Serratia marcescens strain ZNL1 and application thereof

Abstract

The invention belongs to the technical field of agricultural microorganisms, and discloses a serratia marcescens strain ZNL1 as well as a microbial agent, a preparation method and application of the serratia marcescens strain ZNL1. The strain ZNL1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.37364. The strain can efficiently decompose organic nitrogen and inorganic phosphorus, dissolve potassium and zinc and secrete siderophores, has remarkable drought resistance, and can promote plant growth by generating plant hormones, promoting nutrient element absorption and the like. The invention also provides a liquid or solid inoculant containing the strain and a preparation method thereof, and the inoculant can be used for activating soil nutrients, promoting plant growth and improving drought resistance, is especially suitable for cultivation of crops such as fruit trees and the like, and has a wide agricultural application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a Serratia marcescens strain ZNL1 and its applications. Background Technology

[0002] With the development of agricultural modernization, the unreasonable application of chemical fertilizers has led to increasingly prominent problems such as soil fertility decline, nutrient imbalance, and ecological damage. As a green and environmentally friendly agricultural input, microbial preparations can activate nutrients in the soil that are difficult to utilize through the metabolic activities of bacterial strains, promote plant nutrient absorption, and enhance plant stress resistance, playing an important role in improving soil and increasing crop yield and quality.

[0003] However, existing microbial agents still have significant limitations: on the one hand, although some strains of *Serratia* have been shown to have functions such as biological nitrogen fixation, phosphorus solubilization, and secretion of plant hormones, related studies have mostly focused on single functions, lacking a systematic exploration of the multifunctional integration of strains, resulting in single-function and weak targeting in practical applications; on the other hand, compound microbial agents often suffer from reduced efficacy due to incompatibility or antagonism between strains, while general-purpose microbial agents have poor adaptability to the rhizosphere microenvironment of specific crops (such as fruit trees), low colonization efficiency, and difficulty in maintaining stable effects under stress environments such as drought. Therefore, screening and isolating multifunctional, highly active indigenous *Serratia* strains derived from specific habitats, and developing synergistic microbial agents, is of great significance for promoting the development of green agriculture. Summary of the Invention

[0004] The present invention aims to provide a multifunctional Serratia marcescens strain ZNL1, its microbial agent, preparation method and application, in order to solve the problems of single function and weak stress resistance of existing microbial agents, and to achieve the synergistic effect of soil nutrient activation, plant growth promotion and stress resistance enhancement.

[0005] To achieve the above-mentioned technical objectives and effects, the present invention provides the following technical solution:

[0006] A strain of Serratia marcescens, ZNL1, was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37364.

[0007] On the other hand, the present invention also provides a microbial agent of Serratia marcescens strain ZNL1, wherein the agent contains the aforementioned Serratia marcescens strain ZNL1.

[0008] Furthermore, the microbial agent is a liquid microbial agent or a solid microbial agent containing the strain or its fermentation products.

[0009] Furthermore, the solid microbial agent is prepared by mixing and drying a fermentation broth containing the strain ZNL1 with a carrier, wherein the carrier is selected from peat moss, vermiculite, diatomaceous earth or a combination thereof.

[0010] On the other hand, the present invention also provides a method for preparing a Serratia marcescens strain ZNL1 microbial inoculant, comprising the following steps:

[0011] (1) The above-mentioned Serratia strain ZNL1 was inoculated into liquid culture medium for fermentation culture;

[0012] (2) When the number of viable bacteria in the fermentation broth reaches 1×10 9 When the concentration of CFU / mL reaches a certain level, fermentation is terminated to obtain a fermentation broth containing the strain ZNL1.

[0013] Furthermore, it also includes the step of mixing the fermentation broth obtained in step (2) with the carrier and drying it to obtain a solid microbial agent.

[0014] On the other hand, the present invention also provides the application of the above-mentioned Serratia serratia strain ZNL1 in the preparation of microbial preparations for activating soil nutrients.

[0015] Furthermore, the activated soil nutrients include at least one of the following: organic nitrogen solubilizer, inorganic phosphorus solubilizer, potassium solubilizer, zinc solubilizer, and secreted iron carrier.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] 1. Strain ZNL1 has comprehensive functions, combining soil nutrient activation and plant stress resistance and growth promotion, thus solving the problem of the limitations of single-function strains.

[0018] 2. It exhibits outstanding zinc-solubilizing ability, which is 22 times that of the blank control. Its ability to secrete siderophores, solubilize potassium, and resist drought are significantly better than most control strains. Its organic nitrogen solubilizing halo index is ≥2.0, and its application effect is significant.

[0019] 3. The microbial agent preparation process is simple and low-cost, and it is available in both liquid and solid forms to suit different agricultural application scenarios, making it convenient to use.

[0020] 4. The strain originates from the rhizosphere soil of fruit trees, is highly adaptable and environmentally friendly, and can be widely used in agricultural production, contributing to green and sustainable planting. Attached Figure Description

[0021] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a functional diagram of different strains in the embodiment of the present invention for degrading organic nitrogen;

[0023] Figure 2 The halo index of organic nitrogen solubility by different strains in the embodiments of the present invention;

[0024] Figure 3 This is a comparison chart of the phosphate-solubilizing abilities of different strains in the embodiments of the present invention;

[0025] Figure 4 This is a comparison chart of the potassium-solubilizing abilities of different strains in the embodiments of the present invention;

[0026] Figure 5 This is a comparison diagram of the siderophore secretion capabilities of different strains in the embodiments of the present invention;

[0027] Figure 6 The halo index represents the ability of different strains to secrete siderophores in the embodiments of the present invention.

[0028] Figure 7 This is a comparison diagram of the drought resistance of different strains in the embodiments of the present invention;

[0029] Figure 8 This is a comparison chart of the zinc-solubility of different strains in the embodiments of the present invention;

[0030] Figure 9 This is a Gram staining photograph of strain ZNL1 in an embodiment of the present invention;

[0031] Figure 10 These are colony morphology photographs of strain ZNL1 in this embodiment of the invention;

[0032] Figure 11 Phylogenetic analysis of the 16S rRNA gene of strain ZNL1 in this embodiment of the invention;

[0033] Figure 12 Phylogenetic analysis of the gyrB gene of strain ZNL1 in this embodiment of the invention;

[0034] Figure 13 This is a fatty acid profile of strain ZNL1 in an embodiment of the present invention; Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The invention will now be further described with reference to the accompanying drawings.

[0037] 1. Isolation and Validation of Multifunctionality of Strain ZNL1

[0038] 1.1 Strains Isolation

[0039] Sample source: isolated from the rhizosphere soil of healthy plum trees in the main production area of ​​honey plum in Liuma Town, Zhenning County, Guizhou Province.

[0040] Isolation method: The bacteria were spread on TSA medium using the standard dilution plating method and incubated at 28°C for 48 hours. Single colonies were then picked and purified.

[0041] Preservation: The purified strain was named ZNL1 and stored in glycerol at -80°C.

[0042] 1.2 Culture medium

[0043] 10% TSA medium: TSB 3g, agar 5g, H2O to a final volume of 1L. Autoclave at 121℃ for 20 min. Used to ensure strain viability.

[0044] Casein culture medium: 50g skim milk powder, 5.0g trypsin-hydrolyzed casein, 2.5g yeast extract, 1.0g glucose, 12.5g agar, pH 6.8±0.3, diluted to 1L with H2O. Autoclave at 121℃ for 20min. Used for screening active organic nitrogen-mineralizing bacteria.

[0045] Phosphate-solubilizing medium: yeast extract 0.5g, D-glucose 10.0g, tricalcium phosphate 5.0g, ammonium sulfate 0.5g, potassium chloride 0.2g, magnesium sulfate 0.1g, manganese sulfate 0.0001g, ferrous sulfate 0.0001g, agar 15g, and H2O to a final volume of 1L. Autoclave at 121℃ for 20min. Used for screening phosphate-solubilizing bacteria.

[0046] Potassium-soluble medium: Magnesium sulfate 0.5g, calcium carbonate 0.1g, potassium aluminum silicate 2.0g, glucose 5.0g, ferric chloride 0.005g, calcium phosphate 2.0g, agar 15.0g, pH 7.2±0.2, H2O to a final volume of 1L. Autoclave at 121℃ for 20 min. Used for screening potassium-soluble bacteria.

[0047] Zinc-soluble medium: 10.0g glucose, 1.0g ammonium sulfate, 0.2g potassium chloride, 0.1g potassium dihydrogen phosphate, 0.2g magnesium bisulfate (heptahydrate), 1.0g zinc oxide, 15g agar, and H2O to a final volume of 1L. Autoclave at 121℃ for 20 minutes. Used for screening zinc-soluble bacteria.

[0048] CAS medium (g / L) formula: 2g sucrose, 3.0g acid-hydrolyzed casein (casein amino acids), 1.0mL 1mmol / L calcium chloride, 20.0mL 1mmol / L MgSO4-7H2O, 15.0g agar, 1000mL distilled water. After sterilization, add 5.0mL each of sterilized phosphate buffer (pH 6.8) and filtered CAS staining solution to every 100mL of medium at approximately 60℃ to obtain the CAS detection medium.

[0049] The CAS staining solution is prepared as follows: Dissolve 0.079g of chromaine in 50mL of deionized water, then add 10mL of 1mmol / L FeCl3 solution to obtain solution A. Dissolve 0.069g of hexadecyltrimethylammonium bromide in 40mL of deionized water to obtain solution B. Slowly add solution A along the beaker wall to solution B, and stir to mix thoroughly to obtain 100mL of CAS blue detection solution. 0.1mol / L phosphate buffer (pH 6.8): Each 100mL contains 2.427g of Na2HPO4·12H2O, 0.5905g of NaH2PO4·2H2O, 0.075g of KH2PO4, 0.250g of NH4Cl, and 0.125g of NaCl. Dilute 10 times before use. Used for screening siderogenic bacteria.

[0050] Drought-resistant culture medium: A high-osmotic-pressure medium was prepared by adding 40% Sortbitol to a 10% TSA medium. Ninety-six drought-resistant strains were screened based on their growth performance.

[0051] The above culture media, when not containing agar, are liquid culture media and are used for corresponding functional verification experiments.

[0052] 1.3 Functional Verification

[0053] This invention demonstrates the multifunctionality of strain ZNL1 through the following repeatable experiments. The control strains used were rhizosphere bacteria strains R13D5, R26F7, R36G9, and R49G3 from healthy plum trees in the main plum-producing area of ​​Liuma Town, Zhenning County, Guizhou Province. An uninoculated culture medium / medium was used as a blank control (CK). Each strain was treated three times.

[0054] Organic nitrogen decomposition function: The test strain was inoculated onto casein medium with casein (10 g / L) as the sole nitrogen source. For example... Figure 1 As shown, after culturing at 28℃ for 2 days, a clear transparent hydrolysis zone appeared around the colony. Figure 1 The strain R36F11 is the same as strain ZNL1. The ratio of the hydrolysis zone diameter (D) to the colony diameter (d) (D / d) is the halo index ≥ 2.0. Figure 2 ).

[0055] Inorganic phosphorus solubilization function: The tested strain was inoculated into phosphorus-soothing liquid medium. After incubation at 28°C for 5 days, the solubility of the strain in poorly soluble inorganic phosphorus was analyzed. Figure 3 The results showed that the phosphorus solubility of strain ZNL1 was not as good as that of R13D5, R26F7, and R49G3, but significantly better than that of R36G9 and CK.

[0056] Potassium solubility: The tested strain was inoculated into a potassium-soluble liquid culture medium containing sparingly soluble potassium feldspar. After incubation at 28°C for 5 days, the culture medium was filtered to remove the precipitate, and the potassium content of the filtrate was analyzed to characterize the strain's ability to dissolve sparingly soluble potassium feldspar. Figure 4 The results showed that strain ZNL1 had a lower potassium-solubility than R49G3, but significantly higher than R13D5, R26F7, R36G9 and CK.

[0057] Siderophore secretion function: Detected using the CAS blue assay. The test strain was inoculated onto a CAS assay plate, such as... Figure 5 As shown, after culturing at 28℃ for 2 days, a distinct orange-yellow halo appeared around the colony, indicating that it can secrete siderophores (…). Figure 5 Strain R36F11 is equivalent to strain ZNL1. The ratio of halo diameter to colony diameter was used as the halo index to represent its secretory capacity. Results showed that R13D5 and R26F7 lacked siderophore production capacity, while ZNL1's siderophore production capacity was significantly higher than that of R36G9 and R49G3 (e.g., R13D5 and R26F7). Figure 6 (As shown).

[0058] Drought resistance verification: The tested strain was inoculated into 10% TSA liquid medium containing sortbitol to simulate drought stress. After incubation at 30°C and 180 rpm for 24 hours, OD was measured. 600 This demonstrates its survival ability under drought conditions. The results show that strain ZNL1 can still survive under drought stress of 40% sortbitol, with an OD... 600 Although not as good as R13D5, it is significantly better than the other three strains (e.g., Figure 7 (As shown).

[0059] Zinc dissolving ability: The tested strains were inoculated into a zinc dissolving liquid culture medium containing sparingly soluble zinc oxide. After incubation at 30℃ and 180 rpm for 24 hours with shaking, the culture medium was filtered to remove the precipitate, and the zinc content of the filtrate was analyzed to characterize the strains' ability to dissolve sparingly soluble zinc oxide. The results showed that the zinc oxide dissolving capacity of strain ZNL1 was significantly higher than that of the other strains and CK, being 22 times that of CK, exhibiting a particularly outstanding ability to promote zinc dissolution (e.g., Figure 8 (As shown).

[0060] 2. Colony morphology characteristics of strain ZNL1

[0061] Bacterial ZNL1 is Gram-negative, and its cells are short rod-shaped. Figure 9 After culturing on TSA medium for 24 h, the colonies were orange-red, round, and had neat edges. Figure 10 Its morphology and Gram staining characteristics are similar to those of Serratia nematodiphila.

[0062] 3. Identification of strain ZNL1

[0063] 3.1 Identification by Molecular Biological Characteristics

[0064] Templates were obtained using a bacterial genomic DNA extraction kit. The 16S rRNA gene sequence was determined using universal primers 27F and 1492R (27F 5'-GTTTGATCMTGGCTC AG-3'; 1492R 5'-TACGGYTACCTTGTTACGACTT -3'), yielding a 1371 bp gene fragment. Comparison with known type bacteria using the EzbioCloud website (http: / / www.ezbiocloud.net / eztaxon / ) showed that strain ZNL1 had the highest similarity (99.71%) to known type strains *Serratia nematodiphila* DSM 21420T and *Serratia surfactantfaciens* YD25T. Similarity to other type bacteria is shown in Table 1. A phylogenetic tree of strain ZNL1 and related taxa based on the 16S rRNA gene sequence was constructed (see [reference needed]). Figure 11 Using primers gyr-320 and rgyr-1260 (gyr-320CMCCYTCCACCARGTAMAGTTC; rgyr-1260 CMCCYTCCACCARGTAMAGTTC) to determine the conserved gyrB gene, a 916 bp gene fragment was obtained. NCBI alignment showed that strain ZNL1 had the highest similarity (98.92%) to the known model bacterium *Serratia nematodiphil* DSM 21420T. A phylogenetic tree was constructed between strain ZNL1 and its closely related species. Figure 12 As can be seen, strain ZNL1 and Serratia nematodiphila DSM 21420T stably aggregate into the same branch with 100% support.

[0065] Table 1. Comparative analysis of 16S rRNA gene sequences of strain ZNL1

[0066]

[0067] 3.2 Identification of physiological and biochemical characteristics of strain ZNL1

[0068] The fatty acid composition of strain ZNL1 was detected using a rapid microbial fatty acid identification system (MIDI). The results showed that the main fatty acids of the tested strain were C16:0, C17:0 cyclo, C14:0, and C12:0 3OH, with contents of 24.35%, 9.28%, 9.09%, and 6.55%, respectively. Specific fatty acid results are shown in Table 2. Figure 13 This aligns with the major cellular fatty acid characteristics of the genus *Serratia*.

[0069] Table 2 Fatty acid data for strain ZNL1

[0070]

[0071] BIOLOG GENIII test results: Positive reactions included dextran, D-maltose, D-trehalose, sucrose, positive control, pH 6, pH 5, β-Methyl-D glucoside, D-salicylic acid, N-Acetyl-D glucosamine, N-Acetyl-β-D mannosamine, N-Acetyl-D galactosamine, 1% NaCl, 4% NaCl, α-D-glucose, D-mannose, D-fructose, D-galactose, D-trehalose, L-trehalose, inosine, 1% sodium lactate, fusobionic acid, D-serine, D-sorbitol, D-mannitol, myo-inositol, glycerol, D-glucose-6-PO4, D-fructose-6-PO4, D-serine, acetomycin, rifamycin SV, gelatin, Glycyl-L-proline, L-alanine, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, gentamicin, guanidine hydrochloride, sodium tetradecanoate sulfate, pectin, galacturonic acid, L-galactocyanate Gluconic acid, D-gluconic acid, glucuronic acid, glucuronide, vancomycin, tetrazolium violet, tetrazolium blue, p-Hydroxy-phenylacetic acid, L-lactic acid, citric acid, α-Keto-glutaric acid, L-hydroxysuccinic acid, Bromo-succinic acid, lithium chloride, Tween 40, acetic acid, formic acid, aztreonam, sodium butyrate; weakly positive reactions include D-arabinol, dimethylaminetetracycline, pyruvate, D-hydroxysuccinic acid; negative reactions include negative control, D-cellobiose, gentiobiose, D-minobiose, stachyose, D-melatotriose, α-D-lactose, D-melatobiose, N-Acetyl Mannosamine pyruvate, 8% NaCl, 3-Methyl glucose, L-rhamnose, D-aspartic acid, L-arginine, L-pyroglutamic acid, mucoic acid, quinic acid, D-gluconic acid, D-methyl lactate, naphthidinone acid, potassium tellurite, γ-Amino-butyric acid, α-Hydroxy-butyric acid, β-Hydroxy-D,L-butyric acid, α-Keto-butyric acid, acetoacetic acid, propionic acid (see Table 3 for details). This is consistent with the biochemical metabolic characteristics of the genus *Serratia*.

[0072] Table 3. Biologic Genome of strain ZNL1 data

[0073]

[0074] Based on the analysis of the 16S rRNA gene sequence and the conserved gyrB gene sequence, as well as Gram staining and colony morphology, and physiological and biochemical characteristics (whole-cell fatty acids and Biolog), strain ZNL1 was identified as *Serratianematodiphila*. Studies have shown that bacteria of this genus can promote plant growth through biological nitrogen fixation, dissolving insoluble phosphorus, secreting siderophores, and producing plant hormones.

[0075] 4. Basic fermentation and application concept of the strain

[0076] 4.1 Fermentation method of strain ZNL1

[0077] Seed culture preparation: A single colony of ZNL1 was picked and inoculated into LB liquid medium and cultured at 28°C with shaking at 180 rpm for 24 hours until OD500 was reached. 600 It reaches 0.8-1.0.

[0078] Fermentation culture: The seed culture was transferred to the fermentation medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) at an inoculation rate of 2% (v / v).

[0079] Fermentation conditions: Cultured at 28℃ and 180 rpm with shaking for 24 hours, the viable cell count at the fermentation endpoint reached 1×10⁻⁶. 9 CFU / mL.

[0080] 3.2 Application Concept

[0081] Based on the proven functions of strain ZNL1, this invention envisions its specific applications, namely: the application of strain ZNL1 in the preparation of microbial preparations for soil improvement in plum orchards, promoting nutrient absorption in fruit trees and / or enhancing drought resistance in fruit trees.

[0082] The forms of the microbial preparations include, but are not limited to:

[0083] Liquid microbial agent: Fermentation liquid containing strain ZNL1, which can be used directly or after dilution. It can be applied by root irrigation during the budding stage of fruit trees or by root irrigation or foliar spraying during the young fruit stage.

[0084] Solid microbial agent: It is prepared by mixing the fermentation broth or cell of strain ZNL1 with an agriculturally acceptable carrier (e.g., peat moss, vermiculite, diatomaceous earth), and then drying it. It can be applied during the budding or young fruit stage of fruit trees.

[0085] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0086] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A Serratia marcescens strain ZNL1, characterized in that, The strain ZNL1 was deposited at the China General Microbiological Culture Collection Center on January 12, 2026, with accession number CGMCC No. 37364.

2. A microbial inoculant for Serratia marcescens strain ZNL1, characterized in that, The bacterial agent contains the Serratia strain ZNL1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is a liquid microbial agent or a solid microbial agent containing the strain or its fermentation products.

4. The microbial agent according to claim 3, characterized in that, The solid microbial agent is prepared by mixing and drying a fermentation broth containing the strain ZNL1 with a carrier, wherein the carrier is selected from peat moss, vermiculite, diatomaceous earth or a combination thereof.

5. A method for preparing a microbial inoculant of Serratia marcescens strain ZNL1, characterized in that, Includes the following steps: (1) The Serratia strain ZNL1 of claim 1 is inoculated into a liquid culture medium for fermentation culture; (2) When the number of viable bacteria in the fermentation broth reaches 1×10 9 When the concentration of CFU / mL reaches a certain level, fermentation is terminated to obtain a fermentation broth containing the strain ZNL1.

6. The preparation method according to claim 5, characterized in that, It also includes the step of mixing the fermentation broth obtained in step (2) with the carrier and drying it to obtain a solid microbial agent.

7. The use of the Serratia serrata strain ZNL1 as described in claim 1 in the preparation of a microbial preparation for activating soil nutrients.

8. The application according to claim 7, characterized in that, The activated soil nutrients include at least one of the following: organic nitrogen solubilizer, inorganic phosphorus solubilizer, potassium solubilizer, zinc solubilizer, and secreted iron carrier.

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