Yunnan golden camellia root endophytic fungus and application thereof

Abstract

The invention provides Yunnan golden camellia root endophytic fungi and application thereof, and relates to the technical field of crop disease control. The Yunnan camellia nitidissima root endophytic fungus is Lecanicillium vase JHC098 and is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 2025749, and a liquid fermentation liquor crude extract of the Lecanicillium vase can be applied to prevention and treatment of crop diseases. The invention overcomes the defects in the prior art, and the crude extract of the liquid fermentation liquor of the endophytic fungus Lecanicillium viculare JHC098 at the root of Yunnan golden camellia can effectively inhibit the growth of pathogenic bacteria such as citrus canker and tobacco bacterial wilt, thereby achieving the purpose of certain biological control.

Description

Technical Field

[0001] The invention relates to the technical field of crop disease prevention and control, and in particular to an endophytic fungus in the root of Yunnan Camellia chrysantha and an application thereof. Background Art

[0002] Yunnan Camellia chrysantha Camellia fascicularis ), also known as Camellia chrysantha, is one of the precious and rare species with yellow flowers in the genus Camellia of the Theaceae family. It is endemic to Yunnan and is only distributed in Maguan County, Hekou County and Gejiu City, Yunnan Province. Due to its high medicinal, economic and ornamental value, its wild population has been continuously destroyed, and its number in the wild has dropped sharply. It has been listed as a priority protected extremely small population species in the "National Extremely Small Population Rescue and Protection Project Plan (2021-2035)" and the "Yunnan Province Extremely Small Population Wild Plant Protection List (2022 Edition)". Yunnan Camellia chrysantha contains more than ten trace elements and tea polyphenols with important health value to the human body, such as natural organic germanium, selenium, manganese, molybdenum, vanadium, zinc, and essential amino acids for the human body. It has health benefits such as preventing cancer, inhibiting tumor growth, lowering blood lipids, preventing atherosclerosis, and improving the body's immune function.

[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. Xanthomonas citri subsp citri ) is a bacterial disease that harms citrus plants and can be spread by wind, rain, insects, etc. It is more likely to occur in hot and rainy seasons. Ralstonia solanacearum ) mainly harms Solanaceae plants such as tobacco, can survive in the soil, and is spread through irrigation water, agricultural tools, etc. High temperature and high humidity environment can easily induce the disease.

[0005] During crop cultivation, the long-term use of chemicals leads to increased pathogen variability, often leading to the development of resistance to various drugs, which in turn weakens the effectiveness of chemical pest control. Endophytic fungi, with their unique advantages, hold broad application prospects in plant disease and insect pest control. Many endophytic fungi produce antibiotics that enhance the disease resistance of host plants. These active substances not only act against plant pathogens but also inhibit other bacteria, fungi, and some protozoa. Screening for antibiotics from endophytic fungi has become an important source of new antibiotics, with significant implications in medicine, the food industry, and agronomy. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides an endophytic fungus in the roots of Yunnan Camellia chrysantha and its application. The endophytic fungus in the roots of Yunnan Camellia chrysantha belongs to the bottle-shaped wax scale fungus, and the fungus can effectively inhibit the growth of pathogenic bacteria of citrus canker and tobacco bacterial wilt, thereby achieving a certain biological control purpose.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: An endophytic fungus from the roots of Yunnan Camellia chrysantha, wherein the endophytic fungus from the roots of Yunnan Camellia chrysantha is a bottle-shaped wax scale fungus ( Lecanicillium saksenae ) JHC098, and the deposit number is CCTCC NO: M 2025749.

[0008] The endophytic fungi in the roots of Camellia chrysantha can be used for preventing and controlling crop diseases.

[0009] Preferably, the crop disease is citrus canker pathogenic bacteria ( Xanthomonas citri subsp. citri ) and / or tobacco bacterial wilt pathogenic bacteria ( Ralstonia solanacearum ) caused by diseases.

[0010] Preferably, the application method is to use the crude extract of the fermentation broth of the endophytic fungus Lecanopus lecanii JHC098 from the roots of Camellia chrysantha to prepare a bacterial agent or a pharmaceutical composition for preventing and controlling crop diseases.

[0011] Preferably, the method for preparing the crude extract of the fermentation broth of Lecanococcus lecanii JHC098 comprises the following steps: (1) Activating the fungus strain of Lecanococcus lecanii JHC098; (2) The activated fungus JHC098 was inoculated into MSGYD liquid culture medium, and then placed in a constant temperature shaker at 28°C and 150 rpm for 20 days to obtain a fermentation liquid; (3) The fermentation broth was centrifuged to separate the bacterial cells and the fermentation broth, and ethyl acetate was added to the fermentation broth at a volume ratio of 1:1.5, and ultrasonicated for 30 minutes; then the broth was poured into a separatory funnel and allowed to stand for 12 hours 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 formula of the MSGYD liquid culture medium is: 2 g / L magnesium acetate; 20 g / L soy peptone; 20 g / L glucose; 1 g / L yeast powder; 0.5 g / L sodium butyrate, and the balance is water.

[0013] Preferably, the centrifugal speed in step (3) is 3000g and the centrifugal time is 3 minutes.

[0014] The present invention provides an endophytic fungus from the roots of Yunnan Camellia chrysantha and its application, which has the following advantages over the prior art: The present invention obtains a fungus with antibacterial activity by isolating and screening the endophytic fungi from the roots of Yunnan Camellia chrysantha, and the preservation name is Lecanococcus lecanii JHC098. Lecanicillium saksenae JHC098, with a deposit number of CCTCC NO: M2025749. Experiments have shown that the extract from the fermented liquid of the bottle-shaped Lecanococcus lecanii fungus JHC098 has a strong inhibitory effect on the growth of bacteria that cause citrus canker and tobacco bacterial wilt, and can effectively prevent and control plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram showing the mycelial growth of the endophytic fungus Lecanococcus flaskus JHC098 in the roots of Camellia chrysantha in Example 1 of the present invention; Figure 2 This is a micrograph of the hyphae of the endophytic fungus Lecanococcus flaskus JHC098 in the roots of Camellia chrysantha in Yunnan Province 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 Graph showing the inhibitory effect of the flask-type lecan fungus JHC098 on the pathogenic bacteria of citrus canker; 1 is a mycelial extract of the flask-type lecan fungus JHC098; 2 is a crude extract from the liquid fermentation of the flask-type lecan fungus JHC098; 3 is dimethyl sulfoxide; and 4 is a crude extract from the MSGYD liquid culture medium. Figure 5 This figure shows the inhibitory effect of the endophytic fungus Lecanopsis cylindrica JHC098 on the pathogenic bacteria of tobacco wilt; 1: mycelial extract of Lecanopsis cylindrica JHC098; 2: crude extract of liquid fermentation of Lecanopsis cylindrica JHC098; 3: dimethyl sulfoxide; 4: crude extract of MSGYD liquid culture medium. DETAILED DESCRIPTION

[0016] 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.

[0017] The endophytic fungus from the roots of Yunnan Camellia chrysantha provided by the present invention is preserved as Lecanococcus lecanii JHC098. Lecanicillium saksenae JHC098; deposited with the China Center for Type Culture Collection, with the deposit address being Wuhan University, China; received by the collection on April 10, 2025, and identified as alive on April 17, 2025; and the deposit number is CCTCC NO: M 2025749.

[0018] The citrus canker pathogenic bacteria ( Xanthomonas citri subsp. citri )for Xanthomonas citri subsp. citri (ex Hasse) Gabriel et al. 49118 strain, available for purchase at: http: / / www.biovector.net / product / 1249290.html; The pathogenic bacteria of tobacco wilt ( Ralstonia solanacearum )for Ralstonia solanacearum (Smith) Yabuuchi et al. strain BAA-1114, available for purchase at: http: / / www.biovector.net / product / 1228695.html. Example 1:

[0019] Isolation of endophytic fungi from the roots of Camellia chrysantha The roots of Yunnan Camellia chrysantha were collected in Gulinqing Township, Maguan County, Wenshan Prefecture, Yunnan Province. The roots of Yunnan Camellia chrysantha were cut 60 cm from the ground with scissors, placed in a centrifuge tube, and stored at low temperature with ice packs in the field. After being brought to the laboratory, they were rinsed with tap water for 48 hours, cut into 2 cm small pieces, and then transferred to a sterile workbench for disinfection. They were rinsed with 1 L of distilled water, and then soaked in 70% ethanol for 2 minutes. The ethanol was poured out and rinsed 6 times with 50 mL of sterile water. Then, they were soaked in sodium hypochlorite (10%) solution for 1 minute. The sodium hypochlorite solution was poured out and rinsed 10 times with 50 mL of sterile water (50 mL each time).

[0020] Cut 2 cm pieces into 0.5 cm long and wide pieces, place them in a centrifuge tube, add steel balls and 1 mL of sterile water, and disrupt for 2 minutes (180 rpm). Dilute the solution 100-fold and plate it on 10 plates of KASPYM medium (KASPYM: 8 g / L potato starch; 4 g / L yeast extract; 15 g / L maltose; 100 μg / mL karatomycin; 100 μg / mL ampicillin; 100 μg / mL streptomycin; the remainder is water). Incubate for 7 days. Once colonies have grown, select individual colonies and identify them.

[0021] The grown mycelium was transferred to a solid culture medium containing malt extract broth. After culturing for 15 days, the strain was molecularly identified to obtain the bottle-type Lecanococcus JHC098 strain. The solid culture medium contained 15 g / L malt extract broth and 16 g / L agar (the remainder being water).

[0022] 2. Identification of endophytic fungi in the roots of Camellia chrysantha (1) Morphological identification The strain morphology is Figure 1 As shown, the fungus grew well on the malt extract broth solid culture medium, and the colony surface was dense and off-white.

[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 bottle-type lecanthus JHC098 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 of PVP (polyvinyl pyrrolidone). Add 20 μL of 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 r / min 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 10 minutes, discard the supernatant, add 500μL 75% alcohol, turn upside down 2-3 times, let it stand for 3 minutes, and discard the supernatant; ⑥ Add 500 μL of 95% alcohol, invert the tube upside down 2-3 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 fungus JHC098.

[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 morphological and molecular biological identification, the JHC098 strain was identified as Lecanicillium saksenae Bottle-shaped wax scale fungus. Example 2:

[0026] Preparation of crude extracts from endophytic fungi in the roots of Camellia chrysantha by liquid fermentation: 1. Liquid culture The mycelium of the endophytic fungus Lecanococcus lecanii JHC098 from the roots of Camellia chrysantha was inoculated into a 250 mL conical flask containing 100 mL of MSGYD liquid medium (2 g / L magnesium acetate; 20 g / L soy peptone; 20 g / L glucose; 1 g / L yeast powder; 0.5 g / L sodium butyrate, with the balance being water), and the culture was carried out in a constant temperature shaker at 28°C and 150 rpm for 10 days to obtain the fermentation broth.

[0027] 2. Preparation of crude extracts from liquid culture 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 endophytic fungus Lecanopus lecanii JHC098 from the roots of Camellia chrysantha.

[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 minutes to obtain the cells of Lecanopsis lecanii JHC098. The cells were then placed in a conical flask and submerged in 95% ethanol. After sonication for 30 minutes and 12 hours of simmering, the extract was filtered through filter paper and dried under reflux on a rotary evaporator to obtain a crude extract of Lecanopsis lecanii JHC098, an endophytic fungus found in the roots of Camellia chrysantha.

[0030] 4. Preparation of crude extract from MSGYD liquid culture medium Ethyl acetate was added to MSGYD 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 extract of MSGYD liquid medium. Example 3:

[0031] Antimicrobial activity assay of crude extracts from endophytic fungi in the roots of Camellia chrysantha: The crude extract of the liquid fermentation of Lecanopsis lecanii JHC098, the crude extract of the bacterial cells of Lecanopsis lecanii JHC098, and the crude extract of the MSGYD liquid culture medium prepared in Example 2 above were used as samples; 1. Activation of pathogenic bacteria The causative bacteria of citrus canker ( Xanthomonas citri subsp. citri ) and tobacco bacterial wilt pathogenic bacteria ( Ralstonia solanacearum ), 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 0.01 g of crude extract from liquid fermentation of Lecanopsis lecanii JHC098, crude extract from JHC098 cells, and crude extract from MSGYD liquid culture medium were added to 2 mL centrifuge tubes and diluted to 50 mg / mL with 500 μL of DMSO (dimethyl sulfoxide). A DMSO group served as a control. 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 diluted extract from each experimental and control group were placed at the corresponding marked positions on the medium. The medium was incubated upright at 37°C in a constant temperature incubator for 12 h. The appearance of inhibition zones was observed, and the diameter of the inhibition zones was 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 liquid fermentation of the bottle-shaped lecan fungus JHC098 against the pathogenic bacteria of citrus canker is 15.2±0.2 mm ( Figure 4 ),Depend on Figure 5 It can be seen that the inhibition zone of the crude extract of liquid fermentation of lecanthus lecanthus JHC098 against the pathogenic bacteria of tobacco wilt is 15.8±0.2 mm, indicating that the crude extract of liquid fermentation of lecanthus lecanthus JHC098 has significant antibacterial activity against the pathogenic bacteria of citrus canker and tobacco wilt. The crude extract of lecanthus lecanthus JHC098 and the crude extract of MSGYD 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 Yunnan Camellia chrysantha, characterized in that: The endophytic fungus in the root of Yunnan Camellia chrysantha is bottle-shaped wax scale fungus ( Lecanicillium saksenae ) JHC098, and the deposit number is CCTCC NO: M 2025749.

2. Use of the endophytic fungus from the roots of Camellia chrysantha as claimed in claim 1 in the prevention and treatment of crop diseases.

3. The use according to claim 2, characterized in that: The crop disease is citrus canker pathogenic bacteria ( Xanthomonas citri subsp. citri ) and / or tobacco bacterial wilt pathogenic bacteria ( Ralstonia solanacearum ) caused by diseases.

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 Lecanopus lecanii JHC098 from the roots of Yunnan Camellia chrysantha 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 extract of the fermentation broth of the bottle-shaped lecanthus JHC098 comprises the following steps: (1) Activating the fungus strain of Lecanococcus lecanii JHC098; (2) The activated fungus JHC098 was inoculated into MSGYD liquid culture medium, and then placed in a constant temperature shaker at 28°C and 150 rpm for 20 days to obtain a fermentation liquid; (3) The fermentation broth was centrifuged to separate the bacterial cells and the fermentation broth, and ethyl acetate was added to the fermentation broth at a volume ratio of 1:1.5, and ultrasonicated for 30 minutes; then the broth was poured into a separatory funnel and allowed to stand for 12 hours 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 formula of the MSGYD liquid culture medium is: 2 g / L magnesium acetate; 20 g / L soy peptone; 20 g / L glucose; 1 g / L yeast powder; 0.5 g / L sodium butyrate, and the balance is water.

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

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