Burkholderia paragallinarum Ccw389 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and discloses a strain of Paraburkholderia phytofirmware Ccw389 used for growth promotion of coptis chinensis, biological prevention and control of coptis chinensis planting diseases and soil improvement of coptis chinensis planting, and the strain of the Paraburkholderia phytofirmware Ccw389 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 3, 2024, and the preservation number is CGMCC No.31836. The invention further discloses a preparation method of the strain of the Paraburkholderia phytofirmware Ccw389. The Burkholderia paraburica Ccw389 provided by the invention can be used for preventing and treating the root rot of coptis chinensis, is beneficial to reducing the application amount of chemical pesticides in the planting process of coptis chinensis, and has the characteristics of no toxicity, no harm and the like; meanwhile, the strain can promote the healthy growth of coptis chinensis and also has a certain effect on improvement of coptis chinensis planting soil through the processes of dissolving phosphorus, dissolving potassium, fixing nitrogen and the like as well as the modes of generating siderophores, ACC deaminase and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an application of a paraburkholderia Ccw389 in disease prevention and control and growth promotion during the planting process of coptis chinensis. Background Art

[0002] Coptis chinensis, a perennial herb belonging to the Ranunculaceae family, is a commonly used Chinese herbal medicine in my country and is used in 109 kinds of Chinese patent medicines. Coptis chinensis is used as medicine in its dried rhizome, the main components of which are various protoberberines and quaternary ammonium alkaloids, which have the effects of clearing away heat and dampness, purging fire and detoxifying. Modern pharmacological studies have found that Coptis chinensis has anti-cancer, antibacterial and antiviral effects, and can improve cardiovascular diseases, lower blood sugar and blood pressure.

[0003] Coptis chinensis is obviously authentic and has high requirements for its living environment. In recent years, due to the demand for large-scale planting and climate change in the cultivation area, diseases of Coptis chinensis have occurred frequently and become increasingly severe, which has seriously affected the yield and quality of Coptis chinensis. Its diseases mainly occur in the roots and leaves, including root rot, white rot, purple feather disease, leaf spot, powdery mildew and anthracnose, among which root rot is the most common and most serious disease, with an annual incidence of about 40%, and as high as 80-90% in severe plots. Studies have shown that the main pathogenic fungi reported to cause root rot of Coptis chinensis are Fusarium and Ilyonectria fungi.

[0004] At present, the prevention and control of Coptis chinensis root rot still focuses on traditional agricultural prevention and chemical prevention. Agricultural prevention and control methods such as changing the farming mode through crop rotation, intercropping, removal of diseased plants, and soil replacement will not cause pollution, but the effect is minimal. Although the application of chemical agents such as carbendazim, thiophanate-methyl, meconazole, and propiconazole has shown good results in the short term, long-term use will not only cause problems such as pesticide residues and environmental pollution, but more importantly, it will cause hazards such as drug resistance. Therefore, it is urgent to carry out environmentally friendly, green and pollution-free biological control. This experiment aims to develop a pollution-free, environmentally friendly biological strain to achieve green prevention and control of Coptis chinensis diseases by inhibiting the growth of pathogens, producing plant growth-promoting substances, and promoting plant nutrient utilization. Summary of the invention

[0005] Based on the above problems, the present invention provides a microbial strain having biocontrol and growth-promoting effects and its application which can be used for the cultivation of Chinese medicinal materials such as Coptis chinensis.

[0006] The invention screens and obtains a paraburkholderia from rhizosphere soil of coptis chinensis collected from Congjiang, Guangxi. The strain prevents and controls coptis chinensis root rot by inhibiting the growth of pathogenic bacteria, dissolving phosphorus, potassium and nitrogen, producing iron carriers and ACC (aminocyclopropanecarboxylic acid, an ethylene precursor) deaminase, and promotes the healthy growth of coptis chinensis.

[0007] In order to solve the above technical problems, the present invention provides a strain of Burkholderia para

[0008] (Paraburkholderia phytofirmans) Ccw389, the Paraburkholderia phytofirmans (P.phytofirmans) Ccw389 was deposited on September 3, 2024 in the General Microbiology Center of the China Microorganism Culture Collection Administration, abbreviated as CGMCC, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain collection number is CGMCC No.31836.

[0009] In order to solve the above technical problems, the present invention also provides a microbial agent prepared by using Burkholderia parabacterium Ccw389 and a method for promoting healthy growth of plants by using the microbial agent;

[0010] Furthermore, the microbial agent is a fermentation broth of Burkholderia parabacterium Ccw389, and the preparation method of the fermentation broth is as follows: Burkholderia parabacterium Ccw389 is inoculated into TSB medium, shaken and cultured at 28°C and 180r / min for 36 hours (h), and then the bacterial concentration (OD 600 ) and adjust it to OD 600 is 1.0, that is, the bacterial agent is obtained;

[0011] Furthermore, the method of using the microbial agent to promote healthy plant growth is as follows: the agent is used to irrigate the roots of the Coptis chinensis seedlings that have just been transplanted, and the dosage of the agent is 5-10 mL / plant / Coptis chinensis, and then irrigate again every 7 days (d), for a total of 3 times.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The strain Ccw389 provided by the present invention has obvious antagonistic effects on the main pathogenic fungi that are currently reported to cause root rot of Coptis chinensis, Fusarium solani, Fusarium oxysporum and Ilyonectria robusta, indicating that the strain has good application potential in the prevention and control of root rot of Coptis chinensis.

[0014] 2. The strain Ccw389 provided by the present invention has strong phosphorus solubilizing, potassium solubilizing and nitrogen fixing effects. Its phosphorus solubility index on organic phosphorus medium is 1.83, its solubility index on potassium solubilizing medium is 3.2, and it can achieve good growth on nitrogen fixing medium, indicating that the strain has good application prospects in soil improvement.

[0015] 3. The strain Ccw389 provided by the present invention can produce ACC deaminase and siderophore. The ACC deaminase produced by the strain can help plants reduce the ethylene level during growth. After culturing on CAS medium for 7 days, the solubility index of the siderophore produced by the strain reached 3.69, indicating that the strain has a good growth-promoting effect on plants.

[0016] 4. The Burkholderia Ccw389 provided by the present invention is derived from the rhizosphere soil of Coptis chinensis and has strong adaptability in the soil microenvironment of the rhizosphere of Coptis chinensis. Therefore, it can be used for safe and effective prevention and control of Coptis chinensis root rot and growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The antagonistic ability of Burkholderia parahaemolyticus Ccw389 against the main pathogens of Coptis chinensis root rot.

[0018] Figure 2 This is a graph showing the inhibitory effect of strain Ccw389 on the spore germination of Echinococcus robusta.

[0019] Figure 3 Phylogenetic tree of Burkholderia parakholderiae Ccw389 based on 16S rDNA sequences.

[0020] Figure 4 (A) The calculation method of the solubility index and (B) the nitrogen fixation, phosphorus solubilization and potassium solubilization effects of Burkholderia parakholderiae Ccw389.

[0021] Figure 5 (A) The growth and iron carrier production of Burkholderia parakholderiae Ccw389 in CAS detection medium; (B) The growth of Burkholderia parakholderiae Ccw389 on DF and ADF plates.

[0022] Figure 6 This is a picture of the effect of root irrigation with Burkholderia parahaemolyticus Ccw389 on disease prevention and growth promotion of Coptis chinensis seedlings in potted plants. DETAILED DESCRIPTION

[0023] The culture medium used in the examples is as follows:

[0024] (1) TSB medium (1 L): tryptone 17.0 g, NaCl 5.0 g, soy peptone 3.0 g, glucose 2.5 g, anhydrous potassium dihydrogen phosphate 2.5 g, pH 7.3±0.2.

[0025] (2) TSA medium (1 L): 15.0 g tryptone, 5.0 g soy peptone, 5.0 g NaCl, 15 g agar, pH 7.3±0.2.

[0026] (2) PDA medium (1 L): 200 g of peeled potatoes (cut into small pieces and boil for 20 min, then filter through 6-layer gauze to collect the filtrate), 20 g of glucose, and 15 g of agar.

[0027] (3) Montgina organophosphate medium (1 L): glucose 10 g, (NH4)2SO4 0.5 g, KCl 0.3 g, NaCl 0.3 g, FeSO4·7H2O 0.03 g, MgSO4·7H2O 0.3 g, MnSO4·4H2O 0.03 g, CaCO3 5 g, lecithin 0.2 g, agar 15 g, pH adjusted to 7.0-7.5.

[0028] (4) Aleksandrov (potassium-dissolving) medium (1 L): glucose 5 g, MgSO4·7H2O 0.5 g, FeCl3 0.005 g, CaCO3 0.1 g, Ca3(PO4)2 2 g, potassium aluminosilicate (potassium-containing mineral) 2 g, bromothymol blue 0.1 g, agar 15 g.

[0029] (5) Ashby (nitrogen-fixing) medium (1 L): mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 40 g, CaSO4·2H2O 0.2 g, CaCO3 5 g, agar 15 g.

[0030] (6) CAS assay medium (1 L): After sterilizing component ①, cool it to 50-60°C, and add 100 mL of sterile component ② and 100 mL of component ③.

[0031] Ingredients ①: glucose 100g, peptone 20g, MgSO4·7H2O 0.5g, CaCl2 0.5g, agar 15g, pH adjusted to 6.6-7.0;

[0032] Component ②: 10× PBS buffer;

[0033] Ingredients ③: chrome azurol S (CAS) 0.06 g, FeCl3·5H2O 0.0027 g, HDTMA 0.073 g.

[0034] (7) DF medium (1 L): KH2PO4 4 g, Na2HPO4 6 g, MgSO4·7H2O0.2 g, glucose 2 g, glucose 2 g, citric acid 2 g, H3BO3 0.01 mg, MnSO4·H2O0.0112 mg, ZnSO4·7H2O 0.0778 mg, CuSO4·5H2O0.05 mg, MoO3 0.01 mg, FeSO4·7H2O 1.0 mg, agar 15 g.

[0035] (8) ADF medium (1 L): After sterilizing the DF medium, add sterile ACC stock solution and set its final concentration to 5.0 mmol / L.

[0036] Example 1

[0037] Isolation, screening and identification of strains:

[0038] The wild rhizosphere soil of Coptis chinensis collected by the inventor from Congjiang, Guangxi in September 2022 was used for the isolation and screening of target bacteria. The isolation process was carried out by the plate dilution method, which was as follows: 10 g of the collected rhizosphere soil sample was weighed and added to 90 mL of sterile water, and shaken on a shaker at 150 r / min for 30 minutes to suspend the microorganisms in the soil in sterile water as much as possible. The soil suspension was diluted with sterile water by 10 times serial dilution to obtain 10 -3 -10 -5 Dilutions were made in 100 μL dilutions per gradient and smeared on TSA plates. After culturing in a 28°C constant temperature incubator for 48 hours, the growth of the colonies was observed, and single colonies with different morphologies were selected. They were streaked on TSA plates using the plate streak method and cultured under the same conditions for 48 hours. Single colonies were then selected for multiple partition streak purification until pure strains were obtained. Different pure strains were uniformly numbered and transferred to TSA slant culture medium for culture and stored at 4°C for a short period of time. They were also stored in sterilized glycerol with a final concentration of 20% and stored in a -80°C refrigerator for later use.

[0039] According to the biocontrol microorganism screening system established in the laboratory in the early stage, the main pathogenic fungi of Coptis chinensis root rot currently isolated in the laboratory were used as test bacteria, and the plate confrontation method was used to screen bacteria with antagonistic effects on the pathogenic fungi. The specific method is as follows: use an inoculation loop to streak the bacterial solution cultured in TSB for 24 hours in the center of a 60mm PDA plate (through the entire plate), and then place different pathogenic fungal cakes (5mm) cultured for 4 days on one or both sides of the bacteria. After culturing in a constant temperature incubator at 28℃ for 3-7 days, the antibacterial effect was observed. The experiment was repeated 3 times to screen out strains with antagonistic effects on the pathogenic fungi.

[0040] A biocontrol bacterial strain Ccw389 was obtained through the above separation, purification and screening process. The strain Ccw389 has antagonistic effects on the main pathogens of Coptis chinensis root rot reported so far, namely Fusarium solani, Fusarium oxysporum and Ilyonectria robusta. Figure 1 A), and the inhibition rate of strain Ccw389 against different pathogens was further calculated by the following formula, where the calculation method of pathogen colony area was the product of the long diameter and the short diameter of the colony.

[0041]

[0042] The results showed that the inhibition rate of strain Ccw389 against Echinococcus robusta was 43.0%, against Fusarium solani was 45.4%, and against Fusarium oxysporum was 62.5% ( Figure 1 B).

[0043] In addition, we used pure water as the culture medium and tested the effect of Ccw389 on the spore germination of Robusta Trichoderma by co-cultivation. The results showed that when Robusta Trichoderma was cultured alone, its spores began to germinate after 6 hours (red arrows), and its hyphae length became longer and longer as the culture time increased; in contrast, after adding Ccw389 to co-cultivation, the spore germination of Robusta Trichoderma was significantly inhibited, and its spores did not begin to germinate until 72 hours after culture ( Figure 2 ). The above results indicate that this strain has certain application potential in the prevention and control of different soil-borne fungal diseases.

[0044] Morphological identification of the strain showed that it was short rod-shaped, with a bacterial body size of (0.4-0.5) μm×(0.6-1.5) μm. The colonies on TSA medium were round with neat edges, smooth surface and opaque surface.

[0045] The strain was further molecularly identified by PCR amplification of the bacterial 16S rDNA sequence (SEQ ID NO.1) and sequencing comparison.

[0046] The amplification primers were 27F: AGAGTTTGATCMTGGCTCAG, 1492R: TACGGYTACCTTGTTACGACTT;

[0047] The reaction system was: 10× Buffer 2 μL, 2.5 mM dNTP 1.5 μL, Primer1 1 μL, Primer2 1 μL, template 1 μL, enzyme 0.3 μL, water 13.2 μL, total volume 20 μL;

[0048] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min, extension at 72°C for 10 min, and insulation at 4°C forever.

[0049] The PCR products were detected by agarose gel electrophoresis and then sequenced.

[0050] The 16S rDNA sequence of the bacterium was obtained after forward and reverse sequencing and splicing. The sequence was compared with the NCBI database and the EzTaxon database by Blast. The comparison results showed that it had the highest homology with Paraburkholderia phytofirmans PsJN, reaching 99.64%. At the same time, combined with the phylogenetic tree analysis results, it showed that ( Figure 3 ), the strain is a Paraburkholderia phytofirmans, named as Paraburkholderia phytofirmans Ccw389.

[0051] Paraburkholderia phytofirmans Ccw389 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 3, 2024, abbreviated as CGMCC, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31836.

[0052] Example 2

[0053] Nitrogen fixation, phosphorus solubilization and potassium solubilization characteristics of strain Ccw389:

[0054] After the strain Ccw389 was activated and cultured in a TSB liquid test tube for 24 hours, 2 μL of the bacterial solution was taken and inoculated on the Montgena organophosphate plate, Aleksandrov plate and Ashby plate respectively. After the bacterial solution was absorbed by the plate, the plate was inverted and cultured in a 28°C constant temperature incubator in the dark. The results were observed after 7 days. If the strain can grow on the Ashby plate, it means that it has the ability to fix nitrogen spontaneously. If a transparent degradation zone appears on the other two plates, it means that the strain has the ability to dissolve phosphorus or potassium. By measuring the diameter D of the transparent zone and the diameter d of the single colony, the solubility index (D / d) ( Figure 4 A) Phosphate and potassium solubilizing abilities of the strains.

[0055] The results are shown in Table 1 and Figure 4 As shown in B.

[0056] After 7 days of constant temperature culture, strain Ccw389 was able to achieve good growth on the Ashby plate, and the colony color was milky white, indicating that the strain had good self-generated nitrogen fixation ability.

[0057] At the same time, strain Ccw389 can also grow well on Montgena organophosphate plates and Aleksandrov plates, and produce obvious transparent zones.

[0058] The diameter D of the transparent zone of strain Ccw389 on the Montana organophosphate plate was 1.1 cm, the diameter of a single colony was 0.6 cm, and the solubility index of dissolved organophosphate was 1.83 (Table 1).

[0059] The diameter D of the transparent zone of strain Ccw389 on the Aleksandrov plate was 1.6 cm, the diameter of a single colony was 0.5 cm, and the solubility index of dissolved potassium was 3.2 (Table 1).

[0060] Table 1. Nitrogen fixation, phosphorus solubilization and potassium solubilization characteristics of strain Ccw389

[0061]

[0062] The results showed that strain Ccw389 had good nitrogen fixation, phosphorus solubilization and potassium solubilization abilities, indicating that the strain had certain application prospects in soil improvement and plant growth promotion.

[0063] Example 3

[0064] Characteristics of siderophore production by strain Ccw389:

[0065] Solid CAS medium is blue-green. Different strains are inoculated on the plate to obtain single colonies. When the target strain secretes iron carriers, a yellow halo will form around the colony. The larger and darker the yellow halo, the stronger the ability of the strain to produce iron carriers. After activating and culturing strain Ccw389 in a TSB liquid test tube for 24 hours, 2 μL of bacterial solution was inoculated on the CAS plate. After the bacterial solution was absorbed by the plate, the plate was inverted in a 28°C constant temperature incubator for light-proof cultivation. The results were observed after 7 days of cultivation.

[0066] Table 2. Growth and halo formation of strain Ccw389 on CAS plates

[0067]

[0068] The results are as follows Figure 5 A and Table 2.

[0069] After 7 days of cultivation, the colony diameter of strain Ccw389 was 0.65 cm, the halo diameter was 2.4 cm, and the halo color was yellow and transparent. The solubility index calculated from this was 3.69.

[0070] The results showed that strain Ccw389 could promote plant growth by producing siderophores.

[0071] Example 4

[0072] Characteristics of ACC deaminase produced by strain Ccw389:

[0073] ACC deaminase can degrade ACC, the precursor of ethylene, thereby reducing the ethylene level during plant growth and helping plant growth. Take 2 μL of the bacterial solution cultured with TSB liquid medium and spot it on DF plates and ADF plates respectively. Set up 2 parallel replicates for each strain and culture in a 28°C incubator in the dark. After 7 days, observe and compare the growth of the strain on DF and ADF plates. If the growth of the strain on the ADF plate is better than that on the DF plate, it means that the strain has ACC deaminase activity.

[0074] The results are as follows Figure 5 As shown in B. After 7 days of cultivation, the colony diameter of strain Ccw389 on DF plate was 0.65 cm, and the colony diameter on ADF plate was 1.05 cm, which was significantly better than the growth condition on DF plate, indicating that strain Ccw389 has a strong activity of producing ACC deaminase, and thus can achieve the purpose of promoting plant growth.

[0075] Example 5

[0076] Effect of root irrigation with strain Ccw389 on disease prevention and growth promotion of Coptis chinensis seedlings:

[0077] At the Coptis chinensis planting base in Jinshan Village, Xishui County, Guizhou Province, healthy Coptis chinensis seedlings of one year with consistent growth were selected and transplanted into small flower pots, with two plants transplanted per pot. The soil used for potting was fresh soil from the base's nursery shed. The transplanted Coptis chinensis potted plants were placed back into the original nursery shed, and the flower pots were embedded in the soil so that the soil height in the flower pots was consistent with the soil height on the surrounding ground. The bacterial liquid of Burkholderia parasitica Ccw389 was taken after 36 hours of culture, the culture medium was removed by centrifugation, the bacteria were reselected with sterile water, and the OD 600 After the value was adjusted to 1.0, the bacterial solution was used to irrigate the roots of the newly transplanted Coptis chinensis. The amount of bacterial solution was 10 mL / plant·Coptis chinensis. The bacteria were irrigated once every 7 days, and the amount of bacterial solution for the last two times was 8 mL / plant·Coptis chinensis. At the same time, an equal amount of blank sterile water was set as a control for root irrigation. After 30 days, the potted plants were taken out and photographed to record the growth of Coptis chinensis.

[0078] The results are as follows Figure 6As shown. After 30 days, the four pots of Coptis chinensis under the control treatment (CK) showed no obvious growth compared with before transplanting. Only one pot of seedlings grew a new leaf (marked by a blue solid arrow), and two pots of seedlings showed partial leaf wilt (marked by a red dotted arrow). In contrast, the Coptis chinensis seedlings in the treatment group (Ccw389) treated with Ccw389 bacterial solution for three times showed good growth. None of the four pots of seedlings showed disease, all leaves showed a healthy oily green color, and compared with before transplanting, each seedling grew new buds or new leaves (marked by a blue solid arrow). The results show that strain Ccw389 has a good disease prevention and growth promotion effect on Coptis chinensis.

[0079] The above description is only an illustrative embodiment of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by any person skilled in the art without departing from the concept and principle of the present invention shall fall within the scope of protection of the present invention.

Claims

1. Paraburkholderia phytofirmans Ccw389, whose deposit number is CGMCC No.31836.

2. A microbial preparation, the active ingredient of which is the Paraburkholderia phytofirmans Ccw389 described in claim 1.

3. Use of Paraburkholderia phytofirmans Ccw389 according to claim 1 in the preparation of a bacterial agent for antagonizing Fusarium solani, Fusarium oxysporum and / or Ilyonectria robusta.

4. Use of Paraburkholderia phytofirmans Ccw389 according to claim 1 in the preparation of phosphate-dissolving, potassium-dissolving and / or nitrogen-fixing bacterial agents.

5. Use of Paraburkholderia phytofirmans Ccw389 according to claim 1 in the preparation of a siderophore-producing bacterial agent.

6. Use of Paraburkholderia phytofirmans Ccw389 according to claim 1 in the preparation of an ACC deaminase-producing bacterial agent.

7. Use of Paraburkholderia phytofirmans Ccw389 according to claim 1 in the preparation of a disease-preventing and growth-promoting microbial preparation of Coptis chinensis.

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