A ginseng endophyte with high yield of pyrroloquinoline quinone and its application

Abstract

A ginseng endophyte with high PQQ production, characterized in that: the taxonomic name of the ginseng endophyte is Rhodobacter methylophilus Methylorubrum sp.) W7N-70, which is deposited in the China Center for Type Culture Collection (CCTCC), with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC M 20241885, and the deposit date being September 2, 2024. The ginseng endophyte W7N-70 obtained in the present invention has the ability to produce PQQ, and the detected PQQ synthesis amount in its supernatant reaches 75.81 mg / L, which can be effectively applied to the fermentation engineering for synthesizing PQQ. In addition, this strain can produce antibacterial metabolites and can effectively inhibit the growth of various pathogenic bacteria. This strain has excellent effects when applied to promoting plant growth and antibacterial, as well as synthesizing PQQ.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a ginseng endophyte with high yield of pyrroloquinoline quinone and its application. Background Art

[0002] The relationship between plants and microorganisms in nature is close and complex. Microorganisms exist almost on the surface and inside of different plant tissues, and the two co-evolve to form a relatively stable relationship. Endophytes are microorganisms that live inside various tissues and organs of healthy plants at certain stages or all stages of their life cycle, and their presence does not cause any diseases, including multiple microbial groups such as endophytic fungi, endophytic bacteria, and endophytic actinomycetes. Inside plants, the bioactive metabolites produced by endophytes can promote plant growth, inhibit plant pathogens, and improve the tolerance of plants to biotic and abiotic stresses. In addition, some bioactive metabolites synthesized by endophytes can also be used as raw materials for medicines, cosmetics, and foods.

[0003] Ginseng ( Panax ginseng C.A. Meyer) is a perennial herbaceous plant of the genus Panax in the Araliaceae family, mainly distributed in Asia and North America. As a traditional precious Chinese herbal medicine, it has a long medicinal history and is widely used in medicines, functional foods, dietary supplements, cosmetics, etc. Due to over-exploitation in recent years, the wild ginseng resources are becoming increasingly scarce, and cultivated ginseng has gradually become the mainstream in the market. However, compared with other herbaceous medicinal plants, ginseng has a longer growth cycle, generally taking 4 - 6 years to harvest, and has relatively strict environmental requirements. It is easily affected by various factors such as temperature, soil, light, precipitation, and pests and diseases, resulting in problems such as complex cultivation techniques, long production cycles, uncontrollable quality, and unstable yields in ginseng cultivation, which greatly limits the development of the ginseng industry.

[0004] Pyrroloquinoline quinone (PQQ) is a kind of water-soluble orthoquinone compound with reddish-brown color, easily decomposed by light, and having thermal stability. It is the third coenzyme of oxidoreductase after pyridine nucleotide and riboflavin. PQQ has various physiological functions such as participating in the electron transfer of quinoproteinase and respiratory chain, regulating gene expression and protein function, regulating signal transduction and DNA function repair, and regulating the level of free radicals in the body. In addition, as a plant growth regulator, PQQ can improve the physical and chemical properties of soil, enhance the stress resistance of plants, and promote the growth and development of plants, making it have important development prospects in the food industry, light industry, agriculture, and medicine. The synthesis methods of PQQ mainly include chemical synthesis method and microbial fermentation method. Among them, the microbial fermentation method has the advantages of simple operation, low cost, and easy subsequent treatment compared with the chemical synthesis method, and has become the main production method of PQQ. Therefore, screening and obtaining ginseng endophytic bacteria with the ability to synthesize PQQ is of great significance for obtaining new PQQ-producing strains and the development of ginseng microbial agents. Summary of the Invention

[0005] The purpose of the present invention is to provide a ginseng endophytic bacterium with high yield of PQQ. This endophytic bacterium has a high yield of PQQ, and at the same time has an inhibitory effect on various pathogenic bacteria of ginseng. This endophytic bacterium effectively improves the antibacterial ability of ginseng and the growth quality of ginseng, and promotes the development of the ginseng industry.

[0006] Another purpose of the present invention is to provide the application of the above-mentioned ginseng endophytic bacterium.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A ginseng endophytic bacterium with high yield of PQQ, characterized in that: the taxonomic name of the ginseng endophytic bacterium is Rhodobacter methylophilus ( Methylorubrum sp.) W7N-70, deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is CCTCC M20241885, and the deposit date is September 2, 2024.

[0009] The strain W7N-70 belongs to Gram-negative bacteria, with a light pink color, which deepens after culturing for a period of time. The colony is small and round, with a shiny surface, easy to pick, and opaque.

[0010] The 16S rDNA sequence was sequenced, the sequencing results were subjected to Blast comparison analysis in NCBI, and sequence similarity analysis was performed using DNAman. Then a phylogenetic tree was constructed using MEGA, and the strain W7N-70 was identified as Rhodobacter methylophilus ( Methylorubrum sp.).

[0011] Furthermore, after enrichment culture, the screened strain W7N-70 has the ability to produce pyrroloquinoline quinone (PQQ), and the PQQ content detected in its supernatant reaches 75.81 mg / L.

[0012] Furthermore, the above strain W7N-70 also produces metabolites with antibacterial activity against Cylindrocarpon destructans Cylindrocarpon destructans Botrytis cinerea Botrytis cinerea Colletotrichum panacicola Colletotrichum panacicola Rhizoctonia solani Rhizoctonia solani Alternaria panax Alternaria panax Sclerotinia libertiana Sclerotinia sclerotiorum

[0013] In view of the PQQ production ability and the ability to inhibit the growth of pathogenic bacteria of strain W7N-70, this strain can be applied to promote plant growth.

[0014] The application of the above-mentioned Panax ginseng endophyte W7N-70 in promoting plant growth is specifically to promote the growth of Panax ginseng.

[0015] Furthermore, the application of the above-mentioned Panax ginseng endophyte W7N-70 in the fermentation engineering of synthesizing PQQ.

[0016] In addition, the application of the above-mentioned Panax ginseng endophyte W7N-70 in inhibiting the growth of pathogenic bacteria.

[0017] Furthermore, it is the above-mentioned Panax ginseng endophyte W7N-70 that inhibits the growth of at least one of the pathogenic bacteria Cylindrocarpon destructans, Botrytis cinerea, Colletotrichum panacicola, Rhizoctonia solani, Alternaria panax and Sclerotinia sclerotiorum.

[0018] The present invention has the following technical effects:

[0019] The Panax ginseng endophyte W7N-70 obtained in the present invention belongs to the genus Methylobacterium ( Methylorubrum sp.), presumably Methylobacterium zatmanii ( Methylobacterium zatmanii ), which has the ability to produce PQQ, and the detected PQQ synthesis amount in its supernatant reaches 75.81 mg / L, and it can be effectively applied to the fermentation engineering of synthesizing PQQ. In addition, this strain can produce antibacterial metabolites and can effectively inhibit the growth of various pathogenic bacteria. This strain has excellent effects when applied to promoting plant growth and antibacterial, as well as synthesizing PQQ. Description of the Drawings

[0020] Figure 1 : Standard curve corresponding to non-enzymatic detection of PQQ production by strain W7N-70.

[0021] Figure 2 : Observation results of the colony morphology of strain W7N-70.

[0022] Figure 3 : Agarose gel electrophoresis map of the PCR amplification product of the 16S rDNA sequence of strain W7N-70.

[0023] Figure 4 : Phylogenetic tree of strain W7N-70 constructed based on the 16S rDNA sequence.

[0024] Figure 5 : Bacteriostatic effect diagram of strain W7N-70 against 6 kinds of ginseng pathogenic bacteria.

[0025] Figure 6 : Bacteriostatic effect diagram of the supernatant of the culture solution of strain W7N-70 against 6 kinds of ginseng pathogenic bacteria. Specific implementation manners

[0026] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above content of the present invention.

[0027] Example 1

[0028] Isolation and purification of the strain

[0029] 1. Sample collection

[0030] In October 2023, a total of 21 healthy wild ginseng plants were collected in Weishahe Town and Naoshi Town, Linjiang City, Jilin Province, China for the isolation of ginseng endophytes. After collection, tissues such as stems and leaves were removed, only the main roots were retained, the soil adhered to the surface was washed off with running water, the water was blotted dry with absorbent paper, and then packaged in a self-sealing bag and stored at 4 °C for later use. The information of the collected samples is shown in Table 1.

[0031] Table 1:

[0032]

[0033] 2. Isolation and purification of endophytes

[0034] The tissue grinding method was used to isolate endophytes: the surface-sterilized ginseng roots were cut into small pieces, placed in a sterile mortar, ground with an appropriate amount of physiological saline, and the ground tissue was placed on the culture medium. The culture media used were potato dextrose agar (PDA) medium, nutrient broth peptone (NA) medium, and Gao's No. 1 medium.

[0035] After colonies appeared on the culture medium, according to different morphological characteristics, use an inoculation loop to pick colonies and repeat streak culture on the corresponding culture medium 3 times to purify the endophytic bacteria, name and label them, and store them in a -80°C refrigerator for long-term preservation. A total of 206 strains of ginseng endophytic bacteria were isolated, including 97 strains from NA medium, 64 strains from PDA medium, and 45 strains from Gao's No. 1 medium.

[0036] Example 2

[0037] Screening and Identification of Ginseng Endophytic Bacteria

[0038] 1. Screening of Ginseng Endophytic Bacteria with the Ability to Synthesize PQQ

[0039] The 206 strains of endophytic bacteria isolated and purified in Example 1 were respectively inoculated into the enrichment medium, and cultured with shaking at 28°C and 170 rpm for 5 - 6 days to observe the growth of the bacteria. The components of the enrichment medium are as follows: 15 mL of CH3OH, 4 g of (NH4)2SO4, 1.4 g of KH2PO4, 5 g of Na2HPO4, 1 g of MgSO4·7H2O, 0.075 g of CaCl2·2H2O, 0.05 g of FeSO4·7H2O, 1 L of water, pH 6.8.

[0040] The primary screening of the PQQ synthesis ability of ginseng endophytic bacteria was carried out by a non-enzymatic method: Prepare PQQ standard solutions of 100 mg / L, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, 10 mg / L, and 5 mg / L respectively to construct a standard curve. Add 240 μL of the reaction system to a 96-well plate, including 48 μL of the supernatant of the bacterial solution, 32 μL of borate buffer, 160 μL of NBT-potassium glycinate solution, react at 37°C in the dark for 20 min, and detect the OD 530 absorbance value under the conditions. The medium without inoculation was used as a blank control, and each treatment was repeated 3 times. Among the 206 strains of ginseng endophytic bacteria, a strain with the ability to synthesize PQQ was screened out, and this strain was designated as W7N-70 (from a 7-year-old wild ginseng sample in Weishahe Town). The results are shown in Figure 1. The regression equation of the standard curve is y = 0.0077x + 0.1726, R 2 = 0.9954, and the linear relationship is good. A relatively high PQQ content of 75.81 mg / L was detected in the supernatant of the culture solution of strain W7N-70. This strain W7N-70 can synthesize PQQ by fermentation in fermentation engineering, and PQQ can be used as a reagent to promote plant growth in the plant growth process.

[0041] 2. Strain Identification

[0042] The colony morphology of strain W7N-70 cultured on a plate medium is as Figure 2 shown. Strain W7N-70 belongs to Gram-negative bacteria, with a light pink color that deepens after culturing for a period of time. The colonies are small and round, shiny, easy to pick, opaque, and not tightly bound to the medium.

[0043] The genomic DNA of strain W7N-70 was extracted using the Sangon Ezup Column Bacterial Genomic DNA Extraction Kit. Using the extracted genomic DNA as a template, the 16S rDNA sequence was amplified by PCR with universal primers for strain identification.

[0044] The obtained PCR products were detected by 1% agarose gel electrophoresis. The results are as Figure 3 shown. The bands were clear and bright. The bands of appropriate size were cut out and recovered using the Sangon SanPrep Column DNA Gel Extraction Kit, and the gel recovery products were sent to Sangon Biotech Co., Ltd. for sequencing.

[0045] The sequencing results were analyzed by Blast comparison in NCBI. Sequences with high homology were downloaded, and sequence similarity analysis was performed using DNAman and a phylogenetic tree was constructed using MEGA. The results of the phylogenetic tree are shown in Figure 4. The identification results showed that strain W7N-70 is of the genus Rhodobacter methylophilus ( Methylorubrum sp.), and is further speculated to be Rhodobacter zopfii( Methylobacterium zatmanii ).

[0046] The above strain W7N-70 was deposited in the China Center for Type Culture Collection (CCTCC), with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC M 20241885, and the deposit date is September 2, 2024.

[0047] Example 3

[0048] Inhibitory effect of strain W7N-70 on ginseng pathogenic bacteria

[0049] The tested ginseng pathogenic bacteria include: Colletotrichum panacicola ( Cylindrocarpon destructans ), Botrytis cinerea ( Botrytis cinerea ), Colletotrichum ginseng ( Colletotrichum panacicola ), Rhizoctonia solani ( Rhizoctonia solani ), Alternaria panax ( Alternaria panax ) and Sclerotinia sclerotiorum ( Sclerotinia libertiana ). The above tested ginseng pathogenic bacteria were provided by the College of Plant Protection, Jilin Agricultural University.

[0050] Experimental procedure:

[0051] (1)The antibacterial effect of strain W7N-70 against ginseng pathogenic bacteria was studied by the plate confrontation method. Specifically, 6 kinds of ginseng pathogenic bacteria were inoculated in the center of PDA solid medium and cultured in an incubator at 26 °C for 5 days. Then, a bacterial cake was punched with a 5-mm punch and inoculated at the center point of a new PDA medium. Strain W7N-70 was inoculated at four points 2.5 cm away from the center point simultaneously. Only inoculating the pathogenic bacteria was used as a control. It was cultured in an incubator at 26 °C, and relevant data were observed and recorded every 12 h. The cross method was used to measure the colony diameter of the experimental group and the control group to calculate the antibacterial rate. The formula for calculating the antibacterial rate is as follows:

[0052]

[0053] In the formula, L1 is the colony diameter of the control group, and L2 is the colony diameter of the experimental group.

[0054] The results are as Figure 5 shown (A is W7N-70 and Fusarium solani; B is W7N-70 and Colletotrichum gloeosporioides; C is W7N-70 and Rhizoctonia solani; D is W7N-70 and Alternaria alternata; E is W7N-70 and Sclerotinia sclerotiorum; F is W7N-70 and Botrytis cinerea). Strain W7N-70 can effectively inhibit the growth of 6 kinds of ginseng pathogenic bacteria, making their colonies unable to expand outward, and an obvious antibacterial zone is produced. The calculation results of the antibacterial rate are shown in Table 2.

[0055] Table 2: Antibacterial rate of strain W7N-70 against ginseng pathogenic bacteria

[0056]

[0057] Strain W7N-70 has the best antibacterial effect against Colletotrichum gloeosporioides, followed by Rhizoctonia solani, and the antibacterial rates are 56.3% and 42.4% respectively.

[0058] (2)The antibacterial effect of the supernatant of strain W7N-70 culture solution against ginseng pathogenic bacteria was studied by the growth rate method: Specifically, strain W7N-70 was inoculated into a liquid medium. When the OD of the bacterial solution 600When the value is 0.6 - 0.8, centrifuge at 10,000 rpm for 10 min, take the supernatant and place it in a 500 mL beaker. After heating and concentrating it to 50 mL in a water bath, sterilize it at 115℃ for 30 min under high pressure. Then mix the culture supernatant and the PDA medium evenly at a ratio of 1:10 for standby. Use a punch with a 5 mm specification to punch out the test discs of the pathogenic bacteria of ginseng, and inoculate them at the center point of the medium containing the culture supernatant, and culture at 26℃. Observe the growth of the mycelia of the pathogenic bacteria of ginseng every 12 h. The control group uses the same volume of sterile water instead of the concentrated solution. The results are as Figure 6 shown (A is the rust rot pathogen; B is the anthracnose pathogen; C is the damping-off pathogen; D is the black spot pathogen; E is the sclerotinia pathogen; F is the gray mold pathogen). An obvious inhibition zone was formed between the culture supernatant of strain W7N-70 and the 6 kinds of pathogenic bacteria, indicating that the supernatant of strain W7N-70 has a good inhibitory effect on the pathogenic bacteria of ginseng. By calculating the inhibition rate, the results are shown in Table 3.

[0059] Table 3: Inhibition rate of the culture supernatant of strain W7N-70 on the pathogenic bacteria of ginseng

[0060]

[0061] As can be seen from the above table, the culture supernatant of strain W7N-70 also has a good inhibitory effect on the pathogenic bacteria of ginseng. Among them, the inhibitory effect on the damping-off pathogen is better, and the inhibition rate is 32.1%.

[0062] The above tests show that strain W7N-70 and its culture supernatant have a good inhibitory effect on a variety of pathogenic bacteria of ginseng, which further indicates that the endophytic bacterium W7N-70 of ginseng produces metabolites with broad-spectrum antibacterial activity, and can be applied to the fermentation engineering to synthesize metabolites to inhibit pathogenic bacteria.

Claims

1. A ginseng endophyte with high PQQ yield, characterized in that: The taxonomic name of the ginseng endophyte is Rhodobacter methylophilus ([ Methylorubrum Methylorubrum sp.) W7N-70, which is preserved in the China Center for Type Culture Collection (CCTCC). The preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation number is CCTCC M 20241885, and the preservation date is September 2, 2024.

2. Use of the ginseng endophyte W7N-70 as described in claim 1 in promoting plant growth.

3. Use of the ginseng endophyte W7N-70 as described in claim 1 in the fermentation engineering for synthesizing PQQ.

4. Use of the endophytic fungus W7N-70 of Panax ginseng as claimed in claim 1, characterized in that: Use in inhibiting the growth of pathogenic bacteria.

5. The application according to claim 4, characterized in that: Specifically, it inhibits the growth of at least one of the pathogenic bacteria including Cylindrocarpon destructans, Botrytis cinerea, Colletotrichum panacicola, Rhizoctonia solani, Alternaria panax, and Sclerotinia sclerotiorum of ginseng.

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