CN109749959B - Strain HB161398 with nitrogen fixation activity and application thereof - Google Patents

Abstract

The invention discloses a bacterial strain HB161398 with nitrogen fixation activity, wherein the preservation number is CGMCC NO.13939, the preservation date is 2017, 9 and 18 days, the preservation unit is China general microbiological culture Collection center (CGMCC for short), and the preservation address is Beijing City Kogyo Beichen Xilu No.1 institute No. 3, China academy of sciences microbial research institute. The strain belongs to a new genus of Rhodobacteraceae, and has good nitrogen fixation activity. The invention also discloses application of the strain HB161398 in preparation of a fertilizer with a nitrogen fixation effect.

Description

A strain HB161398 with nitrogen-fixing activity and its application

technical field

The invention belongs to the technical field of genetic engineering, in particular to a strain HB161398 with nitrogen-fixing activity.

Background technique

Nitrogen is the main element limiting ecosystem productivity, and biological nitrogen fixation is the main source of nitrogen in natural ecosystems. Biological nitrogen fixation includes three types: symbiotic nitrogen fixation, combined nitrogen fixation and voluntary nitrogen fixation, of which combined nitrogen fixation and voluntary nitrogen fixation are collectively referred to as asymbiotic nitrogen fixation. Compared with symbiotic nitrogen fixation, although the non-symbiotic nitrogen fixation rate is lower, it can survive and fix nitrogen without forming a symbiotic system with other organisms. contribute. As in many tropical forests, especially those lacking a large number of symbiotic nitrogen-fixing plants, voluntary nitrogen fixation is extremely important to meet the high nitrogen requirements of plants and to balance nitrogen losses. In addition, the combined nitrogen-fixing bacteria colonize the roots by gathering on the root surface or through root wounds, exuding organic matter from the rhizosphere obtained from the host as a carbon source and energy source, and supplying its fixed nitrogen and secreted bioactive substances to plants for utilization, Plays an important role in ecosystems dominated by grasses, including grasses and cereals. In addition to providing nitrogen, non-symbiotic nitrogen-fixing bacteria can indirectly promote plant growth by secreting growth hormones, promoting phosphorus solubilization and straw degradation, and enhancing disease resistance and stress resistance. In addition, non-symbiotic nitrogen-fixing bacteria also have the ability to degrade cellulose, hemicellulose and lignin, thereby promoting the degradation of litter and straw, and promoting soil fertilization and nitrogen supplementation. In the face of a series of negative effects such as the decline of soil fertility, non-point source pollution and increase of greenhouse gas emissions caused by the long-term and extensive use of chemical fertilizers, the research and development of non-symbiotic nitrogen fixation is of special significance to the sustainable development of agriculture. However, the current research on non-symbiotic nitrogen-fixing microorganisms is relatively weak, especially the mining and application of excellent strain resources.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a strain HB161398 with nitrogen fixation activity, and the strain has good nitrogen fixation activity.

The purpose of the present invention is also to provide the application of the above-mentioned strain HB161398 in the preparation of fertilizers with nitrogen fixation.

The above-mentioned first object of the present invention is achieved by the following technical solutions: a strain HB161398 with nitrogen-fixing activity, the preservation number of the strain HB161398 is CGMCC NO.13939, the preservation date is September 18, 2017, the depository It is the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short), and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

The strain HB161398 was isolated from the mangrove silt of Qinglan Port, Wenchang, Hainan Province. It was identified as a new species of Mangrovela, a new genus of Rhodobacteraceae family, and named Mangrovela diazotropha.

The acetylene reducing activity (ARA) of nitrogen-fixing bacteria was measured by gas chromatography. The nitrogen-fixing activity of this strain was 178.10±23.42 nmol C ₂ H ₄ ·H ^-1 ·mL ^-1 .

Extract the genomic DNA of strain HB161398 (161398 strain was cultured on a solid plate to pick out a single colony or collect by liquid culture), and use the specific primers nifHf and nifHr for nitrogen-fixing genes to carry out PCR amplification, and the target band was amplified to prove that the strain It has a biological nitrogen fixation gene whose sequence is shown in SEQ ID NO: 2. The primers 27f and 1492r were used for PCR amplification, and the 16S rDNA sequence was obtained by sequencing analysis. The 16S rDNA sequence of the strain was shown in SEQ ID NO: 1.

The gene sequence of the obtained 16S rRNA was selected from the NCBI GenBank and EzBioCloud (http://www.ezbiocloud.net/; (Kim et al. 2012)) database to select a typical strain with high homology as a reference strain, The CLUSTAL X 1.8 program (Thompson et al. 1997) was used for multi-column alignment and the sequence similarity between the test strain and the reference strain was calculated. The MEGA5 software was used to construct a phylogenetic tree (bootstrap values=1000) by neighbor-joining method. ). It can be seen from the phylogenetic tree that it has the closest relationship with Poseidonocella pacifica JCM17310T (AB576005, and the 16S rDNA homology is 96.17%. Combined with the results of physiological and biochemical experiments, it is further shown that the bacterium belongs to a new species of Mangrovela, a new genus of Rhodobacteraceae family, with nitrogen-fixing activity. , named Mangrovela diazotropha.

The advantages of the present invention are: the bacterium has nitrogen-fixing activity and belongs to a new genus; in addition, the strain belongs to non-symbiotic nitrogen-fixing bacteria, can survive and fix nitrogen without forming a symbiotic system with other organisms, has a wide range of applications, and has good nitrogen-fixing activity, It can be used in bacterial fertilizers for vegetables, crops and mangroves.

Description of drawings

Fig. 1 is the morphology of strain HB161398 in Example 1 (electron microscope photo) (2500×);

FIG. 2 is a phylogenetic tree of strain HB161398 in Example 4. FIG.

Detailed ways

The present invention will be further described below through the following specific embodiments, but the content of the present invention is not limited to this at all.

Example 1 Isolation of strain HB161398

无氮培养基的三角瓶中并放置于摇床上以200r/min振荡30min，使土样充分分散。取出样品静置富集5天后取上层液体进行梯度稀释，取10^-3-10^-6稀释度涂布于

无氮培养基平板上，每个梯度2个重复。28℃倒置培养5-7d。挑取菌落较大且呈透明黏稠状的菌株进行平板划线法进行纯化。纯化后进行16s rDNA以及nifH(固氮)基因序列的扩增并鉴定，对菌株编号并于斜面4℃和甘油管-80℃保存。菌株形态如图1所示，菌体呈椭圆形(1.5-2μm长，0.8-1.25μm宽)。Mix the collected mangrove bottom mud (mangrove bottom mud from Qinglan Port, Wenchang, Hainan Province) and weigh 10g in a 100mL container.

The soil samples were fully dispersed in the Erlenmeyer flask of nitrogen-free medium and placed on a shaker at 200 r/min for 30 min. Take out the sample and let it stand for enrichment for 5 days, take the upper layer liquid for gradient dilution, take 10 ^-3 -10 ^-6 dilution and spread it on

2 replicates per gradient on nitrogen-free medium plates. Invert at 28°C for 5-7 days. The strains with larger colonies and transparent viscous shape were picked and purified by streak plate method. After purification, 16s rDNA and nifH (nitrogen fixation) gene sequences were amplified and identified, and the strains were numbered and stored at 4°C in a slant and -80°C in a glycerol tube. The morphology of the strains is shown in Figure 1, and the cells are oval (1.5-2 μm long and 0.8-1.25 μm wide).

无氮培养基(简写：DRN)的成分包括：in

The composition of nitrogen-free medium (abbreviation: DRN) includes:

pH为7.0。

pH was 7.0.

This strain HB161398 has the following microbial properties:

(1) Morphology and culture characteristics

无氮培养基上生长较慢，4-5d后菌落形成，后逐渐扩大，呈黏稠状的圆形半透明菌落。具体特征见表1。Strain HB161398 grows well and fast on nutrient agar, R2A agar and 2216E agar medium, and clear colonies can be seen on 1d. The colonies are round, milky white to light yellow, and the surface is smooth and convex;

The growth on nitrogen-free medium is slow, colonies are formed after 4-5 days, and then gradually expand to form viscous round translucent colonies. The specific characteristics are shown in Table 1.

Table 1 Culture characteristics of strain HB161398

(2) Multiphase classification features

Among the 12 carbon sources of API 20NE, strain HB161398 has strong ability to utilize D-mannitol and maltose, weakly utilizes glucose and N-acetyl-glucosamine, but does not utilize arabinose, mannose, capric acid, and oxalic acid. , citric acid, gluconate, malic acid, phenylacetic acid. Produces cytochrome oxidase, urease, b-glucosidase, nitrate reductase, and weakly produces H ₂ O ₂ enzyme. No starch hydrolase, tyrosine hydrolase, b-galactosidase, arginine dihydrolase, no acidification of glucose, no H ₂ S production, see Table 2 for details. The growth adaptation range of strain salt concentration is 3%-6%. The suitable pH for growth is 5-9, and it can grow weakly at pH 10. Cell wall polysaccharides are ribose, xylose, arabinose and galactose. Polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), glycolipid (GL), phosphatidylmethylethanolamine (PME). The main respiratory quinone of the strain is Q-10. Fatty acids are mainly Summed Feature 8 (C18:1 w7c and/or C18:1w6c) (52.0%), C19:0cyclo ω8c (26.8%), C16:0 (7.0%) and C18:1 ω7c 11-methyl (4.0%) ). The G+C content was 69.06%. The above results combined with phylogenetic analysis showed that strain HB161398 belonged to a new species of the new genus Mangrovela of Rhodobacteraceae family, named Mangrovela diazotropha.

Table 2 Comparison of differences between strain HB161398 and related genera in family Rhodobacteraceae

Strain: 1, HB161398T, 2, Poseidonocella pacifica KMM 9010T (data from Romanenko et al. (2012)), 3, Pseudodonghicola xiamenensis Y-2T (data from Tan et al. (2009) and Hameed et al. (2014)), 4, Roseovarius tolerans DSM 11457T (data from Labrenz et al. (1999)), 5, Donghicola eburneus SW-277T (data from Yoon et al. (2007)), 6, Actibacterium mucosum KCTC 23349T (data from Park et al. (2014) and Lucena et al. (2012).

+Positive, -Negative, w weak reaction, ND No data available. DPG, Diphosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; GL: glycolipid; PG, phosphatidylglycerol; PME, Phosphatidylmethylethanolamine; SQDG, sulfoquinovosyldiacylglycerol; AL, unidentified aminolipids; ; L, unidentified lipids.

Example 2 Cultivation of strain HB161398

The strain HB161398 can be preserved or temporarily stored by slant culture, or the strain HB161398 can be activated or transferred by plate culture, as follows:

(1) Slope culture: From the purified plate, pick a single colony of the strain and cultivate it for 3-5 days. Medium: peptone 10g, beef extract 3g, 50% Chenhai seawater 1000mL, pH 7.2, and stored at 4 ℃ refrigerator storage;

(2) Plate culture: From the purified plate, pick a single colony of the strain and cultivate it for 3-5 days. Medium: peptone 10g, beef extract 3g, 50% Chenhai seawater 1000mL, pH 7.2, and stored at 4 ℃ store in refrigerator.

Example 3 Detection of nitrogen fixation activity of strain HB161398

Inoculate the pure strain stored on the inclined plane in Example 2 into 10 mL of Dobereiner nitrogen-free liquid medium, and shake it at 30 °C for 72 h to measure the OD600. After unified adjustment, take 1 mL of each sterilized 5 mL vial and cover with a rubber stopper. . The rim of the cap is sealed with paraffin. 1 mL of acetylene was injected with a syringe and incubated under the same conditions for 24 h, and then 40 μL of the reacted gas was taken out of it, and the acetylene reducing activity (ARA) of nitrogen-fixing bacteria was measured by gas chromatography.

The calculation method of acetylene reduction activity is as follows, wherein the ratio of the amount of acetylene reduced by acetylene to the fixed nitrogen is converted by using: C ₂ H ₄ : N ₂ =4:1):

ARA[nmol/(mL·h)]=Vst×Cst×Asa×Vtu/Vsa/Ast/h/22.4

where Vst is the volume of injected standard gas (mL), Cst is the concentration of standard gas, Vtu is the volume of the test tube used (mL), Asa is the area of the sample ethylene peak (cm ² ), Vsa is the injected volume of the sample (mL), Ast is the standard gas peak area (cm ² ), h is the cultivation time, and the nitrogen-fixing activity of this strain is 178.10 nmol C ₂ H ₄ ·H ^-1 ·mL ^-1 .

Example 4 Amplification of strain 16S rDNA

Bacterial genomic DNA was extracted using the Bacterial Genomic DNA Isolation Kit (Foregene Biosciences, China). PCR amplification of the 16S rRNA gene was performed using bacterial genomic DNA as a template. The primers for the 16S rRNA gene were 27f (5'-AGAGTTTGATCATGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3') (Weisburg et al. 1991) The PCR amplification procedure is as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing temperature at 55 °C for 45 s, extension at 72 °C for 45 s, and after 30 cycles of extension at 72 °C for 10 min; the 16S rRNA gene amplification product was purified after , and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequence is shown in SEQ ID: NO: 1.

details as follows:

The obtained 16S rRNA gene sequences were selected from the NCBI GenBank and EzBioCloud (http://www.ezbiocloud.net/; (Kim et al. 2012)) database to select typical strains with relatively high homology as reference strains. CLUSTAL X 1.8 program (Thompson et al. 1997) performed multi-column alignment and calculated the sequence similarity between the test strain and the reference strain. The MEGA5 software was used to construct a phylogenetic tree by neighbor-joining method (bootstrap values=1000). , see Figure 2.

From the phylogenetic tree in Figure 2, it can be seen that it is the closest relative to Poseidonocella pacifica JCM 17310 ^T (AB576005, with 16S rDNA homology of 96.17%. Combined with the results of physiological and biochemical experiments, it can be seen that the bacterium belongs to a new species of the new genus Mangrovela of Rhodobacteraceae family. , named Mangroveladiazotropha (Poseidonocella diazotropha on the deposit certificate is a tentative taxonomic name suggested by the deposit center that is closer to this application).

Amplification of the NifH gene of the strain: Bacterial Genomic DNAIsolation Kit (Foregene Biosciences, China) was used for bacterial genomic DNA extraction. The PCR amplification of the nif H gene was carried out using bacterial genomic DNA as a template. The nif H gene primers were nifHf (TGCGAYCC(G/C)AARGCBGACTC) and nifHr(AT(G/C)GCCATCATYTCRCCGGA), and the PCR amplification program was as follows: 94 Pre-denaturation at ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing temperature at 55 ℃ for 30 s, extension at 72 ℃ for 30 s, followed by 30 cycles of extension at 72 ℃ for 10 min. After the Nif H gene amplification product was purified, it was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing, and the sequence was shown in SEQ ID: NO: 2.

details as follows:

Comprehensive above-mentioned physiological and biochemical characteristics, 16SrDNA sequence analysis results, the bacterial strain of the present invention belongs to the new species of Rhodobacteraceae family new genus Mangrovela, named as Mangroveladiazotropha (Poseidonocella diazotropha on the preservation certificate is suggested by the preservation center and This application is relatively close to the tentative classification name).

The isolated and purified strain HB161398 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short), whose address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC NO. .13939, deposited on September 18, 2017.

Embodiment 5 Effectiveness test of bacterial agent—effective test of vegetable planting

The activated single colony (obtained by plate culture in Source Example 2) was cultured in Zobell 2216E liquid medium (5 g of peptone, 1 g of yeast extract, 0.1 g of high iron phosphate, 1000 mL of Chenhai seawater, pH 7.6-7.8) at 28°C for 48 hours , Centrifugal collection of bacterial cells, mixed with 250 grams of coconut bran, inoculated in potted sand (each pot contains 2.5kg sand). Each pot of sand is mixed with 1 liter of fermentation volume.

The cabbage Brassica pekinensis (Lour.) Rupr. was selected for pot experiment. Compared with the uninoculated control after 30 days, the average dry weight of cabbage increased by 28%, and the nitrogen content in the plant increased by 25% on average. Therefore, the strain HB161398 can be used in the preparation of biological nitrogen-fixing bacterial fertilizers to promote the growth of vegetables and other crops.

Embodiment 6 Effectiveness test of inoculum - mangrove plant effectiveness test

Activated single colonies (obtained by plate culture in Example 2) were cultured in 2216E liquid medium (1 liter) at 28° C. for 48 hours, and the cells were collected by centrifugation for use.

The mangrove plant-Red sea lantern was used for pot experiment, and the collected bacteria were resuspended to prepare a bacterial suspension, and the roots of mangroves were soaked for 24 hours and then planted in plastic pots. The plants were directly planted in plastic pots, and the root length, leaf area and total chlorophyll of the plants were measured after 60 days. The results are shown in Table 3 below.

Table 3 The effect of strain HB161398 on red sea lantern embryos treated for 60 days

main root length Leaf area (cm²) total chlorophyll Bacterial suspension soaking 13.54 48.63 22.95 Not soaked in bacterial suspension 11.2 30.56 18.11

From the results in Table 3, it can be seen that the embryos soaked in the bacterial suspension developed faster, and the main root length, leaf area and total chlorophyll were higher than those of the control group, indicating that the strain HB161398 had a promoting effect on the development of the embryos of the red sea lanterns. This was due to the fact that the strain HB161398 has nitrogen-fixing activity, so it can promote the growth of plants, so the strain HB161398 can be used in the preparation of biological nitrogen-fixing bacterial fertilizer for promoting the growth of mangrove plants.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be an equivalent replacement manner, which is included in the protection scope of the present invention.

sequence listing

<110> Hainan Institute of Tropical Oceanography

<120> A strain HB161398 with nitrogen-fixing activity and its application

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Claims (2)

1. a bacterial strain HB161398 with nitrogen-fixing activity is characterized in that: the preservation number of this bacterial strain HB161398 is CGMCCNO.13939, and the preservation date is September 18, 2017, and the preservation unit is the General Microorganism Center of China Microorganism Culture Collection Management Committee, The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. 2. The application of the strain HB161398 of claim 1 in the preparation of fertilizers with nitrogen fixation.

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