Acinetobacter sp. and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a new bacterial strain of Acinetobacter sp. and its use. The bacterial strain is isolated from mangrove soil, is a new species of Acinetobacter sp., is named as Acinetobacter sp. GXMZU3951, and the bacteria have been registered and preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC NO: 36553. The Acinetobacter sp. GXMZU3951 of the present application has iron carrier production activity, can produce higher indole acetic acid, and has the ability to promote plant growth and the potential to resist heavy metals. The present application can be used in the biological remediation of heavy metal contaminated soil and as a green and environment-friendly biofertilizer for improving crop production, and has a good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and environmental bioremediation, specifically involving a new strain of Acinetobacter sp. GXMZU3951 isolated from mangroves. This strain has siderophore production activity, high indoleacetic acid (IAA) synthesis capacity, plant growth promotion and heavy metal tolerance characteristics, and relates to its application in the bioremediation of heavy metal contaminated soil and the preparation of biofertilizers to increase crop yield. Background Technology

[0002] With rapid industrial development, heavy metal pollution has become a global environmental problem, seriously threatening ecological security and human health. Traditional physicochemical remediation methods are costly, prone to causing secondary pollution, and difficult to apply on a large scale. Bioremediation technology has attracted much attention due to its environmental friendliness and cost-effectiveness. Among them, the use of microorganisms with heavy metal tolerance and growth-promoting capabilities for in-situ remediation shows great potential.

[0003] On the other hand, agricultural production faces problems such as soil degradation and environmental pollution caused by excessive use of chemical fertilizers. Overuse of chemical fertilizers not only increases production costs but also damages soil structure and reduces the quality of agricultural products. Microbial fertilizers, as a new type of green and environmentally friendly fertilizer, can promote plant growth through nitrogen fixation, phosphorus solubilization, potassium release, and the secretion of plant hormones, reducing the amount of chemical fertilizers used. This is an important way to achieve sustainable agricultural development.

[0004] Mangroves are mainly distributed in coastal areas of Guangdong, Hainan, and Fujian provinces in my country. As a unique marine-terrestrial ecosystem in the intertidal zone of tropical and subtropical coasts, mangroves possess extreme habitats characterized by high salinity, low oxygen, periodic flooding and exposure, and soils rich in sulfides and organic matter. These habitats foster a diverse array of microbial resources with high biodiversity, strong resilience, and unique metabolic functions. Current research has isolated functional strains of bacteria such as Bacillus, Pseudomonas, and Halomonas, fungi such as Aspergillus and Paecilomyces, and actinomycetes such as Streptomyces and Micromonospora from mangrove habitats. These strains possess both physiological characteristics adapted to the extreme mangrove habitat and diverse biological functions. They can efficiently degrade pollutants such as organic pesticides, secrete halophilic enzymes, novel antibacterial active substances, and biopolysaccharides, and also play roles in plant growth promotion and biocontrol. They demonstrate significant application value in environmental bioremediation, novel biopharmaceutical research and development, industrial enzyme preparation, and ecological agriculture in coastal saline-alkali lands.

[0005] Acinetobacter, belonging to the Moraxellaceae family of the order Moraxellaceae in the class Gammaproteobacteria, is widely distributed in natural environments such as soil, water, and wastewater. Strains in this genus exhibit rich metabolic diversity; some strains can efficiently degrade pollutants such as petroleum hydrocarbons and heavy metals, demonstrating significant application potential in environmental bioremediation. Simultaneously, some strains can secrete industrial enzymes such as lipases and proteases, possessing high economic value in food processing and biocatalysis. However, the exploration of mangrove microbial resources is still in its early stages. Most known Acinetobacter strains show only average performance in terms of heavy metal tolerance, siderophore production, and IAA synthesis capacity, failing to effectively integrate these multiple excellent characteristics, thus limiting their development and application as multifunctional bioremediation agents and biofertilizers. Therefore, screening and identifying a novel Acinetobacter strain possessing high siderophore production, high IAA synthesis, strong heavy metal tolerance, and significant growth-promoting ability is of significant theoretical and practical importance for promoting the bioremediation of heavy metal-contaminated soils and the development of green agriculture. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies and provide a new species of Acinetobacter isolated from mangroves and its applications.

[0007] The first aspect of the present invention is to provide a new species of Acinetobacter isolated from mangrove soil, named Acinetobacter sp. GXMZU3951, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 10, 2025, with accession number CGMCC NO: 36553.

[0008] The second aspect of the present invention is to provide Acinetobacter GXMZU3951 as described in the first aspect of the present invention, characterized in that the strain has siderophore-producing activity and is capable of efficiently chelating iron ions in the environment.

[0009] A third aspect of the present invention is to provide Acinetobacter GXMZU3951 as described in the first aspect of the present invention, characterized in that the strain is capable of producing a high concentration of indoleacetic acid (IAA) under specific culture conditions.

[0010] The fourth aspect of the present invention is to provide Acinetobacter GXMZU3951 as described in the first aspect of the present invention, characterized in that the strain has the ability to promote plant growth and can significantly promote root development and aboveground biomass accumulation in a variety of crops (such as Arabidopsis thaliana, tomato, etc.).

[0011] The fifth aspect of the present invention is to provide Acinetobacter GXMZU3951 as described in the first aspect of the present invention, characterized in that the strain has the potential to be resistant to heavy metals and exhibits a certain degree of tolerance to various heavy metal ions such as zinc (Zn) and nickel (Ni).

[0012] A sixth aspect of the present invention is to provide a microbial inoculant, characterized in that it comprises the Acinetobacter GXMZU3951 strain as described in any one of the first to fifth aspects of the present invention.

[0013] A seventh aspect of the present invention is to provide the microbial agent described in the sixth aspect of the present invention, characterized in that the agent is a liquid agent, a solid microbial fertilizer, or a granular agent composed of other carriers.

[0014] The eighth aspect of the present invention is to provide a method for bioremediation of heavy metal contaminated soil comprising the Acinetobacter GXMZU3951 strain as described in any one of the first to fifth aspects of the present invention or the microbial agent as described in any one of the sixth and seventh aspects of the present invention, characterized by comprising the following steps: (a) applying the microbial agent containing the strain to the heavy metal contaminated soil; (b) colonizing and surviving in the contaminated environment through the heavy metal tolerance characteristics of the strain, and possibly indirectly affecting the bioavailability of heavy metals by changing the physicochemical properties of the rhizosphere microenvironment, thereby reducing their toxic effects on plants.

[0015] The ninth aspect of the present invention is to provide a method for using Acinetobacter GXMZU3951 strain, comprising any one of the first to fifth aspects of the present invention, or a microbial agent, comprising any one of the sixth and seventh aspects of the present invention, as a biofertilizer to increase crop yield, characterized by comprising the following steps: (a) sowing the agent containing the strain as an inoculant together with the seeds, or applying it to the rhizosphere during crop growth; (b) directly stimulating plant root growth through indoleacetic acid produced by the strain, and improving the iron nutrition status of the plant through siderophore activity, thereby synergistically promoting crop growth and development, and increasing crop yield and quality.

[0016] The tenth aspect of the present invention is to provide the use of Acinetobacter GXMZU3951 strain comprising any one of the first to fifth aspects of the present invention or the microbial agent comprising any one of the sixth and seventh aspects of the present invention in the preparation of products for bioremediation of heavy metal contaminated soil.

[0017] The eleventh aspect of the present invention is to provide the use of Acinetobacter GXMZU3951 strain comprising any one of the first to fifth aspects of the present invention or the microbial agent comprising any one of the sixth and seventh aspects of the present invention in the preparation of green and environmentally friendly bio-fertilizer for improving crop yield.

[0018] The *Acinetobacter* sp. GXMZU3951 of this invention, after being cultured in LB medium at 30°C for 48 hours, produced round, milky-white colonies with a moist, glossy surface, neat edges, raised margins, and a strong, pungent odor. It stained red with Gram stain, indicating it is a Gram-negative bacterium. The strain cells were coccobacilli-shaped and could form short chains. The 16S rRNA gene of the strain was amplified by PCR and sequenced using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rRNA gene sequence obtained from sequencing was uploaded to EzBioCloud (https: / / www.ezbiocloud.net) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) for homology searching. A phylogenetic tree was constructed based on the 16S rRNA gene sequence. By comparison, the strain Acinetobacter sp. GXMZU3951 and the type strain Acinetobacter tandoii DSM 14970 were identified. T The highest similarity was 98.69%.

[0019] Whole-genome sequencing analysis was performed on Acinetobacter sp. GXMZU3951, and a whole-genome phylogenetic tree was constructed. The genome size was 3,387,313 bp, with a G+C content of 42.04%. Based on the phylogenetic tree constructed from the 16S rRNA gene and whole genome, closely related species of strain GXMZU3951 were selected. The ANI (mean nucleotide identity), dDDH (digital DNA-DNA hybridization), and AAI (mean amino acid identity) values ​​of strain GXMZU3951 and its closely related species were calculated using EZBioCloud's online tool ANICalculator, the ggdc online website (https: / / ggdc-test.dsmz.de / ggdc.php#), and the EZAAI program. Its ANI, dDDH, and AAI values ​​ranged from 74.80% to 83.13%, 20.40% to 25.5%, and 76.49% to 89.50%, respectively, all below the species classification threshold (ANI < 95%, dDDH < 70%, AAI < 95%), while the AAI values ​​were all above 65%, exceeding the genus classification threshold and conforming to the genus level. This further indicates that Acinetobacters p. GXMZU3951 is a new species of the genus Acinetobacter.

[0020] Biological Preservation

[0021] This invention provides a novel species of Acinetobacter, named Acinetobacter sp. GXMZU3951, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO: 36553, deposit date November 10, 2025, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The Acinetobacter sp. GXMZU3951 of this invention was isolated and screened from mangrove soil in Maoweihai, Qinnan District, Qinzhou City, Guangxi, China. Attached Figure Description

[0022] Figure 1 The images show the cell morphology of Acinetobacter sp. GXMZU3951; where A is a colony morphology image of the bacterium after incubation at 30°C for 48 hours on LB medium, B is an optical microscope image of the bacterium after Gram staining, and C is a scanning electron microscope image of the bacterium.

[0023] Figure 2 This is a neighbor-joining phylogenetic tree of Acinetobacter sp. GXMZU3951 and major type strains of Acinetobacter based on the 16S rRNA gene sequence.

[0024] Figure 3 This is a maximum likelihood phylogenetic tree of Acinetobacter sp. GXMZU3951 and major type strains of Acinetobacter based on the 16S rRNA gene sequence.

[0025] Figure 4 Phylogenetic tree diagram of Acinetobacter sp. GXMZU3951 and closely related Acinetobacter strains based on whole genome sequence.

[0026] Figure 5 The assay was performed to detect the growth-promoting function of Acinetobacter sp. GXMZU3951; where A was the siderophore production capacity assay and B was the IAA production capacity assay.

[0027] Figure 6 Comparative images showing the effects of Acinetobacter sp. GXMZU3951 on tomato growth; where A is a potted tomato plant and B is the root system of the tomato plant.

[0028] Figure 7 Statistical chart showing the effect of Acinetobacter sp. GXMZU3951 on tomato growth.

[0029] Figure 8 To assess the tolerance of Acinetobacter sp. GXMZU3951 to zinc and nickel. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments and the accompanying drawings to provide a better understanding of the invention. However, the scope of protection of the present invention is not limited to the following description. Unless otherwise specified, the experimental methods described below are conventional experimental methods.

[0031] 1. Morphological observation of the strain 1.1 Source of strains Accurately weigh 1g of mangrove soil sample and add it to an Erlenmeyer flask containing 99mL of sterile water. Place the flask in a shaker and shake thoroughly for 24 hours to ensure the bacteria in the soil sample are fully and evenly dispersed. Then, use a 10-fold serial dilution method to dilute the bacterial suspension (to 10). -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7Seven dilutions were used. Different dilutions of bacterial suspension were spread onto the surface of LB agar plates, with three replicates for each dilution. The plates were then incubated at 30°C for 2-3 days. After colonies grew, single colonies with consistent morphological characteristics were selected and repeatedly streaked onto LB agar plates for isolation, repeated three times. The resulting purified single strains were then numbered.

[0032] LB solid medium preparation method: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, 15.0g agar, 1000mL water.

[0033] 1.2 Morphological observation of Acinetobacter sp. GXMZU3951 Strawberry strain GXMZU3951 was streaked on LB agar and incubated at 30°C for 48 hours to obtain single colonies. The colonies were observed to be round, milky white, moist and glossy, with neat, raised edges, and emitting a strong, pungent odor. Optical microscopy revealed that the strain stained red with Gram stain, indicating it is a Gram-negative bacterium. Scanning electron microscopy showed that the strain was coccobacillus-like, could form short chains, and lacked spores and flagella (see instruction manual). Figure 1 Its morphological characteristics are basically consistent with those of the Acinetobacter genus.

[0034] 16S rRNA gene analysis of two strains of Acinetobacter sp. GXMZU3951 The 16S rRNA gene of the strain was amplified by PCR using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified gene was sent to Wuhan Aoke Biotechnology Co., Ltd. for sequencing. The obtained 16S rRNA gene sequence was uploaded to EzBioCloud (https: / / www.ezbiocloud.net) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) for homology searches. The results showed that strain GXMZU3951 is similar to the type strain *Acinetobacter tandoii* DSM 14970. T The highest similarity was 98.69%. Related strains with high homology were selected to obtain their 16S rRNA sequences. A phylogenetic tree based on the 16S rRNA gene was constructed using the neighbor-joining and maximum likelihood methods. The neighbor-joining phylogenetic tree showed that strain GXMZU3951 belongs to the genus *Acinetobacter* and is related to the type strain *Acinetobacter tandoii* DSM 14970. T Most similar (with instruction manual) Figure 2 The maximum likelihood phylogenetic tree showed that strain GXMZU3951 clustered independently into a stable branch (see instruction manual). Figure 3 This indicates that it is similar to the type strain Acinetobacter tandoii DSM 14970. T There are also obvious evolutionary divergences. It is inferred that strain GXMZU3951 is a new species of Acinetobacter and named it Acinetobacter sp. GXMZU3951.

[0035] Whole genome analysis of three strains of Acinetobacter sp. GXMZU3951 The genome sequencing and assembly of strain Acinetobacter sp. GXMZU3951 were performed by Sangon Biotech (Shanghai) Co., Ltd. The results showed that its genome length was 3,387,313 bp, with 17 contigs, all at the chromosome level; the N50 value was 271,470 bp, the G+C content in the DNA was 42.04%, and the average genome sequencing depth was 280×. Other relevant genomic information is shown in Table 1. The relevant genomic data have been uploaded to the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ), accession number JBTLF0000000000. A phylogenetic tree was constructed from the genomes of closely related species similar to strain Acinetobacter sp. GXMZU3951. It was observed that Acinetobacter sp. GXMZU3951 clustered into an independent and stable branch, and was closely related to the type strain Acinetobacter tandoii DSM 14970. T Their close proximity suggests they may be a new species. (Instruction manual attached) Figure 4 )

[0036] Table 1. Genome-wide information of strain Acinetobacter sp. GXMZU3951

[0037] Analysis of ANI, dDDH, and AAI values ​​of the genomes of four Acinetobacter sp. GXMZU3951 strains The ANI (Average Nucleotide Identity), dDDH (Digital DNA-DNA Hybridization), and AAI (Average Amino Acid Identity) values ​​of strain GXMZU3951 and closely related species were calculated using EZBioCloud's online tool ANI Calculator, the ggdc online website (https: / / ggdc-test.dsmz.de / ggdc.php#), and the EZAAI program. Generally, ANI values ​​are ≥95% among strains of the same species; dDDH values ​​are ≥70% among strains of the same species; AAI values ​​are ≥65% among strains of the same genus; and AAI values ​​are ≥95% among strains of the same species. The comparison results showed that strain Acinetobacter sp. GXMZU3951 had an ANI value below 95%, a dDDH value below 70%, and an AAI value below 95% compared to similar strains, all below the species classification threshold; while the AAI value was above 65%, above the genus classification threshold (Table 2). This further confirms that strain Acinetobacter sp. GXMZU3951 is a new species within the genus Acinetobacter.

[0038] Table 2. ANI, dDDH, and AAI values ​​of strain Acinetobacter sp. GXMZU3951 and similar strains.

[0039] 5. Physiological and Biochemical Characteristic Detection 5.1 Determination of growth temperature for strain Acinetobacter sp. GXMZU3951 The strain was streaked onto LB agar and cultured at temperatures of 0℃, 4℃, 15℃, 25℃, 30℃, 37℃, 42℃, and 45℃ for 7 days, and the growth of the strain was observed. The results showed that strain Acinetobacters p. GXMZU3951 grew at 25℃, 30℃, and 37℃, but did not grow at 0℃, 4℃, 15℃, 42℃, and 45℃, with the optimal growth temperature being 30℃.

[0040] 5.2 pH Detection for the Growth of Acinetobacter sp. GXMZU3951 Logarithmic-phase bacterial suspensions were inoculated at 1% (v / v) into LB liquid medium with pH gradients of 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0, respectively, with three replicates for each pH gradient. The cultures were incubated at 30°C and 200 rpm in a shaker for 3 days. The absorbance (OD) of each group of bacterial suspensions at 600 nm was measured using a spectrophotometer. 600 (Value). If OD 600A pH greater than 0.1 indicates that the strain can grow. The results showed that Acinetobacter sp. GXMZU3951 could grow at pH 6.0-9.0, but did not grow at pH 4.0, 5.0, 11.0 and 12.0, and its optimal growth pH was 6.0.

[0041] 5.3 Detection of NaCl concentration during the growth of Acinetobacter sp. GXMZU3951 Logarithmic-phase bacterial suspensions were inoculated at 1% (v / v) into LB liquid medium with NaCl concentrations of 0%, 1%, 2%, 3%, 4%, and 5%, respectively. Three replicates were set up for each concentration. The cultures were incubated at 30℃ and 200 rpm in a shaker for 3 days. The absorbance (OD) of each group of bacterial suspensions at 600 nm was measured using a spectrophotometer. 600 (Value). If OD 600 A value greater than 0.1 indicates that the strain can grow. Results showed that strain Acinetobacter sp. GXMZU3951 could grow at NaCl concentrations of 0-4%, but not at 5% NaCl concentration, with an optimal NaCl concentration of 1% for growth.

[0042] 5.4 Identification of strain Acinetobacter sp. GXMZU3951 by API 50CH and API 20NE biochemical reactions Take Acinetobacter tandoii DSM 14970 T As reference strains, strains *Acinetobacter* sp. GXMZU3951 and *Acinetobacter tandoii* DSM 14970 were tested using API 50CH and API 20NE kits. T The enzymatic properties and carbon source utilization of GXMZU3951 were compared with those of Acinetobacter tandoii DSM 14970. The results are shown in Tables 3 and 4. T There are differences, consistent with the results of genome analysis.

[0043] Table 3 Comparison of API 50CH identification results between strain Acinetobacter sp. GXMZU3951 and the reference strain Note: + indicates positive; - indicates negative.

[0044] Table 4 Comparison of API 20NE identification results between strain Acinetobacter sp. GXMZU3951 and reference strain Note: + indicates positive; - indicates negative.

[0045] 6. Cytochemical Analysis 6.1 Fatty acid composition of strain Acinetobacter sp. GXMZU3951 The test strain was cultured on a large scale, and after collecting sufficient bacterial cells, fatty acids were extracted from the cells through saponification and methylation. Fatty acid analysis was performed by the Yunnan Institute of Microbiology using a gas chromatograph equipped with a MIDI (Microbial Identification System). Peak time and peak area were used to determine the type and relative content of fatty acids in the strain. Table 5 shows that the main fatty acid (content > 5%) of strain Acinetobacter sp. GXMZU3951 is C. 16：0 (31%), C 18：1 ω9c (23.54%), C 14：0 (9.4%), C 18：0 (8.92%), SummaryFeature 3 (C 16：1 ω7c / C 16：1 ω6c)(8.31%), C 12：0 (7.43%), Summed Feature 8(C 18：1 ω7c, C 18：1 ω6c)(5.53%). Compared with reference strain Acinetobacter tandoii DSM 14970 T The dominant fatty acids were consistent with those of the *Acinetobacter* genus, further confirming that strain *Acinetobacter* sp. GXMZU3951 belongs to the *Acinetobacter* genus.

[0046] Table 5. Statistical table of fatty acid content of strain Acinetobacter sp. GXMZU3951 and reference strain. Note: - Not detected

[0047] 6.2 Respiratory quinone composition of strain Acinetobacter sp. GXMZU3951 After the strain was cultured and expanded, the bacterial cells were collected, dried using a freeze dryer, and sent to the Yunnan Provincial Institute of Microbiology for the detection of respiratory quinones. High-performance liquid chromatography (HPLC) was used to determine the main respiratory quinone types of the tested strain based on the relationship between different respiratory quinone components and elution time. The strain *Acinetobacter* sp. GXMZU3951 contained only one methylnaphthoquinone, MK-27(H2), with a content of 100%. In summary, based on 16S rRNA sequencing and phylogenetic analysis, whole-genome sequencing and phylogenetic analysis, morphological observation, physiological and biochemical characterization, and cytochemical analysis, strain Acinetobacters p. GXMZU3951 has been identified as a new species of the genus Acinetobacter. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 36553.

[0048] Detection of the growth-promoting function and effect of 7 strains of Acinetobacter sp. GXMZU3951 7.1 Detection of the growth-promoting function of strain Acinetobacter sp. GXMZU3951 The presence of siderophore-producing function in strains was detected using CAS medium. The appearance of a clear zone indicates siderophore-producing activity. Strawberry strain Acinetobacter sp. GXMZU3951 was streaked onto CAS medium and incubated upside down at 30°C for 3 days. The formation of a clear zone on the CAS medium confirmed that the strain possesses siderophore-producing function (see instruction manual attached). Figure 5 A). The IAA production capacity of the strain was determined using the standard curve method. IAA standard solutions with concentrations of 25, 50, 100, 125, 150, 175, 200, 225, and 25 μg / mL were prepared using methanol. 0.5 mL of each standard solution was added to an equal volume of Salkowsky reagent, mixed well, and incubated in the dark for 30 min. Using an equal volume mixture of methanol and Salkowsky reagent as a blank control, the absorbance (OD) of each standard solution was measured at a wavelength of 530 nm. 530 Plot a standard curve for IAA with IAA concentration on the x-axis and absorbance on the y-axis. After the strain has been cultured in a large-scale manner, 100 μL of a 10-1 IAA concentration was used. 8CFU / mL of bacterial culture was inoculated into LB liquid medium containing tryptophan (0.5 mg / mL) and incubated at 30℃ and 200 rpm for 48 h. The culture was centrifuged at 5000 rpm for 5 min, and the supernatant was collected. 0.5 mL of the supernatant was taken, and an equal volume of Salkowsky reagent was added. After mixing, the mixture was allowed to stand in the dark for 30 min, and the absorbance at 530 nm was measured. Each concentration was tested in triplicate, and the IAA yield was calculated using a standard curve. The experiment showed that strain Acinetobacter sp. GXMZU3951 can produce IAA, with a yield of 131.217 μg / mL, which is relatively high (see instruction manual attached). Figure 5 B).

[0049] 7.2 Detection of the growth-promoting effect of Acinetobacter sp. GXMZU3951 inoculated on tomatoes The tomato variety used in the experiment was Lycopersicon esculentum. Tomato seeds were surface-sterilized with 75% ethanol for 3 minutes each time, repeated 3 times; then rinsed 3 times with sterile water for 3 minutes each time to completely remove residual ethanol. The sterilized seeds were soaked in warm water and placed in a 30℃ incubator for constant temperature treatment. After soaking, the seeds were spread evenly on moist filter paper, covered with another layer of moist filter paper, and placed in the 30℃ incubator for germination. Once the seeds sprouted, they were transplanted into seedling trays and covered with a small amount of substrate (substrate ratio: nutrient soil: vermiculite = 3:1) for seedling cultivation. After about one week of cultivation, when the tomato seedlings had two fully unfolded true leaves, seedlings with uniform growth were selected and transplanted into flowerpots, one seedling per pot. After the seedlings had established themselves, root irrigation was performed. The root irrigation solution for the experimental group was OD... 600 The solution was obtained by diluting Acinetobacter sp. GXMZU3951 bacterial suspension 20 times (=1). Sterile water was used as the blank control group. Each group had three replicates. 20 mL was applied to each plant for root irrigation every 3 days. Tomato growth indicators were measured after 15 days. Root irrigation treatment with Acinetobacter sp. GXMZU3951 showed no toxicity to tomatoes and a significant growth-promoting effect, resulting in more developed root systems in the treated tomatoes (see instruction manual attached). Figure 6 Compared with the control group, the stem height, root length, middle stem diameter, number of leaves, fresh weight, and dry weight of tomatoes increased significantly by 48.67%, 20.06%, 22.58%, 41.44%, 32.85%, and 54.68%, respectively (see instruction manual). Figure 7 ).

[0050] Tests on the tolerance of eight strains of Acinetobacter sp. GXMZU3951 to zinc and nickel. Stock solutions of zinc sulfate (ZnSO4) and nickel sulfate (NiSO4) at concentrations of 100 mM were prepared to test the strain's tolerance to zinc (Zn) and nickel (Ni) heavy metal ions. After sterilization by filtration through a 0.22 μm filter membrane, the stock solutions were added separately to sterilized liquid culture media, and the Zn concentration in the system was adjusted accordingly. 2+ and Ni 2+ Concentration gradient (Zn) 2+ The concentration gradient was set to 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM for Ni. 2+ Concentration gradients were set at 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM. After expansion culture in liquid medium, the strain was inoculated into the aforementioned liquid medium containing heavy metals at a 1% (v / v) inoculum. Liquid medium without heavy metals was used as a positive control, and liquid medium containing heavy metals but without inoculation was used as a negative control (blank control). Each heavy metal concentration gradient was set up in triplicate, and the cultures were incubated at 30℃ and 200 rpm with shaking for 3 days. OD values ​​were then measured. 600 Value, if OD 600 A value greater than 0.1 indicates that the strain can grow. The strain's resistance to Zn is then tested. 2+ and Ni 2+ The minimum inhibitory concentration (MIC) of strain Acinetobacter sp. GXMZU3951 in Zn 2+ It can grow at concentrations of 5mM, 6mM, and 7mM in Zn 2+ It cannot grow at concentrations of 8 mM, 9 mM, and 10 mM, and its MIC for metallic zinc is 7 mM; in Ni 2+ It can grow at concentrations of 2 mM and 3 mM in Ni 2+ It cannot grow at concentrations of 4mM, 5mM, and 6mM. Its MIC for metallic nickel is 3mM (see instruction manual). Figure 8 ).

Claims

1. A new species of Acinetobacter sp., characterized by, The strain was named Acinetobacter sp. GXMZU3951 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 36553.

2. The strain GXMZU3951 according to claim 1, characterized in that, The strain exhibits siderophore-producing activity, enabling it to efficiently chelate iron ions in the environment.

3. The strain GXMZU3951 according to claim 1, characterized in that, The strain is able to produce high concentrations of indoleacetic acid (IAA) under specific culture conditions.

4. The strain GXMZU3951 according to claim 1, characterized in that, The strain has the ability to promote plant growth and can significantly promote root development and aboveground biomass accumulation in a variety of crops (such as Arabidopsis thaliana and tomato).

5. The strain GXMZU3951 according to claim 1, characterized in that, The strain has the potential to tolerate heavy metals and exhibits a certain degree of tolerance to various heavy metal ions such as zinc (Zn) and nickel (Ni).

6. A microbial inoculant, characterized in that, It contains the Acinetobacter GXMZU3951 strain as described in any one of claims 1-5.

7. The microbial agent according to claim 6, characterized in that, The microbial agent is a liquid microbial agent, a solid microbial fertilizer, or a granular agent composed of other carriers.

8. A method for bioremediation of heavy metal contaminated soil using the Acinetobacter GXMZU3951 strain according to any one of claims 1-5 or the microbial agent according to any one of claims 6-7, characterized in that, Includes the following steps: (a) Applying an inoculant containing the strain to soil contaminated with heavy metals; (b) The strain can colonize and survive in polluted environments by means of its heavy metal tolerance, and may indirectly affect the bioavailability of heavy metals by altering the physicochemical properties of the rhizosphere microenvironment, thereby reducing their toxicity to plants.

9. A method for increasing crop yield by using Acinetobacter GXMZU3951 strain according to any one of claims 1-5 or microbial inoculant according to any one of claims 6-7 as biofertilizer, characterized in that, Includes the following steps: (a) Sowing the inoculum containing the strain as an inoculum with the seeds, or applying it to the rhizosphere during crop growth; (b) The indoleacetic acid produced by the strain directly stimulates plant root growth and improves plant iron nutrition status through siderophore activity, thereby synergistically promoting crop growth and development and improving crop yield and quality.

10. The use of Acinetobacter GXMZU3951 strain according to any one of claims 1-5 or the microbial agent according to any one of claims 6-7 in the preparation of products for bioremediation of heavy metal contaminated soil.

11. The use of Acinetobacter GXMZU3951 strain according to any one of claims 1-5 or the microbial agent according to any one of claims 6-7 in the preparation of green and environmentally friendly bio-fertilizers for improving crop yield.

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