A smilax china root endophytic fungus pringheimia fragilis dhj068 and application thereof

Abstract

The present application provides a kind of diandong yam root endophytic fungus fragile fruit pringlea DHJ068 and its application, it is related to crop disease prevention and treatment technical field.The diandong yam root endophytic fungus fragile fruit pringlea DHJ068 is preserved in China typical culture collection, and the preservation number is CCTCC NO:M 2025748, and the fermentation broth extract of the bacteria can be applied to crop disease prevention and treatment.The present application overcomes the shortcomings of the prior art, the fermentation broth extract of diandong yam root endophytic fungus fragile fruit pringlea DHJ068 can effectively inhibit the growth of Chinese cabbage soft rot bacteria and cucumber bacterial angular spot bacteria, and achieve certain biological control purposes.

Description

Technical Field

[0001] This invention relates to the field of crop disease control technology, specifically to a root endophytic fungus, *Principis cristatus* DHJ068, and its applications. Background Technology

[0002] Yunnan Polygonatum ( Polygonatum kingianum It is mainly distributed in Yunnan, Sichuan, Guizhou and other provinces in China. Its rhizome is used medicinally, possessing the effects of 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 weakness, fatigue, dry mouth and poor appetite, dry cough due to lung deficiency, insufficient essence and blood, and internal heat and thirst. Furthermore, in modern medicine, *Polygonatum yunnanense* has various pharmacological effects, including antibacterial, antifungal, and antihypertensive properties.

[0003] Plant endophytic fungi are fungi that exist within healthy plant tissues, spending one or all stages of their life cycle within the plant's tissues and organs without causing obvious infection symptoms in their 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 have unique living environments that promote host plant growth, such as producing antibiotic resistance substances, enhancing drought resistance, and increasing the host plant's tolerance to heavy metals. Terpenoids in plant endophytic fungi exhibit novel structures and significant biological activity. Current research has identified the following chemical components in the secondary metabolites of plant endophytic fungi: terpenoids, steroids, flavonoids, peptides, aliphatic compounds, alkaloids, esters, and phenylpropanoids, most of which possess broad pharmacological activities, such as anti-inflammatory, antitumor, and antibacterial effects.

[0004] Crop diseases and pests are one of the major agricultural disasters in my country. They are characterized by their wide variety, significant impact, and frequent outbreaks, often causing substantial losses to the national economy, particularly agricultural production. One example is the soft rot pathogen of Chinese cabbage (…). Erwinia carotovora The occurrence of this disease is related to the number of wounds caused by field pests, human or natural factors, and black rot. Wounds in Chinese cabbage are mainly classified as natural cracks, insect damage, disease scars, and mechanical injuries. Among these, longitudinal cracks on the petioles are the most common and are the primary route of entry for the disease. In production, prolonged drought followed by rain, excessive seedling hardening, or overwatering can all cause wounds and lead to disease. Waterlogging, a lack of hydrogen in the soil, and the resulting detrimental effects on root development or corking of wounds all exacerbate the disease. The bacterial angular leaf spot pathogen of cucumber (… Pseudomonas syringae pv. lachrymans This disease mainly affects cucumbers, and can affect both seedlings and mature plants, with leaves being the primary site of infection in mature plants. It primarily damages leaves, petioles, tendrils, and fruits, and sometimes also infects stems and vines. When cotyledons are infected, they initially appear as water-soaked, nearly circular, sunken spots, which later become slightly yellowish-brown and dry.

[0005] In the process of crop planting, the pathogenicity of the bacteria is gradually enhanced due to the long-term use of chemicals, and the resistance to various drugs is often generated, resulting in the weakening of the effect of chemical pesticides on pest control. Endophytic fungi have broad application prospects in plant disease and pest control due to their unique advantages. Many endophytic fungi can produce antibiotic substances to improve the disease resistance of host plants. These active substances not only act on plant pathogens, but also inhibit other bacteria, fungi and some protists. Screening of antibiotic substances from endophytic fungi has become an important aspect of new antibiotic sources, which has important significance in medicine, food industry and agronomy. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a kind of diandong yam root endophytic fungus fragile pringlea DHJ068 and its application, diandong yam root endophytic fungus fragile pringlea DHJ068 can effectively inhibit the growth of soft rot pathogenic bacteria and cucumber bacterial angular spot bacteria, and achieve the purpose of biological control.

[0007] To achieve the above object, the technical scheme of the present application is realized by the following technical scheme:

[0008] A kind of diandong yam root endophytic fungus fragile pringlea DHJ068, the fragile pringlea DHJ068 is preserved in China typical culture collection center, and the preservation number is CCTCC NO: M 2025748. Prillingera fragicola )DHJ068, the fragile pringlea DHJ068 is preserved in China typical culture collection center, and the preservation number is CCTCC NO: M 2025748.

[0009] Diandong yam root endophytic fungus fragile pringlea DHJ068 is applied to crop disease control.

[0010] Preferably, the crop disease is caused by soft rot pathogenic bacteria of Chinese cabbage Erwinia carotovora And cucumber bacterial angular spot bacteria Pseudomonas syringae pv. lachrymans .

[0011] Preferably, the application mode is to prepare a microbial agent or a pharmaceutical composition by using the fermentation broth extract of diandong yam root endophytic fungus fragile pringlea DHJ068 to prevent and control crop diseases.

[0012] Preferably, the preparation method of the fermentation broth extract comprises the following steps:

[0013] (1) the fungus of fragile pringlea DHJ068 is activated;

[0014] (2) the activated fragile pringlea DHJ068 fungus is inoculated into WYSP liquid medium, and then placed in a constant temperature shaking bed at 28 DEG C, 150 rpm, and cultured for 20d to obtain fermentation broth;

[0015] (3) the fermentation broth is placed into a 50 ml centrifuge tube, then centrifuged at 3000 g for 3 min, the mycelium and the fermentation broth are separated, ethyl acetate is added into the fermentation broth at a volume ratio of 1:1.5, and ultrasonic treatment is performed for 30 min; then the mixture is poured into a separatory funnel, and after being allowed to stand for 12 h to form layers, the ethyl acetate supernatant is taken out and dried by condensation reflux using a rotary evaporator to obtain the crude extract of the fermentation broth.

[0016] Preferably, the formula of the WYSP liquid medium is as follows: 10 g / L of pea powder, 1 g / L of yeast powder, 2 g / L of sodium acetate, 1 g / L of potassium propionate, and the balance being water.

[0017] The present application provides a kind of diandong Huanggen endophytic fungi fragile fruit pringlea DHJ068 and its application, compared with prior art, the advantages are as follows:

[0018] The present application is isolated and screened from diandong Huanggen endophytic fungi, and a fungus fragile fruit pringlea DHJ068 with antibacterial activity is obtained.The fermentation broth extract of the fungus has strong inhibitory effect on the growth of Erwinia carotovora and Pseudomonas syringae pv.Lactucae, and can effectively prevent and control plant diseases. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the mycelium growth chart of diandong Huanggen endophytic fungi fragile fruit pringlea DHJ068 of the present application embodiment 1;

[0020] Figure 2 It is the mycelium micrograph of diandong Huanggen endophytic fungi fragile fruit pringlea DHJ068 of the present application embodiment 1;

[0021] Figure 3 It is the endophytic fungi phylogenetic tree display chart based on ITS of the present application embodiment 1;

[0022] Figure 4 It is the inhibition chart of diandong Huanggen endophytic fungi fragile fruit pringlea DHJ068 on Erwinia carotovora;1 is the mycelium extract of fragile fruit pringlea DHJ068;2 is the crude extract of fermentation broth of fragile fruit pringlea DHJ068;3 is the crude extract of WYSP liquid medium;4 is DSMO;

[0023] Figure 5 It is the inhibition chart of diandong Huanggen endophytic fungi fragile fruit pringlea DHJ068 on Pseudomonas syringae pv.Lactucae;1 is the mycelium extract of fragile fruit pringlea DHJ068;2 is the crude extract of fermentation broth of fragile fruit pringlea DHJ068;3 is the crude extract of WYSP liquid medium;4 is DSMO. DETAILED DESCRIPTION

[0024] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0025] The endophytic fungus of the roots of Polygonatum yunnanense provided by the present application is named Pringsheimia fragilis Prillingera fragicola DHJ068, which is preserved in China Center for Type Culture Collection, Wuhan University, China, was received by China Center for Type Culture Collection on April 10, 2025, and was detected as alive on April 17, 2025, and the preservation number is CCTCC NO: M 2025748.

[0026] The pathogenic bacteria of Chinese cabbage soft rot bacteria used in the following embodiments are Erwinia carotovora Erwinia carotovora The pathogenic strain is purchased from the following link: http: / / www.biovector.net / product / 210298.html.

[0027] The bacterial angular spot of cucumber used is Pseudomonas syringae pv. lachrymans Pseudomonas syringae pv. lachrymans Pseudomonas syringae pv. cucumbers, and the purchase link is: http: / / www.biovector.net / product / 2096192.html. Embodiment 1

[0028] 1. Isolation of endophytic fungi of the roots of Polygonatum yunnanense

[0029] (1) The roots of Polygonatum yunnanense were collected from Jizhu Mountain in Binchuan County, Dali City, Yunnan Province. The Polygonatum yunnanense was dug out with roots and placed in a self-sealing bag, and kept at low temperature with an ice bag. After being brought back to the laboratory, it was washed with tap water for 48 hours, then cut into small pieces of 2 cm, and then transferred to a sterile workbench for disinfection treatment. 1L of distilled water was used to rinse, then 70% ethanol was used to soak for 2 minutes, the ethanol was poured out, 50mL of sterilized water was used to rinse for 6 times, then sodium hypochlorite (10%) solution was used to soak for 1 minute, the sodium hypochlorite solution was poured out, and 50mL of sterilized water was used to rinse for 10 times (50mL each time).

[0030] ​​(2) Cut 2 cm small pieces into 0.5 cm length and width, put into centrifuge tube, add steel beads, 1 mL sterilized water, crush for 2 minutes (180 rpm). Dilute 100 times and plate 10 PYGKAS media (PYGKAS: 3 g / L of Dioscorea deltoidea powder, 4 g / L of yeast powder, 10 g / L of glucose, 100 μg / mL of carbonyl, 100 μg / mL of ampicillin, 100 μg / mL of streptomycin, and the rest is water). Culture for 10 days, after the growth of colonies, select single colonies; identify the single colonies.

[0031] (3) Transfer the grown mycelium to PDY solid medium for 15 days, then perform molecular identification on the strain to obtain the Pringelina fragilis DHJ068 strain. The PDY solid medium comprises the following components: 15 g / L of potato powder, 5 g / L of glucose, 2 g / L of yeast powder, 13 g / L of agar, and the rest is water.

[0032] 2. Identification of the endophytic fungi in the roots of Dioscorea deltoidea

[0033] (1) Morphological identification

[0034] The strain morphology is shown in Figure 1 and Figure 2 The fungus grows well on malt extract powder solid medium, the colony surface is wrinkled and raised, and the mycelium is light yellow.

[0035] (2) DNA extraction

[0036] ① Before the experiment, heat CTAB (hexadecyl trimethyl ammonium bromide) in a 65°C water bath for 30 min;

[0037] ② Take 50 mg of dried Pringelina fragilis DHJ068 mycelium in a 2 mL centrifuge tube, add 3 small steel beads, put the centrifuge tube into liquid nitrogen for 6 min, and immediately crush it with a crusher for 2 min, add 1 mL of preheated CTAB solution, mix well with a pipette gun, then transfer it into a centrifuge tube containing 200 μL of PVP (polyvinyl pyrrolidone), add 20 μL of β-mercaptoethanol in the fume hood, shake for 15 s to grind thoroughly, then put it in a 65°C water bath for 1.5 h, turn it up and down for 5-6 times every 10 min, after the water bath, centrifuge at 12000 r / min and 4°C for 10 min;

[0038] ③ Take 1 mL of supernatant into a new centrifuge tube, add 500 μL of DNA phenol reagent and chloroform-isopentanol mixture, turn it up and down for 10 min, centrifuge (4°C, 12000 r / min) for 10 min (step ③ is repeated twice);

[0039] IV. Take 900 μL of supernatant into a new centrifuge tube, add 50 μL of 3 mol sodium acetate solution and 900 μL of 95% anhydrous ice ethanol (-20°C), shake well and put into a -20°C refrigerator for precipitation for 3 h;

[0040] V. Centrifuge (4°C, 12000 r / min) for 10 min after precipitation, discard the supernatant, add 500 μL of 75% alcohol, turn up and down for 3 times, stand for 3 min, discard the supernatant;

[0041] VI. Add 500 μL of 95% alcohol, turn up and down for 3 times, stand for 3 min, centrifuge (13000 rpm) for 3 min at room temperature, discard the ethanol and dry;

[0042] VII. Add 40 μL of elution buffer EB, centrifuge (13000 rpm) for 1.5 min at room temperature, and obtain the genomic DNA of Pristinamycetin Streptomyces DHJ068.

[0043] (3) ITS analysis and identification

[0044] The ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') universal primers of fungi were used to amplify the rDNA spacer sequence (containing ITS1 region, 5.8S region and ITS4 region), and the obtained ITS sequencing sequence is shown as SEQ ID No. 1.

[0045] SEQ ID No. 1:

[0046]

[0047] (4) Constructing the development tree

[0048] Based on ITS, the phylogenetic tree of plant endophytic fungi was constructed by MEGA software Figure 3 According to the results of morphological identification and molecular biological identification, the strain DHJ068 was identified as Prillingera fragicola. Example 2:

[0049] Preparation of crude extract of Prillingera fragicola DHJ068 fermentation broth:

[0050] 1. Liquid culture

[0051] The mycelium of Prillingera fragicola DHJ068 in the roots of Polygonatum yunnanense was inoculated into a 250 mL conical flask containing 100 ml of WYSP liquid medium (pea powder 10 g / L; yeast powder 1 g / L; sodium acetate 2 g / L; potassium propionate 1 g / L, and the rest water), and placed in a constant temperature shaker at 28°C and 150 rpm for 20 d.

[0052] 2. Preparation of crude extract of fermentation broth

[0053] The fermentation broth was placed in a 50 ml centrifuge tube, and then centrifuged at 3000 g for 3 min to separate the mycelium and the fermentation broth.

[0054] Ethyl acetate was added to the fermentation broth at a ratio of 1:1.5, and then ultrasonicated for 30 min. Then it was poured into a separatory funnel, and allowed to separate into layers for 12 h to extract the supernatant. The extraction solution was condensed and refluxed to dryness by a rotary evaporator to obtain the crude extract of Prillingera fragicola DHJ068 fermentation broth.

[0055] 3. Preparation of crude extract of mycelium

[0056] The fermentation broth was placed in a 50 ml centrifuge tube, and then centrifuged at 3000 g for 3 min to obtain the mycelium of Prillingera fragicola DHJ068. The mycelium of Prillingera fragicola DHJ068 was placed in a triangular flask, and ethyl acetate was added to submerge the mycelium. After ultrasonication for 30 min, it was allowed to stand for 12 h, and then filtered with gauze to obtain the mycelium extract. The mycelium extract was condensed and refluxed to dryness by a rotary evaporator to obtain the crude extract of Prillingera fragicola DHJ068 mycelium.

[0057] 4. Preparation of crude extract of WYSP liquid medium

[0058] After sterilizing a 250 mL Erlenmeyer flask containing 100 mL of WYSP liquid culture medium, the flask was placed in a constant temperature shaker at 28°C and 150 rpm for 20 days. Ethyl acetate was added to the WYSP liquid culture medium at a volume ratio of 1:1.5, and the mixture was sonicated for 45 min. The mixture was then poured into a separatory funnel and allowed to stand for 12 h to allow for layering. The supernatant was then extracted. The extract was dried under reflux using a rotary evaporator to obtain the crude extract of WYSP liquid culture medium. Example 3:

[0059] Antibacterial activity assay of crude extract from fermentation broth of *Principia fragilis* DHJ068:

[0060] The crude extracts of fermentation broth of Pringles fragilis DHJ068, the crude extracts of Pringles fragilis DHJ068 cells, and the crude extracts of WYSP liquid culture medium prepared in Example 2 above were used as samples.

[0061] 1. Activation of pathogenic bacteria

[0062] The pathogenic bacteria that cause soft rot in cabbage ( Erwinia carotovora ) and cucumber bacterial angular leaf spot ( Pseudomonas syringae pv. lachrymans Take 5 μL and add it to 2 mL centrifuge tubes. Then add 750 μL of LB liquid medium (tryptone 10 g / L + sodium chloride 10 g / L + yeast extract 5 g / L) to each centrifuge tube. Then place the centrifuge tubes in a constant temperature shaker at 37℃ and 180 r / min for 12 h to obtain pathogenic bacteria solution. Store it in a 4℃ refrigerator for later use. Before use, take it out and dilute the activated bacteria to 1 / 10.

[0063] 2. Detection of antibacterial activity using the filter paper disc method

[0064] The filtered mycelium was extracted with ethyl acetate and dried by reflux using a rotary evaporator to obtain a crude extract of *Principia fragilis* DHJ068 mycelium. 0.01 g of the crude extracts from the fermentation broth, mycelium, and WYSP liquid medium of *Principia fragilis* DHJ068 were placed in a 2 mL centrifuge tube, and 500 μL of DMSO (dimethyl sulfoxide) solution was added to dilute to 50 mg / mL. The activated pathogenic bacterial solution was evenly spread on LB solid medium (10 g / L tryptone + 10 g / L sodium chloride + 15 g / L agar + 5 g / L yeast extract). After drying, 5 mm filter paper discs containing dissolved crude fermentation broth extract were placed at the corresponding marked points on the medium. The medium was incubated at 37°C for 12 h, and the appearance of inhibition zones was observed. The diameter of the inhibition zones was measured using the cross-sectional method.

[0065] Depend on Figure 4It can be seen that the crude extract of *Principia fragilis* DHJ068 fermentation broth has an inhibition zone of 16.2 ± 0.2 mm against the pathogenic bacteria causing soft rot of Chinese cabbage. Figure 4 ),Depend on Figure 5 The crude extract of *Principia fragilis* fermentation broth DHJ068 showed an inhibition zone of 16.4 ± 0.2 mm against *Cucumis mellitus* bacterial angular leaf spot, indicating that the crude extract of *Principia fragilis* fermentation broth DHJ068 has significant antibacterial activity against pathogenic bacteria of *Cabbage soft rot* and *Cucumis mellitus* bacterial angular leaf spot. The crude extracts of *Principia fragilis* bacterial cells DHJ068 and WYSP liquid culture medium did not show obvious inhibition zones, therefore they had no antibacterial activity.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An endophytic fungus in the roots of Polygonatum yunnanense, *Principia fragilis* ( ) Prillingera fragicola DHJ068, characterized in that, The Prillingera fragicola DHJ068 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2025748.

2. The application of the Prillingera fragicola DHJ068 in the prevention and treatment of crop diseases caused by the pathogenic bacteria Erwinia carotovora and Pseudomonas syringae pv. lachrymans.

3. Use according to claim 2, characterized in that: The application is to use the fermentation broth of the Prillingera fragicola DHJ068 to prepare a microbial agent or a pharmaceutical composition for the prevention and treatment of crop diseases.

4. Use according to claim 3, characterized in that, The preparation method of the fermentation broth comprises the following steps: (1) activating the Prillingera fragicola DHJ068 fungus; (2) inoculating the activated Prillingera fragicola DHJ068 fungus into a WYSP liquid medium, and then placing it in a constant temperature shaker at 28℃ and 150 rpm for 20 days to obtain a fermentation broth; (3) placing the fermentation broth into a 50 ml centrifuge tube, and then centrifuging it at 3000 g for 3 min to separate the fungus and the fermentation broth, adding ethyl acetate into the fermentation broth at a volume ratio of 1:1.5, and then ultrasonicating for 30 min; immediately pouring it into a separatory funnel, and then standing it for 12 h to make it stratify; taking the ethyl acetate supernatant, and then condensing and refluxing it to dryness by using a rotary evaporator to obtain the fermentation broth.

5. Use according to claim 4, characterized in that: The formula of the WYSP liquid medium is as follows: 10 g / L of pea powder, 1 g / L of yeast powder, 2 g / L of sodium acetate, 1 g / L of potassium propionate, and the rest is water.

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