Flavobacterium phosphoreum and application thereof

Abstract

The application discloses a strain of Frateuria sp. and application thereof. The Frateuria sp. GZRe12 of the application was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on May 17, 2022, and the preservation number is GDMCC No: 62474. The Frateuria sp. disclosed by the application is a new species of the genus, can effectively dissolve the insoluble inorganic phosphorus (Ca3(PO4)2), and the amount of dissolved phosphorus of the culture for 5 days is as high as 495.05+ / -11.58 mg / L, and the phosphorus dissolving performance is significantly better than that of most reported phosphorus dissolving strains. Therefore, the Frateuria sp. GZRe12 disclosed by the application can be used to improve the availability of phosphorus in soil, and has important application potential in promoting the growth and development of plants, preparing microbial agents and biological fertilizers and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganisms, and particularly relates to a phosphate-solubilizing Flavobacterium and application thereof. BACKGROUND

[0002] Phosphorus is the second most essential element for crop growth and plays an important role in plant growth and metabolism. It is an important factor that restricts crop yield and quality. The content of phosphorus in China's cultivated soil is rich, but 95% of the phosphorus exists in mineral form, and the content of available phosphorus that can be absorbed and utilized by crops is extremely low. The utilization rate of exogenous phosphorus fertilizer is only 10%-15%, and most of the phosphorus is chelated and fixed with cations such as Ca 2+ , Fe 3+ , Al 3+ in soil to form insoluble orthophosphate compounds, which cannot be absorbed by plants. In addition, long-term excessive application of phosphorus fertilizer can also cause serious water eutrophication. How to fully activate the fixed phosphorus in the soil, improve the absorption efficiency of phosphorus by crops, and reduce the application of phosphorus fertilizer is an important issue in the development of modern green agriculture.

[0003] Phosphate-solubilizing bacteria are a kind of bacteria that can improve the bioavailability of insoluble phosphorus in soil. They can activate insoluble phosphorus in soil by secreting organic acids and extracellular enzymes, improve the content of available phosphorus in soil, promote the absorption and utilization of phosphorus by plants, and thus improve the yield of plants, and have important application potential in agricultural production. According to statistics, phosphate-solubilizing bacteria account for about 40% of cultivable bacteria in soil. Existing research shows that the phosphate-solubilizing bacteria isolated mainly concentrate in 30 genera of Firmicutes, Proteobacteria and Actinobacteria, such as Bacillus, Panebacillus, Streptomyces, Pseudomonas, Rhizobium, Burkholderia, Microbacterium, etc. These phosphate-solubilizing bacteria not only can improve the absorption and utilization of phosphorus by crops, promote plant growth and development, but also can promote the metabolic activity of beneficial microorganisms in soil and improve the microecological environment of plant rhizosphere. Therefore, it is an excellent way to regulate the phosphorus cycle in soil-plant by using phosphate-solubilizing bacteria in soil.

[0004] It is of great significance to improve the utilization rate of phosphorus fertilizer and improve the phosphorus nutrition of plants by continuously exploring new resources of phosphate-solubilizing bacteria in crop rhizosphere, developing related microbial agents, microbial fertilizers and other products, and converting soil insoluble phosphorus into effective phosphorus that can be absorbed and utilized by plants. SUMMARY

[0005] The purpose of the present application is to provide a new species of phosphate-solubilizing bacteria of Frateuria sp. GZRe12 capable of effectively dissolving inorganic phosphorus, which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on May 17, 2022, located at No. 59, Building 5, 100, Martyrs Road, Yuexiu District, Guangzhou, Guangdong Province, China, with a postal code of 510070, and a preservation number of GDMCC No: 62474.

[0006] The second purpose of the present application is to provide the application of phosphate-solubilizing Frateuria GZRe12 in the preparation of inorganic phosphorus-dissolving preparations.

[0007] The third purpose of the present application is to provide an inorganic phosphorus-dissolving preparation containing phosphate-solubilizing Frateuria GZRe12 as the main active ingredient.

[0008] Preferably, the inorganic phosphorus-dissolving preparation can be a microbial agent or an organic biological fertilizer, etc.

[0009] The present application has the following advantages over the prior art:

[0010] 1) The phosphate-solubilizing Frateuria GZRe12 provided by the present application is a new species of Frateuria different from other phosphate-solubilizing bacteria;

[0011] 2) The amount of inorganic phosphorus dissolved by the phosphate-solubilizing Frateuria GZRe12 in the inorganic phosphorus liquid medium after 5 days of culture is as high as 495±11.58 mg / L, and the phosphate-solubilizing ability is stronger than most reported bacteria. Therefore, the phosphate-solubilizing Frateuria GZRe12 disclosed in the present application can be used to dissolve insoluble inorganic phosphorus in soil and improve the content of soluble phosphorus, and has a wide application prospect in the preparation of microbial agents, organic biological fertilizers and the promotion of crop growth.

[0012] Frateuria sp. GZRe12 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on May 17, 2022, located at No. 59, Building 5, 100, Martyrs Road, Yuexiu District, Guangzhou, Guangdong Province, China, with a postal code of 510070, and a preservation number of GDMCC No: 62474. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1Morphology of Phosphate-dissolving Flavobacterium GZRe12 on R2A plate in the present application.

[0014] Figure 2 Cell transmission electron microscope observation chart of Phosphate-dissolving Flavobacterium GZRe12 in the present application, the scale is 5 μm.

[0015] Figure 3 Phylogenetic tree of 16S rRNA gene of Phosphate-dissolving Flavobacterium GZRe12 in the present application.

[0016] Figure 4 Phylogenetic tree of genome of Phosphate-dissolving Flavobacterium GZRe12 in the present application.

[0017] Figure 5 Phosphorus-dissolving transparent circle of Phosphate-dissolving Flavobacterium GZRe12 on inorganic phosphorus plate in the present application.

[0018] Figure 6 Phosphate-dissolving effect and pH change of culture solution of Phosphate-dissolving Flavobacterium GZRe12 in the present application. DETAILED DESCRIPTION

[0019] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0020] The present application is further illustrated by the following examples, and the test methods in the following examples are all conventional test methods unless otherwise specified, and the experimental reagents and consumables described in the following examples are all from conventional biochemical reagent companies unless otherwise specified.

[0021] Example 1 Isolation and purification of Phosphate-dissolving Flavobacterium GZRe12

[0022] 1. Isolation and screening medium

[0023] R2A agar medium (g / L Qingdao Haibo Biology HB0167-5): yeast extract powder 0.5 g, protein peptone 0.5 g, casein hydrolysate 0.5 g, glucose 0.5 g, agar powder 0.5 g, soluble starch 0.5 g, potassium phosphate dibasic 0.3 g, anhydrous magnesium sulfate 0.024 g, sodium pyruvate 0.3 g, agar 15 g, when used, 15.2 g of the above medium is weighed and dissolved in 1 L of distilled water, sterilized at 121℃ for 15 min by high-pressure steam, and ready for use.

[0024] Inorganic phosphorus bacteria (g / L, Qingdao Haibo Biology, HB8670): glucose 10.0 g, ammonium sulfate 0.5 g, yeast extract powder 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, calcium phosphate 5.0 g, agar powder 15 g, pH 7.0-7.5, when used, 32 g of the above medium is weighed, dissolved in 1 L of distilled water heated to 121°C, high-pressure steam sterilized for 15 min, and ready for use.

[0025] 2. Experimental steps

[0026] (1) Sample collection: soil samples of perennial tomato planting were collected from the farm of South China Agricultural University in Guangzhou, Guangdong Province, and were placed in sampling bags and brought back to the laboratory for natural air drying at room temperature.

[0027] (2) Preparation of soil gradient dilution solution: 10 g of soil was weighed and added to a sterilized flask with glass beads and 90 mL of sterile water, and cultured on a 30°C shaking table at 200 rpm for 30 min. After standing for 5 min, 1 mL of supernatant was taken and added to a test tube containing 9 mL of sterile water, and gradient dilution was performed to 10 -2 -10 -7 g / mL of soil suspension for standby.

[0028] (3) Phosphorus-solubilizing bacteria isolation: 100 μL of diluted soil bacterial suspension (10 -2 -10 -7 g / mL) was taken and uniformly coated on the inorganic phosphorus bacteria culture medium plate using a disposable plastic coating rod, with three replicates for each concentration. After coating, the plate was inverted and placed in a 30°C constant temperature incubator for culture. The next day, the growth of colonies on the plate was observed, and different morphological strains producing transparent circles were picked and streaked on R2A solid plates for strain purification, and finally strain GZRe12 was isolated.

[0029] Example 2. Classification and identification of strain GZRe12

[0030] The isolated strain was identified according to the "Common Bacteria System Identification Manual" (Dong Xiuzhu, Science Press).

[0031] (1) Phenotypic characteristics:

[0032] Strain GZRe12 was inoculated on R2A medium by three-zone streak method, and its colony morphology was observed after 30°C incubation for 3 days. The cultured colonies were picked for Gram staining and morphological observation by optical microscope, and transmission electron microscope was used to observe the cell morphology of the strain. Nitrate reduction, nitrite reduction, indole production, and fermentation of dextrose were tested by API 20NE reagent strips (Merieux Bio, France).

[0033] Strain GZRe12 was dark yellow on R2A medium, and its single colony was round with smooth surface and neat edge without fluidity. Strain GZRe12 was negative for Gram staining, and its cells were short rod-shaped without flagella under transmission electron microscope. Figure 1 Strain GZRe12 was positive for fermentation of dextrose, L-arginine dihydrolase, and urease, and negative for nitrate reduction, nitrite reduction, indole production, β-glucosidase, hydrolysis of gelatin, and β-galactosidase (Table 1).

[0034] Table 1 Phenotypic characteristics of strain GZRe12

[0035]

[0036] (2) Phylogenetic analysis:

[0037] The DNA of the strain GZRe12 was extracted by water boiling method, and the 16S rDNA gene sequence of GZRe12 was amplified by using the 16S rDNA specific primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') and Taq enzyme. The amplification product was analyzed by electrophoresis to produce a band of about 1500 b, and the band was recovered by gel cutting and sent to Suzhou Jinyuzhi Biological Technology Co., Ltd. for sanger sequencing. The sequence obtained by sequencing was spliced by DNAMAN software to obtain the 16S rDNA sequence, and the nucleotide sequence is shown as SEQ ID NO. 1, which was submitted to the EzBioClode website (https: / / www.ezbiocloud.net / ) for identification. The Neighbor Joining phylogenetic tree based on 16S rRNA sequence was constructed by using MEGA 10 software, and the calculation model was Kimura 2-paremeter. The full length of the 16S rRNA gene of the strain GZRe12 obtained by Sanger sequencing is 1421 bp (SEQ ID NO. 1). Sequence alignment found that the similarity between the strain GZRe12 and the published type strain sequence was lower than the critical value of 98.65% for species differentiation (Table 2), and the highest similarity type strain was Frateuria edaphi KACC 16945 T (98.59%). The phylogenetic tree constructed based on 16S rRNA gene sequence and genome sequence showed that the strain GZRe12 belonged to Frateuria, and had the closest genetic relationship with Frateuria edaphi KACC 16945 T , Frateuria soli KACC 16943 T and Frateuria flava MAH-13 T and other type strains. Figure 3 Therefore, according to the results of 16S rRNA gene sequence and morphological characteristics, the strain GZRe12 was identified as a potential new species of Frateuria.

[0038] (3) Comparative genomics analysis:

[0039] The genomic DNA of the strain GZRe12 was extracted using a bacterial genomic DNA extraction kit (Magen), and after 16S rRNA gene amplification verification, the genomic sequencing, double-end library construction and Illumina Hiseq 2500 sequencing were completed by Shanghai Meiji Biological Technology Co., Ltd. The down-machine data was used for genomic sequence splicing assembly by SPAdes (3.13.0) software. The published genomic sequences of the model species to be compared and analyzed were downloaded from the NCBI database, and the average nucleotide identity (ANI) of each strain was calculated based on the genomic sequence using FastANI. The DNA-DNA hybridization value (dDDH) based on the genomic sequence was calculated using the online tool GGDC 2.1 (http: / / hhdc.dsmz.de / home.php). The UBCG was used to construct a genomic ML phylogenetic tree. The genome size of the strain GZRe12 is about 3.59 Mb, and the GC content is 67.8 mol%. The results of genomic evolution analysis are consistent with the results of 16s rRNA gene sequence evolution analysis, showing that the strain GZRe12 belongs to the genus Frateuria Figure 4 ). The results of comparative genomic analysis based on the genomic sequence are shown in Table 2, and the ANI value and dDDH between the strain GZRe12 and the closest published model strain are 78.1%-93.6% and 20.7%-51.1%, respectively, which are significantly lower than the threshold of 95%-96% ANI and 70% dDDH as the species boundary. These results show that the strain GZRe12 represents a different new species of the genus Frateuria, and the present application names the strain GZRe12 as Frateuria sp. GZRe12, which was preserved in the Guangdong Microbial Culture Collection Center on May 17, 2022, at address: No. 100, Martyrs' Road, Yuexiu District, Guangzhou, Guangdong Province, China, Postcode: 510070, and its preservation number is: GDMCC No: 62474.

[0040] Table 2 Comparative genomic analysis of the strain GZRe12 and the published six closely related model species

[0041]

[0042] Example 3: Analysis of the inorganic phosphorus release efficiency of Frateuria sp. GZRe12

[0043] The glycerol bacteria of Flavobacterium heparinum GZRe12 was activated on R2A solid plate, and cultured at 30°C for 2 days. A single colony was inoculated into a test tube containing 5 mL of R2A liquid medium, and then placed in a horizontal shaker at 30°C and 200 rpm. After the bacteria grew to the logarithmic phase, 1% (v / v) of the inoculum was inoculated into 100 mL of inorganic phosphorus liquid medium (phosphorus bacteria phosphorus solubilization efficiency) with Ca3(PO4)2 as the inorganic phosphorus source, and cultured at 30°C and 200 rpm for 5 days. The treatment with sterilized R2A medium without inoculating strain GZRe12 was used as a control, and three biological replicates were set for each treatment. When measuring, 10 mL of sample was centrifuged at 12000 rpm for 2 min, filtered with a 0.22 μm filter to remove bacteria, and then the pH and soluble phosphorus (ws-p) content in the culture solution were measured. In this study, the molybdenum-antimony anti-colorimetric method was used to determine the soluble phosphorus content in the culture solution, and the final phosphorus solubilization amount was calculated by deducting the blank control.

[0044] Inorganic phosphorus medium (without agar, used for determining the phosphorus bacteria phosphorus solubilization efficiency, Norganic Phosphorus Bacteria, g / L, Qingdao Haibo Biology, HB8670-1): glucose 10.0 g, ammonium sulfate 0.5 g, yeast extract powder 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, tricalcium phosphate 5.0 g, pH 7.0-7.5, when used, 17 g of the above medium was weighed, dissolved in 1 L of distilled water by heating, sterilized by high-pressure steam at 121°C for 15 min, and prepared for use.

[0045] The results of the inorganic phosphorus solubilization by Flavobacterium heparinum GZRe12 are shown in Figure 5 It can be seen that a clear transparent circle appeared around the colony, indicating that strain GZRe12 has the ability to dissolve insoluble inorganic phosphorus; the quantitative determination results showed that after 5 days of inoculation and culture, the amount of inorganic phosphorus dissolved by strain GZRe12 was 495.05±11.58 mg / L Figure 6 ).

[0046] SEQ ID NO. 1

[0047]

Claims

1. A strain of Phosphovibrio sp. GZRe12, characterized in that, Frateuria The preservation number is: GDMCC No: 62474. ​ 2. The use of Frateuria phosphatis GZRe12 in the preparation of inorganic phosphorus dissolving preparation according to claim 1.

3. Use according to claim 2, characterized in that, The inorganic phosphorus dissolving preparation is microbial inoculum or biofertilizer.

4. A preparation for dissolving inorganic phosphorus, characterized by comprising, It contains Frateuria phosphatis GZRe12 in claim 1 as active ingredient.

5. The inorganic phosphorus solubilizing agent according to claim 4, characterized by, The inorganic phosphorus dissolving preparation is microbial inoculum or biofertilizer.

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