Indole-3-acetic acid endophytic promiscuous microbacterium and application thereof

Abstract

The application discloses an indole acetic acid producing endophytic microbacterium and application thereof. Microbcaterium The microbacterium (KlspL18) is separated from leaves of Oryza granulata Ohwi, and is preserved in the China Center for Type Culture Collection, and the number is CCTCC M 2022446. The strain of the application can produce IAA in large quantity, has a strong inhibiting effect on rhizoctonia solani, and can obviously promote seed germination and plant growth of rice after being colonized into the rice. The strain provided by the application has advantages of promoting plant growth, preventing and treating rhizoctonia solani and improving soil fertility.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of indole acetic acid endophytic growth promoting microbacterium and its application, belonging to the field of agricultural biotechnology. BACKGROUND

[0002] Plant growth promoting bacteria play an important role in sustainable agricultural production, which can promote plant growth through direct ways such as nitrogen fixation, phosphorus dissolution, and IAA production, and can also reduce the harm of pests and diseases to host plants through indirect ways such as active metabolite production and iron carrier production. It not only has the activities of promoting plant growth and antagonizing plant diseases, but also has the characteristics of improving soil fertility and repairing soil ecological function. In view of the current situation of soil infertility and excessive use of chemical fertilizers and pesticides in agricultural production, it is of great significance to breed plant growth promoting bacteria which can colonize in the body of host plants and promote plant growth and resist plant diseases, and to develop special biological inoculants.

[0003] Rice is a major food crop, and rice diseases and soil infertility seriously affect rice yield. Although the use of chemical pesticides and fertilizers improves food yield, it brings ecological problems such as soil compaction and destruction of soil microbial structure, which seriously restricts the sustainable development of current agriculture. Safe and environmentally friendly biological inoculants have great development potential to solve current problems. Microbacterium is a kind of microorganism widely distributed in nature, which has good application potential in plant pest control, plant growth promotion, and pollutant degradation. Patent 201810534442 reports that the fermentation culture solution of Microbacterium maritypicum can induce soybean seedlings to produce resistance to cyst nematode. The application file with publication number CN 113637616 A introduces that Microbacterium saperdae YJJK-2 has a high colonization rate in saline-alkali soil, has the ability to produce IAA and dissolve phosphorus, and has strong antagonistic effect on the pathogen Fusarium oxysporum of cucumber fusarium wilt. Patent 201911147762.4 reports that Microbacterium oleivorans JWG-G2 can make part of polyethylene terephthalate plastic particles be degraded into hydroxyethyl terephthalate and terephthalic acid which can be directly recycled, and has high application prospect in degrading polyethylene terephthalate. Therefore, the isolation of growth promoting microbacterium strains with disease resistance and high growth hormone production from rice tissues can not only improve the colonization rate of functional bacteria in soil, improve soil ecology, reduce the use of chemical fertilizers, but also enhance the control effect on traditional diseases of rice. SUMMARY

[0004] The isolation of growth promoting microbacterium strains with disease resistance and high growth hormone production from rice tissues can not only improve the colonization rate of functional bacteria in soil, improve soil ecology, reduce the use of chemical fertilizers, but also enhance the control effect on traditional diseases of rice.

[0005] The purpose of the present application is to provide a strain of indole acetic acid endophytic growth-promoting microbacterium and its application method. The endophytic growth-promoting microbacterium (Microbacterium sp. KlspL18) is isolated from the leaf part of Dongxiang wild rice, has the characteristics of high yield of indole acetic acid, promotion of plant growth, antagonism to plant pathogenic fungi (Rhizoctonia solani) and the like, and can be used for producing microbial fertilizer and biological control agent.

[0006] In order to achieve the above purpose, the technical scheme is adopted in the present application:

[0007] An endophytic growth-promoting microbacterium (Microbacterium sp. KlspL18) isolated from the leaf part of Dongxiang wild rice, the strain has been preserved in the China Center for Type Culture Collection, the preservation number is CCTCC M 2022446, and the preservation date is April 21, 2022. The preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The colony and morphological characteristics of the microbacterium KlspL18 strain are as follows: the colony is round, yellow and translucent, the edge is neat, the surface is wet and smooth, and no soluble pigment is present (ISP2 medium plate) Figure 1

[0009] The present application also discloses that the KlspL18 strain of microbacterium can obtain a fermentation broth with high content of indole acetic acid through liquid fermentation culture, and the ethyl acetate extraction phase of the fermentation broth has good inhibitory activity on rice sheath blight.

[0010] The strain of indole acetic acid endophytic growth-promoting microbacterium and its application can include the following steps:

[0011] (1) inoculate the endophytic growth-promoting microbacterium (Microbacterium sp.) KlspL18 in a liquid culture medium and culture for 16h;

[0012] (2) inoculate the above cultured strain in a fermentation medium, the shaking bed rotation speed is 200 rpm, and the culture is carried out for 72-96h, and the fermentation broth and active bacteria are obtained through centrifugation.

[0013] The liquid culture medium is yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, water 1000 mL, and initial pH 7.2.

[0014] ​The liquid fermentation culture is tryptophan 0-3.0 g, yeast extract 4.0 g, peptone 3.0 g, corn starch 10.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, water 1000 mL, initial pH 7.2.

[0015] The content of indole acetic acid in the fermentation liquor is determined by Salkowski colorimetry.

[0016] Further, the fermentation liquor is directly or extracted with ethyl acetate to perform the inhibitory activity experiment on rice sheath blight.

[0017] The application also discloses the application of the Microbacterium sp. KlspL18 strain in promoting the growth of rice seedlings.

[0018] Preferably, the growth-promoting microbacterium is colonized into rice seeds by co-cultivation to promote the growth of rice seedlings.

[0019] Further preferably, the co-cultivation colonization comprises the following steps:

[0020] (1) soaking the rice seeds in 5% NaClO for 10 min for sterilization;

[0021] (2) placing the sterilized rice seeds into a sterile water culture dish with gauze, and culturing at 25 DEG C in the dark until the rice seeds germinate and turn white;

[0022] (3) co-culturing the germinated and white rice seeds obtained in step (2) with the KlspL18 bacterial suspension at 25 DEG C for 24 h as an experimental group, co-culturing the germinated and white rice seeds with sterile water at 25 DEG C for 24 h as a control group, and then transferring to a culture dish for cultivation and observation.

[0023] Further preferably, the temperature for the cultivation in the culture dish in step (3) is 25 DEG C, the relative humidity for the cultivation is 70%, and the light illumination condition for the cultivation is 1600 Lx intensity for 16 h of light illumination and 8 h of dark cultivation.

[0024] The application has the following beneficial effects: the Microbacterium sp. KlspL18 provided in the application has high indole acetic acid yield, can promote the growth of rice seedlings, has strong antagonistic effect on rice sheath blight bacteria, can be made into a biological control agent and a microbial fertilizer, has simple culture conditions, is easy to preserve and produce, has strong colonization ability in rice seedlings, and has wide application prospect in agricultural production. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1: Colony plate and electron microscopic morphological figure of Microbacterium sp. KlspL18;

[0026] Figure 2 : Phylogenetic tree analysis of Microbacterium sp. KlspL18;

[0027] Figure 3 : Colorimetric reaction of Microbacterium sp. KlspL18 for IAA secretion characteristics;

[0028] Figure 4 : Antagonistic effect of Microbacterium sp. KlspL18 fermentation extract on Rhizoctonia solani.

[0029] Figure 5 : Promoting growth test of rice seedlings inoculated with Microbacterium sp. KlspL18. DETAILED DESCRIPTION

[0030] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0031] The present application provides the application of a strain of indole acetic acid endophytic growth promoting microbacterium, which can include the following steps:

[0032] (1) Inoculate the endophytic growth promoting microbacterium (Microbacterium sp.) KlspL18 in the liquid medium and culture for 16h;

[0033] (2) Inoculate the above cultured strain in the fermentation medium and culture at 28-32°C for 72-96h, centrifuge to obtain the fermentation broth and active bacteria.

[0034] The liquid medium is yeast extract 4.0g, malt extract 10.0g, glucose 4.0g, water 1000mL, initial p H 7.2.

[0035] The fermentation medium is tryptophan 0-3.0g, yeast extract 4.0g, peptone 3.0g, corn starch 10.0g, NaCl 0.5g, KNO3 1.0g, K2HPO4·3H2O 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, water 1000mL, initial p H 7.2.

[0036] The fermentation medium can also be tryptophan 0-3.0 g, yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, water 1000 mL, initial pH 7.2.

[0037] The fermentation medium can also be tryptophan 0-3.0 g, glucose 2.5 g, pancreatic casein hydrolysate 15.0 g, soy papain hydrolysate 5.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, water 1000 mL, initial pH 7.2.

[0038] Example 1: Isolation and identification of strain KlspL18

[0039] (1) Strain KlspL18 was isolated from the leaf of Dongxiang wild rice healthy plants. The specific isolation method is as follows: 2.0 g of leaves is first immersed in 3% sodium hypochlorite for 2 min on a clean bench, washed with sterile water for 3 times, then immersed in 75% ethanol for 3 min, and then washed with sterile water for 3-5 times, and dried to obtain the surface sterilized leaf tissue. Then the sterilized leaf tissue is placed in a sterile mortar, 10 mL of phosphate buffer is added, and then it is ground, the supernatant is diluted with sterile water to 10 times, and 100 μL of the original solution and the diluted solution is respectively spread on YIM medium (yeast extract 4.0 g, glucose 4.0 g, malt extract 5.0 g, a little bit of compound vitamin, trace salt 1 mL, agar powder 18.0 g, water 1000 mL, pH 7.2) plate. After incubation at 28 °C for 2-3 days, different size, color and morphology of colonies are picked to the purification medium for streaking purification, and the purified strain is preserved by glycerol preservation method, and the purified colonies are placed in an ultra-low temperature refrigerator for standby.

[0040] (2) Morphological identification of strain KlspL18. The colonies of the strain on ISP2 medium plate are round, yellow and translucent, with neat edges, wet and smooth surface, and no soluble pigment; the strain is cultured in liquid medium to OD 600 0.8, centrifuged to collect the bacterial cells, fixed with glutaraldehyde, dehydrated with gradient ethanol (20%, 40%, 60%, 80%, 90%, 100%) solution, centrifuged to collect the bacterial cells and placed in a freeze dryer for freeze drying; a small amount of dried cells is placed on conductive glue, sprayed with ion sputtering gold after ion sputtering, and the size and shape characteristics of the strain cells are observed by scanning electron microscope. The cells are irregular rods, and the bacterial cells are connected by filaments. The colony plate and electron microscopic morphological diagram are shown in Figure 1

[0041] ​(3) Molecular biological identification of the strain

[0042] The purified strain KlspL18 was inoculated into ISP2 liquid medium and incubated at 30 °C for 2-3 days. The cell culture was collected in a sterile environment. The genomic DNA of the strain was extracted using a column bacterial genomic DNA extraction kit. The PCR amplification system of the obtained 16S rDNA gene sequence of the strain: Taq Mix enzyme 25 μL, genomic DNA 1 μL, forward and reverse primers 27F, 1492R (27F 5-AGAGTTTGATCCTGGCTCAG-3 1492R 5-TACGGCTACCTTGTTACGACTT-3) each 1 μL, ddH2O supplemented to 50 μL. The purified product of PCR amplification was sent to Shanghai Sangon Biological Engineering Co., Ltd. for bidirectional sequencing, and Mega X was used to construct the NJ phylogenetic tree of 16S rRNA gene, as shown in Figure 2 According to the phylogenetic relationship analysis of the 16S rRNA gene sequence, it was determined that the strain KlspL18 was a Microbacterium bacterium.

[0043] Example 2: Fermentation culture and detection of IAA produced by strain KlspL18

[0044] (1) Salkowski colorimetric method for qualitative determination of the ability of the strain to secrete IAA

[0045] Fermentation culture of strain KlspL18: After the strain KlspL18 was inoculated in ISP2 liquid medium and cultured for 16 h (28 °C, 180 r / min), the OD 600 value was adjusted to 0.6 with sterile water, and 100 μL of KlspL18 bacterial suspension was taken into the fermentation medium containing tryptophan (composition: tryptophan 1.0 g, yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, water 1000 mL, initial pH 7.2), and incubated in a temperature-controlled shaker at 30 °C and 180 r / min for 96 h.

[0046] Preparation of Salkowski color developing solution: 4.5 g of FeCl3 was dissolved in 300 mL of distilled water, then 587.4 mL of 98% H2SO4 was slowly added, and after cooling, the volume was adjusted to 1 L. The range of IAA was 5-200 mg / L.

[0047] Salkowski colorimetric qualitative detection of indole acetic acid in fermentation broth: centrifugal collection of fermentation broth as experimental group, positive control is 100 μg / mL tryptophan, blank control is uninfected fermentation broth; according to 1:2 ratio, the above samples and Salkowski colorimetric solution are added to the reaction container, mixed, placed at 40 °C for 30 min, the color change of the reaction solution is observed, if it can change to pink to brown red, it indicates that the strain can secrete IAA, the deeper the color, the more the secretion; no color change indicates no secretion of IAA. The determination results are shown in Table 1. Figure 3 , which shows that the strain KlspL18 can secrete IAA.

[0048] (2) Colorimetric determination of the ability of the strain to secrete IAA

[0049] IAA determination standard curve: prepare IAA stock solution with a concentration of 1.0 mg / mL, and dilute it into concentrations of 0, 25, 50, 75, 100, 125, and 150 μg / mL, respectively, with each concentration repeated three times, and add two volumes of color reagent according to the above method. After the reaction, the absorbance at 530 nm is measured. The standard curve of IAA is drawn with different concentrations of standard IAA solution as the abscissa and the ordinate as the absorbance at 530 nm. The standard curve is Y=0.0056X+0.0435 (R 2 =0.99), and the IAA production ability of the strain is calculated according to the obtained standard curve.

[0050] The strain fermentation broth and Salkowski colorimetric solution are mixed in a test tube at a volume ratio of 1:2, and placed at 40 °C for 30 min. After the reaction, the reaction solution is taken out and placed in a 96-well enzyme-labeled plate. The absorbance of the reaction solution at 530 nm is measured, and the IAA yield of the strain in ISP2 medium is calculated to be 247.8 mg / L.

[0051] Example 3: Effect of medium composition on IAA production of strain KlspL18

[0052] Microbial synthesis metabolites are affected by nutritional factors, and the yield of target products will change under different nutritional components of the medium. In this embodiment, the change of IAA production of strain KlspL18 in different fermentation media is studied under the conditions of culture temperature of 30 °C and rotation speed of 180 r / min. The culture media include:

[0053] Gao No. 1: soluble starch 20.0 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4.7H2O 0.5 g, NaCl 0.5 g, FeSO4 0.01 g, water 1000 mL, initial pH 7.2;

[0054] ISP2: yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, water 1000 mL, initial pH 7.2;

[0055] ISP3: oatmeal 20 g, trace salt solution 1 mL (FeSO4.7H2O 0.1 g, ZnSO4.7H2O 0.1 g, MnCl2.4H2O 0.1 g), water 1000 mL, initial pH 7.2;

[0056] ISP4: soluble starch 10.0 g, K2HPO4 1.0 g, CaCO3 2.0 g, MgSO4.7H2O 1.0 g, (NH4)2SO4 2.0 g, trace salt solution 1 mL (FeSO4.7H2O 0.1 g, ZnSO4.7H2O 0.1 g, MnCl2.4H2O 0.1 g), water 1000 mL, initial pH 7.2;

[0057] MS: soybean powder 20 g, mannitol 20 g, water 1000 mL, initial pH 7.2;

[0058] TSB: glucose 2.5 g, tryptone 15.0 g, soybean papain hydrolysate 5.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4.3H2O 0.5 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.01 g, water 1000 mL, initial pH 7.2;

[0059] Z2 medium: yeast extract 4 g, peptone 3 g, corn starch 10 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4.3H2O 0.5 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.01 g, water 1000 mL, initial pH 7.2;

[0060] All the above-mentioned culture media are added with 1.0 g / L tryptophan as the precursor of IAA synthesis, and the IAA determination is carried out by the method in Example 2. The experimental results are shown in Table 1. The culture media can all produce IAA, but the IAA production of different culture media is obviously different. The yield of Z2 culture medium is the highest, which is 301.1 ± 19.59 mg / L, and the Z2 culture medium is preferably used as the IAA production fermentation medium.

[0061] Table 1: Effect of culture medium on IAA production of strain KlspL18

[0062]

[0063] Example 4: Effect of culture time on IAA production of strain KlspL18

[0064] The synthesis of metabolites by microorganisms is affected by the culture time, and the yield of target product will change at different culture times. In this embodiment, the change of IAA yield in the fermentation broth of strain KlspL18 cultured for 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h in a temperature-controlled shaker at a temperature of 30 °C and a rotation speed of 180 r / min, and the fermentation medium (components: tryptophan 1.0 g, yeast extract 4 g, peptone 3 g, corn starch 10 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, water 1000 mL, initial pH 7.2) is determined by the method in Example 2. The experimental results are shown in Table 2. With the extension of culture time, the yield of IAA shows a trend of first increasing and then decreasing, and the yield reaches the highest of 305.66 ± 16.64 mg / L at 96 h of culture, and 96 h is preferably the optimal fermentation time.

[0065] Table 2: Effect of culture time on IAA production of strain KlspL18

[0066]

[0067] Example 5: Effect of culture temperature on IAA production of strain KlspL18

[0068] The synthesis of microbial metabolites is affected by temperature. To investigate the changes in IAA production by strain Klsp L18 at different culture temperatures, the ability of strain Klsp L18 to produce IAA was detected at a culture temperature of 25°C, 28°C, 30°C, 32°C and 35°C in a fermentation medium (composition: tryptophan 1.0 g, yeast extract 4 g, peptone 3 g, corn starch 10 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, water 1000 mL, initial pH 7.2) at a shaking speed of 180 r / min for 96 h. The experimental results are shown in Table 3. The IAA yield changed little when the culture temperature was in the range of 28-32°C, and the IAA yield showed a large downward trend when the temperature exceeded 35°C. The IAA yield was highest at 30°C, reaching 318.24±11.27 mg / L, and 30°C was the optimal culture temperature.

[0069] Table 3 Effect of culture temperature on IAA production by strain Klsp L18

[0070]

[0071] Example 6: Effect of different tryptophan additions on IAA production by strain Klsp L18

[0072] The microorganism mainly synthesizes IAA by relying on tryptophan as a precursor. The concentration of different tryptophan affects the yield of IAA. In this embodiment, the effect of adding different concentrations of tryptophan (0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL) to the fermentation medium (composition: yeast extract 4 g, peptone 3 g, corn starch 10 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4.3H2O 0.5 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.01 g, water 1000 mL, initial pH 7.2) on the production of IAA by strain KlspL18 was studied to determine the potential of the strain to produce IAA. The fermentation medium with different added tryptophan was placed in a constant temperature shaker at 30 °C and 180 r / min for 96 h. The fermentation broth of different treatments was collected by centrifugation, and the IAA content in the fermentation broth was determined according to the method of quantitative determination of IAA content in fermentation broth in Example 2. The results are shown in Table 4. With the increase of tryptophan, the synthesis of IAA showed a gradual upward trend. When the concentration of tryptophan exceeded 2.0 mg / mL, the production of IAA decreased. The optimal amount of tryptophan added in Z2 medium was 2.0 mg / mL, and the yield was as high as 412.23±15.18 mg / L.

[0073] Table 4 Effect of tryptophan concentration on IAA production by strain KlspL18

[0074]

[0075] Example 7: Determination and verification of the optimal fermentation conditions for IAA production by strain KlspL18

[0076] Based on the fermentation medium, culture time, fermentation temperature and tryptophan addition amount of Examples 3, 4, 5 and 6, the strain KlspL18 was fermented under the following conditions: Z2 medium (yeast extract 4 g, peptone 3 g, corn starch 10 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4.3H2O 0.5 g, MgSO4.7H2O 0.5 g, FeSO4.7H2O 0.01 g, water 1000 mL, initial pH 7.2), tryptophan addition amount 2.0 mg / mL, fermentation time 96 h, fermentation temperature 30 °C, rotation speed 180 r / min. The final yield of IAA under these conditions was 423.78 mg / mL.

[0077] Example 8 Inhibition of rice sheath blight by fermentation broth of strain KlspL18

[0078] (1) Fermentation culture of strain KlspL18: After strain KlspL18 was inoculated in ISP2 liquid medium and cultured for 16 h (30 °C, 180 r / min), the OD value was adjusted to 0.6 with sterile water, and the culture was incubated in a 30 °C, 180 r / min temperature-controlled shaker for 96 h. 600 The OD value was adjusted to 0.6 with sterile water, and the culture was incubated in a 30 °C, 180 r / min temperature-controlled shaker for 96 h.

[0079] (2) Collection and extraction of fermentation broth

[0080] The supernatant was collected by centrifugation and extracted with ethyl acetate, and the extract was concentrated under vacuum to obtain a crude extract for use. The antibacterial activity of the fermentation extract of strain KlspL18 against Rhizoctonia solani was detected by adding the filtered and sterilized fermentation extract to sterile PDA medium to a final concentration of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, and 1 mg / mL, and adding methanol as a blank control. The inhibition rate was shown in Table 5. The inhibition rate against Rhizoctonia solani increased with the increase of the concentration of the extract. When the concentration reached 0.75 mg / mL, the inhibition rate was over 90%, which indicated that the extract contained active substances that inhibited Rhizoctonia solani.

[0081] Table 5 Antagonistic effect of ethyl acetate extract of fermentation broth of strain KlspL18 on Rhizoctonia solani

[0082]

[0083] Example 9 Growth-promoting activity of strain KlspL18 on rice seedlings

[0084] To explore the growth-promoting effect of strain KlspL18 on rice seeds, the strain was colonized on rice seeds, and then the germination of the strain was observed to determine whether it could promote the growth of rice. The specific process is as follows: first, sterile rice seeds were germinated to white, then the cultured strain KlspL18 was centrifuged, and resuspended with sterile water to obtain KlspL18 bacterial suspension, then the germinated seeds were mixed with the bacterial suspension for 24 h of seed soaking and co-culture, and the germinated seeds were co-cultured with sterile water for 24 h as a control group. Finally, the treated and control seed materials were placed on moist filter paper plates in a light incubator for 7 days, and the growth changes of rice seedlings were observed, and the length of shoots, roots and root number of the control and treatment groups were compared, and the experimental results are shown in Figure 5

[0085] Strain KlspL18 has a significant promoting effect on the growth of rice seeds. The length of shoots and roots of the experimental group treated by seed soaking was significantly longer than that of the sterile water treatment group, and the number of roots was also significantly higher than that of the blank control group. The average length of rice seedlings in the experimental group inoculated with KlspL18 and the control group was 8.5±0.92 cm and 6.36±0.99 cm, respectively, with a growth rate of 33.65%. The growth-promoting effect of strain KlspL18 on rice seeds was more significant, and the average length of the root of the rice seedlings treated by inoculation was 5.77±1.10 cm, which was 2.77 times of the root length of the control group (2.08±0.81 cm). The number of root of the experimental group was 2.00 times of that of the control group (Table 6).

[0086] Table 6 Effect of strain KlspL18 on the growth of rice seedlings

[0087] Note: n=10, significant difference between groups: *, p<0.05​

Claims

1. An endophytic, growth-promoting Microbacterium indolicum strain, characterized in that: The endophytic growth-promoting microbes are endophytic growth-promoting microbes ( Microbacterium sp.) KlspL18 has been deposited at the China Center for Type Culture Collection on April 21, 2022, with accession number CCTCC M 2022446.

2. A fermentation broth prepared by using the indole acetic acid endophytic growth promoting microbacterium according to claim 1.

3. Active bacteria prepared by using the indole acetic acid endophytic growth promoting microbacterium according to claim 1.

4. The method for preparing fermentation broth as described in claim 2, characterized in that, comprising the following steps: Endogenous growth-promoting microbes ( Microbacterium sp.) KlspL18 was inoculated in liquid culture medium and cultured for 16 h; the above cultured strain was inoculated in fermentation medium and cultured at 28-32°C for 72-96 h, and the fermentation broth was obtained by centrifugation.

5. The production method according to claim 4, wherein The liquid culture medium is yeast extract 4.0 g, malt extract 10.0 g, glucose 4.0 g, water 1000 mL, initial pH 7.

2.

6. The production method according to claim 4, wherein The fermentation medium is tryptophan 0-3.0 g, yeast extract 4.0 g, peptone 3.0 g, corn starch 10.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, water 1000 mL, initial pH 7.

2.

7. The use of the ethyl acetate extract of the fermentation broth according to claim 2 in the prevention and treatment of rice sheath blight.

8. The use of the active bacteria according to claim 3 in promoting the growth of rice.

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