Kudzuvine root endophytic bacterium JK1700184 and application thereof

Abstract

The invention discloses a kudzuvine root endophytic bacterium JK1700184 and application thereof, and belongs to the technical field of crop biocontrol strains, the kudzuvine root endophytic bacterium JK1700184 is Burkholderia cepacia, and is preserved in Guangdong Microbial Culture Collection Center on April 29, 2024, and the preservation number is GDMCC No: 64582. The invention further discloses a preparation method of the kudzuvine root endophytic bacterium JK1700184. The bacteriostasis rate of the radix puerariae endophytic bacteria JK1700184 obtained through screening on the rice sheath blight disease can reach 66%; compared with a control group, a JK1700184 fermentation liquor treatment experiment group has the advantages that the relative growth rate of the plant height can reach 20% or above, and tillering is generated earlier. The radix puerariae endophytic bacterium JK1700184 obtained through separation and screening can promote rice growth and has an inhibiting effect on rice sheath blight disease, and the radix puerariae endophytic bacterium JK1700184 has a wide application prospect in the aspect of rice yield control.

Description

Technical Field

[0001] The invention relates to the technical field of crop biocontrol strains, and in particular to a kudzu root endophytic bacterium JK1700184 and an application thereof. Background Art

[0002] Rice is one of the most important crops in my country and even in the world. More than half of the world's population relies on rice as food. Due to the continuous growth of the global population and the continuous reduction of grain planting area caused by environmental damage, global food production will soon be insufficient to feed all the people in the world. In order to meet the population's demand for food, food production must be continuously increased. However, rice yields are affected by various diseases, which seriously affects the high and stable yields of rice and directly threatens human production and development. Modern agriculture is over-controlling plant diseases and relies on the use of chemical pesticides. The application of a large amount of chemical pesticides not only has a long-term destructive effect on the ecological environment, but also causes a decline in the quality of agricultural products, excessive pesticide residues, resistance of pathogens, and harm to humans and animals. It is of great significance to find safer and more effective methods for disease prevention and control.

[0003] Rice sheath blight is one of the most common diseases of rice. It is generally more serious in early rice than in late rice. In severe cases, it can cause the plant to fall and die. At present, the main measures for preventing and controlling rice diseases include breeding resistant varieties, chemical control, and biological control. Among them, breeding resistant varieties refers to the use of breeding varieties to prevent and control diseases, but breeding is difficult and specialized resistance is easy to lose; chemical control is convenient, fast, and effective, but it is accompanied by side effects that cannot be ignored, such as chemical residues, environmental pollution, and pest resistance, which obviously does not meet the requirements of sustainable agriculture; biological control refers to an effective strategy to inhibit or kill pathogens using antagonistic microorganisms and metabolites. It is a sustainable and practical plant disease management method and a potential alternative strategy for rice disease control. Therefore, it is of great significance to screen out endophytic bacteria with growth-promoting and antagonistic effects. Screening and then developing and utilizing endophytic bacteria in Pueraria root is also one of the most promising measures to effectively promote rice growth and prevent and control rice sheath blight. Summary of the invention

[0004] The purpose of the present invention is to provide a kudzu root endophytic bacterium JK1700184 and its application to solve the problems existing in the above-mentioned prior art. The kudzu root endophytic bacterium JK1700184 separated and screened by the present invention can promote rice growth and inhibit rice sheath blight.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical Solution 1: A strain of endophytic bacteria JK1700184 from Pueraria lobata, wherein the endophytic bacteria JK1700184 from Pueraria lobata is Burkholderia cepacia, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 29, 2024, with the deposit number GDMCC No: 64582.

[0007] Technical Solution 2: An application of the endophytic bacteria JK1700184 of Pueraria lobata in inhibiting rice sheath blight.

[0008] Technical solution three: An application of the endophytic bacteria JK1700184 of Pueraria lobata in promoting rice growth.

[0009] Technical solution 4: A method for inhibiting the growth of pathogenic bacteria, comprising the following steps:

[0010] The endophytic bacteria JK1700184 or its fermentation liquid is used to treat pathogenic bacteria, thereby inhibiting the growth of pathogenic bacteria; the pathogenic bacteria is Rhizoctonia solani.

[0011] Technical solution 5: A method for preventing and treating rice sheath blight caused by Rhizoctonia solani, comprising the following steps:

[0012] The endophytic bacteria JK1700184 or its fermentation liquid is used to treat rice plants; the OD of the fermentation liquid is 600 =0.7.

[0013] Furthermore, the method for treating rice plants comprises: 600 The rice plants were root-irrigated with the fermentation liquid of endophytic bacteria JK1700184 from Pueraria lobata at a concentration of 0.7.

[0014] Technical Solution 6: A biocontrol agent for preventing and controlling rice sheath blight caused by Rhizoctonia solani, the biocontrol agent comprising the endophytic bacterium JK1700184 of Pueraria lobata or its fermentation liquid.

[0015] The present invention discloses the following technical effects:

[0016] The present invention isolated and screened a uniform strain of endophytic bacteria JK1700184 from the tubers of the medicinal plant Pueraria lobata. The JK1700184 was identified as Burkholderia cepacia, which was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 29, 2024, with a deposit number of GDMCC No: 64582. And by utilizing the endophytic bacteria JK1700184 from Pueraria lobata or its fermentation liquid, a biocontrol agent for preventing and treating rice sheath blight caused by Rhizoctonia solani can be further obtained. After the present invention inoculated the fermentation liquid of JK1700184 for 7 consecutive days on rice seedlings grown for 18 days by the root irrigation method, the growth of the rice seedlings in the experimental group showed obvious growth promotion compared with that in the control group. Figure 1 As shown in Figure 2, the inhibition rate of JK1700184 against rice sheath blight can reach 66%. Figure 6 As shown, the relative growth rate of plant height in the experimental group treated with JK1700184 fermentation liquid was more than 20% compared with the control group, and tillers were produced earlier. The endophytic bacteria JK1700184 of kudzu root screened by the present invention can have a positive effect on the growth of rice seedlings and has outstanding potential in rice cultivation. The strain JK1700184 screened by the present invention has an inhibitory effect on rice sheath blight and has broad application prospects for rice yield control. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The plate confrontation culture of endophytic bacteria JK1700184 from kudzu root (B) and rice sheath blight (A);

[0019] Figure 2 This is a colony morphology characteristic diagram of the endophytic bacteria JK1700184 in Pueraria root;

[0020] Figure 3 This is a diagram showing the bacterial morphology of the endophytic bacterium JK1700184 from Pueraria root;

[0021] Figure 4 Phylogenetic tree constructed based on 16s rDNA sequences for endophytic bacteria JK1700184 and JK1801053 from Pueraria root;

[0022] Figure 5 The germination rate of rice seeds under the treatments of CK group and JK1700184 group;

[0023] Figure 6 This is the growth of rice seedlings under the treatments of CK group and JK1700184 group. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0030] Example 1

[0031] 1 Isolation, screening and identification of strains

[0032] 1.1 Isolation of strains

[0033] The test materials were Guige No. 18, Guige No. 17, and Guige No. 8 planted in the kudzu root experimental field of the College of Agriculture of Guangxi University; the test strains were all provided by the Institute of Plant Pathology of Guangxi University.

[0034] Culture medium: LB culture medium (1L) contains 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, and 15g of agar powder; nitrogen-fixed culture medium, PDA solid culture medium: 200g of potatoes, 18g of sucrose, 15g of agar powder, add distilled water to 1L, sterilize and set aside.

[0035] Surface disinfection: Wash the sample surface three times with sterile water, cut the sample into appropriate sizes with a sterile blade; soak the sample in 75% ethanol for 30 seconds for surface disinfection, and wash it three times with sterile water; soak the sample in 3% sodium hypochlorite solution for 2-8 minutes according to the type and size of the sample for surface sterilization, then quickly wash it 5-7 times with sterile water, and dry the surface for later use. Use the sterile water of the last wash as the control group to confirm the surface disinfection. If there is no colony growth in the control group, it means that the sample surface has been completely disinfected.

[0036] Isolation and purification of strains: Use sterile tweezers to place the surface-sterilized sample in a sterile mortar with sterile phosphate buffer and grind it into a homogenate. Take 50μL of the sample homogenate and evenly spread it on LB medium, nitrogen-fixed medium, and PDA medium plates. Repeat each sample 3 times. Cultivate at 28℃ and observe for 7 days. During this period, observe the shape, size, whether the edges are neat, whether the surface is smooth, the degree of ridges, transparency, colony color and other morphologies every 12 hours. The colonies of different morphologies are separated and purified by streaking until a single colony with a single morphology is obtained. The single colony obtained by separation and purification is inoculated into LB liquid culture medium and cultured in a shaker at 28℃ and 220rpm until the logarithmic growth phase. Take 500μL of bacterial solution and mix it with sterilized 30% glycerol in a ratio of 1:1 and store it in a -80℃ refrigerator.

[0037] The isolated endophytic bacteria of Pueraria root were inoculated on LB solid culture medium plates and cultured at 28°C for 24 hours for use. About 10 mL of PDA culture medium was poured into each culture dish (diameter = 9.0 cm), and the surface moisture was dried and then used. The indicator pathogens stored in the slant test tube were inoculated on the PDA culture medium plate, placed in a static incubator at 28°C for 2-9 days, and healthy indicator pathogens were obtained for use.

[0038] 1.2 Screening

[0039] The plate confrontation method was used to screen the endophytic bacteria in Pueraria lobata.

[0040] Four points were designed to inoculate different endophytic bacteria with equal distance (3.0 cm) from the center of the PDA plate, and 10 μL of bacterial solution was inoculated at each point. After 1 day of culture, the pathogenic bacteria cake of Rhizoctonia solani (diameter = 0.7 cm) was inoculated in the center of the plate, with the mycelium side of the bacteria cake facing down and contacting the culture medium. The plates were co-cultured at 28°C for 1-5 days, and the PDA plates inoculated with only the pathogenic bacteria cake were used as the control group. The plates were observed every 24 hours, and the appearance of the inhibition line was used as the basis to preliminarily judge whether the endophytes had antagonistic ability. Each group of experiments was repeated 3 times.

[0041] The strains that produced the inhibition line obtained in the initial screening were screened twice. The same endogenous bacteria were inoculated at two symmetrical points with an equal distance (3.0 cm) in the center of the PDA plate, and 10 μL of bacterial solution was inoculated at each point. After 1 day of culture, the indicator pathogen cake (diameter = 0.7 cm) was inoculated in the center of the plate and co-cultured at 28°C for 1-5 days. The PDA plate inoculated with only the pathogen cake was used as the control group, and it was observed every 24 hours. The growth size of the pathogen was measured by the cross method, and the inhibition rate was calculated according to the size of the pathogen to determine the antagonistic effect.

[0042] The calculation formula of the ability of endophytic bacteria to inhibit pathogens is as follows:

[0043] Bacterial inhibition rate = (1-(pathogen colony area in experimental group / pathogen colony area in control group))*100%

[0044] The results showed that three endophytic bacteria from kudzu root with strong inhibitory ability against rice sheath blight were obtained, which were named JK1700184, JK1802149 and JK1801053. Among them, the inhibitory rate of the strain named JK1700184 against rice sheath blight was 66%. Figure 1 shown.

[0045] 1.3 Identification

[0046] (I) Morphological characteristics of strains

[0047] The antagonistic strains were inoculated onto LB solid culture medium plates by plate streaking method, and the morphology of single colonies was observed and recorded. Observation of bacterial morphological characteristics The strains cultured on the culture medium were subjected to Gram staining and microscopic examination, and the observed morphological results were photographed under a microscope.

[0048] (II) Strain sequence and phylogenetic analysis

[0049] DNA was extracted from the isolated strains using the bacterial DNA extraction kit (CW05525) from Kangwei Century. The 16SrRNA gene sequence was amplified using universal primers F8 and R1541, which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR primer sequences are shown in Table 1.

[0050] Table 1 PCR primer sequences

[0051]

[0052] PCR products were detected by 1.1% (W / V) agarose gel electrophoresis, with 2000bp DNA ladder as marker. The length of 16SrRNA was about 1500bp. The PCR products with the correct band were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequenced 16SrRNA gene sequence was compared with the GenBank database by BLAST on NCBI (https: / / www.ncbi.nlm.nih.gov / ), and homology analysis was performed with known sequences in the database. Molecular identification of each strain was completed and a phylogenetic tree was established using MAGE7. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0053] Table 2 PCR reaction system

[0054]

[0055] Table 3 PCR reaction conditions

[0056] PCR Pre-denaturation transsexual annealing extend Final extension 16S rRNA 94℃3min 94℃30s 55℃30s 72℃1.5min20cycles 72℃10min

[0057] 1.4 Results:

[0058] (1) Morphological characteristics of strains

[0059] Colony morphology: After streaking culture for 48 hours, the size of a single colony of the antagonistic bacteria JK1700184 is about 2 mm, off-white, slightly moist and slightly raised, with neat edges and round colonies. Figure 2 shown.

[0060] Morphological characteristics of bacteria: After Gram staining, the strain JK1700184 was observed under an optical microscope. The staining was red and the bacteria were rod-shaped. It was determined that the strain was a rod-shaped Gram-negative bacterium. Figure 3 shown.

[0061] (2) 16S rDNA sequence and phylogenetic analysis of strains

[0062] The amplified product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with the GenBank database by BLAST and homology analysis was performed with known sequences in the database. The comparison results showed that JK1700184 had the highest homology with Burkholderia cepacia, with homology and sequence similarity of more than 99%. A phylogenetic tree was established using MAGE7 and the sequencing results of JK1700184. The results of the tree construction are shown in the figure. Figure 4 The strain JK1700184 was preliminarily identified as Burkholderia cepacia, which was deposited in Guangdong Microbiological Culture Collection Center on April 29, 2024, with the deposit number GDMCC No: 64582.

[0063] Example 2

[0064] 1 Evaluation of the effect of bacterial solution on promoting rice growth

[0065] 1.1 Effect of bacterial solution on rice germination rate

[0066] (1) Preparation of bacterial liquid: The active strain JK1700184 obtained in the initial screening was placed on LB solid medium for activation. A 0.6 cm bacterial block was taken and 50 μl of bacterial liquid was inoculated into 75 ml of sterile LB liquid medium. The mixture was cultured in the dark at 28°C and 150 rpm for 1 day and then removed for use.

[0067] (2) Application of bacterial solution: Select rice seeds that are plump and free of disease spots. Use 3% sodium hypochlorite to disinfect the surface of the rice seeds for 5 minutes, and constantly blow and suck to fully disinfect the surface of the seeds; rinse with sterile water repeatedly for 5 times; spread the seeds on sterile filter paper and air-dry the surface moisture. Sterile water is used as the control group, and the endophytic fermentation liquid obtained by fermenting the bacterial solution prepared in step (1) is used as the experimental group: ① sterile water; ② endophytic fermentation liquid (OD 600 =0.7). 50 rice seeds were treated in each group, and each group was repeated 3 times. The rice seeds were completely soaked in the corresponding solution of the experimental group and left to stand for 30 minutes; they were repeatedly rinsed with sterile water for 3 times; the seeds were spread on sterile filter paper and the surface moisture was air-dried. The treated seeds were spread on a culture dish with 2 layers of moist filter paper, with 2 seeds between them. They were cultured at 30℃ and exposed to natural light. During this period, sterile water was added to the culture dish in time to ensure that the filter paper was moist. Germination was considered when the length of the embryo exceeded half of the length of the seed. The germination of seeds was recorded at 5 days.

[0068] 2 Effects of bacterial solution on the growth of rice seedlings

[0069] (1) Preparation of bacterial liquid: The active strain JK1700184 obtained in the initial screening was placed on LB solid medium for activation. A 0.6 cm bacterial block was taken and 100 μl of bacterial liquid was inoculated into 150 ml of sterile LB liquid medium. The mixture was cultured in the dark at 28°C and 150 rpm for 1 day and then removed for later use.

[0070] (2) Soak the seeds in warm water for 24 hours to allow the rice seeds to absorb enough water and remove the unfilled seeds floating on the water surface. Spread the healthy and full seeds flat on the soil and cover the surface of the seeds with a thin layer of soil. Grow them in a greenhouse that can be exposed to the sun. When the rice seedlings grow to about 4 cm, select the seedlings with consistent growth and transplant them into a single cup, with 30 seedlings in each control group and experimental group. Pay attention to daily management during the seedling stage. 14 days after transplanting, the rice seedlings were irrigated with roots. The root irrigation method was applied every other day. The treatment group was applied with 5 ml of bacterial solution (OD 600 =0.7); the control group was given the same amount of sterile water. During the period, attention was paid to management and sterile water was applied to maintain root moisture. After 7 consecutive days of treatment, the growth of rice was observed and recorded.

[0071] 3 Results

[0072] The germination rate of rice seeds after 5 days of culture is as follows Figure 5 As shown in the figure, the fermentation liquid of JK1700184 can promote the germination of rice seeds to a certain extent, and the germination rate is slightly reduced. But overall, there is no significant difference between the germination rate of rice seeds treated with the fermentation liquid of JK1700184 and the control.

[0073] Effect of bacterial solution on the growth of rice seedlings: After the 18-day-old rice seedlings were continuously inoculated with JK1700184 fermentation solution for 7 days by root irrigation, the growth of the rice seedlings in the experimental group showed a significant growth promotion compared with the control group. Figure 6 As shown, the relative growth rate of plant height in the JK1700184 fermentation liquid treatment group was more than 20% higher than that in the control group, and tillers were produced earlier.

[0074] The endophytic bacterium JK1700184 from Pueraria root can have a positive effect on the growth of rice seedlings and has outstanding potential in rice cultivation.

[0075] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An endophytic bacterium of Pueraria root JK1700184, characterized in that: The endophytic bacterium JK1700184 of Pueraria lobata is Burkholderia cepacia, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 29, 2024, with the deposit number GDMCC No: 64582.

2. Use of the endophytic bacterium JK1700184 of Pueraria lobata as claimed in claim 1 in inhibiting rice sheath blight.

3. Use of the endophytic bacterium JK1700184 of Pueraria lobata as claimed in claim 1 in promoting rice growth.

4. A method for inhibiting the growth of pathogenic bacteria, characterized in that: The following steps are involved: The endophytic bacterium JK1700184 of Pueraria lobata or its fermentation liquid as claimed in claim 1 is used to treat pathogens, thereby inhibiting the growth of pathogens; the pathogen is Rhizoctonia solani.

5. A method for preventing and controlling rice sheath blight caused by Rhizoctonia solani, characterized in that: The following steps are involved: The endophytic bacterium JK1700184 of Pueraria lobata or its fermentation liquid according to claim 1 is used to treat rice plants; the OD of the fermentation liquid is 600 =0.

7.

6. The method according to claim 5, characterized in that The method for treating rice plants comprises: 600 The rice plants were root-irrigated with the fermentation liquid of endophytic bacteria JK1700184 from Pueraria lobata at a concentration of 0.

7.

7. A biocontrol agent for preventing and controlling rice sheath blight caused by Rhizoctonia solani, characterized in that: The biocontrol agent comprises the endophytic bacterium JK1700184 of Pueraria lobata according to claim 1 or its fermentation liquid.

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