Corn growth-promoting rhizosphere bacterium enterobacter cloacae LN01 and application thereof

Abstract

The invention discloses a corn rhizosphere growth-promoting bacterium enterobacter cloacae LN01 and an application thereof. The growth-promoting rhizosphere bacterium is named as Enterobacter hormaechei LN01 and is preserved in the China Center for Type Culture Collection, and the preservation number of the growth-promoting rhizosphere bacterium is CCTCC (China Center for Type Culture Collection) NO.M 2024003. Researches find that LN01 has the capabilities of dissolving phosphorus and potassium, fixing nitrogen and secreting indole-3-acetic acid (IAA) and siderophores. The LN01 can promote biomass accumulation of the overground part of the corn and root morphological development such as root length, intersection number, node number and the like. Through antiSMASH analysis, biological gene synthesis clusters of four secondary metabolites, including non-ribosome peptide synthetase, terpene, thiopeptide and aryl polyene, for promoting plant growth are found in the whole genome of LN01. The invention provides a new technical means for research, development and application of the biological fertilizer for promoting corn growth.

Description

Technical Field

[0001] The present invention relates to a corn rhizosphere growth-promoting bacterium and its application, and in particular to a corn rhizosphere growth-promoting bacterium capable of solubilizing phosphorus, potassium, fixing nitrogen, secreting indole-3-acetic acid (IAA) and iron carrier, and its application in promoting corn growth. The present invention belongs to the technical field of agricultural production. Background Art

[0002] As one of the three major staple foods in my country, the current planting situation of corn is not optimistic. Compared with the United States, which ranks first in the world, the yield per hectare is very different. According to the "Grain Increasing Science and Technology Action Plan" proposed by the Chinese Academy of Agricultural Sciences, increasing corn production will be an important scientific research direction in the future. In recent years, many problems such as soil compaction, soil salinization, heavy metal pollution, and destruction of soil microbial diversity caused by the long-term application of fertilizers and pesticides to maintain corn production have become increasingly serious, posing a huge threat to environmental safety and food safety. Therefore, how to solve the above problems urgently needs further research.

[0003] Soil microorganisms participate in soil structure maintenance, soil nutrient cycling, pesticide degradation and pest control, and are an indispensable part of achieving the sustainability of agricultural ecosystems. Bacteria in the rhizosphere of plants that promote plant growth, control plant diseases and increase crop yields are called plant rhizosphere growth-promoting bacteria. They promote and regulate the growth and development of the host through direct or indirect mechanisms of action. Rhizosphere microorganisms are regarded as the second genome of plants, playing an important role in the nutrition and health of host plants, and playing an irreplaceable role in promoting the reduction and efficiency of chemical fertilizers and the green development of agriculture. The application of microbial agents can promote crop growth and improve the soil environment to keep plants healthy and increase soil fertility. The combined use of organic fertilizers and microbial agents can effectively increase agronomic traits such as plant height, number of leaves, and root thickness, and also significantly improve soil properties and the number of microorganisms.

[0004] The inventor of the present invention isolated and purified a strain of Enterobacter hormaechei in the early stage, named Enterobacter hormaechei LN01, and studied its nutrient conversion function and the potential of related genes by means of whole genome information and strain nutrient conversion ability determination. Corn was used as the test crop to study its growth-promoting effect on corn. Summary of the invention

[0005] One of the purposes of the present invention is to provide a newly isolated corn rhizosphere growth-promoting bacterium with the ability to solubilize phosphorus, potassium and fix nitrogen.

[0006] The second purpose of the present invention is to provide the use of the corn rhizosphere growth-promoting bacteria in promoting corn growth.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The invention discloses a kind of corn rhizosphere growth-promoting bacteria isolated and obtained. The corn rhizosphere growth-promoting bacteria is Enterobacter cloacae, named as Enterobacter hormaechei LN01, classified as Enterobacter hormaechei LN01, and deposited in China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit number is CCTCC NO.M2024003, and the deposit time is January 2, 2024.

[0009] Furthermore, the present invention also proposes the use of the corn rhizosphere growth-promoting bacteria in promoting corn growth.

[0010] Among them, preferably, the corn rhizosphere growth-promoting bacteria have the ability to solubilize phosphorus, potassium, fix nitrogen, secrete indole-3-acetic acid (IAA) and iron carriers, and can promote corn growth by promoting the accumulation of corn aboveground parts and the morphological development of roots.

[0011] Among them, preferably, the morphological development of the roots includes root length, number of intersections and number of nodes.

[0012] Among them, preferably, the phosphate solubility, potassium solubilization, nitrogen fixation and IAA secretion of the corn rhizosphere growth-promoting bacteria are 58.33, 15.6, 15.14 and 42.2 mg / L, respectively.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention separates and identifies a strain of corn rhizosphere growth-promoting bacteria, and studies its growth-promoting effect and its genome information. The corn rhizosphere growth-promoting bacteria is named Enterobacter hormaechei LN01, and the genome contains gene clusters involved in iron acquisition (fhuBCDEF, afuABC, efeOBU and fepCDG), phosphate solubilization (pstABCS, phoABER, phnACDEFGHIJKLP and ugpABCE), biological nitrogen fixation (nirBCD, nasA, glnA, gltBD and nrtABC), potassium solubilization (kdpABC, kefBCFG, trkAH and kup) and IAA biosynthesis gene cluster (trpABSCFRGD), and has the ability to solubilize phosphorus, solubilize potassium, fix nitrogen and secrete IAA, which are 58.33, 15.6, 15.14 and 42.2 mg / L respectively. The corn rhizosphere growth-promoting bacteria contain a biogene synthesis cluster of four secondary metabolites that promote plant growth, including non-ribosomal peptide synthetases, terpenes, thiopeptides and aromatic polyenes. Experiments have found that Enterobacter hormaechei LN01 can promote corn growth by promoting corn plant height, dry fresh weight of aboveground and underground parts, root length, number of connections, number of nodes, and promoting the synthesis of auxin. The invention provides a new technical means for the research and development and application of biofertilizers that promote corn growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the sequence alignment result of Enterobacter hormaechei LN01 strain;

[0016] Figure 2 This is a map of the key biosynthetic gene clusters in the genome of Enterobacter hormaechei LN01 strain;

[0017] Figure 3 The growth of Enterobacter hormaechei LN01 strain on phosphate-solubilizing, potassium-solubilizing, nitrogen-fixing and iron-carrier plates respectively;

[0018] Figure 4 It is a gene with growth-promoting properties in the genome of Enterobacter hormaechei LN01 strain;

[0019] Figure 5 The growth-promoting effect of Enterobacter hormaechei LN01 strain on the aboveground and underground parts of corn seedlings;

[0020] in, Figure 5A is a photo of the control group and the LN01 bacterial solution immersion group after 4-5 days of culture; Figure 5 BG shows that the LN01 strain significantly improves corn plant height (B), aboveground dry\fresh weight (D\F), underground dry\fresh weight (E\G), and corn root length, number of forks, number of root tips, number of connections and number of nodes (C). DETAILED DESCRIPTION

[0021] The present invention will be further described below through experiments and examples. It should be understood that these examples are only for illustrative purposes and in no way limit the scope of protection of the present invention.

[0022] Example 1 Isolation and identification of maize rhizosphere growth-promoting bacteria Enterobacter hormaechei LN01

[0023] 1. Materials and methods

[0024] 1.1 Test materials

[0025] The soil used was the rhizosphere soil of maize from the Meilisi Maize Farm of Qiqihar University. The collected rhizosphere soil was transported in an ice box and immediately stored in a -80°C refrigerator when returned to the laboratory.

[0026] 1.2 Isolation of bacteria

[0027] After gradient dilution of rhizosphere soil samples, corn rhizosphere growth-promoting bacteria were isolated using combined nitrogen fixation medium. Whole-genome sequencing of bacteria was performed to identify the strain. The isolated and purified strain was named Enterobacterhormaechei LN01 and deposited in the China Center for Type Culture Collection, Wuhan University. Its microbial collection number is CCTCC NO.M 2024003, and the collection date is January 2, 2024.

[0028] 1.3 Data Analysis

[0029] The sequencing results of the LN01 strain were compared by gene BLAST in the NCBI (National Center of Biotechnology Information) database, and a phylogenetic tree was constructed; the number of biosynthetic gene clusters was predicted using antiSMASH 6.6.0.

[0030] 2. Results and Analysis

[0031] 2.1 Strain taxonomic information

[0032] Based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB and tsf), the obtained sequences were matched with the nucleotide sequences of the GenBank database of the National Center for Biotechnology Information (NCBI) using the local alignment search tool (BlastN). The 19 strains closest at the species level were selected, and the phylogenetic tree was constructed using the NJ (Neighbor-Joining) method using MEGA6.0 software. The results are shown in Figure 1 The isolated strain LN01 had the closest genetic evolutionary distance to Enterobacter hormaechei, and the strain LN01 was identified as Enterobacter hormaechei.

[0033] The isolated and purified strain was named Enterobacter hormaechei LN01 and deposited in the China Center for Type Culture Collection, located at Wuhan University. Its microbial preservation number is CCTCC NO.M2024003, and the preservation date is January 2, 2024.

[0034] 2.2 Overview and characteristics of the Enterobacter hormaechei LN01 genome

[0035] The whole genome analysis of Enterobacter hormaechei LN01 strain showed that the strain genome contained a 5379004bp chromosome and six plasmids, with an average whole genome GC content of 55.1%. The strain genome had 87 tRNAs, 25 rRNAs, 14 gene islands, and 5004 coding sequences (CDSs) (Table 1).

[0036] Table 1

[0037]

[0038]

[0039] 2.3 Secondary metabolite synthesis of Enterobacter hormaechei LN01

[0040] AntiSMASH analysis revealed that strain LN01 has four secondary metabolite biosynthesis gene clusters, including non-ribosomal peptide synthetases, terpenes, thiopeptides, and aryl polyenes ( Figure 2 ). The nonribosomal polypeptide synthetase encoding Turnerbactin showed 30% similarity with BGC0000451. Turnerbactin is a new tricatechin siderophore responsible for iron regulation and absorption. The gene TonB related to Turnerbactin biosynthesis and the gene EntACDEFSH encoding enterobactin synthase and the gene entB encoding the key enzyme isochorinase were found in the genome of strain LN01. The terpene cluster encoding carotenoids showed 100% similarity with BGC0000640. Phytoene desaturase and lycopene cyclase are key enzymes for the synthesis of carotene. The gene crtI encoding phytoene desaturase and the gene lcyB encoding lycopene cyclase were found in the genome of strain LN01. The thiopeptide encoding O-antigen has 14% similarity with BGC0000781, and four genes involved in O-antigen biosynthesis (rpsA, wzzB, wzxC and wzxE) were found in the genome of LN01 strain. The aromatic polyene cluster encoding surface protein has 94% similarity with BGC0002008, and the genes involved in its biosynthesis in the genome of LN01 strain are plsC, fadD, waaE, tesC, loA and FabG.

[0041] Example 2 Determination of Growth-Promoting Properties of Enterobacter hormaechei LN01 Strain and Its Genomic Information

[0042] 1. Materials and methods

[0043] 1.1 Test materials

[0044] 1.1.1 Test strains

[0045] Enterobacter hormaechei LN01 was isolated and identified in Example 1.

[0046] 1.1.2 Seeds for testing

[0047] The tested corn seeds were Kennian No. 1.

[0048] 1.1.3 Culture medium

[0049] Axubei medium: 0.2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 0.2g sodium chloride, 5g calcium carbonate, 0.1g calcium sulfate, 10g mannitol, add water to 1L. For solid medium, add 20g agar.

[0050] Phosphate-dissolving medium: 10.00 g glucose, 0.30 g magnesium sulfate heptahydrate, 0.50 g ammonium sulfate, 0.30 g sodium chloride, 2.00 g calcium phosphate, 0.036 g ferrous sulfite heptahydrate, 0.30 g potassium chloride, 0.03 g manganese sulfate, add water to make up to 1 L. 20 g agar should be added to the solid medium.

[0051] Potassium-dissolving medium: 0.50 g sucrose, 0.10 g calcium carbonate, 0.50 g disodium hydrogen phosphate, 0.05 g ferric chloride, 0.50 g magnesium sulfate, 0.10 g potassium feldspar, add water to make up to 1 L. 20 g agar should be added to the solid medium.

[0052] Beef extract peptone liquid culture medium: peptone 10.0g, beef extract 5.0g, sodium chloride 5.0g, add water to make up to 1L, pH 7.0.

[0053] 1.2 Detection of nitrogen fixation ability of Enterobacter hormaechei LN01

[0054] Take 100 μL of LN01 strain and inoculate it into 150 mL of beef extract peptone liquid medium, and culture it at 30℃ and 180r / min for 24 hours to obtain the seed liquid of LN01. Inoculate the above 3 μL of seed liquid into the Axubei solid medium, and make 3 parallels, and culture it at 30℃ for 48 hours to observe whether the strain can grow.

[0055] The strain was inoculated into 100 mL of Axubei liquid culture medium at a 1% inoculum size, and cultured in a constant temperature shaker at 30°C and 120 r / min for 7 days. The nitrogen fixation ability of strain LN01 was determined using a carbon-nitrogen analyzer.

[0056] 1.3 Detection of potassium-dissolving ability of Enterobacter hormaechei LN01

[0057] The above 3 μL LN01 seed solution was inoculated into potassium-dissolving solid culture medium in triplicate, and cultured at 30°C for 48 h to observe whether the strain could grow.

[0058] The strain was inoculated into 100 mL of Axubei liquid culture medium at a 1% inoculum amount, and after shaking culture in a constant temperature shaker at 30°C and 120 r / min for 7 days, the potassium solubility of the strain LN01 was determined using a flame spectrophotometer.

[0059] 1.4 Detection of phosphate solubilization capacity of Enterobacter hormaechei LN01

[0060] The above 3 μL LN01 seed solution was inoculated into phosphate-dissolving solid culture medium in triplicate, and cultured at 30°C for 48 h to observe whether the strain could grow.

[0061] Determine the absorbance of the standard solution with phosphorus content of 0, 0.2, 0.4, 0.8 and 1.0 mg / L at a wavelength of 700 nm, and draw a standard curve. Inoculate the strain into 100 mL of phosphate-dissolving liquid culture medium at a 1% inoculum amount, shake and culture in a constant temperature shaker at 30°C and 120 r / min for 7 days, and then determine the soluble phosphorus content in the fermentation broth by molybdenum antimony colorimetry. Calculate the phosphorus content of the corresponding sample through the standard curve.

[0062] 1.5 Determination of the ability of Enterobacter hormaechei LN01 to produce siderophores

[0063] Pipette 5 μL of bacterial solution and spot it on the iron carrier production qualitative plate for qualitative detection, and observe whether the target strain can form an orange halo on the CAS blue qualitative detection plate.

[0064] 1.6 Determination of growth hormone (IAA) secretion by Enterobacter hormaechei LN01

[0065] The 200 μg / mL IAA standard solution was diluted to 0, 25, 50, 75, 100, 125, 150 and 175 μg / mL concentrations, and the absorbance was determined by the Salkowski colorimetric method. The IAA standard curve equation was y = 0.0055x - 0.0002, R 2 =0.9967.

[0066] The seed solution of LN01 was inoculated into a liquid beef extract peptone medium containing 200 mg / L tryptophan at a 1% inoculation rate, and cultured at 30°C with shaking at 120 r / min. IAA was measured every 24 hours using the Salkowski colorimetric method.

[0067] 1.7 Determination of the growth-promoting ability of Enterobacter hormaechei LN01 on corn

[0068] Select corn seeds with full grains and no insect holes, disinfect the seeds with 5% sodium hypochlorite and 75% alcohol for 5 minutes, and then wash them with sterile water 3 times, 1 minute each time. Place the seeds on a sterilized plate with filter paper placed on top and bottom, and soak them with sterile water. Place the seeds in an incubator and control the light (16 / 8 hour light / dark cycle), temperature (22±2℃) and relative humidity (60%-70%). After the seeds germinate to 1-2 cm, soak the seeds in a 1×10 8CFU / mL LN01 solution, soaked for 1 hour, and then planted in a nutrient pot filled with non-sterile soil. At the same time, the seeds were soaked in beef extract peptone liquid medium without Enterobacter hormaechei LN01, soaked for 1 hour, and then planted in a nutrient pot filled with non-sterile soil as a control group (CK). The nutrient pots were placed in an incubator and cultured for 4-5 days, and the plant height, dry and fresh weight, and root morphology were measured using a root scanner.

[0069] 1.8 Data Processing

[0070] Microsoft Excel Office 2016 software was used to organize the data; SPSS22 software was used to complete the statistical analysis, and a significant difference of P<0.05 was considered statistically significant; Origin2021 was used to draw the graphs.

[0071] 2. Results and Analysis

[0072] 2.1 Detection of Enterobacter hormaechei LN01 strain characteristics

[0073] Enterobacter hormaechei LN01 strain was inoculated on nitrogen fixation, potassium solubilization, phosphate solubilization, and iron carrier production screening medium, and the growth of the strain was observed. At the same time, its nitrogen fixation, potassium solubilization, and phosphate solubilization abilities as well as IAA secretion were tested. Figure 3 As shown, the strain can grow on the above medium and produce halos, which confirms that the strain has the ability to solubilize phosphate, solubilize potassium, fix nitrogen and produce siderophores. Table 2 shows that the strain's nitrogen fixation, phosphate solubilization, potassium solubilization and IAA secretion are 15.14 mg / L, 58.33 mg / L, 15.6 mg / L and 42.2 mg / L, respectively.

[0074] Table 2

[0075]

[0076] 2.2 Analysis of genes related to growth promotion in Enterobacter hormaechei LN01 strain

[0077] Growth-promoting genes in the genome of Enterobacter hormaechei LN01 strain Figure 4As shown. No complete nitrogenase gene cluster was found in the genome of strain LN01. Regulatory genes nirB, nirC, nirD, nasA, nrtA, nrtB and nrtC were found in the nitrogen metabolism pathway. The protein encoded by nrtB transports extracellular nitrate into the cell and is reduced to nitrate under the control of nasA. Nitrate produces ammonia under the action of nirA. The gene glnA encoding glutamine synthase and the genes gltB and gltD encoding glutamate synthase were found in the genome of strain LN01; genes encoding phosphate transporters (pstS, pstC, pstA and pstB), phosphate transport system proteins (phnA, phnC, phnD, phnE, phnF, phnG, phnH, phnI, phnJ, phnK, phnL, phnN and phnP), alkaline phosphatase (p A group of genes related to potassium solubility were identified in the LN01 genome, including genes encoding potassium transport (kdpA, kdpB and kdpC), potassium efflux system (kefB, kefC, kefF and kefG) and potassium absorption system (trkA, trkH and kup). In addition, the genome of strain LN01 contains genes related to iron carriers, such as encoding ferrous iron uptake proteins (efeO, efeB and efeU), iron-siderophore transport system permease proteins (fepD and fepG), iron-siderophore transport system ATP binding protein (fepC), isohydroxamate iron / heme transport system substrate binding protein (ftsB), iron (III) transport system permease protein (afuB), iron (III) transport system ATP binding protein (afuC), and iron (III) transport system substrate binding protein (afuA). Plant growth hormone IAA has a promoting effect on plant growth. Tryptophan is the precursor of IAA synthesized by bacteria. A group of tryptophan biosynthesis genes (trpA, trpB, trpS, trpCF, trpR and trpGD) genes and ipdC, amiE, patB genes closely related to IAA synthesis were found in the genome of strain LN01. Siderophore transport across the outer membrane of strain LN01 is mediated by an energy transduction complex with proteins (tonB and exbB) and the iron(III)-hydroxamate ABC transporter cluster (fhuB, fhuC, fhuD, fhuE, and fhuF) responsible for the transport of iron, chromium, and other Fe 3+ -Transport of hydroxamic acid compounds.

[0078] 2.3 Growth-promoting effect of Enterobacter hormaechei LN01 on corn

[0079] The growth-promoting effects of Enterobacter hormaechei LN01 on the aboveground and underground parts of maize Figure 5 shown. Figure 5 AG showed that the Enterobacter hormaechei LN01 strain significantly improved maize plant height, aboveground fresh and dry weight, underground fresh and dry weight, and root morphological development such as maize root length, number of forks, number of root tips, number of connections and number of nodes.

[0080] in conclusion:

[0081] Enterobacter hormaechei LN01 promotes corn growth by increasing plant height, aboveground and underground dry and fresh weight, root length, number of connections, number of nodes, and the synthesis of auxin. At the same time, the genome of this strain contains a large number of gene clusters related to the ability to promote plant growth.

Claims

1. A corn rhizosphere growth-promoting bacterium, characterized in that: The corn rhizosphere growth-promoting bacteria is Enterobacter hormaechei, named Enterobacter hormaechei LN01, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO.M 2024003.

2. Use of the corn rhizosphere growth-promoting bacteria according to claim 1 in promoting corn growth.

3. The use according to claim 2, characterized in that The corn rhizosphere growth-promoting bacteria have the ability to solubilize phosphorus, potassium, fix nitrogen, secrete indole-3-acetic acid (IAA) and iron carriers, and can promote corn growth by promoting the accumulation of corn aboveground parts and the morphological development of roots.

4. The use according to claim 3, characterized in that The root morphological development includes root length, number of intersections and number of nodes.

5. The use according to claim 3, characterized in that The phosphate solubility, potassium solubility, nitrogen fixation and IAA secretion of the corn rhizosphere growth-promoting bacteria are 58.33, 15.6, 15.14 and 42.2 mg / L respectively.

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