Application of Lemna-Chromobacterium-X1 in prevention and treatment of rice sheath blight disease

Abstract

The invention discloses an application of a lemna minor endophyte, namely chromobacterium, in the resistance of rhizoctonia solani, and particularly discloses an application of the lemna minor endophyte, namely the chromobacterium, in the resistance of rhizoctonia solani. The chromobacterium Lemna-Chromobacterium-X1 is also used for preventing and / or treating the rice sheath blight disease caused by the rhizoctonia solani, and can also be used for preventing and / or treating the rice sheath blight disease caused by the rhizoctonia solani. The invention further provides a method for preventing and / or treating the rice sheath blight disease caused by the rhizoctonia solani through the chromobacterium Lemna-Chromobacterium-X1. The invention further provides a method for preventing and / or treating the rice sheath blight disease caused by the rhizoctonia solani. The invention provides a green solution for green prevention and control of rice sheath blight disease, and has wide agricultural popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of Lemna-Chromobacterium-X1 of the genus Chromobacterium in the prevention and control of rice sheath blight. Background Art

[0002] Rice sheath blight is a soil-borne fungal disease caused by Rhizoctonia solani, and it is also a rice disease that harms rice all over the world. Rice sheath blight has great harm, strong epidemicity, and is difficult to control, seriously affecting the growth, yield and quality of rice. At present, chemical pesticides are widely used in the control of sheath blight, but long-term use has led to problems such as environmental pollution, increased pathogen resistance, and excessive pesticide residues in agricultural products. Therefore, it is particularly important to develop green, environmentally friendly and efficient biological control methods. Biological control has the advantages of high safety and little impact on the environment.

[0003] Plant endophytic bacteria have the characteristics of promoting plant growth, improving the disease resistance of host plants, and enhancing the stress resistance of plants to the environment. They are a rich and valuable natural resource and have the potential to be developed and utilized by humans. Duckweed (Lemnaspp.) is a widely distributed aquatic plant that symbiotic with a variety of endophytes. These endophytes can secrete a variety of bioactive substances, including antibiotics, enzymes, siderophores and volatile organic compounds (VOCs), and have potential pathogen inhibitory and plant growth-promoting functions. However, there are few reports on the research of duckweed endophytes in disease control at present. Summary of the Invention

[0004] Based on the above problems, the purpose of the present invention is to isolate highly efficient antagonistic strains from duckweed and identify the strains by morphological, physiological and biochemical characteristics and 16S rDNA sequences, in order to provide high-quality endophytes for the biological control of rice sheath blight, and at the same time provide a green solution for rice sheath blight.

[0005] The technical solutions to achieve the above purpose are as follows:

[0006] In the first aspect of the present invention, there is provided the application of Lemna-Chromobacterium-X1 of the genus Chromobacterium in anti-Rhizoctonia solani.

[0007] In the second aspect of the present invention, there is provided the application of Lemna-Chromobacterium-X1 of the genus Chromobacterium in the prevention and / or treatment of rice sheath blight caused by Rhizoctonia solani.

[0008] In some of the embodiments, the 16S rRNA of the aforementioned Lemna-Chromobacterium-X1 is as shown in SEQ ID NO: 1, or has more than 95% homology with SEQ ID NO: 1, or belongs to the same strain as GenBank: OP364816.1.

[0009] In some embodiments, the aforementioned Lemna-Chromobacterium-X1 is screened from duckweed.

[0010] In a third aspect of the present invention, a method for preventing and / or treating rice sheath blight caused by Rhizoctonia solani is provided, wherein a solution containing the Lemna-Chromobacterium-X1 is used to irrigate the rice roots or spray the solution at various growth stages of the rice, such as the seedling stage, tillering stage, jointing stage, booting stage, heading stage, flowering stage, etc. Preferably, the 16S rRNA of the Lemna-Chromobacterium-X1 is as shown in SEQ ID NO: 1, or has more than 95% homology with SEQ ID NO: 1, or belongs to the same strain as GenBank: OP364816.1.

[0011] In some embodiments, the spraying is to spray the solution containing the Lemna-Chromobacterium-X1 on the tillering stage, heading stage and full heading stage of the rice growth period. In particular, the tillering stage is the high-incidence period of rice sheath blight, which requires more prevention.

[0012] In some embodiments, the concentration of the Lemna-Chromobacterium-X1 is 7×10^8 CFU / ml to 9×10^8 CFU / ml (at 8×10^8 CFU / ml, OD600=1).

[0013] A fourth aspect of the present invention is to provide a method for screening the aforementioned Lemna-Chromobacterium-X1, comprising the following steps:

[0014] (1) Collect duckweed, rinse, and then rinse with sterile water;

[0015] (2) blotting the water with sterile filter paper, soaking it with alcohol and sodium hypochlorite solution respectively, and then rinsing it with sterile water, blotting the duckweed with sterile filter paper to obtain a duckweed sample;

[0016] (3) Grind the duckweed samples with a sterilized mortar, dilute the grinding solution with PBS buffer in a 10x-fold gradient, spread each concentration of the diluted solution on LB medium, culture at 25 - 35 °C for 2 - 3 days, and then further purify by streaking and isolation to obtain duckweed endophytes;

[0017] (4) Conduct 16S rRNA molecular biological identification on the duckweed endophytes to obtain Chromobacterium sp. Lemna-Chromobacterium-X1. The 16S rRNA of Chromobacterium sp. Lemna-Chromobacterium-X1 is as shown in SEQ ID NO:1, or has a homology of more than 95% with SEQ ID NO:1, or belongs to the same strain as GenBank: OP364816.1.

[0018] In some of these embodiments, between step (3) and step (4), it further includes: spreading the sterile water used for the last rinse on LB medium as a control to prove that the isolated bacteria are duckweed endophytes.

[0019] The present invention first isolates from duckweed the Chromobacterium sp. strain Lemna-Chromobacterium-X1 that has a significant antagonistic effect against the pathogen of rice sheath blight, Rhizoctonia solani, and identifies the strain using morphology, physiological and biochemical characteristics, and 16S rDNA sequences, providing a high-quality endophyte for the biological control of rice sheath blight. At the same time, it provides a green solution for the green prevention and control of rice sheath blight, and has broad prospects for agricultural promotion and application. Description of the Drawings

[0020] Figure 1 Morphological diagram of Lemna-Chromobacterium-X1 strain.

[0021] Figure 2 Phylogenetic tree of Lemna-Chromobacterium-X1 sequence.

[0022] Figure 3 Inhibitory effect of Lemna-Chromobacterium-X1 on Rhizoctonia solani of rice.

[0023] Figure 4 Protease production (left figure) and amylase production ability (right figure) of Lemna-Chromobacterium-X1.

[0024] Figure 5 Phosphate solubilization (left figure) and chitinase production ability (right figure) of Lemna-Chromobacterium-X1.

[0025] Figure 6 Defense effect of Lemna-Chromobacterium-X1 against sheath blight of rice. In the left figure: antagonistic bacterium + Rhizoctonia solani; in the right figure: Rhizoctonia solani. Specific implementation manners

[0026] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that those skilled in the art can understand the disclosure of the present invention more thoroughly and comprehensively.

[0027] For the experimental methods without specific conditions noted in the following embodiments, they are usually carried out under conventional conditions. For example, the fourth edition of "Molecular Cloning: A Laboratory Manual" edited by Green and Sambrook was published in 2013, or according to the conditions recommended by the manufacturer. All common chemical reagents used in the embodiments are commercially available products.

[0028] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for describing specific embodiments and do not limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0029] Sequence homology is a qualitative concept in biology used to describe whether gene, protein or other biological macromolecule sequences have a common evolutionary origin. Its core lies in judging whether two or more sequences are derived from the same ancestor.

[0030] As used herein, the term "homology" is generally expressed as a percentage and refers to nucleotide sequences that still retain a given degree of identity with each other within the range of at least 40 consecutive nucleotides (such as about 40, 45, 50, 55, 57, 58, 59, 60, 70 or even more consecutive nucleotides) of the 16S rRNA of Chromobacterium Lemna-Chromobacterium-X1. "At least 80%" means about 80% or greater than 80% (such as any value exceeding 80%, advantageously at least 85%, desirably at least 87%, preferably at least 90%, more preferably at least 95%, still more preferably as low as 97% up to 100% sequence homology). The percentage of homology between two nucleotide sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap. In the art, a variety of computer programs and mathematical algorithms can be used to determine the percentage of identity between nucleotide sequences, such as the Basic Local Alignment Search Tool (BLAST) program publicly available at the National Center for Biotechnology Information (NCBI) and described in printed publications.

[0031] The technical advantages of Chromobacterium Lemna-Chromobacterium-X1 for controlling sheath blight of rice include the following:

[0032] Biosafety: The strain is a naturally isolated duckweed endophyte, non-toxic and harmless, and environmentally friendly.

[0033] Highly efficient antibacterial: The strain significantly inhibits pathogenic bacteria through multiple pathways (secretion of antibacterial substances, such as secretion of protease, chitinase, etc.; potential of nitrogen fixation and phosphorus solubilization to promote plant growth to inhibit the invasion of pathogenic bacteria).

[0034] Versatility: The physiological and biochemical parts of the strain were detected by phosphorus solubilization, nitrogen fixation, IAA production, protease production, chitinase production and amylase production, which proved that the strain has the ability of phosphorus solubilization, nitrogen fixation, protease production, chitinase production and amylase production, that is, it has the ability to promote growth. Therefore, the strain can not only directly control diseases, but also promote the growth of rice.

[0035] The following further describes the present invention in detail with specific embodiments.

[0036] Example 1:

[0037] 1. Strain isolation and screening

[0038] (1) Duckweed sample collection: Healthy duckweed plants were collected in a wetland (a wetland area in Guangdong Province), and the surface was rinsed with sterile water to remove attached impurities.

[0039] (2) Isolation of endophytes: Take the collected duckweed, first rinse the surface soil with tap water, and then rinse it 5 - 6 times with sterile water; after rinsing, dry it with sterile filter paper, soak it in 75% alcohol for 2 min and 3% sodium hypochlorite for 5 min for surface disinfection respectively. After discarding the disinfectant solution, rinse it 3 times with sterile water and dry it with sterile filter paper; take 100 μL of the sterile water from the last rinse and spread it on LB medium (tryptone 10 g, yeast extract powder 5 g, sodium chloride 10 g, deionized water 1000 mL, adding 1.8% agar powder makes it a solid medium. Autoclave at 121 °C for 20 min) as a control to verify the disinfection effect and prove that the isolated bacteria are duckweed endophytes rather than other bacteria carried on the surface of duckweed.

[0040] Grind the duckweed sample with a sterile mortar, and gradient dilute the grinding solution with 0.01 mol / L phosphate buffer (PBS) (10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 times). Then take 100 μL from each dilution and spread it on LB medium, culture it at 30 °C for 2 - 3 d, and then further purify it by streaking. Repeat the separation and purification 2 - 3 times to obtain duckweed endophytes.

[0041] Conduct 16S rRNA molecular biological identification on the duckweed endophytes and store them at -80 °C in a refrigerator with a glycerol concentration of 30% for standby.

[0042] (3) Screening of antagonistic strains: Conduct a plate confrontation test between the isolated duckweed endophytes and Rhizoctonia solani, observe the diameter of the inhibition zone, and preliminarily screen out the strains with significant inhibitory effects on the pathogenic bacteria. Then ferment and culture the strains initially screened to have antagonistic effects with LB liquid medium for re-screening. The specific steps are as follows:

[0043] ① Primary screening:

[0044] Use Rhizoctonia solani of rice (purchased from Mingzhou Biological Company) as a biocontrol indicator bacterium, activate it in PDA solid medium. For primary screening, use the spot inoculation method. Inoculate a pathogenic bacterium with a diameter of 1 cm on PDA medium and culture it for 3 - 4 d. Then respectively spot-inoculate the isolated and purified duckweed endophytes at a point 3 cm away from the indicator bacterium, and continue to culture for 2 - 3 d. Repeat each treatment 3 times and observe the antagonistic effect.

[0045] A total of 36 strains with inhibitory ability against Rhizoctonia solani of rice were preliminarily screened out from 218 tested strains, as shown in Table 1.

[0046] Table 1 Determination of the antagonistic ability of the tested strains

[0047]

[0048]

[0049] Note: In the table, the symbol “+” indicates having antagonistic ability, and the symbol “-” indicates having no antagonistic ability

[0050] ② Re-screening: By the plate confrontation method, place a 6-mm diameter rice sheath blight pathogen cake (Rhizoctonia solani) in the center of a PDA plate. Then, use an inoculation loop to dip into the endophyte suspension after primary screening (the concentration of the endophyte suspension is OD600 = 1, that is, the bacterial suspension concentration is about 8×10^8 CFU / ml) and spot-inoculate 2 - 3 kinds of endophytes equidistantly (3 cm from the center of the plate). Incubate in an incubator at 28°C. After 4 days, observe and record the presence and size of the inhibition zone, and repeat 3 times.

[0051] Fourteen strains with the best antagonistic activities were screened out from the 36 strains obtained by primary screening as shown in Table 2, all of which had obvious antagonistic effects. Among them, the effect of strain 102 (X1) was the most obvious and the inhibition rate reached 87.08%. Strain 102 (X1) was re-named Lemna-Chromobacterium-X1.

[0052] Table 2 Statistics of plate antagonistic activities

[0053]

[0054] Note: The data in the table represent the mean ± standard error (n = 3). Different letters after the same-column numbers indicate the significance of differences between treatments (Duncan's method, P = 0.05).

[0055] 2. Identification and characteristic analysis of Lemna-Chromobacterium-X1 strain

[0056] · Morphological observation: Negative rod-shaped, 0.6 - 0.9 μm × 1.5 - 3.5 μm, with blunt ends at both ends, sometimes slightly slender and curved. Single, occasionally in pairs, or extended into short chains. Without a capsule. Without a stationary phase. Gram-negative, often containing striped or lipid inclusions stained at both ends. Can produce pigments. On LB solid medium, produce cheesy pale yellow colonies. The morphology of Lemna-Chromobacterium-X1 strain is shown in Figure 1 .

[0057] · Molecular identification: The genomic DNA of the strain was extracted, the 16S rRNA gene sequence was amplified, and after sequencing, it was identified as Chromobacterium by comparison with the NCBI database and named Lemna-Chromobacterium-X1. See the phylogenetic tree of Lemna-Chromobacterium-X1 in Figure 2 . It is the same strain as the known bacterium GenBank: OP364816.1 in NCBI.

[0058] The 16S rRNA gene sequence of Lemna-Chromobacterium-X1, SEQ ID NO:1, is as follows:

[0059] TGCAGTCGAACGGTAACAGGGTGCTTGCACCGCTGACGAGTGGCGAA

[0060] CGGGTGAGTAATGCATCGGAATGTACCGTGTAATGGGGGATAGCTCGGC

[0061] GAAAGCCGGATTAATACCGCATACGCCCTGAGGGGGAAAGTGGGGGAC

[0062] CGTAAGGCCTCACGTTATACGAGCAGCCGATGTCTGATTAGCTAGTTGG

[0063] TGAGGTAAAGGCTCACCAAGGCGTCGATCAGTAGCGGGTCTGAGAGGA

[0064] TGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG

[0065] GCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCAT

[0066] GCCGCGTGTCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTCCGG

[0067] GAGCAAATCCTAGTGGTTAATAACCGCTGGGTCTGAGAGTACCGGAAG

[0068] AATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGG

[0069] TGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTT

[0070] GTGCAAGTCTGATGTGAAAGCCCCGGGCTTAACCTGGGAACGGCATTG

[0071] GAGACTGCACGACTAGAGTGCGTCAGAGGGGGGTAGAATTCCGCGTGT

[0072] AGCAGTGAAATGCGTAGAGATGCGGAGGAATACCGATGGCGAAGGCA

[0073] GCCCCCTGGGATGACACTGACGCTCATGCACGAAAGCGTGGGGAGCA

[0074] AACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAG

[0075] CTGTTGGGGGTTTGAATCCTTGGTAGCGTAGCTAACGCGAGAAGTTGA

[0076] CCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAAGGAATTGACG

[0077] GGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCG

[0078] AAAAACCTTACCTGGTCTTGACATGTAACGAACGCCGCAGAGATGTGG

[0079] CGGTGCCCGAAAGGGAGCGTTAACACAGGTGCTGCATGGCTGTCGTCA

[0080] GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT

[0081] TGCCATTAGTTGCCATCATTAAGTTGGGCACTCTAATGGGACTGCCGGT

[0082] GACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTA

[0083] TGACCAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTCGCGA

[0084] AGCCGCGAGGTGGAGCCAATCTCATAAAACCGATCGTAGTCCGGATCG

[0085] CACTCTGCAACTCGAGTGCGTGAAGTCGGAATCGCTAGTAATCGCAGA

[0086] TCAGCATGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGT

[0087] CACACCATGGGAGTGAGTTTCACCAGAAGTGGGTAGGCTAACCGTAAGGAGGCC(SEQ ID NO:1)。

[0088] · Antibacterial ability test: The inhibition rate of the supernatant of the liquid fermentation of Lemna - Chromobacterium - X1 against the spores of the pathogen Rhizoctonia solani was as high as 86.52%. See the schematic diagram of the results in Figure 3 。

[0089] Example 2: Analysis of Lemna - Chromobacterium - X1

[0090] Physiological and biochemical analysis of Lemna - Chromobacterium - X1: Through the detection of phosphate solubilization, nitrogen fixation, IAA production, protease production, chitinase production and amylase production, this strain has the ability to solubilize phosphate, fix nitrogen, produce protease, chitinase and amylase.

[0091] 1. Qualitative analysis of IAA production function

[0092] 1 / 2LB solid medium: Weigh 5.0 g of sodium chloride, 25.0 g of yeast extract, and 5.0 g of tryptone, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, add 15 g of agar, sterilize by autoclaving at 121 °C for 30 min, and pour the plate after cooling to 50 °C.

[0093] Salkowski's colorimetric solution for qualitative detection of IAA production by strains: Slowly pour 150 mL of concentrated sulfuric acid along the wall of a beaker into 250 mL of ultrapure water, and continuously stir with a glass rod. Then add 7.5 mL of 0.5 mol / L FeCl3 . 6H2O solution.

[0094] Inoculate Lemna-Chromobacterium-X1 into LB liquid medium containing L-tryptophan (100 mg / L) and place it in a shaker (28 °C, 180 rpm / min) for 36 h. Pipette 50 μL of the cultured bacterial suspension + 50 μL of Salkowski's colorimetric solution into the round holes of a white ceramic plate. The positive control is 50 μL of IAA (50 mg / L) + 50 μL of Salkowski's colorimetric solution. After standing for 30 minutes in a dark and temperature-constant environment, observe that there is no obvious change in its color, indicating that it has no ability to produce IAA.

[0095] 2. Qualitative analysis of phosphorus solubilization function

[0096] Phosphorus-solubilizing bacteria screening medium: Weigh 5 g of tricalcium phosphate, 10 g of glucose, MgSO4·7H2O,

[0097] 0.25 g to MgCl2 . 6H2O 5 g, (NH4)2SO4 0.1 g, KCl 0.2 g, make up to 1000 mL with ultrapure water, adjust the pH value to 7.1 - 7.5, add 15 g of agar, sterilize by autoclaving at 121 °C for 30 minutes, and pour plates after cooling to 50 °C.

[0098] Inoculate Lemna-Chromobacterium-X1 onto the solid medium for detecting the phosphorus solubilization function of the strain. Observe that obvious phosphorus solubilization halos appear on the plate within 2 - 7 days, indicating that it has the ability to solubilize phosphorus, that is, it can dissolve the insoluble phosphorus in the soil and improve the utilization rate of phosphorus in the soil by crops.

[0099] 3. Qualitative analysis of nitrogen fixation function

[0100] 1) Ashby medium: Weigh KH2PO4 0.2 g, mannitol 10 g, NaCl 0.2 mL,

[0101] MgSO4 . 7H2O 0.2 g, CaCO3 5 g, CaSO4 . 2H2O 0.1 g, make up to 1000 mL with ultrapure water, adjust the pH value to 7.0, add 15 g of agar, sterilize by autoclaving at 121 °C for 30 minutes, and pour plates after cooling to 50 °C.

[0102] 2) NFb medium: Weigh 0.5 g of KH2PO4·H2O, 5 g of DL-malic acid, 0.2 g of CaSO4 . ·2H2O, 0.2 g of MgSO4·7H2O, 0.66% EDTA, 0.1 g of NaCl, make up the volume to 1000 mL with ultrapure water, adjust the pH value to 7.0, add 20 g of agar, sterilize by autoclaving at 121 °C for 30 minutes, and pour the plate after cooling to 50 °C.

[0103] Inoculate Lemna-Chromobacterium-X1 onto the solid medium for detecting the nitrogen fixation function of the strain, and observe its growth condition within 2 - 7 days. If it can grow normally, it proves that it has the nitrogen fixation ability, that is, it can promote plant growth.

[0104] 4. Determination of protease production performance: Inoculate Lemna-Chromobacterium-X1 on the protease detection medium, place it in a constant temperature incubator at 30 °C, and culture it in the dark for 48 h. Observe whether there is a clear zone around the colony to judge the protease production ability of the strain. For the example result, see the left figure in Figure 4 . It is observed that there is a clear zone around the colony, which proves that the strain has the ability to produce protease, that is, it has the effect of anti-inflammatory and antibacterial.

[0105] 5. Determination of amylase production performance: Pipette 10 μL of the Lemna-Chromobacterium-X1 bacterial suspension with a concentration of 2×10 7 cfu / mL onto the center of the amylase identification medium, transfer the plate to a constant temperature incubator at 28 °C, and culture it in the dark. After 24 h, add 1 mL of fresh iodine solution to the plate, and let it stand for 1 - 3 min. If a clear zone appears, it proves that the strain has the ability to produce amylase, that is, it can help plants decompose starch organic matter in the soil and promote plant nutrient absorption. For the example result, see the right figure in Figure 4 .

[0106] 6. Determination of chitinase production performance:

[0107] Chitinase medium: 1 g of colloidal chitin, 1 g of peptone, 0.07 g of dipotassium hydrogen phosphate, 0.03 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate heptahydrate, 0.001 g of ferrous sulfate heptahydrate, 0.001 g of zinc sulfate and 1.5 g of agar, make up the volume to 100 mL with deionized water, pH 6.8. Inoculate the Lemna-Chromobacterium-X1 strain on the chitin detection medium, place it in a constant temperature incubator at 30 °C and culture it for 4 - 6 d. Observe whether there is a clear zone around the colony. If so, it proves that the strain has the ability to produce chitinase, that is, it can resist the attack of pathogenic bacteria.

[0108] Example 3: Pot experiment:

[0109] The test soil was the soil of a rice paddy field with severe sheath blight, and the rice variety was Huanghuazhan.

[0110] a. Rice planting

[0111] It was planted in a rice planting basket. There were 2 rows in each basket, 3 plants in each row, the row spacing was 20 cm, and the hole spacing was 20 cm.

[0112] b. Method for culturing the sheath blight pathogen

[0113] The toothpicks were cut into short sticks about 1 cm long and then sterilized at 121 °C under high temperature and high pressure for 20 min. When the PDA medium plate was not solidified, the sterilized short sticks were evenly placed in it and left for use after cooling. A fungal cake was picked and placed in the PDA plate with the short toothpicks placed, and it was cultured in an incubator at 28 °C. After the sheath blight pathogen filled the culture dish (about 4 - 7 d), inoculation was carried out.

[0114] c. Toothpick inoculation method, also called short stick inoculation method. The cultured short toothpicks with hyphae were inoculated into the leaf sheaths of the rice. Note to gently pry open the leaf sheath, put the toothpick in, and then return the leaf sheath to its original position, trying not to damage the original state of the leaf sheath.

[0115] d. The self-bred rice seedlings grew to the 4-leaf stage, and the rice leaves were inoculated in vivo. One fungal cake was inoculated at the leaf sheath of each plant. The test design is as follows:

[0116] Table 3 - 2 Grouping of pot experiment treatments

[0117]

[0118] The pot experiment adopted the root irrigation method and was divided into 2 treatments: inoculating with the sheath blight pathogen of rice; inoculating with the sheath blight pathogen of rice + applying the bacterial liquid of Lemna-Chromobacterium-X1 (the bacterial content was 8×10 8 CFU / mL), and the application amount was 50 ml each time. Each root irrigation treatment was repeated 3 times.

[0119] The results were as Figure 6 shown. The rice in the T2 treatment (right figure) was significantly infected with the sheath blight pathogen, while the rice in the T1 treatment (left figure) showed that Lemna-Chromobacterium-X1 had a good control effect on the sheath blight of rice.

[0120] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Application of Lemna-Chromobacterium-X1 in the fight against Rhizoctonia solani.

2. Application of Lemna-Chromobacterium-X1 in preventing and / or treating rice sheath blight caused by Rhizoctonia solani.

3. The use according to claim 1 or 2, characterized in that: The 16S rRNA of the Lemna-Chromobacterium-X1 is as shown in SEQ ID NO: 1, or has more than 95% homology with SEQ ID NO: 1, or belongs to the same strain as GenBank: OP364816.

1.

4. The use according to claim 1 or 2, characterized in that: The Lemna-Chromobacterium-X1 was screened from duckweed.

5. A method for preventing and / or treating rice sheath blight caused by Rhizoctonia solani, characterized in that: The solution containing the Lemna-Chromobacterium-X1 is used to irrigate rice roots or spray rice at various growth stages. Preferably, the 16S rRNA of the Lemna-Chromobacterium-X1 is as shown in SEQ ID NO: 1, or has more than 95% homology with SEQ ID NO: 1, or belongs to the same strain as GenBank: OP364816.

1.

6. The method for preventing and / or treating rice sheath blight caused by Rhizoctonia solani according to claim 5, characterized in that: The solution containing the Lemna-Chromobacterium-X1 is sprayed on the tillering stage, heading stage, and heading stage of the rice growth period.

7. The method according to claim 5, characterized in that: In the solution, the concentration of Lemna-Chromobacterium-X1 is 7×10^8 CFU / ml to 9×10^8 CFU / ml.

8. The method according to claim 5, characterized in that: In the solution, the concentration of Lemna-Chromobacterium-X1 is 7.5×10^8 CFU / ml to 8.5×10^8 CFU / ml.

9. A method for screening Lemna-Chromobacterium-X1, characterized in that: The following steps are involved: (1) Collect duckweed, rinse, and then rinse with sterile water; (2) using sterile filter paper to absorb water, soaking with alcohol and solution respectively, and then rinsing with sterile water, and absorbing duckweed with sterile filter paper to obtain duckweed samples; (3) Grinding duckweed samples with a sterilized mortar, diluting the grinding solution 10x in a gradient manner with PBS buffer, applying each concentration of the dilution solution to LB medium, culturing at 25-35°C for 2-3 days, and then streaking and separating to obtain duckweed endophytes; (4) The duckweed endophyte was identified by 16S rRNA molecular biology to obtain Lemna-Chromobacterium-X1, wherein the 16S rRNA of the Lemna-Chromobacterium-X1 is as shown in SEQ ID NO: 1, or has more than 95% homology with SEQ ID NO: 1, or belongs to the same strain as GenBank: OP364816.

1.

10. The screening method according to claim 9, characterized in that: Between step (3) and step (4), the method further includes: applying the sterile water obtained after the last flushing on LB culture medium as a control to prove that the isolated bacteria are endophytes of duckweed.

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