CN114045247A - Bacillus marinus and application thereof in production of salinization farmland - Google Patents
Bacillus marinus and application thereof in production of salinization farmland Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a seawater bacillus and application thereof in production of salinization farmland, belonging to the technical field of salt-tolerant microorganisms. The seawater Bacillus is a seawater Bacillus (Bacillus aquimaris) JES4 strain, and the preservation number is CGMCC NO. 20301. The seawater bacillus JES4 strain can obviously promote wheat germination, seedling emergence and wheat biomass accumulation in a salinity environment with the salt concentration of 0.2-0.8%, and has wide application prospect in agricultural production of salinized soil.
Description
Technical Field
The invention belongs to the technical field of salt-tolerant microbes, and particularly relates to a bacillus marinus and application thereof in production of salinized farmlands.
Background
Globally, over 8.33 billion hectares are affected by salt. The saline soil has huge area and various varieties, and has very serious influence on agricultural production and ecological environment. In recent years, the restoration and efficient utilization of saline soil play an important role in guaranteeing grain safety, providing agricultural yield and optimizing industrial structure. However, the saline soil treatment is a serious challenge due to the characteristics of high salt content, complex soil structure, low microbial activity and the like. The microorganisms are the driving force for the transformation of substances in the soil, and the improvement of the activity of soil samples through the remodeling and the regulation of the microorganisms is a very effective and simple path.
The salinized soil is special in environment, is in dynamic change, is fragile in ecological environment, single in vegetation type, low in variety of species and low in exogenous organic matter input amount, and is also a key factor for limiting the microbial activity of the soil. At present, the products for improving soil microorganisms are more, but the application range is less, and the products are difficult to popularize in areas with higher salinity. By supplementing the microbial agent, the soil microbial community structure and the soil property are improved, the utilization efficiency of the salinized soil is improved, and the key is to obtain the functional strain which is suitable for and improves the salinized soil. At present, the high-efficiency and salt-tolerant functional strains related to the salinized soil are few, and research and development work for improving the production capacity and benefit of the salinized soil through a microbial technology is urgent.
Disclosure of Invention
Aiming at the problems in the prior art, one of the purposes of the invention is to provide a seawater Bacillus, wherein the seawater Bacillus is a seawater Bacillus (Bacillus aquimaris) JES4 strain, and the preservation number is CGMCC NO. 20301.
The second purpose of the invention is to provide the application of the seawater bacillus with the preservation number of CGMCC NO.20301 in the production of salinized farmlands, in particular to improve the germination rate and the emergence rate of crops in the salinized farmlands, promote the accumulation of biomass and improve the yield.
The invention also aims to provide a microbial preparation for producing the salinized farmland, which comprises seawater bacillus with the preservation number of CGMCC NO.20301, and the microbial preparation is used for improving the germination rate and emergence rate of crops in the salinized farmland, promoting the accumulation of biomass and improving the yield.
The fourth purpose of the invention is to provide a method for improving the germination rate and emergence rate of crops in a salinized farmland, promoting the accumulation of biomass and improving the yield, and particularly to soak seeds with the fermentation liquor of the seawater bacillus with the preservation number of CGMCC NO.20301 or inoculate the fermentation liquor of the seawater bacillus with the preservation number of CGMCC NO.20301 into planting soil.
In a specific embodiment, the bacteria concentration of the fermentation liquor of the seawater bacillus with the preservation number of CGMCC NO.20301 is more than or equal to 2.5x109 CFU/mL。
In a specific embodiment, the fermentation liquid of the marine bacillus with the preservation number of CGMCC NO.20301 is prepared by the following method: inoculating the lawn preserved on the slant culture medium into LB liquid culture medium, performing shake culture (120 rpm) at 37 deg.C, transferring to new LB liquid culture medium when it reaches logarithmic phase, and culturing under the same conditions until logarithmic phase to obtain the desired fermentation liquid.
In a specific embodiment, the seed soaking is to mix the fermentation liquor of the marine bacillus with the preservation number of CGMCC NO.20301 and the crop seeds to be sown according to the volume-mass ratio of 1:1 and perform soaking treatment.
In a specific embodiment, the fermentation liquor for inoculating the marine bacillus with the preservation number of CGMCC NO.20301 into the planting soil is inoculated according to the proportion of inoculating 2.5mL of zymophyte liquid into every 50g of soil and is uniformly mixed.
The technical scheme of the invention has the advantages that:
an important mechanism of the Bacillus marinus (Bacillus aquimaris) JES4 strain capable of promoting the growth of wheat in salinized soil is to promote the growth of wheat root systems and improve the adaptability and tolerance to salt environments.
The seawater Bacillus (Bacillus aquimaris) JES4 with the preservation number of CGMCC NO.20301 can obviously promote wheat germination, seedling emergence and wheat biomass accumulation in a salinity environment with the salt concentration of 0.2-0.8 percent, and has wide application prospect in agricultural production of salinized soil.
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FIG. 1 is a colony morphology of JES4 strain;
FIG. 2 phylogenetic analysis of 16S rDNA of JES4 strain;
FIG. 3 shows the growth variation of Bacillus marinus (Bacillus aquimaris) JES4 in liquid culture media with different salinity;
FIG. 4 is a graph showing the effect of JES4 strain on wheat germination and emergence rate under salt stress conditions
FIG. 5 is a graph showing the effect of JES4 strain on wheat seedling stage (14d) biomass in saline soil;
FIG. 6 is a graph showing the effect of JES4 strain on wheat yield in saline soil.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
Separation, screening and identification of Bacillus marinus (Bacillus aquimaris) JES4
Taking 10g coastal salinized rhizosphere soil (coastal saline-alkali soil polluted by coastal state), adding the soil with the soluble salt content of 0.9% into a triangular flask filled with 100mL sterile water, oscillating at high speed for 20min, diluting the soil mixed solution according to gradient, and selecting the concentration gradient of 10–2、10–3、10–4、10–5、10–6The method comprises the following steps of coating a soil mixed solution, sucking 100 mu L of bacterial suspension by using a pipette gun, coating the bacterial suspension on an LB solid culture medium of 8% sodium chloride, selecting a single colony which can grow on a plate, separating and purifying, screening according to the single colony growing on the plate, activating and inoculating the screened single colony into wheat, screening a strain capable of promoting the accumulation of wheat biomass, obtaining a monoclonal strain JES4, and storing the purified strain in a refrigerator at the temperature of-80 ℃ by using 40% of glycerol.
Morphological observation of colonies and cells of the JES4 strain:
as shown in figure 1, the diameter of a colony of the strain cultured on an S-G plate for 3d is 2-3 mm, and the colony is circular, smooth, low-bulge, opaque and orange-yellow. The thallus is straight rod-shaped, chain-shaped and is provided with periphytic flagellum which is gram-positive.
Physiological and biochemical characteristics of JES4 strain:
the growth temperature is as follows: 4-40% of the total nutrient solution, and is aerobic, wherein the NaCl tolerance is 0-11.7%. The catalase test, the methylrubine test and the oxidase test are positive, glucose, fructose and mannitol can be used as carbon sources, xylose cannot be used, ammonium nitrate and ammonium sulfate can be used, starch and gelatin can be hydrolyzed, and hydrogen sulfide and hydrogen peroxide are not generated.
16S rDNA gene sequence determination of JES4 strain:
collecting 1mL of bacterial liquid in the log phase of bacterial growth for extracting genome DNA, performing PCR amplification by using a 16S rRNA gene universal primer after the genome DNA is extracted, and handing over sequence to Shanghai Biotechnology Co., Ltd. The sequence of the 16S rDNA gene of the JES4 strain is shown as SEQ ID NO: 1 is shown in the specification; and comparing the sequencing results, and obtaining high-matching sequence and species through an NCBI (https:// blast. Phylogenetic analysis was constructed on the obtained sequences based on MEGA 6. Phylogenetic results show (figure 2) that the 16S rRNA gene sequence of the JES4 strain is 1449bp in length, the similarity of the sequence comparison results and the difference of Bacillus in an online database is more than 97%, and the JES4 strain and Bacillus aquimaris belong to the same branch. Therefore, the colonies and cells were identified as Bacillus aquimaris by combining their morphological and physiological biochemical characteristics.
The strain JES4 is preserved in China general microbiological culture Collection center (CGMCC) at 7.06.2020, with the preservation number of CGMCC NO.20301, the preservation name of Bacillus aquimaris JES4 and the preservation address of Beijing Shang Yang Kong West Lu No. 1 Hospital No. 3.
SEQ ID NO：1
Example 2
Salt tolerance of Bacillus marinus (Bacillus aquimaris) JES4
Utilizing an inorganic salt culture medium, adding 0.1% (m/V) yeast powder, and setting four NaCl treatments with different concentration gradients: 0. 5.0%, 8.0%, 10.0%, 12.0% (m/V), the medium of each treatment group was inoculated with 2% of JES4 cell suspension, and after inoculation, the cells were cultured in a constant temperature incubator (37 ℃) for 48 hours, and the absorbance of the cell suspension was measured and repeated three times, and the results are shown in FIG. 1.
As shown in the figure 3, under the condition of NaCl salinity of 0-8.0%, the JES4 strain can normally grow, when the salinity is increased to 10.0%, the growth amount of the JES4 strain is about one fourth of the maximum growth amount, the salinity is continuously increased, the JES4 strain is remarkably inhibited from growing, and the JES4 strain cannot grow in the environment with the salt concentration of 12.0%. Therefore, the seawater Bacillus (Bacillus aquimaris) JES4 is a halotolerant bacterium, and the salt tolerance of the seawater Bacillus JES4 in a liquid culture medium can reach 12.0%.
Example 3
Growth promotion effect of seawater Bacillus (Bacillus aquimaris) JES4
The cultured JES4 strain fermentation liquor reaching the logarithmic growth phase is prepared by the following method: inoculating thallus Porphyrae on slant culture medium in LB liquid culture medium, performing shake culture (120 rpm) at 37 deg.C, transferring to new LB liquid culture medium when it reaches logarithmic phase, culturing under the same conditions until it reaches logarithmic phase, and culturing at concentration of 2.5 × 109CFU/mL is the required bacterial liquid.
1. Promoting germination.
Taking a wheat variety 'Jimai No. 22' as a material, setting an experimental group and a control group, infiltrating absorbent paper with 0.8% NaCl solution, placing 50 wheat seeds, inoculating 300 mu L of the bacterial liquid of JES4 prepared by the method to the experimental group, carrying out constant-temperature culture for 5 days without treatment on the control group under the condition that the optimal germination temperature is 25 ℃, setting three times of repetition, and measuring the germination rate of the wheat, wherein the result is shown in figure 4. From FIG. 4, it can be seen that inoculation with JES4 strain can increase the germination rate of wheat at a salt concentration of 0.8%.
2. And (4) promoting seedling emergence.
Collecting salinized soil (the salt content is 0.16%), adding NaCl to enable the salt content of the soil to reach 0.8%, setting an experimental group and a control group, adding cultured JES4 strain fermentation liquor which reaches the logarithmic growth phase into the experimental group, adding 2.5mL JES4 strain fermentation liquor into every 50g of soil, sieving and uniformly mixing, filling 500g of soil into a culture pot, adding deionized water to adjust the water content of the soil to reach 60%, planting 20 Jimai No. 22 wheat varieties, measuring the rate of emergence after ten days, not inoculating JES4 strains into the control group, and repeating the treatment for three times, wherein the result is shown in figure 4. As can be seen from FIG. 4, after the JES4 strain is inoculated, the emergence rate of wheat in high-salinity soil is remarkably improved.
3. Promoting biomass accumulation.
Collecting salinized soil (the salt content is 0.16%), adding NaCl to enable the salt content of the soil to reach 0.5%, setting a test group and a control group, adding cultured JES4 strain fermentation liquor which reaches the logarithmic growth phase into the test group, adding 2.5mL of JES4 strain fermentation liquor into every 50g of soil, sieving and uniformly mixing, filling 500g of soil into a culture pot, adding deionized water to adjust the water content of the soil to reach 60%, planting 20 Jimai No. 22 wheat strains, not inoculating JES4 strains into the control group, setting three times for each group, weighing and counting the underground biomass of the wheat and the above-ground biomass after two weeks, wherein the result is shown in figure 5. As can be seen in FIG. 5, the underground biomass and the above-ground biomass of the wheat are obviously increased after the JES4 strain is inoculated. As can be seen, the JES4 strain has an obvious effect of promoting biomass accumulation.
Example 4
Application of Bacillus marinus (Bacillus aquimaris) JES4 in actual production
Selecting a salinized land with the soil salinity of 0.29%, setting an experimental group and a control group: the JES4 strain-treated group was an experimental group, the non-inoculated JES4 strain-treated group was a control group, each treatment set was repeated 3 times, and the area was 66.7m2. The JES4 strain is treated by a seed soaking method, fermentation liquor (the fermentation liquor prepared by the method in example 3) of the JES4 strain in a logarithmic growth phase is obtained through culture, and then wheat seeds (Jimai No. 22) and bacterial liquid are mixed uniformly according to the proportion of 1: 1(m/V), soaking for 60 minutes, taking out, draining, and sowing in a test field. The control group was treated in the same manner as the control group except that it was not subjected to the bacterial liquid immersion treatment. Wheat yield was measured at harvest and the results are shown in figure 6. As shown in FIG. 6, in the salinized soil with the salt content of 0.29%, the yield of the wheat seeds treated by the JES4 strain is remarkably improved. Therefore, the JES4 strain is adopted for treatment, so that the productivity of the salinized soil can be effectively improved.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Binzhou college
<120> seawater bacillus and application thereof in production of salinization farmland
<160> 1
<170> SIPOSequenceListing 1.0
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<213> Bacillus marinus (Bacillus aquimaris)
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ctcgtggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga 120
tccgcgatta ctagcgattc cagcttcatg taggcgagtt gcagcctaca atccgaactg 180
agaacggttt tatgggattg gctaaacctc gcggtcttgc tgccctttgt accgtccatt 240
gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc 300
ctccggtttg tcaccggcag tcatcttaga gtgcccaact gaatgctggc aactaagatc 360
aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc 420
atgcaccacc tgtcactctg tcccccgaag gggaaagccc tatctctagg gttgtcagag 480
gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg 600
agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcgcc 720
tcagtgtcag ttacagacca gaaagtcgcc ttcgccactg gtgttcctcc aaatatctac 780
gcatttcacc gctacacttg gaattccact ttcctcttct gcactcaagt cccccagttt 840
ccaatgaccc tccacggttg agccgtgggc tttcacatca gacttaagga accacctgcg 900
cgcgctttac gcccaataat tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtaccgcc ctattcgaac 1020
ggtacttgtt cttccctaac aacagagttt tacgatccga aaaccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gtcgccttgg tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag 1260
tgatagctaa aagccacctt tcaacatttc ctcatgcgag gaaatgagtt atccggtatt 1320
agccccggtt tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc 1380
cgtccgccgc tgactgatgg gagcaagctc ccatcaatcc gctcgacttg gcatgtatag 1440
cagccgcct 1449
Claims (10)
1. A strain of seawater Bacillus is characterized in that the seawater Bacillus is a strain of seawater Bacillus (Bacillus aquimaris) JES4, and the preservation number is CGMCC NO. 20301.
2. Application of seawater bacillus with preservation number of CGMCC NO.20301 in production of salinization farmland.
3. The use of claim 2, wherein the germination rate and emergence rate of the crops in the salinized farmland are increased, biomass accumulation is promoted, and yield is increased.
4. A microbial preparation for production of salinization farmland is characterized by comprising seawater bacillus with the preservation number of CGMCC NO. 20301.
5. The microbial preparation for salinized farmland as claimed in claim 4, which is used for improving germination rate and emergence rate of crops in salinized farmland, promoting biomass accumulation and improving yield.
6. A method for increasing germination rate and emergence rate of crops in salinized field, promoting accumulation of biomass and increasing yield features that the fermented liquid of seawater bacillus with CGMCC NO.20301 is used to immerse seeds or inoculate the fermented liquid of seawater bacillus with CGMCC NO.20301 to planting soil.
7. The method for improving germination rate and emergence rate of crops in salinized farmland as claimed in claim 6, promoting biomass accumulation and improving yield, wherein the strain concentration of the fermentation liquor of the seawater bacillus with preservation number of CGMCC NO.20301 is not less than 2.5x109CFU/mL。
8. The method for improving the germination rate and emergence rate of crops in the salinized farmland, promoting the accumulation of biomass and improving the yield as claimed in claim 7, wherein the fermentation broth of the seawater bacillus with the preservation number of CGMCC NO.20301 is prepared by the following method: inoculating the lawn on the slant culture medium into LB liquid culture medium, performing shake culture at 37 deg.C, transferring to new LB liquid culture medium when logarithmic phase is reached, and culturing under the same conditions to logarithmic phase to obtain the desired fermentation liquid.
9. The method for improving the germination rate and emergence rate of crops in the salinized farmland, promoting the accumulation of biomass and improving the yield according to any one of claims 6 to 8, wherein the seed soaking is carried out by mixing the fermentation liquor of the seawater bacillus with the preservation number of CGMCC NO.20301 and the crop seeds to be sown according to the volume-to-mass ratio of 1: 1.
10. The method for improving the germination rate and emergence rate of crops in the salinized farmland, promoting the accumulation of biomass and improving the yield according to any one of claims 6 to 8, wherein the fermentation liquor for inoculating the marine bacillus with the preservation number of CGMCC NO.20301 into the planting soil is inoculated according to the proportion of inoculating 2.5mL of zymocyte liquid into every 50g of soil and is uniformly mixed.
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| CN114045247B (en) | 2023-04-25 |
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