Efficient phosphate-solubilizing salt-tolerant growth-promoting pseudomonas deliciosa and application thereof

Abstract

The invention discloses a high-efficiency phosphate-solubilizing salt-tolerant growth-promoting pseudomonas deliciosa and application thereof, and relates to the technical field of microorganisms, and the technical key points are as follows: the pseudomonas deliciosa HX144 is preserved in China General Microbiological Culture Collection Center (CGMCC) in October 2024, and the preservation number of the pseudomonas deliciosa HX144 is CGMCC NO.32292. The Pseudomonas deliciosa HX144 provided by the invention is high in phosphate solubilizing activity and strong in salt tolerance, the dissolved available phosphorus content of the Pseudomonas deliciosa HX144 is up to 195.75 + / -0.5 mg / L, and the Pseudomonas deliciosa HX144 can tolerate a culture medium environment in which the NaCl concentration is up to 6%. And the strain has a good inhibition effect on plant pathogenic bacteria and human and / or animal pathogenic bacteria. In addition, the strain also has the functions of producing protease, plant growth hormone, siderophore and the like. The strain has a growth promoting effect on wheat crops and can remarkably improve indexes such as plant height, fresh weight and root length of plants. HX144 is an excellent salt-resistant, disease-preventing and growth-promoting pseudomonas strain, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a highly efficient phosphorus-solubilizing, salt-tolerant, and growth-promoting Tehran Pseudomonas and an application thereof. Background Art

[0002] Soil salinization is one of the most important issues threatening agricultural production, food security and sustainability in arid and semi-arid regions worldwide. Soil salinization will reduce agricultural productivity, water quality and soil biodiversity. Salinized soil reduces the ability of crops to absorb water and the availability of trace elements and causes soil structure degradation, seriously affecting crop growth. Strengthening the transformation and improvement of saline-alkali land and effectively curbing the trend of salinization of cultivated land are important strategic issues in my country at present. In addition, phosphorus as a nutrient element is crucial in the growth and development of plants, but most of the phosphorus in the soil is insoluble mineral phosphorus, which is difficult for plants to absorb and utilize. Phosphorus fertilizer applied to the soil is easy to combine with Fe, Al, and Ca to form insoluble Fe-P, Al-P, and Ca-P precipitation, resulting in a very low utilization rate of phosphorus by crops. Moreover, excessive application of phosphorus fertilizer also brings serious ecological and environmental pollution problems. In actual production, the growth of crops is often invaded by pathogens. The diseases caused by these pathogens seriously threaten my country's food safety production. Chemical control as the main prevention and control means often leads to problems such as drug resistance, environmental pollution and food safety. Therefore, exploring environmentally friendly, green and efficient disease prevention and control strategies and soil improvement methods is of great significance to the sustainable and green and high-quality development of my country's agriculture.

[0003] The development of green inputs using beneficial microorganisms is one of the important measures to achieve green disease prevention and control and improve soil quality. Among them, Pseudomonas spp. is rich in species and widely distributed, especially in the rhizosphere environment of plants. It has strong rhizosphere colonization ability and growth-promoting and disease-preventing characteristics, and has become a star strain for the development of microbial agents. Studies have shown that the application of Pseudomonas microbial agents can improve soil fertility, promote the release of insoluble mineral nutrients, improve the absorption and utilization rate of fertilizers, and enhance the ability of crops to resist diseases and pests and cold, drought, lodging, and salinity. However, at present, Pseudomonas agents have the problem of relatively single commercial preparations, and their mechanism of action is also single. Therefore, exploring new beneficial Pseudomonas and enriching microbial germplasm resources not only provide an important resource basis for the development and utilization of new and efficient microbial green inputs, but also lay an important foundation for the green and high-quality development of agriculture.

[0004] Therefore, the present invention provides a highly efficient phosphate-solubilizing, salt-tolerant, and growth-promoting Pseudomonas Tehran HX144 and application thereof. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems, to provide a highly efficient phosphate-solubilizing and salt-tolerant growth-promoting Pseudomonas Tehran HX144 and its application, and to clarify its growth-promoting effect on plants, its stimulation of plant immunity and its inhibitory effect on tobacco bacterial wilt by exploring the specific growth-promoting and antagonistic functions of Burkholderia, so as to provide an important resource basis for the research and development of highly efficient disease-preventing and growth-promoting microbial preparations, and to provide an important reference for exploring new green prevention and control strategies for plant diseases.

[0006] In order to achieve the above-mentioned object, the technical scheme of the present invention is as follows: a Pseudomonas Tehran HX144, which has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration in October 2024, and its deposit number is: CGMCC NO.32292, and the 16S rRNA and rpoD gene sequences of the Pseudomonas Tehran HX144 strain are shown in SEQ ID No.1 and SEQ ID No.2.

[0007] Another object of the present invention is to provide a method for culturing Pseudomonas Tehran HX144, which comprises the following steps: activating strain HX144 on KB solid culture medium; picking a fresh single colony and inoculating it into KB liquid culture medium, and shaking and culturing it overnight at 28°C and 200rpm; transferring it to LB liquid culture medium with a dilution of 1:1000, and shaking and culturing it for 48h under the same culture conditions to obtain a fermentation liquid.

[0008] Another object of the present invention is to provide a method for preparing metabolites of Pseudomonas tehranensis HX144, the method comprising the following steps: inoculating strain HX144 into KB liquid culture medium, and shaking overnight at 28°C and 200rpm; transferring the culture into LB liquid culture medium, and continuing to shake for 48h under the same culture conditions; the obtained fermentation broth contains metabolites of strain HX144.

[0009] Another object of the present invention is to provide a use of Pseudomonas Tehran HX144 or its metabolites, wherein the strain has a good inhibitory effect on plant pathogenic bacteria and human and / or animal pathogenic bacteria Salmonella and Staphylococcus aureus.

[0010] The present invention is further configured as follows: the plant pathogenic bacteria include but are not limited to tomato bacterial wilt pathogen Ralstonia solanacearum, tomato bacterial canker pathogen Bacillus michiganensis, rice white leaf blight pathogen Xanthomonas oryzae, rice stripe pathogen Xanthomonas oryzae, crucifer black rot pathogen Xanthomonas campestris, tomato bacterial leaf spot pathogen Pseudomonas syringae, oat acidophilus, watermelon acidophilus, and carrot soft rot Pectinobacterium.

[0011] Another object of the present invention is to provide a use of Pseudomonas tehranensis HX144 or its metabolites, wherein the strain or its metabolites are used for: inhibiting plant pathogenic bacteria; hydrolyzing organic phosphorus and inorganic phosphorus; producing protease, ammonia, plant growth hormone and siderophore; promoting plant growth and increasing plant biomass; and improving plant salt tolerance.

[0012] Another object of the present invention is to provide a microbial preparation using Pseudomonas tehranensis HX144, wherein the preparation comprises the above-mentioned strain HX144 or its metabolites.

[0013] Another object of the present invention is to provide an application of Pseudomonas Tehran HX144 in agriculture, wherein the strain is used to prepare a microbial fertilizer or biological preparation containing the above-mentioned microbial fertilizer.

[0014] Another object of the present invention is to provide an application of Pseudomonas tehranensis X144 or its metabolites in promoting wheat growth. The strain or its metabolites are used for root irrigation of wheat to significantly improve the growth index of wheat.

[0015] Compared with the prior art, the beneficial effects of this scheme are as follows: the colony of the strain Pseudomonas tehranensis HX144 of the present invention is round, with neat edges, opaque, light yellow on the front, and smooth surface. Experiments have shown that the Pseudomonas tehranensis HX144 of the present invention is a stable, efficient and broad-spectrum plant disease prevention and growth-promoting bacterium. The strain has a good inhibitory effect on pathogenic bacteria such as Ralstonia solanacearum, rice stripe pathogen Xanthomonas oryzae, carrot soft rot pectin bacteria, rice Xanthomonas oryzae white leaf blight pathogenic variants, wild rapeseed Xanthomonas, Staphylococcus aureus, etc. The strain Pseudomonas tehranensis HX144 has a dissolved effective phosphorus content of up to 195.75±0.5mg / L, has a strong phosphorus solubilizing ability, and the strain can tolerate a culture medium environment with a NaCl concentration of up to 6%. In addition, the strain also has the functions of producing protease, secreting plant auxin (IAA), and producing siderophore, which can effectively improve the soil nutrient environment and promote plant growth. The results showed that Pseudomonas tehranensis HX144 is a plant rhizosphere probiotic with the advantages of high phosphate solubilization activity, strong salt tolerance, good growth-promoting properties, easy cultivation, good preservation, environmental friendliness and harmlessness to humans and animals. It has the prospect of being developed and applied as a new type of microbial resource. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the phylogenetic tree constructed based on 16S rRNA and rpoD gene sequences in Example 1 of the present invention;

[0017] Figure 2The antagonistic ability of Pseudomonas tehranensis HX144 to pathogenic bacteria in Example 2 of the present invention is determined. Among them, A is Ralstonia solanacearum; B is Xanthomonas oryzae; C: Pectinobacterium; D: Xanthomonas oryzae pv. oryzae; E: Xanthomonas campestris; F: Staphylococcus aureus; G: Bacterial canker of tomato; H: Bacterial leaf spot of tomato; I: Acidophilus avenae; J: Acidophilus citrullus; K: Salmonella;

[0018] Figure 3 The protease production assay of Pseudomonas tehranensis HX144 in Example 3 of the present invention;

[0019] Figure 4 The determination of inorganic phosphorus hydrolysis by Pseudomonas tehranensis HX144 in Example 4 of the present invention;

[0020] Figure 5 The determination of organic phosphorus hydrolysis by Pseudomonas tehranensis HX144 in Example 5 of the present invention;

[0021] Figure 6 The quantitative determination of the phosphate solubilization ability of the strain of Pseudomonas tehranensis HX144 in Example 6 of the present invention;

[0022] Figure 7 The ammonia production determination of Pseudomonas tehranensis HX144 in Example 6 of the present invention;

[0023] Figure 8 The secretion of auxin by Pseudomonas tehranensis HX144 in Example 7 of the present invention is measured;

[0024] Fig. 9 The siderophore production of Pseudomonas tehranensis HX144 in Example 8 of the present invention is determined;

[0025] Fig.10 The salt tolerance of Pseudomonas tehranensis HX144 in Example 9 of the present invention is determined;

[0026] Fig.11 The method is to determine the wheat growth-promoting ability of Pseudomonas tehranensis HX144 in Example 10 of the present invention;

[0027] Fig.12 This is a comparison chart of physiological indicators of the effect of the Pseudomonas tehranensis HX144 bacterial suspension on wheat growth in Example 11 of the present invention. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The pathogens used in the following implementation examples can be collected from the wild by the public, or obtained from the Institute of Agricultural Resources and Agricultural Zoning of the Chinese Academy of Agricultural Sciences to repeat the experiments of this application.

[0031] Quote strain explanation:

[0032] 1) Xanthomonas oryzae pv. oryzicola, which causes rice bacterial leaf streak disease (Ye Wenxin, Chang Zheng, Liu Qianqian, et al. Mutation analysis of the xrvA gene encoding a Xanthomonas virulence regulatory protein in Xanthomonas oryzae pv. oryzicola [J / OL]. Molecular Plant Breeding: 1-17 [2024-07-01]), hereinafter referred to as Xanthomonas oryzae pv. oryzicola.

[0033] 2) Ralstonia solanacearum, which causes tobacco bacterial wilt (isolated from tobacco rhizosphere soil in our laboratory), hereinafter referred to as Ralstonia solanacearum.

[0034] 3) Pectobacterium carotovorum (Su Z, Liu X, Guo Q, et al. Insights into complex infection by two Pectobacterium species causing potato blackleg and soft rot. Microbiological Research. 2022 Aug; 261: 127072. DOI: 10.1016 / j.micres.2022.127072. PMID: 35594651.), which causes carrot soft rot, hereinafter referred to as Pectobacterium carotovorum.

[0035] 4) Xanthomonas oryzae pv. oryzae that causes rice bacterial leaf blight (Yang Yayun, Zhang Dunyu, Chen Ling, Chen Yue, Yin Fuyou, Jiang Chunmiao, Xiao Suqin, Ke Xue, Yu Tengqiong, Wang Bo, Fu Jian, Zhong Qiaofang, Chen Gongyou, Cheng Zaiquan Identification of resistance of Yunnan medicinal wild rice to four major rice diseases. Acta Phytopathologica Sinica, 2019, 1: 101-112), hereinafter referred to as Xanthomonas oryzae pv. oryzae.

[0036] 5) Xanthomonas campestris pv. carnpestris that causes black rot of crucifers (Lan Nihan, Su Deshan, Zhou Le, Qin Xia, Huang Peifang, Yang Mei, Jiang Bole. Identification of small molecule compounds that inhibit the type III secretion system of black rot pathogen in host plants. Acta Phytopathologica Sinica, 2019, 49(02): 262-270), hereinafter referred to as black rot pathogen of crucifers.

[0037] 6) Staphylococcus aureus was purchased from China Agricultural Microbiological Culture Collection Center with strain number ACCC 01334, hereinafter referred to as Staphylococcus aureus.

[0038] 7) Pseudomonas syringaepv.tomato that causes bacterial leaf spot of tomato (Wang Ruona, Ma Yinan, Li Junzhou, et al. Analysis of the enzymatic function of HrpH, a component of the type III secretion system of Pseudomonas syringae [J]. Acta Phytopathologica Sinica, 2022, 52(03): 334-340.), hereinafter referred to as bacterial leaf spot pathogen of tomato.

[0039] 8) Salmonella sp. was purchased from China Agricultural Microbiological Culture Collection Center with strain number ACCC 01321, hereinafter referred to as Salmonella.

[0040] 9) Acidovorax avenae, which causes bacterial fruit blotch of melon (Lin Tao, Ma Guobin, Jiang Shouyang, et al. Screening of fungicides for controlling seed-borne pathogens of bacterial fruit blotch of melon. Shanghai Journal of Agriculture, 2023, 39(03): 55-60.), hereinafter referred to as Acidovorax avenae.

[0041] 10) Acidovorax citrulli, which causes bacterial fruit spot of watermelon (Xu Xiulan, Song Shunhua, Zhang Zhiyong, Wu Xuehong, Geng Lihua. Research progress on important seed-borne diseases of watermelon. Plant Protection, 2016, 42(01): 12-18), hereinafter referred to as Acidovorax citrulli.

[0042] 11) Clavibacter mic higanensis, which causes bacterial canker of tomatoes, hereinafter referred to as tomato bacterial canker pathogen.

[0043] The following is the method for preparing the culture medium used in the examples:

[0044] Luria-Bertani (LB) solid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 12 g agar, distilled water to 1000 mL, pH 7.0-7.2.

[0045] Luria-Bertani (LB) liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, distilled water to 1000 mL, pH 7.0-7.2.

[0046] King's B (KB) solid culture medium: 20 g of peptone No. 3, 0.4 g of MgSO4·7H2O, 10 mL of glycerol, 1.5 g of K2HPO4, 12 g of agar, and distilled water to make up to 1000 mL.

[0047] King's B (KB) liquid culture medium: 20 g of peptone No. 3, 0.4 g of MgSO4·7H2O, 10 mL of glycerol, 1.5 g of K2HPO4, and distilled water to make up to 1000 mL.

[0048] NutrientAgar (NA) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, 12 g agar, pH 7.0, distilled water to 1000 mL.

[0049] Nutrient Broth (NB) medium: 3 g beef extract, 5 g peptone, 2.5 g glucose, pH 7.0, distilled water to 1000 mL.

[0050] Trypticase SoyAgar (TSA) medium: 15 g of casein tryptic digest, 5 g of soy flour papain digest, 5 g of sodium chloride, 15 g of agar, distilled water to 1000 mL, pH 7.0-7.5.

[0051] Protease detection medium: peptone 10g, NaCl 5g, CaCl2 0.1 g, skim milk 100mL (add when the temperature of the culture medium drops to 45°C after sterilization), agar 12g, and distilled water to 1000mL.

[0052] Montgina organophosphorus medium: glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, FeSO4·7H2O 0.03 g, MnSO4·4H2O 0.03 g, egg yolk lecithin 0.2 g, CaCO3 5 g, yeast extract 0.4 g, agar 15 g, pH 7.0, distilled water to 1000 mL.

[0053] Inorganic phosphorus culture medium: 10g glucose, 0.5g (NH4)2SO4, 0.2g NaCl, 0.2g KCl, 5.0g Ca3(PO4)2, 0.03g MgSO4·7H2O, 0.03g MnSO4, 0.003g FeSO4, 0.5g yeast extract, 1.2% agar, pH 6.8-7.0, distilled water to 1000mL.

[0054] Peptone ammoniation medium: peptone 5g, K2HPO4 0.5 g, NaCl 0.25g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, distilled water to 1000mL. Chrome azurol S (CAS) blue qualitative detection medium:

[0055] 0.012 g of chrome azurol sulphonate (CAS) was dissolved in 10 mL of deionized water and mixed with 2 mL of 1 mmol / LFeCl3 solution to obtain solution a;

[0056] Take 0.015 g of hexadecyl trimethyl ammonium bromide (HDTMA) and dissolve it in 8 mL of deionized water to obtain solution b;

[0057] Slowly add liquid a into liquid b and mix thoroughly to obtain dye solution c;

[0058] 10 mL of 0.1 mol / L phosphate solution (2.427 g Na2HPO4·12H2O, 0.5905 g NaH2PO4·2H2O, 0.075 g KH2PO4, 0.125 g NaCl, 0.25 g NH4Cl, 100 mL of deionized water, diluted 10 times when used) and 6.04 g of piperazine diethanolsulfonic acid (pipes) were added into a clean conical flask containing 150 mL of distilled water, mixed well, and the pH was adjusted to 6.8 with 50% NaOH solution, and finally 4 g of agar powder was added to obtain medium d;

[0059] Sterilize dye solution c, culture medium d, 1mmol / L CaCl2, 1mmol / L MgSO4·7H2O, 20% glucose, and 10% casamino acids (115°C, 20 min). Measure 0.2mL of the above 1mmol / L CaCl2, 4mL of 1mmol / L MgSO4·7H2O, 6mL of 10% casamino acids, and 2mL of 20% glucose solution and add them to culture medium d. Then slowly add dye solution c and shake well to obtain a blue qualitative detection culture medium.

[0060] The present invention will be described in detail below with reference to embodiments.

[0061] Example 1: Isolation and identification of Pseudomonas tehranensis HX144

[0062] The Pseudomonas tehranensis HX144 described in the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC) in October 2024, with a deposit number of CGMCC No. 32292, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing (Institute of Microbiology, Chinese Academy of Sciences), with a zip code of 100101. P. tehranensis HX144 is an aerobic Gram-negative bacterium that can produce fluorescence on King's B medium. The 16SrRNA and rpoD gene sequences of Pseudomonas HX144 strains are shown in Seq ID No. 1 and Seq ID No. 2.

[0063] In the present invention, Pseudomonas tehranensis HX144 or metabolites of Pseudomonas tehranensis HX144 are P. tehranensis HX144 cells and substances secreted into the culture medium by the cells; the preparation method can be as follows: activating P. tehranensis HX144 on KB solid culture medium, picking fresh single colonies and inoculating them into KB liquid culture medium, shaking and culturing them at 28° C. and 200 rpm overnight; transferring them into LB liquid culture medium (1:1000), shaking and culturing them under the same culture conditions for 48 hours to obtain a fermentation broth.

[0064] The culture of P.tehranensis HX144 has at least one of the following functions B1-B20:

[0065] B1, inhibiting Ralstonia solanacearum;

[0066] B2, inhibiting the rice leaf streak pathogen Xanthomonas oryzae;

[0067] B3, inhibiting pectin bacteria;

[0068] B4. Inhibition of Xanthomonas oryzae bacteria:

[0069] B5, inhibiting Xanthomonas campestris;

[0070] B6, inhibit Staphylococcus aureus;

[0071] B7, inhibits tomato bacterial canker bacteria;

[0072] B8, inhibit bacterial leaf spot of tomatoes;

[0073] B9, inhibits Acidophilus avenae;

[0074] B10, inhibits acidophilus bacteria in watermelon;

[0075] B11, inhibiting Salmonella;

[0076] B12, hydrolyze organic phosphorus;

[0077] B13, hydrolyze inorganic phosphorus;

[0078] B14, produces protease;

[0079] B15, ammonia production;

[0080] B16, secretion of auxin (IAA);

[0081] B17, siderophore production

[0082] B18, promote the formation of plant lateral roots;

[0083] B19, increase plant biomass;

[0084] B20, salt-tolerant (can tolerate culture medium with a NaCl concentration of 6%).

[0085] Any of the following applications of the fermentation broth also falls within the protection scope of the present invention:

[0086] C1. Application of the fermentation broth in inhibiting Ralstonia solanacearum;

[0087] C2. Application of the fermentation liquid in inhibiting Xanthomonas oryzae, a pathogenic bacteria of rice;

[0088] C3. Application of the fermentation broth in inhibiting pectinobacterium;

[0089] C4. Application of the fermentation liquid in inhibiting the pathogenic bacteria of Xanthomonas oryzae:

[0090] C5. Application of the fermentation broth in inhibiting Xanthomonas campestris;

[0091] C6. Use of the fermentation broth in inhibiting Staphylococcus aureus;

[0092] C7. Application of the fermentation liquid in inhibiting tomato bacterial canker pathogens;

[0093] C8. Application of the fermentation liquid in inhibiting bacterial leaf spot of tomatoes;

[0094] C9. Application of the fermentation broth in inhibiting acidophilic bacteria of oats;

[0095] C10, application of the fermentation liquid in inhibiting acidophilic bacteria of watermelon;

[0096] C11. Application of the fermentation broth in inhibiting Salmonella;

[0097] C12. Application of the fermentation broth in hydrolyzing organic phosphorus;

[0098] C13. Application of the fermentation broth in hydrolyzing inorganic phosphorus;

[0099] C14, use of the fermentation broth in producing protease;

[0100] C15. Use of the fermentation broth in producing ammonia;

[0101] C16, application of the fermentation broth in secreting plant auxin (IAA);

[0102] C17, use of the fermentation broth in producing siderophore;

[0103] C18. Application of the fermentation liquid in promoting lateral root formation of plants;

[0104] C19. Application of the fermentation broth in increasing plant biomass.

[0105] C20. Use of the fermentation broth in a salt-tolerant culture medium (a culture medium that can tolerate a NaCl concentration of 6%).

[0106] Furthermore, in the present invention, the plant may be a monocotyledonous plant, the monocotyledonous plant may be a cereal plant, the cereal plant may be a plant of the genus Triticum, and the plant of the genus Triticum may be wheat.

[0107] 1. Isolation of strain HX144

[0108] Weigh 1g of soil sample, and repeat three times for each sample; add 10mL PBS solution and 2-3 2.4mm sterile stainless steel balls, vortex for 15-30s, and evenly take 1ml of sample in a 1.5ml centrifuge tube. Then centrifuge at 600rmp for 1min; the supernatant is the stock solution. Take 100uL of the stock solution and add it to a 1.5ml centrifuge tube containing 900uL PBS solution, blow and mix evenly, and make 10 -1 System solution; then take 100uL10 -1 Add the system solution into a 1.5ml centrifuge tube containing 900uL PBS solution, blow evenly to make 10 -2 System solution, repeat the above operation, and then get 10 -3 , 10 -4 System solution. 100uL of soil dilution was respectively applied to KB medium, incubated at 28°C for 2-3 days, and then the colonies with fluorescent morphology under the irradiation of UV analyzer were picked out, and the strain HX144 was obtained after purification.

[0109] 2. Identification of strain HX144

[0110] The strain HX144 was inoculated on LB solid medium and cultured at 28°C for 2 days. A trace amount of monoclonal colonies were picked as templates and 16S rRNA and rpoD genes were amplified using the 16S PCR amplification system and rpoD amplification system, respectively. Figure 1 shown.

[0111] 16S PCR reaction system: 30 μL

[0112]

[0113]

[0114] Total PCR reaction system 50 μL

[0115] PCR amplification procedure: step 1: pre-denaturation at 94°C for 5 min; step 2: denaturation at 94°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 1 min, 35 cycles; step 3: full extension at 72°C for 5 min; storage at 4°C.

[0116] rpoD reaction system: 50 μL

[0117]

[0118] Total PCR reaction system 50 μL

[0119] PCR amplification procedure: step 1: pre-denaturation at 94°C for 3 min; step 2: denaturation at 94°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 1.5 min, 34 cycles; step 3: full extension at 72°C for 5 min; storage at 4°C.

[0120] According to the comparison and analysis of 16S rRNA and rpoD (Seq ID No.1 and Seq ID No.2) of HX144 in EZBioCloud, the similarity between HX144 and Pseudomonas tehranensis reached 99.86%. Based on the comparison and analysis of 16S rRNA sequence (Seq ID No.1) and rpoD sequence (Seq ID No.2) of HX144 in EZBioCloud and the construction of phylogenetic tree, it was found that the strain had a similarity with Pseudomonas tehranensis. T They were clustered into one branch with a close relationship, so strain HX144 was identified as Pseudomonas tehranensis.

[0121] Example 2 Determination of the antibacterial spectrum of Pseudomonas hyaluroides HX144

[0122] The information of the tested pathogenic bacteria, pathogenic fungi and oomycetes is shown in Table 1. The antagonistic ability of strain HX144 against 4 pathogenic bacteria, 4 pathogenic fungi and oomycetes was tested.

[0123] Table 1 Information on pathogenic bacteria and fungi tested

[0124]

[0125] The antagonistic ability of strain HX144 against the above-mentioned pathogenic bacteria was detected by plate antagonism test. The experimental method is as follows: After melting the NA / LB / KB medium with an agar content of 1%, cool it to 45°C at room temperature. Add the pathogenic bacteria that have been shaken overnight in the NA / LB / KB liquid medium to the stable growth phase to the cooled NA / LB / KB medium at a ratio of 1:1000 and pour the plate. After drying, inoculate 10μL of strain S6330 culture solution (LB liquid shake culture) on the plate. After drying, invert the plate in a 28°C incubator for culture, use the plate inoculated with only pathogens as the control, and set up 3 replicates for each treatment. Observe and measure the diameter of the inhibition zone after 48 hours of culture.

[0126] The plate confrontation test was used to detect the antagonistic ability of strain HX144 against pathogenic fungi and oomycetes: a 0.7 cm diameter puncher was used to take the pathogenic fungi and oomycete cakes freshly cultured on the PDA plate at 25°C, and the cakes were picked up with an inoculation needle, and the mycelium was inoculated at the center of the fresh PDA plate with the mycelium facing down, and then 10 μL of the culture solution of strain S6330 (LB liquid shaking culture) was inoculated at a distance of about 2.5 cm from the cake. After drying, the plates were inverted and cultured in a 25°C incubator, and the plates inoculated with only pathogens were used as controls. Each treatment was repeated 3 times. After 5-7 days of culture, the width of the inhibition zone was observed and measured.

[0127] The results showed that strain HX144 had a good antagonistic effect on pathogenic bacteria such as Xanthomonas oryzae and Ralstonia solanacearum. Figure 2 As shown, the above results indicate that strain HX144 has a highly efficient and targeted resistance to plant pathogens and can be used in a targeted manner in the biological control of plant diseases.

[0128] Example 3 Pseudomonas tehranensis HX144 has the ability to produce protease

[0129] The activated strain HX144 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 200rpm. 10μl of the culture solution was spotted on the protease detection medium plate, and 10μL of LB liquid was spotted as the control treatment. Each treatment was repeated 3 times. After drying, it was inverted and cultured at 28°C for 2 days to observe whether a transparent circle appeared. The results showed that there was no change in the control plate, while the detection plate inoculated with the culture solution of the strain HX144 had an obvious hydrolysis transparent circle around the inoculated colony, as shown in the attached Figure 3 As shown, the protein in the plate was decomposed, proving that strain HX144 has the ability to produce protease.

[0130] Example 4: Determination of the ability of Pseudomonas tehranensis HX144 to hydrolyze inorganic phosphorus

[0131] The activated strain HX144 was inoculated into KB liquid culture medium and cultured overnight at 28°C and 220rpm. 10μL of culture solution was spotted onto the inorganic phosphorus culture medium plate, and 10μL of KB liquid culture medium was spotted as the control treatment. Each treatment was repeated 3 times. After drying, the plate was inverted and cultured in a 28°C incubator for 5 days to observe whether a hydrolysis halo appeared. The results showed that there was no change in the control plate, while an obvious hydrolysis halo appeared around the inoculated colony on the plate inoculated with the culture solution of strain P683, as shown in the attached Figure 4 As shown, it indicates that strain HX144 has the ability to hydrolyze inorganic phosphorus.

[0132] Example 5: Determination of the ability of Pseudomonas tehranensis HX144 to hydrolyze organic phosphorus

[0133] The activated strain HX144 was inoculated into KB liquid culture medium and cultured overnight at 28°C and 220rpm. 10μL of the culture solution was spotted on the Montkina organophosphate culture medium plate, and 10μL of KB liquid culture medium was spotted as the control treatment. Each treatment was repeated 3 times. After drying, it was inverted and cultured in a 28°C incubator for 5 days to observe whether a hydrolysis halo appeared. The results showed that there was no change in the control group plate, while the plate inoculated with the culture solution of the strain HX144 had an obvious hydrolysis halo around the inoculated colony, as shown in the attached Figure 5 As shown, it indicates that strain HX144 has the ability to hydrolyze organic phosphorus.

[0134] Example 6: Quantitative determination of the ability of Pseudomonas tehranensis HX144 to hydrolyze phosphorus

[0135] Drawing of phosphorus standard curve: Weigh potassium dihydrogen phosphate to make a 100mg / L phosphorus standard stock solution, and place it in a brown reagent bottle for use. Before the determination, take 5mL of phosphorus standard stock solution into a 100mL volumetric flask, and dilute it with distilled water to make a 5mg / L phosphorus standard working solution. Pipette 0, 1, 2, 3, 4, 5, and 6mL of phosphorus standard working solution into 7 50mL volumetric flasks, add distilled water to about 15-20mL, then add 1 drop of 2,4-dinitrophenol indicator, and then add sulfuric acid solution dropwise until the solution is nearly colorless, add 0.75mL of ascorbic acid solution, mix well, add 5mL of molybdate solution after 30s, dilute to 50mL with water, and mix well. After leaving it at room temperature for 30min, use a 10nm cuvette to compare the color at a wavelength of 700nm, and use deionized water as a reference to measure the absorbance. Use the reagent blank correction absorbance as the ordinate and the corresponding phosphorus concentration as the abscissa to draw the phosphorus standard curve, as shown in the attached figure. Figure 6 The phosphorus concentrations in this standard series are 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg / L.

[0136] Scrape one loop of bacterial moss from the preserved slant and culture it in LB liquid. After culturing at 30°C for 24 hours, use it as seed solution. Inoculate the seed solution into a conical flask (150 mL) containing 50 mL of PVK liquid culture medium at a ratio of 1% (v / v). Inoculate an equal volume of sterilized seed solution (replaced with LB liquid) in the control group. Culture the solution at 30°C and 150 r / min for 3 days with shaking. Then, centrifuge the culture solution at 8000 r / min for 10 min, and take the supernatant to determine the available phosphorus content.

[0137] After a series of operations according to the above method, it was observed that the color of the HX144 strain sample was significantly darker than that of the CK control group. Figure 7 As shown, the HX144 sample OD was measured. 700 The standard curve was used to determine that the dissolved effective phosphorus content of Pseudomonas tehranensis HX144 strain was as high as 195.75±0.5 mg / L, proving that the HX144 strain had a strong ability to solubilize phosphorus.

[0138] Example 7: Quantitative determination of ammonia production capacity of Pseudomonas tehranensis HX144. Freshly cultured Pseudomonas tehranensis HX144 was transferred to peptone ammoniation medium and cultured at 28°C and 200 rpm for 48 hours. 200 μL of culture solution was dropped onto a white ceramic plate, and the control was a peptone ammoniation medium without bacteria. Three drops of Nessler's reagent were added to the culture medium and peptone ammoniation medium. The presence of a yellow or brownish-red precipitate indicated that the strain had the ability to produce NH3. The results showed that after adding Nessler's reagent to the culture medium of Pseudomonas tehranensis HX144, an obvious brownish-red precipitate appeared, as shown in the attached Figure 8 As shown, Pseudomonas tehranensis HX144 has significant ammonia production ability.

[0139] Example 8: Pseudomonas tehranensis HX144 has the ability to produce IAA. The strain Pseudomonastehranensis HX144 was inoculated into LB liquid culture medium containing 100 mg / L tryptophan, and cultured at 28°C and 200 rpm for 2 days with shaking, followed by centrifugation at 8000 r / min for 2 min, the supernatant was discarded, and the bacterial suspension was resuspended with an equal amount of physiological saline. Next, 100 μL of the bacterial suspension was dropped onto a white ceramic plate, and 100 μL of Salkowski colorimetric solution was added at room temperature and protected from light for 30 min before observation. If the color turns red, it means that IAA can be secreted, and the darker the color, the stronger the IAA production ability; if it does not change color, it means that IAA cannot be secreted. The strain Pseudomonas tehranensis HX144 turns pink in the dark, indicating that Pseudomonas tehranensisHX144 has the ability to secrete IAA, thereby promoting the germination of plant seeds and the growth of seedlings, as shown in the attached Fig. 9 shown.

[0140] Example 9: Pseudomonas tehranensis HX144 has the ability to produce siderophore

[0141] The activated strain S6330 was inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm. 10 μL of the culture solution was spotted on a CAS blue qualitative detection culture medium plate and cultured inverted in a 28°C incubator for 3 days to observe whether a yellow halo is produced around the colony. Since the siderophore competes for the iron ions chelated by EDTA in the culture medium, the culture medium changes from blue to yellow, so the appearance of a yellow halo around the colony indicates the production of siderophore. The results showed that a yellow halo appeared around the colony of strain S6330, proving that strain HX144 has the ability to produce siderophore, as shown in the attached Fig.10 shown.

[0142] Example 10: Determination of salt tolerance of Pseudomonas tehranensis HX144

[0143] Prepare LB liquid culture medium with 2%, 4%, 6%, 8%, and 10% Nacl concentration in advance, and use LB culture medium with 1% Nacl concentration as a control; pick a single colony of HX144 and inoculate it in KB culture medium, culture it at 28°C and 200rpm for 24 hours, then transfer the HX144 single colony to LB culture medium with different salt concentrations according to the 1% inoculation amount, and continue to culture it at 28°C and 200rpm for 24 hours. Take out 1ml from the culture bottle every 12 hours to measure its growth. As can be seen from the figure, the HX144 strain has a strong salt tolerance. HX144 grows better at a concentration of 4% Nacl than at a concentration of 2%, and has the potential to be a halophilic bacterium. At a concentration of 6% Nacl, HX144 can grow steadily with the increase of days. The strain HX144 can tolerate and grow in a high-concentration salt environment, such as the attached Fig.11 shown.

[0144] Example 11: Pseudomonas tehranensis HX144 significantly promotes the growth of wheat

[0145] Mix nutrient soil, vermiculite and natural soil (1:1:1) evenly and add them into eight rows of flower pots. Soak them with water, sow about 4 wheat seeds in each hole, cover them with a small amount of soil, and place them in a greenhouse with a temperature of 25°C and a humidity of 65% to 75%. After about a week, the wheat will grow, and wheat seedlings with consistent growth will be selected for growth promotion experiments.

[0146] Growth promotion experiment: A single colony of HX144 was selected and inoculated into KB medium. After culturing at 28°C and 200 rpm for 24 h, it was transferred to 50 mL KB medium at a 1% inoculum and continued to be cultured at 28°C and 200 rpm for 24 h. After the culture was completed, HX144 was prepared with distilled water to a concentration of 1×10 8 CFU·mL -1 The HX144 bacterial suspension was then poured into the roots of wheat with 10 mL of the HX144 bacterial suspension. Each treatment had 40 plants and was repeated 3 times. The distilled water for root irrigation was used as the control. The growth was observed and recorded every day. On the 21st day after root irrigation, the fresh weight, root length, and plant height of the wheat were measured. The wheat treated with the HX144 bacterial suspension grew vigorously, with an average fresh weight of 0.42±0.05g, which was significantly higher than the fresh weight of the control group by 1.31 times. The average plant height was 30.73±0.5cm, which was significantly higher than the plant height of the control group by 1.41 times. The average root length was 24.32±0.5g, which was significantly higher than the root length of the control group by 1.83 times. Fig.12 shown.

[0147] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A Tehran Pseudomonas HX144, characterized in that: The Pseudomonas Tehranensis HX144 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration in October 2024, and its deposit number is: CGMCC NO.32292. The 16S rRNA and rpoD gene sequences of the Pseudomonas Tehranensis HX144 strain are shown in SEQ ID No.1 and SEQ ID No.

2.

2. A method for culturing Pseudomonas tehranensis HX144 as claimed in claim 1, characterized in that: The method comprises the following steps: activating strain HX144 on KB solid culture medium; picking a fresh single colony and inoculating it into KB liquid culture medium, shaking and culturing it overnight at 28°C and 200rpm; transferring it into LB liquid culture medium with a dilution of 1:1000, shaking and culturing it for 48h under the same culture conditions to obtain fermentation liquid.

3. A method for preparing the metabolite of Pseudomonas tehranensis HX144 as claimed in claim 1, characterized in that: The method comprises the following steps: inoculating strain HX144 into KB liquid culture medium, and shaking culture overnight at 28°C and 200rpm; transferring the culture into LB liquid culture medium, and continuing to shake culture for 48h under the same culture conditions; and obtaining a fermentation broth containing metabolites of strain HX144.

4. The use of Pseudomonas tehranensis HX144 or its metabolites prepared as claimed in claim 2 or 3, characterized in that: The strain has a good inhibitory effect on plant pathogenic bacteria and human and / or animal pathogenic bacteria Salmonella and Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The plant pathogenic bacteria include but are not limited to tomato bacterial wilt pathogen Ralstonia solanacearum, tomato bacterial canker pathogen Bacillus michiganensis, rice white leaf blight pathogen Xanthomonas oryzae, rice streak pathogen Xanthomonas oryzae, crucifer black rot pathogen Xanthomonas campestris, tomato bacterial leaf spot pathogen Pseudomonas syringae, Acidophilus avenae, Acidophilus watermelon, and Pectobacterium carrotosum.

6. A use of Pseudomonas tehranensis HX144 or its metabolites prepared as claimed in claim 2 or 3, characterized in that: The strain or its metabolites are used for: inhibiting plant pathogenic bacteria; hydrolyzing organic phosphorus and inorganic phosphorus; producing protease, ammonia, plant growth hormone and siderophore; promoting plant growth and increasing plant biomass; and improving plant salt tolerance.

7. A microbial preparation using Pseudomonas tehranensis HX144, characterized in that: The preparation comprises the strain HX144 or a metabolite thereof according to claim 1.

8. An application of Pseudomonas tehranensis HX144 in agriculture, characterized in that: The strain is used for preparing the microbial fertilizer or biological preparation containing the microbial fertilizer or biological preparation according to claim 7.

9. Use of Pseudomonas tehranensis X144 or its metabolites prepared as claimed in claim 2 or 3 in promoting wheat growth, characterized in that: The strain or its metabolite is used for root irrigation of wheat, thus significantly improving the growth index of wheat.

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