A biocontrol growth-promoting bacterium GNLJ-7 and its application

Abstract

This invention provides a biocontrol and growth-promoting bacterium, GNLJ-7, and its applications, relating to the field of microbial technology. The biocontrol and growth-promoting bacterium GNLJ-7 described in this invention is classified as *Gordonia didemni*, with the preservation number CCTCC NO: M20241529. The biocontrol and growth-promoting bacterium GNLJ-7 of this invention possesses nitrogen-fixing activity and inhibits seed germination and parasitism of the weed *Orobanche deserticola*, while promoting root growth in the host tomato. It has potential application as a novel biocontrol strain in the control of *Orobanche deserticola* weed.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically a biocontrol and growth-promoting bacterium GNLJ-7 and its applications. Background Technology

[0002] Branched ointment ( Orobanche ramosa L. is a parasitic weed commonly found in fields at altitudes of 140-1400 meters. It mainly harms crops such as tomatoes, sunflowers, melons, and peppers, and can have a serious impact on agricultural production.

[0003] Traditional weed control methods mainly include chemical and physical weeding. Chemical weeding typically involves spraying herbicides, but the use of herbicides must be tailored to the environment; excessive application can easily cause pollution, lead to herbicide resistance in weeds, and cause toxicity to non-target organisms. Physical weeding, such as manual weeding and mechanized tillage, also has limitations such as high labor costs, low efficiency, and potential damage to crop growth. With increasing attention to environmental and ecological balance, biological control methods are gradually gaining importance. Biological control methods include protecting crops in the field through natural enemies, pathogens, and microorganisms.

[0004] The application of biocontrol and growth-promoting bacteria in agriculture mainly manifests in promoting plant growth, increasing crop yield, enhancing plant resistance to diseases, pests, and weeds, and improving soil quality. *Gordonella* spp. are a rare type of actinomycetes, with 41 effective species isolated from habitats such as soil, animal manure, and compost. In recent years, their biodegradation and bioactive substance production capabilities have attracted widespread attention in the research field, but their application as biocontrol and growth-promoting bacteria in agriculture has been rarely reported. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a biocontrol and growth-promoting bacterium GNLJ-7 and its application. This biocontrol and growth-promoting bacterium has nitrogen-fixing activity and inhibits the seed germination and parasitism of the weed *Orobanche deserticola*, while promoting the growth of the root system of the host tomato.

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

[0007] This invention provides a biocontrol growth-promoting bacterium, GNLJ-7, whose taxonomic name is *Goldenella*. Gordonia didemni The accession number is CCTCC NO: M20241529.

[0008] The present invention also provides a bacterial solution comprising the biocontrol growth-promoting bacterium GNLJ-7.

[0009] The present invention also provides a method for preparing the bacterial solution, comprising the following steps: picking colonies of biocontrol and growth-promoting bacteria GNLJ-7 and inoculating them into NB medium, and culturing them at a constant temperature of 30~35℃ for 18~25h to obtain the bacterial solution.

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

[0011] The present invention also provides the application of the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution described herein in the control of branching broomrape weeds.

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

[0013] Preferably, the biocontrol and growth-promoting bacteria GNLJ-7 or the bacterial solution can inhibit the degree of parasitism of broomrape in plant roots.

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

[0015] Preferably, the plant includes tomatoes, sunflowers, melons, or peppers.

[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 advantages:

[0018] This invention provides a biocontrol growth-promoting bacterium, GNLJ-7, which is classified as *Goldenella*. Gordonia didemni This bacterium has nitrogen-fixing properties and can inhibit the germination and parasitism of Orobanche deserticola seeds, while promoting the root growth of tomato seedlings. As a novel biocontrol strain, it has potential for application in the control of Orobanche deserticola weed.

[0019] The biocontrol and growth-promoting bacterium GNLJ-7 described in this invention can inhibit the germination of branched broomrape seeds under in vitro culture conditions (1.0 × 10⁻⁶). 6A CFU / mL concentration of the bacterial agent reduced the seed germination rate of *Broomria thunbergii* from 91.93% to 13.18%. Under pot conditions, the biocontrol and growth-promoting bacterium *Goldenella* GNLJ-7, when inoculated into soil containing *Broomria thunbergii* through root irrigation, significantly promoted tomato root growth, reduced the degree of *Broomria thunbergii* parasitism, increased superoxide dismutase (SOD) content, and decreased proline (PRO) and malondialdehyde (MDA) content. This invention, by developing novel biocontrol and growth-promoting bacteria, can reduce the use of chemical pesticides, decrease environmental pollution, and save costs. Therefore, this invention provides a good strain resource for the future development of specialized microbial biocontrol agents and inoculants for the control of *Broomria thunbergii*.

[0020] Biological Preservation Information

[0021] The biocontrol and growth-promoting bacteria GNLJ-7 described in this invention is classified and named Gordon's bacterium. Gordonia didemni Depository: China Center for Type Culture Collection; Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Accession Number: CCTCC NO: M20241529; Date of Deposit: July 10, 2024. Attached Figure Description

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

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

[0024] Figure 3 The phylogenetic tree of the biocontrol and growth-promoting bacterium GNLJ-7 constructed for this invention;

[0025] Figure 4 The effect of the biocontrol and growth-promoting bacterium GNLJ-7 of the present invention on the root system of tomato parasitized by Orobanche deserticola is shown in the figure from left to right: application of DNKAS to tomato roots, application of GNLJ-7 to tomato roots, and application of water to tomato roots.

[0026] Figure 5 The figure shows the effect of the biocontrol and growth-promoting bacterium GNLJ-7 of the present invention on the superoxide dismutase (SOD) activity of the host tomato. This means p < 0.01. This represents p < 0.001;

[0027] Figure 6 The figure shows the effect of the biocontrol growth-promoting bacterium GNLJ-7 of the present invention on the proline (PRO) content of the host tomato. This represents p < 0.001;

[0028] Figure 7The figure shows the effect of the biocontrol and growth-promoting bacterium GNLJ-7 of the present invention on the malondialdehyde (MDA) content of the host tomato. This means p < 0.05. This means p < 0.001. Detailed Implementation

[0029] This invention provides a biocontrol growth-promoting bacterium, GNLJ-7, whose taxonomic name is *Goldenella*. Gordonia didemni The biocontrol and growth-promoting bacterium GNLJ-7 described in this invention was isolated from processing tomato fields in Changji region, Xinjiang Uygur Autonomous Region. After culturing GNLJ-7 on NA plates for 24 hours, the resulting colonies were orange-red in color, round or nearly round in shape, and had a smooth surface. The bacteria were rod-shaped, without spores or flagella, and had a length of 2–2.5 µm and a width of 0.5–0.8 µm.

[0030] The physiological and biochemical characteristics of the biocontrol growth-promoting bacterium GNLJ-7 described in this invention are as follows: Gram-positive, aerobic, catalase-positive, oxidase-negative, and can grow at 10~45℃, pH 4.0~10.5 and 0~8% NaCl. The optimal growth temperature is 30℃, the optimal growth pH is 6.5~7.5, and the optimal NaCl is 0.5%.

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

[0032] This invention also provides a bacterial suspension comprising the biocontrol growth-promoting bacterium GNLJ-7. This invention further provides a method for preparing the bacterial suspension, preferably comprising the following steps: picking colonies of the biocontrol growth-promoting bacterium GNLJ-7 and inoculating them onto NB medium, then culturing at a constant temperature of 30-35℃ for 18-25 hours to obtain the bacterial suspension. More preferably, it comprises the following steps: picking colonies of the biocontrol growth-promoting bacterium GNLJ-7 and inoculating them onto NB medium, then culturing at a constant temperature of 35℃ for 20 hours to obtain the bacterial suspension. In this invention, the NB medium is obtained from the following raw material ratio: 10.0 g peptone, 3.0 g beef extract powder, 5.0 g NaCl, 1000 mL distilled water, pH 7.0-7.3.

[0033] This invention also provides an application of the biocontrol and growth-promoting bacterium GNLJ-7 or the bacterial solution described above in plant nitrogen fixation. In this invention, the activated GNLJ-7 strain was transferred to Assoubai medium to test its nitrogen-fixing ability. Preliminary screening showed that the GNLJ-7 strain of this invention could grow normally on Assoubai medium and form colonies, indicating that the GNLJ-7 strain obtained by this invention has nitrogen-fixing activity and has certain application prospects in plant nitrogen fixation.

[0034] This invention also provides the application of the biocontrol growth-promoting bacterium GNLJ-7 or the bacterial solution described above in the control of *Broomrape lanceolata* weeds. Preferably, the biocontrol growth-promoting bacterium GNLJ-7 or the bacterial solution described above can inhibit the seed germination of *Broomrape lanceolata*. Experimental results show that the biocontrol growth-promoting bacterium GNLJ-7 described above can inhibit the seed germination of *Broomrape lanceolata* under in vitro culture conditions, at a concentration of 1.0 × 10⁻⁶. 6 A CFU / mL concentration of the inoculant can reduce the germination rate of branched broomrape seeds from 91.93% to 13.18%.

[0035] In this invention, the biocontrol growth-promoting bacterium GNLJ-7 or the bacterial solution can inhibit the parasitism of broomrape in plant roots, and the plant is preferably tomato. Experiments have shown that, under potted conditions, the biocontrol growth-promoting bacterium GNLJ-7, when inoculated into soil containing broomrape through root irrigation, can significantly promote tomato root growth, reduce the parasitism of broomrape, increase the content of superoxide dismutase (SOD) in the plant, and decrease the content of proline (PRO) and malondialdehyde (MDA).

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

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

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0039] Example 1

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

[0041] This invention utilizes a gradient dilution method to isolate growth-promoting microorganisms from processing tomato fields in Changji, Xinjiang Uygur Autonomous Region, obtaining a total of 52 isolates. The purified bacteria were numbered, and single colonies were picked and transferred to NA slant agar for later storage (NA medium: 10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1000 mL distilled water, pH 7.0–7.3). The activated strain was transferred to Assumption medium to test its nitrogen-fixing ability (Assumption 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). After incubation at 35℃ for 72h, colony formation on the medium was observed. Following preliminary screening, the nitrogen-fixing activity of strain GNLJ-7 was determined to be... Figure 1 ).

[0042] 2. Colony characteristics and colony morphology

[0043] After culturing the GNLJ-7 strain on NA plates for 24 hours, the colonies formed were orange-red in color. Figure 2 The colonies are round or nearly round with a smooth surface. The bacteria are rod-shaped, without spores or flagella, and are 2–2.5 µm long and 0.5–0.8 µm wide.

[0044] 3. Physiological and biochemical characteristics

[0045] According to the test results, GNLJ-7 is Gram positive, aerobic, catalase positive, and oxidase negative. It can grow at 10~45℃, pH 4.0~10.5 and 0~8% NaCl. The optimal growth temperature is 30℃, 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 strain GNLJ-7 was aligned with sequence data in the GenBank database using MEGA software's proximity method. (See attached image.) Figure 3 The results showed that strain GNLJ-7 was related to... G.didemni Located on the same branch, its 16S rDNA sequence is similar to... G.didemni The similarity to B204 (JN615417) reached 98.53%. Based on colony morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis, it was identified as… Gordonia didemni .

[0048] Example 2: Germination Experiment of Branched Orobanche Seeds

[0049] Those proven to be effective against ointmentia Bacillus subtilis The DNKAS strain (provided by Baoding Kelvfeng Biochemical Technology Co., Ltd.) was used as a positive control. Single colonies of GNLJ-7 and DNKAS obtained from Example 1 were picked and inoculated into NB medium and cultured at 35℃ for 20 h at a rotation speed of 150 r / min. The negative control (CK) was NB liquid medium without microorganisms. The initial concentration of the microbial liquid culture was diluted to 1.0 × 10⁻⁶ by adjusting the absorbance value at 600 nm. 8 CFU / mL.

[0050] Prepare a disposable culture dish and line it with a sterile glass fiber filter paper with a diameter of 8.0 mm. Using a pipette, add 30 μL of cell fermentation filtrate or NB medium (dilution gradient 10) to the filter paper. -2 10 -3 Then, using sterilized tweezers, a pre-cultured *Orobanche deserticola* seed was transferred onto a glass fiber filter paper. 30 μL of GR24 solution (0.1 mg / L) was added to each seed using a pipette. To maintain humidity within the culture dish, a moistened qualitative filter paper was folded three times evenly and placed in the center of the dish, and the dish was sealed with a sealing film to prevent drying. Six replicates were set up for each condition. The culture dishes were wrapped in two layers of black plastic bags and incubated at a constant temperature of 25°C. After 7 days, the germ tube germination and growth of the *Orobanche deserticola* seeds were observed using a stereomicroscope, and the number of germinated seeds and the total number of seeds were recorded. Germination was defined as the germ tube breaking through the seed coat of the *Orobanche deserticola* seed.

[0051] Table 1. Effects of GNLJ-7 strain on the germination inhibition rate of Orobanche deserticola.

[0052]

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

[0054] As can be seen from Table 1, when the bacterial agent is diluted to 1.0 × 10⁻⁶… 6 At CFU / mL, compared with the control in NB medium of the same concentration, the germination rate of *Orobanche deserticola* seeds decreased from 91.93% to 13.18%, a significant difference. Its inhibition rate reached 86.47%, 15.22% higher than the widely used DNKAS strain. When the inoculum was diluted to 1.0 × 10⁻⁶ CFU / mL, the germination rate of *Orobanche deserticola* seeds decreased from 91.93% to 13.18%, a significant difference. 5At CFU / mL, the inhibition rates of both GNLJ-7 and DNKAS strains decreased due to the reduced concentration, but the inhibition rates were still higher than those without the strain, and the inhibition effect of GNLJ-7 was slightly higher than that of DNKAS. The results indicate that the biocontrol and growth-promoting bacterium GNLJ-7 described in this invention has a strong inhibitory effect on the germination of Orobanche deserticola seeds.

[0055] Example 3: Potted experiment with branched Orobanche deserticola

[0056] The collected farmland soil for processing tomatoes was sieved through a 2mm×2mm sieve. The sieved soil, peat moss, and vermiculite were mixed in a 2:1:1 volume ratio to prepare the culture medium. 0.25g of Cucurbita seeds were added per kilogram of culture medium and mixed thoroughly to prepare the inoculation medium. 100g of the inoculation medium was placed in a 7cm×7cm×11cm seedling pot. Tomato seedlings with uniform growth of 3-4 leaves were transplanted into the seedling pot, one seedling per pot. GNLJ-7 and DNKAS bacterial solutions (both at a concentration of 1.0×10⁻⁶) were applied weekly via root drenching. 8 The concentration of CFU / mL was added to the nutrient pots with water, with a root irrigation volume of 0.5 mL / pot / time. The control group (CK) received an equal volume of water. Each treatment was replicated 12 times. Watering was carried out as needed. After 50 days, the degree of parasitism and plant growth were assessed, and the activities of SOD, PRO, and MDA in the host crop were measured. SOD activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method, PRO content was determined using the acidic ninhydrin colorimetric method, and MDA content was determined using the thiobarbituric acid colorimetric method. All physiological indicators were measured using kits from Suzhou Keming Biotechnology Co., Ltd.

[0057] Table 2 Effects of GNLJ-7 application on tomato growth

[0058]

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

[0060] B. subtilis DNKAS strain is a commercially available biocontrol agent for controlling broomrape. Under the same cultivation conditions, as shown in Table 2, the application of GNLJ-7 bacterial solution also promoted tomato plant growth, especially in promoting root development, with effects exceeding those of DNKAS. B. subtilis DNKAS bacteria increased root length and root weight by 30% and 134.95%, respectively.

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

[0062] Table 3. Effects of GNLJ-7 application on orobanche deserticola in tomato roots.

[0063]

[0064] Note: Different letters 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 was significantly increased after root irrigation with GNLJ-7 and DNKAS bacterial solutions, compared with the control. SOD is one of the important protective enzymes for scavenging superoxide free radicals in plants, and the increase in SOD enzyme activity indicates the enhancement of the biocontrol activity of tomatoes.

[0066] from Figure 6 It can be seen that the proline content in tomato leaves significantly decreased and significantly increased after root irrigation treatment with GNLJ-7 and DNKAS bacterial solutions, respectively. Proline is an important osmotic regulator that plays a crucial role in tomatoes' response to biological stress. It can maintain cell osmotic pressure, stabilize protein structure, and scavenge free radicals. Orobanche stress causes oxidative stress or other forms of damage to the host crop. The addition of DNKAS bacteria may help alleviate stress and increase proline content through its growth-promoting and stress-resistance properties. The decrease in proline content in tomatoes after application of GNLJ-7 bacterial solution may be because this bacterium alleviates the effects of orobanche stress on the crop by improving nutrient absorption and promoting root growth, thus reducing the need for tomatoes to accumulate excessive proline to cope with the stress.

[0067] from Figure 7 It can be seen that the malondialdehyde (MDA) content in tomato leaves was significantly reduced after root irrigation with GNLJ-7 and DNKAS bacterial solutions. A decrease in MDA content usually indicates a reduction in lipid peroxidation of plant cell membranes, which is important for maintaining the normal structure and function of plant cells. Simultaneously, the decrease in MDA content also reflects an enhanced resistance of tomatoes to broomrape parasites, helping tomatoes maintain stable growth and development under adverse conditions.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

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

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

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

4. The use of the biocontrol growth promoting bacteria GNLJ-7 according to claim 1 or the bacterial liquid according to claim 2 in the prevention and treatment of O. hamata.

5. Use according to claim 4, characterized in that, The biocontrol growth promoting bacteria GNLJ-7 or the bacterial liquid can inhibit the seed germination of O. hamata.

6. Use according to claim 4, characterized in that, The biocontrol growth promoting bacteria GNLJ-7 or the bacterial liquid can inhibit the parasitism of O. hamata in the root system of tomato.

7. The use of the biocontrol growth promoting bacteria GNLJ-7 according to claim 1 or the bacterial liquid according to claim 2 in promoting the growth of tomato.

8. Use according to claim 7, characterized in that, The biocontrol growth promoting bacteria GNLJ-7 or the bacterial liquid can promote the growth and development of the root system of tomato.

Images