CN117063943B - Application of bacillus W25 in improving plant salt tolerance and improving salinized soil - Google Patents
Application of bacillus W25 in improving plant salt tolerance and improving salinized soil Download PDFInfo
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- CN117063943B CN117063943B CN202311153775.8A CN202311153775A CN117063943B CN 117063943 B CN117063943 B CN 117063943B CN 202311153775 A CN202311153775 A CN 202311153775A CN 117063943 B CN117063943 B CN 117063943B
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- bacillus amyloliquefaciens
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- soil
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- salinized soil
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
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- 241000186063 Arthrobacter Species 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 229930182847 D-glutamic acid Natural products 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
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- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
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- 244000052769 pathogen Species 0.000 description 1
- LQPLDXQVILYOOL-UHFFFAOYSA-I pentasodium;2-[bis[2-[bis(carboxylatomethyl)amino]ethyl]amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC(=O)[O-])CCN(CC([O-])=O)CC([O-])=O LQPLDXQVILYOOL-UHFFFAOYSA-I 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
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- 239000003175 pesticide synergist Substances 0.000 description 1
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- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
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Classifications
-
- 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
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/40—Soil-conditioning materials or soil-stabilising materials containing mixtures of inorganic and organic compounds
-
- 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
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Soil Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Dentistry (AREA)
- Biomedical Technology (AREA)
- Agronomy & Crop Science (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides application of bacillus W25 in improving plant salt tolerance and improving salinized soil, belongs to the technical field of agricultural microorganism application, and belongs to the technical field of agricultural microorganism application, wherein bacillus W25 is preserved in China general microbiological culture Collection center (CGMCC) No.20042 in 6-8 days of 2020, and has the effect of promoting plant growth in a salt environment, and meanwhile, the salinized soil can be improved, so that the utilization rate and the productivity of salinized soil are improved, and the crop yield is further improved.
Description
Technical Field
The invention relates to the technical field of agricultural microorganism application, in particular to application of bacillus W25 in improving plant salt tolerance and salinized soil improvement.
Background
The soil salinization areas in China are wide, and at present, the problems of soil fertility reduction, crop yield reduction, ecological damage and the like are caused by the factors of improper long-term irrigation, poor drainage, drought climate and the like; further expansion of saline-alkali soil area seriously affects development of agricultural production and grain safety.
gamma-PGA (Poly-gamma-Glutamic acid) is a nontoxic and harmless high molecular polymer formed by polymerization of bacillus with D-Glutamic acid and/or L-Glutamic acid through gamma-amide bond formed between alpha-amino and gamma-carboxyl; gamma-PGA has wide application in agriculture, for example as a plant pesticide synergist, which inhibits the growth of pests and pathogens; as a protective agent, a layer of protective film can be formed on the surface of the plant, so that drought resistance and salt resistance of the plant are improved; as a soil restoration agent, a metal ion or the like is adsorbed by using a carboxyl functional group which is abundant.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W25 strain is described in patent CN 111718870A, and has the capacity of producing gamma-PGA, and can reduce the content of Cd (cadmium) in crops, increase the Vc content of plants, increase the content of plant soluble proteins, increase the abundance of soil microorganisms, increase the activity of soil urease and the like.
In order to solve the problem of soil salinization in China, the microbial agent has the advantages of environmental friendliness, low investment, simplicity and convenience in operation and the like, and the microbial agent is used for improving the salt tolerance of plants, improving the soil quality, improving the utilization rate and the productivity of salinized lands and meeting the requirements of modern ecological agriculture and sustainable development of agriculture; in order to find out more microbial agents capable of improving the salt tolerance of plants and improving salinized soil, the invention aims to take the bacillus amyloliquefaciens W25 (hereinafter referred to as bacillus W25) as a test object and research the application of the bacillus amyloliquefaciens W25 in the field of crop production.
Disclosure of Invention
In view of the above problems, the present invention provides the use of bacillus W25 for improving plant salt tolerance and improving salinized soil, which can promote plant growth in salt environment and improve salinized soil.
The technical scheme of the invention is as follows:
the application of bacillus (Bacillus amyloliquefaciens) W25 in improving the salt tolerance of plants, wherein the bacillus W25 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20042 in the year 6 and 8 of 2020.
Preferably, bacillus W25 is used to reduce Na of plants in salt environments + Content of Ca is increased 2+ 、K + The content is as follows.
Preferably, bacillus W25 is used to increase the content of IAA, SOD, POD, proline, vc, soluble sugars and soluble proteins of plants in salt environments.
Preferably, bacillus W25 is used to reduce MDA content of plants in salt environments.
The bacillus (Bacillus amyloliquefaciens) W25 is applied to improvement of salinized soil.
Preferably, the bacillus W25 is used for reducing the quick-acting sodium content of the salinized soil and improving the content of gamma-PGA, quick-acting phosphorus and quick-acting potassium in the salinized soil.
Preferably, the bacillus W25 is used for promoting the generation of beneficial environmental factors in the salinized soil and reducing the conductivity of the salinized soil; wherein the beneficial environmental factors comprise organic matters, organic carbon and NO 3 -N、NH 4 -N, etc.
Preferably, bacillus W25 is used to increase microbial diversity and microbial abundance in salinized soil.
Preferably, bacillus W25 is used to promote the proliferation of microorganisms in salinized soil.
Preferably, bacillus W25 is used to promote the proliferation of Pseudomonas in salinized soil.
Preferably, the application method is that bacillus W25 is prepared into a microbial inoculum and applied in a salt environment with the sodium chloride concentration less than or equal to 300mM, and the application amount is 1-2 Kg/mu.
Preferably, the preparation method of the microbial inoculum comprises the following steps: the viable count is more than or equal to 10 8 And (5) drying the bacillus W25 bacterial liquid of each/mL to obtain the bacterial agent.
The beneficial effects are that:
the invention provides application of bacillus W25 in improving plant salt tolerance and salinized soil, which can promote plant growth in salt environment, improve salinized soil, effectively avoid the problem of crop yield reduction in salt environment and improve the utilization rate and productivity of salinized soil.
Drawings
FIG. 1 shows the number of cells (OD) in a culture medium inoculated with a W25 microbial inoculum in a saline and non-saline environment 600 Representation), pH and γ -PGA concentration changes; in the figure: the different letters on each column represent 5% significant levels between treatments (LSD-test, P<0.05)；
FIG. 2 shows K in a culture solution inoculated with a W25 microbial inoculum in a saline and non-saline environment + And Na (Na) + Concentration change bar graph and gamma-PGA versus K under salt environment + And Na (Na) + An adsorption amount change line graph of (a); in the figure: the different letters represent 5% between treatmentsSignificant level (LSD-test, P<0.05)；
FIG. 3 shows the effect of W25 inoculum inoculation in saline and non-saline environments on Na/K ratio (panels A and B), na/Ca ratio (panels C and D), IAA content (panels I and J) and on MDA content (panel E), proline content (panel F), POD activity (panel G), SOD activity (panel H) in lettuce roots and leaves; in the figure: the different letters on each column represent 5% significant levels between treatments (LSD-test, P < 0.05);
FIG. 4 is the effect of inoculation with W25 inoculum on the Alpha diversity of lettuce rhizosphere soil microflora; in the figure: the different letters on each column represent 5% significant levels between treatments (LSD-test, P < 0.05);
FIG. 5 is the effect of inoculation with W25 inoculum on the Beta diversity of lettuce rhizosphere soil microflora;
FIG. 6 shows the relative abundance of bacteria and fungi at the phylum level, genus level, respectively, in soil after inoculation with W25 inoculum;
FIG. 7 is a RDA and clearman related heat map of dried vegetable weight, soil physicochemical properties, and relative abundance of the first 20 dominant bacteria and fungi.
Detailed Description
The following description is made in connection with specific embodiments:
experimental material sources:
ELISA kit: the product numbers JM-110029P1, JM-09865P2, JM-01683P 2, JM-01185P2, JM-06093402 are all available from Jiangsu Beijing Biometrics;
FastDNA SPIN kit: goods number 116560-200, purchased from an Anbei medical instrument trade company;
inductively coupled plasma emission spectrometer: model ICP-OES, optima 2100DV, available from Perkin Elmer instruments Co., ltd;
olsen spectrophotometer: model ICO-2600, available from Shanghai instruments, china;
basic analyzer: model Flash EA 1112, available from Siemens Feier technologies;
spectrophotometers: model NanoDrop 1000, available from sameimers technologies;
the bacillus (Bacillus amyloliquefaciens) W25 used in the following examples was deposited in the China general microbiological culture Collection center with a deposit number of CGMCC No.20042, at 8/6 of 2020.
Example 1
Preparation of bacillus W25 microbial inoculum:
inoculating the W25 strain on a solid culture medium in a slant way, and culturing for 3d at 30 ℃; then selecting full and sticky W25 colony to inoculate in liquid culture medium, shaking and culturing at 32 deg.C and 180rpm for 20h; transferring the fermentation liquor into a sterile centrifugal bottle, centrifuging at 5000rpm for 5min, discarding the supernatant, and collecting thalli; washing thalli with sterile deionized water and re-suspending to obtain bacterial suspension, so that the viable count in the bacterial suspension reaches 10 8 Drying the bacterial suspension to prepare bacillus W25 bacterial agent;
wherein the solid culture medium comprises the following components: sucrose 10.0g/L, (NH) 4 ) 2 SO 4 0.5g/L、Na 2 HPO 4 2.0g/L、KCl 0.191g/L、MgSO 4 ·7H 2 O0.5 g/L, agar 20g/L, and water in balance;
wherein, the liquid culture medium comprises the following components: sucrose 10.0g/L, (NH) 4 ) 2 SO 4 0.5g/L、Na 2 HPO 4 2.0g/L、KCl 0.191g/L、MgSO 4 ·7H 2 O0.5 g/L, and the balance water.
Example 2
The bacillus W25 microbial inoculum is used for controlling the content of gamma-PGA and Na in the salty soil filtrate + And K + Effect of concentration:
(1) Adding 9L of deionized water into 3kg of soil, oscillating at 180rpm for 48h, centrifuging at 6000rpm for 15min, and collecting supernatant; filtering the supernatant through a microporous filter membrane (0.45 mu m pore size), sterilizing, and then mixing with a sterile basic fermentation medium according to a volume ratio of 2:1, uniformly mixing to obtain a mixed solution; wherein, the components of the sterile basic fermentation medium are as follows: beef extract 3.0g, peptone 5.0g, glucose 10.0g, distilled water 1.0L, pH7.0.
(2) Adding 200mL of the mixed solution obtained in the step (1) into a triangular flask, adding 5g of NaCl into the mixed solution as an experimental group, enabling the final concentration of NaCl to be 25g/L, and setting 3 groups to be parallel; taking the mixed solution without adding NaCl as a control group;
(3) Inoculating the bacillus W25 microbial inoculum prepared in the example 1 into the experimental group and the control group in the step (2) respectively, wherein the inoculation amount is 0.2g, so as to obtain a culture solution; culturing the culture solution at 32deg.C and 180rpm, sampling at 0h, 48h, 96h, 144h, 192h, 240h, and measuring cell number (OD) 600 ) pH, gamma-PGA content and Na + And K + The content is as follows;
the method for detecting the content of the gamma-PGA is described in the article by Wei Zeng et al, and is described in detail in An integrated high-throughput strategy for rapid screening of poly (gamma-glutamic acid) -producing bacteria. Appl Microbiol Biotechnol (2013) 97:2163-2172; na (Na) + And K + The content detection method comprises the following steps: determination of Na by inductively coupled plasma emission spectrometer + And K + Is a concentration of (3).
The detection results are shown in fig. 1-2:
as can be seen from graph a in fig. 1, on day 2 after inoculation with the W25 inoculum, there was a significant increase in pH in the culture solution, but with increasing culture time, the pH increase was small, and there was no significant difference in pH values in the experimental and control groups; OD in culture solution after inoculation of W25 microbial inoculum 600 Continuously rising and starting to stabilize on day 4, and there was no significant difference in pH in the experimental and control groups;
as can be seen from the B-panel in fig. 1, the γ -pga content in the culture broth after inoculation with the W25 inoculum increases significantly with increasing culture time, and reaches the maximum peak on the sixth day; and it can be seen that the experimental group γ -pga concentration is always higher than the control group at high concentration (2.5%) of salt stress;
as shown in FIG. 2, the culture time increased, and the culture medium was inoculated with the W25 microbial inoculum for 2 to 6 days + The content is obviously increased, and Na in the culture solution of the experimental group + The content is obviously reduced; compared with a control group, in an experimental group under the condition of high concentration (2.5%) salt stress, the W25 microbial inoculum can obviously improve K in the culture solution + Content and decrease Na + The content is as follows.
Example 3
Application of bacillus W25 microbial inoculum in lettuce planting in salt environment:
the W25 microbial inoculum is applied to lettuce potting experiments, and the specific method is as follows:
2.5kg of soil is filled in the flowerpots, then NaCl with different dosages is respectively added, so that the final concentration of Na < + > in the soil of each flowerpots is respectively 0mM/L, 100mM/L, 200mM/L and 300mM/L, 3 repeats are arranged in each group, and then NaCl and the soil are fully mixed and then balanced for 30 days; sowing full lettuce seeds with sterilized surfaces in the flowerpot, and thinning to 10 plants/pot after germination; diluting W25 bacteria with sterile deionized water to 1×10 8 Digging a ditch (1-2 cm deep) around the root part in a third leaf stage of the raw vegetable, adding the bacterial suspension of the W25 microbial inoculum into the ditch, wherein the liquid adding amount is 50 mL/basin, and adding the same dose of sterile deionized water as a control group; potted plants are cultivated in a greenhouse (the temperature is 10-22 ℃ and the relative humidity is 30-45 percent, and the illumination is normal) for 45 days; after the cultivation is finished, collecting the root and the overground part (lettuce leaves) of each lettuce basin and carrying out subsequent analysis on rhizosphere soil;
lettuce roots and edible tissues were washed sequentially with 0.01M sodium ethylenediamine tetraacetate (EDTA-2 Na) and distilled water and divided into two parts: inactivating a part of the dried lettuce roots and edible tissues at 105 ℃ for 30min, drying at 60 ℃ to constant weight, weighing, grinding and digesting the dried lettuce roots and the edible tissues to determine the content of Na, K and Ca; the other part adopts ELISA kits (with the product numbers of JM-110029P1, JM-09865P2, JM-01683P 2 and JM-01185P 2) to respectively measure the Vc (vitamin C) content, the soluble sugar (glucose, fructose, maltose and sucrose) content, the soluble protein content, the MDA (malondialdehyde) content, the proline (proline) content, the SOD (superoxide dismutase) activity and the POD (peroxidase) activity in fresh leaves, and the IAA (indoleacetic acid) content in roots and the leaves; wherein, the Vc content in the leaf is measured by adopting a 2, 6-dichlorophenol titration method (AOAC), the soluble sugar content in the leaf is measured by adopting a sulfuric acid-anthryl colorimetric method (620 nm), and the proline content in the leaf is measured by adopting a spectrophotometry (520 nm) after the color reaction under the acidic condition;
the measurement results are shown in table 1 and fig. 3, respectively:
TABLE 1 lettuce root and leaf dry weight and Vc, soluble sugar and soluble protein content in leaf
As can be seen from table 1, under both salt and non-salt environments, biomass (dry weight) in roots and leaves increased, and it can be seen that the W25 inoculant can promote lettuce growth; under the conditions of 100mM and 200mM salt stress, compared with a control group, the method can obviously improve the Vc, soluble protein and soluble sugar content in lettuce leaves after being inoculated with the W25 microbial inoculum; under the condition of high-concentration salt stress (300 mM), although the growth of lettuce is inhibited, compared with a control group, the dry weight of the lettuce root after being inoculated with the W25 microbial agent is obviously increased by 30.0 percent, and the dry weight of the leaf blade is increased by 59.5 percent;
as can be seen from graphs A to D in FIG. 3, when the soil salt concentration is 100mM, 200mM and 300mM, na/K and Na/Ca in lettuce roots and leaves are increased; however, compared with the control group, the Na/K and Na/Ca in the lettuce roots and leaves after being inoculated with the W25 microbial agent in the salt environment are obviously reduced, which proves that the Na in the lettuce tissues in the salt environment is reduced to a certain extent after being inoculated with the W25 microbial agent + The content of Ca is increased 2+ 、K + The content is as follows;
as can be seen from the graphs E to J in fig. 3, compared with the no-salt control group: MDA content in lettuce leaves is obviously reduced after the W25 microbial inoculum is inoculated under the salt concentration conditions of 200mM and 300mM, proline and POD content in lettuce leaves is obviously improved after the W25 microbial inoculum is inoculated under the salt concentration conditions of 100mM, 200mM and 300mM, SOD content in lettuce leaves is obviously improved under the salt concentration conditions of 100mM, 200mM and 300mM, IAA content in lettuce leaves and roots is slowly reduced under the salt concentration conditions of 100mM, 200mM and 300mM, but IAA content in lettuce leaves and roots after the W25 microbial inoculum is inoculated is still higher than that in a control group; the detection result reflects bacillus W25 to a certain extent, can effectively improve the antioxidation capability of lettuce in a salt environment, reduces the generation of peroxide in leaves, and improves the lettuce quality.
Example 4
Influence of bacillus W25 inoculant on related physicochemical properties such as enzymatic activity in saline soil:
example 3 rhizosphere soil in lettuce pot was collected, urease activity, phosphatase activity, DTPA-5Na (pentasodium diethylenetriamine pentaacetate) content, pH (water to soil volume ratio 2.5:1 at pH) in soil was measured, and NO in soil was measured 3 -N (nitrate nitrogen), NH 4 -N (ammonium nitrogen), ANa (Ex-Na, fast-acting sodium), AP (fast-acting phosphorus) and AK (fast-acting potassium);
the determination method of the urease activity, the phosphatase activity and the DTPA-Na content is described in detail in the article "Soil Sci Ch.Acad,1980; S.Guan et al, 1986;
na, K, P content determination: by oxidation with potassium dichromate and potassium sulfate using 1.0M ammonium acetate (NH 4 OAc, ph 7.0) to determine ANa and AK contents, and determining AP contents by ICP-OES using an Olsen spectrophotometer;
NH 4 -N and NO 3 -N content determination: fresh soil samples were extracted with 2M potassium chloride solution (soil: potassium chloride=1:5) and NH determined with a basic analyzer 4 -N and NO 3 -N content;
IAA content measurement: ELISA kit was used, and the method was the same as in example 3;
the measurement results are shown in the following table 2:
TABLE 2 detection of soil-related physicochemical Properties such as rhizosphere soil enzymatic Activity
As can be seen from table 2, compared with the control group, inoculation of W25 inoculant significantly increased urease activity (24-37% increase) and phosphatase activity (33-59% increase) in the saline soil, and IAA content significantly increased by 7-9%, which also corresponds to IAA reduction in lettuce tissue in example 3;
in addition, compared with a control group, the inoculated W25 microbial inoculum can obviously reduce the content of quick-acting sodium in the saline soil, so that the organic matter content (OM) in the soil is obviously improved by 4.0-9.1%, the Cation Exchange Capacity (CEC) is obviously improved by 12.1-21.0%, the organic carbon content (ESP) is obviously reduced by 38.1-41.9%, and NO 3 The contents of N, AP and AK are obviously improved by 23.7 to 26.1 percent, 29.7 to 49.3 percent and 16.8 to 37.6 percent respectively, and the EC value (conductivity) of soil is obviously reduced by 12.6 to 19.2 percent, which indicates that bacillus W25 can promote the production of beneficial environmental factors in the soil and has positive effect on maintaining lower EC value in the salinized soil, thereby reducing the conductivity of the salinized soil; the soil conductivity is an index for measuring water-soluble salts of soil, the water-soluble salts of soil are an important index for mineral nutrition in surface soil which can be rapidly utilized by plants, and are factors for judging whether salt ions in the soil limit crop growth, and normal EC values of the soil can prevent the plants from being damaged or causing death of plant root systems due to high-salt environments.
Example 5:
influence of bacillus W25 inoculant on microbial diversity in saline soil:
total DNA was extracted from 0.5g of fresh salted rhizosphere soil and control soil without NaCl in example 3 using FastDNA SPIN kit, and the amount and quality of total DNA was determined by spectrophotometry and gel electrophoresis, respectively;
PCR amplification of the extracted total DNA was performed using bacterial universal primers 338F and 806R, targeting the V4 region of bacterial 16s rRNA, and high throughput sequencing was done in Illumina Hiseq 2000 (Illumina Inc., san Diego, USA); wherein, the primer sequence of 338F is: 5'-ACTCCTAGGGGGCAGCA-3';806R primer sequences were: 5'-GGACTACHVGGGTWTCTAAT-3';
specific detection results are shown in fig. 4 to 7 respectively:
FIG. 4 shows the effect of the inoculation of W25 inoculum on the diversity of the bacterial (panels A and B) and fungal (panels C and D) rhizosphere soil microflora Alpha of lettuce, and it can be seen that the inoculation of W25 inoculum can significantly improve the shannon index and the simpson index of the bacteria in both saline and non-saline environments compared with the control group; as can be obtained from the A graph and the B graph, under the salt concentration conditions of 100mM (S1), 200mM (S2) and 300mM (S3), the inoculation of the W25 microbial inoculum can obviously improve the shannon index and the simpson index of bacteria compared with a control group; from panels C and D, it can be seen that inoculation of the W25 inoculum at 200mM (S2) and 300mM (S3) salt concentration significantly improved the shannon index and the simpson index of the fungus compared with the control group.
FIG. 5 is a non-metric multidimensional scale (NMDS) ordering of genus-level bacterial (panel A) and fungal (panel B) community composition based on Bray-Curtis distance similarity, inoculating a W25 inoculant on the Beta diversity of lettuce rhizosphere soil microbial communities; it can be seen that at the bacterial level, there is a significant difference between the bacterial community in the lettuce rhizosphere soil environment after inoculation with the W25 inoculant and the control group, which to some extent reflects the change in the microbial community structure of the salinized soil by the bacillus W25 treatment, and this change has a tendency in different salt concentration treatments.
FIG. 6 is a graph showing the relative abundance of bacteria (A) and fungi (B) at the portal level and the relative abundance of bacteria (C) and fungi (D) at the genus level; as can be seen from the graph A, actinobacteria, proteobacteria and Acidobacteria in the bacteria are dominant bacteria phylum and account for more than 70% of the total bacterial community; as can be seen from the diagram B, ascomycota in fungi is the dominant phylum; as can be seen from the graph C, the relative abundance of Pseudomonas and Bacillus in the soil after inoculation with the W25 microbial inoculum is improved to a different extent compared with the control group at salt concentrations of 100mM, 200mM and 300mM, wherein the proliferation effect of the W25 microbial inoculum on Pseudomonas is particularly remarkable; from the graph D, the addition of the W25 microbial inoculum obviously reduces the relative abundance of Fusarium (Fusarium) and cephalosporin (Cephalosporins) in all samples, and the abundance of the Fusarium (Fusarium) and the cephalosporin (Cephalosporins) in the soil environment is positively correlated with the pollution degree of soil mould.
FIG. 7 RDA and sporman correlation of dried weight, soil physicochemical properties of raw vegetables with the relative abundance of the first 20 dominant bacteria (panels A and C) and fungi (panels B and D)A heat map; as can be seen from A, B, the rhizosphere soil sample of the control group which is not inoculated with the W25 microbial inoculum, EC and NH 4 + N, ex-Na, ESP and the like are positively correlated, and on the contrary, the rhizosphere soil sample inoculated with the W25 microbial inoculum is directly correlated with NO 3 - N, IAA, AP, AK, CEC, OM the phosphatases and ureases are positively correlated, RDA also shows that lettuce dry weight is associated with soil AP, AK, OM, CEC, phosphatases, ureases, IAAs and NO 3 -N is positively correlated but negatively correlated with soil Ex-Na (fast-acting sodium) and ESP; furthermore, as can be seen from the C, D plot, plant biomass is positively correlated with pseudomonas, saccharomyces, arthrobacter, but negatively correlated with fungus abundance in fusarium, cephalosporin, penicillium, and the like; fig. 7 further verifies the experimental results in examples 2 to 4 described above.
In conclusion, the bacillus W25 can be applied to improvement of plant salt tolerance and salinized soil, so that the problem of yield reduction of crops in a salt environment is effectively avoided, and the utilization rate and the productivity of salinized soil are improved.
Claims (9)
1. The application of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W25 in improving the salt tolerance of plants, wherein the bacillus amyloliquefaciens W25 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 20042 in the month-6 and 8-2020.
2. The use according to claim 1, wherein the bacillus amyloliquefaciens W25 is used for reducing Na of plants in salt environments + Content of Ca is increased 2+ 、K + The content is as follows.
3. The use according to claim 1, wherein the bacillus amyloliquefaciens W25 is used for increasing the IAA, SOD, POD, proline, vc, soluble sugar and soluble protein content of plants in a salt environment and decreasing the MDA content of plants in a salt environment.
4. Use of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) W25 according to claim 1 for improvement of salinized soil.
5. The use according to claim 4, wherein the bacillus amyloliquefaciens W25 is used for reducing the quick-acting sodium content of salinized soil and increasing the content of gamma-PGA, quick-acting phosphorus and quick-acting potassium in the salinized soil.
6. The use according to claim 4, wherein the bacillus amyloliquefaciens W25 is used for promoting the production of beneficial environmental factors in the salinized soil and reducing the conductivity of the salinized soil; wherein the beneficial environmental factor is NO 3 -N。
7. The use according to claim 4, wherein the bacillus amyloliquefaciens W25 is used for promoting the proliferation of pseudomonas in salinized soil.
8. The method according to claim 4, wherein the bacillus amyloliquefaciens W25 is prepared into a microbial inoculum, and the microbial inoculum is applied in a salt environment with a sodium chloride concentration of less than or equal to 300mM, and the application amount is 1-2 Kg/mu.
9. The use of claim 8, wherein the preparation method of the microbial inoculum comprises: the viable count is more than or equal to 10 8 And (5) drying the bacillus amyloliquefaciens W25 bacterial liquid with the concentration of one/mL to obtain the bacterial agent.
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