CN104277994B - One strain Sinorhizobium SCAUs65 and application thereof - Google Patents

Abstract

The invention discloses a strain of Sinorhizobium SCAUs65 and its application. The strain is isolated and purified from fresh soybean nodules. The strain was deposited in the Chinese Type Culture Collection Center of Wuhan University on March 14, 2014, with the preservation number CCTCCNO: M2014084, and the classification name was Sinorhizobium fredii. The strain was applied to the production of Sichuan soybean. The Sinorhizobia SCAUs65 of the present invention is an excellent broad-spectrum soybean rhizobium strain with strong symbiotic nitrogen fixation ability, wide adaptability to Sichuan soybean varieties, and ability to dissolve inorganic phosphorus, organic phosphorus and potassium, and strong stress resistance . And it has a good matching affinity with the main bean varieties in Sichuan. In different ecological zones, no nitrogen fertilizer is applied, and SCAUs65 is inoculated to increase soybean yield by more than 38.9%, which is significantly different from the control without inoculation.

Description

A strain of Sinorhizobium SCAUs65 and its application

technical field

The invention relates to the field of microorganisms, in particular to a strain of Sinorhizobium SCAUs65 and its application.

Background technique

Chemical nitrogen fertilizers have made great contributions to increasing crop yields, but they have high production costs, low utilization rates, and pollute the ecological environment. Biological nitrogen fixation has the advantages of low cost, large amount of nitrogen fixation, continuous nitrogen fixation process, and no pollution, which is conducive to the protection of the ecological environment and the sustainable development of agriculture, and is an effective means of saving costs and increasing efficiency in agricultural production. Great efforts are needed to develop environmentally friendly biological nitrogen fixation technologies in food production. The rhizobia-legume nitrogen fixation system is the most efficient in biological nitrogen fixation. Artificial inoculation of rhizobia in leguminous plants is an important agricultural measure to increase crop yield and quality and improve the ecological environment. Inoculation of rhizobia is an internationally recognized biological nitrogen fixation technology. The symbiotic system of soybean and soybean rhizobia is a typical representative of biological nitrogen fixation. The symbiosis between soybean and soybean rhizobia is the result of interaction and mutual selection in long-term evolution, and also the result of adapting to the environment. Other soybean-growing countries in the world have great advantages in applying the technology of soybean rhizobia. However, my country is the origin of soybeans and has a long history of planting soybeans. The co-evolution of rhizobia and soybeans has made the indigenous rhizobia with low nitrogen fixation efficiency in the soil have a strong matching affinity with soybean varieties, thus greatly reducing the efficiency of nitrogen fixation. The effect of rhizobia inoculants. Therefore, it is of great practical value to study the compatibility between rhizobia and soybean varieties.

To improve symbiotic nitrogen fixation, visible strains and soybean varieties should be two fundamental factors. However, the population distribution of rhizobia has geographical limitations. In the screening of rhizobia, it is necessary to pay attention to its adaptability to the environment of the application area. Generally speaking, the most effective rhizobia in a certain area often come from strains in this area or areas with similar conditions to this area. Therefore, in the selection of rhizobia, it is necessary not only to carry out research on the matching combination of rhizobia and soybean varieties, but also to pay attention to the regional characteristics of bacterial agent application.

Soybean is the fourth largest crop in China. It is deeply loved by the Chinese people. It is eaten in a variety of ways and has a wide range of uses. It is an important food crop and oil crop in my country. Therefore, it is very meaningful to carry out the screening and application technology research of excellent soybean rhizobia strains in our country. The northeast soybean production area is the largest soybean production area in my country, and the Huanghuaihai area is the second largest soybean production area in my country. There are relatively many studies on excellent strains that match the main soybean varieties and ecological environments of these two largest production areas. For the arid Northwest soybean producing area, the screening of excellent soybean rhizobia has also been reported. For the first production area of soybean in my country: the Northeast production area, Du Yinghui and others isolated from Heilongjiang an excellent strain that is well matched with the Northeast soybean variety and suitable for the Northeast region, and was granted an invention patent in June 2012. Aiming at the acidic soil in South China, Cao Guiqin and others selected and bred three rhizobium strains resistant to acid and aluminum for the phosphoric acid-deficient red soil in South China, and applied for three invention patents in December 2007. However, there are few reports on excellent rhizobia that match the soybean varieties and ecological environment in Sichuan soybean production areas in southern China.

Soybean in Sichuan has always been a minor crop, with scattered planting, low yield and small planting area. In recent years, soybean production in Sichuan has developed rapidly, with large-scale and large-scale contiguous planting, and the production level has increased significantly. It has become one of the main food crops in Sichuan Province, and its sown area ranks sixth in the country. In the Twelfth Five-Year Plan for Planting Industry Development (2011-2015), the Ministry of Agriculture listed Sichuan and Southwest Interleaved edible soybeans as one of the three major production areas in the country, and the National Development and Reform Commission included Sichuan-Chongqing soybeans in the National Twelfth Five-Year Plan Sichuan soybean, the main food crop, has attracted the attention of governments at all levels in the country and Sichuan Province. The soybean cultivation method in Sichuan is mainly intercropping and intercropping with corn, accounting for more than 90% of the soybean planting area in the province. Therefore, inoculation of good rhizobia in Sichuan soybean production, the intercropping system of Sichuan soybean can also alleviate the "ammonia repression" of nitrogenase activity of rhizobia. The leading varieties of intercropping soybeans in Sichuan are "Nandou 12" and "Gongxuan 1". The intercropping soybeans in Sichuan are mainly planted in the hilly areas of Sichuan, and the Panxi region, which is rich in light and heat resources, mainly produces spring soybeans or fresh soybeans. At present, no good soybean rhizobia that matches the main soybean varieties in Sichuan and is suitable for different ecological environments in Sichuan has not been screened out. For this reason, the selection of excellent soybean strains is carried out according to the main planting varieties in Sichuan and the main ecological environment for planting soybeans, so as to give full play to the biological nitrogen fixation effect, improve the application effect of biological nitrogen fixation in production practice, and reduce the application of chemical fertilizers, which is very important for ecological protection. It has important practical value to protect the environment, save energy and resources, and promote the sustainable development of agriculture.

Contents of the invention

The technical problem to be solved by the present invention is to provide a strain of Sinorhizobium SCAUs65 and its application in view of the deficiencies of the prior art.

Technical scheme of the present invention is as follows:

A strain of Sinorhizobium SCAUs65, named Sinorhizobium fredii SCAUs65 Sinorhizobiumfredii SCAUs65, was deposited in the Chinese Type Culture Collection Center of Wuhan University on March 14, 2014, and its preservation number is CCTCC NO: M2014084.

The Sinorhizobium SCAUs65 is applied to the production of soybeans in Sichuan.

The Sinorhizobia SCAUs65 of the present invention is an excellent broad-spectrum soybean rhizobium strain with strong symbiotic nitrogen fixation ability, wide adaptability to Sichuan soybean varieties, and ability to dissolve inorganic phosphorus, organic phosphorus and potassium, and strong stress resistance . And it has a good matching affinity with the main soybean varieties in Sichuan. In different ecological zones, no nitrogen fertilizer is applied, and SCAUs65 is inoculated to increase soybean yield by more than 38.9%, which is significantly different from the control without inoculation.

Description of drawings

Figure 1 is the colony morphology of Sinorhizobium SCAUs65 on YMA medium.

Fig. 2 is a phylogenetic diagram of the 16rRNA gene of Sinorhizobium SCAUs65.

Fig. 3 is a phylogenetic diagram jointly constructed by the three housekeeping genes glnII, atpD and recA of Sinorhizobium SCAUs65.

detailed description

The present invention will be described in detail below in conjunction with specific embodiments.

Example 1 Isolation, purification and preservation of Sinorhizobium SCAUs65

From the soybeans planted on the purple soil in the hilly area of Jingyang District, Deyang City, Sichuan Province, select the large and plump red root nodules on the main roots of robust plants, scrub the root nodules clean, pick off some root barks, dry them with paper and put them away. In a vial filled with anhydrous calcium chloride and covered with absorbent cotton. After soaking the collected root nodules in sterile water for imbibition in the laboratory, soak in 95% ethanol for 30 seconds to eliminate surface tension, then disinfect the surface with 0.1% (m/v) mercury chloride for 5 minutes, and then rinse with sterile water 6 times, in the case of aseptic operation, the single root nodule was clipped and then added to the YAM medium with Congo red (mannitol 10g, yeast powder 0.8g, KH ₂ PO ₄ 0.25g, MgSO ₄ .7H ₂ O0. 2g, CaCl ₂ .6H ₂ O0.1g, NaCl0.1g, 1% (m/v) sodium molybdate 2ml, 1% (m/v) boric acid 2ml, 1% (m/v) Congo red 2.5ml, pH6 .8-7.0, agar 18-20g, water 1000ml) and cultured in an incubator at 28°C.

After the colonies grow out, select colonies that do not absorb red and look like Rhizobium bacteria from the plate, dilute and streak on the plate for culture. Observe the colony shape at about 3 days, and observe until about 15 days, because Bradyrhizobium takes 6-15 days to appear colonies. Repeat dilution and streak separation until purification. Preliminary determination of whether it is rhizobia is based on the following two aspects: (1) Colony morphology on YMA medium added with Congo red: no red absorption, round, milky white, raised, neat edges without spreading, smooth surface, and relatively viscous , Wetter. The fast-growing rhizobium bacteria grow colonies after 3-5 days of culture, and the brady-rhizobia bacteria grow colonies after 6-10 days of culture. (2) Cell morphology: mark the confirmed rhizobia colonies, and prepare slides for Gram staining. The microscopic examination results of rhizobia show that the cells are small rod-shaped and consistent in shape, without spores, and often contain β-hydroxybutyrate in the cells Sour and ring-shaped, Gram-negative (G-). If the above-mentioned marked colony has the characteristics of the above two aspects, then the colony is inserted into the test tube slant of YMA medium for culture and preservation.

The Sinorhizobium SCAUs65 obtained by the separation and purification of this embodiment is a fast-growing rhizobia. When cultured on the YMA medium with Congo red, the bacterium does not absorb red, and the colonies are larger, round, milky white, with higher bulges, and moist , Slightly transparent, colonies grow around 4d. Microscopic examination by Gram staining was G-, showing a small rod shape.

The back grafting and matching test of embodiment 2 Sinorhizobium SCAUs65

The backgraft test of Sinorhizobia SCAUs65 was carried out by hydroponics. The soybean variety used in the test was "Nandou 12", a leading variety in Sichuan. ) and harvested after 46 days of planting. After successful back-grafting with "Nandou 12", the compatibility test with other main soybean varieties was carried out, and the hydroponics method was also carried out. The soybean variety used in the test: another main soybean variety "Gongxuan 1" for intercropping ", the main spring soybean variety "Nandou 8", and the main fresh soybean variety "Chunfengzao". After being cultivated in the above-mentioned light chamber for 1 week, they were cultured under normal light and temperature conditions, and harvested 41 days after planting. Regularly supplement sterile micro-nitrogen nutrient solution. Different combinations of Sinorhizobium SCAUs65 and the above-mentioned soybean varieties were formed. The hydroponic device used a 250ml ampoule (glass infusion bottle for hospital use), and soybean plants of the same variety that were not inoculated with Sinorhizobium SCAUs65 were used as a control. After harvest, the nodule number and plant dry weight of soybean plants were used to evaluate the inoculation effect of Sinorhizobium SCAUs65. The bacterial culture, seed germination, hydroponics production and planting methods of the back-grabbing test and the matching test are the same.

(1) Bacterial culture: Inoculate the above-mentioned Sinorhizobium SCAUs65 into YMA liquid medium, place on a shaker at 120 rpm/min, and culture at 28°C until the logarithmic growth phase (about 4 days).

(2) Germination of seeds: Select large, plump soybean seeds without damage, soak them in 95% alcohol for 5 minutes, pour off the alcohol, and add 0.1% (m/v) mercuric chloride solution to disinfect the surface for 5 minutes. Finally, wash with sterile water 4 to 6 times, 5 minutes each time. Accelerate germination at 28°C, and sow when the main root grows to about 2 to 3 cm and the fibrous roots do not grow.

(3) Production of the hydroponic device: Use a 250ml ampoule (glass infusion bottle for hospital use) as the hydroponic device. First make sterile micro-nitrogen nutrient solution, formula of micro-nitrogen nutrient solution: 0.46g calcium sulfate, 0.075g potassium chloride, 0.06g magnesium sulfate, 0.03g calcium nitrate, 0.075g ferric citrate, 0.136g dipotassium hydrogen phosphate, 1000ml Distilled water, 1ml trace elements (2.86g boric acid, 0.02g molybdic acid, 0.8g copper sulfate, 0.22g ZnSO ₄ zinc sulfate, 1.81g manganese sulfate, add distilled water to 1000ml). Pour the prepared micro-nitrogen nutrient solution into the cleaned bottle, cover the bottle mouth with a layer of kraft paper, open a small hole (about 0.6cm in diameter) in the center of the bottle mouth, plug the small hole with cotton, and cover it with a layer of high temperature resistant Plastic film, sterilized at 121°C for later use.

(4) Planting and measurement indicators: Put the germinated seeds in a sterile petri dish and soak them in the bacterial solution for 15 minutes, insert the roots of the seedlings into the small holes of the hydroponic device with sterile tweezers, one plant per bottle, and then plant Add 1ml of bacterial solution to the seedlings, and plug the cotton in the original hole around the seeds to prevent dust from falling into the bottle and causing pollution. In addition, the plants of the same variety without inoculation treatment were set as the control (CK). When planting, plant CK first. Each treatment was repeated 3 times. The results of the hydroponic experiments are listed in Table 1.

The results in Table 1 show that the Sinorhizobium SCAUs65 has good matching affinity with the four tested soybean varieties, and all show better nodulation ability and symbiotic nitrogen fixation ability; SCAUs65 can significantly increase the plant dry weight of each soybean variety, which is 24%-55% higher than the control not inoculated with Sinorhizobium SCAUs65. It can be seen that the Sinorhizobium SCAUs65 is an excellent broad-spectrum strain with good compatibility with Sichuan soybean varieties.

Table 1 Hydroponic test results of Sinorhizobium SCAUs65

Note: The data are the average value of three replicates; ** and * indicate that the dry weight between the inoculated treatment and the corresponding control reaches a significant level of 1%, 5%, respectively.

The stress resistance of embodiment 3 Sinorhizobium SCAUs65

The stress resistance of Sinorhizobium SCAUs65 was mainly determined by acid and alkali tolerance, salt tolerance and growth greenhouse range. The YMA medium was used as the basal medium, and the YMA plate cultured at pH 7 and 28°C for 7 days was used as the positive control. The above-mentioned YMA slant culture of Sinorhizobium SCAUs65 was scraped and washed with sterile water for use. The point inoculation method was adopted and repeated 3 times. YMA medium was used as the basal medium for the determination of acid and alkali resistance, and the pH value was adjusted with HCl and NaOH. The pH value was 4.0, 5.0, 6.0, 8.0, 9.0, 10.0, 11.0, and 12.0. Salt tolerance determination also uses YMA medium as the base medium, and the strains are spot-inoculated on a plate containing NaCl, and the mass volume fraction of NaCl is 1%, 2%, 3%, 4% and 5%. Plates for acid and alkali resistance and salt resistance tests were cultured at 28°C for 7 days, and the results were observed and recorded. For the determination of the growth temperature range, the strains were inoculated on YMA medium, and 5 temperature treatments were set up, and they were cultured in biochemical incubators at 4°C, 10°C, 37°C, and 40°C for 30d, 10d, 7d, and 7d, respectively. After heat shock treatment at 60°C for 30 minutes, transfer to 28°C for 7 days. The test results show that Sinorhizobium SCAUs65 has strong stress resistance ability and can grow on the plate with pH5～12, but the colony on the plate with overacid or overalkali is smaller than the positive control with pH7, which shows that overacid and overalkali have a certain effect on its growth. Inhibitory effect; it has certain salt tolerance and can grow on YMA plates with 1% NaCl; the growth temperature range is wide, and it can grow in the temperature range of 10-37 °C, and the strain can still survive after heat shock treatment at 60 °C for 30 minutes. Can survive, indicating that the strain can tolerate high temperature for a short time.

The growth-promoting ability of embodiment 4 Sinorhizobium SCAUs65

74% of the cultivated land in China is deficient in phosphorus, and more than 95% of the phosphorus in the soil is difficult for plants to directly absorb and utilize. However, the utilization rate of artificially applied phosphate fertilizers in the current season is only 5% to 25%, and most of them are quickly chemically fixed by the soil to form closed-storage insoluble phosphates. This "high input, low output" ecological chain not only causes a lot of waste of phosphorus resources, but also increases the environmental pollution caused by the consumption of resources. Although China's soluble potassium resources are scarce, potassium-containing rocks and potassium-containing mineral particles in soil are very rich, but the potassium in these potassium-containing minerals mainly exists in the form of insoluble potassium-containing silicates, and cannot be directly used for crops. Absorb and utilize. Therefore, improving the utilization efficiency of atmospheric nitrogen and soil phosphorus and potassium by screening functional microorganisms such as efficient nitrogen fixation, phosphorus dissolution and potassium dissolution has been recognized as a cheap and efficient biological measure with great environmental protection significance.

The growth-promoting ability of Sinorhizobium SCAUs65 mainly examined its ability to dissolve phosphorus and potassium.

(1) The ability to dissolve organic phosphorus and inorganic phosphorus

Use the phosphorus-dissolving circle method. The organic phosphorus source is lecithin, and the inorganic phosphorus source is calcium phosphate (Ca ₃ (PO ₄ ) ₂ ), aluminum phosphate (AlPO ₄ .2H ₂ O), and iron phosphate (FePO ₄ .2H ₂ O), all of which are commercially available for analysis Pure reagents.

Montkina medium for measuring the ability to dissolve organic phosphorus, formula (g/L): 10g glucose, 0.5g(NH ₄ ) ₂ SO ₄ , 0.3g NaCl, 0.3g KCl, 0.03g FeSO ₄ .7H ₂ O, 0.03g MnSO ₄ .4H ₂ O, 0.2g lecithin, 5g CaCO ₃ , 0.4g yeast powder, 20g agar, 1000ml distilled water, pH 6.8-7.0. Among them, the lecithin is heated and dissolved with 75% alcohol, sterilized separately, mixed with the sterilized medium cooled to about 60°C, and poured onto a plate.

PKO medium for measuring the ability of dissolving inorganic phosphorus, formula (g/L): 10g glucose, 3.0g of the above inorganic phosphorus source material, 0.5g (NH ₄ ) ₂ SO ₄ , 0.2g NaCl, 0.2g KCl, 0.03 g MgSO ₄ .7H ₂ O, 0.03g MnSO ₄ , 0.003g FeSO ₄ .7H ₂ O, 0.5g yeast powder, 20g agar, 1000ml distilled water, pH 6.8-7.0. Among them, calcium phosphate, aluminum phosphate, and iron phosphate were crushed with a mortar and passed through a 300-mesh sieve, and after dry heat sterilization alone, they were mixed with the culture medium whose sterilization temperature was lowered to about 60°C and poured onto a plate for use. The strain preparation and spot inoculation methods were the same as those in Example 3 for the stress resistance test, and were repeated 3 times. After culturing in an incubator at 28°C for 7 days, observe whether the strain grows and whether there is a phosphorus-dissolving circle. The results showed that Sinorhizobium SCAUs65 did not grow on the aluminum phosphate plate, and had certain solubility to calcium phosphate, iron phosphate and lecithin. The diameter ratios are 1.17, 1.13, 1.08, respectively.

(2) Potassium dissolving ability

Medium for measuring potassium dissolving ability (g/L): 5.0g sucrose, 2.0g Na ₂ HPO ₄ , 0.05g MgSO ₄ .7H ₂ O, 0.05g FeCl ₃ , 0.1g CaCO ₃ , 5g potassium feldspar powder, 20.0g agar , distilled water 1000ml. Among them, the potassium feldspar powder is ground into a powder, passed through a 300-mesh sieve, washed with deionized water to remove water-soluble K and Si plasma, and dried in the shade. After dry heat sterilization alone, mix with the sterilized medium cooled to about 60°C, pour it on a plate, and set it aside. The strain preparation and spot inoculation method were the same as the above-mentioned determination of phosphorus dissolving ability, repeated 3 times. After culturing in an incubator at 28°C for 7 days, observe whether the strain grows, because the medium uses potassium feldspar powder as the only potassium source, and if it grows, it indicates that the strain has the ability to dissolve potassium. The test results showed that Sinorhizobium SCAUs65 grew normally on the plate with potassium feldspar powder as the only potassium source, indicating that Sinorhizobium SCAUs65 had a certain ability to dissolve potassium.

Example 5 Amplification and phylogenetic analysis of the 16S rRNA gene and other housekeeping genes glnII, atpD, recA of Sinorhizobium SCA Us65

The total DNA of the strain was extracted, and the above-mentioned 4 genes were amplified by PCR with the primers shown in Table 2. The PCR reaction was carried out with a Bio-RAD MyCycler TM instrument. After the PCR amplification product was detected on 1.0% agarose gel electrophoresis, it was sent to Go to the handsome company for sequence determination. The calculation of gene sequence similarity was carried out with the software DNAman6.0.

Table 2 PCR primers used in this test

Note: Y=CorT,H=A,CorT,R=AorG,S=CorG,K=GorT,N=A,C,GorT,I=inosine,M=AorC,N=anybase..

(1) Amplification of 16SrRNA gene and construction of phylogenetic tree

Using the total DNA as a template, the 16S rRNA gene was amplified with the general primers P1 and P6 in Table 2. PCR reaction system (50 μl): 25 μl of 2×PCR Mix, 1 μl of primers P1 and P6 (20 μM), 1 μl of DNA template, add ultrapure water to make up to 50 μl. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 56°C for 30 s, extension at 72°C for 1 min, and 30 cycles; final extension at 72°C for 10 min. The amplified product was detected by the above method, and the result of sequencing by Yingjun Company is shown as SEQ ID No1.

SEQIDNo1 sequence:

TACAGCGGGCAGGCTTACACATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCTTTTCTACGGAATAACGCAGGGAAACTTGTGCTAATACCGTATGAGCCCTTCGGGGGAAAGATTTATCGGGAAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGACATTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGGTGTCTAGAGTCCGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCGGTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTTTACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATCCCGATCGCGGATACGAGAGATCGTATCCTTCAGTTCGGCTGGATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTCTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGGTAGGTCAGCGAACGT

The obtained sequence results were compared at the National Center for Biological Information (NCBI), and it was found that the model strain with the highest similarity to the 16SrRNA gene sequence of Rhizobium SCAUs65 was Sinorhizobium fredii USDA205 ^T , with a similarity of 99.4%. Using the results of sequence alignment on NCBI, the type strains with high similarity were selected as reference strains to construct a phylogenetic tree. The 16S rRNA gene phylogenetic tree was constructed using the Neighbor-joining method in the Mega5 software, and the bootstrap value (bootstrap) was 1000. The phylogenetic tree is shown in Figure 2.

(2) Construction of joint phylogenetic tree of multi-locus gene sequences

In order to further and more accurately determine the taxonomic status of Sinorhizobium SCAUs65, another three loci of housekeeping genes atpD, recA and glnII sequences were selected to construct a joint phylogenetic tree.

Primers recAF1 and recAR1 were used to amplify recA, primers atpDF1 and atpDR were used to amplify atpD, and primers GSII-1 and GSII-2 were used to amplify glnII. The primer sequences are shown in Table 2. The reaction system is 50 μl, and the composition of the reaction solution is as follows: the reaction system (50 μl) is: 2×PCR Mix 25 μl; 10 mM forward primer and reverse primer 0.5 μl each; DNA template 1 μl; ddH ₂ O 23 μl. (1) The PCR amplification reaction procedures of recA and atpD were the same: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 59°C for 45 s, extension at 74°C for 1.5 min, and 30 cycles; final extension at 74°C for 6 min. (2) glnII amplification conditions: pre-denaturation at 92°C for 3 min; denaturation at 94°C for 1 min, annealing at 55°C for 1.5 min, extension at 72°C for 2 min, and 30 cycles; final extension at 72°C for 10 min. The amplified products were detected by the above method and then sent to Yingjun Company for sequencing. Each gene was sequenced in two directions (sequence of forward and reverse primers), and then the sequences of forward and reverse primers were spliced with DNAman6.0 software, and the sequences of forward and reverse primers were removed. After sequencing, the atpD sequence size is 496nt, the sequence result is SEQ ID No2, the glnII sequence size is 578nt, the sequence result is SEQ ID No3, the recA sequence size is 499nt, and the sequence result is SEQ ID No4.

SEQ ID No2 gene sequence:

CATCGGCGAGCCGGTTGACGAAGCCGGCCCGCTCAAGACCTCGGCCCGCCGTGCGATCCACCAGGACGCTCCCTCCTACGTCGAACAGTCGACGGAAGCGCAGATCCTCGTCACCGGCATCAAGGTCGTCGACCTGCTTGCGCCTTACGCAAAGGGCGGCAAGATCGGTCTCTTCGGTGGCGCGGGCGTCGGCAAGACCGTTCTGATCATGGAACTGATCAACAACGTCGCCAAGGCGCACGGTGGTTATTCGGTCTTCGCCGGTGTGGGTGAACGTACCCGCGAAGGCAACGACCTTTATCACGAAATGATCGAATCGGGCGTGAACAAGCACGGCGGCGGCGAAGGCTCCAAGGCCGCTCTCGTCTACGGCCAGATGAACGAACCGCCGGGCGCGCGTGCTCGCGTTGCTCTGACCGGCCTGACGGTTGCCGAACAGTTCCGTGACGAAGGTCAGGACGTTCTCTTCTTCGTCGACAACATATTCCGCTTCACC

SEQ ID No3 gene sequence:

ACGCGTTCCCGGCGCTCGAGCAGCTTCCGCTCTGGGGTTTCGACGGCAGCTCGACGAACCAGGCCGAAGGCCGCAGCTCCGATTGCGTGCTGAAGCCGGTTGCCGTCTATCCGGATCCGGTCCGCACCAACGGCGTACTGGTCATGTGCGAAGTCATGATGCCGGACGGCAAGACGCCGCATCCGTCGAACAGCCGCGCCACGATCCTCGACGACGAAGGCGCCTGGTTTGGCTTCGAGCAGGAATACTTCTTCTACAAGAACGGCCGCCCGCTCGGCTTCCCGGAGCAGGGCTATCCGGCGCCGCAGGGCCCGTACTACACCGGTGTCGGCTATTCGAATGTCGGTGATGTCGCCCGCAAGATTGTCGAAGAGCATCTCGACATCTGCCTGGCCGCCGGCATCAACCATGAAGGCATCAACGCCGAAGTCGCCAAGGGCCAGTGGGAATTCCAGGTCTTCGGCAAGGGCTCCAAGAGGGCTGCCGACGAAGTGTGGATGGCCCGCTACCTGCTGCAGCGCCTGACCGAAAAATACGGCATTGACGTCGAGTACCACTGCAAGCCGCTCGGCGACACC

SEQ ID No4 gene sequence:

CAAAAGCAAGGCACTTGAAGCGGCTCTTTCCCAGATCGAACGTTCGTTCGGCAAGGGATCGATCATGAAGCTCGGATCGAAGGACAGCGTCATCGAGATCGAAACCGTCTCGACCGGATCGCTCGGCCTCGATATCGCGCTCGGCATCGGCGGCCTGCCAAAGGGGCGCATTGTTGAGATTTACGGGCCGGAAAGCTCGGGCAAGACGACGCTGGCCCTGCAGACCATTGCCGAGGCGCAAAAGAAAGGCGGAATCTGCGGCTTTGTCGACGCCGAACATGCGCTCGATCCGATTTATGCGCGCAAACTGGGCGTCGACCTCGAAAACCTGCTGATCTCGCAACCCGATACGGGCGAGCAGGCGCTCGAAATCACCGACACCCTGGTTCGCTCCGGCGCAATCGATGTGCTCGTCGTCGACTCGGTTGCAGCACTCGTGCCGCGTGCCGAAATCGAAGGTGAGATGGGCGACAGCCTGCCGGGCATGCAAGCCCGCCTG

The obtained sequence results were compared at the National Center for Bioinformatics (NCBI), and it was found that the type strain with the highest sequence similarity to the housekeeping genes atpD, glnII and recA of Sinorhizobium SCAUs65 was Sinorhizobium fredii USDA205 ^T , and the similarities with the model strain were 99.8%, 98.2%, 100%, respectively. Using the results of comparison of each gene sequence on NCBI, the type strain with high similarity with the three genes was selected as the reference strain for tree construction.

Construction of 3-gene (atpD, glnII, and recA) joint phylogenetic tree: Firstly, the sequences of atpD, glnII, and recA 3 housekeeping genes were respectively compared with the corresponding gene sequences of the reference strain, compared with MEGA5, and cut with the minimum length as the standard. The trimmed sequences were saved in FASTA format. The lengths of the trimmed three gene sequences were 462nt (atpD), 545nt (glnII), and 384nt (recA). Open in Notepad format and stitch the 3 sequences together, use the neighbor-joining method (Neighbor-joining) in MEGA5 software to construct the joint phylogenetic tree, the bootstrap value (bootstrap) is 1000, atpD, glnII, recA joint phylogenetic tree As shown in Figure 3.

Figure 2 and Figure 3 show that the 16S rRNA gene of SCAUs65 and the joint sequence of the three housekeeping genes atpD, glnII, and recA are on the same branch node as the type strain Sinorhizobium fredii USDA205 ^T of Sinorhizobium fischeri. Also as analyzed above, the strain has a high similarity with the four genes of the type strain Sinorhizobium fredii USDA205 ^T , indicating that the strain SCAUs65 belongs to Sinorhizobium fredii (Sinorhizobium fredii).

Embodiment 6 field inoculation effect

The main production area of soybeans in Sichuan is the hilly area of Sichuan. The soybean variety is "Gongxuan No. 1", the main plant variety in Sichuan, which is mainly used as summer soybean and autumn soybean varieties. The habitats of the Panxi area, which is rich in light and heat resources, are quite different from the hilly areas. The Panxi area is mainly dominated by spring soybeans. In recent years, soybeans "Shishi 8" and "Chunfengzao" have been introduced as the main soybean varieties in this ecological area. . The field inoculation effect test of the strains was carried out in a typical major soybean producing area in Sichuan - the hilly area of Yucheng District, Ya'an City, Sichuan Province, and in Panxi area - Renhe District, Panzhihua City.

(1) Hilly area - field inoculation test in Yucheng District, Ya'an City, Sichuan

In this experiment, two treatments were set up, inoculated with Sinorhizobium SCAUs65 and the control treatment (CK) without inoculation, and the soybean variety "Gongxuan No. 1" was selected as the main cultivar in Sichuan. No chemical fertilizers were applied during planting. The experiment was conducted from June to October 2012. The prepared Rhizobium inoculum (the number of viable bacteria: 5.1×10 ⁸ CFU/g inoculum) was mixed with soybean seeds, dried in the shade and sown in holes, 6 grains per nest, 2 seedlings, the area of the plot was 4.2m ² , and the distance between litters was 40cm. The row spacing is 35cm, and CK is sown first when sowing, so as to avoid the influence of CK treatment by inoculating rhizobia. Sampling was carried out at the full flowering stage (64 days of growth), and the plant height, number of nodules, and dry weight of aboveground plants were measured; the yield was measured at the harvest stage (131 days of growth). The management during this period is carried out according to the routine management of soybean planting by farmers.

Table 3 Field inoculation effect in Yucheng District (hilly area) of Ya'an City

In the treatment of rhizobia inoculation, the plant height and dry weight of plants at the full flowering stage were higher than those of the control without inoculation, but not at a significant level; the number of nodules was significantly increased compared with CK (F=8.341*); the yield was significantly increased compared with CK (F =23.360**), an increase of 38.9%. It can be seen that the inoculated excellent rhizobia can promote the growth of plants after the full flowering stage is more obvious. The Sinorhizobium fredii SCAUs65 is an excellent rhizobia suitable for the production of "Gongxuan No. 1" in this ecological zone (Sichuan hilly area).

(2) Indirect field experiment in Panxi area

In this experiment, the main bean variety "Yu 8" was selected as the local bean variety, and two treatments were set up, one was inoculated with Sinorhizobium SCAUs65 and the other was not inoculated with control (CK), and three repetitions were set up. Before planting, apply the base fertilizer according to P ₂ O ₅ 45kg/ha, K ₂ O 45kg/ha, and put it in at one time according to the unified standard. The phosphate fertilizer is calcium superphosphate, and the potassium fertilizer is potassium chloride. The experiment adopts random block design, ridge planting, 4 ridges per plot, ridge width 60cm, interval between ridges 20cm, ridge length 5m. Two rows were planted on the ridge, the row spacing was 40cm, the nest spacing was 30cm, and 32 litters were planted on each ridge. The experiment was conducted from May to September 2013. The Sinorhizobium SCAUs65 bacterial agent was mixed with the soybean "Shi No. 8", and after drying in the shade, it was planted in holes, with 5 grains per litter and 2 seedlings. When sowing, CK was also sown first. Sampling was carried out at the initial pod stage (growth period 67 days), and the plant height, number of nodules, and dry weight of aboveground plants were measured; the yield was measured at the harvest stage (growth period 98 days). The management during this period is carried out according to the conventional management of soybean planting by local farmers.

Table 4 Field inoculation effect in Panxi area

In the treatment of rhizobia inoculation, the plant height (F=8.234*), plant dry weight (F=9.183*), root nodule number (F=50.944**) and yield (F=35.401**) at the initial pod stage were compared with those of Significant increase in non-vaccinated controls (CK). It can be seen that the Sinorhizobium fredii SCAUs65 has a good match with the main soybean variety "Shi No. 8" in Panxi, Sichuan, and is an excellent rhizobium suitable for soybean production in the Panxi ecological environment.

Field inoculation experiments in two typical ecological areas in Sichuan found that after inoculation of the Sinorhizobium SCAUs65, the dry weight of the plant, the number of nodules and the yield were all higher than those of the control without inoculation, and there was a very significant increase in yield, which could increase yield by 39% %above. It can be seen that the Sinorhizobium SCAUs65 isolated by the present invention can be widely used in soybean production in Sichuan.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present invention.

Claims (2)

1. A strain of Sinorhizobium SCAUs65, characterized in that its preservation number is CCTCC NO: M2014084. 2. The application of Sinorhizobium SCAUs65 according to claim 1, characterized in that it is applied to the production of soybeans in Sichuan.