Shigella-like bacterium in the rhizosphere of wheat and application thereof

Abstract

The application discloses a Schumannella sp. M29 and application thereof. The Schumannella sp. M29 is isolated from the rhizosphere soil of winter wheat in Moyu County, Hetian region, Xinjiang, and the strain can tolerate 3% NaCl, has the activities of dissolving insoluble inorganic phosphorus and organic phosphorus, and has urease and weak cellulose degradation activity. The application of the Schumannella sp. M29 to the preparation of a plant growth-promoting phosphorus-dissolving bacterial agent can not only reduce the use amount of chemical fertilizers, but also promote the growth of plants, and has a wide development and application prospect in agricultural biological fertilizers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel rhizosphere strain of *Schumannii* from wheat, its isolation method, and its application. Background Technology

[0002] Phosphorus is one of the three essential nutrients for plant growth, and phosphorus in the soil mainly exists in a poorly soluble form. For farmland ecosystems, applying phosphate fertilizer to the soil is the main way to supply phosphorus. However, existing studies have shown that the utilization rate of phosphate fertilizer after it is applied to the soil is only 10-25% in the current season, with the majority of the remainder forming poorly soluble phosphorus compounds such as calcium phosphate, iron phosphate, and aluminum phosphate.

[0003] Soil microorganisms drive the cycling of various elements in the soil. For example, some microorganisms can convert insoluble or fixed phosphorus into a form that plants can absorb and utilize; these microorganisms are called phosphate-solubilizing microorganisms (PSMs). Compared to the root zone and bare soil, the rhizosphere of crops typically contains a large number of phosphate-solubilizing microorganisms. Utilizing their unique ecological functions, several phosphate-solubilizing bacterial groups have been isolated from the soil, including *Bacillus*, *Burkholderia*, *Enterobacter*, and *Erwinia*. Some strains have been developed into growth-promoting phosphate-solubilizing agents and successfully applied in agricultural production.

[0004] Currently, although there are already several microbial strains with phosphorus-solubilizing functions in existing technologies, isolating and screening new, highly active plant rhizosphere phosphorus-solubilizing bacteria and studying their phosphorus-solubilizing mechanisms and growth-promoting effects on plants are of great significance for reducing fertilizer use and ensuring the green and sustainable development of agriculture. Summary of the Invention

[0005] In view of the current situation regarding low phosphate fertilizer utilization, scarcity of highly active rhizosphere phosphate-solubilizing bacteria, and lack of literature and patent reports on the application of Schumannella sp. in the preparation of growth-promoting phosphate-solubilizing agents, this invention aims to provide a novel Schumannella sp. M29 and its applications. This invention isolated a novel Schumannella sp. M29 from the rhizosphere soil of winter wheat in Moyu County, Hotan Prefecture, Xinjiang. This bacterium can tolerate 3% NaCl, possesses activity in dissolving insoluble inorganic and organic phosphorus, and exhibits urease and weak cellulose-degrading activity. This new strain, Schumannella sp. M29, and its growth-promoting phosphate-solubilizing agent have broad development and application prospects in plant growth promotion. The Schumannella sp. M29 growth-promoting phosphate-solubilizing agent can not only reduce the amount of chemical fertilizer used but also promote plant growth, showing broad development and application prospects in agricultural bio-fertilizers.

[0006] To achieve the above technical effects, the present invention is implemented through the following technical solution.

[0007] A growth-promoting phosphate-solubilizing agent of a new strain of Schumannella sp. M29, which is prepared by fermentation of the new strain of Schumannella sp. M29.

[0008] The novel *Schumannella* sp. M29 provided by this invention was isolated from the rhizosphere soil of winter wheat in Moyu County, Hotan Prefecture, Xinjiang. Phylogenetic and morphological analysis of its 16S rRNA sequence confirmed that strain M29 belongs to the *Schumannella* genus. Gene sequencing of this strain and BLAST alignment analysis on the NCBI website showed that the 16S rRNA gene sequence of strain M29 exhibited the highest homology (96.6%) with *Schumannella luteola KHIA* (AB362159). Phylogenetic trees of 16S rRNA were constructed using different methods based on highly homologous sequences. Strain M29 showed the closest phylogenetic relationship to *Schumannella luteola KHIA* (AB362159), with confidence levels of 91%, 73%, and 70%, respectively. Serial polymorphic classification identification confirmed that strain M29 is a novel *Schumannella* sp. species, exhibiting typical characteristics of a new species.

[0009] The Schumannella sp. M29 provided by this invention has undergone molecular-level identification using well-known and recognized strain systems in the art. Combined with morphological identification and physiological and biochemical characteristic multiphase classification system identification and analysis, the Schumannella sp. M29 involved in this invention differs from the standard type strain of the genus with the closest homology in several aspects. This confirms that Schumannella sp. M29 belongs to a new species of the genus Schumannella. It has been named Schumannella sp. M29 and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24257 and deposit date of January 6, 2022.

[0010] The gene sequence of the above-mentioned new Schumannella sp. M29 is shown in SEQ ID NO:1.

[0011] In this invention, the isolation and activation medium for the new strain of Schumannella sp. M29 is as follows (g / L): glucose 0.5, yeast extract 0.5, peptone 0.5, acid-hydrolyzed casein 0.5, soluble starch 0.5, sodium pyruvate 0.3, dipotassium hydrogen phosphate 0.3, magnesium sulfate 0.05, agar 1, and distilled water to a final volume of 1000 mL, pH 7.2.

[0012] In this invention, the purification culture medium for the new Schumannella sp. M29 is as follows: glucose 10.0, ammonium sulfate 0.5, yeast extract 0.5, NaCl 0.3, KCl 0.3, magnesium sulfate 0.3, ferrous sulfate 0.03, manganese sulfate 0.03, calcium phosphate 5.0, pH 7.0-7.5, and agar 15.0.

[0013] Meanwhile, this invention provides a method for preparing a Schumannella sp. M29 growth-promoting phosphate-solubilizing agent, specifically including the following steps: Inoculating a single colony of Schumannella sp. M29 into NB medium and culturing it at 30°C and 180 rpm for 2 days to obtain a Schumannella sp. M29 seed culture; Inoculating the Schumannella sp. M29 seed culture into NB liquid medium at a volume ratio of 1% and fermenting it at 30°C and 180 rpm for 3 days to prepare the Schumannella sp. M29 growth-promoting phosphate-solubilizing agent.

[0014] Furthermore, this invention provides an application of a novel Schumannella sp. M29 strain in plant growth promotion, which can promote plant growth and has broad development and application prospects in plant growth promotion.

[0015] Meanwhile, this invention provides an application of a new Schumannella sp. M29 growth-promoting phosphate-solubilizing agent in plant growth promotion, which can promote plant growth and has broad development and application prospects in plant growth promotion.

[0016] Through the above technical solutions, the present invention achieves the following technical effects:

[0017] (1) This invention provides a new strain of Schumannella sp. M29. After molecular-level identification by a well-known and recognized strain system in the field, combined with identification and analysis based on morphological identification, physiological and biochemical characteristics, chemical characteristics and other multi-phase classification system, Schumannella sp. M29 has many differences from the standard type strain of the genus with the closest homology. It is indeed a new strain of Schumannella. It is confirmed that the strain number M29 obtained in the Schumannella category is a typical new strain. Therefore, it is necessary to preserve it in accordance with legal requirements.

[0018] (2) This invention provides a new strain of Schumannella sp. M29 that can tolerate 3% NaCl and has the activity of dissolving insoluble organic and inorganic phosphorus. It can dissolve various insoluble phosphorus compounds such as calcium phytate and calcium phosphate in the soil. It can be used to prepare plant growth-promoting phosphorus-solubilizing bacteria agents and has broad development and application prospects. The Schumannella sp. M29 growth-promoting phosphorus-solubilizing bacteria agent can not only reduce the amount of chemical fertilizers used, but also promote plant growth. It has broad development and application prospects in agricultural bio-fertilizers. Attached Figure Description

[0019] Figure 1 The diagram shows a phylogenetic tree of strain M29 constructed using the neighbor-joining method based on the 16S rRNA gene sequence.

[0020] Figure 2 The diagram shows a phylogenetic tree of strain M29 constructed using the maximum parsimony method based on the 16S rRNA gene sequence.

[0021] Figure 3 The diagram shows a phylogenetic tree of strain M29 constructed using the maximum likelihood method based on the 16S rRNA gene sequence.

[0022] Figure 4 The image shows the morphological characteristics of strain M29. Detailed Implementation

[0023] The present invention will now be illustrated with examples; however, the present invention is not limited to the examples described below. All raw and auxiliary materials used in the present invention, as well as the selected microbial culture methods, are well known in the art. All percentages mentioned in the present invention are volume percentages unless otherwise specified.

[0024] The NB culture medium used in the preparation of the Schumannella sp. M29 growth-promoting phosphate-solubilizing agent of this invention is (g / L): peptone 10, beef extract powder 3, sodium chloride 5, deionized water 1L, pH value 7.4±0.2.

[0025] Example 1: Isolation, purification and identification of Schumannella sp. M29

[0026] (I) Separation and purification

[0027] Soil samples were collected from the rhizosphere of winter wheat in Hotan Prefecture, Xinjiang Uygur Autonomous Region, using the plate dilution method. The samples were sealed in plastic bags and brought back to the laboratory for storage at 4℃ until use. 2g of soil sample was accurately weighed, and 18mL of sterile water was added. The mixture was shaken at 30℃ and 180rpm for 30min. The concentration was then diluted to 10 with sterile water. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 For gradient culture, 200 μL of sample suspension was placed on bacterial inorganic phosphorus medium plates and incubated at 30°C for 7 days. Single colonies were picked and streaked on the same medium for purification. The resulting pure cultures were then frozen and stored in 20% skim milk powder.

[0028] (II) Identification of 16S rRNA gene

[0029] 1. DNA extraction

[0030] Schumannella sp. M29 was inoculated into purified liquid culture medium and cultured at 30°C for 3 days to obtain bacterial cells. Genomic DNA was extracted using a rapid bacterial genomic DNA extraction kit.

[0031] 2. PCR amplification

[0032] 16S rRNA sequence primers:

[0033] 27F: 5′-AGA GTT TGATCC TGG CTC-3′;

[0034] 1492R: 5′-CGG CTACCT TGT TAC GAC TT-3′.

[0035] Reaction system and conditions:

[0036]

[0037] 3. Sequencing

[0038] The PCR amplification product was sequenced after electrophoresis and purification. The sequence length was 1514 bp, and the sequencing result is shown in SEQ ID No: 1. A BLAST homology search was performed using NCBI, and a phylogenetic tree was constructed using the Neighbor-Joining method (1000 replicates) with MEGA 7.0 software, a commonly used tool in this field. The results are shown in the appendix. Figure 1-3 As shown, the obtained sequences were compared and analyzed on the NCBI website. The results showed that the 16S rRNA gene sequence of strain M29 had the highest homology with *Schumannella luteola* KHIA (AB362159), with a similarity of 96.6% (see Table 1 for details). In phylogenetic trees constructed using different methods based on the 16S rRNA gene sequence, the 16S rRNA sequence of strain M29 was most closely related to *Schumannella luteola* KHIA (AB362159). Strain M29 clustered with *S. luteola* KHIA and formed an independent branch, with confidence levels of 91%, 73%, and 70%, respectively. This indicates that strain M29 has extremely high support as a new species and exhibits excellent stability in the phylogenetic tree. Through a comprehensive assessment of species similarity and homology, the obtained species number M29 within the *Schumannella* genus is confirmed to be a typical new species of *Schumannella*.

[0039] Table 1: Homology of the strains of this invention

[0040]

[0041]

[0042] Example 2: Classification and identification of Schumannella sp. M29

[0043] (I) Colony morphology characteristics of Schumannella sp. M29

[0044] The strain M29 to be observed was inoculated onto NB plates and incubated at 30°C for 4 days. Once the colonies had fully grown onto the plate, their characteristics were observed, recorded, and photographed. Scanning electron micrographs of the colonies were also recorded. The results are shown in the appendix. Figure 4 As shown.

[0045] From the appendix Figure 4 The results showed that strain M29, when cultured in NB medium at 30℃ for 4 days, produced colonies with a diameter of about 1 mm, neat edges, a raised center, and a yellow color. The cells were rod-shaped, with a diameter of about 0.3 μm and a length of about 0.8-0.9 μm.

[0046] Based on the above biological characteristics, strain M29 was identified as *Schumannella* sp. This strain is deposited at the Budapest Treaty International Collection Unit for Microbial Cultures: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, postcode: 100101, deposit date: January 6, 2022, accession number: CGMCC No. 24257.

[0047] (II) Physiological and biochemical characteristics of Schumannella sp. M29

[0048] Strain M29 was inoculated onto NB medium, and its physiological and biochemical characteristics were tested. Analysis of characteristics such as growth temperature, salinity, pH range, gelatin liquefaction, nitrate reduction, hydrogen peroxide production, urease, amylase, esterase, cellulose decomposition, and carbon source utilization was primarily based on the "Handbook of Systematic Identification of Common Bacteria".

[0049] Strain M29 grows well on TSA and NB differential media, but poorly on NA and R2A differential media. It can grow on media containing 0-3% NaCl, with an optimal concentration of 0%; it can grow at 25-40℃, with an optimal growth temperature of 30℃; it can grow at pH 6-9, with an optimal pH of 7.0. It is positive for milk coagulation, negative for gelatin liquefaction, and negative for milk peptonization; it is urease positive, positive for Tween 20 and 80 hydrolysis, and has weak starch hydrolytic activity; it does not degrade cellulose; it can utilize glucose, sucrose, lactose, raffinose, D-xylose, rhamnose, acetic acid, melibiose, mannitol, galactose, succinate, oxalic acid, malic acid, sorbitol, and ribose, but cannot utilize mannose, cellobiose, arabinose, succinic acid, citric acid; maltose, trehalose, cellobiose, lactose, mannose, glycerol, aspartic acid, glutamic acid, and histidine. It can utilize cysteine, glutamic acid, glutamine, L-tryptophan, L-aspartic acid, xanthine, hydroxyproline, glycine, adenine, L-arginine, L-tryptophan, histidine, leucine, lysine, and L-alanine.

[0050] Using the plate staining method, strain M29 was spotted onto a phosphorus bacteria culture medium containing insoluble lecithin or calcium phosphate. After incubation at 30°C for 3 days, a clear zone was observed around the colony, indicating that strain M29 has the activity of dissolving insoluble organic and inorganic phosphorus.

[0051] Therefore, strain M29 has similar physiological and biochemical characteristics to the closest homologous strain in the genus, Schumannella luteola KHIA (AB362159), but there are several differences, making it a potential new species of Schumannella.

[0052] Based on the comprehensive analysis of 16S rRNA gene sequence homology, morphological identification, and physiological and biochemical characteristics, it is clearly different from the standard type strain with the closest homology. This strain M29 is identified as a new species of Schumannella and named Schumannella sp. M29 according to its isolation source.

[0053] Example 3: Preparation of Schumannella sp. M29 growth-promoting phosphate-solubilizing agent

[0054] Based on Examples 1-2, this embodiment describes a method for preparing a phosphate-solubilizing agent of *Schumannella sp.* M29, specifically including the following steps: A single colony of *Schumannella sp.* M29 is inoculated into NB medium and cultured at 30°C and 180 rpm for 2 days to obtain a seed culture of *Schumannella sp.* M29; the seed culture of *Schumannella sp.* M29 is then inoculated into NB liquid medium at a volume ratio of 1% and fermented at 30°C and 180 rpm for 3 days to prepare the phosphate-solubilizing agent of *Schumannella sp.* M29.

[0055] Example 4: Application of Schumannella sp. M29

[0056] Following the method described in standard NY / T 1847-2010, M29 bacteria were inoculated into Erlenmeyer flasks containing 10.0 g / L calcium phosphate, aluminum phosphate, ferric phosphate, lecithin, or calcium phytate as phosphate-soluble bacterial liquid culture medium, and incubated at 30°C and 200 rpm for 72 h on a shaker. After incubation, the available phosphorus content in the supernatant was determined, and the specific test results are detailed in Table 2. The phosphate-soluble liquid culture medium consisted of: glucose 0.5 g, yeast extract 0.5 g, peptone 0.5 g, acid-hydrolyzed casein 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g, dipotassium hydrogen phosphate 0.3 g, magnesium sulfate 0.05 g, agar 15 g, and distilled water to a final volume of 1000 mL, pH 7.2.

[0057] Table 2: Phosphate-solubilizing activity of strain M29

[0058]

[0059] As shown in Table 2, the strain M29 provided by this invention exhibits a solubility of 7.37 mg / L for calcium phosphate, 4.03 mg / L for aluminum phosphate, and 7.92 mg / L for ferric phosphate; a solubility of 29.04 mg / L for calcium phytate and 0.52 mg / L for lecithin. This indicates that the strain M29 provided by this invention has significant activity in dissolving insoluble organic and inorganic phosphorus compounds.

[0060] Strain M29 was inoculated into activation medium and cultured in shake flasks at 30°C for 2 days to obtain activated bacterial suspension of *Schumannii* M29 from the wheat rhizosphere. Phosphate-solubilizing liquid medium was prepared. The *Schumannii* M29 bacterial suspension was inoculated into the phosphate-solubilizing liquid medium at a ratio of 1:100 and cultured at 30°C and 180 rpm for 3 days. The phosphate-solubilizing ability of the phosphate-promoting bacterial agent was determined using the molybdenum-antimony colorimetric method. The M29 phosphate-solubilizing bacterial agent was inoculated into Erlenmeyer flasks containing 10.0 g / L calcium phosphate, aluminum phosphate, ferric phosphate, lecithin, or calcium phytate, respectively, and cultured on a shaker at 200 rpm and 30°C for 72 hours. After the incubation, the available phosphorus content in the supernatant was measured. Specific results are shown in Appendix Table 3. The phosphate-solubilizing liquid culture medium consisted of: 0.5g glucose, 0.5g yeast extract, 0.5g peptone, 0.5g acid-hydrolyzed casein, 0.5g soluble starch, 0.3g sodium pyruvate, 0.3g dipotassium hydrogen phosphate, 0.05g magnesium sulfate, 15g agar, and distilled water to a final volume of 1000mL, with a pH of 7.2.

[0061] Table 3: Phosphate-solubilizing activity of the phosphate-solubilizing agent of strain M29

[0062]

[0063] As shown in Table 3, the growth-promoting phosphate-solubilizing agent of strain M29 provided by this invention has a solubility of 7.59 mg / L for calcium phosphate, 4.27 mg / L for aluminum phosphate, and 8.16 mg / L for ferric phosphate; a solubility of 31.21 mg / L for calcium phytate and 0.65 mg / L for lecithin. This indicates that the growth-promoting phosphate-solubilizing agent of strain M29 provided by this invention has significant activity in dissolving insoluble organic and inorganic phosphorus.

[0064] The above data analysis shows that the new Schumannella sp. M29 provided by this invention can tolerate 3% NaCl and dissolve insoluble phosphorus in the soil, especially organic phosphorus phytate calcium. It can increase the content of soluble phosphorus in the soil to a certain extent, promote the absorption and utilization of phosphorus by plants, thereby promoting plant growth and reducing the amount of phosphate fertilizer used. Its application in the preparation of growth-promoting phosphorus-solubilizing bacteria can not only reduce the amount of chemical fertilizer used, but also promote plant growth, which has good ecological and social benefits and has broad development and application prospects in agricultural bio-fertilizers.

Claims

1. A strain of Schumannellasp. M29, characterized in that, The Schumannellasp. M29 strain is deposited at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 24257.

2. A phosphate-solubilizing agent for Schumannellasp. M29, characterized in that, This growth-promoting phosphate-solubilizing agent was obtained by fermentation of Schumannellasp. M29.

3. A method for preparing the Schumannellasp. M29 growth-promoting phosphate-solubilizing agent as described in claim 2, characterized in that, The preparation method includes the following steps: a single colony of strain Schumannellasp. M29 is inoculated into NB medium and cultured in a shake flask at 30°C for 2 days to obtain Schumannellasp. M29 seed culture; the Schumannellasp. M29 seed culture is inoculated into NB liquid medium at an inoculation rate of 1% by volume and fermented at 30°C and 180 rpm for 3 days to obtain Schumannellasp. M29 growth-promoting phosphate-solubilizing agent.

4. The application of Schumannellasp. M29 as described in claim 1 in increasing the soluble phosphorus content in soil.

5. The application of the Schumannellasp. M29 growth-promoting phosphate-solubilizing agent as described in claim 2 in the activity of dissolving organic and inorganic phosphorus.

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