CN114891658B - Common vetch root nodule strain HBUJ010033 and application thereof - Google Patents
Common vetch root nodule strain HBUJ010033 and application thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Soil Sciences (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a common vetch root nodule bacterial strain HBUJ010033 and application thereof, wherein the common vetch root nodule bacterial strain is sophora japonica sp HBUJ010033, the preservation unit is the Guangdong province microorganism strain preservation center, the preservation address is the No. 5 building of No. 59 of the 100 university of Mitsui, the Guangzhou city, the preservation date is 2021, 1 month and 7 days, and the preservation number is GDMCC No.61423. The application of the strain HBUJ010033 in the planting and production of common vetch in inner Mongolian. The strain is a good vetch rhizobium strain with good affinity for the spring vetch No. 3 of the inner vetch, obvious yield increase, IAA secretion and phosphorus dissolution capacity, and strong stress resistance, and when no nitrogen fertilizer is applied and the strain is only inoculated, the yield and dry weight of the vetch are increased by 52.1%, and the plant height, nodulation number and crude protein content are also increased.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a common vetch rhizobium strain HBUJ010033 and application thereof.
Background
Common vetch (Vicia sativa L.), an aliased herb, perk, safflower, a herb belonging to the genus Vicia (Vicia L.) of the order Fabales), belonging to the genus vines. The common vetch plant She Liangduo is soft in stems and branches and good in palatability, can carry out biological nitrogen fixation with rhizobia, can obtain nitrogen nutrition for self-demand, and is an excellent feed with rich protein. The arrow peas are warm and cool in climate, especially suitable for cold areas such as Tibet, are good in growth in early spring and late autumn, short in growth period, quite high in biomass and nutritive value, and after the plants die, a large amount of root residues and fallen leaves can increase humus of soil, so that the content of organic matters in the soil is remarkably increased, and therefore the arrow peas are high-quality green fertilizer crops, the arrow peas and rhizobia symbiotic nitrogen fixation not only increase the nutrition of soil nitrogen, but also play the roles of improving the soil structure and improving the soil fertility, and the arrow peas and rhizobia symbiotic nitrogen fixation are main green fertilizer crops in northern China. The common vetch also has the characteristics of drought resistance, barren resistance and strong stress resistance, and is the previous work of various crops.
The fertilizer value and the feed value of the leguminous green manure are higher than those of the non-leguminous green manure. The leguminous green manure has biological nitrogen fixation effect, can provide nitrogen fertilizer for crops and increase crop yield, thereby reducing the use amount of chemical synthesis nitrogen fertilizer in agriculture and reducing environmental pollution. But leguminous plants need to interact with corresponding rhizobia to form a symbiotic matching relationship so as to play the advantages of a symbiotic nitrogen fixation system, in recent years, green manure crops are effectively utilized by adopting the measures of turning and returning straws to fields, the growth of succeeding crops is greatly promoted, and the soil activity and the contents of nitrogen, phosphorus and the like of the soil are enhanced.
Inoculating rhizobia to common vetch can increase plant biomass and nodulation number, and the seedlings grow well (duckweed, 2016; wang Xuecui, etc., 2016). The mixed sowing of the common vetch and the oat is one of measures for constructing high-yield and high-quality pasture, can relieve the shortage of forage grass supply, especially in alpine pasture areas, and is beneficial to improving the yield and quality of the pasture (Qin Yan, 2020). The breeding research of the high-efficiency rhizobia strain of the common vetch is less, and no matching research of varieties exists. Rhizobia strains have the characteristics of environmental adaptation selectivity and specificity for plant species, so that the selection of suitable rhizobia in a specific environment is very necessary, a certain plant is planted for the first time, the corresponding matched rhizobia is lacking, leguminous plants cannot exert the effects of fully fixing nitrogen and improving soil nutrients (Chen Wenxin, 2004; 2011), and currently, available strains of rhizobia of common vetch are few.
The existing research shows that besides nitrogen fixation, few rhizobia have the growth promoting characteristics of dissolving phosphorus and secreting growth hormone (IAA), so that the application value of the rhizobia is increased. Rhizobium sojae has stable phosphate solubilizing ability in different soils (Wang Jinsheng, 2020), and strains of common vetch were tested for phosphate and potassium solubilizing and IAA secreting growth promoting ability (Xu Kai, not, 2016).
Disclosure of Invention
The invention aims to provide a common vetch rhizobium strain HBUJ010033 and application thereof, and provides a high-efficiency nitrogen fixation rhizobium strain HBUJ010033 matched with a spring pea No. 3 main cultivated variety of an inner Mongolian green manure, so as to solve the problem of insufficient high-efficiency common vetch rhizobium strain in production in the prior art. The strain of the invention has IAA secretion and certain phosphate dissolving and potassium dissolving capacity, and has good field application prospect.
The technical scheme of the invention is as follows: a strain of a root nodule of common vetch is HBUJ010033, which is a sophora rhizobium (Rhizobium sophorae) HBUJ010033, the preservation unit is Guangdong microorganism strain preservation center, the preservation address is No. 59 building 5 of 100 university of Migo, guangzhou City, the preservation number is GDMCC No.61423, and the preservation date is 2021, 1 and 8 days.
The application of the vetch root nodule strain HBUJ010033 in the planting production of vetch in inner Mongolia.
Seed dressing is carried out on the rhizobium japonicum HBUJ010033 bacterial agent and the common vetch, and then sowing is carried out.
The number of viable bacteria contained in the rhizobium viciae HBUJ010033 microbial inoculum is 10 7 ～10 9 CFU/g。
When the rhizobium viciae HBUJ010033 microbial inoculum is prepared, the strain culture conditions are as follows: the temperature is 25-28 ℃, and the pH is 6-9.
When the rhizobium viciae HBUJ010033 microbial inoculum is prepared, the salt concentration of a strain culture medium is less than or equal to 3.0wt%.
The beneficial effects of the invention are as follows:
the vetch rhizobium strain HBUJ010033 is a superior vetch rhizobium strain with strong symbiotic nitrogen fixation capability, good affinity for the vetch spring vetch No. 3 of the vetch, obvious yield increase, IAA secretion and phosphorus dissolution capability and strong stress resistance, and when no nitrogen fertilizer is applied, the yield dry weight of the vetch is increased by 52.1%, and the plant height, the nodulation number and the crude protein content are also increased when the vetch rhizobium strain is only inoculated.
Drawings
FIG. 1 shows colony morphology of the vetch nodule strain HBUJ010033 on YMA medium.
FIG. 2 is a phylogenetic tree of the 16S rRNA gene sequence of the vetch root nodule strain HBUJ 010033.
FIGS. 3-5 are phylogenetic trees constructed from the combination of three housekeeping genes recA, atpD and glnA of the Kja sativa rhizobium strain HBUJ 010033.
Detailed Description
The invention is further illustrated by the following examples, in which the processes and methods not described in detail are conventional and well known in the art, and in which the starting materials or reagents used are commercially available unless otherwise indicated.
Example 1 isolation, purification and preservation of the common vetch root nodule strain HBUJ 010033.
1. The method comprises the steps of collecting vetch root nodules from the county of Wuchuan in inner Mongolia, firstly selecting plants with developed strong root systems, taking root nodules which are large and full and are ground on main roots, cutting off the root systems of the root nodules, cleaning, cutting off part of the root nodules with the root, and then preserving the color-changing silicone tube of the root nodules. After the root nodule is brought back into the room, soaking the root nodule in sterile water for 0.5-2h, and then sequentially carrying out surface disinfection: soaking in 95% ethanol for 5min to remove surface tension, sterilizing with 3.2% NaClO for 3min, and washing with sterile water for 5-7 times. The root nodule is placed in a sterile culture dish under a sterile environment, the front end of the root nodule is crushed by a sterile toothpick, bacterial liquid in the root nodule flows out, and an inoculating loop is used for dipping the bacterial liquid to streak and inoculate on a YMA flat plate. Culturing at 28deg.C for 2-3 days.
Wherein the YMA medium formula: mannitol 10.00g, yeast powder 0.80g, K 2 HPO 4 0.25g、KH 2 PO 4 0.25g、MgSO 4 0.2g, naCl 0.10g, agar powder 18g, 1000mL of distilled water, pH6.8-7.0.
2. After single colony is grown, bacterial colonies similar to rhizobia in morphology are selected from a plate for gram staining, microscopic examination, gram staining is negative, bacterial rod-shaped bacterial colonies are subjected to dilution streak plate separation for purification, and pure cultures are obtained for 16S rRNA gene sequencing. Determining whether the isolate is rhizobia from: (1): colony morphology on YMA medium: the white and semitransparent colony is round, the edge is thin, the middle is thick, the colony is thin, and the size of the colony is 3-5mm; the surface is smooth, moist and sticky. Colonies were grown by incubation at 28℃for 3-5 days. (2) cell morphology: labeling the identified rhizobia colonies, staining the rhizobia colonies with a gram, and microscopic examination of the rhizobia colonies to give cellsThe two ends of the particle are slightly round, the shape is more consistent, the size is 2-3 mu m multiplied by 3-5 mu m, no spore exists, the cell often contains beta-hydroxybutyrate particles, the particle occupies more than 1/3 volume of the cell, the refractive property is strong, and the particle is gram negative (G - ). (3) And (3) extracting bacterial DNA from the 16S rRNA gene, and amplifying and sequencing the 16S rRNA gene sequence, wherein the 16S rRNA gene belongs to rhizobium related genus. If the labeled colony has the characteristics of the three aspects, the colony is inoculated into a test tube YMA slant for culture and preservation.
EXAMPLE 2 tieback screening assay for rhizobia
The tieba inoculation test adopts sand culture, and the common vetch variety used in the test is spring vetch No. 3, which is a northern general variety and is derived from an inner Mongolian autonomous region. Inoculating the common vetch root nodule strain HBUJ010033 to germinated spring vetch No. 3 seedlings, then planting in Sha Peiqi, adopting a plastic cup of 400mL for Sha Peiqi, adopting vermiculite as a substrate, and repeating each treatment for three times by taking the same plant without inoculating the root nodule strain HBUJ010033 as a Control. Culturing at 25deg.C for 8 hr, planting for 50 days, and periodically supplementing sterilized nitrogen-free nutrient solution. The inoculation effect of the vetch root nodule strain HBUJ010033 was evaluated after harvest with dry weight, nodule number, chlorophyll content and crude protein content of the vetch plants.
(1) Preparation of bacterial liquid: inoculating the common vetch rhizobium strain HBUJ010033 into YMA slant culture medium, culturing at 28 ℃ to logarithmic growth phase, and collecting thallus on the slant with sterile water to obtain bacterial suspension.
(2) Accelerating germination of seeds: selecting large and full undamaged spring pea No. 3 seeds, treating the seeds with concentrated sulfuric acid for 10min, sterilizing the seeds with sterile water for 2 to 3 times, sterilizing the seeds with 3.2 percent NaClO for 5min, and then washing the seeds with sterile water for 5 to 7 times. Finally, the seeds are placed on 0.8 percent water agar, placed for three days at the temperature of 4 ℃ in the dark, and then transferred to the temperature of 25 ℃ for germination, and the seeds with young roots grow to 0.5 cm to 1cm for standby.
(3) Sha Peiqi preparation: 400mL of disposable sterilized plastic cup is selected as Sha Peiqi, vermiculite is sterilized for three times in the night, then the bottom of the plastic cup is perforated, gauze strips with the length twice as long as the cup is penetrated into the plastic cup for absorbing water, then the plastic cup is filled with the vermiculite, and 500mL of glass bottle (the glass bottle is sterilized in advance) containing 250mL of sterile low-nitrogen nutrient solution is clamped in the plastic cup.
(4) The formula of the nitrogen-free nutrient solution comprises the following steps: na (Na) 2 HPO 4 ·2H 2 O 0.15g，CaCl 2 ·2H 2 O 0.10g，MgSO 4 ·7H 2 O 0.12g，KH 2 PO 4 0.10g，FeC 6 H 5 O 7 ·H 2 O0.005 g, trace element 1mL, distilled water 1000mL.
(5) The formula of the trace element solution comprises the following steps: HBO (heterojunction bipolar transistor) 3 2.86g，MnCl 2 ·4H 2 O 1.81g，ZnSO 4 ·7H 2 O 0.22g，MgSO 4 ·4H 2 O 2.03g，Na 2 MoO 4 ·2H 2 O0.13 g, distilled water 1000mL.
(6) Planting and measuring indexes: inoculating the prepared bacterial suspension to a germination accelerating seedling for 30min in a sterile culture dish, then planting the seedling in a plastic cup containing vermiculite, and sucking bacterial liquid by a pipetting gun to lightly beat near the root of common vetch. The same plant species without inoculation treatment is also set as a Control. When in planting, the control plants are planted first, so that inoculation pollution is avoided, and each treatment is repeated for 3 times.
(7) The inoculated plant culture device is placed in a greenhouse at 25 ℃, red and blue light LED lamps are irradiated for 8 hours every day, sterilizing nitrogen-free nutrient solution is supplemented regularly, plants grow for 50 days, vermiculite is cleaned, the plants are taken out, and the root nodule number, dry weight, chlorophyll content and crude protein content of single plants of the plants are detected. The sand culture test results are shown in table 1.
The results in Table 1 show that the vetch root nodule strain HBUJ010033 and the spring vetch No. 3 of the vetch variety to be tested show better nodulation capability and symbiotic nitrogen fixation capability; compared with a control without inoculating the common vetch root nodule strain HBUJ010033, the strain can obviously improve the dry weight, the root nodule number and the chlorophyll and crude protein content of the plant of spring vetch No. 3. The dry weight is improved by 136.1% compared with the non-inoculated control. It can be seen that the vetch rhizobium strain HBUJ010033 is a good and efficient strain that matches best with spring vetch No. 3.
TABLE 1 sand culture test of the vetch root nodule strain HBUJ010033
Note that: data are the average of three replicates.
Example 3 stress resistance of the common vetch root nodule Strain HBUJ010033
(1) The stress resistance of the common vetch root nodule strain HBUJ010033 is mainly measured in the salt resistance, acid and alkali resistance and suitable growth temperature range. YMA plates incubated for 5d at pH7 and 28℃were used as positive controls (controls) in the basal medium. YMA slant cultures of the common vetch root nodule strain HBUJ010033 are prepared into bacterial suspensions for later use by scraping with sterile water. Each treatment was repeated 3 times using the spot seeding method. Plates for acid and alkali resistance test were each incubated at 28℃for 3d, and the results were recorded. The formula of the culture medium for acid and alkali resistance determination comprises the following components: the YMA culture medium is taken as a basic culture medium, and the pH value of the culture medium is adjusted by HCl and NaOH to be 4.0,5.0,6.0,8.0,9.0, 10.0 and 11.0 in sequence.
(2) The formula of the culture medium for salt tolerance determination comprises: the concentration of NaCl 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 based on YMA medium from which NaCl was removed.
Test results show that the vetch rhizobium strain HBUJ010033 has strong acid and alkali resistance, can grow on a plate with the pH value of 6-9, but has a certain inhibition effect on the growth of peracid or peracid-peralkali by positive control with the colony diameter smaller than pH7 on the plate with the peracid or the peracid-peralkali; the salt tolerance is also stronger, and the growth can be carried out on YMA flat plates with NaCl concentration of 0.2% -3%.
Example 4 growth-promoting ability of the common vetch root nodule strain HBUJ 010033.
(1) Determination of the ability to secrete auxin
Qualitative determination: 50mL of modified TY liquid medium (yeast powder 3.0g, tryptone 5.0g, caCl) 2 ·2H 2 0.7g of O, 18.0g of agar, 1000mL of distilled water, 7.0 of pH and 0.1g of tryptophan) Filling into 150mL triangular flasks, sterilizing at 121 ℃ for 20min, cooling, and respectively inoculating 500 mu L of strain bacterial suspension to be detected into each triangular flask. 3 replicates per strain were used as controls with minimal medium without strain inoculation. Placing the triangular flask of the inoculated strain on a shaking table at 28deg.C and 200r/min, shaking and culturing for 12d, dripping 100 μl of the bacterial suspension onto a white ceramic plate, and adding 50 μl of colorimetric solution (composition of colorimetric solution: 4.5g FeCl) 3 Dissolving in 300ml distilled water, adding H 2 SO 4 580mL, constant volume to lL). The white ceramic plate was allowed to react at room temperature for 15min, and then the color change was observed. The pink color shows that the strain can secrete IAA, the darker the color shows that the secretion capacity is stronger, and the non-discolored one shows that the strain does not produce IAA. And (3) taking another glass plate while measuring the strain to be tested, respectively dripping a proper amount of IAA (IAA) of 5mg/L, 10mg/L, 15mg/L and 20mg/L, then adding a colorimetric solution, reacting for 15min at room temperature, and preliminarily judging the content of the secreted auxin of each strain to be tested by observing the change degree of the color and comparing the color with the color of the strain to be tested which is positive after the colorimetric solution is added.
Quantitative determination: centrifuging the culture solution for 12d at 10000r/min and 4 ℃ for 10min, taking 1mL of supernatant, adding 1mL of colorimetric solution, standing in darkness for 30min, rapidly colorimetrically measuring absorbance at 530nm wavelength by using a spectrophotometer, and calculating IAA content according to the absorbance. Standard curves were made with pure indoleacetic acid.
And (3) standard curve establishment: IAA of 5, 10, 15 and 20mg/l is prepared, IAA with different concentrations is mixed with colorimetric solution 1:1 to measure OD, and distilled water is mixed with the colorimetric solution for zeroing.
As a result, it was found that the vetch rhizobium strain HBUJ010033 has an IAA-secreting ability and can secrete 9.93mg/L of IAA.
(2) Capacity to dissolve organic and inorganic phosphorus
The capacity of the strain to dissolve organic phosphorus and inorganic phosphorus is determined by a phosphorus dissolving ring method. The organic phosphorus source is lecithin, and the inorganic phosphorus source is Ca 3 (PO 4 ) 2 All of the above reagents are commercially available analytically pure reagents. Determination of the ability to dissolve organic phosphorus Using Meng Jinna Medium, inorganic phosphorus was dissolvedThe capacity assay uses PKO medium. The inoculation was performed by spot inoculation, and the bacterial suspension was formulated as in example 3 stress resistance test, and each treatment was repeated 3 times. After 5d cultivation in an incubator at 28℃it was observed whether the strain grew or not and whether a phosphate solubilizing loop appeared or not.
1) Determination of the organic phosphorus dissolving Capacity Using Meng Jinna Medium, formulation (g/L): 10g of glucose, caCO 3 5g，(NH 4 ) 2 SO 4 0.5g，NaCl 0.3g，KCl 0.03g，FeSO 4 ·7H 2 O 0.03g，MnSO 4 ·4H 2 O0.03 g, lecithin 0.2g, yeast extract 0.4g, agar 20g, distilled water 1.000 mL, pH 7.0-7.2. Wherein lecithin is selected from diluted ovum gallus Domesticus flavus, and is mixed with sterilized culture medium cooled to about 60deg.C, and poured into flat plate.
2) Determination of the ability to dissolve inorganic phosphorus PKO Medium, formulation (g/L): glucose 10g, ca 3 (PO 4 ) 2 5g，(NH 4 ) 2 SO 4 0.5g，NaCl 0.2g，KCl 0.2g，MgSO 4 ·7H 2 O 0.03g，MnSO 4 0.03g，FeSO 4 0.003g, yeast extract 0.5g, agar 20g, 1000mL of distilled water and pH value of 6.8-7.0. Wherein Ca is 3 (PO 4 ) 2 Grinding with mortar, sieving with 300 mesh sieve, sterilizing, cooling with sterilized culture medium to about 60deg.C, mixing, and pouring into flat plate.
The results show that the common vetch root nodule strain HBUJ010033 has certain dissolving capacity on organic phosphorus source substances and calcium phosphate. The ratio of the diameter of the phosphate solubilizing ring to the diameter of the colony, which is measured on the lecithin and calcium phosphate plates, is 1.33,1.32, which shows that the vetch rhizobium strain HBUJ010033 has certain capabilities of solubilizing organic phosphorus and calcium phosphate.
Example 5 amplification and phylogenetic analysis of the 16S rRNA Gene of the Amaranthus sonchifolius strain HBUJ010033 and the other housekeeping genes recA, atpD and glnA
The total DNA of the strain was extracted, the 4 genes were amplified by PCR using the primers shown in Table 2, and the PCR amplification reaction was performed by using a Biometra-Tgradient instrument, and the PCR amplification product was detected by electrophoresis on 1.0% agarose gel, and then sent to Shanghai Biotechnology Co., ltd for sequencing, and the phylogenetic tree was constructed by using software Mega7.0.
TABLE 2 amplification of primer sequences required for Rhizobium vicina
Note that: s=c or G
(1) Amplification of 16S rRNA Gene and construction of phylogenetic Tree
Extracting DNA of the tested strain by adopting a GUTC method,
(a) The activated strain is inoculated on TY solid slant culture medium and cultured for 2-3d at 28 ℃.
(b) 1mL of sterile physiological saline is added for washing, the solution is centrifuged for 2min at 12000r/min, and the bacterial liquid is poured into a 1.5mL centrifuge tube.
(c) The supernatant was discarded by centrifugation at 12000r/min three times with 1 XDE buffer.
(d) Adding 600 μl GUTC buffer, shaking, standing at room temperature for 15min, centrifuging at 12000r/min for 2min, and discarding supernatant.
(e) 60 μl of the diatomaceous earth suspension was added and shaken, left at room temperature for 15min, centrifuged at 12000r/min for 2min, and the supernatant was discarded.
(f) Adding 500 μl GUTC buffer, shaking, standing at room temperature for 15min, centrifuging at 12000r/min for 2min, and discarding supernatant.
(g) 600 μl of wash buffer was added and washed twice, and the supernatant was discarded by centrifugation at 12000r/min for 2min.
(h) 600 μl of 75% ethanol was added and washed once, and the supernatant was discarded by centrifugation at 12000r/min for 2min.
(i) The precipitate was dried on an ultra clean bench (until the diatomaceous earth became white), and was incubated with 50. Mu.l of TE buffer at 55-65℃for 5-7min, and centrifuged at 12000r/min for 2min.
(j) The supernatant was collected into a 1.5mL centrifuge tube and stored at-20℃until use.
The 16S rRNA gene was amplified using the general primers P1 and P6 of Table 2 using the total DNA as a template. PCR reaction System (20. Mu.L): 2 XPCRMIX 10. Mu.L, 1. Mu.L each of primers P1 and P6 (20. Mu.M), 1. Mu.L of DNA template, and 20. Mu.L of ultrapure water were added. PCR reaction conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min for 30s,30 cycles; finally, the extension is carried out for 10min at 72 ℃.
Mixing 2.5 mu L of 16S rDNA amplification product with 0.6 mu L of 10 xLoding Buffer, loading the mixture into 1% agarose gel for sample application, taking 100bp xLoding Ladder as a marker, and carrying out voltage electrophoresis detection with the Loading amount of 2.5 mu L and 5V/cm; scanning and photographing in a gel imager after electrophoresis is finished, checking the length and concentration of amplified fragments, and preserving at-20 ℃ for standby. The amplified product is sent to Shanghai Biotechnology Co.Ltd for sequencing, and the result is seqID1.
16S rRNA Gene sequence seqID1
>GGATCGGAAGGCTTACCATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACG GGTGAGTAACGCGTGGGAATCTACCCTTGACTACGGAATAACGCAGGGAAACTTGTGCTAATACCGTATGTGTCCTTCGGGAGAAAGATTTATCGGTCAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGAGAAGATAATGACGGTATCCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATGCCCGGCTACTTGCAGAGATGCAAGGTTCCCTTCGGGGACCGGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTAAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGGTAGGTTCAGGCGACCT
Results of the sequences obtainedComparing EzTaxon (http:/www.ezbiocloud.net/ezTaxon), selecting model bacteria with high similarity as reference strain, constructing phylogenetic tree, and finding that model bacteria with highest similarity to 16S rRNA gene sequence of rhizobium HBUJ010033 is Rhizobium sophoraeCCBAU 03386 T The similarity is 99.99%, the 16S rRNA gene phylogenetic tree is constructed by using the maximum likelihood method (Maximun likelihood method) of Mega7.0 software by using the comparison result of the sequence on EzTaxon, the self-expanding value (boottrap) is 1000, the base substitution frequency of each position is 1%, and the phylogenetic tree is shown in figure 2.
(2) Construction of phylogenetic tree of Multi-site Gene sequences
To further more accurately determine the taxonomic status of rhizobia veticus HBUJ010033, the phylogenetic status was determined by additionally selecting the sequences of the 3-locus housekeeping genes recA, atpD and glnA. Primers rec41F and rec640R for recA, atpD352F and atpD871R for atpD, glnA 144F and glnA1142R for glnA, and the primer sequences are shown in Table 2. The reaction system was 25. Mu.L. The composition of the reaction solution was as follows: 2 XPCRMIX 10. Mu.L, 1. Mu.L each of the upper primer and the lower primer (10. Mu.M), 1. Mu.L of the DNA template, and 25. Mu.L of ultrapure water were added.
1) recA amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 45s,30 cycles; finally, the extension is carried out for 5min at 72 ℃.
2) atpD amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 64℃for 1min, extension at 72℃for 1min for 30s,30 cycles; finally, the extension is carried out for 5min at 72 ℃.
3) glnA amplification conditions: pre-denaturation at 95℃for 10min; denaturation at 94℃for 30s, annealing at 61℃for 1min, extension at 72℃for 1min30s,30 cycles; finally, the extension is carried out for 10min at 72 ℃.
The amplified products are detected by the method and sent to Shanghai Biotechnology Inc. for sequencing, each gene is subjected to bidirectional sequencing (sequences of forward and reverse primers), the sequencing results are spliced by DNAman6.0, and the recA sequence is 487bp, the sequence result is seqID2, the atpD sequence is 515bp, the sequence result is seqID3, the glnA sequence is 972bp, and the sequence result is seqID4.
recA gene sequence seqID2:
>GCCTGTGGTGCATGGTGTTCGACTTGGAGATCGAAGCGGTGAGCTTGCGCAGCGC CTGGCTCATCAGGCGCGCCTGAAGACCGGGAAGGCTATCGCCCATCTCGCCCTCGATTTCGGCACGCGGCGTCAGTGCGGCGACCGAGTCAACGACGAGAACGTCGACGGCGCCGGAGCGCACCAGCGTATCGGTGATTTCGAGCGCTTGCTCGCCGGTATCGGGCTGCGAGATCAGAAGGTTCTGCAGGTCGACGCCAAGCTTGCGGGCATAGACGGGATCGAGCGCATGTTCGGCATCGACGAAGGCGCAGATGCCACCCTTCTTCTGCGCTTCGGCAATCGTCTGTAGTGCAAGCGTCGTCTTGCCGGAGCTTTCCGGCCCGTAGATTTCGATGATGCGGCCCCTCGGCAGGCCACCAACGCCGAGTGCAATATCGAGGCCAAGCGAGCCGGTCGAGATCGTCTCGATCTCGACAACTACCACGCGAAA
atpD gene sequence seqID3:
>GGGACGATGACGAGCGGTCCGCTGGTCACTGCTCACAAGCGCGCCATCCACCAG GATGCGCCGTCTTATGTCGAGCAGTCGACGGAATCGCAGATCCTCGTCACCGGCATCAAGGTCGTCGATCTTCTGGCTCCTTACGCACGCGGCGGCAAGATCGGCCTGTTCGGCGGCGCCGGCGTCGGCAAGACCGTTCTGATCATGGAACTGATCAACAACGTCGCCAAGGCGCATGGTGGTTACTCGGTTTTTGCCGGTGTCGGTGAACGCACGCGCGAAGGCAACGACCTCTATCACGAAATGATCGAATCGAACGTCAACAAGCATGGCGGCGGCGAAGGCTCGAAGGCGGCACTTGTTTACGGTCAGATGAACGAGCCGCCGGGCGCCCGCGCCCGCGTCGCCCTGACCGGCCTGACGGTCGCCGAACACTTCCGCGACCAGGGCCAGGACGTTCTGTTCTTCGTCGACAACATCTTCCGCTTCACGCAGGCAGGTTCTGAAGTGTTCGGCTCTTA
glnA gene sequence seqID4:
>TGGAATCGTTGGAGCATCACGAGTCCGACATGGTGCTGATGCCCGATACCGAAAC GGTGCATATGGACCCGTTTTTCGCCCAGTCGACCATGGTCATCGTCTGCGACATCCTCGATCCGGTTTCCGGCGAGGCCTATAACCGCGATCCGCGCGGCACCGCCAAGAAGGCCGAAGCCTATCTCAAGGCCTCCGGCATCGGCGATACGGTCTTCGTCGGCCCCGAAGCCGAATTCTTCGTCTTCGACGACGTCAAGTACAAGGCCGATCCCTACAATACCGGCTTCAAGCTCGACTCGACCGAATTGCCATCGAACGACGATACCGATTACGAGACCGGCAACCTCGGCCATCGCCCGCGCGTCAAGGGCGGTTATTTCCCTGTTCCTCCCGTCGACAGCGCCCAGGACATGCGTTCGGAAATGCTGACGGTGCTCTCAGAAATGGGCGTCGTCGTCGAAAAGCATCACCATGAAGTCGCGGCCGCCCAGCACGAACTCGGCATCAAGTTCGATACGCTGGTGCGCAACGCCGACAAGATGCAGATCTACAAATACGTCGTGCACCAGGTGGCCAACGCCTACGGCAAGACGGCGACCTTCATGCCGAAGCCGATCTTCGGCGACAATGGCTCGGGCATGCATGTGCACCAGTCGATCTGGAAGGGCGGCAAGCCGACCTTTGCCGGCGACGAATATGCCGGCCTTTCCGAGAGCTGCCTGTTCTACATCGGCGGCATCATCAAGCATGCCAAGGCGATCAACGCCTTCACAAATCCGTCGACGAACTCCTACAAGCGTCTCGTCCCGGGTTACGAAGCCCCTGTGCTGCTTGCCTATTCGGCCCGCAACCGCTCGGCTTCCTGCCGCATTCCGTTCGGCTCCAATCCGAAGGCCAAGCGCGTCGAAGTCCGCTTCCCGGATCCGACCGCCAACCCCTATCTCGCCTGCCGCCAGCCGCATATGCAAGACCGTA
the obtained sequence results are aligned in the National Center for Biological Information (NCBI), and 5 model strains with higher similarity with 3 genes are selected as reference strains for tree construction by using the alignment results of each gene sequence in the NCBI.
Phylogenetic tree construction of 3 genes (recA, atpD and glnA) was identical to the 16S rRNA gene sequence. The phylogenetic tree is shown in figures 3-5, and figure 3 is a phylogenetic tree of the rhizobium vicina recA gene, wherein the Bootstrap value is 1000, and the replacement frequency of bases at each position is 5%. FIG. 4 is a phylogenetic tree of the Rhizobium vicina atpD genes, with Bootstrap values of 1000 and base substitutions at each locus of 2%. FIG. 5 is a phylogenetic tree of the GlnA gene of Rhizobium vicina of common vetch, bootstrap value of 1000 and base substitution frequency of 2% per site. From the figure, it was found that the model strain having the highest similarity to the atpD and glnA gene sequences of the vetch rhizobium strain HBUJ010033 was Rhizobium laguerreae, the similarity was 97% and 98%, the model strain having the highest similarity to the recA gene was Rhizobium leguminosarum, the similarity was 97.3%, and the similarity to Rhizobium sophorae was 97%.
Example 6 Amaranthus sonchifolius root nodule strain HBUJ010033 genomic information
The strain HBUJ010033 was sent to a norhe source for sequencing of the bacterial frame map and the sequencing result information is shown in table 3.
TABLE 3 genomic information of strain HBUJ010033
As can be seen from the above table, the genome of this root nodule strain is 7.87Mb, the G+C content is 60.82%, and it has 1 rRNA operon and 51 tRNA's.
Example 7 strain comparison test for vermiculite culture
The strain was subjected to vermiculite screening tests at laboratory temperature.
The experiment was designed with the seed of the rhizobium viciae strain HBUJ010033 and the seed of the rhizobium viciae strain 77 strain without the seed of the Control (Control) and other isolated plants from the field. The seed is spring-type pea No. 3, and the test is carried out in the application and research key laboratory of the microorganism in Hebei province of the university of Hebei's life sciences of Hebei in 2020.7-2020.8 months. Seed germination, inoculation and plant cultivation were all performed as in example 2, and after 30 days of plant growth, the number of nodules, dry weight of the plant and chlorophyll and crude protein content were determined.
Isolation of field plant rhizobia:
root nodule sampling points are selected from regions such as inner Mongolia, gansu, qinghai and the like respectively, a 5-point sampling method is adopted every time one place is reached, plants with better growth vigor are selected, large and powdery root nodules are selected for each plant, and 77 strains are obtained from the root nodules of the pea of the arrow in total through separation, culture and preservation of the root nodule bacteria in the same manner as in example 1.
The comparative test results are shown in Table 4.
TABLE 4 influence of rhizobia on the growth trait of spring arrow 3 (enumerating partial results)
The Tukey test was used, with letters a b c, etc. being a different subset, and the above data being the average of the parallel runs for each strain. The control group was not inoculated with rhizobia, and the other experimental conditions were the same as those of the experimental group.
The dry weight of the spring pea No. 3 is measured and is improved by 136.1 percent compared with the control, which proves that the biomass and nodulation rate of the common vetch can be greatly improved by inoculating rhizobium HBUJ 010033.
Example 8 field inoculation comparative test Effect
The field inoculation effect test of the strain is carried out on inner Mongolia, gansu and Qinghai test bases.
For each trial, 11 treatments were applied, inoculated with the vetch rhizobium strain HBUJ010033, and no Control (Control) and 9 other Control strains. Inner MongoliaThe planting method is characterized in that the planting method comprises the steps of planting common vetch in a clear water river, planting common vetch in Qinghai, planting common vetch in west herry 333, planting common vetch in Gansu province, and applying no chemical fertilizer and organic fertilizer in a test land which is a sample land of the common vetch without planting common vetch. The field random block arrangement is adopted. Experiments were performed at 2021.3.15 to 2021.7.1. The prepared rhizobium arrow (viable count 5.0X10) 8 CFU/g microbial inoculum) and common vetch seeds, and disseminated after drying in the shade. Cell area 2m 2 Repeated 5 times. Sampling in the vigorous growth period (150 d in the growing period), measuring the root nodule number, the fresh weight of the overground part plant and the fresh weight of the underground part root, and the yield of fresh grass, and the inoculation results in each province are shown in tables 5-7. The management of the period is carried out according to the conventional management of the peas planted by peasant households.
TABLE 5 Effect of Rhizobium japonicum inoculation on vetch biomass
TABLE 6 Effect of Rhizobium inoculation in Qinghai on vetch biomass
TABLE 7 Effect of Rhizobium inoculation in Gansu on the biomass of vetch
The biomass of the vetch in the full bloom stage is measured, and according to the results in the table, the yield of the inoculated strain is improved compared with that of the control fresh grass, the yield of the inoculated strain HBUJ010033 vetch is improved most, and the dry weight improvement rate is 16.7%; in Qinghai, the strain dry weight increase of the inoculated HBUJ010033 strain is inferior to that of the strain HBUJ010038 (the dry weight increase rate is 32.6%), and the dry weight increase rate is only 0.05%; in Gansu, the inoculation strain HBUJ010033 is the strain, and the dry weight improvement rate is 11.2 percent which is inferior to the inoculation effect of inner Mongolia. In the above description, the inoculation of the vetch root nodule strain HBUJ010033 directly or indirectly promotes the growth of vetch in inner mongolia, has regional advantage (p=0.004 < 0.01), may be affected by soil or climate in other areas, and has an effect inferior to that of the application in inner mongolia.
The isolated vetch root nodule bacterial strain HBUJ010033 can be specially used in the production of vetch with inner Mongolia and can be popularized and applied in a large area.
Claims (6)
1. A common vetch root nodule strain HBUJ010033 is characterized in that the common vetch root nodule strain is sophora japonica root nodule strainRhizobium sp.) HBUJ010033, the preservation unit is the microorganism strain preservation center of Guangdong province, the preservation address is building 5 of No. 59 of Mitsui 100 of Guangzhou City, the preservation date is 2021, 1 month and 7 days, and the preservation number is GDMCC No.61423.
2. Use of the vetch root nodule strain HBUJ010033 of claim 1 in the production of vetch plants.
3. Use according to claim 2, characterized in that the rhizobia of vetch HBUJ010033 inoculant is mixed with vetch seeds, followed by sowing.
4. The use according to claim 3, wherein the rhizobium veticus HBUJ010033 inoculant comprises a viable count of 10 7 ~ 10 9 CFU/g。
5. The use according to claim 3, wherein the strain culture conditions for the preparation of the rhizobium veticus HBUJ010033 microbial inoculum are: the temperature is 25-28 ℃, and the pH is 6-9.
6. The use according to claim 3, wherein the salt concentration of the strain medium is less than or equal to 3.0wt% when the rhizobium viciae HBUJ010033 inoculant is prepared.
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| CN106967652A (en) * | 2017-05-09 | 2017-07-21 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium and its application that one plant of promotion Jian Kuo pea grows |
| CN107881134A (en) * | 2017-12-26 | 2018-04-06 | 四川农业大学 | A kind of Jian Kuo rhizobium leguminosarum strains VS5 1 and its application |
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| CN106967652A (en) * | 2017-05-09 | 2017-07-21 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium and its application that one plant of promotion Jian Kuo pea grows |
| CN107881134A (en) * | 2017-12-26 | 2018-04-06 | 四川农业大学 | A kind of Jian Kuo rhizobium leguminosarum strains VS5 1 and its application |
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