Fungus trichoderma FNJHC048 in roots of camellia kintinensis and application thereof

Abstract

This invention provides a root endophytic fungus, *Verticillium* FNJHC048, from the roots of the *Camellia chrysantha* tree in Funing and its applications, relating to the field of crop disease control technology. The *Verticillium* root endophytic fungus is protected under the name *Verticillium* FNJHC048. Verticillium sp. FNJHC048, deposited at the China Center for Type Culture Collection (CCTCC) NO: M20251252, and its crude extract from liquid fermentation broth can be used for crop disease control. This invention overcomes the shortcomings of existing technologies; the crude extract from the liquid fermentation broth of Verticillium FNJHC048 can effectively inhibit the growth of pathogenic bacteria causing citrus canker and bacterial angular leaf spot of cucumber, achieving a certain degree of biological control.

Description

Technical Field

[0001] This invention relates to the field of crop disease control technology, specifically to the endophytic fungus Verticillium FNJHC048 in the roots of a Camellia chrysantha var. funingensis and its applications. Background Technology

[0002] Camellia mingii, a shrub, grows to 2-4 meters tall. Young branches are cylindrical, brown to dark brown, densely covered with light yellow spreading hairs. It mainly grows in evergreen broad-leaved forests in the limestone areas of Yunnan. Current pharmacological research on Camellia mingii focuses on its anti-tumor, anti-inflammatory, antioxidant, lipid-regulating, blood sugar-lowering, anti-obesity, blood pressure-lowering, anti-allergic, and anti-aging effects. Endophytic fungi exist in healthy plant tissues, mutually benefiting the host plant and promoting its growth and enhancing its resistance. Their secondary metabolites include terpenes, steroids, and flavonoids, possessing anti-inflammatory, anti-tumor, and antibacterial pharmacological activities.

[0003] 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 It belongs to the domain Bacteria, phylum Proteobacteria, class Gamma-Proteobacteria, order Xanthomycetes, family Xanthomycetes, and genus Xanthomonas. Xanthomonas citri subsp citri It is the main pathogenic bacterium causing citrus canker. Citrus canker pathogens are among the most destructive bacterial diseases of citrus worldwide, posing a serious threat to the citrus industry. Cucumber bacterial angular leaf spot (… Pseudomonas syringae (pv. Lachrymans) is a disease that mainly occurs on cucumbers, affecting both seedlings and mature plants, but primarily the leaves of mature plants. It mainly 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 turn slightly yellowish-brown and dry out.

[0004] In crop cultivation, the long-term use of chemical products has increased the variability of pathogens, leading to a weakening of the effectiveness of chemical control. Endophytic fungi, due to their unique advantages, show great promise in the control of plant diseases and pests. Many endophytic fungi produce antibiotics that not only enhance the disease resistance of host plants but also inhibit other bacteria, fungi, and protozoa. Screening for antibiotics from endophytic fungi has become an important source of new antibiotics, with significant implications for the fields of medicine, food industry, and agronomy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a root endophytic fungus, Verticillium FNJHC048, from the Funing Camellia chrysantha tree and its applications. Verticillium FNJHC048 can effectively inhibit pathogenic bacteria causing citrus canker. Xanthomonas citri subsp.citri With cucumber bacterial angular leaf spot Pseudomonas syringae The growth of pv. Lachrymans can achieve a certain degree of biological control.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] Verticillium FNJHC048, an endophytic fungus in the roots of a Camellia chrysantha var. fungus from Funing, has the preservation number CCTCC NO: M 20251252.

[0008] Verticillium FNJHC048 is used for the control of crop diseases, specifically the pathogenic bacteria of citrus canker. Xanthomonas citri subsp. citri With cucumber bacterial angular leaf spot Pseudomonas syringae Diseases caused by pv. Lachrymans.

[0009] Preferably, the application method is to use the crude extract of Verticillium FNJHC048 fermentation broth to prepare bacterial agents or drug compositions for the prevention and control of crop diseases.

[0010] Preferably, the method for preparing the crude extract of the fermentation broth of Verticillium FNJHC048 includes the following steps:

[0011] S1. The mycelium of Verticillium FNJHC048 was inoculated into GNK2 liquid medium and then placed in a constant temperature shaker at 28℃ and 150rpm for 20 days to obtain the fermentation broth.

[0012] S2. Filter the fermentation broth with gauze to separate the bacterial cells and the fermentation broth. Add ethyl acetate to the fermentation broth at a volume ratio of 1:1.5 and sonicate for 30 min. Then pour the mixture into a separatory funnel and let it stand for 12 h to allow it to separate into 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.

[0013] Preferably, the formulation of the GNK2 liquid culture medium is as follows: glucose 15 g / L, sodium nitrate 0.5 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium acetate 0.5 g / L, potassium propionate 0.5 g / L, sodium butyrate 1 g / L, with water added to make up to 1L.

[0014] This invention provides a root endophytic fungus, Verticillium FNJHC048, from the Funing golden camellia tree and its applications. Compared with existing technologies, its advantages are:

[0015] This invention obtained a fungus with antibacterial activity by isolating and screening endophytic fungi from the roots of Camellia chrysantha var. fungi in Funing, and its preservation name is Verticillium FNJHC048. Verticilliumsp. FNJHC048, with accession number CCTCC NO: M20251252. Experiments revealed that the crude extract of Verticillium FNJHC048 liquid fermentation broth exhibits activity against bacteria causing citrus canker. Xanthomonas citri subsp. citri With cucumber bacterial angular leaf spot Pseudomonas syringae pv. Lachrymans has a strong inhibitory effect on growth and can effectively prevent and control plant diseases. Attached Figure Description

[0016] Figure 1 This is a diagram showing the mycelial growth of the endophytic fungus *Verticillium* FNJHC048 in the roots of the *Camellia chrysantha* tree in Example 1 of the present invention.

[0017] Figure 2 Micrograph of the hyphae of Verticillium FNJHC048, an endophytic fungus in the roots of Camellia chrysotricha from Funing, as described in Example 1 of this invention;

[0018] Figure 3 This is a diagram illustrating the phylogenetic tree of endophytic fungi constructed based on ITS in Embodiment 1 of the present invention.

[0019] Figure 4 This diagram illustrates the inhibitory effect of the endophytic fungus *Verticillium* FNJHC048 on the pathogenic bacteria of citrus canker. 1 represents *Verticillium* FNJHC048 mycelial extract; 2 represents *Verticillium* FNJHC048 crude extract obtained through liquid fermentation; 3 represents crude extract obtained from GNK2 liquid culture medium; and 4 represents DSMO.

[0020] Figure 5 This diagram illustrates the inhibitory effect of the endophytic fungus *Verticillium* FNJHC048 on bacterial angular leaf spot of cucumber. 1 represents *Verticillium* FNJHC048 cell extract; 2 represents crude extract of *Verticillium* FNJHC048 fermentation broth; 3 represents crude extract of GNK2 liquid culture medium; and 4 represents DSMO. Detailed Implementation

[0021] 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.

[0022] The endophytic fungus in the roots of Camellia chrysanthemi provided by this invention is preserved under the name Verticillium FNJHC048. Verticilliumsp. FNJHC048; deposited at the China Center for Type Culture Collection, Wuhan University, China; deposit date: June 4, 2025; accession number: CCTCC NO: M 20251252.

[0023] The following examples use bacteria that cause citrus canker ( Xanthomonas citri subsp. citri )for Xanthomonas citri subsp The strain *Citri* (ex Hasse) Gabriel et al. 49118 can be purchased via the following link: http: / / www.biovector.net / product / 1249290.html.

[0024] The bacterial angular leaf spot fungus used in cucumbers ( Pseudomonas syringae pv. Lachrymans) is Pseudomonas syringae pv. Lachrymans (Smith and Bryan 1915) Young, Dye and Wilkie 1978, purchase link: http: / / www.biovector.net / product / 2096192.html. Example 1:

[0025] 1. Isolation of endophytic fungi from the roots of Camellia chrysantha var. funingensis.

[0026] (1) After collecting the roots of the Funing golden camellia tree 30cm above the ground, put them in a foam box containing ice packs and keep them at a low temperature until they arrive at the laboratory. Rinse them with clean water for 10 minutes, then cut them into 2cm pieces. Then transfer them to a sterile workbench for disinfection. Soak them in 15% hydrogen peroxide for 3 minutes, then soak them in sterile water 3 times for 2 minutes each time. Use sterilized filter paper to absorb the water from the surface of the root sample.

[0027] (3) Cut the 2cm pieces into tissue blocks with a length and width of 0.5cm, place them in centrifuge tubes, add steel balls and 1mL of sterile water, and crush for 2 minutes (180rpm). Dilute 1000 times and plate 10 GNMKAS agar plates (GNMKAS: glucose 15 g / L, sodium nitrate 0.5 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 1 g / L, 200μg / mL kanamycin, 200μg / mL ampicillin, 200μg / mL streptomycin, 15g / L agarose, balance water). Incubate for 10 days. After bacterial growth, select single colonies and identify them.

[0028] After the mycelia were transferred to PDY solid medium and cultured for 15 days, the strain was molecularly identified, yielding Verticillium FNJHC048. The PDY solid medium consisted of the following components: 10 g / L glucose, 15 g / L potato starch, 5 g / L yeast extract, 1 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 16 g / L agar, with the remainder being water.

[0029] 2. Identification of endophytic fungi in the roots of Camellia chrysantha var. funingensis.

[0030] (1) Morphological identification

[0031] The morphology of this strain is as follows Figure 1 As shown, the fungus grows well on PDY solid medium, with fluffy light brown hyphae on the colony surface, and the micrograph of the hyphae is shown in the figure. Figure 2 As shown.

[0032] (2) DNA extraction

[0033] ① Before the experiment, heat CTAB (hexadecyltrimethylammonium bromide) in a 65℃ water bath for 30 minutes;

[0034] ② Take 50 mg of dried Verticillium FNJHC048 mycelium 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℃ water bath for 1.5 h, turning it upside down 6 times every 10 min. After the water bath, centrifuge at 12000 r / min and 4℃ for 10 min.

[0035] 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).

[0036] 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.

[0037] 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;

[0038] 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.

[0039] ⑦ Add 40 μL of elution buffer EB, centrifuge at room temperature (13000 rpm) for 1.5 min to obtain the genomic DNA of the fungus Verticillium FNJHC048.

[0040] (3) ITS analysis and identification

[0041] 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.

[0042] SEQ ID No. 1:

[0043]

[0044] (4) Constructing a developmental tree

[0045] 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 FNJHC048 was identified as... Verticillium sp. Example 2:

[0046] Preparation of crude extract from liquid fermentation of Verticillium FNJHC048 strain:

[0047] 1. Liquid culture

[0048] The mycelium of Verticillium FNJHC048 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of GNK2 liquid medium (15 g / L glucose, 0.5 g / L sodium nitrate, 1 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium acetate, 0.5 g / L potassium propionate, 1 g / L sodium butyrate, with the remainder being water), and incubated in a constant temperature shaker at 28 °C and 150 rpm for 20 days.

[0049] 2. Preparation of crude extract from liquid culture

[0050] The fermentation broth was filtered through gauze to separate the bacteria from the fermentation broth.

[0051] 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 phase separation. The supernatant was then extracted. The extract was dried by reflux using a rotary evaporator to obtain the crude extract of Verticillium FNJHC048 liquid fermentation.

[0052] 3. Preparation of crude bacterial extract

[0053] The fermentation broth was filtered through a filter cloth to obtain Verticillium FNJHC048 cells. The Verticillium FNJHC048 cells were placed in an Erlenmeyer flask, and 95% ethanol was added to submerge the cells. After sonication for 30 minutes, the mixture was allowed to stand for 12 hours, and then filtered through filter paper to obtain a cell extract. The cell extract was dried by reflux using a rotary evaporator to obtain a crude extract of Verticillium FNJHC048 cells.

[0054] 4. Preparation of crude extract from GNK2 liquid culture medium

[0055] A 250 mL Erlenmeyer flask containing 100 mL of GNK2 liquid culture medium was placed in a constant temperature shaker at 28 °C and 150 rpm for 20 days. Ethyl acetate was added to the liquid culture medium at a volume ratio of 1:1.5, and the mixture was stirred thoroughly and 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 GNK2 liquid culture medium. Example 3:

[0056] Antibacterial activity assay of the crude extract of Verticillium FNJHC048 liquid fermentation:

[0057] The crude extracts of Verticillium FNJHC048 fermentation broth, Verticillium FNJHC048 cells, and GNK2 liquid culture medium prepared in Example 2 above were used as samples.

[0058] 1. Activation of pathogenic bacteria

[0059] bacteria that cause citrus canker Xanthomonas citri subsp . citri and cucumber bacterial angular leaf spot ( Pseudomonas syringae As the pathogen, 5 μL of the pathogen was added to 2 mL centrifuge tubes, and then 750 μL of LB liquid medium (10 g / L tryptone + 10 g / L sodium chloride + 5 g / L yeast extract) was added to each centrifuge tube. The centrifuge tubes were then placed in a constant temperature shaker at 37°C and 180 r / min for 12 h to obtain the pathogenic bacterial solution. The solution was stored in a 4°C refrigerator for later use. Before use, the activated bacteria were taken out and diluted to 1 / 10.

[0060] 2. Detection of antibacterial activity using the filter paper disc method

[0061] The filtered bacterial cells were extracted with ethanol and dried by reflux using a rotary evaporator to obtain a crude extract of Verticillium FNJHC048 cells. 0.01 g of the liquid fermentation crude extract of Verticillium FNJHC048, the crude extract of Verticillium FNJHC048 cells, and the crude extract from GNK2 liquid medium 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 the dissolved crude extract of the fermentation broth were placed at the corresponding marked positions on the medium. The medium was then incubated upright in a 37°C incubator for 12 h. The presence of inhibition zones was observed, and their diameter was measured using the cross-sectional method.

[0062] like Figure 4 and Figure 5As shown, the liquid fermentation crude extract of Verticillium FNJHC048 is effective against Citrus canker pathogens (… Xanthomonas citri subsp . citri) and cucumber bacterial angular leaf spot ( Pseudomonas syringae Both *pv. lachrymans* and *pv. lachrymans* exhibited significant antibacterial activity, with inhibition zone diameters of 25.1 ± 0.2 mm and 23.5 ± 0.2 mm, respectively. In contrast, no obvious inhibition zones were observed in the crude extracts of the bacteria and the crude extracts from GNK2 liquid medium, indicating that they lacked antibacterial activity.

[0063] 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, Verticillium, on the roots of a Camellia chrysantha var. fungus from Funing ( Verticillium sp.)FNJHC048, characterized in that: The preservation number of the Verticillium FNJHC048 is CCTCC NO: M 20251252.

2. The application of Verticillium FNJHC048 as described in claim 1 in the control of crop diseases, characterized in that: The crop disease mentioned is Xanthomonas aureus, a citrus-pathogenic strain (… Xanthomonas citri subsp. citri ) and the causative agent of cucumber angular leaf spot by *Pseudomonas syringae* ( Pseudomonas syringae pv Lachrymans Diseases caused by ).

3. The application according to claim 2, characterized in that: The application method involves using the crude extract of Verticillium FNJHC048 fermentation broth to prepare bacterial agents or drug compositions for the prevention and control of crop diseases.

4. The application according to claim 3, characterized in that: The method for preparing the crude extract of the fermentation broth of Verticillium FNJHC048 includes the following steps: S1. The mycelium of Verticillium FNJHC048 was inoculated into GNK2 liquid medium and then placed in a constant temperature shaker at 28℃ and 150rpm for 20 days to obtain fermentation broth. The formula of the GNK2 liquid medium is: glucose 15 g / L, sodium nitrate 0.5 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium acetate 0.5 g / L, potassium propionate 0.5 g / L, sodium butyrate 1 g / L, and water was added to make up to 1L. S2. Filter the fermentation broth with gauze to separate the bacterial cells and the fermentation broth. Add ethyl acetate to the fermentation broth at a volume ratio of 1:1.5 and sonicate for 30 min. Then pour the mixture into a separatory funnel and let it stand for 12 h to allow it to separate into 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.

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