Polygonatum kingianum root endophytic fungus Princasa crispa DHJ068 and application thereof

Abstract

The invention provides a polygonatum kingianum root endophytic fungus Princasa crispa DHJ068 and application thereof, and relates to the technical field of crop disease control. The polygonatum kingianum root endophytic fungus Princephala crispa DHJ068 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2025748, and a fermentation liquor crude extract of the Princephala crispa DHJ068 can be applied to prevention and treatment of crop diseases. The invention overcomes the defects in the prior art, and the fermentation liquor crude extract of the polygonatum kingianum root endophytic fungus Prringtonia crispa DHJ068 can effectively inhibit the growth of Chinese cabbage soft rot bacteria and cucumber bacterial angular leaf spot bacteria, thereby achieving the purpose of certain biological control.

Description

Technical Field

[0001] The present invention relates to the technical field of crop disease prevention and control, and in particular to an endophytic fungus Pulingerella fragilis DHJ068 from the roots of Polygonatum cyrtonema and applications thereof. Background Art

[0002] Polygonatum yunnanensis ( Polygonatum kingianum Yunnan Polygonatum sibiricum is primarily distributed in Yunnan, Sichuan, and Guizhou provinces in China. Its rhizomes are used as medicine, boasting benefits such as tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It can be used to treat symptoms such as spleen and stomach deficiency, fatigue, dry mouth and poor appetite, lung deficiency, dry cough, deficiency of essence and blood, and internal heat and thirst. Furthermore, in modern medicine, Yunnan Polygonatum sibiricum has various pharmacological properties, including antibacterial, antifungal, and antihypertensive effects.

[0003] Endophytic fungi are fungi that reside in healthy plant tissues, spending some or all stages of their life cycle within plant tissues and organs without causing overt symptoms of infection in the host plant. Endophytic fungi are ubiquitous in plant tissues and have a positive impact on plant growth, forming a mutually beneficial symbiotic relationship with their host plants through long-term co-evolution. Endophytic fungi thrive in unique environments and can promote the growth of their host plants by producing antibiotic resistance, enhancing drought resistance, and improving tolerance to heavy metals. Terpenoids in endophytic fungi exhibit novel structures and significant biological activity. Current research indicates that secondary metabolites of endophytic fungi have been classified into the following chemical categories: terpenes, steroids, flavonoids, peptides, aliphatics, alkaloids, esters, and phenylpropanoids. Most of these compounds possess a wide range of pharmacological activities, including anti-inflammatory, anti-tumor, and antibacterial properties.

[0004] Crop diseases and insect pests are one of the major agricultural disasters in my country. They are characterized by a wide variety of species, great impact, and frequent outbreaks. Their scope and severity often cause significant losses to my country's national economy, especially agricultural production. Erwinia carotovora ) is related to the number of wounds caused by field pests, man-made or natural causes, and black rot. Cabbage wounds are mainly divided into natural cracks, insect wounds, disease scars, and mechanical wounds. Among them, the natural cracks on the petiole are mostly longitudinal cracks, which are the main way for the disease to invade. In production, long-term drought followed by rain, or excessive seedling squatting and excessive watering will cause wounds and cause the disease. Waterlogging on the surface and lack of hydrogen in the soil are not conducive to the development of the cabbage root system, or the wounds become corky, which will cause severe disease. Cucumber bacterial angular leaf spot ( Pseudomonas syringae pv. lachrymans ) primarily affects cucumbers, affecting both seedlings and mature plants, with leaves being the primary target. It primarily harms leaves, petioles, tendrils, and fruit, sometimes also infecting stems. The disease initially appears on cotyledons, with water-soaked, nearly circular, sunken spots appearing, eventually becoming slightly yellowish-brown and drying up.

[0005] During crop cultivation, the long-term use of chemicals has led to increased pathogen variability, often resulting in resistance to various drugs, which has weakened the effectiveness of chemical pest control. Endophytic fungi, with their unique advantages, have broad application prospects in plant disease and insect pest control. Many endophytic fungi can produce antibiotic substances that enhance the disease resistance of host plants. These active substances not only act against plant pathogens but also have inhibitory effects on other bacteria, fungi, and some protozoa. Screening for antibiotic substances from endophytic fungi has become an important aspect of the source of new antibiotics, with important significance in medicine, the food industry, and agronomy. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides an endophytic fungus, Pullingella brittle fruit DHJ068, from the roots of Polygonatum sibiricum and its application. The endophytic fungus, Pullingella brittle fruit DHJ068, from the roots of Polygonatum sibiricum can effectively inhibit the growth of pathogenic bacteria of soft rot and bacterial angular leaf spot of cucumber, thereby achieving a certain biological control purpose.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions: An endophytic fungus from the roots of Polygonatum yunnanensis, Pulingerella fragilis ( Prillingera fragicola ) DHJ068, the Pulingerella crispa DHJ068 is preserved in the China Center for Type Culture Collection with the accession number of CCTCC NO: M 2025748.

[0008] The endophytic fungus Pulingerella brittlebone DHJ068 from the roots of Polygonatum cyrtonema is used to prevent and control crop diseases.

[0009] Preferably, the crop disease is caused by the pathogenic bacteria of cabbage soft rot Erwinia carotovora Cucumber bacterial leaf spot Pseudomonas syringae pv. lachrymans Diseases caused.

[0010] Preferably, the application method is to use the crude extract of the fermentation broth of the endophytic fungus Pulingella fragilis DHJ068 from the roots of Polygonatum cyrtonema to prepare a bacterial agent or a pharmaceutical composition for preventing and controlling crop diseases.

[0011] Preferably, the method for preparing the crude fermentation broth extract comprises the following steps: (1) Activating the fungus strain Pulingerella crispa DHJ068; (2) The activated P. crispatus DHJ068 fungus was inoculated into WYSP liquid culture medium, and then placed in a constant temperature shaker at 28°C and 150 rpm for 20 days to obtain a fermentation broth; (3) The fermentation broth was placed in a 50 ml centrifuge tube and then centrifuged at 3000 g for 3 min to separate the bacteria and the fermentation broth. Ethyl acetate was added to the fermentation broth at a volume ratio of 1:1.5 and ultrasonicated for 30 min. The broth was then poured into a separatory funnel and allowed to stand for 12 h to separate the layers. The ethyl acetate supernatant was taken and condensed and refluxed on a rotary evaporator to obtain the crude extract of the fermentation broth.

[0012] Preferably, the WYSP liquid culture medium has a formula of: 10 g / L pea flour; 1 g / L yeast powder; 2 g / L sodium acetate; 1 g / L potassium propionate, and the balance is water.

[0013] The present invention provides an endophytic fungus from the roots of Polygonatum cyrtonema, Pringlesia crispa DHJ068, and its application, which has the following advantages over the prior art: The present invention obtains a fungus Pringlesia crispa DHJ068 with antibacterial activity by isolating and screening endophytic fungi from the roots of Polygonatum yunnanensis. The fermentation broth extract of the fungus has a strong inhibitory effect on the pathogenicity of cabbage soft rot fungi and the growth of cucumber bacterial angular leaf spot fungi, and can effectively prevent and control plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram showing the mycelial growth of the endophytic fungus Pringlesia crispa DHJ068 in the roots of Polygonatum cyrtonema according to Example 1 of the present invention; Figure 2 This is a micrograph of the hyphae of the endophytic fungus Pringlesia crispa DHJ068 in the roots of Polygonatum cyrtonema according to Example 1 of the present invention; Figure 3 This is a diagram showing the phylogenetic tree of endophytic fungi constructed based on ITS in Example 1 of the present invention; Figure 4 1 is an extract of mycelium of P. brittlebone DHJ068; 2 is a crude extract of fermentation broth of P. brittlebone DHJ068; 3 is a crude extract of WYSP liquid culture medium; 4 is DSMO; Figure 5 1 is an extract of mycelium of P. brittlebone DHJ068; 2 is a crude extract of fermentation broth of P. brittlebone DHJ068; 3 is a crude extract of WYSP liquid culture medium; 4 is DSMO. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0016] The endophytic fungus of the root of Polygonatum sibiricum provided by the present invention is preserved as Pulingerella fragilis Prillingera fragicola DHJ068; deposited with the China Center for Type Culture Collection, Wuhan University, China; received by the China Center for Type Culture Collection on April 10, 2025, and tested alive on April 17, 2025; deposit number: CCTCC NO: M 2025748.

[0017] The cabbage soft rot pathogenic bacteria used in the following examples ( Erwinia carotovora )for Erwinia carotovora Pathogenic strains, purchase link: http: / / www.biovector.net / product / 210298.html.

[0018] The bacterial leaf spot of cucumber ( Pseudomonas syringae pv. lachrymans )for Pseudomonas syringae pv. lachrymans The cucumber pathogenic strain of Pseudomonas syringae can be purchased at: http: / / www.biovector.net / product / 2096192.html. Example 1:

[0019] 1. Isolation of endophytic fungi from the roots of Polygonatum sibiricum (1) The roots of Polygonatum sibiricum were collected from Jizu Mountain, Binchuan County, Dali City, Yunnan Province. After being dug out with the roots, Polygonatum sibiricum was placed in a ziplock bag and kept at low temperature with an ice pack. After being brought back to the laboratory, it was rinsed with tap water for 48 hours, cut into 2 cm small pieces, and then transferred to a sterile workbench for disinfection. It was rinsed with 1L of distilled water, then soaked in 70% ethanol for 2 minutes, the ethanol was poured out, and it was rinsed 6 times with 50mL of sterile water. It was then soaked in sodium hypochlorite (10%) solution for 1 minute, the sodium hypochlorite solution was poured out, and it was rinsed 10 times with 50mL of sterile water (50mL each time).

[0020] (2) Cut the 2 cm small pieces into tissue blocks with a length and width of 0.5 cm each, place them in a centrifuge tube, add steel balls and 1 mL of sterile water, and crush for 2 minutes (180 rpm). Dilute 100-fold and plate on 10 PYGKAS culture medium plates (PYGKAS: 3 g / L Yunnan Polygonatum powder, 4 g / L yeast powder, 10 g / L glucose, 100 μg / mL karanomycin, 100 μg / mL ampicillin, 100 μg / mL streptomycin, and the balance is water). Cultivate for 10 days. After colonies grow, select single colonies and identify them.

[0021] (3) The grown mycelia were transferred to PDY solid medium and cultured for 15 days. The strain was molecularly identified and the P. crispatus DHJ068 strain was obtained. The PDY solid medium contained the following components: 15 g / L potato flour, 5 g / L glucose, 2 g / L yeast powder, 13 g / L agar, and the balance was water.

[0022] 2. Identification of endophytic fungi in the roots of Polygonatum yunnanensis (1) Morphological identification The strain morphology is Figure 1 and Figure 2 As shown, the fungus grew well on the malt extract broth solid culture medium, the colony surface had wrinkles and ridges, and the hyphae were light yellow.

[0023] (2) DNA extraction ① Before the experiment, heat CTAB (cetyltrimethylammonium bromide) in a 65℃ water bath for 30 minutes; ② Take 50 mg of dried Pullingella crispa DHJ068 mycelium in a 2 mL centrifuge tube, add 3 small steel balls, put the centrifuge tube into liquid nitrogen for 6 minutes, and immediately crush it with a crusher for 2 minutes. Add 1 mL of preheated CTAB solution, pipette and mix it, and then transfer it all into a centrifuge tube containing 200 μL PVP (polyvinyl pyrrolidone). Add 20 μL β-mercaptoethanol in the air in a fume hood, shake for 15 seconds to fully grind, and then place it in a 65°C water bath for 1.5 hours, turning it upside down 5-6 times every 10 minutes. After the water bath, centrifuge it at 12000 rpm and 4°C for 10 minutes; ③ Take 1 mL of the supernatant and transfer it to a new centrifuge tube. Add 500 μL each of DNA phenol reagent and chloroform-isoamyl alcohol mixture. Turn the tube upside down for 10 minutes and centrifuge (4°C, 12,000 rpm) for 10 minutes. (Repeat step ③ twice.) ④ Take 900 μL of the supernatant and place it in a new centrifuge tube. Add 50 μL of 3M sodium acetate solution and 900 μL of 95% anhydrous ice ethanol (-20°C). Shake well and place in a -20°C refrigerator to precipitate for 3 hours. ⑤ After precipitation, centrifuge (4℃·12000r / min) for 10min, discard the supernatant, add 500μL 75% alcohol, invert upside down 3 times, let it stand for 3min, and discard the supernatant; ⑥ Add 500 μL of 95% alcohol, invert the tube upside down 3 times, let it stand for 3 minutes, centrifuge at room temperature (13000 rpm) for 3 minutes, discard the ethanol, and set aside to dry; ⑦ Add 40 μL of elution buffer EB and centrifuge at room temperature (13000 rpm) for 1.5 min to obtain the genomic DNA of P. crispatus DHJ068.

[0024] (3) ITS analysis and identification The fungal universal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used to amplify the fungal rDNA spacer sequence (including the ITS1 region, 5.8S region, and ITS4 region). The resulting ITS sequencing sequence is shown in SEQ ID No. 1.

[0025] SEQ ID No. 1: (4) Constructing a developmental tree The phylogenetic tree of plant endophytic fungi was constructed using MEGA software based on ITS ( Figure 3 ), based on the results of strain morphology and molecular biology identification, the DHJ068 strain was identified as Prillingera fragicola. Example 2:

[0026] Preparation of crude extract of P. crispatus DHJ068 fermentation broth: 1. Liquid culture The mycelium of the endophytic fungus Pulingerella brittle fruit DHJ068 from the roots of Polygonatum cyrtonema was inoculated into a 250 mL conical flask containing 100 mL of WYSP liquid medium (10 g / L pea flour; 1 g / L yeast powder; 2 g / L sodium acetate; 1 g / L potassium propionate, and the balance was water), and the flask was placed in a constant temperature shaker at 28°C and 150 rpm for 20 days.

[0027] 2. Preparation of crude fermentation broth extract The fermentation broth was placed in a 50 ml centrifuge tube and then centrifuged at 3000 g for 3 min to separate the bacteria and the fermentation broth.

[0028] Ethyl acetate was added to the fermentation broth at a ratio of 1:1.5, followed by sonication for 30 minutes. The broth was then poured into a separatory funnel and allowed to stand for 12 hours to allow for separation. The supernatant was then extracted. The extract was condensed and dried under reflux on a rotary evaporator to obtain a crude extract of the fermentation broth of the endophytic fungus Pringella crispa DHJ068 from the roots of Polygonatum cyrtonema.

[0029] 3. Preparation of crude bacterial extract The fermentation broth was placed in a 50 ml centrifuge tube and centrifuged at 3000 g for 3 min to obtain the P. crispatus DHJ068 bacterial cells. The P. crispatus DHJ068 bacterial cells were placed in a conical flask and ethyl acetate was added to submerge the cells. After sonication for 30 min and 12 h of stagnation, the bacterial extract was filtered through gauze and dried under reflux on a rotary evaporator to obtain a crude extract of the endophytic fungus P. crispatus DHJ068 from the roots of Polygonatum cyrtonema.

[0030] 4. Preparation of crude extract from WYSP liquid culture medium A 250 mL Erlenmeyer flask containing 100 mL of WYSP liquid medium was sterilized and placed in a thermostatic shaker at 28°C and 150 rpm for 20 days. Ethyl acetate was added to the WYSP liquid medium at a volume ratio of 1:1.5, mixed thoroughly, and sonicated for 45 minutes. The mixture was then poured into a separatory funnel and allowed to stand for 12 hours to allow for separation. The supernatant was then extracted. The extract was condensed and dried under reflux on a rotary evaporator to obtain a crude WYSP liquid medium extract. Example 3:

[0031] Antimicrobial activity test of crude extracts from fermentation broth of P. crispatus DHJ068: The crude extract of the fermentation broth of P. cruzi DHJ068, the crude extract of the bacterial cells of P. cruzi DHJ068, and the crude extract of the WYSP liquid culture medium prepared in Example 2 above were used as samples; 1. Activation of pathogenic bacteria The pathogenic bacteria of cabbage soft rot ( Erwinia carotovora ) and Cucumber Angular Leaf Spot Fungus ( Pseudomonas syringae pv. lachrymans ), take 5 μL and add it into 2 mL centrifuge tubes respectively, then add 750 μL of LB liquid culture medium (tryptone 10 g / L + sodium chloride 10 g / L + yeast extract 5 g / L) to each centrifuge tube, and then place the centrifuge tube in a constant temperature shaker at 37°C and a speed of 180 r / min for 12 hours to obtain the pathogenic bacteria solution, which was placed in a 4°C refrigerator for use. Take it out before use and dilute the activated bacteria to 1 / 10.

[0032] 2. Detection of antibacterial activity by filter paper method The filtered mycelium was extracted with ethyl acetate and dried under reflux on a rotary evaporator to obtain a crude mycelial extract of P. crispatus DHJ068. 0.01 g of the crude extract from the fermentation broth, mycelium, and WYSP liquid medium was placed in a 2 mL centrifuge tube and diluted to 50 mg / mL with 500 μL of DMSO (dimethyl sulfoxide). The activated pathogenic bacterial suspension was evenly spread on LB solid medium (10 g / L tryptone, 10 g / L sodium chloride, 15 g / L agar, and 5 g / L yeast extract). After the smear dried, 5 mm filter paper discs containing the dissolved fermentation broth extract were placed at the corresponding marks on the medium. The medium was incubated at 37°C for 12 hours. The appearance of inhibition zones was observed and their diameters were measured using the cross-hatch method.

[0033] Depend on Figure 4 It can be seen that the inhibition zone of the crude extract of the fermentation broth of P. crispus DHJ068 against the pathogenic bacteria of cabbage soft rot is 16.2±0.2 mm ( Figure 4 ),Depend on Figure 5 It can be seen that the inhibition zone of the crude extract of the fermentation broth of P. crumberiae DHJ068 against bacterial angular leaf spot of cucumber is 16.4±0.2 mm, indicating that the crude extract of the fermentation broth of P. crumberiae DHJ068 has significant antibacterial activity against the pathogenic bacteria of cabbage soft rot and bacterial angular leaf spot of cucumber. The crude extract of P. crumberiae DHJ068 and the crude extract of WYSP liquid culture medium have no obvious inhibition zone, and therefore have no antibacterial activity.

[0034] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An endophytic fungus from the roots of Polygonatum yunnanensis, Pulingerella crispa ( Prillingera fragicola ) DHJ068, characterized in that The Pulingera crispa DHJ068 is preserved in the China Center for Type Culture Collection with the collection number of CCTCC NO: M2025748.

2. A use of the endophytic fungus Pulingerella brittlebone DHJ068 from the roots of Polygonatum cyrtonema as claimed in claim 1 in the prevention and control of crop diseases.

3. The use according to claim 2, characterized in that: The crop disease is caused by the pathogenic bacteria of cabbage soft rot Erwinia carotovora Cucumber bacterial leaf spot Pseudomonas syringae pv. lachrymans Diseases caused.

4. The use according to claim 2, characterized in that: The application method is to use the crude extract of the fermentation liquid of the endophytic fungus Pulingerella brittle fruit DHJ068 from the roots of Polygonatum cyrtonema to prepare a bacterial agent or a pharmaceutical composition for preventing and controlling crop diseases.

5. The use according to claim 4, characterized in that The preparation method of the crude fermentation broth extract comprises the following steps: (1) Activating the fungus strain Pulingerella crispa DHJ068; (2) The activated P. crispatus DHJ068 fungus was inoculated into WYSP liquid culture medium, and then placed in a constant temperature shaker at 28°C and 150 rpm for 20 days to obtain a fermentation broth; (3) The fermentation broth was placed in a 50 ml centrifuge tube and then centrifuged at 3000 g for 3 min to separate the bacteria and the fermentation broth. Ethyl acetate was added to the fermentation broth at a volume ratio of 1:1.5 and ultrasonicated for 30 min. The broth was then poured into a separatory funnel and allowed to stand for 12 h to separate the layers. The ethyl acetate supernatant was taken and condensed and refluxed on a rotary evaporator to obtain the crude extract of the fermentation broth.

6. The use according to claim 5, characterized in that: The WYSP liquid culture medium has a formula of: 10 g / L pea flour; 1 g / L yeast powder; 2 g / L sodium acetate; 1 g / L potassium propionate, and the balance is water.

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