New species F1-18 of endophytic growth-promoting microbacterium of lavender and application of new species F1-18

Abstract

The invention discloses a new endophytic growth-promoting microbacterium species F1-18 of lavender and application of the new endophytic growth-promoting microbacterium species The invention provides an endophyte F1-18 separated from a Xinjiang Yili lavender seed. The endophyte F1-18 is a novel microbacterium sp. Strain with a plant growth promoting function, and the endophyte F1-18 is an endophyte F1-18 separated from the Xinjiang Yili lavender seed. The strain F1-18 has no hemolytic activity, has the capabilities of dissolving inorganic phosphorus, producing protease and secreting indoleacetic acid, and has strong tolerance to acid-base and high-salt environments. After the strain F1-18 is treated, the germination rate of lavender and corn seeds can be remarkably increased, the growth and development of lavender, corn and lettuce plants are promoted, the diversity of soil microorganisms is improved, and the microbial community structure is improved. The strain is expected to be developed into a novel agricultural microbial agent, and has important significance in reducing the use of chemical fertilizers, activating soil nutrients, improving the soil quality and improving the plant yield and quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology and biotechnology, in particular to a new species of lavender endophytic Promicromonospora F1-18 and its application. BACKGROUND

[0002] Compared with many soil microorganisms, endophytic bacteria in seeds have a colonization advantage of highly adapting to plant tissue life (Cope-Selby et al, 2017). In addition, seed endophytes have multiple plant growth-promoting functions, including nitrogen fixation, phosphorus solubilization, micronutrient supply, photosynthetic efficiency enhancement, plant defense response induction, heavy metal form transformation, and organic pollutant degradation, thereby significantly improving the environmental adaptability of the host (Compant S et al, 2010). The absence of endophytes can significantly inhibit seed germination and seedling development, while their presence can significantly increase seed germination rate and promote seedling growth (Verma S et al, 2017). As an important part of the plant microbiome, seed endophytes can be transmitted between generations through vertical transmission and play a key role in seed germination and seedling establishment. Therefore, in-depth study of the diversity, function and interaction mechanism of seed endophytes with plants is of great significance for the development of new microbial inoculants and optimization of crop production (Li P et al, 2024). At present, researchers have described and summarized many aspects of seed endophyte research and development, systematically described the growth-promoting mechanisms and disease resistance mechanisms of seed endophytes, and studied the functional mechanisms of seed endophytes in enhancing the resistance of host plants under heavy metal stress (Chen KX et al, 2021). Based on the development of new microbial fertilizers from seed endophytes has become a current hot field and has significant development potential and broad application prospects (Titir et al, 2024). These studies show that endophytes have great application potential in agricultural production.

[0003] The unique soil and climate conditions in the Yili River Valley region of Xinjiang provide rich resource advantages for the development of excellent indigenous microorganisms. Lavender (Lavandula angustifolia Mill.) is a perennial herb of the Lamiaceae family, which is widely cultivated in Xinjiang and has a high economic value. The roots and rhizosphere of lavender are rich in endophytes, which play an important role in the growth and development of lavender. However, the diversity and function of lavender endophytes have not been systematically studied. Lavandula angustifoliaLamiaceae Lavandula, known for its unique ornamental value and extensive medicinal value, is praised as the "aromatic medicinal herb after". Lavandula was introduced and cultivated in Ili region in 1964 from France. After more than 50 years of introduction, domestication, breeding and application promotion, Ili region has become one of the three major lavender producing areas in the world, and lavender has become an economic characteristic pillar industry in Ili region. Due to the continuous expansion of lavender planting area, it is inevitable to appear the phenomena of mixed varieties, degeneration of species, serious diseases and so on. The perennial continuous planting of lavender affects the rhizosphere microenvironment and causes the yield and quality of lavender to decrease; at present, there is a great deficiency in the exploration of lavender endophytic bacterial resources, and the separation of strains with growth promoting function from the endophytic environment of lavender is beneficial to the development of microbial fertilizer and other products, the improvement of soil microbial community structure, and the promotion of the growth of crops such as lavender and the reduction of the use amount of chemical fertilizers and pesticides in the process of lavender planting. SUMMARY

[0004] The purpose of the present application is to provide a new species of lavender endophytic growth promoting microbacterium F1-18 with plant growth promoting function and its application.

[0005] In order to achieve the purpose of the present application, in the first aspect, the present application provides a strain isolated from the endophytic environment of lavender seeds in Ili, Xinjiang, i.e. a new species of lavender endophytic growth promoting microbacterium F1-18, which is a new species of microbacterium genus and is classified and named as Microbacterium sp. by polyphase classification and identification, and the strain F1-18 has been preserved in Guangdong Microbial Culture Collection Center (GDMCC), located at No. 59 Building, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Guangzhou, Guangdong, China, with a postal code of 510070, with a preservation number of GDMCC No: 65647 and a preservation date of December 16, 2024.

[0006] The present application screens the endophytic bacteria F1-18 with good growth promoting function, and the strain F1-18 has the abilities of degrading inorganic phosphorus, producing protease and secreting IAA. It is of great significance to improve the yield and quality of plants and develop new agricultural microbial agents.

[0007] In the second aspect, the present application provides an agent or biological fertilizer prepared from the microbacterium, fermentation broth or crude extract of fermentation broth.

[0008] In the third aspect, the present application provides a plant growth promoting agent, agricultural fertilizer, phosphorus activator or soil activator prepared from the microbacterium or its agent.

[0009] In the fourth aspect, the present application provides any one of the following applications of the microbacterium or its agent: 1) for dissolving phosphorus; 2) for producing IAA (auxin); 3) for producing protease; 4) for promoting plant growth or preparing plant growth promoter; 5) for reducing the use of chemical fertilizer, increasing soil nutrient content, increasing soil microbial diversity, or preparing soil activator.

[0010] Further, the phosphorus in 1) is inorganic phosphorus (such as calcium phosphate).

[0011] Further, the method for promoting plant growth in 4) comprises seed soaking treatment of plants with a bacterial liquid of the microbacterium, or applying the bacterial liquid to the rhizosphere soil of plants.

[0012] Further, the plants include lavender, corn and lettuce.

[0013] Further, the increase of soil nutrient content in 5) includes the increase of soil available nitrogen, available phosphorus and the like, and the increase of soil microbial diversity includes the increase of the community diversity of soil bacteria and fungi.

[0014] The present application provides a strain of endophyte F1-18 isolated from lavender seeds, which is a new species of microbacterium with plant growth promoting function. The strain F1-18 has no hemolytic activity, has the ability to dissolve inorganic phosphorus, produce protease and secrete indole acetic acid, and has strong tolerance to acid-base and high salt environment. The seed soaking treatment of the strain F1-18 can significantly improve the germination rate of lavender and corn seeds, promote the growth and development of lavender and corn plants, and increase the soil available nitrogen, available phosphorus and the like. The strain is expected to be developed as a new type of agricultural microbial agent, which has important significance for activating soil nutrients and improving the yield and quality of plants. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is the colony morphology of the strain F1-18 of the present application.

[0016] Figure 2 The figure is the 16S rRNA gene phylogenetic tree of the strain F1-18 of the present application.

[0017] Figure 3 The figure is the whole genome-based phylogenetic tree of the strain F1-18 of the present application.

[0018] Figure 4 The figure is the Wayne diagram of the orthologous genes of the strain F1-18 of the present application and its close relatives.

[0019] Figure 5 The figure is the phosphorus-dissolving (a) and protease-producing (b) ability of the strain F1-18 in the preferred embodiment of the present application.

[0020] Figure 6Promoting effect of strain F1-18 on lavender seeds in the preferred embodiments of the present application.

[0021] Figure 7 Promoting effect of strain F1-18 on corn seeds in the preferred embodiments of the present application.

[0022] Figure 8 Promoting effect of strain F1-18 on potted corn in the preferred embodiments of the present application.

[0023] Figure 9 Promoting effect of strain F1-18 on field-grown lettuce in the preferred embodiments of the present application.

[0024] Figure 10 Promoting effect of strain F1-18 on growth traits of field-grown lettuce in the preferred embodiments of the present application.

[0025] Figure 11 Effect of strain F1-18 on rhizosphere soil microbial diversity index of field-grown lettuce in the preferred embodiments of the present application. A and B are chao index and ace index diversity of fungi in the rhizosphere soil of lettuce, respectively, and C and D are chao index and ace index diversity of bacteria in the rhizosphere soil of lettuce, respectively. * P<0.01, ** P<0.005, *** P<0.001.

[0026] Figure 12 Effect of strain F1-18 on rhizosphere soil microbial community structure of field-grown lettuce in the preferred embodiments of the present application. A is the Venn diagram of fungal phylum in the rhizosphere soil of lettuce, B is the Venn diagram of fungal genus in the rhizosphere soil of lettuce, C is the Venn diagram of bacterial phylum in the rhizosphere soil of lettuce, and D is the Venn diagram of bacterial genus in the rhizosphere soil of lettuce. DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

[0028] The percentage sign "%" involved in the present application, if not specifically stated, refers to the mass percentage. However, the percentage of the solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0029] Example 1: Isolation and screening of lavender seed endophyte F1-18 1. Isolation and screening of strain F1-18 In 2023, 'French Blue' lavender seeds were collected from Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region. One g of lavender seeds were first soaked in 75% ethanol for 30 seconds for surface disinfection, then soaked in sterile water for 30 seconds, and rinsed three times to ensure disinfection effectiveness. The sterile water from the last rinse was spread on LB agar plates (yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar 15 g / L) to observe for colony formation, verifying the surface disinfection effect. The surface moisture of the disinfected lavender seeds was blotted dry with sterile filter paper, and the seeds were placed in a sterile mortar with 1.5 mL of sterile physiological saline for grinding. After standing for 10 minutes, the supernatant was used as the stock solution of endophytic bacteria suspension. The stock solution was then diluted 10-fold in a 10-fold gradient to 10 ... -1 10 -2 10 -3 For each dilution, 200 μL was plated onto LB agar plates and incubated at 28 °C for 72 h, with each treatment repeated three times. Colonies with different morphological characteristics were picked, purified by streak plating, and stored at 4 °C for later use. Single colonies of endophytic bacteria were picked and activated in LB tubes, and the bacterial culture was mixed with 40% (v:v) sterile glycerol at a 1:1 ratio and stored at -80 °C.

[0030] Following the above isolation process, a total of 55 endophytic bacteria strains were isolated from 'French Blue' lavender seeds. Among them, strain F1-18, which has good plant growth-promoting ability, was selected.

[0031] 2. Polyphasic taxonomic identification of strain F1-18 (1) Analysis of colony and cell morphology characteristics Strain F1-18 was Gram-positive. After incubation on LB medium at 28°C for 24 h, the colonies were orange-yellow, round, with neat edges, and a moist, glossy surface. Figure 1 ).

[0032] (2) Phylogenetic analysis of 16S rRNA gene of strain F1-18 Genomic DNA was extracted from bacterial strain F1-18 using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for bacterial 16S rRNA. The PCR reaction system consisted of 12.5 μL of 2×Taq PCR HiFi Mix and 10 μmol / L... -1Each 1 μL of primer, 2.0 μL of DNA template, and sterile deionized water were added to make up to 8.5 μL; the PCR amplification procedure was as follows: 95 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 25 s, 55 ℃ rehydration for 25 s, 72 ℃ extension for 25 s, 35 cycles; 72 ℃ extension for 5 min. After the PCR product was detected by agarose gel electrophoresis, it was sent to Shangon Biotech (Shanghai) Co., Ltd. for sequencing. After splicing the obtained 16S rRNA gene sequence, 16S rRNA sequence similarity comparison analysis was carried out through NCBI BLAST and EzBioCloud (www.ezbiocloud.net), and MEGA 11 software was used to construct a 16S rRNA gene phylogenetic tree by the neighbor-joining method (Neighbour-Joining) for preliminary identification of the bacterial strain.

[0033] The results showed that the 16S rRNA gene sequence similarity of the strain F1-18 was the highest with the strains of the genus Microbacterium, including M. arabinogalacti-lucum DSM 20754 (99.30%), M. testaceum DSM 20530 (99.09%) and M. radiotolerans NE2HP2 (98.79%). The phylogenetic tree results also showed that the strain F1-18 was clustered with GIMN1.002, DSM 20754, DSM 20530 and NE2HP2 in the same branch, with the closest genetic distance. The 16S rRNA gene sequence of the strain F1-18 is shown as SEQ ID NO: 1. Microbacterium ) strains, including M. arabinogalacti-lucum DSM 20754 (99.30%), M. testaceum DSM 20530 (99.09%) and M. radiotolerans NE2HP2 (98.79%). The phylogenetic tree results also showed that the strain F1-18 was clustered with GIMN1.002, DSM 20754, DSM 20530 and NE2HP2 in the same branch, with the closest genetic distance. The 16S rRNA gene sequence of the strain F1-18 is shown as SEQ ID NO: 1. Microbacterium arborescens DSM 20754 T Microbacterium imperiale DSM 20530 T Microbacterium radiodurans GIMN1.002 T Microbacterium radiodurans GIMN1.002 T , Microbacterium arborescens DSM 20754 T , Microbacterium imperiale DSM 20530 T and Microbacterium plantarum NE2HP2 T . Figure 2 The 16S rRNA gene sequence of the strain F1-18 is shown as SEQ ID NO: 1.

[0034] (3) Whole genome sequencing and basic genomic characteristics of the strain F1-18 After the extracted genomic DNA of the strain F1-18 was detected to be qualified, it was entrusted to Shanghai Shengong Bioengineering Co., Ltd. for sequencing whole genome framework sequencing. Illumina platform was used for sequencing, SPAdes software was used for splicing the second-generation sequencing data, and NCBI-PGAP software was used for predicting gene elements CDS, tRNA and rRNA, etc.

[0035] ​​​The N50 of the genome sequencing of strain F1-18 was 920,398 bp, the genome size was 3.34 Mb, the G+C content was 69.8 mol%, a total of 3110 coding genes were predicted, the coding region accounted for 92.96% of the genome, and a total of 5 rRNAs and 54 tRNAs were annotated (Table 1).

[0036] Table 1 Basic information of the genome of strain F1-18

[0037] (4) Genome phylogenetic analysis of strain F1-18 The results of species identification based on genome by the Type (Strain) Genome Server (TYGS, https: / / tygs.dsmz.de) showed that strain F1-18 belonged to a potential new species. The phylogenetic tree based on genome sequence was drawn by TYGS, and the results showed that strain F1-18 was close to Microbacterium arborescens DSM 20754 T and Microbacterium plantarum NE2HP2 T gathered in a branch, and the phylogenetic relationship was close Figure 3 .

[0038] (5) Nucleotide similarity analysis of strain F1-18 The Average Nucleotide Identity (ANI) of strain F1-18 and other type strains of the genus Microbacterium was calculated by JSpeciesWS (https: / / jspecies.ribohost.com / jspeciesws), and the digital DNA-DNA hybridization (dDDH) value was calculated by GGDC.

[0039] The results showed that the nucleotide similarity of strain F1-18 was the highest with Microbacterium arborescens DSM 20754 T and Microbacterium plantarum NE2HP2 T , the ANI values were 88.82% and 88.41% respectively, and the dDDH values were 37.0% and 36.5% respectively (Table 1), and both ANI and dDDH were lower than the species threshold (ANI=95~96%, dDDH=70%), indicating that strain F1-18 was a new species of the genus Microbacterium (Table 2).

[0040] Table 2 Part of the sequences used for genome analysis and ANI and dDDH values of strain F1-18 and its close relatives

[0041] (6) Physiological and biochemical characteristics of strain F1-18 Strain F1-18 is facultative anaerobic, catalase positive, oxidase negative and no hemolytic reaction. The API 20NE identification card (BioMerieux) test results showed that strain F1-18 cannot reduce nitrate, does not produce indole, and does not ferment glucose; arginine dihydrolase and urease are negative, while beta-galactosidase and beta-glucosidase are positive, and does not hydrolyze esculin, but can hydrolyze gelatin; can utilize D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, gluconate and Malate, but cannot utilize Caprate, Adipate, Citrate and Phenylacetic acid, etc. (Table 3). Compared with the close relatives Microbacterium plantarum NE2HP2 T and Microbacterium arborescens DSM 20754 T Strain F1-18 has unique characteristics in arginine dihydrolase, esculin and gelatin hydrolysis, D-mannose, N-acetyl-glucosamine and citrate utilization, etc. (Table 3).

[0042] Table 3. Differences in physiological and biochemical characteristics between strain F1-18 and close relatives

[0043] Note: nd means no data retrieved, "+" means positive, "w" weakly positive, "-" negative.

[0044] * Type strain M. plantarum NE2HP2 T and M. arborescens DSM 20754 T The physiological and biochemical data of the type strain Microbacterium plantarum sp. nov. and Microbacterium thalli sp. nov., two endophytic metal-resistant bacteria isolated from Sphaeralcea angustifolia (Cav.) G. Don and Prosopis laevigata(Humb. et Bonpl. ex Willd) M.C. Johnston. Int J Syst Evol Microbiol. 2023;73:006052. API 50CH identification results showed that strain F1-18 could utilize 24 kinds of carbon sources to produce acid, including mannitol, L-arabinose, D-xylose, methyl-β-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, mannitol, methyl-α-D-mannopyranoside, N-acetylglucosamine, amygdalin, ARBULIN, aesculin, salicin, D-cellubiose, D-maltose, D-sucrose, D-trehalose, D-erntrinose, D-erntrinose, potassium gluconate (weak positive), 5-ketogluconate potassium (Table 4).

[0045] Table 4 Carbon source utilization characteristics of strain F1-18 (API 50CH identification card)

[0046] Example 2 Stress resistance of strain F1-18 Strain F1-18 was inoculated on TSA medium (BD Bacto) plates and incubated at 4°C, 28°C, 37°C, 40°C, 42°C and 45°C for 4 days, respectively, to observe the growth of the strain. TSB culture solution containing 4%, 5%, 6%, 7%, 8%, 9%, 10% NaCl (w:v) was prepared, and strain F1-18 seed liquid was inoculated at 1% inoculation amount and incubated at 28°C, 150 rpm for 4 d, and the OD 600 value was measured. The pH value of the TSB culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 with HCl and NaOH, and incubated at 28°C, 150 rpm for 4 d, and the OD 600 value was measured. A control group (CK) without inoculation was set up, and each treatment was repeated 3 times to determine the salt and acid-base tolerance of strain F1-18.

[0047] The results showed that strain F1-18 could grow at temperatures ranging from 4-45°C, and could tolerate 10% NaCl and pH 4-11 acid-base environment. Compared with the closely related strains Microbacterium plantarum NE2HP2 T and Microbacterium arborescens DSM 20754 T , strain F1-18 had higher tolerance to temperature and salt (Table 5).

[0048] Table 5 Stress resistance characteristics of strain F1-18

[0049] According to the above morphological characteristics, genotypic characteristics, and physiological and biochemical characteristics, strain F1-18 is identified as a new species of Microbacterium, which has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) with the accession number GDMCC No: 65647. Microbacterium

[0050] Example 3: Homologous gene analysis of strain F1-18 and related bacteria OrthoVenn3 (https: / / orthovenn3.bioinfotoolkits.net) was used to analyze the homologous genes of strain F1-18 and its four related bacteria. The results showed that there were 2202 orthologous genes in the genomes of the five species, indicating that the genomic structure of Microbacterium species was conservative, and F1-18, M. plantarum NE2HP2 T , M. arborescens DSM 20754 T , M. imperiale DSM 20530 T , M. thalli NM3R9 T respectively had 2, 6, 12, 6, and 4 unique gene clusters. Figure 4

[0051] Example 4: Secondary metabolic function genes of strain F1-18 antiSMASH 8.0 (https: / / antismash.secondarymetabolites.org) was used to analyze the secondary metabolic product synthesis gene clusters of strain F1-18. The results are shown in Table 6, and a total of 6 secondary metabolic gene clusters were predicted. Among them, Region 1.1 and Region 2.4 encode ribosome natural products lanthipeptide, Region 2.4 has high similarity with the synthesis gene cluster of antibacterial lipoprotein Microvionin, while Region 1.1 does not have similar sequences, which may encode new products. Region 2.2 encodes terpene, which has certain sequence similarity with carotenoid synthesis gene cluster. T3PKS and betalactone do not have similar sequences, and the types of these metabolites need to be further verified.

[0052] Table 6: Prediction results of secondary metabolic gene clusters of strain F1-18​​

[0053] Note: antiSMASH analysis with strict mode, nd means no data.

[0054] Example 5 Detection of the plant growth promoting characteristics of strain F1-18 (1) Detection of the phosphate solubilizing ability of strain F1-18 After the strain F1-18 was activated on LB medium, it was inoculated on the inorganic phosphate (calcium phosphate) detection medium plate using filter paper piece method, 3 replicates were inoculated on each plate, and incubated at 28°C for 7 days. Whether there was a phosphate solubilizing transparent circle around the colonies was observed. The molybdenum antimony anti colorimetric method was used to quantitatively determine the solubilization ability of strain F1-18 to calcium phosphate, and the phosphorus content in the culture solution was calculated by the phosphorus standard curve. The results showed that on the inorganic phosphate detection plate, there was a clear transparent circle around the colonies of strain F1-18 Figure 5 a), and the phosphate solubilizing index UP value was 1.33. The total amount of inorganic phosphorus dissolved by strain F1-18 after 7 days of culture was 2.89 mg / L (Table 7) measured by molybdenum antimony anti colorimetric method.

[0055] Table 7 Plant growth promoting characteristics of strain F1-18

[0056] Note: The data in the table are the average values ± standard deviations of 3 replicates.

[0057] (2) Protease production ability of strain F1-18 The activated F1-18 strain was inoculated on the protease detection medium (tryptone 5.0 g, yeast extract 3.0 g, glucose 1.0 g, agar 15.0 g, distilled water 1000 mL, pH 7.0, 121°C high pressure sterilization for 30 min. When the sterilized detection medium was cooled to about 50°C, sterile skim milk was added to the medium at a proportion of 10% and mixed well, then poured into a culture dish, and cooled for standby), 3 replicates, 28°C incubation for 2 d, and observation of whether there was a transparent circle. The results showed that there was a hydrolysis circle around the colonies of strain F1-18, indicating that it had the ability to produce protease, and the corresponding UP value was 56.69 Figure 5 b, Table 7).

[0058] (3) IAA production ability of strain F1-18 The activated F1-18 strain was inoculated into the IAA detection DF+Try medium (peptone 5.0 g, yeast extract 1.5 g, beef extract 1.5 g, NaCl 5.0 g, tryptophan 0.5 g, distilled water 1000 mL, pH 7.0, 121°C high-pressure steam sterilization for 30 min) at an amount of 1%, and was placed in a 28°C, 150 rpm shaking culture for 7 days, with 3 repeats. After 7 days, the bacterial liquid was centrifuged at 12000 rpm for 5 min, and the supernatant was taken to measure the IAA content in the F1-18 bacterial liquid by Salkowkin colorimetry, which was as high as 18.54 mg / L (Table 7).

[0059] Example 6 Promoting effect of strain F1-18 on lavender seed germination

[0060] The F1-18 single colony was inoculated into LB culture solution and was placed in a 28°C, 150 rpm culture for 12 h, and was then transferred to a LB flask at an inoculation amount of 1% and was placed in a 28°C, 150 rpm culture for 24 h. The bacterial liquid was centrifuged to collect the bacterial bodies, the supernatant was discarded, the bacterial bodies were washed with sterile water for 3 times and were resuspended, and the OD value of the bacterial liquid was adjusted to 0.8 as the test bacterial suspension. 600

[0061] The F1-18 single colony was inoculated into LB culture solution and was placed in a 28°C, 150 rpm culture for 12 h, and was then transferred to a LB flask at an inoculation amount of 1% and was placed in a 28°C, 150 rpm culture for 24 h. The bacterial liquid was centrifuged to collect the bacterial bodies, the supernatant was discarded, the bacterial bodies were washed with sterile water for 3 times and were resuspended, and the OD value of the bacterial liquid was adjusted to 0.8 as the test bacterial suspension. 600 The F1-18 single colony was inoculated into LB culture solution and was placed in a 28°C, 150 rpm culture for 12 h, and was then transferred to a LB flask at an inoculation amount of 1% and was placed in a 28°C, 150 rpm culture for 24 h. The bacterial liquid was centrifuged to collect the bacterial bodies, the supernatant was discarded, the bacterial bodies were washed with sterile water for 3 times and were resuspended, and the OD value of the bacterial liquid was adjusted to 0.8 as the test bacterial suspension.

[0062]

[0063] The results are shown in Table 8. Figure 6 ​As shown in Table 8, F1-18 bacterial solution treatment significantly promoted the germination rate and growth of lavender seeds during the germination period. Compared with the sterile water treatment control, F1-18 bacterial solution treatment increased the germination rate of lavender seeds by 54.8%, stem length by 18.9%, root length by 24.6%, and fresh weight by 25%. Figure 6 (Table 8).

[0064] Table 8. Growth-promoting effect of strain F1-18 on lavender seeds.

[0065] Note: Different letters indicate that the F1-18 bacterial culture treatment group and the control group are significantly different at the P<0.05 level.

[0066] Example 7: The promoting effect of strain F1-18 on maize seed germination

[0067] Pick a single colony of F1-18 and inoculate it into LB broth. Incubate at 28°C with shaking at 150 rpm for 12 h. Transfer the inoculum to an LB shake flask at a 1% inoculation rate and incubate at 28°C with shaking at 150 rpm for 24 h. Adjust the OD of the bacterial culture with sterile LB broth. 600 The bacterial solution was prepared by adjusting the concentration to 0.8. Plump, healthy corn seeds were selected, rinsed with sterile water, then disinfected by soaking in 75% ethanol for 15 seconds, and finally rinsed three times with sterile water. For each treatment, 50 corn seeds (Jinchong No. 1 variety) were placed in a beaker containing the bacterial solution and soaked for 4 hours. The control group was soaked in an equal volume of LB broth and placed in a petri dish with filter paper moistened by the bacterial solution on both sides. This process was repeated three times. After 3 days of cultivation, the number of germinated seeds was observed and counted, and the germination rate was calculated.

[0068] The results of the maize seed germination experiment showed that soaking in F1-18 bacterial solution promoted maize seed germination to a certain extent, with the germination rate increasing by 4.4% compared to the control group. However, F1-18 inoculation significantly promoted maize growth during the seed germination period, increasing the plumule length by 30% and the radicle length by 16%. Figure 7 (Table 9).

[0069] Table 9. Growth-promoting effect of strain F1-18 on maize seeds.

[0070] Note: Different letters indicate that the difference between the inoculation treatment group and the control group is significant at the P<0.05 level.

[0071] Example 8: The promoting effect of strain F1-18 on the growth of greenhouse potted maize.

[0072] Select plump, healthy corn seeds, rinse them thoroughly with sterile water, then soak them in 75% ethanol for 15 seconds for disinfection. Finally, rinse them three times with sterile water for surface disinfection. Place them in a 28℃ incubator to germinate until the seeds show white sprouts. Mix garden soil, vermiculite, perlite, and sand in a volume ratio of 3:1:1:1, filling each pot with 1.8 kg of the mixture, ensuring consistent compaction. Add OD to each pot. 600 200 mL of F1-18 bacterial suspension with a pH of 0.8 was mixed evenly with soil for treatment. Each treatment was replicated in 6 pots, with a water control included. Three corn seeds were sown in each pot. After 7 days, the seedlings were thinned out. After 28 days of cultivation in a greenhouse, the corn seedlings were removed, and the roots were washed with clean water. The growth indicators of corn plants, such as plant height, stem diameter, above-ground fresh weight, below-ground fresh weight, above-ground dry weight, and below-ground dry weight, were measured. The total root length, average diameter, root surface area, root volume, and number of root tips were measured using a root scanner. The nutrient content of the rhizosphere soil was also measured.

[0073] The results are as follows Figure 8 As shown in Table 8, strain F1-18 significantly promoted maize growth. After inoculation with F1-18, the aboveground and root growth of maize was significantly higher than that of the control group, with plant height increasing by 11.5%, stem diameter by 11.8%, aboveground fresh weight and dry weight increasing by 24.0% and 26.9%, respectively, and underground fresh weight and dry weight increasing by 11.9% and 23.5%, respectively. Inoculation with F1-18 also significantly promoted root development in maize seedlings; compared with the control group, total root length, average diameter, root surface area, root volume, and number of root tips increased by 3.8%, 4.7%, 8.6%, 3.7%, and 4.4%, respectively. Figure 8 (Table 10). In addition, the available nitrogen content in the soil of the F1-1 bacterial solution treatment group increased by 17.5% and the available phosphorus content increased by 25.2%, indicating that inoculation with strain F1-18 is beneficial to soil nutrient activation (Table 10).

[0074] Table 10. Growth-promoting effect of strain F1-18 on potted maize

[0075] Note: Different letters indicate the difference between the F1-18 inoculation treatment group and the control group. P The difference was significant at the <0.05 level.

[0076] Example 9: Application effect of strain F1-18 in field lettuce cultivation

[0077] From April to May 2025, a field cultivation experiment of lettuce was conducted in a vegetable greenhouse in Dadamutu Town, Yining City, Xinjiang. Base fertilizer was applied before planting. Ten days after transplanting, chemical fertilizer or microbial inoculant treatment was applied. Three control groups were established: a control group without fertilizer (CK0), a control group with conventional fertilizer (CK1, 110 kg / mu of urea), and a group treated with F1-18 microbial inoculant (55 kg / mu of urea, with OD245 added). 600 5 L / mu of F1-18 bacterial suspension with a pH of 0.8 (diluted 100 times with water before root irrigation). A randomized block design was used, with each plot 5m long and 2m wide, for a total area of ​​10m². 2 Each treatment was replicated three times, for a total of nine plots. Standardized field management was implemented according to farmers' routine practices, and harvesting took place 30-40 days after transplanting. Ten lettuce plants were randomly selected from each plot, and plant height was measured for each treatment. Leaves from the same spatial location on each lettuce plant were selected, and chlorophyll content was measured using a chlorophyll meter. The aboveground and underground parts of each sample were separated, cleaned, and their fresh weights were weighed separately. The lettuce root system was scanned using a root scanner. Each sample was then placed in an oven at 105℃ for 30 minutes to blanch, and then dried at 80℃ to constant weight. The dry weights of the aboveground and underground parts were measured. Simultaneously, rhizosphere soil samples were collected and stored at -80℃ for microbial diversity analysis using 16S and ITS amplicon sequencing technologies.

[0078] The results showed that root irrigation with F1-18 bacterial solution had a significant growth-promoting effect on lettuce, reflected in indicators such as plant height, biomass, number of leaves, and chlorophyll content. Overall, an application level of 5 L / acre of bacterial solution could achieve a 50% reduction in urea application. Figure 9 Compared with the urea-treated group (CK1), the F1-18 bacterial solution treatment significantly increased lettuce plant height and chlorophyll content by 40% and 63.3% respectively (P<0.05), while there was no significant difference in leaf number and biomass. Figure 10 Furthermore, the F1-18 bacterial solution treatment increased the microbial community diversity and affected the community structure of the rhizosphere soil of lettuce. Compared with CK0 and CK1, the species richness indices of rhizosphere bacteria and fungi, ace and chao, were significantly increased after the F1-18 bacterial solution treatment. Figure 11 Furthermore, significant differences were observed in the distribution of rhizosphere fungi and bacteria at the phylum and genus levels. Figure 12 ).

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A new species of endophytic microbacterium promoting lavender growth, which is a microbacterium Microbacterium sp. F1-18, accession number GDMCC No: 65647.

2. A microbial agent or bio-fertilizer prepared from the microbacterium described in claim 1, its fermentation broth, or a crude extract of the fermentation broth.

3. Plant growth promoters, agricultural fertilizers, phosphorus activators, or soil activators prepared from the microbacterium or its inoculum as described in claim 1.

4. Any of the following applications of the microbacterium or its inoculum according to claim 1: 1) Used for phosphorus dissolution; 2) Used for producing IAA; 3) Used for protease production; 4) Used to promote plant growth or to prepare plant growth promoters; 5) Used to reduce fertilizer use, increase soil nutrient content, improve soil microbial diversity, or prepare soil activators.

5. The application according to claim 4, characterized in that, The phosphorus mentioned in 1) is inorganic phosphorus.

6. The application according to claim 4, characterized in that, The plant growth promotion method described in 4) includes: soaking the plants in the bacterial solution of the microbacterium, or applying the bacterial solution to the plant rhizosphere soil.

7. The application according to claim 4 or 6, characterized in that, The plants mentioned include lavender, corn, and lettuce.

8. The application according to claim 4, characterized in that, 5) The improvement of soil nutrient content includes increasing the content of available nitrogen and available phosphorus in the soil, and the improvement of soil microbial diversity includes increasing the community diversity of soil bacteria and fungi.

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