-pressure steam for 20 min, and cool to prepare modified Czapek's medium (MM1020). Then inoculate Cytospora chrysosperma, and the inoculation method is the same as that in Application Example 1. Incubate it on a constant temperature shaker at 25 °C with an oscillation speed of 150 rpm for 10 days. At the same time, prepare 200 ml of modified Czapek's medium without inoculating Cytospora chrysosperma, and co-culture it as a medium control (used to prove that the medium itself is non-toxic). After the cultivation is completed, filter the cultivation product of the medium with 8 layers of gauze to filter out the bacteria, and further filter it with a microporous filter membrane to filter out the spores.

[0062] (a) Observe the growth and development of Cytospora chrysosperma in three different media as shown in Table 1 and Figure 2 as follows.

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

[0064]

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

[0066] (b) Collect 1 L of the culture solutions of Cytospora chrysosperma in the 50 g / L poplar bark powder medium, the potato dextrose broth medium, and the modified Czapek's medium respectively, and 200 ml of the culture solutions of the corresponding blank 50 g / L poplar bark powder medium, the blank potato dextrose broth medium, and the blank modified Czapek's medium. Then use a rotary evaporator to concentrate them by rotary evaporation. After concentration, conduct a toxicity determination experiment on poplar leaves. Specifically: Use an inoculation needle to puncture and inoculate on poplar leaves, and drop 5 μl of the concentrated solution at the inoculation site. Place the treated poplar leaves in a petri dish for moisturizing culture, observe and record the size of the necrotic spots caused by the concentrated solutions of the three culture solutions on the poplar leaves, and evaluate the toxin production induction effect of each medium. The results are shown in Figure 3 .

[0067] From Figure 3 the results, it can be seen that the concentrated solution of the 50 g / L poplar bark powder culture solution of Cytospora chrysosperma caused necrotic spots, while the concentrated solutions of the modified Czapek's medium and the potato dextrose broth medium of Cytospora chrysosperma did not cause obvious necrotic spots. Therefore, it is considered that the components of the bark powder medium are superior to those of the Czapek's medium and the potato dextrose broth medium.

[0068] (c) Collect 5 L of the culture solution of Cytospora chrysosperma in the 50 g / L poplar bark powder medium and 200 ml of the culture solution of the corresponding blank 50 g / L poplar bark powder medium. Use ethyl acetate to extract the above culture solutions respectively, finally combine the organic phases, and use a rotary evaporator to concentrate the organic phases to obtain the crude secondary metabolites toxin of Cytospora chrysosperma and the extract of the 50 g / L poplar bark powder medium. Dissolve the crude toxin in a mixture of dichloromethane and methanol. Use the leaf puncture method to conduct a biological activity determination experiment on the crude secondary metabolites toxin of Cytospora chrysosperma. Specifically: Use an inoculation needle to puncture and inoculate on poplar leaves, and drop 5 μl of the crude toxin and the extract of the 50 g / L poplar bark powder medium at the inoculation site respectively. Place the treated poplar leaves in a petri dish for moisturizing culture, observe and record the necrosis phenomenon caused by the crude secondary metabolites toxin of Cytospora chrysosperma on the poplar leaves. The results are shown in Figure 4 .

[0069] From Figure 4 the results, it can be seen that obvious necrotic spots appeared at the inoculation site of the crude toxin 24 h after inoculation, and the leaf structure at the inoculation site of the extract of the 50 g / L poplar bark powder medium was intact without necrotic spots.

[0070] The above experimental results show that: the liquid medium and the corresponding culture method provided by the present invention can induce Cytospora chrysosperma to produce a large amount of toxic metabolites that are harmful to host plants, while the liquid medium itself has no harmful effect on poplar trees. The liquid medium provided by the present invention can also be applied to the induced culture of secondary metabolites of other trunk-type (canker disease) pathogenic fungi, which will not be elaborated here.

[0071] The above content is only an example and illustration of the present invention. Those skilled in the art of this technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, as long as they do not deviate from the invention or exceed the scope defined by the claims of this patent, they should fall within the protection scope of the present invention.

Claims

1. A liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites, characterized in that: Taking the total volume of the culture medium as 1L, the culture medium includes the following components in mass concentrations: 25-300g / L poplar bark powder, 2-6g / L yeast extract, 5-10g / L sucrose, and 1000-2000g distilled water.

2. The liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites according to claim 1, characterized in that: Taking the total volume of the culture medium as 1L, the culture medium includes the following components in mass concentrations: 25-150g / L poplar bark powder, 2-4g / L yeast extract, 5-8g / L sucrose, and 1000-1500g distilled water.

3. A method for preparing a liquid culture medium for inducing branch and trunk pathogenic fungi to produce secondary metabolites as claimed in claim 1 or 2, characterized in that: The following steps are involved: A1. Add poplar bark powder into distilled water according to the formula, boil, filter, and the obtained filtrate is poplar bark powder juice; A2. Add yeast extract and sucrose to poplar bark juice and mix. Then add distilled water to make up to volume and sterilize with high pressure steam.

4. A method for inducing the cultivation of branch and trunk pathogenic fungi to produce secondary metabolites using the liquid culture medium as claimed in claim 1 or 2, comprising the following steps: 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; S2. Use a cork puncher to punch fresh fungus blocks growing on the edge of the activated cultured branch pathogenic fungus hyphae plate, inoculate them into the center of the PDA agar plate, and then place it in a 25°C constant temperature incubator for 2 to 3 days; S3, using a puncher to punch out fungus blocks from the fresh mycelium growth area on the branch and trunk pathogenic fungus plate after culturing for 2 to 3 days, and inoculating the obtained fungus blocks into a liquid culture medium, and then culturing in a constant temperature incubator at 25° C., 100-150 rpm for 10 to 20 days to obtain a culture product containing branch and trunk pathogenic fungus mycelium and bacterial liquid; S4. Use gauze to filter the culture product containing branch and trunk pathogenic fungi mycelium and bacterial liquid for the first time to remove the branch and trunk pathogenic fungi mycelium, and then use a microporous filter membrane to filter out the spores for the second time, extract, and obtain the secondary metabolites of the branch and trunk pathogenic fungi.

5. The method for inducing and culturing branch and trunk pathogenic fungi to produce secondary metabolites according to claim 4, characterized in that: In step S1, the bacterial strain activation culture time is 4 to 5 days.

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