CN116925952A - Screening and identifying method and application of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus - Google Patents
Screening and identifying method and application of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus Download PDFInfo
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Classifications
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- 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
- A01G20/00—Cultivation of turf, lawn or the like; Apparatus or methods therefor
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Abstract
The invention relates to a screening and identifying method and application of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus, wherein the preservation number of the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus is as follows: CCTCCNO: m20221929, which is inserted with SEQ ID No:1, wherein the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus is used for improving the growth of alfalfa seedlings and the germination rate of seeds.
Description
Technical Field
The invention relates to the field of microorganisms and agriculture, in particular to a screening and identifying method and application of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus. The bacillus pumilus strain used in the invention is screened in rhizosphere soil of suaeda salsa, and is easy to culture, high in production safety and good in phosphorus dissolving effect. The effective viable count of the bacillus pumilus LPJ5-3-1 is up to 102 hundred million cfu/mL (the national standard liquid type microbial inoculum requires the effective viable count (cfu) to be more than or equal to 2.0 hundred million cfu/mL), and the bacillus pumilus LPJ has higher application value. Seeds and seedlings of alfalfa are inoculated by soaking the strain, and the biomass is higher than that of alfalfa without inoculating the strain. The invention not only improves the growth of alfalfa seedlings and the germination rate of seeds, but also can help the alfalfa of the leguminous plant dissolve phosphorus, thereby better promoting the absorption of nitrogen elements by the leguminous plant.
Background
Suaeda salsa (Suaeda salsa) school name: suaeda salsa (L.) pall. Alias Yan Hao, herba Begoniae Laciniatae, also known as Suaeda heteroptera, belongs to the genus Suaeda of the Chenopodiaceae, the annual herbaceous plants are mainly distributed in Europe, asia, northeast China, north China and northwest China. The salt suaeda salsa in the Shandong province, jiangsu and Zhejiang province has high yield, and the salt suaeda salsa in partial areas can be used as food and has the medical value of heat and food retention elimination. Meanwhile, the suaeda salsa is an important plant resource for repairing the polluted saline-alkali soil, can improve the soil fertility, and has important ecological value.
In general, the soil growth environment of most plants is very complex, and a large number of microorganisms exist in the rhizosphere area of the plants, and the microorganisms can be classified into beneficial microorganisms, harmful microorganisms and neutral microorganisms according to the effect on the plants. Studies have shown that plants can selectively recruit and symbiotic with a population of microorganisms that are beneficial to their growth, some of which are plant growth-promoting bacteria, which can colonize the plant rhizosphere, alter the hormone levels of the plant host, promote plant nutrient uptake salts, and thereby induce the plant host to develop "systemic resistance" against biotic and abiotic stresses.
Alfalfa medical sativa l is a leguminous grass widely planted worldwide, and 30% of the world land is reported to be used for grass production and herbivore production, and alfalfa is also a best quality leguminous grass with the largest planting area in China, but with the rapid development of animal husbandry, the quality of domestic alfalfa products cannot meet the current market demand, and alfalfa grass products imported from abroad every year have a trend of rapid growth. On the premise of not touching red lines of 18 hundred million mu cultivated land, other grassland resources need to be searched for to increase the yield and quality of high-quality pastures, and 5 hundred million mu of available saline-alkali land resources just can make up for the resource gap, but the alfalfa is poor in salt tolerance, and the salt stress seriously influences the nutrient absorption, photosynthesis and growth and development of the alfalfa, so that the expansion of the planting range, the improvement of the yield and the improvement of the quality of the alfalfa are limited. The current research on the suaeda salsa microorganisms is only focused on the influence on the suaeda salsa, and few researches on utilizing the salt tolerance of the saline-alkali soil microorganisms and adding the saline-alkali soil microorganisms to the rhizosphere of other salt-tolerant plants and promoting the growth of the saline-alkali soil microorganisms are reported.
Bacillus pumilus is a gram-positive bacillus cereus with high growth speed and relatively simple nutrition requirement, and can degrade non-starch polysaccharide, so that the utilization rate of livestock feed is improved, the meat-to-feed ratio is reduced, and the immune function and the stress capability of animals are enhanced. When acting on plants, the bacillus pumilus can also be used for preventing and controlling wheat root rot, strawberry gray mold and the like, and the bacillus pumilus has obvious promotion effect on the growth of plants such as arabidopsis thaliana, tobacco, soybeans, peppers and the like, and is characterized in that seed germination is promoted and growth of rhizosphere, buds and seedlings is promoted. We have found that Bacillus pumilus also promotes germination of alfalfa seeds and growth of seedlings.
Bacillus pumilus was studied in terms of arabidopsis thaliana and soybean seeds, qu Fa reported an influence study of bacillus pumilus on tomato seed germination and seedling growth, and found that a phosphorus-soluble bacillus pumilus NMCC46 strain isolated from the root zone soil of tibetan tricuspidata was shown to have an obvious promoting effect on the growth of arabidopsis thaliana and soybean, and that after treatment of tomato seeds or seedlings with fermentation broth of bacillus pumilus NMCC46 strain at different concentrations, the effect on seed germination, root bud growth and seedling plant height and plant weight was obvious (Qu Fa, yu Mingli, zhang Zhuqi, yuan Ding, & Li Ming.2014. Influence of bacillus pumilus on tomato seed germination and seedling growth [ J ] northern gardening (15): 109-111.). In fact, bacillus pumilus is not applied to research for promoting germination of alfalfa seeds, and reports for promoting germination of alfalfa seeds and seedling growth by using Bacillus pumilus fermentation broth are not found at present.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a screening and identifying method of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus and application thereof, wherein the strain is easy to culture, high in production safety and capable of dissolving phosphorus, the germination rate of alfalfa seeds treated by the strain is 4.5% higher than that of a control, the difference is obvious, as shown in a figure 1 (P < 0.05), and the seedling length and the seedling weight of alfalfa seedlings treated by fermentation liquor of the strain are obviously improved.
The Bacillus pumilus of the invention for solving the technical problems is classified and named as Bacillus pumilus (Bacillus pumilus LPJ 5-3-1), and the preservation number is CCTCC NO: m20221929, the preservation date is 2022, 12 and 12, the preservation unit is China center for type culture Collection, and the preservation address is eight paths of Lopa nationality in Wuchang district of Wuhan, hubei province.
The invention relates to suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus, which is characterized in that: which is inserted with SEQ ID No:1, and a gene sequence shown in the specification. The gene sequence fragment can be found in a GenBank database established by NCBI, and according to the found related information, the fragment in the Bacillus pumilus LPJ5-3-1 sequence is matched with the salt-tolerant gene fragment in the GenBank database, which shows that the strain has salt-tolerant property, and the strain is prepared into microbial fertilizer in future to have the potential of being capable of being applied back to saline-alkali soil.
The application of the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus disclosed by the invention is characterized in that: can help the alfalfa of the leguminous plant dissolve phosphorus, thereby better promoting the absorption of nitrogen elements by the leguminous plant and improving the yield and quality of the leguminous high-quality pasture.
The application of the invention is characterized in that: the application is used for improving the growth of alfalfa seedlings and the germination rate of seeds.
The screening method of the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus disclosed by the invention is characterized by comprising the following steps of: the method comprises the following steps:
(1) Sampling: sampling saline-alkali soil (37 DEG 51 '32.78' N,117 DEG 49 '39.66' E) in the chiese county of the coastal state of Shandong province, selecting rhizosphere soil of suaeda salsa, and filling the rhizosphere soil into a sterile sampling bag;
(2) Screening: preparing soil suspension, diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Respectively coating the soil suspension in NA culture medium for screening culture, and selecting dominant bacteria on each plate for separation and purification;
(3) And (3) re-screening: inoculating the strain to NBRIP phosphate dissolving culture medium;
(4) And collecting fermentation liquor, measuring the phosphorus dissolving amount of the fermentation liquor, and selecting strains with higher phosphorus dissolving amount.
The screening method of the invention comprises the following steps: the NA culture medium in the step 2 comprises the following components in parts by weight: beef extract 3%, tryptone 5%, agar 18-20%, distilled water 1000ml, pH=7.2-7.4,% all represent g/1000mL.
The screening method of the invention comprises the following steps: the NBRIP phosphate solubilizing culture medium in the step 3 comprises the following components in parts by weight and the dosage: glucose 10.0g, mgCl 2 ·6H 2 O 5.0g,（NH 4 ) 2 SO 4 0.1g，MgSO 4 ·7H 2 0.25g of O, 0.2g of KCl, 18-20 parts of agar, 1000mL of distilled water and pH=7.0-7.5.
The screening method of the invention comprises the following steps: the determination of the dissolved phosphorus in the step 4 is specifically as follows:
A. inoculating the strain obtained by primary screening into a seed liquid culture medium, and performing shake culture for 24-48 hours at the temperature of 28-30 ℃ and at the speed of 150-180 r/min to obtain seed liquid;
B. taking 100mL of NIBPR culture based on a 250mL conical flask, sterilizing and cooling, inoculating 2-4% of seed liquid into a sterile ultra-clean workbench, and then placing the seed liquid into a shaking table for shake culture at 28-30 ℃ and 120-180 r/min for 3-5 days;
C. after incubation, the OD value at a wavelength of 600 was measured by a spectrophotometer, and was measured according to the OD 600 The amount of dissolved phosphorus can be obtained by the value.
The screening method of the invention comprises the following steps: the formula and the weight proportion of the seed liquid culture medium in the step A are as follows: beef extract 3%, peptone 5%, naCl5%, distilled water 1000mL, pH=7.2-7.4,% all represent g/1000mL.
The salt content of the soil grown from the suaeda salsa collected by the invention can reach 5.45g kg -1 Therefore, the salt tolerance is strong. The strain of the invention is passed for a plurality of times, still inherits stably, and no obvious variant strain appears in the process of passage at present.
The bacillus pumilus strain screened from the suaeda salsa rhizosphere is easy to culture, high in production safety and good in phosphorus dissolving effect. By adopting the strain to soak and inoculate alfalfa seeds and seedlings, the biomass (seedling length and seedling weight) is obviously improved compared with that of the strain which is not inoculated, the average seedling length and average seedling weight respectively reach 1.848cm and 0.1820g, and the biomass is respectively improved by 19.38 percent and 30.3 percent compared with NB culture medium (control) (1.548 cm and 0.1397 g). The difference is obvious, as shown in figures 2 and 3 (P < 0.05), the invention not only can help the alfalfa of the leguminous plant dissolve phosphorus, but also can improve the growth of the alfalfa seedling and the germination rate of the seeds, thereby better promoting the absorption of nitrogen elements by the leguminous plant and having higher application value. Bacillus is one of the most widely studied and most deeply known phosphorus-solubilizing microorganisms (Li Haiyun, kong Weibao, wen Yan, niu Shi. Soil phosphorus-solubilizing microorganism research progress [ J ]. Biological bulletins, 2013,48 (07): 1-5.).
Drawings
FIG. 1 germination rate of alfalfa seeds treated with the strain of the present invention was 4.5% higher than control, and the difference was significant (P < 0.05).
FIG. 2 shows that the biomass (seedling length) of alfalfa seeds and seedlings inoculated with this strain was 19.38% higher than that without the strain, and the difference was significant (P < 0.05).
FIG. 3 shows that the biomass (seedling weight) of alfalfa seeds and seedlings inoculated with this strain was increased by 30.3% compared to that without the strain, and the difference was significant (P < 0.05).
FIG. 4 shows that the strain LPJ5-3-1 is closely related to Bacillus pumilus (accession number: KF 515665.1).
Detailed Description
The invention will be further described with reference to the following specific embodiments, all of which are commercially available from the reagent company:
the experimental methods used in the examples below are conventional, unless otherwise specified
The Bacillus pumilus (Bacillus pumilus LPJ-3-1) is a strain separated from the rhizosphere of the collected suaeda salsa, is identified by morphological and physiological and biochemical characteristics, and is finally identified as Bacillus pumilus (Bacillus pumilus) by comparing the 16SrRNA ribosome database, wherein the strain with the highest homology is LJ15, YS193, HIS2200905, CLC-F32 and LTW 29. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2022, 12 months and 12 days, and the preservation number is CCTCC NO: m20221929.
Example 1 screening of Bacillus pumilus
(1) Sampling: sampling saline-alkali soil (37 DEG 51 '32.78' N,117 DEG 49 '39.66' E) in the chiese county of the coastal state of Shandong province, selecting rhizosphere soil of suaeda salsa, and filling the rhizosphere soil into a sterile sampling bag;
(2) Screening: preparing soil suspension, diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Respectively coating the soil suspension in a culture medium for screening culture, and selecting dominant bacterial groups on each plate for separation and purification;
(3) And (3) re-screening: inoculating the strain to NBRIP phosphate dissolving culture medium;
(4) And collecting fermentation liquor, measuring the phosphorus dissolving amount of the fermentation liquor, and selecting strains with higher phosphorus dissolving amount.
In a preferred embodiment, the NBRIP is phosphate solubilizing cultureThe base comprises the following components in parts by weight: glucose 10.0g, mgCl 2 ·6H 2 O 5.0g,（NH 4 ) 2 SO 4 0.1g，MgSO 4 ·7H 2 0.25g of O, 0.2g of KCl, 18-20 parts of agar, 1000mL of distilled water and pH=7.0-7.5.
The determination of the dissolved phosphorus in the step (4) is specifically as follows:
A. inoculating the strain obtained by primary screening into a seed liquid culture medium, and placing the seed liquid culture medium into a shaking table for culturing at 28-30 ℃ for 24 days at 150-180 r/min to obtain seed liquid;
B. taking 100mL of NIBPR culture based on a 250mL conical flask, sterilizing and cooling, inoculating 2-4% of seed liquid into a sterile ultra-clean workbench, and then placing the seed liquid into a shaking table for shake culture at 28-30 ℃ and 120-180 r/min for 2-5 days; if the inoculation amount is too small, the total amount of thalli in the early stage of fermentation is too small, and the strain cannot reach a certain strain concentration in a short period; if the inoculation amount is too large, the initial concentration of the strain may be too high, and a large amount of nutrients in the culture medium are consumed for the growth and propagation of the bacterial strain. These may all have an effect on the strain's dissolved phosphorus source.
C. After incubation, the OD value at a wavelength of 600 was measured by a spectrophotometer, and was measured according to the OD 600 The value can be used for obtaining the phosphorus dissolving amount.
EXAMPLE 2 preparation of Bacillus pumilus LPJ5-3-1 liquid microbial inoculant
1. Strain activation
The Bacillus pumilus LPJ5-3-1 strain isolated in example 1 was transferred to NA solid medium and incubated at 30℃for 48h for further use.
2. Preparation of seed liquid
A single colony is picked, inoculated into a 250mL conical flask with 100mL NB culture medium, placed in a controlled Wen Yaochuang shake, cultured for 24 hours at 30 ℃ and 180r/min for later use.
3. Preparation of fermentation Medium
The fermentation medium adopts a fermentation medium special for Bacillus pumilus which is improved in the earlier stage of the subject group: the fermentation culture medium is prepared by using 3.0% of beef extract, 5.0% of tryptone and the balance of water, wherein the pH value of the culture medium is 7.2-7.4, and the liquid loading amount is 100/250mL conical flask. % are expressed in g/1000mL.
4. Bacterial fermentation culture
Inoculating the seed liquid obtained in the step 2 into the fermentation culture medium prepared in the step 3 according to the inoculum size of 2% (volume ratio), wherein the rotation speed of a shaking table is 180r/min, the culture temperature is 28 ℃, and the fermentation liquid obtained after 32h culture is the liquid microbial agent.
5. Liquid microbial agent
The effective viable count of the bacillus pumilus LPJ5-3-1 in the liquid microbial agent obtained in the step 4 is up to 102 hundred million cfu/mL (the national standard liquid microbial agent requires the effective viable count (cfu) to be more than or equal to 2.0 hundred million cfu/mL).
Example 3 growth-promoting effect of Bacillus pumilus LPJ5-3-1 liquid microorganism on alfalfa
1. Material
Alfalfa (Medicago sativa l.) seed is alfalfa No. 3, from beijing-baster grass company limited. After seed cleaning, the seeds are put into envelope paper bags and are placed into a refrigerator with the temperature of 4 ℃ for refrigeration.
Liquid microbial agent the liquid microbial agent of Bacillus pumilus LPJ5-3-1 prepared in example 2.
2. Seed pretreatment
The test selects 4 bags (about 350 bags) of alfalfa seeds with full seeds and uniform sizes, firstly, soaking the alfalfa seeds in 75% ethanol for 5min, stirring the alfalfa seeds at an indefinite time, then, flushing the alfalfa seeds with distilled water for 3 times, flushing the alfalfa seeds with sterile water for 3-5 times until the alfalfa seeds are non-sticky, and placing the alfalfa seeds in a beaker for standby after cleaning.
3. Design of experiment
The germination test of alfalfa is carried out by water culture, and a completely random design is adopted.
The treatment is respectively control (NB culture medium), sterile water, liquid microbial agent of Bacillus pumilus LPJ5-3-1, and filtration, and liquid microbial agent of Bacillus pumilus LPJ5-3-1 is not filtered.
The 4 bags of purified seeds are respectively put into 4 treated triangular flasks to be soaked for 30min.
The NB medium formula was: beef extract 3.0%, tryptone 5.0%, distilled water 1000mL. % are expressed in g/1000mL. The pH is adjusted to 7.2-7.4. The culture medium is sterilized by an autoclave at 121 ℃ for 20min.
The soaked seeds were uniformly placed in a culture dish (13 cm. Times.13 cm. Times.1 cm) filled with a double layer of sterile filter paper and 10mL of sterile water was added for germination, 50 grains per dish.
The experimental conditions were light/dark (16/8 h,25/20 ℃). Recording the germination number for 1-7 days, and measuring the seedling length and the seedling weight.
Preserving: preserving one part of screened bacillus pumilus by using a test tube inclined plane, wherein the inclined plane is a seed liquid solid culture medium; the other part is preserved in a refrigerator at-80 ℃ with 50% glycerol as a protective agent.
The resulting strain sequences were submitted to other homologous sequences in the NCBI-established GenBank database for comparison, and phylogenetic trees were constructed in MEGA10.0 software using the Neighbor-joining Method. The phylogenetic tree constructed is shown in the figure, and FIG. 4 shows that the strain LPJ5-3-1 is closely related to Bacillus pumilus (accession number: KF 515665.1)
While the basic principles and main features of the present invention and advantages thereof have been shown and described, the foregoing embodiments and description are merely illustrative of the principles of the present invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Qingdao university of agriculture
<120> Bacillus pumilus for dissolving phosphorus in suaeda salsa rhizosphere and application thereof
<160>1
<170>
<210>1
<211>1448
<212>RNA
<213> artificial sequence
<220>
<223>
<400>1
1 cggacacgta ctgtctgcag tcgagcggac agaagggagc ttgctcccgg atgttagcgg
61 cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc
121 ggagctaata ccggatagtt ccttgaaccg catggttcaa ggatgaaaga cggtttcggc
181 tgtcacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag
241 gcgacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc
301 agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc
361 aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa
421 gtgcaagagt aactgcttgc accttgacgg tacctaacca gaaagccacg gctaactacg
481 tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag
541 ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca
601 ttggaaactg ggaaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa
661 atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg
721 ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa
781 acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag
841 cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc
901 acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga
961 catcctctga caaccctaga gatagggctt tcccttcggg gacagagtga caggtggtgc
1021 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc
1081 ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg
1141 aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct
1201 acaatggaca gaacaaaggg ctgcgagacc gcaaggttta gccaatccca caaatctgtt
1261 ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg
1321 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg
1381 agagtttgca acacccgaag tcggtgaggt aacctttatg gagccagccg cccgaagggt
1441 tgttcagg 1448
Claims (9)
1. Bacillus pumilus with a preservation number of suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus: cctccc NO: m20221929.
2. The suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus of claim 1, wherein the method comprises the following steps of: which is inserted with SEQ ID No:1, and a gene sequence shown in the specification.
3. The use of the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus of claim 1, characterized in that: is used for improving the growth of alfalfa seedlings and the germination rate of seeds.
4. The use according to claim 3, characterized in that: the application can help alfalfa in the leguminous plant dissolve phosphorus, so that the absorption of nitrogen elements by the leguminous plant is better promoted.
5. The method for screening the suaeda salsa rhizosphere phosphorus-dissolving bacillus pumilus according to claim 1, which is characterized in that: the method comprises the following steps:
(1) Sampling: sampling saline-alkali soil (37 DEG 51 '32.78' N,117 DEG 49 '39.66' E) in the chiese county of the coastal state of Shandong province, selecting rhizosphere soil of suaeda salsa, and filling the rhizosphere soil into a sterile sampling bag;
(2) Screening: preparing soil suspension, diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Respectively coating the soil suspension in NA culture medium with the same pH as the original soil environment for screening culture, and selecting dominant bacteria on each plate for separation and purification;
(3) And (3) re-screening: inoculating the strain to NBRIP phosphate dissolving culture medium;
(4) And collecting fermentation liquor, measuring the phosphorus dissolving amount of the fermentation liquor, and selecting strains with higher phosphorus dissolving amount.
6. The screening method according to claim 5, wherein: the NA culture medium in the step 2 comprises the following components in parts by weight: beef extract 3%, tryptone 5%, agar 18-20%, distilled water 1000ml, pH=7.2-7.4,% all represent g/1000mL.
7. The screening method according to claim 5, wherein: the NBRIP phosphate solubilizing culture medium in the step 3 comprises the following components in parts by weight and the dosage: glucose 10.0g, mgCl 2 ·6H 2 O 5.0g,（NH 4 ) 2 SO 4 0.1g，MgSO 4 ·7H 2 0.25g of O, 0.2g of KCl, 18-20 parts of agar, 1000mL of distilled water and pH=7.0-7.5.
8. The screening method according to claim 5, wherein: the determination of the dissolved phosphorus in the step 4 is specifically as follows:
A. inoculating the strain obtained by primary screening into a seed liquid culture medium, and performing shake culture for 24-48 hours at the temperature of 28-30 ℃ and at the speed of 150-180 r/min to obtain seed liquid;
B. taking 100mL of NIBPR culture based on a 250mL conical flask, sterilizing and cooling, inoculating 2-4% of seed liquid into a sterile ultra-clean workbench, and then placing the seed liquid into a shaking table for shake culture at 28-30 ℃ and 120-180 r/min for 3-5 days;
C. after incubation, the OD value at a wavelength of 600 was measured by a spectrophotometer, and was measured according to the OD 600 The amount of dissolved phosphorus can be obtained by the value.
9. The screening method according to claim 8, wherein: the formula and the weight proportion of the seed liquid culture medium in the step A are as follows: beef extract 3%, peptone 5%, naCl5%, distilled water 1000mL, pH=7.2-7.4,% all represent g/1000mL.
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