A strain of Bacillus daimoniae that efficiently produces siderophore and its application

Abstract

The present invention discloses a strain of Dai's bacteria C257 that efficiently produces iron carriers and its application, belonging to the technical field of agricultural microorganisms. The strain C257 is identified as Dai's bacteria ( Dyella ) and named Dyella sp. C257, which has been deposited in Guangdong Microbial Culture Collection Center (GDMCC) with a deposit number of GDMCC No: 64889. The Daishi sp. disclosed in the present invention can efficiently produce iron carriers. Therefore, the Daishi sp. C257 disclosed in the present invention has important application potential in promoting iron absorption and growth of crops.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to a strain of Dai's bacterium capable of efficiently producing iron carriers and application thereof. Background Art

[0002] There are a large number of microbial groups in the soil that are beneficial to plant growth, such as plant growth-promoting rhizobacteria (PGPR). They are a general term for a large class of bacteria that can promote plant growth through various mechanisms, including organic and inorganic phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, siderophore-producing bacteria, IAA-producing bacteria, and ACC deaminase-producing bacteria. Siderophore-producing bacteria are a widely reported type of PGPR. They can promote the dissolution of sparingly soluble iron oxides in the soil by secreting siderophores, thereby enhancing the absorption of iron by crops and ultimately promoting crop growth. In addition, siderophore-producing bacteria can also effectively decompose selenium-iron oxide complexes by secreting siderophores into the surrounding environment, releasing plant-available selenium. Recent studies have found that synthetic communities constructed using siderophore-producing bacteria can increase the effectiveness of soil selenium (up to 68.7%) and promote selenium absorption by pomelo plants (up to 92.2%). Therefore, siderophore-producing bacteria can not only improve the iron nutrition of crops, but also have the potential to enhance crop selenium absorption. They are a type of microbial group with great development and application value.

[0003] South China is the main grain producing area in my country and also the concentrated area of ​​acidic red soil in my country, with an area of ​​about 21.8% of the total land area of ​​my country. Due to the characteristics of high temperature and high rainfall in the region and the role of soil desiliconization and iron and aluminum enrichment, the content of iron and aluminum oxides in the soil in South China is very rich, leading to problems such as soil acidification and heavy stickiness. Iron is not only a trace element necessary for plant growth, but also a trace element necessary for human and animal growth. Activating the plant-unusable iron in the soil through microbial technology can not only improve the physical and chemical properties of the soil, but also reduce environmental pollution and production costs caused by additional fertilization. Siderophore-producing bacteria are an effective way to achieve soil iron recycling, especially in the acidic red soil area of ​​South China, which is rich in iron oxides. Therefore, it is very important to explore and develop the resources of efficient siderophore-producing bacteria in the acidic red soil area of ​​South China. Summary of the invention

[0004] The present invention aims to provide a new species of the genus Daishi ( Dyella sp.) C257, providing a new microbial resource for utilizing soil iron and promoting crop iron absorption and growth.

[0005] To achieve the above-mentioned purpose, the new species C257 of the genus Daishila provided by the present invention is isolated from tomato rhizosphere soil. The strain is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), located at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with a deposit number of GDMCC NO: 64889 and a deposit date of July 18, 2024.

[0006] Identification characteristics of the described Dai's bacteria:

[0007] Strain C257 is a Gram-negative bacterium. On TSA medium, the single colony is round, light yellow, smooth, translucent, and has neat edges. The 16S rRNA gene sequence of strain C257 is 1545 bp long. Through sequence comparison analysis, the strain is Dyella marensis The strain C257 has the highest similarity of 98.59%. Based on the results of comparative genome analysis, the average nucleotide identity of strain C257 and the closest published type strain of Dai's bacteria is 80.41%~84.59%, and the DNA-DNA hybridization value is 23.30%~28.00%. The results show that strain C257 represents a new species of Dai's bacteria.

[0008] Siderophore production capacity:

[0009] The present invention found that strain C257 has the ability to produce high iron carriers. Through quantitative analysis of the iron carriers produced by bacteria, it was found that the relative content of the iron carriers produced by the strain C257 was as high as 76.26%.

[0010] The present invention also provides a microbial fertilizer, characterized in that it contains the Dai's bacteria C257, or its fermentation liquid, or the supernatant of its fermentation liquid as an active ingredient.

[0011] The present invention also provides the use of the above-mentioned Dai's bacteria C257 or the above-mentioned microbial fertilizer in producing iron carriers.

[0012] The present invention has the following advantages over the prior art:

[0013] The present invention provides a potential new species of the genus Daishi ( Dyella sp.) C257, which has strong siderophore-producing activity, is stronger than most reported bacteria. Therefore, the discovery of this strain enriches the beneficial microbial resources used for crop growth promotion in my country and has important application prospects in promoting crop iron absorption and growth.

[0014] Dyellasp. C257 is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), located at Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, with the deposit number GDMCC NO: 64889 and the deposit date of July 18, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The colony morphology of Dai's bacteria C257 on TSA medium. (A) The overall state of the colonies on the plate; (B) The state of a single colony. The scale bar is 500 μm.

[0016] Figure 2 This is the phylogenetic tree of the genome of D. difficile C257. DETAILED DESCRIPTION

[0017] The following are specific implementation examples of the present invention. It should be noted that these embodiments are only examples and do not constitute any limitation to the scope of the present invention. Modifications and replacements made to the details and forms of the implementation scheme within the scope of the present invention fall within the protection scope of the present invention.

[0018] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified.

[0019] Example 1: Isolation of tomato rhizosphere soil bacteria

[0020] Tomato rhizosphere soil was collected from Jintang Town, Maonan District, Maoming City, Guangdong Province. The high-throughput isolation, culture and identification technology of bacteria was used to isolate rhizosphere soil bacterial strains, which was mainly divided into the following four steps: (1) high-throughput bacterial culture based on the limiting dilution method; (2) labeling library construction and high-throughput sequencing; (3) bioinformatics analysis of sequencing data; (4) pure strain isolation and preservation. Specifically, 1 g of fresh soil was taken, diluted 5000 times with sterile magnesium chloride solution, and then diluted 2000 times with 10% TSB liquid medium. 160 μL of the dilution was added to 48 96-well cell culture plates. After 2 weeks of dark culture at 30°C, 10 μL of bacterial solution was aspirated from each well of the 96-well cell culture plate and transferred to a PCR tube for alkaline lysis method to extract bacterial DNA. 140 μL of 80% (v / v) glycerol was added to the remaining bacterial solution and stored as bacterial glycerol seed. The first round of PCR amplification was performed using unlabeled primers 799F and 1193R (V5-V7 region of 16S rRNA gene, product size 394 bp) and Green Taq Mix from Novogene. The first round of PCR products were diluted 120 times and used as templates for the second round of PCR. PCR amplification was performed using primers 799F and 1193R with sequencing adapters (Illumina adapters) and barcodes. The second round of PCR products were electrophoresed, and a band of about 500 bp was cut out for purification and the product concentration was determined. Each 96-well plate was prepared according to 100 ng, and the mixed library of 48 plates totaled 4800 ng. High-throughput sequencing was performed at Novogene, with a sequencing depth of 1 million raw tags. The sequencing data was analyzed using the analysis pipeline Culturome (https: / / github.com / YongxinLiu / Culturome) to determine the location of cultured bacteria. Take 25 μL of bacterial solution from the corresponding well of the 96-well cell culture plate and streak it on 1 / 2 TSA medium to activate the bacterial solution. Purify it by multiple streaking method, mix the purified bacterial strain with 25% glycerol (v / v) and store it at -20 o C. Refrigerator.

[0021] Example 2: Screening of siderophore-producing bacteria

[0022] A single colony on the TSA medium was inoculated into a triangular flask containing 50 mL of TSB liquid medium. Three biological replicates were set up, and 1 mL of the bacterial liquid cultured at 200 rpm for 2 days at 30°C was taken. The supernatant was collected by high-speed centrifugation at 12000 rpm for 5 min. The iron carrier production was determined by the CAS method. 100 μL of the supernatant was added to a 96-well cell culture plate containing an equal volume of CAS detection solution and allowed to stand for 1 h under dark conditions. The absorbance at a wavelength of 630 nm was measured using an ELISA reader, and the results were expressed as As. The sterile TSB liquid medium was used as a control, and the absorbance at a wavelength of 630 nm was Ar. The relative content of iron carriers = (1-As / Ar) × 100%.

[0023] Studies have shown that the relative content of iron carriers in strain C257 among the purified bacterial strains screened was as high as 76.26%.

[0024] Example 3: Identification of the siderophore-producing strain C257

[0025] Gram staining confirmed that strain C257 was a Gram-negative bacterium. Strain C257 was inoculated on TSA medium and cultured at 30°C for 3 days for morphological observation. On TSA medium, the single colony was round, light yellow, smooth, translucent, and had neat edges ( Figure 1 ).

[0026] The genomic DNA of strain C257 was extracted using a bacterial DNA extraction kit. The genomic DNA was used as a template and the bacterial universal primers 27F / 1492R were used to amplify its 16S rRNA gene. The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1, with a length of 1545 bp. The sequence obtained by sequencing was subjected to homology comparison analysis on the EzBioCloud website. The results showed that strain C257 was similar to the model strain. Dyella marensis CS5-B2 T The highest similarity is 98.59%; the 16S rRNA gene sequence similarity with other type strains of the genus Daishi is lower. These analyses show that strain C257 is similar to the type strain Dyella marensis CS5-B2 T According to the results of 16S rRNA gene sequence comparison, strain C257 belongs to the genus Daishi, but its taxonomic status cannot be accurately determined.

[0027] Example 4: Genome analysis of D. daimoniae C257

[0028] The genome of strain C257 extracted in Example 3 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing using the Illumina Novaseq platform. SPAdes v3.15.3 software was used for genome sequence splicing, and contig sequences of low quality and less than 500 bp in length were removed. CheckM v1.1.3 software was used to perform quality inspection and analysis on the integrity and contamination of the genome. The analysis results showed that the total length of the strain C257 genome was 5.07 Mbp, the length of N50 was 303880 bp, the G+C content of genomic DNA was 65.64%, the genome completeness was 100.00%, and the contamination was 0.91%.

[0029] The phylogenetic tree of strain C257 based on the genome was constructed using UBCG v3.0 software. Figure 2 As shown, strain C257 and the model strain D. marensis JCM 14959 T and D. subtropica RXD159 T Belong to the same branch, indicating that strain C257 and the type strain D. marensis JCM 14959 T and D. subtropica RXD159 T The online tools ANI Calculator (http: / / www.ezbiocloud.net / tools / ani) and Genome-to-Genome Distance Calculator v3.0 (https: / / ggdc.dsmz.de / ggdc.php#) were used to calculate the average nucleotide identity (ANI) and DNA-DNA hybridization value (DDH) between the genome sequences of strain C257 and the model strains of closely related species. The results showed that strain C257 and closely related model strains D. marensis JCM 14959 T and D. subtropica RXD159 T The ANI values ​​of the strains were 84.59% and 80.41%, and the DDH values ​​were 28.00% and 23.30%, respectively. Currently, the ANI value of 95% to 96% and the DDH value of 70% are defined as the thresholds for defining new prokaryotic species. Therefore, these results indicate that strain C257 belongs to the genus Dai Dyella). The strain has been deposited in Guangdong Microbiological Culture Collection Center (GDMCC), Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, with the deposit number GDMCC No: 64889 and the deposit date July 18, 2024.

[0030] 16S rRNA gene sequence of strain C257 (SEQ ID NO.1)

[0031]

[0032] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Dai's bacteria Dyella sp.) C257, deposited with GDMCC No: 64889.

2. A microbial fertilizer, characterized in that: The invention contains the Dai'erella C257 described in claim 1 as an active ingredient.

3. Use of the Daishima sp. C257 described in claim 1 or the microbial fertilizer described in claim 2 in producing siderophores.

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