CN115895954A - Pseudomonas laurophilus sulfate CNBG-PGPR-8 and application thereof - Google Patents
Pseudomonas laurophilus sulfate CNBG-PGPR-8 and application thereof Download PDFInfo
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- 241000589516 Pseudomonas Species 0.000 title claims abstract description 92
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 title claims abstract description 32
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- 241000196324 Embryophyta Species 0.000 claims description 25
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Abstract
The invention discloses a Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8, which has the preservation date of 2021 year, 1 month and 28 days and is preserved in China center for microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.21753; the growth is fast, the stability is good, the culture can be amplified in a large-volume device, and the self salt tolerance is strong. The invention also discloses a microbial agent or a preparation. The invention also discloses application of the bacterium or microbial agent or preparation in promoting plant growth. The invention also discloses application of the bacterium or microbial agent or preparation in improving salt tolerance of plants. The invention also discloses a method for obtaining the plant with high salt tolerance.
Description
Technical Field
The invention relates to a strain and application thereof, in particular to a Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfativorans) CNBG-PGPR-8 strain and application thereof, belonging to the field of microorganisms.
Background
The importance of saline-alkali soil treatment and agricultural utilization in the aspects of ensuring basic self-sufficiency of grains, ensuring absolute safety of grains, keeping the stability of the existing cultivated land, strictly preventing the red line of the dead-guard cultivated land and the like is further highlighted, and the problem of soil salinization becomes important content under the global change research framework.
Soil bioremediation is a technology for restoring soil microenvironment by using life metabolic activities of microorganisms, and has the advantages of low cost, remarkable effect and small influence on the environment. Among them, the microbial fertilizer has excellent soil improvement, plant growth promotion and environment affinity for green and safety, so that the application of the microbial fertilizer becomes an effective means for solving the problems of saline-alkali soil. The microbial resources are huge, but the strain resources for producing microbial fertilizers are still limited.
Pseudomonas is the largest genus of gamma proteobacteria. The pseudomonas is well known for its flexible degradation ability in soil, activated sludge, plant surfaces and habitats such as fresh water or marine environments, and the related reports show that the pseudomonas participates in biotransformation and bioremediation widely. However, pseudomonas has fewer functional studies on salt tolerance and growth promotion, and has few related functions capable of improving the salt tolerance of plants.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a strain of Pseudomonas lauriformis (Pseudomonas lauriformis) CNBG-PGPR-8, wherein the preservation date is 2021 year, 1 month and 28 days, the strain is preserved in China center for culture Collection of microorganisms (CGMCC), and the preservation number is CGMCC No.21753.
The invention also provides a microbial agent or a preparation, wherein the microbial agent or the preparation contains the Pseudomonas laurifolii sulfate (CNBG-PGPR-8).
The invention also aims to solve the technical problem of providing the application of the Pseudomonas lauriformis (Pseudomonas lauriformis) CNBG-PGPR-8 or the microbial agent or preparation in promoting the growth of plants.
The invention also aims to solve the technical problem of providing the application of the Pseudomonas lauriformis Laurelsulfovorans CNBG-PGPR-8 or the microbial agent or preparation in improving the salt tolerance of plants.
The technical problem to be solved by the invention is to provide a method for obtaining plants with high salt tolerance, which comprises the following steps: administering said Pseudomonas laurifolii sulfate (Pseudomonas laurifolii) CNBG-PGPR-8 or said microbial agent or formulation to a plant.
The technical scheme is as follows: in order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a Pseudomonas lauriformis (Pseudomonas lauriformis) CNBG-PGPR-8, which has the preservation date of 2021 year, 1 month and 28 days and is preserved in China center for culture Collection of microorganisms (CGMCC) with the preservation number of CGMCC No.21753.
The invention also provides a microbial agent or a preparation, which contains the Pseudomonas laurifolii sulfate (Pseudomonas laurifolii) CNBG-PGPR-8.
The invention also provides application of the Pseudomonas laurifolii sulfate (Pseudomonas laurifolii) CNBG-PGPR-8 or the microbial agent or preparation in promoting plant growth.
The invention also provides application of the Pseudomonas lauriformis (Pseudomonas lauriformis) CNBG-PGPR-8 or the microbial agent or the preparation in improving the salt tolerance of plants.
Wherein the salt is NaCl solution with the mass concentration of 0.5-5%.
Preferably, the salt is NaCl solution, and the effect of improving the salt tolerance of the plants is best when the mass concentration is 3%.
The invention also provides a method for obtaining the plant with high salt tolerance, which comprises the following steps: applying said Pseudomonas laurifolii sulfate (CNBG-PGPR-8) or said microbial agent or formulation to a plant.
Wherein, the steps also comprise applying a salt solution to the plants, wherein the salt solution is a NaCl solution, and the mass concentration is 0.5-5%.
Wherein the concentration of the Pseudomonas lauriformis Laurelsufativorans CNBG-PGPR-8 is 10 7 ～10 9 CFU/ml。
Wherein, the plant includes but is not limited to tomato, and other crops are also suitable.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
1. the Pseudomonas lauriformis (Pseudomonas lauriformis) CNBG-PGPR-8 CGMCC No.21753 has fast growth and good stability, and can be amplified and cultured in a large-volume device;
2. in the aspect of salt tolerance, pseudomonas lauriformis (Pseudomonas laurylsulfaltorans) CNBG-PGPR-8 can normally grow in a liquid culture medium containing 3% of sodium chloride at most, can normally grow in a solid culture medium containing 3% of sodium chloride, and has strong salt tolerance;
3. when the Pseudomonas laurifolii sulfate (Pseudomonas laurifolii sulfate) CNBG-PGPR-8 is applied to tomatoes under the condition of salt stress, the stress of salt on tomato seedlings can be remarkably reduced, the plant height, fresh weight and the like of the tomato seedlings are improved, and the Pseudomonas laurifolii sulfate is used for relieving the stress of salt on the tomatoes, improving various indexes of the tomato plants and promoting the growth of the tomato plants.
Drawings
FIG. 1 shows the colony morphology of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 on a plate;
FIG. 2 shows the morphology of Pseudomonas lauriformis (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 cells under a microscope at 100 times magnification;
FIG. 3 is a phylogenetic tree of Pseudomonas laurifolii (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8;
FIG. 4 shows the growth of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 in liquid medium containing different concentrations of salts;
FIG. 5 shows the growth of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 in solid media containing different salt concentrations;
FIG. 6 shows the effect of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 on tomato growth and development under salt stress conditions;
FIG. 7 is a graph showing the effect of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 on fresh weight of tomato seedlings under salt stress conditions;
FIG. 8 shows the effect of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 on the plant height of tomato seedlings under salt stress conditions.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1 isolation and identification of Pseudomonas laurifolii sulfate (CNBG-PGPR-8)
(1) Sample dilution and coating
Orchard taken from Xuzhou Feng county of Jiangsu province(N34 ℃ 20 '41', E117 ℃ 4 '23') surrounding soil, placing about 0.5g of orchard planting soil sample in 4.5mL of sterile physiological saline for suspension oscillation, namely 10 -1 Gradient (ten-fold dilution), taking 0.5mL from the dilution and suspending and shaking in 4.5mL sterile physiological saline to obtain 10 -2 Gradient (hundred fold dilution), and so on until dilution to 10 -6 And (4) gradient. mu.L of each of the 6 dilutions was pipetted into 6 dilutions, spread on Agar Nutrient Agar (NA) solid medium plates, prepared 2 portions in parallel, inverted, and incubated in 30 ℃ incubator for 24-36h with regular observation.
(2) Strain isolation and purification
Taking out all plates with colonies growing under the same sample, selecting a dilution plate with obvious single colonies and small quantity, picking colonies with different forms on the plate to a new NA plate, carrying out partition purification, repeating purification until the single colonies appear, and then repeating streak culture for at least 5 times.
(3) Dyeing and preservation
Selecting a single bacterial colony of a strain to be detected in the central area of a glass slide, carrying out primary dyeing by using an ammonium oxalate-crystal violet dye solution, mordanting by using an iodine solution, decoloring by using 75% ethanol and carrying out redyeing by using safranin, carrying out microscopic observation, and recording a dyeing result; and (3) picking the single colony which is purified and dyed from the NA solid culture medium plate to a sterile NA liquid culture medium, and culturing at 30 ℃ for 24 +/-2 h at 200 r/m. Shaking up with shaking, mixing 60% sterile glycerol: bacterial liquid is prepared according to the following steps of V: v = 1: 1, subpackaged in sterile tubes, labeled and stored in a-80 ℃ refrigerator.
(4) Identification of strains
Taking out the plate containing the strain with the corresponding number from a refrigerator at 4 ℃, selecting a single colony as a DNA template, and carrying out PCR amplification and 16S rDNA sequencing and sequence comparison, wherein the specific operations are as follows:
(1) amplification system 50 μ L:
the 50 mu L system consists of the following five parts: taq enzyme Mix (25. Mu.L), forward primer 27F (1. Mu.L), reverse primer 1492R (1. Mu.L), DNA template (1. Mu.L), ddH2O (22. Mu.L). Wherein, the sequence of the upstream primer 27F is as follows: 5'-AGA GTT TGA TCM TGG CTC AG-3', and a downstream primer 1492R has the sequence as follows: 5'-GGY TAC CTT GTT ACG ACT T-3'. The length of the amplified fragment is 1401bp, and the amplified nucleotide sequence is shown as SEQ ID NO. 1.
(2) Conditions for amplification
Pre-denaturation temperature: 105 ℃ C
First-step denaturation: 7min at 95 ℃; 30s at 95 DEG C
And a second step of annealing: 30s at 55 DEG C
And a third step of extension: 90s at 72 DEG C
Cycle number: 30s at 95 deg.C, and 33 cycles
The fourth step is finally extended: 5min at 72 DEG C
The fifth step is that: 10min at 12 DEG C
(3) Agarose gel electrophoresis
Weighing 1g of agarose, dissolving in 100mL of TBE electrophoresis buffer solution, heating by microwave until the solution is clear and transparent, adding 1 per thousand of GelRed nucleic acid dye, shaking uniformly, standing until no bubbles exist, slowly pouring into a gel plate, standing for about 1h, taking out a solidified gel block, placing the gel block in an electrophoresis tank, adding 4 muL of PCR amplification product into each gel hole, running gel for 120V 20min, taking out the gel block, placing the gel block in a gel imager, selecting UV illumination and taking a picture, recording the corresponding sample number with clear strips at 1500bp positions on the gel block, and placing the residual PCR amplification product of the sample in a refrigerator at 4 ℃.
(4) DNA purification
The PCR amplification product stored in the refrigerator at 4 ℃ was taken out, and the PRC product was purified using AxyPrepTM PCR clean Kit nucleic acid purification Kit (cat. AP-PCR-4G) from AxyGen to obtain a resuspended DNA.
(5) Sequence identification
Sending the heavy suspension DNA obtained in the step to Nanjing Ongjingkscience and technology Limited company for sequencing, inputting the sequence returned by the mail into a sequence search column of an NCBI website for searching, and comparing results to show that the strain provided by the invention is Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfutorans).
The Pseudomonas laurifolii sulfate (Pseudomonas lauriformis) CNBG-PGPR-8 strain is preserved in China general microbiological culture Collection center (CGMCC) at 28 days 1 month 2021, with the preservation address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, the accession number of the strain is: CGMCC No.21753, classified and named as Pseudomonas laurylsulfaltovans.
The colony morphology of Pseudomonas laurifolii (Pseudomonas laurifolii) CNBG-PGPR-8 (preservation number: CGMCC No. 21753) is shown in figures 1 and 2, and the colony on the surface of the NA plate culture medium is thick, round, convex lens-shaped, light yellow or beige, smooth and opaque, complete in edge, uniform in texture and milky. The thallus of the Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathionans) CNBG-PGPR-8 observed under a 100-fold microscope is in a ball-rod shape, has the length of 0.5-1.0 μm and the width of 1.0-2.0 μm, and is arranged in a free state or a diad state. The gram staining result is red, namely the gram-negative bacteria.
By comparing the 16S sequence of the microbial inoculum of the invention, a phylogenetic tree is prepared for analysis (FIG. 3), and the genetic relationship with Pseudomonas morganii (Pseudomonas mohnii) is closer.
Example 2 determination of the salt tolerance of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8
(1) Determination of salt tolerance in liquid culture media
Preparing a salt-tolerant liquid culture medium: peptone 1% (mass fraction); 1 percent of beef extract (mass fraction); sodium chloride is 0.5 percent, 2 percent, 3 percent, 4 percent and 5 percent (mass fraction) respectively, and the balance is water; sterilizing at 120 deg.C for 30min, and packaging in 50ml centrifuge tube.
Selecting a single colony of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 to be activated in an NA liquid culture medium, activating for 2 generations according to the inoculum size of 5 percent, inoculating into a subpackaged salt-tolerant liquid culture medium according to the inoculum size of 5 percent, culturing at 30 ℃ under 180rpm, sampling every 12 hours, and determining the growth condition of the strain by measuring the OD value (light absorption value) at the 600nm wavelength of a bacterial liquid. As shown in FIG. 4, the strain of Pseudomonas laurifolii (Pseudomonas laurylsulfathionans) CNBG-PGPR-8 can grow normally in a liquid medium containing 0.5%, 2% and 3% sodium chloride, and when the salinity reaches 4%, the growth rate of Pseudomonas laurifolii (Pseudomonas laurylsulfathionans) CNBG-PGPR-8 decreases, but can still reproduce normally; when the salinity reaches 5 percent, the CNBG-PGPR-8 of the Pseudomonas lauriformis (Pseudomonas laurylsulfathivorans) does not grow basically.
(2) Determination of salt tolerance in solid media
Preparing a salt-tolerant solid culture medium: peptone 1% (mass fraction); 1 percent of beef extract (mass fraction); 2% of agar; 0.5 percent of sodium chloride, 2 percent of sodium chloride, 3 percent of sodium chloride, 4 percent of sodium chloride, 5 percent of sodium chloride (mass fraction), and the balance of water; sterilizing at 120 deg.C for 30min, cooling the sterilized culture medium to about 75 deg.C, and storing in refrigerator at 4 deg.C for a long time.
Selecting a single colony of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 to be activated in an NA liquid culture medium, dipping the bacterial liquid by using an inoculation rod, coating the bacterial liquid on a salt-tolerant solid culture medium, culturing for 48 hours at the temperature of 30 ℃, and photographing to observe whether the bacterial strain can normally grow or not after the culture medium is finished. As shown in 5, the Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 can normally grow at the salinity of 0.5%, 2% and 3%; at 4% salinity, the bacteria are stressed, the growth rate is reduced, but the normal physiological function and the reproductive function of the bacteria are not influenced, the cell viability is not irreversibly inhibited, and the bacteria can still normally reproduce, although at 4% salt concentration, the growth rate is reduced, and the activity of the bacteria can be restored by being inoculated into a normal culture medium again. Example 3 Effect of Pseudomonas laurifolii sulfate (Pseudomonas laurylsulfathivorans) CNBG-PGPR-8 on tomato growth and development under salt stress conditions
Salt stress growth promotion experiments are carried out in laboratories of the plant research institute of Chinese academy of sciences of Jiangsu province. 4 treatments were set for the experiment, treatment 1 being a clear water Control (CK); treatment 2 (T1) was 200mM NaCl salt stress treatment (concentration by mass about 1%); the treatment 3 (T2) is clear water plus Pseudomonas laurifolii sulfate (CNBG-PGPR-8 fermentation liquid)(ii) a Treatment 4 (T3) was 200mM NaCl salt stress (concentration about 1% by mass) + Pseudomonas laurylsulfate (CNBG-PGPR-8 broth). The experimental object is tomato seedlings, the tomatoes are firstly sowed in seedling-raising trays, the tomato seedlings with the same growth vigor are selected and transplanted into the plug tray after 2 weeks, 9 plug trays are used for carrying out the experiment for one treatment, the bacteria are poured in one week after the transplantation, 20ml of 10-concentration water is poured in each pot in the T2 and T3 treatment 7 Pouring 20mL of NA culture medium into each pot for the treatment of CK and T1 by CFU/mL bacterial liquid, performing salt treatment after the bacterial pouring, pouring 100mL of 200mM NaCl solution into each pot for the treatment of T1 and T3, pouring 100mL of clear water into each pot for the treatment of CK and T2, and treating once a week. After about one month harvesting was performed, and the post-harvest test indices included the plant height and fresh weight difference of tomatoes (fig. 6).
As can be seen from FIGS. 7 and 8, under the condition of non-salt stress, the fresh weight and the plant height of the tomato seedlings are obviously improved by 32.5 percent and the plant height is improved by 8 percent when the fungicide of the invention is applied compared with CK; after the tomato seedlings are treated by 200mM NaCl and have obvious salt stress, the plant height and fresh weight of the tomato seedlings are obviously reduced; after the bacterial liquid is applied, the salt stress effect is obviously improved, compared with T1, the fresh weight is increased by 40.6%, and the plant height is increased by 32.6%, which shows that the bacterial liquid has an obvious effect of increasing the salt stress of tomato seedlings. The concentration of CNBG-PGPR-8 (10) in the process of increasing the concentration of Pseudomonas lauriformis Laurelsulfovorans (Pseudomonas lauriformis) is increased 8 CFU/ml、 10 9 CFU/ml), with a concentration of 10 7 The plant height and fresh weight of the tomato are changed equivalently and slightly.
Claims (9)
1. The Pseudomonas lauriformis Laurelsulfovorans CNBG-PGPR-8 is characterized in that the Pseudomonas lauriformis Laurelsulfovorans CNBG-PGPR-8 has the preservation date of 2021 year, 1 month and 28 days and is preserved in China center for culture Collection of microorganisms (CGMCC) with the preservation number of CGMCC No.21753.
2. A microbial agent or formulation comprising Pseudomonas laurylsulfate (CNBG-PGPR-8) according to claim 1.
3. Use of the Pseudomonas laurifolii sulfate (Pseudomonas lauriformis) CNBG-PGPR-8 according to claim 1 or the microbial agent or preparation according to claim 2 for promoting plant growth.
4. Use of the Pseudomonas laurifolii sulfate (Pseudomonas laurifolii sulfate) CNBG-PGPR-8 according to claim 1 or the microbial agent or preparation according to claim 2 for improving salt tolerance of plants.
5. The use according to claim 4, wherein the salt is NaCl solution with a mass concentration of 0.5-5%.
6. A method for obtaining plants with high salt tolerance, comprising the steps of: applying to a plant the microorganism bacterium agent or preparation described in claim 2 or Pseudomonas laurifolii sulfate (CNBG-PGPR-8) described in claim 1.
7. The method of claim 7, wherein the steps further comprise applying a salt solution to the plants, wherein the salt solution is a NaCl solution and has a concentration of 0.5-5% by mass.
8. The method as claimed in claim 7, wherein the Pseudomonas laurylsulfate (Pseudomonas laurylsulfativorans) CNBG-PGPR-8 is administered at a concentration of 10 7 ～10 9 CFU/ml。
9. Use according to claim 3 or 4, method according to any one of claims 6 to 8, wherein the plant comprises tomato.
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