CN107904194B - Common vetch rhizobium strain VS13-1 and application thereof - Google Patents

Abstract

A common vetch Rhizobium strain VS13-1 is obtained by separating and purifying from fresh common vetch Rhizobium and belongs to Rhizobium rhizuiense strains. The strain is preserved in China center for type culture Collection of Wuhan university at 23.10.2017 with the preservation number: CCTCC No: m2017607. The common vetch rhizobia VS13-1 disclosed by the invention is applied to production of common vetch in Sichuan, is an excellent common vetch rhizobia strain which is strong in symbiotic nitrogen fixation capacity, obvious in yield increase effect on common vetch, and strong in IAA secretion capacity, phosphorus dissolution capacity, potassium dissolution capacity and stress resistance, and can increase the yield of fresh common vetch by more than 76.5% and increase the yield of fresh root by 68.7% under the condition that nitrogen fertilizer is not applied and other fertilizers are only inoculated with rhizobia VS 13-1.

Description

Common vetch rhizobium strain VS13-1 and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to a vicia sativa strain VS13-1 and application thereof.

Background

Common vetch (Vicia sativa L.), also called vetch, belongs to a cultivated variety of Vicia genus of Leguminosae (Leguminosae), has large main roots, shallow soil, multiple root nodules and common vetch leaves, is named as common vetch, has the original production place in southern Europe and western Asia, is introduced into China for 20 th century and cultivated in the beginning, and has the planting range of China up to 29 provinces (regions) such as Qinghai, Jiangsu, Hunan, Taiwan and the like (Hongkuxing and the like, 1985). Common vetch is used as an excellent crop combining feed and green manure, has high nutritive value, tender stems and branches, a large amount of leaves and good palatability, and is a relatively common green manure germplasm resource used in production (Lubielin et al 2015; Zhao Jing 2015). The common vetch also has the characteristics of drought resistance, barren resistance and strong stress resistance, can inhibit soil degradation through the biological nitrogen fixation effect and save the use amount of nitrogen fertilizer, and is good forward cropping of various crops. In the production process of leguminous plants, the fertilizer is not applied, and only the high-efficiency rhizobia is used for inoculation so as to achieve the purpose of increasing the yield of crops. For example, soybeans planted in Brazil are inoculated with only effective rhizobia without applying nitrogen fertilizer, the yield is equivalent to 400kg of fertilizer per hectare, and the saved nitrogen fertilizer per year is up to 25 hundred million dollars. By inoculating rhizobia in the United states, the annual biological nitrogen fixation amount is about 620 ten thousand t, which accounts for more than 55% of the total nitrogen fertilizer consumed in the United states in the same year. Therefore, artificial inoculation of rhizobium is an important agricultural measure for promoting the growth of leguminous plants, increasing the yield of leguminous crops and improving the ecological environment.

At present, screening research reports about root nodule bacteria of common vetch are mostly concentrated in northwest areas such as Qinghai and Gansu. Wangxuecui (2016) collects 61 rhizobia from common vetch in Qinghai main producing area to be separated and screened for a water culture tieback test and in-vitro salt tolerance evaluation, and 5 rhizobia are found to have good nodulation effect but different salt tolerance, wherein the salt tolerance limit of the 5 rhizobia is 0.9% -1.2%, and finally two rhizobia J3-12-1 and J5-5-3 which have both good nitrogen fixation effect and salt tolerance are obtained through a pot culture test. Duckweed (2015) select 3 a vicia sativa root nodule bacteria to carry out its nodulation azotobacter of experimental study of cultivated in a pot to 3 vicia sativa varieties, discover to compare with not inoculating the fungus and handle, inoculate root nodule bacteria and can show improvement vicia sativa plant height, the fresh weight of root nodule, the full nitrogen content of plant and azotase activity. A field verification experiment is carried out in summer river county of Gansu south China, and it is found that rhizobium inoculation obviously promotes the growth of common vetch, the ratio of hay yield to CK is increased by 21.2% -28%, and the selected strain can promote the growth of common vetch. However, the population distribution of rhizobia has geographical limitations, and in the efficient screening of rhizobia, attention needs to be paid to the adaptability of rhizobia to the environment of an application area (old and new, et al, 2004). In general, the rhizobia that is most effective in a certain area is often derived from a local or a strain in a region similar to the local conditions (Jia et al, 2008; Chengxin et al, 2011). At present, the research of the high-efficiency rhizobia of common vetch suitable for the ecological environment in Sichuan is almost blank.

The existing research shows that few rhizobia also have the growth promoting characteristics of dissolving phosphorus, secreting growth hormone (IAA) and the like. St.zuki (2007) and the like preliminarily screens 730 strains of Rhizobium meliloti to obtain 29 strains of Rhizobium meliloti with better growth promoting capability, only 10 strains of Rhizobium meliloti with strong IAA secreting capability are screened from the 29 strains of Rhizobium meliloti, and the determination of organic phosphorus and inorganic phosphorus dissolving capability of the 29 strains is carried out, so that the results show that all strains have no inorganic phosphorus dissolving capability, the 29 strains of Rhizobium meliloti can dissolve organic phosphorus, but the difference of phosphorus dissolving capability is large, only 8 strains have stronger organic phosphorus dissolving capability, and the rest strains have weak or weak organic phosphorus dissolving capability. More than 200 rhizobia sojae atricolor strains are separated and screened from Sichuan, and 4 of the 4 strains of the rhizobia sojae atricolor have the advantages of good growth promoting capabilities of dissolving phosphorus, dissolving potassium, secreting IAA and the like and strong stress resistance, so that the 4 strains apply for 4 invention patents and are authorized. Therefore, the screening of high-quality and high-efficiency multifunctional growth-promoting rhizobium strains is particularly important, so that the rhizobium is developed from nitrogen fixation and growth promotion to nitrogen fixation, phosphorus dissolution and IAA secretion and multifunctional growth promotion.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a high-efficiency nitrogen-fixing rhizobium strain VS13-1 matched with a main Sichuan cultivar-common vetch, which has IAA secretion, certain phosphorus and potassium dissolving capacity and better field production and application prospects.

The technical scheme of the invention is as follows:

a Rhizobium victoriae strain VS13-1 is classified and named as Rhizobium anhuiensis VS13-1 and is preserved in the China center for type culture Collection of Wuhan university in 2017 at 23 and 10 months, and the preservation numbers are as follows: CCTCC NO: M2017607.

Common vetch rhizomatous strain VS13-1 is applied to production of common vetch in Sichuan.

The invention has the beneficial effects that:

the common vetch rhizobium VS13-1 disclosed by the invention is an excellent common vetch rhizobium strain which has strong symbiotic nitrogen fixation capacity, good compatibility to common vetch, obvious yield increase, IAA secretion capacity, phosphorus dissolving and potassium dissolving capacity and strong stress resistance, and can increase the yield of fresh common vetch by more than 76.5% and increase the yield of fresh root by 68.7% when no nitrogen fertilizer is applied and only VS13-1 is inoculated

Drawings

FIG. 1 shows the colony morphology of Rhizobium victoriae VS13-1 on YMA medium;

FIG. 2 is a phylogenetic diagram of the 16S rRNA gene sequence of Rhizobium victoriae VS 13-1;

FIG. 3 is a phylogenetic diagram of recA, atpD and glnII combined construction of rhizobium japonicum VS13-1 of common vetch.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1 isolation, purification and preservation of Rhizobium victoriae VS13-1

Selecting a big and full red root nodule on the main root of a robust plant from a wild common vetch collected from Rexingchengmen Zhen Ganmui of Ying county, Ming city, Sichuan province, cleaning the root nodule, collecting partial root bark, and performing surface disinfection: soaking in 95% ethanol for 5min to remove surface tension, sterilizing with mercuric chloride (0.1% m/v) for 5min, and washing with sterile water for 6-8 times. Placing the root nodule in a sterilized white porcelain plate under sterile environment, smearing the root nodule on a flat plate of YMA culture medium by using a bamboo stick to clamp the root nodule, carrying out streak purification, and culturing in an incubator at 28 ℃. YMA culture medium formula: KH (Perkin Elmer)₂PO₄0.25g，MgSO₄·7H₂O 0.2g，CaCl₂·6H₂O0.1g, NaCl 0.1g, ammonium molybdate (1%) 2mL, boric acid (1%) 2mL, Congo red (1%) 2.5mL, mannitol 10g, yeast powder 0.8g, agar 18-20 g, water 1000mL, pH 6.8-7.0.

After the fungus grows out, selecting the shape from the flat platecarrying out dilution streak culture on a single colony which does not absorb red and is similar to rhizobia in state on a plate, observing the colony morphology about 3d until about 7d is observed, and purifying the colony because the bradyrhizobia needs 6-15 d, and carrying out dilution streak plate separation method to preliminarily judge whether the colony is rhizobia or not from the following two aspects of (1) carrying out milky white colony morphology, no absorption of red, round colony morphology, uplift, regular and non-spreading edge, smooth surface, moistness and viscosity on the colony, culturing the colony which grows out for 3-5 d to be fast rhizobia, culturing the colony which grows out for 6-10 d to be slow rhizobia, and (2) carrying out cell morphology, marking the confirmed rhizobia colony, carrying out gram staining, wherein the microscopic examination result of the rhizobia is that the cell is in a small rod shape and consistent in shape, has no spore, and the cell often contains β -hydroxybutyric acid and is in a ring shape, and is gram negative (G)^-). If the marked colony has the characteristics of the two aspects, the colony is inoculated into YMA culture medium for slant culture in a test tube and stored.

The strain VS13-1 obtained by separation and purification in the embodiment is fast-growing rhizobia, and is cultured on a YMA culture medium added with congo red, so that the thallus does not absorb red, the bacterial colony is small, round, milky white, viscous, high in degree of swelling, and slightly transparent, and the bacterial colony grows out after 2-3 days. Gram stained as G^-It is in the shape of a small rod.

Example 2 tieback test of Rhizobium

The sand culture method for the rhizobium tieback test is characterized in that a common vetch variety used in the test is a common vetch of Sichuan province, and is derived from Yangjiang district wild gooses of Sichuan Yangyang city and rattles in water villages. And planting in an illumination room (controlling the temperature to be 22-24 ℃, controlling the illumination intensity to be 2800 lux and keeping the sunshine time to be 14h) for 46d and harvesting. Regularly supplementing a sterilizing nitrogen-free nutrient solution, inoculating common vetch rhizobia VS13-1 into a sand culture device, wherein the sand culture device adopts a 300mL plastic cup, vermiculite is selected as a substrate, the same variety of plants which are not inoculated with common vetch rhizobia VS13-1 are used as a control CK, and each treatment is repeated three times. The inoculation effect of common vetch rhizobia VS13-1 was evaluated with the number of nodules and the dry weight of the plants after harvesting.

(1) Bacterial liquid culture: the rhizobium victoriae VS13-1 was inoculated into YMA liquid medium, and cultured on a shaker at a rotation speed of 120rpm/min at 28 ℃ until logarithmic phase (about 3 d).

(2) Accelerating germination of seeds: selecting big, plump and undamaged common vetch seed, soaking with 95% alcohol for 5min, pouring out alcohol, adding mercuric chloride (0.1% m/v), soaking the surface, and sterilizing for 5 min. And finally, washing with sterile water for 6-8 times, and 5min each time. And finally, scattering the seeds in the sterilized wood chips for accelerating germination, wherein the accelerating germination temperature is 28 ℃, and sowing is carried out when the main roots grow to be about 2-3 cm and the fibrous roots do not grow.

(3) Manufacturing the sand culture device: a300 mL plastic cup is selected as a sand culture device, sterilized vermiculite is added into the sterilized plastic cup to occupy 3/4 of the whole cup, sterile nitrogen-free nutrient solution is added to keep the vermiculite moist, the plastic cup and the vermiculite are sterilized respectively, quartz sand with the grain diameter of 2-4mm is selected to be paved for isolating mould pollution, and the quartz sand is also sterilized.

(4) The formula of the nitrogen-free nutrient solution comprises the following components: 0.46g calcium sulfate, 0.136g dipotassium phosphate, 0.075g potassium chloride, 0.075g ferric citrate, 0.06g magnesium sulfate, 0.03g calcium nitrate, 1000mL distilled water, 1mL trace element solution

(5) The formula of the trace element solution comprises the following components: 2.86g boric acid, 1.81g manganese sulfate, 0.8g copper sulfate, 0.22g zinc sulfate, 0.02g molybdic acid plus distilled water to 1000 mL.

(6) Planting and measuring indexes: and planting the germination accelerating seeds into a plastic cup filled with vermiculite, and slightly pumping 2mL of bacterial liquid to the vicinity of the root of common vetch by using a liquid transfer gun. The same variety of plants not treated with inoculation was set as Control (CK). When planting, CK is planted firstly to avoid inoculation pollution. Each treatment was repeated 3 times. The hydroponic test results are shown in table 1.

The results in Table 1 show that the common vetch rhizobium VS13-1 and the tested common vetch show better nodulation capacity and symbiotic nitrogen fixation capacity; compared with a control without inoculation of common vetch rhizobia VS13-1, VS13-1 can obviously improve the dry weight of the common vetch plant in Sichuan, and is improved by 65.7% compared with the control without inoculation of rhizobia. As can be seen, the common vetch rhizobium VS13-1 is an excellent and efficient strain which is well matched with common vetch in the northwest China.

TABLE 1 Sand culture test results of common vetch rhizobia VS13-1

Note: data are the average of triplicates;

example 3 stress resistance of Rhizobium victoriae VS13-1

The stress resistance of the common vetch rhizobium VS13-1 is mainly measured for salt resistance and acid and alkali resistance. YMA medium was used as a basal medium, and YMA plates cultured at 28 ℃ and pH7 for 7 days were used as positive Controls (CK). The YMA slant culture of the above rhizobium victoriae VS13-1 was scraped with sterile water to prepare a bacterial suspension for use. Each treatment was repeated 3 times using the point inoculation method. The acid and alkali resistance and salt resistance test plates are cultured at 28 ℃ for 3d, and the recorded results are observed.

(1) The formula of the medium for acid and alkali resistance determination is as follows: YMA medium was used as a basal medium, and pH was adjusted with HC1 and NaOH to 4.0, 5.0, 6.0, 8.0, 9.0, 10.0, and 11.0 in this order.

(2) The formula of the culture medium for measuring the salt tolerance comprises the following components: the NaCl concentration was set to 0.2%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% (mass volume fraction) in this order, using the NaCl-free YMA medium as a basal medium.

Test results show that the rhizobium victoriae VS13-1 has strong acid and alkali resistance and can grow on a flat plate with the pH value of 4-11, but the diameter of a bacterial colony on the flat plate with peracid or over-alkali is smaller than the positive control of pH7, which indicates that the over-alkali and peracid have a certain inhibition effect on the growth; the salt tolerance is also stronger, and the YMA flat plate with 0.2-0.5% of NaCl can grow.

Example 4 growth promoting ability of Rhizobium victoriae VS13-1

The growth promoting capability of the rhizobium victoriae VS13-1 mainly examines the capabilities of secreting auxin (IAA), dissolving phosphorus and dissolving potassium.

(1) Determination of the ability to secrete auxins

Determining the ability of rhizobia to secrete auxin (IAA) by colorimetry, and determining the culture medium harvestAn improved Congo red liquid culture medium is used, and the culture medium comprises: 10g of mannitol, 1g of yeast extract and 1g of NH₄NO₃、0.5g K₂HPO₄·3H₂O、0.2gMgSO₄·7H₂O, 0.1g NaCl, 10mL congo red (0.25%), 100mg L-tryptophan, 1000mL distilled water, pH 7.0. The formula of the colorimetric solution is as follows: 0.5M FeCl₃1mL, concentrated H₂SO₄30mL and distilled water 50 mL.

Inoculating the strain into a triangular flask containing 50mL of culture medium, placing the flask in a shaking table with the rotation speed of 160rpm/min and the temperature of 28 ℃, repeating for 3 times, after culturing for 7 days, centrifuging at 8000rpm/min for 10min, taking 10mL of bacterial liquid, adding 10mL of colorimetric solution, keeping out of the sun for 30min, and determining the OD value by selecting the wavelength of 530nm on an ultraviolet spectrophotometer. And (5) converting the IAA content according to the standard curve. Preparing standard yeast: IAA stock solution is used to prepare 0, 5, 10, 20 and 40mg/L series of standard solutions as working solution. Colorimetric result determination shows that rhizobium VS13-1 secretes IAA 8.92mg/L, which indicates that rhizobium VS13-1 has the capability of secreting IAA.

(2) Capacity of dissolving organic and inorganic phosphorus

The capacity of the strain for dissolving organic phosphorus and inorganic phosphorus is measured by a phosphorus dissolving ring method. The organic phosphorus source is lecithin, and the inorganic phosphorus source is Ca₃(PO₄)₂、AlPO₄·2H₂O、FePO₄·2H₂O, the above reagents are all commercially available analytical pure reagents. Monkina culture medium and PKO culture medium are prepared into plates, strain preparation and point inoculation methods are the same as those of the stress resistance test in example 3, and each treatment is repeated for 3 times. After culturing for 7d in an incubator at 28 ℃, observing whether the strain grows and whether a phosphorus-dissolving ring appears.

1) The formula (g/L) of Monkina culture medium for the nutrient medium with the capacity of dissolving organic phosphorus is as follows: 10g glucose, 5g CaCO₃，0.5g(NH₄)₂SO₄0.4g of yeast powder, 0.3g of KCl, 0.2g of lecithin, 0.3g of NaCl, 0.03g of MnSO₄·4H₂O，0.03gFeSO₄·7H₂O, 20g of agar, 1000mL of distilled water and the pH value of the mixture is 6.8-7.0. Wherein lecithin is dissolved in 75% ethanol under heating, sterilized separately, mixed with sterilized culture medium cooled to about 60 deg.C, and poured into flat plate.

2) The formula (g/L) of the culture medium for dissolving inorganic phosphorus is as follows: 10g of glucose, 3.0g of the above inorganic phosphorus source substance (Ca)₃(PO₄)₂、AlPO₄·2H₂O、FePO₄·2H₂O)，0.5g(NH₄)₂SO₄，0.2g KCl，0.2g NaCl，0.03gMnSO₄，0.03g MgSO₄·7H₂O，0.003g FeSO₄·7H₂O, 0.5g of yeast powder, 20g of agar and 1000mL of distilled water, wherein the pH value is 6.8-7.0. Wherein the calcium phosphate, the aluminum phosphate and the iron phosphate are crushed by a mortar and sieved by a 300-mesh sieve, and after being independently dried and heat sterilized, the calcium phosphate, the aluminum phosphate and the iron phosphate are mixed with a culture medium with the sterilization temperature reduced to about 60 ℃ and poured into a flat plate.

The result shows that the common vetch rhizobium VS13-1 has certain dissolving capacity on organic phosphorus source substances and calcium phosphate and does not grow on an aluminum phosphate and iron phosphate culture medium. The ratios of the diameters of the phosphorus-dissolving rings to the diameters of the colonies measured on the lecithin and calcium phosphate plates are 1.34 and 1.6 respectively, which shows that rhizobium VS13-1 has certain capacity of dissolving organic phosphorus and calcium phosphate.

(3) Capacity of dissolving potassium

The potassium-dissolving capacity was determined by flame photometry. The potassium source is selected from washing potash feldspar ore powder (800 meshes). Potassium-free medium (1L): 10g of cane sugar, 0.5g of MgSO4.7H2O, 0.1g of NaCl, 0.5g of yeast extract and pH of 7.2-7.4.

A250 mL triangular flask is used as a shaking flask, 100mL of potassium-free basic liquid culture medium is filled, 0.5g of potassium feldspar mineral powder (800 meshes) is added, 5mLVS13-1 bacterial suspension is inoculated, the same amount of sterile water is inoculated in a contrast manner, 3 times of repetition are set, the culture is carried out at the temperature of 28 ℃ and at the speed of 160rpm/min for 7d, 10mL of culture solution is taken, the culture solution is centrifuged at 8000rpm/min for 10min, the supernatant is taken, and the content of soluble potassium in the supernatant is measured by a flame photometer method. Preparing standard yeast: potassium standard solution is used to prepare 0, 5, 10, 20, 30, 40 and 50mg/L series of standard solution as working solution. The potassium dissolving capacity of the rhizobium VS13-1 is 3.38mg/L as determined by the result of the computer, which indicates that the rhizobium VS13-1 has the potassium dissolving capacity.

Example 5 amplification and phylogenetic analysis of the 16S rRNA Gene and the other housekeeping genes recA, glnII, atpD of Rhizobium victoria VS13-1

Extracting total DNA of the strain, performing PCR amplification on the 4 genes respectively by using primers shown in Table 2, detecting PCR amplification products on 1.0% agarose gel electrophoresis by using a Bio-RAD MyCyclerTM instrument, and then sending the products to Chengdu Hingxi biotechnology Limited company for sequence determination. The software DNAman 6.0 is used for calculating the similarity of gene sequences.

TABLE 2 PCR primers used in this experiment

Note: y ═ C or T, H ═ a, C or T, R ═ a or G, S ═ C or G, K ═ G or T, N ═ a, C, G or T, I ═ inosine, M ═ a or C, N ═ an base.

(1) Amplification of 16S rRNA gene and construction of phylogenetic tree

The 16S rRNA gene was amplified using total DNA as a template and using universal primers P1 and P6 of Table 2. PCR reaction (50. mu.l): mu.l of 2 XPCRMix 25. mu.l each of primers P1 and P6 (10. mu.M), 1. mu.l of DNA template, and made up to 50. mu.l with ultrapure water. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 1min, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min, and circulation for 30 times; final extension at 72 ℃ for 10 min. The sequencing result of Chengdu Hingxi biotechnology Limited company detected by the amplification product according to the method is shown as SEQIDNo 1.

16S rRNA gene sequence SEQ ID No 1:

GGGGCGCTGCTTACCGTGCGAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCTTGACTACGGAATAACGCAGGGAAACTTGTGCTAATACCGTATGTGTCCTTCGGGAGAAAGATTTATCGGTCAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATGCCCGGCTACTTGCAGAGATGCAAGGTTCCCTTCGGGGACCGGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGGTAGATCAGCC

the obtained sequence results are compared in EzTaxon (http:// www.ezbiocloud.net/EzTaxon), a model strain with high similarity is selected as a reference strain, and a phylogenetic tree is constructed. The model strain found to have the highest similarity to the 16SrRNA gene sequence of Rhizobium anhuiense CCBAU 23252 of VS13-1 is^TThe similarity was 98.87%. Using the results of the alignment of the sequences on EzTaxon, the construction of a phylogenetic tree of the 16S rRNA gene was carried out using the Neighbor-joining method (Neighbor-joining) in the Mega7 software, with a bootstrap value (bootstrap) of 1000, and the phylogenetic tree is shown in FIG. 2.

(2) Construction of a Joint phylogenetic Tree of Multi-site Gene sequences

In order to further more accurately determine the classification status of the rhizobium victoriae VS13-1, other 3 sites of sequences of housekeeping genes recA, glnII and atpD are selected for constructing a combined phylogenetic tree.

The primers recAF2 and recAR2 for amplifying recA, the primers atpDF1 and atpDR for atpD, and the primers GSII-5 and GSII-6 for glnII are shown in table 2. The reaction system is 30 mul, and the reaction solution comprises the following components: the reaction system (30. mu.l) was: 2 XPCR Mix 15. mu.l; 0.5. mu.l each of 10mM forward and reverse primers; 1. mu.l of DNA template; ddH₂O13μl。

1) recA amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 1min, annealing at 60 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 2 min.

2) glnII amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 1min, annealing at 56 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 7 min.

3) atpD amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 45s, annealing at 57.5 deg.C for 1min, extension at 74 deg.C for 1.5min, and circulation for 30 times; final extension at 74 ℃ for 5 min.

The amplification product is detected according to the method and then sent to Du Zhixi biotechnology Limited company for sequencing, each gene is subjected to two-way sequencing (sequences of a forward primer and a reverse primer), then the sequences of the forward primer and the reverse primer are spliced by DNAman 6.0 software, the sequences of the forward primer and the reverse primer are removed, and then atpD sequence size of 496nt, sequence results such as SEQID No2, glnII sequence size of 637nt, sequence results such as SEQID No3, recA sequence size of 493nt and sequence results such as SEQID No4 are respectively obtained.

atpD gene sequence SEQ ID No 2:

CATCGGCGAGCCGGTCGACGAAGCCGGTCCGCTGGTCACCGCTCATAAGCGCGCCATCCACCAGGATGCGCCGTCCTATGTCGAGCAGTCGACGGAATCGCAGATTCTCGTCACCGGCATCAAGGTCGTTGACCTTCTCGCTCCCTATGCACGCGGCGGCAAGATCGGCCTCTTCGGCGGCGCTGGCGTCGGCAAGACCGTTTTGATCATGGAACTGATCAACAACGTCGCCAAGGCGCATGGTGGTTATTCGGTTTTTGCGGGCGTCGGTGAACGCACCCGCGAAGGCAACGACCTCTACCACGAAATGATCGAATCGAACGTCAACAAGCATGGCGGCGGCGAAGGTTCGAAGGCCGCGCTGGTTTACGGCCAGATGAACGAGCCGCCGGGCGCCCGCGCCCGCGTCGCCCTGACCGGCCTGACGGTCGCTGAACATTTCCGCGACCAGGGCCAGGACGTTCTGTTCTTCGTCGACAACATCTTCCGCTTCACG

the sequence of glnII gene SEQ ID No 3:

CGATGGGTACACTCCGGTACCGAACCTGCGTGGCAAGACGCAGATCAAGGAATTCGACGCATTCCCGACGCTGGAACAGCTTCCGCTCTGGGGCTTTGACGGCTCCTCGACGCAGCAGGCTGAAGGCCGCAGCTCCGATTGCGTGCTGAAGCCGGTCGCCATCTATCCCGACCCGGCCCGCACCAACGGCGCTCTCGTCATGTGCGAAGTCATGATGCCGGATGGGGTCACGCCGCACGCATCGAATGCCCGCGCCACCATCCTCGACGACGAAGATGCCTGGTTCGGCTTCGAGCAGGAATATTTCTTCTACCAGAACGGCCGTCCGCTCGGCTTCCCCGAGCAGGGCTACCCGGCTCCGCAGGGTCCCTACTACACCGGCGTCGGCTATTCGAATGTCGGCGACGTCGCCCGCGAAATCGTCGAAGAACATCTCGACCTCTGCCTCGCTGCCGGCATCAATCACGAAGGCATCAATGCCGAAGTGGCCAAGGGCCAGTGGGAATTCCAGATTTTCGGCAAGGGCTCCAAGAAGGCCGCCGACCAAATCTGGATGGCACGCTACCTCTTGCAGCGCCTGACTGAAAAGTACGGCATCGACATCGAGTATCACTGCAAGCCGCTCGGTGACACCGAC

recA gene sequence SEQ ID No 4:

CTCGATTATGAAACTCGGCTCCAACGAGAATGTTGTCGAGATCGAGACGATCTCGACCGGCTCGCTTGGCCTCGATATCGCACTTGGTGTCGGCGGCCTGCCGAGGGGCCGCATCATCGAAATTTACGGACCGGAAAGCTCCGGTAAAACGACGCTTGCGCTGCAGACAATTGCCGAGGCGCAGAAAAAGGGCGGTATCTGCGCCTTCGTCGATGCCGAACATGCGCTCGATCCTGTCTATGCCCGCAAGCTTGGCGTCGATCTGCAGAACCTTCTGATCTCGCAGCCCGATACCGGCGAGCAGGCGCTTGAAATCACCGATACGCTGGTGCGCTCCGGCGCCGTCGACGTCCTCGTCGTCGACTCGGTCGCCGCACTGACGCCACGCGCCGAAATCGAAGGCGAGATGGGCGACAGCCTTCCCGGCCTGCAGGCGCGCCTGATGAGCCAGGCGCTGCGCAAGCTCACCGCCTCGATCTCGAAGTCGAACACC

comparing the obtained sequence results in the National Center for Biological Information (NCBI) of the United states, applying the comparison result of each gene sequence in the NCBI, and selecting 6 model strains with high similarity with 3 genes as reference strains for tree building.

Construction of 3 genes (recA, glnII and atpD) in combination with phylogenetic trees: firstly, sequences of 3 housekeeping genes of recA, atpD and glnII are respectively compared with corresponding gene sequences of a reference strain by MEGA7, the sequences are aligned by taking the minimum length as a standard, the aligned sequences are stored in a FASTA format, the lengths of the three gene sequences after alignment are 342nt, 350nt and 438nt respectively, 3 sequences are spliced together by WORD opening, and a combined phylogenetic tree is constructed by using a Neighbor-joining method (Neighbor-joining) in MEGA7 software, wherein the self-expanding value (bootstrap) is 1000, and the combined phylogenetic tree of atpD, glnII and recA is shown in figure 3.

The model strains which are found to have the highest sequence similarity with the housekeeping genes at three sites of recA, glnII and atpD of Rhizobium victoriae VS13-1 are all Rhizobium anhuiense CCBAU 23252^TThe degrees of similarity with this model strain were 98.25%, 99.54%, 98.57%, respectively.

FIGS. 2 and 3 show that the 16S rRNA gene of VS13-1 and the combined sequences of recA, glnII, atpD 3 housekeeping genes and the model strain Rhizobium anhuiense CCBAU 23252^TOn the same branch node. Again, as previously analyzed, the strain was compared to the model strain Rhizobium anhuiense CCBAU 23252^TThe similarity of these 4 genes was very high, and the similarity of 16SrRNA, recA, glnII, atpD was 98.87%, 98.25%, 99.54%, 98.57%, respectively, indicating that the strain VS13-1 belongs to Rhizobium anhuiense.

Example 6 Effect of field inoculation

The field inoculation effect test of the strain is carried out by selecting a modern agricultural base of Alnus Quanzhen Sichuan agricultural university in Sichuan Chong State.

Two treatments are established altogether in this experiment, inoculate common vetch rhizobium VS13-1 and not inoculate contrast processing (CK), and the common vetch main cultivar "Sichuan common vetch" is selected to the bean kind, does not apply any chemical fertilizer and fertilizer. And random block arrangement in the field is adopted. The test was carried out in 2016, 9 and 30 months, to 2017, 4 and 15 months. The prepared common vetch rhizobium VS13-1 microbial inoculum (viable count is 4.3 multiplied by 10)⁸CFU/g microbial inoculum) and common vetch seed dressing, and sowing after drying in the shade. Cell area 7m²And (3) sowing the CK first during sowing so as to avoid the CK treatment from being influenced by the inoculation of rhizobia. Sampling is carried out in the full-bloom stage (the growth stage is 197d) of the plants, and the effective number of nodules, the total number of nodules, the fresh weight of the overground part of the plants, the fresh weight of the underground part of the roots and the yield of fresh grass of the plants are determined. Management during the period is performed according to conventional management of a farmer planting vicia sativa.

TABLE 3 field inoculation Effect of common vetch inoculated with Rhizobium VS13-1

Note: data are mean of triplicates

The inoculation of the common vetch rhizobium VS13-1 results in higher effective nodule number, total nodule number and fresh grass yield than the non-inoculation control in the full-bloom period, the fresh grass yield is increased by 76.5%, and the fresh root yield is increased by 68.7% compared with CK. At present, reports about rhizobium of common vetch are few, and only the research (duckweed, 2015) of rhizobium inoculation in Gansu area on the growth influence of common vetch is found, and the rhizobium inoculation treatment yield is increased by 21.2-28% compared with the rhizobium inoculation-free (control CK). There is not any relevant report of influence of rhizobium of common vetch on growth and yield of common vetch. Therefore, the common vetch rhizobium VS13-1 is an excellent efficient rhizobium suitable for large-area popularization and application in production of common vetch.

According to the field inoculation test research of alder spring town of Chongzhou city in Sichuan, after the vetch rhizobium is inoculated with VS13-1, the effective number of tumors, the total number of the tumors and the yield of fresh grass are all higher than those of an uninoculated control, the yield of the fresh grass is increased by 76.5%, the yield of the fresh root is increased by 68.7% compared with CK, and the increase of the biomass of the overground underground part shows that rhizobium VS13-1 promotes the growth of vetch. Therefore, the rhizobium victoriae VS13-1 obtained through separation can be applied to large-area popularization and application in production of vetch.

Sequence listing

<110> Sichuan university of agriculture

<120> common vetch rhizobium strain VS13-1 and application thereof

<141>2017-12-26

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1381

<212>DNA

<213> Rhizobium (rhizobium anhuiense)

<400>1

ggggcgctgc ttaccgtgcg agtcgagcgc cccgcaaggg gagcggcaga cgggtgagta 60

acgcgtggga atctaccctt gactacggaa taacgcaggg aaacttgtgc taataccgta 120

tgtgtccttc gggagaaaga tttatcggtc aaggatgagc ccgcgttgga ttagctagtt 180

ggtggggtaa aggcctacca aggcgacgat ccatagctgg tctgagagga tgatcagcca 240

cattgggact gagacacggc ccaaactcct acgggaggca gcagtgggga atattggaca 300

atgggcgcaa gcctgatcca gccatgccgc gtgagtgatg aaggccctag ggttgtaaag 360

ctctttcacc ggagaagata atgacggtat ccggagaaga agccccggct aacttcgtgc 420

cagcagccgc ggtaatacga agggggctag cgttgttcgg aattactggg cgtaaagcgc 480

acgtaggcgg atcgatcagt caggggtgaa atcccagggc tcaaccctgg aactgccttt 540

gatactgtcg atctggagta tggaagaggt gagtggaatt ccgagtgtag aggtgaaatt 600

cgtagatatt cggaggaaca ccagtggcga aggcggctca ctggtccatt actgacgctg 660

aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 720

atgaatgtta gccgtcgggc agtatactgt tcggtggcgc agctaacgca ttaaacattc 780

cgcctgggga gtacggtcgc aagattaaaa ctcaaaggaa ttgacggggg cccgcacaag 840

cggtggagca tgtggtttaa ttcgaagcaa cgcgcagaac cttaccagcc cttgacatgc 900

ccggctactt gcagagatgc aaggttccct tcggggaccg ggacacaggt gctgcatggc 960

tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aaccctcgcc 1020

cttagttgcc agcattcagt tgggcactct aaggggactg ccggtgataa gccgagagga 1080

aggtggggat gacgtcaagt cctcatggcc cttacgggct gggctacaca cgtgctacaa 1140

tggtggtgac agtgggcagc gagcacgcga gtgtgagcta atctccaaaa gccatctcag 1200

ttcggattgc actctgcaac tcgagtgcat gaagttggaa tcgctagtaa tcgcggatca 1260

gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1320

tggttttacc cgaaggtagt gcgctaaccg caaggaggca gctaaccacg gtagatcagc 1380

c 1381

<210>2

<211>496

<212>DNA

<213> housekeeping Gene (atpd)

<400>2

catcggcgag ccggtcgacg aagccggtcc gctggtcacc gctcataagc gcgccatcca 60

ccaggatgcg ccgtcctatg tcgagcagtc gacggaatcg cagattctcg tcaccggcat 120

caaggtcgtt gaccttctcg ctccctatgc acgcggcggc aagatcggcc tcttcggcgg 180

cgctggcgtc ggcaagaccg ttttgatcat ggaactgatc aacaacgtcg ccaaggcgca 240

tggtggttat tcggtttttg cgggcgtcgg tgaacgcacc cgcgaaggca acgacctcta 300

ccacgaaatg atcgaatcga acgtcaacaa gcatggcggc ggcgaaggtt cgaaggccgc 360

gctggtttac ggccagatga acgagccgcc gggcgcccgc gcccgcgtcg ccctgaccgg 420

cctgacggtc gctgaacatt tccgcgacca gggccaggac gttctgttct tcgtcgacaa 480

catcttccgc ttcacg 496

<210>3

<211>637

<212>DNA

<213> housekeeping Gene (glni)

<400>3

cgatgggtac actccggtac cgaacctgcg tggcaagacg cagatcaagg aattcgacgc 60

attcccgacg ctggaacagc ttccgctctg gggctttgac ggctcctcga cgcagcaggc 120

tgaaggccgc agctccgatt gcgtgctgaa gccggtcgcc atctatcccg acccggcccg 180

caccaacggc gctctcgtca tgtgcgaagt catgatgccg gatggggtca cgccgcacgc 240

atcgaatgcc cgcgccacca tcctcgacga cgaagatgcc tggttcggct tcgagcagga 300

atatttcttc taccagaacg gccgtccgct cggcttcccc gagcagggct acccggctcc 360

gcagggtccc tactacaccg gcgtcggcta ttcgaatgtc ggcgacgtcg cccgcgaaat 420

cgtcgaagaa catctcgacc tctgcctcgc tgccggcatc aatcacgaag gcatcaatgc 480

cgaagtggcc aagggccagt gggaattcca gattttcggc aagggctcca agaaggccgc 540

cgaccaaatc tggatggcac gctacctctt gcagcgcctg actgaaaagt acggcatcga 600

catcgagtat cactgcaagc cgctcggtga caccgac 637

<210>4

<211>493

<212>DNA

<213> housekeeping Gene (reca)

<400>4

ctcgattatg aaactcggct ccaacgagaa tgttgtcgag atcgagacga tctcgaccgg 60

ctcgcttggc ctcgatatcg cacttggtgt cggcggcctg ccgaggggcc gcatcatcga 120

aatttacgga ccggaaagct ccggtaaaac gacgcttgcg ctgcagacaa ttgccgaggc 180

gcagaaaaag ggcggtatct gcgccttcgt cgatgccgaa catgcgctcg atcctgtcta 240

tgcccgcaag cttggcgtcg atctgcagaa ccttctgatc tcgcagcccg ataccggcga 300

gcaggcgctt gaaatcaccg atacgctggt gcgctccggc gccgtcgacg tcctcgtcgt 360

cgactcggtc gccgcactga cgccacgcgc cgaaatcgaaggcgagatgg gcgacagcct 420

tcccggcctg caggcgcgcc tgatgagcca ggcgctgcgc aagctcaccg cctcgatctc 480

gaagtcgaac acc 493

Claims (3)

1. The utility model provides a common vetch rhizobia strain VS13-1 which characterized in that: the culture is preserved in China center for type culture Collection of Wuhan university in 2017, 10 and 23 months, with the preservation number: CCTCC NO: M2017607.

2. The use of rhizobium victoriae strain VS13-1 according to claim 1, wherein: this root nodule bacterial strain is applied to the production of Sichuan common vetch.

3. The use of a vetiveria vicia strain VS13-1 according to claim 2, characterized in that: will contain living thingsBacterial count 4.3X 10⁸The CFU/g common vetch rhizobium VS13-1 microbial inoculum and the common vetch are mixed for seed dressing, dried in the shade and then sowed.

Images