CN108239615B - Rhizobium fabae strain Bga2-2 and application thereof - Google Patents

Abstract

A broad bean Rhizobium strain Bga2-2 is obtained by separating and purifying fresh broad bean Rhizobium and belongs to Rhizobium Rhizobium anhuiense 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: M2017613. The rhizobium strain Bga2-2 is an excellent broad-spectrum rhizobium fabae strain which has strong nitrogen-fixing capability, wide application range to Sichuan fabae varieties, IAA secretion capability, inorganic phosphorus and organic phosphorus dissolving capability and strong stress resistance; the broad bean product has good matching affinity with main planted broad bean varieties in Sichuan, nitrogen fertilizer is not applied in the production of broad beans of different varieties in Sichuan, the broad beans are inoculated with Bga2-2, the yield of the broad beans is increased by more than 31.8 percent, and the difference with non-inoculated contrast reaches a remarkable level.

Description

Rhizobium fabae strain Bga2-2 and application thereof

Technical Field

The invention relates to the field of microorganisms, and in particular relates to a broad bean rhizobium strain Bga2-2 and application thereof.

Background

China is the largest broad bean producing country, the yield of dry broad beans accounts for 35 percent of the world (FAO2014), the dry broad beans are important agricultural product resources of the traditional foreign trade in China, and the export quantity of the dry broad beans is the second place in the coarse cereals in China (Liuyujian, etc., 2002). The sowing area of broad beans in Sichuan province is about 30 hectares, which is second to Yunnan, and the total yield reaches 6 hundred million kilograms (Yang Wuyun, 2003). Broad beans are famous all over the country, and the demand is large, and the quantity of the broad beans required per year is up to 12 ten thousand tons, but the broad beans are mainly imported from other places at present. In addition to harvesting dry broad beans, fresh broad beans can be harvested as vegetables with higher economic value and can also be used as green manure, so that broad beans planted in Sichuan have great potential, but in recent years, due to the fact that a large amount of nitrogen fertilizer is put into use, production area is reduced year by year, yield is reduced compared with that of the western countries, the nitrogen fertilizer is high in production cost and low in utilization rate, ecological environment is polluted, and protection of the ecological environment and sustainable development of agriculture are not facilitated.

Rhizobia with high efficiency in inoculation and matching of leguminous crops is an internationally recognized effective biological nitrogen fixation technology, biological nitrogen fixation is high in quality and utilization rate, the nitrogen release process is continuous, the after effect is long, the waste is little, the environment is not polluted (Ge Cheng, 2007), and the ecological environment protection and the agricultural sustainable development are facilitated. Inoculation of rhizobia in Qinghai province by Hanmei et al (2016) can significantly improve yield of broad beans and increase soil fertility; the biomass of Wangwonli and the like (2010) is increased by 6.48 to 12.86 percent compared with the biomass treated by non-inoculation after the inoculation of rhizobia in the production of broad beans in Gansu province. In the face of the great scientific and technological requirements of zero increase of chemical fertilizers and reduced application and efficiency, the production of broad beans can realize high quality and high yield by using matched efficient rhizobia.

Due to geographical limitations of the population distribution of rhizobia, attention needs to be paid to their adaptability to the environment of the 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 area or a strain in an area similar to local area conditions (Yaqi, 2014; Chenwining et al, 2011). Therefore, in the screening of the rhizobia high-efficiency nitrogen-fixing strain, not only the matching property between rhizobia and leguminous plant varieties needs to be considered, but also the regionality of the application of the microbial inoculum needs to be considered.

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, 730 more strains of Rhizobium meliloti were studied, only 10 strains of Rhizobium meliloti with strong IAA secretion ability were selected from the 29 strains of Rhizobium meliloti, and the phosphorus-dissolving ability of the 29 strains was measured, and it was found that all the strains had no inorganic phosphorus-dissolving ability, and that the 29 strains of Rhizobium meliloti were able to dissolve organic phosphorus, but the difference in phosphorus-dissolving ability was large, only 8 strains had strong organic phosphorus-dissolving ability, and the remaining strains had weak or weak organic phosphorus-dissolving ability.

At present, the screening application of the broad bean high-efficiency rhizobia is not reported except that the areas of Qinghai and Gansu (cunningmin, 2002; Hanmei, etc., 2010; Wangwnli, etc., 2010) and Hebei (Wangru, 2016) have relevance reports. Therefore, broad-spectrum excellent strains of the broad beans are screened aiming at main cultivars of Sichuan, the biological nitrogen fixation effect is fully exerted in the production of the Sichuan broad beans, the excellent strain resources are screened, the application of chemical fertilizers or no nitrogen fertilizers is reduced, and the method has important practical value for protecting the ecological environment and promoting the sustainable development of agriculture.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a broad bean rhizobium strain Bga2-2 capable of efficiently fixing nitrogen, which has IAA secretion and certain phosphorus dissolving capacity and has better field production and application prospects.

The technical scheme adopted by the invention is as follows:

a Rhizobium fabae strain Bga2-2, which is classified and named as Rhizobium anhuiensis Bga2-2, is preserved in the China center for type culture Collection of Wuhan university at 23 months 10 and 2017, and has the preservation number: CCTCC NO: M2017613.

The broad bean rhizobium strain Bga2-2 is applied to production of broad beans in the Chengdu plain area of the Sichuan broad bean main production area.

The invention has the beneficial effects that:

the rhizobium strain Bga2-2 is an excellent broad-spectrum rhizobium fabae strain which has strong nitrogen-fixing capability, wide application range to Sichuan fabae varieties, IAA secretion capability, inorganic phosphorus and organic phosphorus dissolving capability and strong stress resistance; the broad bean product has good matching affinity with main planted broad bean varieties in Sichuan, nitrogen fertilizer is not applied in the production of broad beans of different varieties in Sichuan, the broad beans are inoculated with Bga2-2, the yield of the broad beans is increased by more than 31.8 percent, and the difference with non-inoculated contrast reaches a remarkable level.

Drawings

FIG. 1 shows the colony morphology of Rhizobium fabae strain Bga2-2 on YMA medium;

FIG. 2 is a phylogenetic diagram of the 16S rRNA gene sequence of Rhizobium fabae strain Bga 2-2;

FIG. 3 is a phylogenetic diagram of the combined construction of three housekeeping genes glnII, atpD and recA of Rhizobium fabae strain Bga 2-2.

Detailed Description

Example 1 isolation, purification and preservation of Rhizobium

From broad beans planted in yellow loam soil of the neighborhood of Water county, Guangan City, Sichuan province, robust plants are selected to have large and full red nodules on their main roots, the nodules are scrubbed clean, partial root skins are harvested, the roots are sucked dry by paper and placed in a small tube filled with anhydrous calcium chloride and covered with absorbent cotton. Soaking and swelling the collected root nodule in sterile water in laboratory, treating with 95% ethanol for 5min, sterilizing with 0.1% m/v mercuric chloride surface for 5min, washing with sterile water for 6 times, and under aseptic condition, crushing single root nodule, and adding YMA culture medium (mannitol 10g, yeast powder 0.8g, KH) containing congo red₂PO₄0.25g，MgSO₄.7H₂O0.2g，CaCl₂.6H₂0.1g of O, 0.1g of NaCl, 2ml of sodium molybdate (1%), 2ml of boric acid (1%), 2.5ml of Congo red (1%), 18-20 g of agar, 1000ml of water and pH6.8-7.0) are streaked and cultured in an incubator at 28 ℃.

After the bacteria grow out, the bacteria colony which does not absorb red and is similar to rhizobium in shape is selected from the plate and is diluted and streaked on the plate for culture. And observing the colony morphology about 3 days, and observing about 15 days until the colony appears about 7-15 days because of the slow rhizobium. The dilution and streaking were repeated for repeated separations until purification. Whether the rhizobia is rhizobium is preliminarily judged according to the following two aspects: (1) colony morphology on congo red-added YMA medium: no red absorption, round, milky white, raised colony, regular and non-spreading edge, and surfaceSmooth, viscous and moist, fast rhizobia and slow rhizobia grow out of the colony after 3-5 days of culture, (2) cell morphology, namely marking the confirmed rhizobia colony, and performing gram staining on the slide, wherein the microscopic examination result of the rhizobia is that the cell is in a small rod shape, has no spores, and is in a ring shape and gram negative (G) because the cell usually contains β -hydroxybutyric acid^-). 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 Bga2-2 obtained by separation and purification in the embodiment is a fast rhizobium, and is cultured on a YMA culture medium added with congo red, so that the thallus does not absorb red, the colony is small, round, milky white, viscous, high in swelling degree and slightly transparent, and the colony grows out after 3-4 days. Gram stained as G^-It is in the shape of a small rod.

Example 2 tieback and matchability test of Rhizobium

The broad bean variety used for the rhizobium tieback test is broad bean, and is planted in an illumination room (the temperature is controlled to be 22-24 ℃, the illumination intensity is 2700-3000 lux, and the sunshine time is 14 hours) and harvested for 46 days. After the broad beans are successfully grafted back, the compatibility test is carried out on the broad beans with other main planted broad bean varieties (adult fiddle 14, adult fiddle 15 and Panzhihua broad beans), and the compatibility test is also carried out by a water culture method. The culture was cultured in the above illumination chamber for 41d for harvest. Periodically supplementing sterile nitrogen-free nutrient solution. The fast-growing broad bean rhizobium Bga2-2 and the broad bean varieties form different combinations, a 250ml narrow-necked bottle (glass infusion bottle for hospitals) is adopted as a water culture device, and plants of the same variety without being inoculated with the broad bean rhizobium are used as a control. After harvesting, the inoculation effect of the broad bean rhizobium Bga2-2 was evaluated by the number of root nodules and the dry weight of the plants. The bacterial liquid culture, the seed germination acceleration, the water culture device manufacturing and planting methods of the tie-back test and the matching test are consistent.

(1) Bacterial liquid culture: inoculating the rhizobium fabae Bga2-2 into a YMA liquid culture medium, placing on a shaker, and culturing at the rotation speed of 120rpm/min at the temperature of 28 ℃ until the logarithmic phase (about 3-4 d).

(2) Accelerating germination of seeds: selecting large, full and undamaged broad bean seeds, soaking the seeds in 95% alcohol for 5min, pouring out the alcohol, adding 0.1% mercuric chloride solution to sterilize the surfaces for 5min, finally cleaning the seeds with sterile water for 4-6 times, accelerating germination at the temperature of 28 ℃ for 5min each time, and sowing the seeds when main roots grow to about 2-3 cm and fibrous roots do not grow.

(3) And (3) manufacturing a water culture device: a250 ml narrow-neck bottle (glass infusion bottle for hospitals) is used as a water culture device. Firstly, preparing a nitrogen-free nutrient solution, injecting the prepared nitrogen-free water culture nutrient solution into a cleaned bottle, covering a layer of kraft paper on the bottle mouth, forming a small hole (the diameter is about 1cm) in the center of the bottle mouth, plugging cotton in the small hole, covering a layer of high-temperature-resistant plastic film on the small hole, and sterilizing at the temperature of 121 ℃ for later use.

(4) Planting and measuring indexes: the germination accelerating seeds are placed in an aseptic culture dish and soaked in the bacterial liquid for 15min, the roots of the seedlings are inserted into small holes of a water culture device by using aseptic tweezers, 1 strain is added into each bottle, then 1ml of bacterial liquid is added into each seedling, and the periphery of the seeds is plugged by cotton in the original holes, so that dust is prevented from falling into the bottles to cause pollution. The same variety of plants not treated with inoculation was set as Control (CK). CK was planted first during planting, and each treatment was repeated 3 times. The hydroponic test results are shown in table 1.

The results in Table 1 show that the rhizobium fabae Bga2-2 has good matching affinity with 4 broad bean varieties to be tested (Sichuan main cultivated varieties), and shows good nodulation capacity and symbiotic nitrogen fixation capacity; compared with the treatment without rhizobium inoculation, the treatment of rhizobium inoculation Bga2-2 improves the dry weight of plants by 30.8-71.3%. As can be seen, the broad bean rhizobia Bga2-2 is an excellent broad-spectrum strain with good matching property with Sichuan broad bean varieties, but no related report is found on the rhizobia which is efficiently matched with Sichuan main broad bean varieties at present.

TABLE 1 Rhizobium fabae Bga2-2 hydroponic test results

Note: data are the average of three replicates.

Stress tolerance of Rhizobium Bga2-2 described in example 3

The stress resistance of the rhizobium Bga2-2 is mainly determined by acid and alkali resistance, salt resistance and greenhouse growth range. YMA culture medium was used as a basal medium, and YMA plates cultured at 28 ℃ and pH7 for 7 days were used as positive controls. The YMA slant culture of Bga2-2 described above was scraped with sterile water for use. The method comprises the following steps of adopting a point inoculation method, repeating for 3 times, taking YMA culture medium as a basic culture medium for acid and alkali resistance measurement, and adjusting the pH value with HC1 and NaOH, wherein the pH value is 4.0, 5.0, 6.0, 8.0, 9.0, 10.0 and 11.0 in sequence. Salt tolerance assay the strains were spotted on plates containing NaC1, again using YMA medium as basal medium, at mass volume fractions of NaC1 of 0.2%, 0.4%, 0.6%, 1.0%, 1.5%, 2.5%, 3.5% and 4.5%. The acid and alkali resistance and salt resistance test plates are cultured at 28 ℃ for 7d, and the recorded results are observed.

Measuring the growth temperature range, inoculating the strain on YMA culture medium, performing 5 temperature treatments, respectively culturing in biochemical incubator at 8 deg.C, 15 deg.C, 37 deg.C, and 45 deg.C for 30d, 10d, 7d, and 7d, performing heat shock treatment at 60 deg.C for 30min, and culturing at 28 deg.C for 7 d. Test results show that the rhizobium Bga2-2 has strong stress resistance and can grow on a flat plate with the pH value of 4-11, and the acid and alkali resistance of the strain is good; salt tolerance is general, and the salt tolerance can grow on a YMA flat plate with 0.6% NaCl; the growth temperature range is wide, the strain can grow within the temperature range of 10-28 ℃, and the strain can survive after being subjected to heat shock treatment at 60 ℃ for 30min, which shows that the strain can endure high temperature in a short time.

Growth promoting ability of Rhizobium Bga2-2 described in example 4

The growth promoting capability of the rhizobium Bga2-2 mainly considers the secretion of auxin (IAA) (colorimetric method) and phosphorus dissolving capability (phosphorus dissolving ring method).

(1) Determination of the ability to secrete auxins

The ability of rhizobia to secrete auxin (IAA) was determined colorimetrically (cheshanli et al, 2007), and the medium was determined using a modified congo red liquid medium, medium composition: 0.5g K₂HPO₄.3H₂O、0.2g MgSO₄.7H₂O, 0.1g NaCl, 1g yeast extract, 10g mannitol, 10ml 0.25% Congo red and 1g NH₄NO₃100mg of L-tryptamineAcid, 1000ml distilled water, pH 7.0. The formula of the colorimetric solution is as follows: 0.5M FeCl₃1ml of concentrated H₂SO₄30ml and distilled water 50 ml.

Inoculating the strain into a triangular flask containing 50ml of culture medium, culturing on a shaking table at the rotation speed of 125rpm/min and the temperature of 28 ℃, repeating for 3 times, culturing for 12 days, taking 100 mu l of rhizobium suspension, placing on a white plastic colorimetric plate, adding 100 mu l of colorimetric solution, and observing the color change after 15 min. Pink is positive, which indicates that the strain can secrete IAA, and the deeper pink indicates that the IAA secretion capacity is larger; colorless is negative, indicating that the strain is unable to secrete IAA. The colorimetric solutions were added with 10mg/L (CK1), 30mg/L (CK2) and 50mg/L (CK3) IAA in equal amounts as positive controls to compare the pink color depth (Shelinqiao et al, 2005; Shishanli et al, 2007). The results showed that the colorimetric reaction of the rhizobium Bga2-2 was pale pink, indicating that the amount of IAA secreted by the rhizobium Bga2-2 was less than 10 mg/L.

(2) Capacity of dissolving organic and inorganic phosphorus

The phosphorus ring dissolving method is used. The organic phosphorus source is lecithin, and the inorganic phosphorus source is calcium phosphate (Ca)₃(PO₄)₂) Aluminum phosphate (AlPO)₄.2H₂O), iron phosphate (FePO)₄.2H₂O), are all commercially available analytical pure reagents.

Monkina culture medium for measuring the capacity of dissolving organic phosphorus, and the formula (g/L) is as follows: 10g glucose, 0.5g (NH)₄)₂SO₄，0.3g NaCl，0.3g KCl，0.03g FeSO_4.7H₂O，0.03g MnSO₄.4H₂O, 0.2g lecithin, 5g CaCO₃0.4g of yeast powder, 20g of agar and 1000ml of distilled water, and the pH value 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.

The culture medium for measuring the inorganic phosphorus dissolving capacity is PKO culture medium, and the formula (g/L): 10g of glucose, 3.0g of the above inorganic phosphorus source, 0.5g of (NH)₄)₂SO₄，0.2g NaCl，0.2g KCl，0.03g MgSO_4.7H₂O， 0.03g MnSO₄，0.003g FeSO_4.7H₂O, 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 are independently subjected to dry heat sterilization, and then are mixed with a culture medium with the sterilization temperature reduced to about 60 ℃ and are poured into a flat plate for later use. The strain preparation and the point inoculation method are the same as those in the stress resistance test of the example 3 and are repeated for 3 times. And (5) after culturing for 7d in an incubator at 28 ℃, observing whether the strain grows and whether a phosphorus dissolving ring appears. The result shows that the fast growing rhizobium Bga2-2 has certain dissolving capacity on two inorganic phosphorus sources of calcium phosphate and aluminum phosphate and organic phosphorus source substances, but cannot dissolve the iron phosphate.

Therefore, the strain Bga2-2 not only can efficiently fix nitrogen, but also has the capability of dissolving calcium phosphate, aluminum phosphate and organophosphorus lecithin, and the growth promotion effects of phytohormone (IAA) and the like, and meanwhile, the strain has a wider growth temperature range and strong acid and alkali resistance.

Amplification and phylogenetic analysis of 16S rRNA Gene and other housekeeping genes glnII, atpD, recA of Rhizobium Bga2-2 described in example 5

Extracting total DNA of strain, performing PCR amplification on the above 4 genes with primers shown in Table 2, respectively, and performing PCR reaction with Bio-RAD MyCycler^TMIn the instrument, PCR amplification products are detected on 1.0% agarose gel electrophoresis, sent to Invitrogen company for sequence determination, and 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＝any 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 XPCR Mix, 1. mu.l each of primers P1 and P6 (10. mu.M), 1. mu.l of DNA template, 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 result of the sequencing of the amplification product by Invitrogen company after detection by the above method is shown as SEQ ID No 1.

SEQ ID No116S rRNA gene sequence:

CTGCGGCGGCTTACCATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTG AGTAACGCGTGGGAATCTACCCTTGACTACGGAATAACGCAGGGAAACTTGTGCTAA TACCGTATGTGTCCTTCGGGAGAAAGATTTATCGGTCAAGGATGAGCCCGCGTTGGAT TAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAG GATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGT GGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAA GGCCCTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAG AAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGT TCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATC CCAGGGCTCAACCCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGT GAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGG CGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTC GCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATGCCCGGCTACTTGC AGAGATGCAAGGTTCCCTTCGGGGACCGGGACACAGGTGCTGCATGGCTGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTT GCCAGCATTCAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGT GGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAAT GGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTC AGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCG GATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACC ATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCAC GGTAGTCT*

the sequence results obtained were compared with EzTaxon (http:// www.ezbiocloud.net/EzTaxon), and the highest similarity of the 16S rRNA gene sequence of Rhizobium Bga2-2 was found in the model strain Rhizobium anhuiensis CBAU 23252^TThe similarity is 100%. Using the results of the alignment of the sequences on NCBI, a model strain with high similarity was selected asAnd (5) constructing a phylogenetic tree by referring to the strain. Construction of a phylogenetic tree of the 16S rRNA gene, bootstrap (bootstrap)1000, was carried out by the Neighbor-joining method in Mega5 software, 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 determine the classification status of the rhizobium fastidiosum Bga2-2 more accurately, 3 additional loci of housekeeping genes atpD, recA and glnII sequences were selected for the construction of a joint 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 50 mul, and the reaction solution comprises the following components: the reaction system (50. mu.l) was: 2 XPCR Mix 25. mu.l; 0.5. mu.l each of 10mM forward and reverse primers; 1. mu.l of DNA template; ddH₂O 23μl。

(1) recAPCR amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 45s, annealing at 59 deg.C for 45s, extension at 74 deg.C for 1.5min, and circulation for 30 times; final extension at 74 ℃ for 6 min.

(2) PCR amplification conditions for atpD: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 45s, annealing at 57.5 deg.C for 45s, extension at 74 deg.C for 1.5min, and circulation for 30 times; final extension at 74 ℃ for 6 min.

(3) glnII amplification conditions: pre-denaturation at 92 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 1.5min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 10 min.

The amplified product is detected according to the method and sent to Invitrogen company for sequencing, each gene is subjected to two-way sequencing (sequences of a positive primer and a negative primer), then the sequences of the positive primer and the negative primer are spliced by DNAman 6.0 software, the sequences of the positive primer and the negative primer are removed, so that atpD, glnII and recA sequences with the sizes of 496nt, 637nt and 483nt are respectively obtained, and the sequence results are respectively shown as SEQID No2, SEQID No3 and SEQID No 4.

SEQ ID No2 atpD gene sequence:

CATCGGCGAGCCGGTCGACGAAGCCGGTCCGCTGGTCACCGCTCACAAGCGCGCCAT CCACCAGGATGCGCCGTCCTATGTCGAGCAGTCGACGGAATCGCAGATTCTCGTCAC CGGCATCAAGGTCGTTGACCTTCTCGCTCCCTATGCACGCGGCGGCAAGATCGGCCTG TTCGGCGGCGCTGGCGTCGGCAAGACCGTTTTGATCATGGAACTGATCAACAACGTC GCCAAGGCGCATGGTGGTTATTCGGTTTTTGCGGGCGTCGGTGAACGTACCCGCGAA GGCAACGACCTCTATCACGAAATGATCGAATCGAACGTCAACAAGCATGGCGGCGGC GAAGGTTCGAAGGCTGCGCTGGTTTACGGCCAGATGAACGAACCGCCGGGCGCCCGC GCCCGTGTCGCCCTGACCGGCCTGACGGTCGCTGAACATTTCCGCGACCAGGGCCAG GACGTTCTGTTCTTCGTCGACAACATCTTCCGCTTCACG

SEQ ID No3 glnII gene sequence:

CGATGGGTACACTCCGGTACCGAACCTGCGTGGCAAGACGCAGATCAAGGAATTCGA CGCATTCCCGACGCTGGAACAGCTTCCGCTCTGGGGCTTTGACGGCTCCTCGACGCAG CAGGCTGAAGGCCGCAGCTCCGATTGCGTGCTGAAGCCGGTCGCCATCTATCCCGAC CCGGCCCGCACCAACGGCGCTCTCGTCATGTGCGAAGTCATGATGCCGGATGGCGTC ACGCCGCACGCATCGAATGCCCGCGCCACCATCCTCGACGACGAAGATGCCTGGTTC GGCTTCGAGCAGGAATATTTCTTCTACCAGAACGGCCGTCCGCTCGGCTTCCCCGAGC AGGGCTACCCGGCTCCGCAGGGTCCCTACTACACCGGCGTCGGCTATTCGAATGTAG GCGACGTCGCCCGCGAAATCGTCGAAGAACATCTCGACCTCTGCCTCGCTGCCGGCA TCAATCACGAAGGCATCAATGCCGAAGTGGCCAAGGGCCAGTGGGAATTCCAGATTTTCGGCAAGGGCTCCAAGAAGGCCGCCGACCAAATCTGGATGGCACGCTACCTCTTGC AGCGCCTGACCGAAAAGTACGGCATCGACATCGAGTATCACTGCAAGCCGCTCGGTG ACACCGAC

SEQ ID No4 recA gene sequence:

TATGACTCGGCTCACGAGATGTTGTCGAGATCGAGACGATCTCGACCGGCTCGCTTGG CCTCGATATCGCACTTGGCGTCGGCGGCCTGCCGAGGGGCCGCATCATCGAAATTTAC GGACCGGAAAGCTCCGGTAAAACGACGCTTGCGCTGCAGACCATTGCCGAAGCGCAG AAAAAGGGCGGTATCTGCGCCTTCGTCGATGCCGAACATGCGCTCGATCCTGTCTATG CCCGCAAGCTTGGCGTCGATCTGCAGAACCTTCTGATCTCGCAGCCCGATACCGGCGA GCAGGCGCTTGAAATCACCGATACGCTGGTGCGCTCCGGCGCCGTCGACGTCCTGGT CGTCGACTCGGTCGCCGCACTGACGCCACGCGCCGAAATCGAAGGCGAGATGGGCGA CAGCCTTCCCGGCCTGCAGGCGCGCCTGATGAGCCAGGCGCTGCGCAAGCTCACCGC TTCGATCTCGAAGTCGAACACC

the obtained sequence results were compared at the National Center for Biotechnology Information (NCBI) of the United states, and it was found that the model strains having the highest sequence similarity with the atpD, glnII and recA housekeeping genes at three sites of Bga2-2 were all Rhizobium anhuiensis CCBAU 23252^TThe similarity with the model strain is 100%, 99.1% and 98.8%, respectively. The comparison result of each gene sequence on NCBI is used to select 3 genes with high similarityThe model strain of (3) as a reference strain for tree construction.

Construction of 3 genes (atpD, glnII and recA) in combination with phylogenetic trees: the sequences of atpD, glnII and recA 3 housekeeping genes are respectively compared with the corresponding gene sequences of a reference strain by MEGA5, the sequences are cut up by taking the minimum length as a standard, the cut-up sequences are stored in a FASTA format, and the lengths of the three gene sequences are respectively 350nt, 460nt and 341 nt. The 3 sequences were spliced together in the notebook format and the tree was constructed using the Neighbor-joining method in MEGA5 software (bootstrap-joining), with a self-expanding value (bootstrap) of 1000 and atpD, glnII, recA as shown in FIG. 3.

FIGS. 2 and 3 show that the 16S rRNA gene of Bga2-2, and atpD, glnII, and recA 3 housekeeping gene combined sequences with the model strain Rhizobium anhuiense CCBAU 23252^TOn the same branch node, the similarity is 99.3%, and the international standard for the classification is 97% similarity. Again, as previously analyzed, the strain was compared to the model strain Rhizobium anhuiense CCBAU 23252^TThe similarity of these 4 genes was more than 97%, indicating that the strain Bga2-2 belongs to Rhizobium anhuiense.

Example 6 Effect of field inoculation

The field inoculation effect test of the strain is carried out in Chongzhou city in Chengdu plain region.

The test is carried out in 2016 for 10 months to 2017 for 4 months by adopting random block arrangement in the field, wherein two treatments are carried out in total, namely rhizobium Bga2-2 inoculation and no inoculation contrast treatment (CK), and the main Sichuan variety of the fennel is selected as the bean seed, no chemical fertilizer and organic fertilizer are applied. The prepared rhizobium inoculant (viable count is 5.8 multiplied by 10)⁸CFU/g microbial inoculum) and broad beans, drying in the shade, and hole sowing, wherein each nest has 3 granules, 2 seedlings are fixed, and the area of a cell is 10.8m²The distance between the pits is 30cm, the distance between the rows is 50cm, and CK is sown firstly during sowing so as to avoid the influence of rhizobium inoculation on CK treatment. Sampling in the full-bloom stage (growth period 105d) of the plant, and measuring the plant height, the root nodule number and the dry weight of the overground part of the plant; yield was determined during harvest (growth period 200 d). The management in the period is carried out according to the conventional management of broad bean planting of farmers.

TABLE 3 effects of inoculation in the field

At present, related researches on the field application effect of the broad bean rhizobia only relate to the nitrogen fixation effect and the yield influence of different rhizobia microbial inoculum on a broad bean/gramineae intercropping system, and the results show that the yield of the broad bean is increased by 13.7% after the rhizobia is inoculated compared with the yield of the broad bean which is not inoculated with the rhizobia (2009 ), and related reports on the nitrogen fixation effect and the yield influence of the broad bean rhizobia on a broad bean single-cropping system are not available at present. In the research of the subject, after the rhizobia is inoculated, the plant height, the plant dry weight and the root nodule number are all higher than those of the rhizobia which is not inoculated in the full-bloom period, the yield is obviously increased than CK, and the yield is increased by 31.8 percent, so that the inoculated excellent rhizobia has more obvious effect on the growth of the plants after the full-bloom period, and the rhizobia Rhizobium anhuiense Bga2-2 is the excellent rhizobia suitable for the ecological region.

Sequence listing

<110> Sichuan university of agriculture

<120> Rhizobium fabae strain Bga2-2 and application thereof

<141>2017-12-25

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1377

<212>DNA

<213> Rhizobium (Rhizobium anhuiense)

<400>1

ctgcggcggc ttaccatgca 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 gtagtct 1377

<210>2

<211>496

<212>DNA

<213> housekeeping Gene (atpd)

<400>2

catcggcgag ccggtcgacg aagccggtcc gctggtcacc gctcacaagc gcgccatcca 60

ccaggatgcg ccgtcctatg tcgagcagtc gacggaatcg cagattctcg tcaccggcat 120

caaggtcgtt gaccttctcg ctccctatgc acgcggcggc aagatcggcc tgttcggcgg 180

cgctggcgtc ggcaagaccg ttttgatcat ggaactgatc aacaacgtcg ccaaggcgca 240

tggtggttat tcggtttttg cgggcgtcgg tgaacgtacc cgcgaaggca acgacctcta 300

tcacgaaatg atcgaatcga acgtcaacaa gcatggcggc ggcgaaggtt cgaaggctgc 360

gctggtttac ggccagatga acgaaccgcc gggcgcccgc gcccgtgtcg 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 gatggcgtca cgccgcacgc 240

atcgaatgcc cgcgccacca tcctcgacga cgaagatgcc tggttcggct tcgagcagga 300

atatttcttc taccagaacg gccgtccgct cggcttcccc gagcagggct acccggctcc 360

gcagggtccc tactacaccg gcgtcggcta ttcgaatgta ggcgacgtcg cccgcgaaat 420

cgtcgaagaa catctcgacc tctgcctcgc tgccggcatc aatcacgaag gcatcaatgc 480

cgaagtggcc aagggccagt gggaattcca gattttcggc aagggctcca agaaggccgc 540

cgaccaaatc tggatggcac gctacctctt gcagcgcctg accgaaaagt acggcatcga 600

catcgagtat cactgcaagc cgctcggtga caccgac 637

<210>4

<211>482

<212>DNA

<213> housekeeping Gene (reca)

<400>4

tatgactcgg ctcacgagat gttgtcgaga tcgagacgat ctcgaccggc tcgcttggcc 60

tcgatatcgc acttggcgtc ggcggcctgc cgaggggccg catcatcgaa atttacggac 120

cggaaagctc cggtaaaacg acgcttgcgc tgcagaccat tgccgaagcg cagaaaaagg 180

gcggtatctg cgccttcgtc gatgccgaac atgcgctcga tcctgtctat gcccgcaagc 240

ttggcgtcga tctgcagaac cttctgatct cgcagcccga taccggcgag caggcgcttg 300

aaatcaccga tacgctggtg cgctccggcg ccgtcgacgt cctggtcgtc gactcggtcg 360

ccgcactgac gccacgcgcc gaaatcgaag gcgagatggg cgacagcctt cccggcctgc 420

aggcgcgcct gatgagccag gcgctgcgca agctcaccgc ttcgatctcg aagtcgaaca 480

cc 482

Claims (3)

1. A Rhizobium fabae strain Bga2-2 is 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: M2017613.

2. The use of a Rhizobium fabae strain Bga2-2 according to claim 1, wherein: the broad bean rhizobium strain is applied to production of broad beans in the Chengdu plain area of the Sichuan broad bean main production area.

3. The use of fava bean rhizobium strain line Bga2-2 according to claim 2, wherein: the number of viable bacteria contained in the culture medium is 5.8 multiplied by 10⁸CFU/g broad bean rhizobium inoculant Bga2-2 and broad bean seeds are mixed and sowed in holes after being dried in the shade.

Images