Gentiana internal fungus promoting growth and disease resistance fermentation liquor, and preparation method and application thereof

Abstract

This invention belongs to the field of biocontrol technology of plant endophytic fungi and their metabolites, specifically relating to a growth-promoting and disease-resistant fermentation broth of a gentian endophytic fungus, its preparation method, and its application. The fermentation broth of this invention is prepared by activating and fermenting the endophytic fungus *Trichoderma* sp. 3711 from gentian. Antibacterial and growth-promoting experiments have demonstrated that the fermentation broth of this invention has significant broad-spectrum antimicrobial effects against plant pathogens and promotes plant (especially crop) growth. Furthermore, the preparation method of the fermentation broth based on *Trichoderma* sp. 3711 isolated from gentian is simple, inexpensive, and has good economic value and broad market prospects.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology of plant endophytic fungi and their metabolites, specifically involving a growth-promoting and disease-resistant fermentation broth of a gentian endophytic fungus, its preparation method, and its application. Background Technology

[0002] Currently, the extensive use of chemical pesticides and fertilizers has many drawbacks, such as increasingly serious environmental pollution, significantly improved pest resistance, and threats to human and animal safety, resulting in a multitude of ecological problems. Furthermore, public demand for food safety is growing stronger. Therefore, the development of safe, efficient, green, and environmentally friendly new pesticides and fertilizers is urgently needed.

[0003] Microbial fermentation, with its simple process, relatively low cost, low likelihood of inducing resistance in pests and diseases, and mildness to humans and the environment, has become a hot topic in the development of novel biopesticides and fertilizers. Plant endophytes, as a type of microorganism, have attracted considerable attention due to their extremely broad prospects in the development of new pesticides and fertilizers. Plant endophytes refer to a class of fungi that have co-evolutionary interactions with their host plants and have formed a mutually beneficial symbiotic relationship. This mutually beneficial symbiotic relationship between plant endophytes and their host plants manifests in the following ways: photosynthetic products produced by the host plant provide nutrients for the endophytes; and metabolic products produced by the endophytes stimulate the growth and development of the host plant while enhancing its resistance to adverse conditions. Therefore, the metabolic products of plant endophytes provide abundant raw materials and broad scope for the discovery of new compounds and the screening of new drugs, making plant endophytes a research hotspot in multiple fields and disciplines.

[0004] Gentian is the dried root and rhizome of *Gentiana manshurica* Kitag., *Gentiana ascabra* Bge., *Gentiana triflora* Pall., or *Gentiana rigescens* Franch., all belonging to the Gentianaceae family. Researchers both domestically and internationally have isolated various components from gentian, including schizocarpine glycosides, polysaccharides, and alkaloids. Gentian is bitter and cold in nature, and is a commonly used traditional Chinese medicine for clearing damp-heat in the liver and gallbladder, and purging stagnant fire. Clinically, gentian is often used for its choleretic, stomachic, hepatoprotective, and central nervous system stimulant effects; its anti-inflammatory and antibacterial effects are also significant. Based on previous research, it is speculated that endophytic fungi with strong anti-plant pathogenic activity may exist in gentian, and their secondary metabolites may have growth-promoting effects. Therefore, research on endophytic fungi in gentian has theoretical and practical reference value for the development and use of pesticides and fertilizers in the new era.

[0005] However, there are currently no pesticides or plant growth promoters derived from endophytic fungi of gentian that simultaneously promote growth and resist disease. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems and fill the technical gaps in related fields, this invention is based on the endophytic fungus Trichoderma sp. 3711 isolated from Gentiana, and the metabolites obtained by fermentation culture are developed into products mainly used for broad-spectrum resistance to plant pathogens and promotion of plant growth.

[0007] Specifically, the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a gentian endophytic fungus, which is used to prepare a fermentation broth with anti-plant pathogenic and plant growth-promoting effects. The gentian endophytic fungus is *Trichoderma* sp. 3711, an endophytic fungus of the genus *Trichoderma*. The microbial accession number of *Trichoderma* sp. 3711 is CCTCC NO: M 20211203, the accession date is September 24, 2021, the accession location is the China Center for Type Culture Collection (CCTCC), and the accession unit address is Wuhan University, Wuhan, China.

[0009] In a second aspect, the present invention provides a fermentation broth that has the effects of resisting plant pathogens and promoting plant growth, said fermentation broth being prepared from the gentian endophytic fungus described in the first aspect above.

[0010] Alternatively, the plant pathogens in the fermentation broth may include one or more of the following: Pythium aphanidermatum (FM), Rhizoctonia solani Kühn (WK), and Fusarium oxysporum f.sp.cucumerinum (KW).

[0011] In a third aspect, the present invention provides a method for preparing the fermentation broth described in the second aspect above, which has the effects of resisting plant pathogens and promoting plant growth, the preparation method comprising the following steps:

[0012] (1) Activation of strains: Under aseptic conditions, the strains of endophytic fungi of gentian were transferred to sterilized PDA medium plates and incubated at a constant temperature of 24-30℃ for 3-6 days to obtain activated strains.

[0013] (2) Fermentation culture: Under aseptic conditions, the mycelium cake is punched out with a punch and inoculated into an Erlenmeyer flask containing PD medium. The flask is sealed with a sealing film and placed in a constant temperature shaking incubator. It is then cultured on a shaker for 4-8 days. The resulting fermentation broth is filtered through 4 layers of gauze to obtain the fermentation broth of the endophytic fungus of Gentiana scabra.

[0014] As an optional method, in the above preparation method, the PDA culture medium used in step (1) consists of 200g potato, 20g glucose, 15g agar, and 1000mL distilled water.

[0015] As an optional method, in the above preparation method, the PD culture medium used in step (2) is 200g of potato, 20g of glucose, and 1000mL of distilled water.

[0016] As an optional method, in the above preparation method, in step (1), the temperature in the incubator is kept constant at 28°C for 4 days.

[0017] As an optional method, in the above preparation method, in step (2), the culture is carried out on a shaker for 4-6 days, preferably 6 days.

[0018] Preferably, in step (2), the diameter of the punch is 1 cm, the shaking incubation conditions are pH = 4-8, 20-32℃, 120 r / min, and the liquid volume of the conical flask is 50 mL-150 mL / 250 mL.

[0019] More preferably, in step (2), the diameter of the punch is 1 cm, the shaking incubation conditions are pH = 4-6, 24-28℃, 120 r / min, and the liquid volume of the conical flask is 50 mL-100 mL / 250 mL.

[0020] Most preferably, in step (2), the diameter of the punch is 1 cm, the shaking incubation conditions are pH = 6, 28℃, 120 r / min, and the liquid volume of the conical flask is 50 mL or 100 mL / 250 mL.

[0021] In a fourth aspect, the present invention provides the use of the fermentation broth described in the second aspect above, which has the effects of resisting plant pathogens and promoting plant growth, or the fermentation broth prepared by the preparation method described in the third aspect above, in the preparation of agricultural adjuvants, wherein the agricultural adjuvants have broad-spectrum effects of resisting plant pathogens and promoting plant growth.

[0022] Alternatively, in the above-mentioned uses, the agricultural adjuvant is a pesticide or a plant growth promoter.

[0023] Alternatively, in the above-mentioned uses, the agricultural adjuvant is a fertilizer-pesticide.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] Currently, commercially available pesticides and fertilizers generally contain more than ten ingredients, and none utilize endophytic fungal fermentation broth as a single raw material to combat plant pathogens and thus promote plant growth. However, antibacterial and growth-promoting experiments have demonstrated that the fermentation broth described in this invention has significant broad-spectrum anti-plant pathogen effects and promotes plant (especially crop) growth. Furthermore, the fermentation broth prepared using the endophytic fungus *Trichoderma* sp. 3711 isolated from *Gentiana* as a base, according to this invention, is simple to prepare, inexpensive, and possesses good economic value and broad market prospects. Attached Figure Description

[0026] Figure 1 Effect of fermentation temperature on the inhibition rate of endophytic fungi 3711 fermentation broth.

[0027] Figure 2 Effect of fermentation days on the inhibition rate of endophytic fungi 3711 fermentation broth.

[0028] Figure 3 Effect of liquid volume on the antibacterial rate of endophytic bacteria 3711 fermentation broth.

[0029] Figure 4 Effect of initial pH on the inhibition rate of endophytic fungi 3711 fermentation broth. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0033] 1. An exemplary preparation method of the gentian endophytic bacteria fermentation broth of the present invention:

[0034] (1) Activation of microbial strains:

[0035] Under aseptic conditions, the endophytic fungus *Trichoderma* sp. 3711 of *Gentiana* was transferred to sterilized PDA agar plates and incubated at 28°C for 4 days to obtain activated strains.

[0036] The PDA culture medium consisted of 200g potato, 20g glucose, 15g agar, and 1000mL distilled water.

[0037] (2) Fermentation culture:

[0038] All the following steps were performed in a sterile operating table. Four mycelial cakes were punched using a 1cm diameter punch in the sterile operating table and inoculated into 250mL Erlenmeyer flasks containing 100mL of PD medium. The flasks were sealed with film and placed in a constant temperature shaking incubator at pH 4-6, 24-28℃, and 120rpm for 4-6 days. The resulting fermentation broth was filtered through four layers of gauze to obtain the fermentation broth of *Trichoderma* sp. 3711, an endophytic fungus of *Trichoderma* genus, as described in this invention.

[0039] The PD culture medium consisted of 200g potato, 20g glucose, and 1000mL distilled water.

[0040] 2. Test on the resistance of the gentian endophytic bacteria fermentation broth to plant pathogens of this invention:

[0041] 2.1 Plant pathogens

[0042] The three pathogens were identified as Pythium aphanidermatum (FM), Rhizoctonia solani Kühn (WK), and Fusarium oxysporum f.sp.cucumerinum (KW), from the Department of Pesticide Science, College of Plant Protection, Shenyang Agricultural University, and were identified by Associate Professor Gu Zumin.

[0043] 2.2 Pathogenic microorganism culture

[0044] Using an inoculation loop, pick up 1cm of plant pathogen cake and inoculate it into a petri dish containing PDA medium for activation. Incubate in a constant temperature incubator (28℃) until the medium covers the entire surface.

[0045] 2.3 Determination of the inhibitory effect of 3711 strain fermentation broth on pathogenic fungal mycelial growth

[0046] The growth rate method was used, with the leaf spot pathogen as an indicator for FM, WK, and KW activity detection. Antimicrobial activity against pathogenic fungi was assessed using an antimicrobial assay. Under aseptic conditions, 10-15 mL of fermentation broth from the endophytic fungus *Trichoderma* sp. 3711 was mixed thoroughly with 90-85 mL of sterilized, melted PDA culture medium at 60-70℃. The mixture was poured into petri dishes to prepare 15% (V / V) fermentation broth plates. Sterile water was used as a control, with three replicates per group. After the culture medium cooled and solidified, it was sterilized under UV light for 30 min. A 1 cm diameter plant pathogen pellet was inoculated into each petri dish and incubated at 28℃ in the dark. After several days, the diameter of pathogen colonies in each petri dish was measured, and the inhibition rate (inhibition rate of pathogens) of the endophytic fungal fermentation broth was calculated as an indicator of its antimicrobial activity.

[0047] The formula is as follows:

[0048] Antibacterial rate = (diameter of control group - diameter of drug-containing group) / diameter of control group;

[0049] Inhibition rate (inhibition rate of pathogens): The probability that pathogen colonies are inhibited by the liquid fermentation products of endophytic fungi;

[0050] Control group diameter: The diameter of pathogenic fungal colonies in the control group plate minus 1 (cm);

[0051] Diameter of drug-containing group: The diameter of pathogenic fungal colonies in the drug-containing plate is 1 cm.

[0052] 2.4 Experimental Results

[0053] 2.4.1 Effect of temperature on the antibacterial activity of 3711 fermentation broth

[0054] Four 1cm diameter 3711 bacterial discs were inoculated into Erlenmeyer flasks containing PD medium (100mL / 250mL). The flasks were incubated at 20℃, 24℃, 28℃, 32℃, and 36℃ with shaking at 120 r / min for 4 days, with three replicates for each treatment. The inhibition rate of the fermentation broth against pathogenic bacteria was investigated according to section "2.3". The results are shown below. Figure 1 .

[0055] The results showed that as the temperature increased, the inhibitory effect of the fermentation broth of endophytic fungus 3711 on four plant pathogens first increased and then decreased; when the temperature reached 36℃, it was no longer suitable for the growth and fermentation of endophytic fungus 3711; in the range of 20℃-32℃, the fermentation broth of 3711 had antibacterial activity, and at a temperature of 28℃, the fermentation product achieved the highest inhibition rate against the four pathogens.

[0056] 2.4.2 Effect of fermentation days on the antibacterial activity of 3711 fermentation broth

[0057] Four 1cm diameter 3711 bacterial discs were inoculated into Erlenmeyer flasks containing PD medium (100mL / 250mL). The flasks were incubated at 28℃ with shaking at 120 rpm for 2, 3, 4, 5, and 6 days, with three replicates for each treatment. The inhibition rate of the fermentation broth against pathogenic bacteria was investigated according to section "2.3". The results are shown below. Figure 2 .

[0058] The results showed that the inhibitory effect of the endophytic fungus 3711 fermentation broth on four plant pathogens increased with the increase of fermentation days; on the 5th and 6th day of fermentation, the inhibition rate of the endophytic fungus 3711 fermentation broth on pathogens was not much different, but the inhibition rate on KW, WK and FM was relatively high.

[0059] 2.4.3 Effect of liquid volume on the antibacterial activity of 3711 fermentation broth

[0060] Four 1cm diameter 3711 bacterial discs were inoculated into 250mL Erlenmeyer flasks with PD (distillation) solution volumes of 50mL, 100mL, 150mL, and 200mL, respectively. The flasks were incubated at 28℃ and 120rpm for 5 days with shaking. Each treatment was replicated in triplicate. The inhibition rate of the fermentation broth against pathogenic bacteria was investigated according to section "2.3". The results are shown below. Figure 3 .

[0061] The results showed that the inhibitory effect of the fermentation broth of endophytic fungus 3711 on four plant pathogens weakened with the increase of liquid volume. When the liquid volume in the 250mL conical flask was 50mL and 100mL, the inhibition rate of the fermentation broth of endophytic fungus 3711 on the pathogens was similar; when the liquid volume was greater than 100mL, the inhibition rate of the fermentation broth decreased significantly.

[0062] 2.4.4 Effect of pH value on the antibacterial activity of 3711 fermentation broth

[0063] Four 1cm diameter 3711 bacterial discs were inoculated into Erlenmeyer flasks containing PD medium (100mL / 250mL Erlenmeyer flasks). The pH values ​​were adjusted to 2, 4, 6, 8, and 10 (using 1mol / L NaOH and 1mol / L HCl solutions), respectively. The flasks were incubated at 28℃ with shaking at 120 rpm for 5 days, with three replicates per treatment. The inhibition rate of the fermentation broth against pathogenic bacteria was investigated according to section "2.3". The results are shown below. Figure 4 .

[0064] The results showed that as the initial pH of the culture increased, the antibacterial activity of the fermentation broth of endophytic fungus 3711 against the four pathogens first increased and then decreased. Within the pH range of 4-6, the fermentation broth had high antibacterial activity against the four pathogens, and the fermentation broth obtained under the condition of initial pH 6 had the highest inhibition rate against the four pathogens.

[0065] 3. Experiment on the plant growth promotion effect of the gentian endophytic fungal fermentation broth:

[0066] 3.1 Preparation of drug solution

[0067] Dispense 5-15 mL of 3711 fermentation broth into 250 mL conical containers containing 85-95 mL of pre-weighed purified water, totaling 100 mL per container. Seal the containers and autoclave at 121°C for 30 minutes. Let them cool before use.

[0068] 3.2 Experiment on the effect of 3711 bacteria fermentation broth on cucumber seed growth promotion

[0069] The paper germination method was used to test cucumber seeds (which spread all over the ground). Hebei Dayu Seed Industry Co., Ltd. treated the surface of cucumber seeds with 1% sodium hypochlorite for 10 minutes, followed by treatment with 75% alcohol for 5 minutes. Afterward, the seeds were rinsed three times with sterile water. Twenty seeds were placed in each petri dish lined with sterile filter paper. A 15% fermentation broth was prepared, and 5 mL of broth was added evenly to each dish. Sterile water was used as a control group, with three replicates per group. The seeds were cultured at 25℃ and 30% humidity. Seed germination rate was measured on day 3 of culture, and root weight, root length, stem weight, and stem length of cucumber seedlings were measured after 5 days.

[0070] 4.3 Experimental Results

[0071] The experimental results are shown in Tables 1 and 2 below. Compared with the control group, the group containing the fermentation broth improved the seed germination rate to a certain extent, and also increased the stem length, root length, stem weight, and root weight of cucumber seedlings. These results indicate that the 3711 bacteria fermentation broth has the effect of promoting the growth of cucumber seedlings.

[0072] Table 1: Effects of the fermentation broth of strain 3711 of the present invention on the growth of cucumber seedlings

[0073]

[0074]

[0075] Table 2: Effect of the fermentation broth of strain 3711 of the present invention on the germination rate of cucumber seeds

[0076]

[0077] In summary, antibacterial and growth-promoting experiments have demonstrated that the fermentation broth of Trichoderma sp. 3711 described in this invention exhibits significant broad-spectrum antimicrobial activity against plant pathogens and promotes plant growth. Furthermore, the fermentation broth prepared using Trichoderma sp. 3711, an endophytic fungus isolated from Gentiana, is simple to prepare and inexpensive, possessing good economic value and broad market prospects.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Gentiana endophytic fungus, characterized in that: The gentian endophytic fungus is an endophyte Trichoderma of the genus Trichoderma Trichoderma The microbial preservation number of the Trichoderma sp. 3711 is CCTCC NO: M20211203, the preservation time is September 24, 2021, the preservation place is China Center for Type Culture Collection (CCTCC), and the preservation unit address is Wuhan University, Wuhan, China. Trichoderma The gentian endophytic fungus is used for preparing a fermentation liquor with the effects of resisting plant pathogenic fungi and promoting plant growth, the plant pathogenic fungi are rice sheath blight pathogenic fungi ( Rhizoctonia solani Kühn ), and the effect of promoting plant growth is to improve the germination rate of cucumber seeds and promote the growth of cucumber seedlings.

2. A fermentation broth having an anti-phytopathogenic fungus and a plant growth promoting effect, characterized by: The fermentation liquid is prepared from the endophytic fungus of gentiana described in claim 1, the plant pathogenic bacteria is rice sheath blight pathogen (Rhizoctonia solani Kuhn) Rhizoctonia solani Kühn ), and the plant growth promoting effect is to improve the seed germination rate of cucumber and promote the growth of cucumber seedlings. The method for preparing the fermentation broth comprises the following steps: (1) strain activation: under sterile conditions, the strain of gentian endophytic fungi is transferred to a sterilized potato dextrose agar solid medium (PDA medium) plate, and is placed in a culture box at a constant temperature of 28°C for 5 6 days to obtain activated strains; (2) fermentation culture: under sterile conditions, the fungus cake is punched by a puncher and inoculated into a conical flask containing potato dextrose liquid medium (PD medium), and then the conical flask is sealed with a sealing film and placed in a constant temperature shaking incubator for 4 8 days of shaking bed culture, and the obtained fermentation liquor is filtered through 4 layers of gauze to obtain the fermentation liquor of the gentian endophytic fungus, the diameter of the puncher is 1 cm, the shaking bed culture conditions are pH = 4 6, 20 32℃, 120 r / min, and the liquid loading amount of the conical flask is 50 mL 100 mL / 250mL.

3. The method for preparing the fermentation broth having an anti-phytopathogenic fungi and plant growth promoting effect according to claim 2, characterized by: The preparation method comprises the following steps: (1) strain activation: under sterile conditions, the strain of gentian endophytic fungi is transferred to a sterilized potato dextrose agar solid medium (PDA medium) plate, and is placed in a culture box at a constant temperature of 28°C for 5 6 days to obtain activated strains; (2) Fermentation culture: under sterile conditions, the fungus cake is punched by a puncher and inoculated into a conical flask containing potato dextrose liquid medium (PD medium), and then the conical flask is sealed with a sealing film and placed in a constant temperature shaking incubator for 4 8 days of shaking bed culture, and the obtained fermentation liquor is filtered through 4 layers of gauze to obtain the fermentation liquor of the gentian endophytic fungus. The diameter of the puncher is 1 cm, the shaking bed culture conditions are pH = 4 6, 20 32℃, 120 r / min, and the liquid loading amount of the conical flask is 50 mL 100 mL / 250mL.

4. The method of claim 3, wherein: The PDA culture medium used in step (1) is potato 200 g, glucose 20 g, agar 15 g, and distilled water 1000 mL.

5. The method of claim 3, wherein: The PD culture medium used in step (2) is potato 200 g, glucose 20 g, and distilled water 1000 mL.

6. Use of the fermentation broth having an anti-phytopathogenic fungi and plant growth promoting effect according to claim 2 or the fermentation broth prepared by the preparation method according to any one of claims 3 to 5 for the preparation of an agricultural adjuvant, characterized in that: The agricultural adjuvant has the effects of resisting plant pathogens and promoting plant growth. The plant pathogen is rice sheath blight pathogen (…). Rhizoctonia solani Kühn The plant growth-promoting effect mentioned above is to increase the germination rate of cucumber seeds and promote the growth of cucumber seedlings.

7. Use according to claim 6, characterized in that: The agricultural adjuvant is a pesticide and a plant growth promoter.

8. Use according to claim 7, characterized in that: The agricultural adjuvant is a pesticide and a plant growth promoter.

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