Potassium-dissolving strain for tomatoes as well as fungicide and application of potassium-dissolving strain

Abstract

The invention discloses a potassium-dissolving bacterial strain for tomatoes as well as a bacterial agent and application of the potassium-dissolving bacterial strain, the potassium-dissolving bacterial strain is Acinetobacter pittii, the potassium-dissolving bacterial strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20252080. The bacterial agent remarkably improves the fresh weight, dry weight and potassium content of tomato plants in a pot experiment, and has the effect of promoting tomato growth; the microbial inoculum can effectively dissolve potassium in soil, significantly increase the content of rapidly available potassium in the soil, and promote absorption and utilization of potassium by tomatoes. In addition, the acinetobacter peteri B6 strain also has multiple beneficial functions of solubilizing phosphorus, producing siderophores, secreting indoleacetic acid (IAA) and the like, and the comprehensive potential of the acinetobacter peteri B6 strain in the aspect of promoting plant growth is further enhanced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a potassium-dissolving bacterial strain for tomatoes, a bacterial agent thereof and application. BACKGROUND

[0002] Potassium is an important nutrient element necessary for plant growth and development, and plays a key role in various physiological processes. It not only promotes photosynthesis, but also actively participates in the transport of assimilation products, carbohydrate metabolism, protein synthesis and other key metabolic pathways. At the same time, potassium helps maintain cell osmotic pressure balance and enhance cell wall mechanical strength, thereby significantly improving the drought resistance and stress resistance of tomato plants, and having a direct impact on crop yield and quality formation.

[0003] Potassium in soil mainly exists in inorganic and organic forms, of which inorganic potassium is the main source of plant absorption and utilization. However, in inorganic potassium, only 0.1%-2% of the total soil potassium content is available potassium (including water-soluble potassium and exchangeable potassium) that can be directly absorbed and utilized by plants, while 90%-95% of the total soil potassium content is mineral potassium (such as potassium feldspar, mica, etc.) that exists in the form of insoluble silicate as a "potential potassium reservoir" and is difficult to be directly utilized by plants. This occurrence state leads to the fact that although the total amount of potassium in soil is abundant, the proportion of available potassium is extremely low, and the biological availability of potassium becomes a key factor limiting plant potassium nutrition. Therefore, how to effectively activate mineral potassium in soil and improve the content of available potassium in soil has become an important problem to be solved in the sustainable development of agriculture.

[0004] Potassium-solubilizing bacteria (KSB) are a kind of soil beneficial microorganisms that can decompose potassium-containing minerals. They convert insoluble mineral potassium in soil into available potassium that can be absorbed by plants through the production of organic acids, extracellular polysaccharides and other metabolites, thereby effectively improving the content of available potassium in soil and the biological availability of potassium, and playing an important role in improving crop yield and improving soil fertility. In recent years, the development of microbial fertilizers using potassium-solubilizing bacteria has become a research hotspot in the field of agriculture.

[0005] Tomato is an important vegetable crop in China, not only with high nutritional value but also with significant economic value. Its growth and development status is closely related to the potassium supply level, and it is a typical potassium-sensitive plant. Studies have shown that the high demand of tomato for potassium may drive the rhizosphere microbial community to evolve stronger potassium-dissolving ability, so it is more likely to obtain functional strains with specific growth-promoting effects on tomato by screening high-efficiency potassium-dissolving bacteria from the rhizosphere soil of tomato.

[0006] Although existing studies have shown that applying potassium-soluble bacteria as microbial fertilizer can effectively increase the content of available potassium in the soil and reduce the amount of chemical potassium fertilizer applied, the growth-promoting effects of different potassium-soluble bacteria on host plants vary significantly, and most reported potassium-soluble bacteria have relatively singular functions. Therefore, isolating and screening potassium-soluble strains with highly efficient potassium-promoting functions and multiple growth-promoting characteristics from the tomato rhizosphere has important theoretical value and application prospects for improving potassium use efficiency in tomatoes and promoting green tomato production. Currently, regarding Acinetobacter pietrain (… Acinetobacter pittii There are no systematic reports on its use as a potassium-soluble bacterium for tomatoes, and its comprehensive functions in potassium solubilization, phosphorus solubilization, iron production, and secretion of plant growth hormones still need to be explored in depth. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a potassium-soluble strain for tomatoes, its inoculant, and its application.

[0008] To achieve the above objectives, the present invention provides a potassium-soluble bacterium for tomatoes, wherein the strain is Acinetobacter pietrogensis B6 (… Acinetobacter pittii The accession number is CCTCC NO: M20252080, and the accession date is September 22, 2025.

[0009] The present invention also provides a potassium-soluble bacterial agent prepared from the above-mentioned Acinetobacter piezoides B6 strain.

[0010] Furthermore, the bacterial agent is a liquid bacterial agent with a viable count of not less than 1.0 × 10⁻⁶. 8 CFU / mL.

[0011] The present invention also provides a method for preparing the above-mentioned potassium-soluble bacterial agent, comprising the following steps: Strain activation: Inoculate Acinetobacter piezoides B6 strain onto LB solid medium and incubate at 25-30℃ for 48 hours; Liquid fermentation: Activated single colonies were inoculated into LB liquid medium and cultured with shaking at 25-30℃ and 120-180 r / min for 72 hours to obtain OD. 600 Bacterial solutions with a value ≥ 1.0.

[0012] The present invention also provides the application of the potassium-soluble bacterial agent as described above in promoting tomato growth.

[0013] The present invention also provides the application of the potassium-soluble bacterial agent as described above in increasing the potassium content of tomato plants.

[0014] The present invention also provides the application of the potassium-soluble bacterial agent as described above in increasing the available potassium content in soil.

[0015] The present invention also provides a method for promoting tomato growth, comprising the step of applying an effective amount of the above-mentioned potassium-soluble bacterial agent to the tomato rhizosphere, wherein 10 mL of bacterial agent is applied per 500 g of soil.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The Acinetobacter piezoides B6 provided by this invention can effectively promote the growth of tomato plants and exhibits excellent potassium-solubilizing ability, converting insoluble potassium minerals in the soil into readily available potassium that plants can utilize.

[0017] This strain possesses multiple growth-promoting functions, including the ability to dissolve potassium minerals, dissolve phosphorus, secrete the plant growth hormone indoleacetic acid, and produce siderophores. This multifunctionality forms a synergistic growth-promoting mechanism, collectively promoting tomato growth and development through multiple pathways, such as improving soil nutrient availability, stimulating plant growth, and enhancing iron absorption.

[0018] Biological Preservation Instructions Acinetobacter piezophyllum B6 ( Acinetobacter pittii (), deposited at the China Center for Type Culture Collection, date of deposit: September 22, 2025, accession number: CCTCC NO.M20252080. Attached Figure Description

[0019] Figure 1 This is a characterization diagram of the potassium dissolution zone of Acinetobacter piezoides B6 strain on Aleksandrov solid medium (plate method). Figure 2 Figure 1 shows the potassium solubilization effect (KE value) and potassium release capacity of Acinetobacter piezoides B6 strain in liquid culture. Figure 3 This image shows the colony morphology of Acinetobacter pitteri B6 strain on LB solid medium. Figure 4 A graph representing the siderogenic capacity of Acinetobacter pituitaria B6 strain on CAS detection medium; Figure 5 A graph representing the phosphate-solubilizing ability of Acinetobacter pietrogen B6 strain on inorganic phosphate-solubilizing medium; Figure 6 The graph shows the effect of Acinetobacter pituitaria B6 inoculum on the fresh and dry weight of tomato plants; where A represents the fresh weight of the whole plant and B represents the dry weight of the whole plant. Figure 7 Figure showing the effect of Acinetobacter pituitaria B6 inoculum on potassium absorption in tomato plants; Figure 8 The figure shows the results of the determination of the effect of Acinetobacter pituitaria B6 inoculum on the available potassium content in the soil. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0022] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0023] Example 1: Screening and determination of potassium-solubilizing strains LB solid medium was used to isolate and culture microorganisms from soil samples collected from the rhizosphere of tomatoes in the previous stage. Strains were picked from the primary culture medium and subjected to multiple streak separation and purification processes until single colonies were obtained.

[0024] The purified single strain was inoculated onto Aleksandrov agar plates used for screening potassium-solubilizing bacteria and incubated upside down at 28°C for 4 days. Strains with a clear zone around the colony were preliminarily identified as having potassium-solubilizing ability. Colonies with clear zones were picked and repeatedly streaked on 1 / 10 TSA agar plates for purification three times, finally obtaining pure cultured single colonies of potassium-solubilizing bacteria, named B6.

[0025] To quantitatively evaluate the potassium-solubilizing ability of the strain, purified single colonies were picked and inoculated onto Aleksandrov solid medium. After incubation at 28°C for 7 days, the diameter of the clear zone (H) and the colony diameter (C) were measured, and the potassium-solubilizing effect (KE = (H + C) / C) was calculated. The larger the KE value, the stronger the potassium-solubilizing ability of the strain.

[0026] The potassium-solubility effect was further verified using liquid culture: the purified strain was inoculated into LB liquid medium and cultured at 30°C with shaking at 150 r / min until the bacterial concentration reached 10. 8 CFU / mL (OD) 600 ≥1). Take 1 mL of bacterial culture and inoculate it into Aleksandrov liquid medium, using uninoculated medium as a blank control. Incubate at 28±1℃ and 150 r / min with shaking for 7 days. After the culture is completed, take the fermentation broth and centrifuge it at 5000 r / min for 10 min. Determine the potassium content in the supernatant by flame spectrophotometry.

[0027] like Figure 1 As shown, a distinct clear zone appears around the colony of Acinetobacter piezoides B6, indicating that this strain has potassium-soluble capabilities. Figure 2As shown, the potassium-solubilizing effect value (KE) of Acinetobacter piezoides B6 reached 3.5±0.2, and the potassium release in liquid culture reached 45.6±2.1 mg / L, indicating that this strain is a highly efficient potassium-solubilizing strain.

[0028] Culture medium formulation: LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, agar 15.0 g / L (solid medium), pH=7.3±0.2.

[0029] 1 / 10 TSA medium: 1.5 g / L tryptone, 0.5 g / L soybean peptone, 0.5 g / L sodium chloride, 15.0 g / L agar (solid medium), pH=7.3±0.2.

[0030] Aleksandrov medium: glucose 3.5 g / L, MgSO4·7H2O 0.5 g / L, CaCO3 0.1 g / L, FeCl3 0.5 mg / L, Ca3PO4 2.0 g / L, insoluble mica powder 1.0 g / L (potassium source), agar 15.0 g / L (solid medium).

[0031] Example 2: Identification and Characterization of Strains This example illustrates the identification process of Acinetobacter piterum B6 strain.

[0032] (1) Morphological and physiological biochemical identification Purified Acinetobacter piezoides B6 strain was selected and cultured on LB agar using the streak plating method at 28°C for 2 days. Colony morphology was observed as follows. Figure 3 As shown: the colonies are round with neat edges, white in color, smooth and moist on the surface, uniform in texture, and opaque.

[0033] Simultaneously, Gram staining, starch hydrolysis, and gelatin liquefaction tests were performed to identify strain B6, and the results are shown in Table 1. Strain B6 is a Gram-negative bacterium with the ability to hydrolyze starch and liquefy gelatin.

[0034] Table 1. Colony morphology and physiological and biochemical assays of Acinetobacter piezoides B6 Note: A Gram stain "-" indicates Gram-negative bacteria, while "+" indicates that the strain possesses this function. (2) Molecular biological identification The isolated and purified strain was subjected to molecular biological identification. A 1430 bp 16S rRNA gene sequence was obtained through DNA extraction, PCR amplification, and 16S rRNA gene sequencing, and its nucleotide sequence is shown in SEQ ID No. 1. The obtained 16S rRNA sequence was compared with the NCBI database for nucleic acid sequence homology (Blastn), revealing that this potassium-soluble bacterium strain is similar to *Acinetobacter pietroides*. Acinetobacter pittii The homology of the strain reached 99.8%, and phylogenetic analysis showed that it belonged to the same branch as *Acinetobacter pitterii*. Based on morphological and physiological-biochemical characteristics, the strain was identified as *Acinetobacter pitterii*. Acinetobacter pittii ).

[0035] SEQ ID No. 1: Example 3: Determination of the strain's ability to produce IAA, siderophores, and solubilize phosphorus. 1. Determination of IAA production capacity of the strain A single colony of purified Acinetobacter pituitaria B6 was inoculated into LB liquid medium containing 100 μg / mL L-tryptophan and incubated at 30°C and 180 rpm in the dark for 72 hours. One mL of the culture was mixed with 2 mL of Salkowski chromogenic reagent and incubated in the dark for 30 min. A blank control was also performed. The culture medium of strain B6 turned pink, indicating that this strain has the ability to produce IAA.

[0036] The culture medium of strain B6 was centrifuged at 10,000 rpm for 10 min, and 1 mL of the supernatant was reacted with 2 mL of colorimetric reagent for 30 min. The OD value was measured at a wavelength of 530 nm. The IAA production capacity of strain B6 was determined to be 22.36 ± 1.42 μg / mL by calculation using the IAA standard curve.

[0037] 2. Determination of the strain's ability to produce siderophores A single colony of purified Acinetobacter pitera B6 was picked and inoculated onto a CAS assay plate and incubated upside down at 28°C for 7 days. Figure 4 As shown, a distinct orange-yellow halo appears around the colony, indicating that this strain has a strong ability to produce siderophores.

[0038] 3. Determination of the phosphate-solubilizing ability of the strain A single colony of purified Acinetobacter piezoides B6 was picked and inoculated onto an inorganic phosphate-solubilizing medium plate, and incubated upside down at 28°C for 7 days. Figure 5 As shown, a clear halo appears around the colony, indicating that the strain has phosphate-solubilizing ability.

[0039] Example 4: Preparation of microbial agent and verification of its growth-promoting effect on potted plants 1. Preparation of microbial agents Single colonies of the purified B6 strain were picked and inoculated into LB liquid medium. The culture was carried out at 28°C and 180 r / min with shaking for 72 hours to obtain the KSB fermentation broth. The fermentation broth was centrifuged at 8000 r / min for 10 min, and the bacterial pellet was collected. The pellet was washed three times with sterile distilled water, and finally resuspended in sterile distilled water. The OD of the bacterial suspension was adjusted. 600 When the concentration reaches 1.0, KSB liquid bacterial agent is obtained.

[0040] 2. Pot experiment The experiment consisted of two treatments: an experimental group (inoculated with B6 inoculant) and a control group (inoculated with an equal volume of sterile distilled water). Each group had three replicates, with six pots per replicate (n=18). The tomato variety used was "Dongnong 708". Seeds were disinfected with 10% trisodium phosphate for 5 min before germination and then routinely raised as seedlings. When the seedlings had two true leaves, they were transplanted into plastic pots (10×10 cm) containing 500 g of mixed soil (field soil: sand = 1:2, available potassium content 125.3±8.6 mg / kg).

[0041] Seven days after transplanting, each plant in the experimental group was irrigated with 10 mL of KSB bacterial suspension (10 mL). 8 The control group was watered with an equal volume of sterile distilled water (CFU / mL). To prevent leakage, the bottom of all pots was sealed with polypropylene plastic film. Samples were taken 14 days after transplanting to determine the fresh weight, dry weight, and potassium concentration of the plants.

[0042] like Figure 6 As shown, the fresh weight of the tomato plant in the treatment group inoculated with Acinetobacter pylori B6 was (4.60±0.34) g and the dry weight was (0.44±0.05) g, which were 78.8% and 61.5% higher than those of the control group (2.57±0.11) g and (0.27±0.02) g, respectively, with extremely significant differences (***P<0.001, Student's t-test, n=3).

[0043] Figure 7 The results showed that the potassium uptake of the treated plants reached (21.29±0.92) mg / plant, which was 37.6% higher than that of the control group (15.47±0.49) mg / plant, and the difference was extremely significant (***P<0.001).

[0044] The above results confirm that the microbial agent significantly increases plant biomass by enhancing the absorption of potassium by tomatoes.

[0045] Example 5: Effect of microbial agents on soil available potassium content The experiment consisted of two treatments: an experimental group (inoculated with KSB inoculant) and a control group (inoculated with an equal volume of sterile distilled water). Each treatment had three replicates, with each replicate containing six pots (n=18). Each pot was a 10×10 cm plastic pot containing 500 g of field soil (available potassium content 132.7±9.2 mg / kg), with the bottom sealed with polypropylene plastic film to prevent leakage. No tomatoes were planted in any of the pots; only inoculant treatment was administered: each pot in the experimental group was watered with 10 mL of KSB inoculant suspension (concentration 10...). 8 The control group was treated with an equal volume of sterile water (CFU / mL). Samples were taken 14 days after treatment to determine the available potassium content in the soil.

[0046] like Figure 8As shown, 14 days after soil inoculation with the bacterial agent under plant-free conditions, the available potassium content in the treated soil was (468.6±14.8) mg / kg, which was 134% higher than that in the control group (200.0±7.9) mg / kg, and the difference was highly significant (***P<0.001, Student's t-test, n=3). This indicates that the bacterial strain can directly activate soil mineral potassium independently of plants, significantly improving potassium availability.

[0047] Example 6: Study on the stability of microbial agents during storage The prepared liquid bacterial agent was stored at 4℃, room temperature (25℃), and 37℃, and the changes in viable bacterial count were monitored periodically. The results showed that after 30 days of storage at 4℃, the viable bacterial count remained at 1.0 × 10⁻⁶. 8 CFU / mL or higher; viable count decreased to 8.5 × 10⁻⁶ after 15 days of storage at room temperature. 7 CFU / mL; after storage at 37℃ for 7 days, the viable bacterial count dropped sharply to 3.2 × 10⁻⁶. 6 CFU / mL. It is recommended to store the bacterial agent at 4°C; the shelf life can reach 30 days.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A potassium-soluble bacterium for tomatoes, characterized in that, The strain was Acinetobacter pitera B6 ( Acinetobacter pittii The accession number is CCTCC NO: M20252080, and the accession date is September 22, 2025.

2. A potassium-soluble bacterial agent prepared from the Acinetobacter piezoides B6 strain as described in claim 1.

3. The potassium-soluble bacterial agent according to claim 2, characterized in that, The bacterial agent is a liquid bacterial agent with a viable count of not less than 1.0 × 10⁻⁶. 8 CFU / mL.

4. A method for preparing the potassium-soluble bacterial agent according to claim 2 or 3, characterized in that, Includes the following steps: Strain activation: Inoculate Acinetobacter piezoides B6 strain onto LB solid medium and incubate at 25-30℃ for 48 hours; Liquid fermentation: Activated single colonies were inoculated into LB liquid medium and cultured with shaking at 25-30℃ and 120-180 r / min for 72 hours to obtain OD. 600 Bacterial solutions with a value ≥ 1.

0.

5. The application of the potassium-soluble bacterial agent as described in claim 2 in promoting tomato growth.

6. The application of the potassium-dissolving bacterial agent as described in claim 2 in increasing the potassium content of tomato plants.

7. The application of the potassium-soluble bacterial agent as described in claim 2 in increasing the available potassium content in soil.

8. A method for promoting tomato growth, characterized in that, The step includes applying an effective amount of the potassium-soluble bacterial agent as described in claim 2 or 3 to the tomato rhizosphere, wherein 10 mL of the bacterial agent is applied per 500 g of soil.

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