A yunnan golden camellia root endophytic fungus and application thereof

Abstract

The application provides a Yunnan golden camellia root endophytic fungus and application thereof, and relates to the technical field of crop disease prevention and treatment. The Yunnan golden camellia root endophytic fungus is Lecanicillium attenuatum JHC098, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 2025749. The liquid fermentation broth extract of the fungus can be applied to crop disease prevention and treatment. The application overcomes the defects of the prior art. The liquid fermentation broth extract of the Yunnan golden camellia root endophytic fungus Lecanicillium attenuatum JHC098 can effectively inhibit the growth of citrus canker and tobacco bacterial wilt pathogenic bacteria, so that the purpose of biological prevention is achieved.

Description

Technical Field

[0001] This invention relates to the field of crop disease control technology, specifically to an endophytic fungus in the roots of a Camellia yunnanensis plant and its application. Background Technology

[0002] Yunnan golden camellia ( Camellia fascicularis Camellia yunnanensis, also known as the golden camellia, is a rare and precious species of yellow-flowered camellia in the genus Camellia of the Theaceae family. It is endemic to Yunnan Province, found only in Maguan County, Hekou County, and Gejiu City. Due to its high medicinal, economic, and ornamental value, its wild population has been continuously destroyed, leading to a sharp decline in its numbers. It has been listed as a priority protected species under the "National Project for the Rescue and Protection of Extremely Small Populations (2021-2035)" and the "List of Wild Plants with Extremely Small Populations in Yunnan Province (2022 Edition)". Camellia yunnanensis contains more than ten trace elements with important health benefits, including natural organic germanium, selenium, manganese, molybdenum, vanadium, and zinc, as well as tea polyphenols and essential amino acids. It is believed to have health benefits such as anti-cancer properties, inhibiting tumor growth, lowering blood lipids, preventing atherosclerosis, and improving the body's immune function.

[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 diversity, significant impact, and frequent outbreaks, often causing substantial losses to the national economy, particularly agricultural production. Citrus canker is caused by bacteria (…). Xanthomonas citri subsp citri Tobacco bacterial wilt is a bacterial disease that harms citrus plants. It can be spread by wind, rain, and insects, and is more likely to occur during hot and rainy seasons. The pathogenic bacteria for tobacco bacterial wilt (…) Ralstonia solanacearum This disease primarily harms plants in the Solanaceae family, such as tobacco. It can survive in the soil and spread through irrigation water and agricultural tools. High temperature and humidity environments easily induce the disease.

[0005] In crop cultivation, the long-term use of chemical products has led to increased variability in pathogens, frequently resulting in resistance to various pesticides and weakening the effectiveness of chemical pest control. Endophytic fungi, with their unique advantages, hold great promise for plant disease and pest control. Many endophytic fungi can produce antibiotic-like substances to enhance the disease resistance of host plants. These active substances not only act on plant pathogens but also inhibit other bacteria, fungi, and some protozoa. Screening for antibiotic-like substances from endophytic fungi has become an important aspect of new antibiotic sources, holding significant importance in medicine, the food industry, and agronomy. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an endophytic fungus found in the roots of Camellia yunnanensis and its applications. This endophytic fungus belongs to the flask-shaped scale fungus family, and it can effectively inhibit the growth of pathogenic bacteria causing citrus canker and tobacco bacterial wilt, thus achieving a certain degree of biological control.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An endophytic fungus on the roots of a Camellia yunnanensis plant, wherein the endophytic fungus on the roots of the Camellia yunnanensis plant is *Vallisneria natans* (a type of fungus). Lecanicillium saksenae JHC098, and the accession number is CCTCC NO: M 2025749.

[0009] The endophytic fungi in the roots of the Yunnan golden camellia can be used for the prevention and control of crop diseases.

[0010] Preferably, the crop disease is caused by the bacteria that cause citrus canker (Citrus canker). Xanthomonas citri subsp. citri ) and / or the bacteria that cause tobacco bacterial wilt ( Ralstonia solanacearum Diseases caused by ).

[0011] Preferably, the application method involves using the crude extract of the fermentation broth of *Camellia yunnanensis* root endophytic fungus *Varicaria yunnanensis* JHC098 to prepare a fungal agent or drug composition for the prevention and control of crop diseases.

[0012] Preferably, the method for preparing the crude extract of the fermentation broth of the bottle-shaped scale fungus JHC098 includes the following steps:

[0013] (1) Activate the fungus JHC098 of the bottle-shaped wax scale;

[0014] (2) The activated bottle-shaped wax scale fungus JHC098 was inoculated into MSGYD liquid medium and then placed in a constant temperature shaker at 28℃ and 150rpm for 20 days to obtain fermentation broth;

[0015] (3) Centrifuge the fermentation broth to separate the cell bodies and fermentation broth, add ethyl acetate to the fermentation broth at a volume ratio of 1:1.5, and sonicate for 30 min; then pour into a separatory funnel and let stand for 12 h to separate the layers; take the supernatant of ethyl acetate and dry it by reflux using a rotary evaporator to obtain the crude extract of the fermentation broth.

[0016] Preferably, the MSGYD liquid culture medium is formulated as follows: magnesium acetate 2g / L; soybean peptone 20g / L; glucose 20g / L; yeast extract 1g / L; sodium butyrate 0.5g / L, with the remainder being water.

[0017] Preferably, in step (3), the centrifugation speed is 3000g and the centrifugation time is 3min.

[0018] This invention provides an endophytic fungus on the roots of Camellia yunnanensis and its applications. Compared with existing technologies, its advantages are:

[0019] This invention obtained an antibacterial fungus by isolating and screening endophytic fungi from the roots of Camellia yunnanensis, with the preservation name *Valviformis ventricosa* JHC098. Lecanicillium saksenae JHC098, with accession number CCTCC NO: M2025749, was found through experiments to have a strong inhibitory effect on the growth of pathogenic bacteria causing citrus canker and tobacco bacterial wilt, thus effectively controlling plant diseases. Attached Figure Description

[0020] Figure 1 This is a diagram showing the mycelial growth of *Camellia yunnanensis* endophytic fungus *Varicaria pyriformis* JHC098, which grows on the roots of *Camellia yunnanensis* according to Example 1 of the present invention.

[0021] Figure 2 Micrograph of the mycelium of *Camellia yunnanensis* root endophytic fungus *Varicaria natans* JHC098, as described in Example 1 of this invention;

[0022] Figure 3 This is a diagram illustrating the phylogenetic tree of endophytic fungi constructed based on ITS in Embodiment 1 of the present invention.

[0023] Figure 4 This diagram illustrates the inhibitory effect of *Cyclocarya spp.* JHC098 on pathogenic bacteria causing citrus canker. 1 represents *Cyclocarya spp.* JHC098 mycelial extract; 2 represents *Cyclocarya spp.* JHC098 crude extract obtained through liquid fermentation; 3 represents dimethyl sulfoxide; and 4 represents MSGYD crude extract obtained through liquid culture medium.

[0024] Figure 5The diagram shows the inhibitory effect of the endophytic fungus *Phytophthora ventricosa* JHC098 on the pathogenic bacteria causing tobacco bacterial wilt. 1 is a mycelial extract of *Phytophthora ventricosa* JHC098; 2 is a crude extract of *Phytophthora ventricosa* JHC098 obtained through liquid fermentation; 3 is dimethyl sulfoxide; and 4 is a crude extract of MSGYD liquid culture medium. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The endophytic fungus on the roots of Camellia yunnanensis provided by this invention is preserved under the name *Vesselia spp.* JHC098. Lecanicillium saksenae JHC098; deposited at the China Center for Type Culture Collection, Wuhan University, China; received by the Center on April 10, 2025, and identified as viable on April 17, 2025; accession number CCTCC NO: M 2025749.

[0027] The following examples use the pathogenic bacteria of citrus canker ( Xanthomonas citri subsp. citri )for Xanthomonas citri subsp. citri (ex Hasse) Gabriel et al. strain 49118, purchase link: http: / / www.biovector.net / product / 1249290.html;

[0028] The bacteria causing bacterial wilt of tobacco used ( Ralstonia solanacearum )for Ralstonia solanacearum (Smith) Yabuuchi et al. strain BAA-1114, can be purchased via the following link: http: / / www.biovector.net / product / 1228695.html. Example 1:

[0029] Isolation of endophytic fungi from the roots of Camellia yunnanensis

[0030] The roots of *Camellia yunnanensis* were collected from Gulinqing Township, Maguan County, Wenshan Prefecture, Yunnan Province. The roots, 60cm above the ground, were cut with scissors and placed in centrifuge tubes. They were then stored in the field with ice packs at low temperatures. Upon arrival at the laboratory, the roots were rinsed with tap water for 48 hours, cut into 2cm pieces, and then transferred to a sterile workbench for disinfection. They were rinsed with 1L of distilled water, then soaked in 70% ethanol for 2 minutes, the ethanol was discarded, and the roots were rinsed 6 times with 50mL of sterile water. Next, they were soaked in a 10% sodium hypochlorite solution for 1 minute, the sodium hypochlorite solution was discarded, and the roots were rinsed 10 times with 50mL of sterile water each time.

[0031] Cut the 2cm tissue pieces into 0.5cm length and width blocks, place them in centrifuge tubes, add steel balls and 1mL of sterile water, and crush for 2 minutes (180rpm). Dilute 100 times and plate 10 KASPYM agar plates (KASPYM: potato starch 8g / L; yeast extract 4g / L; maltose 15g / L; 100μg / mL kanamycin; 100μg / mL ampicillin; 100μg / mL streptomycin, the remainder being water). Incubate for 7 days. After bacterial growth, select single colonies and identify them.

[0032] The grown mycelia were transferred to malt extract broth solid medium and cultured for 15 days. Molecular identification of the strain revealed *Valva vesicularis* strain JHC098. The malt extract broth solid medium consisted of the following components: 15 g / L malt extract broth + 16 g / L agar (the remainder being water).

[0033] 2. Identification of endophytic fungi in the roots of Camellia yunnanensis

[0034] (1) Morphological identification

[0035] The morphology of this strain is as follows Figure 1 As shown, the fungi grew well on malt extract broth solid medium, with dense, off-white colonies.

[0036] (2) DNA extraction

[0037] ① Before the experiment, heat CTAB (hexadecyltrimethylammonium bromide) in a 65℃ water bath for 30 minutes;

[0038] ② Take 50 mg of dried bottle-shaped scale insect mycelium JHC098 into a 2 mL centrifuge tube, add 3 small steel balls, place the centrifuge tube in 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, and then transfer the mixture into a centrifuge tube containing 200 μL of PVP (polyvinylpyrrolidone). Add 20 μL of β-mercaptoethanol while the tube is suspended in a fume hood, shake for 15 s to grind thoroughly, and then place it in a 65°C water bath for 1.5 h, turning it upside down 5-6 times every 10 min. After the water bath, centrifuge at 12000 r / min, 4°C for 10 min.

[0039] ③ Take 1 mL of supernatant into a new centrifuge tube, add 500 μL each of DNA phenol reagent and chloroform-isoamyl alcohol mixture, invert for 10 min, and centrifuge (4℃, 12000 r / min) for 10 min (repeat step ③ twice).

[0040] ④ Take 900 μL of the supernatant and put it into a new centrifuge tube. Add 50 μL of 3 mol sodium acetate solution and 900 μL of 95% anhydrous ice ethanol (-20℃). Shake well and place in a -20℃ refrigerator for precipitation for 3 hours.

[0041] ⑤ After precipitation, centrifuge (4℃·12000r / min) for 10min, discard the supernatant, add 500μL of 75% alcohol, invert 2-3 times, let stand for 3min, and discard the supernatant;

[0042] ⑥ Add 500 μL of 95% ethanol, invert the container 2-3 times, let stand for 3 minutes, centrifuge at room temperature (13000 rpm) for 3 minutes, discard the ethanol, and let it dry.

[0043] ⑦ Add 40 μL of elution buffer EB, centrifuge at room temperature (13000 rpm) for 1.5 min to obtain the genomic DNA of fungus JHC098.

[0044] (3) ITS analysis and identification

[0045] The spacer sequence (containing the ITS1 region, 5.8S region, and ITS4 region) of fungal rDNA was amplified using the universal fungal primers ITS1 (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The resulting ITS sequencing sequence is shown in SEQ ID No. 1.

[0046] SEQ ID No. 1:

[0047]

[0048] (4) Constructing a developmental tree

[0049] A phylogenetic tree of plant endophytic fungi was constructed using MEGA software based on ITS. Figure 3 Based on the combined morphological and molecular biological identification results, strain JHC098 was identified as... Lecanicillium saksenae Bottle-shaped wax scale fungus. Example 2:

[0050] Preparation of crude extract from liquid fermentation of endophytic fungi in the roots of Camellia yunnanensis:

[0051] 1. Liquid culture

[0052] Mycelium of the endophytic fungus *Varicaria yunnanensis* JHC098 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of MSGYD liquid medium (2 g / L magnesium acetate; 20 g / L soybean peptone; 20 g / L glucose; 1 g / L yeast extract; 0.5 g / L sodium butyrate, with the remainder being water). The flask was placed in a constant temperature shaker at 28 °C and 150 rpm and cultured for 10 days to obtain the fermentation broth.

[0053] 2. Preparation of crude extract from liquid culture

[0054] Place the fermentation broth into a 50 ml centrifuge tube, then centrifuge at 3000 g for 3 min to separate the bacterial cells and the fermentation broth.

[0055] Ethyl acetate was added to the fermentation broth at a ratio of 1:1.5, followed by sonication for 30 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 by reflux using a rotary evaporator to obtain the crude liquid fermentation extract of *Camellia yunnanensis* endophytic fungus *Varicella yunnanensis* JHC098.

[0056] 3. Preparation of crude bacterial extract

[0057] The fermentation broth was placed in a 50 ml centrifuge tube and centrifuged at 3000 g for 3 min to obtain *Phytophthora ventricosa* JHC098 cells. The *Phytophthora ventricosa* JHC098 cells were then placed in an Erlenmeyer flask, and 95% ethanol was added to submerge the cells. After sonication for 30 min and standing for 12 h, the extract was filtered through filter paper to obtain the cell extract. The extract was dried by reflux using a rotary evaporator to obtain a crude extract of *Phytophthora ventricosa* JHC098 cells, an endophytic fungus found in the roots of *Camellia yunnanensis*.

[0058] 4. Preparation of crude extract from MSGYD liquid culture medium

[0059] Ethyl acetate was added to MSGYD liquid 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 MSGYD liquid medium. Example 3:

[0060] Antibacterial activity detection of crude extract from liquid fermentation of endophytic fungi in the roots of Camellia yunnanensis:

[0061] The crude extracts of *Bottle-type wax scale* JHC098 prepared in liquid fermentation, crude extracts of *Bottle-type wax scale* JHC098 cells, and crude extracts of MSGYD liquid culture medium prepared in Example 2 above were used as samples.

[0062] 1. Activation of pathogenic bacteria

[0063] The pathogenic bacteria of citrus canker ( Xanthomonas citri subsp. citri ) and the bacteria that cause tobacco bacterial wilt ( Ralstonia solanacearum 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.

[0064] 2. Detection of antibacterial activity using the filter paper disc method

[0065] 0.01g of crude extracts from liquid fermentation of *Bacillus cereus* JHC098, crude extracts of *Bacillus cereus* JHC098 cells, and crude extracts from MSGYD liquid medium were used as materials for each experimental group. These were added to 2mL centrifuge tubes, followed by 500μL of DMSO (dimethyl sulfoxide) solution to dilute to 50mg / mL. A DMSO group served as a control group. The activated pathogenic bacterial solution was evenly spread onto LB solid medium (10g / L tryptone + 10g / L sodium chloride + 15g / L agar + 5g / L yeast extract). After drying, 5mm filter paper discs containing the diluted materials from each experimental group and control group were placed at the corresponding marked positions on the medium. The medium was then incubated upright in a 37℃ incubator for 12h. The presence of inhibition zones was observed, and their diameter was measured using the cross-sectional method.

[0066] Depend on Figure 4 It was found that the crude extract of *Cyclocarya paliurus* JHC098, obtained through liquid fermentation, exhibited an inhibition zone of 15.2 ± 0.2 mm against the pathogenic bacteria causing citrus canker. Figure 4 ),Depend on Figure 5The inhibition zone of the crude extract of *Vallisneria natans* JHC098 obtained through liquid fermentation against the pathogens causing tobacco bacterial wilt was 15.8 ± 0.2 mm, indicating that the crude extract of *Vallisneria natans* JHC098 obtained through liquid fermentation has significant antibacterial activity against the pathogens causing citrus canker and tobacco bacterial wilt. The crude extracts of *Vallisneria natans* JHC098 cells and the crude extracts obtained from MSGYD liquid culture medium did not have obvious inhibition zones and therefore had no antibacterial activity.

[0067] 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. A strain of endophytic fungus in the root of Camellia yunnanensis, characterized in that, The endophytic fungus in the roots of the Yunnan golden camellia is *Vallisneria natans* (also known as *Vallisneria natans*). Lecanicillium saksenae JHC098, and the accession number is CCTCC NO: M 2025749.

2. The application of the endophytic fungus in the root of Yunnan golden flower tea in the prevention and treatment of crop diseases according to claim 1, characterized by: The crop disease is a disease caused by citrus canker pathogenic bacteria ( Xanthomonas citri subsp. citri ) and / or tobacco bacterial wilt pathogenic bacteria ( Ralstonia solanacearum ); and the application mode is to use the fermentation broth of Lecanicillium attenuatum JHC098, an endophytic fungus in the roots of Yunnan golden tea, to prepare a microbial agent or a pharmaceutical composition for preventing and treating crop diseases.

3. Use according to claim 2, characterized in that, The preparation method of the fermentation broth crude extract of the Ampelomyces quisqualis JHC098 includes the following steps: (1) activating the fungus of the Ampelomyces quisqualis JHC098; (2) inoculating the activated Ampelomyces quisqualis JHC098 fungus into MSGYD liquid medium, and then placing it in a constant temperature shaker at 28℃ and 150rpm for 20d to obtain fermentation broth; (3) centrifuging the fermentation broth to separate the bacteria and the fermentation broth, adding ethyl acetate to the fermentation broth at a volume ratio of 1:1.5, and ultrasonicating for 30min; then pouring it into a separatory funnel, standing for 12h to make it stratify, and then taking the ethyl acetate supernatant and condensing it to dryness by rotary evaporation to obtain the fermentation broth crude extract.

4. Use according to claim 3, characterized in that: The formula of the MSGYD liquid medium is: magnesium acetate 2g / L; soybean peptone 20g / L; glucose 20g / L; yeast powder 1g / L; sodium butyrate 0.5g / L, and the rest is water.

5. Use according to claim 3, characterized in that: The centrifugal speed in the step (3) is 3000g, and the centrifugal time is 3min.

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