CN117511769A - Sword bacterium for promoting growth and nodulation of sesbania in saline-alkali soil and application thereof - Google Patents
Sword bacterium for promoting growth and nodulation of sesbania in saline-alkali soil and application thereof Download PDFInfo
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- CN117511769A CN117511769A CN202311320641.0A CN202311320641A CN117511769A CN 117511769 A CN117511769 A CN 117511769A CN 202311320641 A CN202311320641 A CN 202311320641A CN 117511769 A CN117511769 A CN 117511769A
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Classifications
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- 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
-
- 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
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- 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
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- 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
-
- 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
Abstract
The invention discloses a sword fungus Ensifer sp.KSU03 for promoting the growth and nodulation of sesbania in saline-alkali soil and a sword fungus microbial agent prepared from the same. The strain KSU03 is separated from sesbania root nodules in sesbania test areas of saline-alkali lands in karis of Xinjiang, is resistant to saline and alkali, can dissolve no/organic phosphorus, has the characteristics of secreting plant growth hormone, synthesizing 1-aminocyclopropane-1-carboxylic acid deaminase and producing extracellular polysaccharide. Sesbania is treated by Ensifer sp.KSU03 and the prepared sword bacteria microbial agent, so that sesbania nodule can be effectively promoted, and the number of nodule nodules reaches 15.07; the sesbania plant height, the stem thickness and the dry weight on the ground are obviously improved; the nitrogen fixation amount of sesbania single plant and the chlorophyll content of leaf blades are also obviously improved, and compared with the original soil and a control group, the total salt content of the detected soil is respectively reduced by 36.05 percent and 20.2 percent, which shows that the sword fungus Ensifer sp.KSU03 and the sword fungus microbial agent prepared by the invention have wide development and application prospects in the aspect of sesbania growth promotion in saline-alkali soil.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganism application, and particularly relates to a sword fungus Ensifer sp.KSU03 for promoting growth and nodulation of sesbania in saline-alkali soil and a sword fungus microbial agent prepared from the same and the technical field of application.
Background
Soil salinization is one of typical abiotic stress factors, seriously affects the operation and distribution of soil nutrients, and inhibits the normal reproductive physiological process of plants. Soil salinization causes that most crops cannot be produced normally, and becomes a main factor for limiting the stability and sustainable development of an agricultural ecological system. The saline-alkali soil in China has large area and wide distribution, and especially causes such factors as population quantity surge, climate change, improper agricultural water and fertilizer management and the like in nearly half century, the soil salinization and secondary salinization trend are increasingly aggravated, and the large-area farming land is in a degradation state. The restoration of the saline-alkali soil makes the saline-alkali soil become a reserve cultivated land resource, and has important economic and social significance. The research shows that the biological inoculation method, namely, inoculation of beneficial flora for plants, can effectively relieve the adverse effect of saline-alkali stress on plants, and has positive effects on promoting the growth of the plants in saline-alkali soil and improving the soil properties. The improvement of the saline-alkali soil based on the transplantation of the soil beneficial bacteria is a low-impact and high-benefit mode conforming to the development of technology.
The genus Sinorhizobium (Ensifer) is a class of gram-negative bacilli widely distributed in soil of the Rhizobiaceae family and is synonymous with Sinorhizobium. Symbiotic nitrogen fixation systems of leguminous plants and rhizobia are always focused on hot spots, and the symbiotic systems can solve the problems of soil structure deterioration, soil nutrient deficiency and the like caused by pollution and degradation. The exogenous Ensifer strain is used for promoting nodulation and nitrogen fixation of leguminous plants, so that the growth of the leguminous plants can be promoted, the nitrogen nutrition in barren soil can be improved and supplemented, and a new way is hopefully explored for improving saline-alkali soil.
Sesbania (Sesbania cannabina) is annual herbaceous halophyte of sesbania of Leguminosae, and has strong stress resistance under saline-alkali, barren and other stress environments. Sesbania root system is developed, root nodule is many and big, nitrogen fixation capability is strong, fresh grass yield is high, nutrient content is rich, and the characteristics make it become pioneer plant for saline-alkali soil improvement and reformation. The rhizobia can invade the sesbania body and form rhizobium, and establish a symbiotic nitrogen fixation system with the rhizobia, so that the growth of the sesbania is promoted, and meanwhile, the nitrogen nutrient of the soil can be supplemented. Therefore, inoculating highly effective rhizobia is an effective strategy for promoting sesbania symbiotic nitrogen fixation and improving the nutritional status of sesbania. At present, a small number of researches on sesbania are reported: the invention patent CN110063161A discloses a method for promoting the growth of andrographis paniculata and improving the content of active ingredients by utilizing sesbania stem nodule azorhizobia; the invention patent CN113179882A discloses a method for promoting the nodulation and nitrogen fixation of stem nodule mustard root by utilizing sesbania nitrogen fixation rhizobia. However, the existing similar microbial inoculum products have no wide saline-alkali adaptability and cannot be popularized and applied for arid and semiarid region saline-alkali regions. Therefore, there is a need to develop microbial inoculant products for promoting sesbania nodulation and growth for arid and semiarid regions of saline-alkali soil to promote the application of the microbial inoculant products as green manure in improving the soil of the saline-alkali soil.
Disclosure of Invention
Aiming at the technical situation that no microbial agent for promoting sesbania nodulation and growth in arid and semiarid areas of saline-alkali soil exists in the prior art, development of a microbial strain for efficiently promoting sesbania growth and nodulation in the saline-alkali soil is needed to promote application of the microbial strain as green manure in improving soil in the saline-alkali soil, the invention aims at a sword bacterial Ensifer sp.KSU03 for promoting the sesbania growth and nodulation in the saline-alkali soil, a sword bacterial microbial agent prepared by the same and application of the sword bacterial agent, and the sword bacterial Ensifer sp.KSU03 is separated from sesbania nodulation in a sesbania test area in the saline-alkali soil in Xinjiang karst. The strain KSU03 is resistant to saline and alkaline, can dissolve no/organic phosphorus, and has the characteristics of secreting plant growth hormone (IAA), synthesizing 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase and producing extracellular polysaccharide. The sesbania is treated by Ensifer sp.KSU03 and the prepared sword bacteria microbial agent, so that sesbania nodule can be effectively promoted, and the number of nodule is up to 15.07; the sesbania plant height, the stem thickness and the dry weight on the ground are obviously improved; the nitrogen fixation amount of sesbania single plant and the chlorophyll content of leaf blades are also obviously improved, and compared with the original soil and a control group, the total salt content of the detected soil is respectively reduced by 36.05 percent and 20.2 percent, which shows that the Ensifer sp.KSU03 and the prepared sword microbial agent thereof have wide development and application prospects in the aspect of promoting the growth of sesbania in saline-alkali soil, and not only can reduce the fertilizer consumption, but also can promote the growth of sesbania.
In order to achieve the technical effects, the invention is realized by the following technical scheme.
The invention provides a strain Ensifer sp.KSU03 for promoting the growth and nodulation of sesbania in saline-alkali soil, wherein the strain Ensifer sp.KSU03 is preserved in China Center for Type Culture Collection (CCTCC) No: m2023823.
The invention provides a sword fungus Ensifer sp.KSU03 which is screened and separated from sesbania root nodules in sesbania test areas of saline-alkali lands in karis of Xinjiang, and the strain KSU03 belongs to Ensifer through 16Sr RNA sequence phylogenetic analysis and morphological analysis. The sequence obtained by sequencing the gene of the strain is subjected to BLAST comparison analysis on NCBI website, and the 16Sr RNA gene sequence of the strain KSU03 is compared with the standard strain Ensifer sesbaniae CCBAU 65729 T (GenBank No: JF 834143) has the highest homology, and the similarity is 98.69%. Selecting sequences with high homology to construct 16Sr RNA phylogenetic tree, and strains KSU03 and Ensifer morelensis Lc04 T (AY 024335) the strain was collected as a branch, and the relationship was recent. The strain KSU03 can be determined to be a new strain of Ensifer through series multiphase classification identification, and has the characteristic of the representativeness of the new strain.
The gene sequence of the Sword Ensifer sp.KSU03 is shown as SEQ ID NO. 1.
In the invention, the separation culture medium of the sword bacteria Ensifer sp.KSU03 is:mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O0.004 g/L, naCl 0.1g/L, congo red solution 0.25% 10mL/L, agar 20g/L.
In the invention, the purification culture medium of the sword bacteria Ensifer sp.KSU03 is: mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O 0.004g/L，NaCl 0.1g/L。
The invention provides an Ensifer sp.KSU03 sword microbial agent, which is prepared by fermenting Ensifer sp.KSU03.
Meanwhile, the invention provides a preparation method of Ensifer sp.KSU03 sword microbial agent, which specifically comprises the following steps: inoculating a single colony of the Ensifer sp.KSU03 into YMA culture medium, and culturing for 48 hours at 28 ℃ and 180rpm to obtain Ensifer sp.KSU03 seed solution; inoculating Ensifer sp.KSU03 seed solution into special liquid culture medium according to the inoculum size of 2% by volume, fermenting and culturing at 28deg.C and 180rpm for 48 hr, and regulating OD 600 0.8, ensifer sp.KSU03 Sword microbial agent was obtained.
The special liquid culture medium is as follows: 7.5g/L sucrose, 1.5g/L yeast powder, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O0.004 g/L, naCl 0.1g/L, initial pH value 8, and liquid loading amount 40%.
The invention provides an application of a sword fungus Ensifer sp.KSU03 in sesbania growth and nodulation in saline-alkali soil, which can promote sesbania growth and has wide development and application prospects in plant growth promotion.
The invention provides application of Ensifer sp.KSU03 sword microbial agent in sesbania growth and nodulation in saline-alkali soil, which can promote sesbania growth and has wide development and application prospects in plant growth promotion.
Through the technical scheme, the invention has the following technical effects:
(1) The invention provides a strain Ensifer sp.KSU03, which is proved to be a typical new strain in the field of the acquisition of the Sword strain with the strain number of KSU03 through molecular level identification of a strain system well known and accepted in the field and combining with multiphase classification system identification analysis according to morphological identification, physiological and biochemical characteristics and the like, wherein the strain KSU03 has a plurality of differences from a standard mode strain with the closest homology to the genus, namely the new strain Ensifer sp.
(2) The invention provides an Ensifer sp.KSU03 which is fermented to obtain a rapier microbial agent, and the seed and seedling of sesbania are soaked to be inoculated with the microbial agent, so that the plant height, the stem thickness and the dry weight of sesbania are increased by 29.5%, 24.1% and 56.6% compared with the control; the nitrogen fixation amount of sesbania single plant can reach 5.56+/-1.98 mmol C 2 H 4 And (h), the chlorophyll content of sesbania leaves is obviously higher than that of a control group, and is increased by 33.6% compared with that of the control group, so that the sesbania leaves can effectively promote the growth of sesbania and the accumulation of biomass.
(3) The Ensifer sp.KSU03 sword microbial agent is adopted to soak sesbania seeds and inoculated seedlings, the average number of sesbania nodule is 15.07, and no nodule is formed in the contrast, so that the sesbania nodule can effectively promote the growth of sesbania nodule.
(4) The invention provides a new bacterium and new bacterium microbial agent which can reduce the salt content of soil in saline-alkali soil, increase the content of quick-acting nitrogen and nutrient in soil and has wide application prospect, wherein the quick-acting nitrogen content of the soil is 52.8% higher than that of control after sesbania is treated by the microbial agent, and the total salt content of the soil is 36.05% lower than that of original soil.
Drawings
FIG. 1 shows a phylogenetic tree constructed for strain KSU03 based on the 16Sr RNA gene sequence.
FIG. 2 shows a plate colony morphology of Ensifer sp.KSU03 of the present invention.
FIG. 3 shows a graph of the effect of temperature on the growth of Ensifer sp.KSU03 according to the invention.
FIG. 4 shows the effect of different salt concentrations on the growth of Ensifer sp.KSU03 according to the invention.
FIG. 5 shows the effect of different pH values on the growth of Ensifer sp.KSU03 according to the invention.
FIG. 6 shows a graph of a one-factor optimization test of Ensifer sp.KSU03 medium composition and conditions.
FIG. 7 shows a graph of the conditions for the response surface method optimized Ensifer sp.KSU03 fermentation.
FIG. 8 shows the effect of different tieback modes of Ensifer sp.KSU03 microbial agents of the present invention on sesbania growth and physiological index. Wherein A is the influence of different tie-back modes on the plant height of sesbania; b is the influence of different tie-back modes on sesbania stems; c is the influence of different tieback modes on the overground dry weight of sesbania; d is the influence of different tie-back modes on the number of sesbania root nodule formation; e is the influence of different tie-back modes on the nitrogen fixation amount of sesbania individual root nodule living bodies; f is the influence of different tieback modes on the chlorophyll content of sesbania.
FIG. 9 shows the effect of different tieback modes of Ensifer sp.KSU03 microbial agents of the present invention on soil quick-acting nitrogen and total salt content; wherein A is the influence of different tie-back modes on the quick-acting nitrogen content of sesbania cultivation soil; b is the influence of different tie-back modes on the total salt content of sesbania cultivation soil.
FIG. 10 shows the effect of the different tieback modes of the Ensifer sp.KSU03 microbial agents of the present invention on sesbania growth and its nodule characteristics, wherein A is the condition of sesbania growth in the different tieback modes of Ensifer sp.KSU03; b and C are sesbania nodule characteristics.
Detailed Description
The present invention will be described with reference to the following examples, but the present invention is not limited to the examples. All the raw materials and auxiliary materials selected in the invention, as well as the selected strain culture methods are well known in the art, and all percentages referred to in the invention are by volume unless otherwise indicated.
The YMA culture medium adopted in the preparation of the sword bacteria Ensifer sp.KSU03 microbial agent is (g/L): mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O 0.004g/L，NaCl 0.1g/L，pH5-10.0。
Example 1: separation, purification and identification of Sword Ensifer sp.KSU03
(one) separation and purification
Separating by adopting a flat plate dilution method, collecting samples from root nodules on sesbania root systems in a sesbania test area in Xinjiang, selecting fresh and full root nodules, cleaning the root nodules with sterile water in a biosafety cabinet, removing surface soil, disinfecting with 75% alcohol for 3min, disinfecting with 3% NaClO solution for 5min, and then washing with sterile water; diluting with sterile water to 10 -4 Uniformly coating 0.2mL of each on a separation plate culture medium, coating 10 plates on each dilution gradient, inversely culturing at 28 ℃, picking single colony, streaking and purifying, wherein the separation culture medium is as follows: mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O0.5g/L，FeCl 3 ·3H 2 O0.004 g/L, naCl 0.1g/L, congo red solution 10mL/L and agar 20g/L; the purified medium was: mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O 0.004g/L，NaCl 0.1g/L。
(II) identification of 16Sr RNA Gene
Extraction of DNA
Ensifer sp.KSU03 was inoculated into a purified liquid medium, cultured at 28℃for 2 days to obtain bacterial cells, and genomic DNA extraction was performed using a bacterial genomic DNA rapid extraction kit.
PCR amplification
16Sr RNA sequence primer:
27F：5′-AGAGTTTGATCCTGGCTCAG-3′
1492R：5′-TACGACTTAACCCCAATCGC-3′。
reaction system and conditions: the PCR reaction system was 50. Mu.L: 2 XPCR Mix 25mL, template DNA 2. Mu.L, 1. Mu.L each of the upstream and downstream primers, ddH 2 O was made up to 50. Mu.L. The reaction conditions are 94 ℃ pre-denaturation for 3min,94 ℃ denaturation for 30s,55 ℃ annealing for 30s and 72 DEG CExtending for 1min, 30 cycles, and maintaining at 72 ℃ for 10min.
3. Sequencing
The PCR amplified product is sequenced after electrophoresis detection and purification, the sequence length is 1384bp, and the sequencing result is shown in SEQ ID No:1, submitting the 16Sr RNA gene sequence of the strain to EzBioCloud database for comparison and analysis, selecting and downloading a standard strain with higher homology according to the comparison result, establishing a phylogenetic tree (repeated sampling 1000 times) by using MEGA 7.0 software commonly adopted in the field through a Neighbor-Joing method, and comparing and analyzing the obtained sequence in NCBI website to find that the 16Sr RNA gene sequence of the strain KSU03 is compared with the standard strain Ensifer sesbaniae CCBAU 65729, wherein the result is shown in figure 1 T (GenBank No: JF 834143) has the highest homology of 98.69%, and in a phylogenetic tree constructed by 16Sr RNA gene sequence, the 16Sr RNA gene sequence of the strain KSU03 is identical to Ensifer morelensis Lc04 T The relativity of the strain (AY 024335) is recent, which shows that the strain KSU03 has extremely high support rate as a new strain, shows extremely good stability in an evolutionary tree, and confirms that the obtained Ensifer belongs to a typical new strain in the category of the strain number KSU03 through comprehensive judgment of the similarity and the homology of the strain.
Example 2: multiphase classification of Ensifer sp.KSU03
Colony morphology characterization of Ensifer sp.KSU03
The strain KSU03 to be observed is inoculated on a YMA plate without Congo red, and is cultured for 3 days at 28 ℃, and after the colony grows over the whole plate, the colony characteristic record is observed, the record is photographed and stored, and meanwhile, a scanning electron microscope photograph of the colony is recorded, and the record result is shown in the figure 2.
As can be seen from the results of FIG. 2, the strain KSU03 was cultured at 28℃for 3 days in YMA medium without Congo red, and the colony was in the form of a micro-transparent gel, irregular in shape, and milky white streak, and the colony diameter was 0.4-0.8cm when cultured at 28℃for 3 days.
Based on the above biological characteristics, the strain KSU03 was identified as Ensifer sp. The strain has been deposited in the Budapest treaty International deposit unit of microorganisms: china Center for Type Culture Collection (CCTCC), address: chinese, wuhan, university of Wuhan, postal code: 430072, date of deposit: 2023, 5 months and 26 days, with a preservation number of CCTCC No: m2023823.
Physiological and biochemical characteristics of Ensifer sp.KSU03
Strain KSU03 was inoculated onto YMA medium and each physiological and biochemical characteristic was detected separately. The characteristic analysis of growth temperature, salinity, pH value range and the like is mainly referred to a general bacterial System identification Manual, and specific results are shown in figures 3-5.
Inoculating Ensifer sp.KSU03 single colony into YMA liquid culture medium without Congo red, shaking culturing at 28deg.C and 180rpm for 48 hr, transferring to fresh YMA liquid culture medium again at 2% inoculum size, shaking culturing at 180rpm for 48 hr under conditions of 4deg.C, 8deg.C, 12deg.C, 16deg.C, 20deg.C, 25deg.C, 28deg.C, 32deg.C, 37deg.C, 42 deg.C and 45deg.C, respectively, and measuring the OD of the culture solution 600 The absorbance at nm was repeated for each temperature. As can be seen from the data of figures 3-5, the strain KSU03 can normally grow at 12-37 ℃, and the optimal growth temperature range is 28-37 ℃; the strain KSU03 can grow normally under the concentration of 5% NaCl, grow obviously slowly under the concentration of 6% NaCl and 7% NaCl, and culture solution OD after 48 hours of culture 600 The absorbance at nm was 49.8% and 20.2% at NaCl concentration of only 1%. Does not grow at NaCl concentration of 8% or more. The growth conditions are not different when the NaCl concentration is below 4%; the strain KSU03 can grow in the pH range of 5-10, but the growth is obviously weaker at the pH of 5 and 10, and the optimal growth pH is 6-9; the strain KSU03 is resistant to saline and alkaline, can dissolve non-organic phosphorus, has the function of secreting plant growth hormone (IAA), synthesizes 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase, and generates extracellular polysaccharide.
The bacterial strain KSU03 is determined to be a new Ensifer sp strain, and is named Ensifer sp.KSU03 according to the separation source.
Example 3: preparation of Ensifer sp.KSU03 Sword microbial agent
This example is based on examples 1-2 Ensifer sp.KSThe preparation method of the U03 sword bacteria microbial agent specifically comprises the following steps: inoculating single colonies of Ensifer sp.KSU03 into YMA culture medium, and culturing at 28 ℃ and 180rpm for 48 hours to obtain Ensifer sp.KSU03 seed solution; inoculating Ensifer sp.KSU03 seed solution into special liquid culture medium according to the inoculum size of 2% by volume, fermenting and culturing at 28deg.C and 180rpm for 48 hr, and regulating OD 600 Ensifer sp.KSU03 microbial agent was obtained at 0.8.
Example 4: ensifer sp.KSU03 culture condition optimization
Medium composition and condition single factor optimization
1. Influence of carbon source species on bacterial density: based on the purified culture medium, glucose, sucrose, lactose and soluble starch with final addition of 10g/L are used as carbon source instead of mannitol, seed solution is respectively inoculated with 2% of inoculation amount, and cultured for 48h at 28deg.C and 180rpm, three repeats are set for each culture medium, the liquid loading amount is 100mL/250mL conical flask, and OD is measured by turbidimetry 600 Bacterial density of culture solution at nm.
2. Influence of carbon Source concentration on bacterial Density: and finally determining sucrose as the optimal carbon source according to an influence test of the carbon source type on the number of living bacteria and considering economic cost. Adding sucrose with final concentration of 2g/L, 4g/L, 6%, 8g/L, 10g/L, 12g/L and 14g/L into purified culture medium without changing other components, inoculating seed solution with 2% inoculum size, culturing at 28deg.C and 180rpm for 48 hr, repeating three steps, adding 100mL/250mL conical flask, and turbidimetry to determine OD 600 Bacterial density of culture solution at nm.
3. Effect of nitrogen source concentration on bacterial density: in the culture medium for determining optimal carbon source concentration, respectively regulating yeast powder concentration to 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L and 3.5g/L to obtain culture media with different nitrogen source concentrations, respectively inoculating seed solution with 2% inoculum size, culturing at 28deg.C and 180rpm for 48 hr, each culture medium is provided with three repeated conical flasks with liquid loading volume of 100mL/250mL, and turbidimetry measuring OD 600 Bacterial density of culture solution at nm.
4. Effect of initial pH on bacterial density: at the position ofIn the culture medium with optimal carbon source and nitrogen source concentration, regulating pH value of the culture medium to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 with HCl and NaOH of 1mol/L respectively, inoculating seed liquid with 2% inoculum size respectively, culturing at 28deg.C and 180rpm for 48 hr, setting three repeats for each culture medium, measuring OD with liquid loading amount of 100mL/250mL conical flask, and measuring OD by turbidimetry 600 Bacterial density of culture solution at nm.
5. Influence of liquid loading on bacterial density: setting liquid loading amounts of 10%, 20%, 30%, 40%, 50% and 60% (250 mL conical flask) respectively under optimal carbon source concentration, nitrogen source concentration and initial pH, inoculating seed liquid respectively with 2% inoculum size, culturing at 28deg.C and 180rpm for 48 hr, setting three repetitions for each culture medium, and measuring OD by turbidimetry 600 Bacterial density of culture solution at nm.
The results of the 6.Ensifer sp.KSU03 culture medium composition and condition single-factor optimization test are shown in fig. 6, in the carbon source type screening test, mannitol and sucrose can well promote Ensifer sp.KSU03 fermentation, and the bacterial density of the culture solution is not obviously different, but the sucrose is used as the carbon source and is only one fifth of that of mannitol in the economic cost, so that the sucrose is determined to be the optimal carbon source; in the carbon source concentration screening test, the bacterial density of the culture solution obtained after adding 10g/L sucrose as the final concentration is the highest; the culture solution obtained by adding 2g/L yeast powder in the nitrogen source concentration screening test has the maximum bacterial density; the bacterial density of the culture solution obtained is the largest when the initial pH value is 7.5; the culture medium obtained had the greatest bacterial density at a liquid loading level of 30% (250 mL Erlenmeyer flask).
7. Response surface method optimizes Ensifer sp.KSU03 fermentation conditions: the optimal carbon source concentration, the nitrogen source concentration, the initial pH and the liquid loading amount determined by a single factor are taken as independent variables, the bacterial density of the culture solution is taken as a response value, and a response surface analysis test of a level of 4 factors and 3 is designed to be shown in a table 1 and a figure 7 in detail so as to further optimize the Ensifer sp.KSU03 culture condition; ensifer sp.KSU03 fermentation conditions 4 factor 3 level response surface assay.
Table 1: response surface analysis test design
| Factors of | -1 | 0 | 1 |
| Carbon source concentration (g.L) -1 )(X 1 ) | 7.5 | 10 | 12.5 |
| Concentration of Nitrogen Source (g.L) -1 )(X 2 ) | 1.5 | 2 | 2.5 |
| Initial pH (X) 3 ) | 7.0 | 7.5 | 8.0 |
| Liquid loading (%) (X) 4 ) | 20 | 30 | 40 |
Carrying out regression analysis on the table data by using Design-Expert statistical software, establishing a regression model, and taking the bacterial density of the culture solution of Ensifer sp.KSU03 as a response value (R1) to obtain a secondary regression equation:
R1＝1.78+0.05*A+0.05*B+0.016667*C+0.05*D+0.075*A*B+0.075*A*C+0.1*A*D+0.05*B*C+0.075*B*D+0.075*C*D-0.09*A 2 -0.115*B 2 -0.165*C 2 -0.14*D 2 。
further experimental verification determines that the optimal culture conditions of Ensifer sp.KSU03 are: 7.5g/L of sucrose, 1.5g/L of yeast powder, 8 initial pH value and 40 percent of liquid loading.
Example 5: application of Ensifer sp.KSU03 sword microbial agent
The present example was based on examples 1 to 3, and the Ensifer sp.KSU03 Sword microbial agent provided by the present invention was used.
Experiment design: the strain Ensifer sp.KSU03 tieback sesbania test design is (1) sesbania seed tieback; (2) inoculating a seedling with a bacterial solution; (3) sesbania seed soaking and seedling inoculation bacterial liquid; (4) none of the four treatments (CK).
(1) Seed soaking by microbial agent: selecting sesbania seeds with full particles and basically consistent sizes, sterilizing for 15 minutes by using 3% NaClO solution, and then washing the sesbania seeds with sterile water; placing the sterilized sesbania seeds in a clean beaker, adding the Ensifer sp.KSU03 microbial agent provided by the invention, wrapping the beaker by using a sterile air-permeable sealing film with a microporous filter membrane when the sesbania seeds are used, standing at 28 ℃ for soaking overnight, and soaking the sesbania seeds in warm water at 37 ℃ or less after sterilizing the sesbania seeds in a control group.
(2) Root irrigation by microbial agent: inoculating Ensifer sp.KSU03 sword microbial agent provided by the invention when sesbania seedlings grow to 4-6 stage, and treating by root irrigation, namely OD 600 5mL of Ensifer sp.KSU03 sword microbial agent with the concentration of 0.8 is added into a 50mL clean centrifuge tube, 15mL of sterile water is added for uniform mixing, and the mixture is poured into the root periphery of sesbania seedling.
(3) Microbial agent seed soaking and root irrigation: the sesbania seeds immersed by the Ensifer sp.KSU03 sword microbial agent of the invention germinate to a 4-6 leaf period, and then the sesbania seeds are inoculated with OD by root irrigation 600 5mL of Ensifer sp.KSU03 Sword microbial agent was used.
(4) No Control (CK) was inoculated: selecting sesbania seeds with full particles, sterilizing for 15 minutes by using 3% NaClO solution, and then washing the sesbania seeds with sterile water; placing the sterilized sesbania seed in a clean beaker, adding warm water with temperature not exceeding 37 ℃ and soaking overnight.
Cultivation of sesbania: taking saline-alkali soil (soil pH8.6, total salt content 1.83 g.kg) -1 The contents of alkaline hydrolysis nitrogen, quick-acting potassium and effective phosphorus are 23.42, 104.15 and 17.25 mg.kg respectively -1 The organic matter content is 15.30 g.kg -1 ) Proper amount of the components are sterilized by high temperature damp heat and then dried at 70 ℃, and the weight ratio is 8:2 mixing the mixture with clean vermiculite in proportion, filling the mixture into a flowerpot, adjusting the water content of soil to 70-80% by using sterile water, and sowing the soil, wherein the size of the flowerpot is 21cm in caliber, 14cm in bottom diameter and 17.5cm in height. The seeding mode is specifically as follows: (1) And (3) 3 sesbania seeds soaked with Ensifer sp.KSU03 sword microbial agent are sown for each pot, and (2) 3 seeds soaked with warm water at 37 ℃ or less are sown for each pot.
Sesbania cultivation and management: culturing the sown sesbania in an illumination incubator, wherein parameters are set to 28 ℃, illumination is 14h,18 ℃ and darkness is 10h. And (3) uniformly and quantitatively supplementing water at regular intervals, and randomly adjusting the positions of the flowerpots to ensure that the flowerpots are uniformly irradiated. And (3) sprouting sesbania until 3-leaf period seedlings are obtained, 1 seedling is reserved in each pot, and 15 pots are reserved in each treatment. (2) And (3) the treatment group completes root filling inoculation of the bacterial liquid according to the requirement in the period 4-6.
And (3) measuring a growth index: harvesting plant after sesbania seedling grows for 70 days, respectively measuring plant height, stem thickness, dry matter weight, chlorophyll content, root nodule number, living root nodule nitrogen fixation capacity and other indexes, airing soil, and measuring quick-acting nitrogen content
The changes of sesbania growth in saline-alkali soil, root nodule formation, sesbania chlorophyll content, quick-acting nitrogen content in soil and total salt content in saline-alkali soil under different treatment conditions are examined, and specific results are shown in figures 8-10.
As can be seen from the data of the figure 8, compared with CK, after being treated by different tie-back modes of the Ensifer sp.KSU03 sword microbial inoculum, the sesbania seed soaking and seedling inoculation bacterial liquid treatment group is remarkably higher than CK in terms of sesbania seed height, stem thickness and overground dry weight, and 29.5%, 24.1% and 56.6% are respectively increased compared with CK; seed soaking of sesbania seeds and inoculation of seedlings with bacterial liquidAfter the treatment, the number of sesbania root nodules is up to 15.07, the CK group does not form root nodules, the sesbania root living root nodules treated by the Ensifer sp.KSU03 sword microbial inoculum of the invention in different tiebaging modes are measured by an acetylene reduction method, the acetylene reduction capability is shown, the nitrogen fixation amount of sesbania single plants is evaluated by calculating the ethylene production amount in a reaction system, and the nitrogen fixation amount of sesbania single plants treated by seed soaking and seedling inoculation bacterial liquid of sesbania seeds is up to 5.56+/-1.98 mmol C 2 H 4 /(strain h); the sesbania leaf treated by sesbania seed soaking and seedling inoculation bacterial liquid has the highest chlorophyll content, which is increased by 33.6% compared with CK; the invention proves that the Ensifer sp.KSU03 sword microbial agent can effectively promote sesbania growth and biomass accumulation, can effectively promote sesbania nodule, and obviously increases the nitrogen fixation amount of a single plant and the chlorophyll content of sesbania leaves.
As can be seen from the data of FIGS. 9-10, the quick-acting nitrogen content (23.42 mg.kg) -1 ) The quick-acting nitrogen content of the soil is increased after sesbania is cultivated for 70 days, and the quick-acting nitrogen content of the soil after the Ensifer sp.KSU03 sword microbial agent is inoculated in different modes is obviously different from CK (P is less than 0.05), and the quick-acting nitrogen content of the soil after sesbania seed soaking and seedling inoculation bacterial liquid treatment is highest and is 52.8% higher than CK; compared with the total salt content of the original soil (1.83 g.kg) -1 ) The total salt content of the soil is obviously reduced after sesbania is cultivated for 70 days, the total salt content of the soil after the Ensifer sp.KSU03 sword microbial agent is inoculated in different modes is obviously different from CK, the total salt content of the soil after sesbania seed soaking and seedling inoculation bacterial liquid treatment is most reduced, and the total salt content of the soil is respectively reduced by 36.05 percent and 20.2 percent compared with original soil and CK; the invention shows that the Ensifer sp.KSU03 sword microbial agent can obviously increase the quick-acting nitrogen content of soil and effectively reduce the salt content of the soil.
From the data, the invention separates a strain of sword bacteria Ensifer sp.KSU03 from sesbania root nodules in sesbania test areas of saline-alkali soil in Xinjiang Kaisha. The strain Ensifer sp.KSU03 is salt and alkali resistant, can dissolve no/organic phosphorus, has the characteristics of secreting plant growth hormone (IAA), synthesizing 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase and producing extracellular polysaccharide. Ensifer sp.KSU03 and a rapier microbial agent prepared by the Ensifer sp.KSU03 are used for soaking sesbania seeds, and sesbania cultivated in saline-alkali soil is subjected to root irrigation treatment, so that the Ensifer sp.KSU03 and the rapier microbial agent prepared by the Ensifer sp.KSU03 can effectively promote sesbania nodule, and the number of nodule reaches 15.07 after the sesbania seeds are soaked and the seedling inoculation bacterial liquid is treated; after being treated by the microbial agent provided by the invention, sesbania plant height, stem thickness and overground dry weight are all obviously improved; the nitrogen fixation amount of sesbania single plant and the chlorophyll content of leaf blades are also obviously improved, and compared with the original soil and a control group, the total salt content of the detected soil is respectively reduced by 36.05 percent and 20.2 percent, so that the Ensifer sp.KSU03 and the prepared sword microbial agent thereof have wide development and application prospects in the aspect of sesbania growth promotion in saline-alkali soil, the Ensifer sp.KSU03 sword microbial agent not only can reduce the fertilizer consumption, but also can promote sesbania growth, and have wide development and application prospects in the aspect of agricultural biofertilizer.
In summary, the foregoing examples are merely illustrative of preferred embodiments of the present test, and are not intended to limit the scope of the present test, and various modifications and improvements made by those skilled in the art to the technical solution of the present test should fall within the protection scope determined by the present test without departing from the spirit of the design of the present test.
Claims (8)
1. The strain Ensifer sp.KSU03 for promoting the growth and nodulation of sesbania in saline-alkali soil is characterized in that the strain Ensifer sp.KSU03 is preserved in China Center for Type Culture Collection (CCTCC) No: m2023823.
2. The rapier sp.ksu03 for promoting the growth and nodulation of sesbania in saline-alkali soil according to claim 1, wherein the gene sequence of the strain Ensifer sp.KSu03 is shown in SEQ ID NO: 1.
3. The rapier sp.ksu03 for promoting the growth and nodulation of sesbania in saline-alkali soil according to claim 1, wherein the isolation medium of the strain insefer sp.ksu03 is: mannitol (mannitol)10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O0.004 g/L, naCl 0.1g/L, congo red solution 0.25% 10mL/L, agar 20g/L.
4. The rapier sp.ksu03 for promoting the growth and nodulation of sesbania in saline-alkali soil according to claim 1, wherein the purification medium of the strain insefer sp.ksu03 is: mannitol 10.0g/L, yeast powder 1g/L, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O 0.004g/L，NaCl 0.1g/L。
5. An Ensifer sp.KSU03 sword microbial agent for promoting the growth and nodulation of sesbania in saline-alkali soil according to claim 1, wherein the microbial agent is obtained by the following steps: inoculating single colonies of Ensifer sp.KSU03 into YMA culture medium, and culturing at 28deg.C and 180rpm for 48 hr to obtain new bacteria Ensifer sp.KSU03 seed solution; inoculating Ensifer sp.KSU03 seed solution into special liquid culture medium according to the inoculum size of 2% by volume, fermenting and culturing at 28deg.C and 180rpm for 48 hr, and regulating OD 600 0.8, ensifer sp.KSU03 Sword microbial agent was obtained.
6. The Ensifer sp.KSU03 sword microbial agent according to claim 5, wherein in the preparation of the microbial agent, the special liquid culture medium is: 7.5g/L sucrose, 1.5g/L yeast powder, mgSO 4 ·7H 2 O 0.2g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，FeCl 3 ·3H 2 O0.004 g/L, naCl 0.1g/L, initial pH value 8, and liquid loading amount 40%.
7. The use of an Ensifer sp.KSU03 sword microbial inoculant according to claim 5 for sesbania growth promotion and nodulation in saline-alkali soil.
8. Use of the sword bacteria Ensifer sp.KSU03 for promoting the growth and nodulation of sesbania in saline-alkali soil according to claim 1.
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