Biocontrol growth-promoting bacterium GNLJ-7 and application thereof

Abstract

The invention provides a biocontrol growth-promoting bacterium GNLJ-7 and application thereof, and relates to the technical field of microorganisms. The classification name of the biocontrol growth-promoting bacterium GNLJ-7 disclosed by the invention is Gordonia discni, and the preservation number of the biocontrol growth-promoting bacterium GNLJ-7 is CCTCC (China Center For Type Culture Collection) NO: M20241529. The biocontrol growth-promoting bacterium GNLJ-7 has nitrogen fixation activity, has an inhibiting effect on seed germination and parasitism of weed Orobanche coerulescens, has a promoting effect on growth of host tomato root systems, and has application potential in the aspect of prevention and control of Orobanche coerulescens weed damage as a novel biocontrol strain.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a biocontrol and growth-promoting bacterium GNLJ-7 and an application thereof. Background Art

[0002] Orobanche ramosa L. is a parasitic weed that is commonly found in fields at an altitude of 140 to 1,400 meters. It mainly harms crops including tomatoes, sunflowers, melons, and peppers, and can easily have a serious impact on agricultural production.

[0003] Traditional weed control methods are mainly chemical weed control and physical weed control. Chemical weed control is generally achieved by spraying chemical herbicides, and the use of chemical herbicides must be applied reasonably according to the environment. Once excessive, it is easy to cause pollution, produce drug resistance in field weeds, and toxic effects on non-target organisms. Physical weed control, such as manual weeding and mechanical tillage, also has the limitations of high labor costs, low efficiency, and easy damage to crop growth. With the increasing attention to the environment and ecological balance, biological control methods have gradually received attention. Biological control methods include protecting field crops through natural enemies, pathogens and microorganisms.

[0004] The application of biocontrol and growth-promoting bacteria in agriculture is mainly reflected in promoting plant growth, increasing crop yields, enhancing plant resistance to pests and diseases, and improving soil quality. Gordonia actinomycetes are a rare class of actinomycetes, and 41 effective species have been isolated from habitats such as soil, animal manure, and compost. In recent years, its ability to biodegrade and produce active substances has attracted widespread attention in its research field, but its application as a biocontrol and growth-promoting bacteria in agriculture has rarely been reported. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a biocontrol and growth-promoting bacterium GNLJ-7 and its application, which has nitrogen fixation activity, and has an inhibitory effect on the seed germination and parasitism of the weed Orobanche deserticola, and promotes the growth of the host tomato root system.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a biocontrol and growth-promoting bacterium GNLJ-7. The classification name of the biocontrol and growth-promoting bacterium GNLJ-7 is Gordonia didemni, and the preservation number is CCTCC NO: M20241529.

[0008] The present invention also provides a bacterial liquid comprising the biocontrol and growth-promoting bacteria GNLJ-7.

[0009] The present invention also provides a method for preparing the bacterial solution, comprising the following steps: selecting a colony of the biocontrol and growth-promoting bacteria GNLJ-7 and inoculating it into an NB culture medium, and culturing it at a constant temperature of 30 to 35° C. for 18 to 25 hours to obtain a bacterial solution.

[0010] The present invention also provides an application of the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution in plant nitrogen fixation.

[0011] The present invention also provides an application of the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution in preventing and controlling branched Orobanche weeds.

[0012] Preferably, the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the germination of Oleander seeds.

[0013] Preferably, the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the parasitism of branched mycelium in the root system of plants.

[0014] The present invention also provides a use of the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution in promoting plant growth.

[0015] Preferably, the plant comprises tomato, sunflower, melon or pepper.

[0016] More preferably, the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can promote the growth and development of plant roots.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention provides a biocontrol and growth-promoting bacterium GNLJ-7. The biocontrol and growth-promoting bacterium GNLJ-7 is classified and named Gordonia didemni. The bacterium has a nitrogen-fixing property and can inhibit the germination and parasitism stages of Orobanche seeds, and promote the growth of the root system of tomato seedlings. As a new biocontrol strain, the bacterium GNLJ-7 has application potential in the prevention and control of Orobanche weed damage.

[0019] The biocontrol and growth-promoting Gordonia GNLJ-7 of the present invention can inhibit the germination of Orobancha ciliata seeds under the conditions of culture in a dish, 1.0×10 6The bacterial agent with a CFU / mL concentration can reduce the germination rate of Orobanche seed from 91.93% to 13.18%. Under potted conditions, the biocontrol and growth-promoting Gordonia GNLJ-7 is inoculated into the soil containing Orobanche by root irrigation, which can significantly promote the growth of tomato roots, reduce the parasitic degree of Orobanche, increase the superoxide dismutase (SOD) content of the plant, and reduce the content of proline (PRO) and malondialdehyde (MDA). The present invention can reduce the use of chemical pesticides, reduce pollution to the environment, and save costs by developing new biocontrol and growth-promoting bacteria. Therefore, the present invention provides a good strain resource for the future development of special microbial biocontrol agents and fertilizers for preventing and controlling Orobanche.

[0020] Biological deposit information

[0021] The biocontrol and growth-promoting bacteria GNLJ-7 described in the present invention is classified and named Gordonia didemni, and the preservation unit is China Center for Type Culture Collection; the preservation unit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation number is CCTCC NO: M20241529; the preservation date is July 10, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention on the Axubei culture medium;

[0023] Figure 2 This is a colony morphology diagram of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention;

[0024] Figure 3 A phylogenetic tree of the biocontrol and growth-promoting bacteria GNLJ-7 constructed for the present invention;

[0025] Figure 4 The effect of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention on the tomato root system parasitized by Oleander, from left to right in the figure, DNKAS was applied to the tomato root, GNLJ-7 was applied to the tomato root, and water was applied to the tomato root;

[0026] Figure 5 The effect of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention on the superoxide dismutase (SOD) activity of host tomatoes, ** in the figure represents p<0.01, *** represents p<0.001;

[0027] Figure 6 This is the effect of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention on the proline (PRO) content of host tomatoes, and *** in the figure represents p<0.001;

[0028] Figure 7This figure shows the effect of the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention on the malondialdehyde (MDA) content of host tomatoes. In the figure, * represents p<0.05, and *** represents p<0.001. DETAILED DESCRIPTION

[0029] The present invention provides a biocontrol and growth-promoting bacterium GNLJ-7, the classification name of the biocontrol and growth-promoting bacterium GNLJ-7 is Gordonia didemni, and the preservation number is CCTCC NO: M20241529. The biocontrol and growth-promoting bacterium GNLJ-7 of the present invention is isolated and obtained from a processing tomato planting field in Changji, Xinjiang. The present invention cultured the biocontrol and growth-promoting bacterium GNLJ-7 on a NA plate for 24 hours to form a colony color of orange-red, a round or nearly round colony shape, and a smooth surface. The bacterial body is rod-shaped, without spores and flagella, and the length of the bacterial body is 2 to 2.5 μm and the width is 0.5 to 0.8 μm.

[0030] The biocontrol and growth-promoting bacteria GNLJ-7 of the present invention has the following physiological and biochemical characteristics: Gram-positive, aerobic, catalase-positive, oxidase-negative, can grow under the conditions of 10-45°C, pH 4.0-10.5 and 0-8% NaCl, the optimum growth temperature is 30°C, the optimum growth pH is 6.5-7.5, and the optimum NaCl is 0.5%.

[0031] The present invention compares the 16S rDNA sequence of the biocontrol and growth-promoting bacteria GNLJ-7 with the sequences in the GenBank database, and the results show that GNLJ-7 and G. didemni are on the same branch, and the similarity of its 16S rDNA sequence with G. didemni B204 (JN615417) is 98.53%. Combining the colony morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis, it is identified as Gordonia didemni.

[0032] The present invention also provides a bacterial liquid including the biocontrol and growth-promoting bacteria GNLJ-7. The present invention also provides a method for preparing the bacterial liquid, preferably comprising the following steps: picking colonies of the biocontrol and growth-promoting bacteria GNLJ-7 and inoculating them into NB culture medium, and culturing them at a constant temperature of 30 to 35°C for 18 to 25 hours to obtain a bacterial liquid. More preferably, it comprises the following steps: picking colonies of the biocontrol and growth-promoting bacteria GNLJ-7 and inoculating them into NB culture medium, and culturing them at a constant temperature of 35°C for 20 hours to obtain a bacterial liquid. In the present invention, the NB culture medium is obtained by the following raw material ratio: 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of NaCl, 1000 mL of distilled water, and pH 7.0 to 7.3.

[0033] The present invention also provides an application of the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution in plant nitrogen fixation. The present invention transfers the activated GNLJ-7 strain to the Axubei culture medium to detect the nitrogen fixation ability of the strain. After preliminary screening, the GNLJ-7 strain of the present invention can grow normally in the Axubei culture medium and form colonies, indicating that the GNLJ-7 strain screened by the present invention has nitrogen fixation activity and has certain application prospects in plant nitrogen fixation.

[0034] The present invention also provides a use of the biocontrol growth-promoting bacteria GNLJ-7 or the bacterial solution in controlling the branched Orobanche weed. Preferably, the biocontrol growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the germination of Orobanche seeds. The experimental results show that the biocontrol growth-promoting bacteria GNLJ-7 of the present invention can inhibit the germination of Orobanche seeds under the conditions of culture in a dish, 1.0×10 6 CFU / mL concentration of the inoculant can reduce the germination rate of Orobancha seeds from 91.93% to 13.18%.

[0035] In the present invention, the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the parasitism of Orobanche in the root system of a plant, and the plant is preferably a tomato. Experiments have shown that under potted conditions, the biocontrol and growth-promoting bacteria GNLJ-7 can be inoculated into the soil containing Orobanche by root irrigation, which can significantly promote the growth of tomato roots, reduce the parasitism of Orobanche, increase the superoxide dismutase (SOD) content of the plant, and reduce the proline (PRO) and malondialdehyde (MDA) content.

[0036] The present invention also provides an application of a biocontrol and growth-promoting bacterium GNLJ-7 or the bacterial solution in promoting plant growth. In the present invention, the plants include but are not limited to tomatoes, sunflowers, melons and peppers. More preferably, the biocontrol and growth-promoting bacterium GNLJ-7 or the bacterial solution can promote the growth and development of plant roots. Experimental results show that the application of the biocontrol and growth-promoting bacterium GNLJ-7 described in the present invention has a promoting effect on the growth of tomato plants, especially in promoting root development, which can increase root length and root weight by 30% and 134.95%, respectively.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0039] Example 1

[0040] 1. Screening process of biocontrol and growth-promoting bacteria GNLJ-7

[0041] The present invention uses a gradient dilution method to isolate growth-promoting microorganisms from processing tomato planting fields in Changji, Xinjiang, and obtains a total of 52 isolates. The purified bacteria are numbered, and single colonies are picked and transferred to NA slant medium for storage (NA medium: peptone 10.0g, beef extract powder 3.0g, NaCl 5.0g, agar 15.0g, distilled water 1000mL, pH 7.0-7.3). The activated strain was transferred to Axubei medium to detect the nitrogen fixation ability of the strain (Axubei medium: glucose 10g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, CaCO3 5.0g, agar 20g, distilled water 1000mL, pH 6.8-7.0), cultured at 35℃ for 72h to observe the colony formation on the medium. After preliminary screening, the nitrogen fixation activity of the strain numbered GNLJ-7 was ( Figure 1 ).

[0042] 2. Colony characteristics and colony morphology

[0043] After culturing GNLJ-7 on NA plates for 24 h, colonies were formed with an orange-red color ( Figure 2 ), the colony is round or nearly round in shape, with a smooth surface. The bacteria are rod-shaped, without spores and flagella, with a length of 2 to 2.5 μm and a width of 0.5 to 0.8 μm.

[0044] 3. Physiological and biochemical characteristics

[0045] It was determined that GNLJ-7 is Gram-positive, aerobic, catalase-positive, oxidase-negative, and can grow at 10-45°C, pH 4.0-10.5, and 0-8% NaCl. The optimal growth temperature is 30°C, the optimal growth pH is 6.5-7.5, and the optimal NaCl is 0.5%.

[0046] 4. 16S rDNA sequence analysis

[0047] The 16S rDNA sequence of GNLJ-7 strain was compared with the sequence data in the GenBank database using the MEGA software proximity method. Figure 3 The results showed that the strain GNLJ-7 was on the same branch as G. didemni, and its 16SrDNA sequence had a similarity of 98.53% with G. didemniB204 (JN615417). Combining the colony morphology, physiological and biochemical characteristics and 16SrDNA sequence analysis, it was identified as Gordonia didemni.

[0048] Example 2: Experiment on germination of Orobancha spp. seeds

[0049] The Bacillus subtilis DNKAS strain (provided by Baoding Kelufeng Biochemical Technology Co., Ltd.), which has been proven to have the effect of controlling broomrape, was set as a positive control. Single colonies of GNLJ-7 and DNKAS obtained in Example 1 were picked and inoculated into NB medium, and cultured at a constant temperature of 35°C for 20 hours at a rotation speed of 150 r / min. The negative control (CK) used NB liquid medium without inoculation of microorganisms. The initial concentration of the microbial liquid culture was diluted to 1.0×10 8 CFU / mL.

[0050] Prepare a disposable culture dish and lay a piece of sterile glass fiber filter paper with a diameter of 8.0 mm on it. Use a pipette to add 30 μL of cell fermentation filtrate or NB medium (dilution gradient of 10 -2 , 10 -3 ). Then, use sterilized tweezers to transfer a pre-cultured Oleander seed on the glass fiber filter paper. Use a pipette to add 30 μL of GR24 solution (concentration of 0.1 mg / L) to each seed. In order to maintain the humidity environment in the culture dish, fold a moist qualitative filter paper evenly three times and place it in the middle of the culture dish, and wrap the culture dish with a sealing film to prevent internal drying. Six repeated experimental samples were set up for each condition. After wrapping the culture dish with two layers of black plastic bags, it was placed at a constant temperature of 25°C for culture. After 7 days, the germination and growth of the germ tubes of the Oleander seeds were observed using a stereo microscope, and the number of germinated seeds and the total number of seeds were recorded. The germination standard is: the germ tubes of the Oleander seeds break through the seed coat, which is considered to be successfully germinated.

[0051] Table 1 Effect of GNLJ-7 strain on germination inhibition rate of Orobancha ciliata

[0052]

[0053] Note: Different letters in a, b, and c in the table indicate significant differences at the P<0.05 level

[0054] It can be seen from Table 1 that when the bacterial agent is diluted to 1.0×10 6 CFU / mL, compared with the NB medium control with the same concentration, the germination rate of Orobanche aspergillus seeds decreased from 91.93% to 13.18%, with a significant difference. Its inhibition rate reached 86.47%, which was 15.22% higher than the widely used DNKAS strain. 5CFU / mL, due to the decrease in concentration, the inhibition rates of GNLJ-7 and DNKAS strains decreased, but the inhibition rates were still higher than those without bacteria, and the inhibition effect of GNLJ-7 bacteria was slightly higher than that of DNKAS strain. The results show that the biocontrol and growth-promoting bacteria GNLJ-7 of the present invention has a strong inhibitory effect on the germination of Orobanche asper seeds.

[0055] Example 3: Parasitic potted plant experiment of Orobancha fascicularis

[0056] The collected processing tomato farm soil was sieved through a 2mm×2mm sieve, and the sieved soil, peat and vermiculite were mixed in a volume ratio of 2:1:1 to prepare the culture soil. 0.25g of guaylan seeds were added to each kilogram of culture soil to prepare the inoculation soil for later use. 100g of inoculation soil was placed in a 7cm×7cm×11cm nutrient pot, and 3-4 leaf tomato seedlings with uniform growth were moved into the nutrient pot, 1 plant / pot, and GNLJ-7 and DNKAS bacterial solutions (both at a concentration of 1.0×10 8 CFU / mL) were poured into the nutrient pots with water, and the root irrigation volume was 0.5mL / pot / time. The control group (CK) was added with an equal amount of clean water. Each treatment was set up with 12 replicates, and watering was done as needed. After 50 days, the parasitism degree of Orobancha and the growth of the plants were counted, and the SOD, PRO, and MDA activities of the host crops were detected. Among them, the SOD activity was determined by the nitroblue tetrazolium (NBT) photoreduction method, the PRO content was determined by the acid ninhydrin colorimetric method, and the MDA content was determined by the thiobarbituric acid colorimetric method. The above physiological indicators were all determined using the kits of Suzhou Keming Biotechnology Co., Ltd.

[0057] Table 2 Effect of applying GNLJ-7 bacteria on tomato growth

[0058]

[0059] Note: Different letters in a and b in the table indicate significant differences at the P<0.05 level

[0060] B.subtilis DNKAS is a commercialized biocontrol bacterium for the prevention and treatment of branched broomrape. Under the same cultivation measures, according to the results in Table 2, it can be seen that the application of GNLJ-7 bacterial solution also promotes the growth of tomato plants, especially in promoting root development, with an effect exceeding that of B.subtilis DNKAS, increasing root length and root weight by 30% and 134.95% respectively.

[0061] After carefully cleaning the tomato roots, the number and weight of parasites on the roots were counted. The results are shown in Table 3. Figure 4 The treatment group using the GNLJ-7 bacterial solution of the present invention reduced the number of parasites of Orobanche by 47.62%, and the weight of Orobanche was reduced by 37.90%, and the parasitic situation was significantly alleviated.

[0062] Table 3 Effects of GNLJ-7 on Orobanche parasitism on tomato roots

[0063]

[0064] Note: Different letters in a and b in the table indicate significant differences at the P<0.05 level

[0065] from Figure 5 It can be seen that the SOD enzyme activity of tomato leaves after root irrigation with GNLJ-7 and DNKAS bacterial solution increased significantly compared with the control. SOD is one of the important protective enzymes in plants for removing superoxide free radicals. The increase in SOD enzyme activity indicates the enhancement of tomato biocontrol activity.

[0066] from Figure 6 It can be seen that the proline content of tomato leaves after root irrigation with GNLJ-7 and DNKAS bacterial solution was significantly reduced and significantly increased, respectively. Proline is an important osmotic regulating substance and plays an important role in tomatoes coping with biological adversity. It can maintain cell osmotic pressure, stabilize protein structure, and scavenge free radicals. Orobanche stress causes oxidative stress or other forms of damage to host crops. The addition of DNKAS bacteria may help crops alleviate stress and increase proline content through its growth-promoting and stress-resistant properties. The application of GNLJ-7 bacterial solution reduced the proline content of tomatoes, which may be because the bacteria alleviated the impact of Orobanche stress on crops by improving nutrient absorption and promoting root growth, so that tomatoes do not need to accumulate too much proline to cope with adversity.

[0067] from Figure 7 It can be seen that the malondialdehyde content in tomato leaves was significantly reduced after root irrigation with GNLJ-7 and DNKAS bacterial solution. The reduction in MDA content usually means a reduction in the degree of lipid peroxidation in plant cell membranes, which is of great significance for maintaining the normal structure and function of plant cells. At the same time, the reduction in MDA content also reflects the enhanced resistance of tomatoes to Orobanche parasitism, which helps tomatoes maintain the stability of growth and development under adverse conditions.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biocontrol and growth-promoting bacterium GNLJ-7, characterized in that: The classification name of the biocontrol and growth-promoting bacteria GNLJ-7 is Gordonia didemni, and the preservation number is CCTCC NO: M20241529.

2. A bacterial liquid comprising the biocontrol and growth-promoting bacteria GNLJ-7 according to claim 1.

3. The method for preparing the bacterial solution according to claim 2, characterized in that: The method comprises the following steps: picking colonies of the biocontrol and growth-promoting bacteria GNLJ-7 and inoculating them into NB culture medium, and culturing them at a constant temperature of 30-35°C for 18-25h to obtain bacterial liquid.

4. Use of the biocontrol and growth-promoting bacteria GNLJ-7 according to claim 1 or the bacterial solution according to claim 2 in plant nitrogen fixation.

5. Use of the biocontrol and growth-promoting bacteria GNLJ-7 according to claim 1 or the bacterial solution according to claim 2 in controlling the branched Orobanche weed.

6. The use according to claim 5, characterized in that: The biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the germination of Oleander seeds.

7. The use according to claim 5, characterized in that: The biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the parasitism of branched schizophylla in the root system of plants.

8. Use of the biocontrol and growth-promoting bacteria GNLJ-7 according to claim 1 or the bacterial solution according to claim 2 in promoting plant growth.

9. The use according to claim 8, characterized in that: The plants include tomatoes, sunflowers, melons or peppers.

10. The use according to claim 8 or 9, characterized in that: The biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can promote the growth and development of plant roots.

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