CN107904193B - Rhizobium V14-2 and application thereof - Google Patents
Rhizobium V14-2 and application thereof Download PDFInfo
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Abstract
The invention discloses Rhizobium (Rhizobium anhuiense) V14-2 and application thereof, wherein the Rhizobium (Rhizobium anhuiense) V14-2 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2017611. The rhizobium V14-2 is an excellent rhizobium strain of vetch, which has strong symbiotic nitrogen fixation capacity, obvious yield increase of vetch in Sichuan, IAA secretion capacity, organic phosphorus dissolving capacity, potassium dissolving capacity and strong stress resistance. The fresh grass yield of the vetch can be increased by 29.5 percent and the fresh root yield can be increased by 79.3 percent without applying nitrogen fertilizer and inoculating rhizobium V14-2.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to rhizobium V14-2 and application thereof.
Background
Vetch (Viciavilososal) is a main leguminous green manure crop in the rice region of China, is a main vetch variety used for cultivation of green manure and forage grass, and is a high-quality organic fertilizer (Duoyanzhu, 2017; Caowendong, etc., 2010). The green feed has the characteristics of tender stem juice, more leaves, high yield, good palatability, rich nutrition and the like, is a high-quality green feed, is a good substitute and a compound feed raw material of concentrated feed, and is deeply popular with farmers in breeding industries of Sichuan, Yunnan, Guizhou and the like (Guotaere, 2013; Shiwanqing, 2016). The purpose of increasing the yield of the vetch is achieved by applying chemical fertilizers generally in the planting process of the vetch, and researches show that the purpose of stabilizing the yield and increasing the yield can be achieved by applying 300kg of farmyard manure and 10kg of phosphate fertilizer to each mu of the vetch planted by farmers in Vietnamese county (Wang De Meng, 2006). However, the application of the fertilizer has the problems of high production cost, low utilization rate, ecological environment pollution and the like. And the yield of the leguminous crops can be increased only by inoculating the high-efficiency rhizobia without applying a chemical fertilizer in the production process due to the nitrogen fixation characteristic of the leguminous crops and the rhizobia. 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, the research on the screening and application of the high-efficiency rhizobia is mostly concentrated in leguminous crops with higher economic value, such as soybeans, and the like, and the research report on the application of the high-efficiency rhizobia of the vetch is few. The rhizobia group (1974) of Nanjing soil research institute of Chinese academy of sciences carries out screening of excellent strains of the rhizobia veticus in Jiangsu province between 1971 and 1973, and 7 rhizobia strains with fast growth, early nodulation and strong nitrogen fixation capacity are screened from 162 separated rhizobia strains; in 1972, a vetch inoculation rhizobium test is carried out in Zhang Gji public society of Cushan county of Jiangsu province in 9 months, and the vetch rhizobium inoculated under the same fertilizing condition can improve the fresh grass yield of the vetch in the initial bud period and increase the yield by 6.2-39.1%. 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 area or a strain in an area with similar conditions to the local area (Jianetal, 2008; Chengxin, et al, 2011). At present, the research of the high-efficiency vetch rhizobium suitable for the ecological environment of 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.
It is understood that the actual effect of rhizobia is closely related to the species of leguminous plants and the environment of the growing area of leguminous plants, and that rhizobia having a high effect on one leguminous plant is not always effective on another leguminous plant, and that rhizobia having a high effect on a certain area is not always effective on another area.
Therefore, it is particularly important to screen high-quality and high-efficiency multifunctional root nodule growth promoting strains suitable for specific regions and specific plants.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a high-quality and high-efficiency rhizobium suitable for planting of vetch in Sichuan area.
The technical scheme of the invention is as follows: rhizobium (Rhizobium anhuiense) V14-2, deposited in the China center for type culture Collection in Wuhan university in Wuhan City, Hubei province, China within 10 months and 23 days of 2017, with the deposit numbers: CCTCC NO: m2017611.
The rhizobium V14-2 is applied to planting vetch with smooth leaves in the Sichuan area.
The invention separates and purifies rhizobia from the root nodule of the fair-town wild vetch in the Wen-river region of the urban area of the city of Sichuan province, selects the bacterial colony characteristic of the rhizobia with the gram staining result of bacterial strain with small rod-shaped and gram-negative cells.
And performing a water culture tie-back test on the initially selected strain of the vetch variety, and screening out a strain which can nodulate and has strong nodulation capability and good symbiotic nitrogen fixation effect as a target strain rhizobium V14-2. Compared with the method without inoculating rhizobia, the dry weight of the sweet potato plant is improved by 37.9 percent.
Extracting total DNA from rhizobium V14-2, sequencing a plurality of housekeeping genes, constructing a phylogenetic tree, and determining the classification status of the target strain rhizobium V14-2. It was revealed that Rhizobium V14-2 belongs to Rhizobium anhuiense.
Rhizobium V14-2 is used for performing stress resistance tests such as acid and alkali resistance, salt resistance, growth temperature range and the like, growth promotion tests of IAA secretion capacity and phosphorus dissolution capacity and field inoculation tests. The results show that: the rhizobium V14-2 has strong acid and alkali resistance and can grow on a plate with the pH of 4-11, but the diameter of a bacterial colony on the plate with peracid or overbasing is smaller than the positive control of the pH of 7, which indicates that the peracid and the overbasing have a certain inhibition effect on the growth of the plate; the salt tolerance is also strong, and the YMA flat plate with 0.2-1.5% of NaCl can grow; the growth temperature range is wide, and the growth can be carried out within the temperature range of 20-28 ℃. Rhizobium V14-2 secreted IAA 8.13mg/L, indicating that Rhizobium V14-2 also had the ability to secrete IAA. Rhizobium V14-2 has certain dissolving capacity to organic phosphorus source matter and calcium phosphate, and the ratio of the phosphorus dissolving ring diameter to the colony diameter measured on lecithin and calcium phosphate plate is 1.34 and 1.19 respectively. The rhizobium V14-2 dissolves potassium by 1.88mg/L, which indicates that the rhizobium V14-2 has the capacity of dissolving potassium. The inoculation of the rhizobium V14-2 in the field shows that compared with the inoculation without the rhizobium, the fresh grass yield of the vetch can be increased by 29.5 percent, and the fresh root yield can be increased by 79.3 percent.
Compared with the prior art, the invention has the following beneficial effects:
the rhizobium V14-2 is an excellent rhizobium strain of vetch, which has strong symbiotic nitrogen fixation capacity, obvious yield increase of vetch in Sichuan, IAA secretion capacity, organic phosphorus dissolving capacity, potassium dissolving capacity and strong stress resistance. The fresh grass yield of the vetch can be increased by 29.5 percent and the fresh root yield can be increased by 79.3 percent without applying nitrogen fertilizer and inoculating rhizobium V14-2.
Preservation description:
rhizobium (Rhizobium anhuiense) V14-2, deposited in the China center for type culture Collection in Wuhan university in Wuhan City, Hubei province, China within 10 months and 23 days of 2017, with the deposit numbers: CCTCC NO: m2017611.
Drawings
FIG. 1 shows the colony morphology of Rhizobium V14-2 on YMA medium;
FIG. 2 is a phylogenetic diagram of the 16rRNA gene sequence of Rhizobium V14-2;
FIG. 3 is a phylogenetic diagram of the joint construction of three housekeeping genes, recA, atpD and glnII, of Rhizobium V14-2.
Detailed Description
Example 1 isolation, purification and preservation of Rhizobium V14-2
From wild sweet potatoes collected in the fair town of the Wen-river district of the urban area of the Sichuan province, selecting big and full red root nodules on the main root of a robust plant, cleaning the root nodules, collecting partial root skins, and performing surface disinfection: soaking in ethanol (95%) for 5min, pouring off, soaking in mercuric chloride (0.1%) for 5min, and washing with sterile water for 6-8 times. Placing the root nodule on a sterilized white porcelain plate in an ultraclean workbench, clamping the root nodule with a bamboo stick, smearing the root nodule on a flat plate of a YMA culture medium, scribing, purifying, and culturing in a thermostat at 28 ℃. YMA culture medium formula: KH (Perkin Elmer)2PO40.25g，MgSO4`7H2O 0.2g，CaCl2`6H20.1g of O, 0.1g of NaCl, 2mL of ammonium molybdate (1%), 2mL of boric acid (1%), 2.5mL of Congo red (1%), 10g of mannitol, 0.8g of yeast powder, 18-20 g of agar, 1000mL of water and pH 6.8-7.0.
After the plate grows out of the bacteria, the single bacterial colony which does not absorb red and is similar to rhizobium in shape is selected from the plate and is subjected to dilution streak culture on the plate. And observing the colony morphology about 3 days, and observing about 7 days until the slow rooting tumor bacteria appear in 6-10 days. Purification was performed by dilution-streaking plate separation. And preliminarily judging whether the bacterial colony is rhizobium or not according to the following two aspects: (1) colony morphology on YMA medium: milky white, not red, round, raised colony, neat and non-spreading edge, smooth surface, wetter and sticky. Fast-growing rhizobia for culturing colonies growing for 3-5 daysand (2) culturing the rhizobia to grow colonies for 6-10 days, wherein the cell morphology is slow rhizobia, (2) marking the confirmed rhizobia colonies, and performing gram staining on the section, wherein the microscopic examination result of the rhizobia shows that the cells are in small rod shapes, consistent in morphology, free of spores, and ring-shaped and gram-negative (G) cells usually contain 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 rhizobia V14-2 obtained by separation and purification in the embodiment is fast rhizobia, and is cultured on YMA culture medium added with congo red, so that the thalli do not absorb red, are milky white, have small bacterial colonies, are round, viscous, have high swelling degree and are slightly transparent, and the bacterial colonies grow 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 rhizobium inoculation test adopts a sand culture method, and the vetch variety used in the test is smooth-leaf vetch which is purchased from Chengdu green grass garden breed limited responsibility company. 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. Periodically supplementing a sterilized nitrogen-free nutrient solution, inoculating rhizobium V14-2 into a sand culture device, wherein the sand culture device adopts a 300mL plastic cup, a matrix adopts vermiculite, and each treatment is repeated three times by taking a plant of the same variety without inoculation of rhizobium V14-2 as a control CK. After harvesting, the inoculation effect of rhizobia V14-2 is evaluated by the root nodule number and the plant dry weight of the vetch plant.
(1) Bacterial liquid culture: rhizobium V14-2 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 large, plump and undamaged seed of vetch, soaking in 95% alcohol for 5min, removing alcohol, adding mercuric chloride (0.1%), soaking 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 sucking 2mL of bacterial liquid by using a liquid transfer gun and lightly beating the bacterial liquid near the roots of the vetch. 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 rhizobium V14-2 and the tested vetch show better nodulation capability and symbiotic nitrogen fixation capability; compared with the control without inoculation of rhizobia V14-2, V14-2 can obviously improve the dry weight of the plant of the vetch with smooth leaves and improve the dry weight by 37.9 percent compared with the control without inoculation of rhizobia. Therefore, the rhizobium V14-2 is an excellent and efficient strain well matched with the vetch.
TABLE 1 hydroponic test results of Rhizobium V14-2
Note: data are the average of triplicates;
example 3 stress tolerance of Rhizobium V14-2
The stress resistance of rhizobium V14-2 is mainly measured by 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). YMA slant culture of the above Rhizobium V14-2 was scraped with sterile water to prepare a bacterial suspension for use. Each treatment was repeated 3 times using the point inoculation method. Acid and alkali resistance and salt tolerance tests, and the results are observed and recorded after the plates suitable for measuring the growth temperature range are cultured for 4 days at 28 ℃.
(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: NaCl was added to YMA medium without NaCl as a basal medium, and NaCl concentrations were set so that NaCl0.2%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, 3%, 3.5% and 4% of the medium were present.
Test results show that rhizobia V14-2 has strong acid and alkali resistance and can grow on a plate with the pH of 4-11, but the diameter of a bacterial colony on the peracid or alkali-passing plate is smaller than that of a positive control with the pH of 7, which indicates that peracid and alkali-passing have a certain inhibition effect on the growth of the rhizobia V14-2; the salt tolerance is also stronger, and the YMA flat plate with 0.2-1.5% of NaCl can grow.
Example 4 growth promoting ability of Rhizobium V14-2
The growth promoting capability of rhizobium V14-2 mainly considers the capabilities of secreting auxin (IAA), dissolving phosphorus and potassium.
(1) Determination of the ability to secrete auxins
The capacity of rhizobium to secrete auxin (IAA) is measured by a colorimetric method, and the measurement culture medium adopts an improved Congo red liquid culture medium, and comprises the following components: 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 the rhizobium V14-2 secretes IAA 8.13mg/L, which indicates that the rhizobium V14-2 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.
the formula (g/L) of Monkina culture medium for dissolving organophosphorus is 10g of glucose and 5g of CaCO3，0.5g(NH4)2SO40.4g of yeast powder, 0.3g of KCl, 0.2g of lecithin, 0.3g of NaCl and 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 PKO culture medium formula (g/L) for culture medium capable of dissolving inorganic phosphorus 10g of glucose and 3.0g of the above-mentioned 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 results show that rhizobium V14-2 has certain dissolving capacity for organic phosphorus source substances and calcium phosphate, and the ratio of the phosphorus dissolving ring diameter to the bacterial colony diameter measured on lecithin and calcium phosphate plates is 1.34 and 1.19 respectively.
(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): sucrose 10g, MgSO4.7H20.5g of O, 0.1g of NaCl, 0.5g of yeast extract and pH 7.2-7.4.
A250 mL triangular flask is used as a shake flask, 100mL of potassium-free basic liquid culture medium is filled, 0.5g of potassium feldspar mineral powder (800 meshes) is added, 5mL of V14-2 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 the rpm/min for 7 days, 10mL of culture solution is taken, the culture solution is centrifuged at 8000r/min for 10min, the supernatant is taken, and the content of soluble potassium in the supernatant is measured by a flame photometry method. The rhizobium V14-2 dissolves potassium by 1.88mg/L, which indicates that the rhizobium V14-2 has the capacity of dissolving potassium.
Example 5 amplification and phylogenetic analysis of 16S rRNA Gene and other housekeeping genes glnII, recA of Rhizobium V14-2
Extracting total DNA of the strain, performing PCR amplification on the 3 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 ═ 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 sequencing result of Chengdu Hingxi biotechnology Limited company detected by the amplification product according to the method is shown as SEQIDNo.1.
The sequence results obtained were compared with EzTaxon (http:// www.ezbiocloud.net/EzTaxon), and the model strain having the highest similarity to the 16S rRNA gene sequence of Rhizobium V14-2 was found to be Rhizobium anhuiensis ECCBAU 23252TThe similarity was 99.00%. And (3) selecting a model strain with high similarity as a reference strain by using a result of comparing the sequence on the EzTaxon to construct a phylogenetic tree. Construction of a phylogenetic tree of the 16S rRNA gene, with a bootstrap value (bootstrap) of 1000, was carried out by the Neighbor-joining method in Mega7 software, and the phylogenetic tree is shown in FIG. 1.
(2) Construction of a Joint phylogenetic Tree of Multi-site Gene sequences
In order to further determine the classification status of rhizobium V14-2 more accurately, sequences of housekeeping genes recA and glnII of 2 sites are selected for constructing a combined phylogenetic tree.
The primers recAF2 and recAR2 for amplifying recA 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; ddH2O13. mu.l. (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. 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 the sequence size of glnII is 637nt, the sequence result is SEQID No.2, the sequence size of recA is 493nt and the sequence result is SEQID No. 3.
The obtained sequence results were compared with 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 housekeeping genes at the glnII and recA sites of Rhizobium V14-2 were both RhizobiumTThe similarity with the model strain was 100% and 98.17%, respectively. The results of the alignment of each gene sequence at NCBI are used to select 6 model strains with high similarity with 2 genes as reference strains for tree building.
Construction of a joint phylogenetic tree of 2 genes (recA and glnII): firstly, the sequences of the 2 housekeeping genes of recA and glnII are respectively compared with the corresponding gene sequences of a reference strain by MEGA7, 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 sequences of the recA and glnII are 342nt and 438nt respectively after the sequences are cut up. The 2 sequences were spliced together by WORD opening, and the construction of a joint phylogenetic tree was performed by using the Neighbor-joining method (Neighbor-joining) in MEGA7 software, with a bootstrap (bootstrap) of 1000 and a glnII, recA joint phylogenetic tree as shown in FIG. 3.
FIGS. 2 and 3 show that the 16S rRNA gene of V14-2, and the combination sequence of recA, glnII2 housekeeping genes, combined with the model strain Rhizobium anhuiense CCBAU 23252TOn the same branch node. Again, as previously analyzed, the strain was compared to the model strain Rhizobium anhuiense CCBAU 23252TThe similarity of these 2 genes is very high, and the similarity of recA and glnII is 98.17% and 100% respectively, which indicates that Rhizobium V14-2 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.
The test is provided with two treatments, namely inoculation of rhizobium V14-2 and no inoculation of control treatment (CK), the bean species is selected from a Sichuan vetch main cultivated variety 'smooth leaf vetch', and no chemical fertilizer or organic fertilizer is applied. And random block arrangement in the field is adopted. The test was carried out in 2016, 9 and 30 months, to 2017, 4 and 22 months. The prepared rhizobium V14-2 microbial inoculum (viable count is 6.4 multiplied by 10)8CFU/g microbial inoculum) and vetchDressing the seeds, drying in the shade and then sowing. 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 in the full-bloom stage (growth stage 204d) of the plant, and measuring the root nodule number of the plant, the fresh weight of the plant on the overground part, the fresh weight of the root on the underground part and the yield of fresh grass. The management in the period is carried out according to the conventional management of farmers for planting the sweet potatoes. The results of the field tests are shown in Table 3.
TABLE 3 field inoculation Effect of vetch seed inoculated with Rhizobium V14-2
Note: data are mean of triplicates
The inoculation of rhizobium V14-2 results in higher effective nodule number, total nodule number and fresh grass yield than those of un-inoculated control in full-bloom stage, and the fresh grass yield is 29.5% higher than CK and 79.3% higher than CK. Therefore, the inoculated excellent rhizobia has obvious effect on increasing the yield of the vetch. The rhizobia V14-2 is an excellent rhizobia suitable for the production of the vetch.
The field inoculation test research of the alder spring town of Chongzhou city in Sichuan shows that after the rhizobium V14-2 is inoculated, the number of plant red nodules, the total number of the rhizobium and the yield of fresh grass are all higher than those of an uninoculated control, the yield of the fresh grass is increased by 29.5%, the yield of the fresh root is also higher than that of the uninoculated control treatment, the yield is increased by 79.3%, and the increase of the biomass of the ground and underground parts indicates that the rhizobium V14-2 promotes the growth of the vetch seeds. Therefore, the rhizobia V14-2 obtained by separation can be widely popularized and applied in the production of the Sichuan vetch.
The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.
Claims (2)
1. Rhizobium anhuiense V14-2, which is preserved in China center for type culture Collection with the preservation number of CCTCC M2017611.
2. The use of the Rhizobium anhuiense V14-2 in planting vetch-broom vetch in Sichuan area according to claim 1.
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