CN119242537B - A strain of Pseudomonas capable of enhancing peanut immunity and promoting growth and its application - Google Patents
A strain of Pseudomonas capable of enhancing peanut immunity and promoting growth and its application Download PDFInfo
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- CN119242537B CN119242537B CN202411762983.2A CN202411762983A CN119242537B CN 119242537 B CN119242537 B CN 119242537B CN 202411762983 A CN202411762983 A CN 202411762983A CN 119242537 B CN119242537 B CN 119242537B
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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Abstract
本发明涉及微生物技术领域，特别涉及一株具有增强花生免疫力及促生作用的假单胞菌及其应用。本发明提供了保藏编号为CCTCC NO:M 2024598的假单胞菌（Pseudomonas sp.），能有效拮抗病原菌、显著增强植物免疫力并发挥显著的促生作用。
The present invention relates to the field of microbial technology, and in particular to a strain of Pseudomonas sp. having the effects of enhancing peanut immunity and promoting growth and its application. The present invention provides a strain of Pseudomonas sp. with a deposit number of CCTCC NO: M 2024598, which can effectively antagonize pathogens, significantly enhance plant immunity and play a significant role in promoting growth.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to pseudomonas with the functions of enhancing plant immunity and promoting growth and application thereof.
Background
Along with the large-scale and intensive development of peanut (Arachis hypogaea) production, the peanuts are susceptible to being infected by various diseases such as southern blight, root rot, fruit rot and the like in the planting process, wherein the peanut southern blight caused by sclerotium rolfsii (Sclerotium rolfsii sacc.) severely restricts the yield and quality of the peanuts, and is one of the most serious peanut soil-borne fungal diseases. In addition, due to the large amount of chemical pesticides, on one hand, the ecological balance and biodiversity of soil microorganism groups are destroyed, and on the other hand, the food safety problems such as pesticide residues are caused, so that the search for effective alternative products is urgent.
Biological control measures are one of the effective strategies for replacing traditional fertilizers and pesticides. Researchers visually culture plant rhizosphere microorganisms, screen out strains which promote plant growth and prevent plant diseases, and re-connect the strains back to plant roots after large-scale fermentation culture, so that the strains promote plant growth and prevent plant diseases by secreting biomass-promoting substances, antibacterial substances, inducing plant immunity and the like.
Pseudomonas is widely present in soil, water, plants and human and animal intestinal tracts, occupying a large number of niches. In recent years, studies have shown that the isolation and identification of Pseudomonas bacteria with good biocontrol effects from different plants, which are capable of secreting various secondary metabolites to antagonize plant pathogens (such as siderophores, phenazine-1-carboxylic acid, luteolin, etc.), can control various plant fungi and bacterial diseases. Although these secondary metabolites are capable of controlling plant diseases directly or indirectly, plants are also primarily dependent on the autoimmune system when they are resistant to pathogenic bacterial infestation. Some root microorganisms have been shown to induce systemic acquired resistance (Systemic acquired resistance, SAR) to protect plants from attack in addition to direct antagonism of the pathogenic bacteria by secreted metabolites. Is mainly characterized by defensive reactions such as Reactive Oxygen Species (ROS) burst, callose (Callose deposition) and Lignin (Lignin) deposition.
Various biocontrol products exist, but in practical use, the effect is greatly compromised, and although the application method and environment are related, the effect is more caused by the diversity and complexity of soil pathogen genes. If part of the genotype of the pathogenic bacteria may be tolerant to antagonistic bacteria, the strain in the biological agent is hard to resist the attack of the pathogenic bacteria through antagonistic function. Therefore, the separation and screening of the multifunctional bacterial strain resources with the functions of antagonizing pathogenic bacteria, enhancing plant immunity and promoting growth from the specific host body has important significance.
Disclosure of Invention
In view of the above, the invention provides a pseudomonas strain with plant immunity enhancing and growth promoting effects and application thereof, and the pseudomonas strain has multiple functions, can effectively antagonize pathogenic bacteria, remarkably enhance plant immunity and exert remarkable growth promoting effects, and well meets the various challenges.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Pseudomonas (Pseudomonas sp.) with a preservation number of CCTCC NO 2024598.
The invention also provides a fermentation product of the above Pseudomonas sp.
The invention also provides the use of the above Pseudomonas sp or the above fermentation product in at least one of the following:
(A1) A siderophore producing carrier;
(A2) Decomposing organic phosphorus;
(A3) Decomposing inorganic phosphorus;
(A4) Producing amylase;
(A5) A catalase;
(A6) Producing a bacterial biofilm;
(A7) Inhibiting or antagonizing pathogenic bacteria.
In some embodiments of the invention, the pathogenic bacteria used above include sclerotium rolfsii (Sclerotium rolfsii sacc.).
The invention also provides the use of the above Pseudomonas sp or the above fermentation product in at least one of the following:
(B1) Promoting plant leaves to generate ROS;
(B2) Promoting the deposition of callose in plant root system;
(B3) Increasing plant growth rate and aboveground biomass.
In some embodiments of the invention, the plants used above include peanut;
the peanuts include colorful peanuts.
The invention also provides the use of the above Pseudomonas sp or the above fermentation product in at least one of the following:
(C1) Preventing, reducing or eliminating plant diseases;
(C2) Promoting plant growth and development;
(C3) Enhancing plant immunity or disease resistance;
(C4) And preparing a product for planting.
In some embodiments of the invention, the plants used above include peanuts;
the peanuts include colorful peanuts.
In some embodiments of the invention, the disease applied above includes southern blight.
In some embodiments of the invention, the aforementioned application of the southern blight includes peanut southern blight.
In some embodiments of the invention, the aforementioned application of the peanut southern blight is peanut southern blight caused by sclerotium rolfsii (Sclerotium rolfsii sacc.).
The invention also provides a formulation comprising:
(D1) Pseudomonas as described above (Pseudomonas sp.); or (b)
(D2) And the fermentation product.
In some embodiments of the invention, the formulation further comprises MgCl 2.
In some embodiments of the invention, the preparation method of the above formulation comprises the step of resuspending the above pseudomonas (pseudomonasp.) with a MgCl 2 solution.
The invention also provides a product for planting comprising:
(E1) Pseudomonas as described above (Pseudomonas sp.); or (b)
(E2) Or the fermentation product
(E3) And the preparation.
In some embodiments of the invention, the product comprises a microbial fertilizer.
The invention also provides a culture method of the Pseudomonas sp.
In some embodiments of the invention, the above-described culture method comprises the step of inoculating the above-described Pseudomonas sp.
The invention also provides a planting method of crops, which is based on any one of the following planting methods:
(F1) Pseudomonas as described above (Pseudomonas sp.); or (b)
(F2) Or the fermentation product
(F3) And the preparation.
In some embodiments of the invention, the crop plants include peanuts;
the peanuts include colorful peanuts.
The strain and the application of the invention have the following effects:
On the one hand, the plant is selectively enabled to colonize the strain in the tissue under the influence of plant species, variety and regional environment, most strains can only colonize the soil around the rhizosphere, in the process of re-inoculation, the strain is easy to be affected by the environment and has low survival rate, the strain separated from the host tissue has high specificity, and the strain has the advantage in the process of re-inoculation and colonization, can enter the plant tissue more easily, establishes symbiotic relation with the plant and plays a role. The strain is separated from the colorful peanut root system tissue, and when the strain is re-inoculated back to the root of a host in the form of bacterial liquid or biological bacterial agent, the strain can more easily colonize in plant tissues to establish symbiotic relation with plants.
On the other hand, because of the diversity and complexity of plant pathogenic bacteria genes, single-function strains are difficult to counter, so that strain resources with multiple functions (such as antagonism of pathogenic bacteria, enhancement of plant immunity and growth promotion) are excavated, and the influence on the aspects of pathogenic bacteria diversity and the like can be better dealt with. The strain has multiple functions, can effectively antagonize pathogenic bacteria, remarkably enhance plant immunity and exert remarkable growth promoting effect, thereby well coping with challenges brought by various diversity. Specifically:
(1) The strain is an endophytic bacterium separated from a colorful peanut root system of a special homeland variety in Yunnan, and can better establish a symbiotic relationship with plants so as to reduce rejection reaction.
(2) The root system of the colorful peanut plant is soaked with 6 h in the bacterial liquid OD 600 0.2.2, and the colorful peanut plant has better effective control rate on the colorful peanut southern blight (sclerotium rolfsii).
(3) After 7 days of inoculation bacterial liquid (OD 600 0.5.5) at the root of the colorful peanut plant, under the stimulation of 500 mug/mL chitin, the living tissue of the plant leaf can generate a large amount of ROS, and a large amount of callose is deposited in the root system, so that the immunity of the plant is effectively enhanced.
(4) Root 6 h of the root system of the colorful peanut plant is immersed in the bacterial liquid OD 600 0.2.2, and the growth and development of the colorful peanut are promoted to a certain extent under the condition of no pathogenic bacterial stress.
(5) Pseudomonas B1207 has the functions of producing siderophores, organic phosphorus, inorganic phosphorus, amylase, H 2O2 enzyme, and biological membrane, and has important roles in colonisation, disease resistance and growth promotion.
(6) The strain provided by the application has multiple functions of disease resistance, growth promotion, plant immunity enhancement and the like, and provides strain resources for development of living vaccines of plants.
Description of biological preservation
The culture name is Pseudomonas B1207 (Pseudomonas sp.B1207), which is preserved in China center for type culture Collection, with the preservation address of China university of Wuhan and Wuhan, and the preservation number of CCTCC NO: M2024598 at the year 2024, and the month 04 and 01.
The B1207 is a strain with a preservation number of CCTCC NO: M2024598.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a morphology map of Pseudomonas sp.B 1207;
FIG. 2 shows the homologous sequence alignment;
FIG. 3 shows a sclerotium rolfsii pathogenicity test;
FIG. 4 shows antagonistic strain selection results;
FIG. 5 shows the morphological effects of antagonistic strains on control Bai Juan of disease plants;
FIG. 6 shows the morphological effects of antagonistic strains to control Bai Juan root stems;
FIG. 7 shows the results of the antagonistic strain ROS burst test;
FIG. 8 shows the deposition of callose from the B1207 strain, wherein a shows a 50-fold magnification of the CK root system, B shows a 100-fold magnification of the CK root system, c shows a 200-fold magnification of the CK root system, d shows a 50-fold magnification of the B1207 root system, d shows a 250-fold magnification of the B1207 root system, e shows a 100-fold magnification of the B1207 root system, f shows a 200-fold magnification of the B1207 root system, and f shows a 50-fold magnification of the B1207 root system;
FIG. 9 shows a B1207 16s rDNA extraction amplification gel assay;
FIG. 10 shows B1207 results of the efficacy-enhancing effect test;
FIG. 11 shows the results of a siderophore production capability test;
FIG. 12 shows the results of an organic phosphorus dissolution test;
FIG. 13 shows the results of an inorganic phosphorus dissolving ability test;
FIG. 14 shows amylase producing ability test results;
FIG. 15 shows the results of an H 2O2 enzyme production capability test;
fig. 16 shows the results of the biofilm production test.
Detailed Description
The invention discloses pseudomonas with the functions of enhancing plant immunity and promoting growth and application thereof, and the technical parameters can be properly improved by a person skilled in the art by referring to the content of the pseudomonas. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention screens out a pseudomonas with the functions of preventing and treating the sepia-sinensis southern blight and promoting the growth, and the identification results are as follows:
1. Morphological identification:
The strain is cultured for 24 hours at 30 ℃ on TSA solid culture medium [ formula: 17 g/L casein (pancreatic hydrolysate), 3 g/L soybean peptone, 5 g/L NaCl, 2.5 g/L K 2HPO4, 2.5 g/L glucose (Dextrose), 20 g/L agar powder, pH 7.2 with NaOH, and then constant volume to 1L, sterilized at 121 ℃ and 15 min ], the colony diameter is 2.0-3.0 mm, the colony shape is nearly circular, micro-bulge, the color is gray, opaque, and the obligate aerobic, as shown in figure 1. The strain is cultured in TSB liquid culture medium [ formula: 17 g/L casein (pancreas zymolyte), 3 g/L soybean peptone, 5 g/L NaCl, 2.5 g/L K 2HPO4, 2.5 g/L glucose (Dextrose), 20. 20 g/L agar powder, pH 7.2 is regulated by NaOH, volume is fixed to 1L, and after a plurality of days of sterilization at 121 ℃ 15 min ], the color of the culture medium is gradually changed into dark green, a large amount of biological film is generated, and the bacterial liquid is viscous. The cells are observed to be in a rod shape under a microscope, the cell size is (1.5-3.0) mu m x (0.5-0.8) mu m, and the cells are arranged singly or in pairs, have 1-3 flagella, are active in movement and have no spores, can form capsules, are easy to dye by common dyes and are gram-negative.
2. Molecular characterization
Based on the 16S rDNA sequence, a phylogenetic tree is constructed by adopting MEGA 7.0 software, and is shown in figure 2, so that the strain has the closest relationship with Pseudomonas aeruginosa.
Morphological characteristics and homologous sequence analysis showed that the above strain belongs to the bacterial kingdom, pseudomonas sp. Aeruginosa (Pseudomonas aeruginosa). The strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university, with addresses of eight paths 299 of Wuchang district of Wuhan, hubei province, postal code 430072, preservation date 2024, 04, 01, and preservation number of CCTCC NO M2024598, and named as Pseudomonas sp B1207.
The invention provides Pseudomonas sp (Pseudomonas sp.) B1207 with functions of preventing and treating seven-color peanut southern blight and promoting growth and application thereof.
The pseudomonas B1207 provided by the invention has stronger colonization capability to colorful peanuts, can effectively prevent and treat southern blight caused by sclerotium rolfsii, and has the effect of promoting plant growth and development.
The invention comprises a method for separating and obtaining pseudomonas B1207, which is a strain separated from a colorful peanut root system collected by Mengyan county in Yunnan and is obtained by adopting a flat dilution method.
The invention comprises application of the pseudomonas B1207 in preventing and controlling the sepia esculenta southern blight caused by sclerotium rolfsii and a method thereof.
The invention comprises application of the pseudomonas B1207 in promoting the growth of colorful peanut plants and a method thereof.
Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all commercially available and commercially available.
The invention is further illustrated by the following examples.
Example 1 isolation and disease resistance screening of Pseudomonas B1207
(1) Sample collection, namely collecting a seven-color peanut root system sample in 8 months and 17 days of 2023 in Mengli county of Yunnan, washing the sample with sterile water, putting the sample into a 50 mL centrifuge tube containing 25% of glycerol, and preserving the sample in a dry ice barrel at low temperature for later use.
(2) And separating and purifying, namely washing the sample in an ultra-clean workbench with sterile water, sterilizing the sample with 75% alcohol for 30 s times, rinsing the sample with sterile water for 3 times, sterilizing the sample with 3% sodium hypochlorite for 2 min times, rinsing the sample with sterile water for 3 times, and finally sucking the water by sterilizing filter paper, and then quickly baking the sample back and forth on the flame of an alcohol lamp for 2-3 times. The surface of the sample is sufficiently sterilized. And (3) putting the mixture into a sterilizing mortar, adding a small amount of sterile water for grinding, diluting the slurry into suspensions with different gradients, taking 100 mu L of the suspension, coating the suspension on a TSA solid culture medium, and culturing the suspension in a constant-temperature incubator at 30 ℃ for 24-72 h. Selecting single colonies with different shapes, colors and sizes, streaking on a TSA plate, culturing at 30 ℃ for 24-48 hours, repeating the above operation until purified colonies are obtained, numbering the purified colonies, storing the purified colonies in 25% glycerol after liquid culture, and storing the purified colonies in a-80 ℃ refrigerator for the next step of screening antagonistic strains.
(3) The cultivation of the potted colorful peanut sterile seedlings comprises the steps of sterilizing a potting matrix with a mixture of vermiculite and organic matter matrix at a temperature of 4:1 at a high temperature of 121 ℃ under high pressure for 90 min for later use, selecting colorful peanut seeds with full shapes, soaking and sterilizing the seeds with 75% absolute ethyl alcohol for 30 s, washing the seeds with sterile water for 3 times, soaking and sterilizing the seeds with 2.5% silver hydrogen peroxide ion disinfectant and 0.01% tween-20 for 48 h, soaking and washing the seeds with sterile water for 4 times, sowing the seeds in the sterile matrix, and growing the seeds in a greenhouse with a temperature of 30 ℃ per 25 ℃ and a light quantum flux of about 200 mu mol m -2·s-1 for about 1 week to obtain the sterile peanut seedlings.
(4) Pathogenic bacteria (sclerotium rolfsii) pathogenicity test, namely, the strain is separated from a colorful peanut continuous cropping test field with serious disease Bai Juan in the early stage of a laboratory, is determined to be the sclerotium rolfsii through morphological characteristics and molecular identification, and is stored in a refrigerator at the temperature of-80 ℃ in the laboratory.
Activating the strain of the sclerotium rolfsii in PDA culture medium (formula: 6 g/L potato powder, 20 g/L glucose, 20 g/L agar), adjusting pH to 5.6 with NaOH, fixing volume to 1L, sterilizing at 121deg.C for 15 min), and culturing in dark for more than 45 days to obtain sclerotium rolfsii.
Selecting aseptic peanut seedlings, removing cotyledons, transplanting the peanut seedlings into a circular glass flowerpot of 18X 35 cm, sealing a bottle mouth by using a 0.45 mu m filter membrane, growing the peanut seedlings in a greenhouse with a day and night mode of 16 h/8 h, a temperature of 30 ℃/25 ℃ and a humidity of more than 90% and a luminous flux of about 200 mu mol m -2·s-1 for 3 days, and then inoculating sclerotium (3-4 sclerotium or 3mm fungus cakes) into the greenhouse to observe and count the morbidity of the plants, wherein the morbidity is shown in figure 3.
The pathogenicity test results show that: the root and stem parts of the plants show lesions of different degrees after 4 days of inoculation of sclerotium (or cake) of sclerotium (sclerotium) with the passage of time. The main symptoms of the plants are irregular spots, constriction symptoms, yellowing leaves, wilting and the like of the rhizome parts, and the plant has the same incidence as the field incidence symptoms, has the incidence rate of 100 percent and has stronger infectivity and pathogenicity. According to Ke Hefa, the root and stem parts of the disease in the experiment were placed in a PDA plate to separate the strains, and the obtained strains were identical to the inoculated strains in shape, which proved that the inoculated strains and the disease strain were identical strains (sclerotium rolfsii).
(5) Antagonistic bacteria primary screening, namely evaluating antagonistic activity of each isolated strain on the southern blight pathogenic bacteria, screening out antagonistic strains, and determining radius of the tested strain and inhibition zone formed between the tested strain and the southern blight pathogenic bacteria by an antagonistic test. The smaller the radius of the southern blight pathogen, the stronger the strain tested against the southern blight pathogen. 3 replicates of each strain were independently tested for antagonism, the pathogen strain was inoculated in the middle of the PDA plate, the test strain was inoculated at about 2cm on both sides of the pathogen, and the PDA plate inoculated with sterile water was used as the CK treatment. Antagonistic activity of each tested strain was evaluated when CK just grown over the whole plate. The percentage inhibition efficiency was calculated using the following formula:
Percentage of inhibition efficiency = (radius of southern blight pathogen in CK-radius of southern blight pathogen in test strain)/radius of southern blight pathogen in CK x 100%;
8 strains with obvious antagonistic effect on the aligned micronucleus were obtained in total, and are shown in FIG. 4 and Table 1.
TABLE 1 counter experiment results of different strains and Rhizoctonia cerealis
(6) The antagonistic bacterial strain is re-screened, the strain with inhibition effect on pathogenic bacteria in the primary screening is re-screened, the re-screened strain is firstly inoculated into a TSB liquid culture medium of 2mL according to the proportion of 0.5 percent for resuscitation, then inoculated into a TAB culture medium of 50mL according to the proportion of 2 percent, the strain is cultivated to logarithmic growth phase (OD 600 value of 0.8) under the conditions of 30 ℃ and 120 rpm, the thalli are collected after centrifugation, and after resuspension by using 10 mmol/L sterile MgCl 2 solution, the bacterial solution is diluted to OD 600 value of 0.2, sterile peanut seedlings with uniform morphology size are selected, each treatment is carried out for 20 pieces, root systems are soaked in the re-screened bacterial solution for 12h (the control is soaked in sterile MgCl 2), the root systems are transplanted into a round glass flowerpot of 18 multiplied by 35 cm, the bottle mouth is sealed by a filter membrane of 0.45 mu m, the temperature is 30 ℃ and 25 ℃, the humidity is above 90%, the bacterial cake is controlled to be about 200 mu mol per -2·s-1 m, the bacterial strain is well aligned with the bacterial strain which shows about three-phase growth rate of 6B or the bacterial strain which is well aligned with the bacterial strain of 120B, and the bacterial strain is observed to be the bacterial strain with the bacterial strain, and the bacterial strain is more than about 3 days, and the bacterial strain is more than about 80B, and the bacterial strain is observed to have the bacterial strain is more than about 80B and is more than about 3 to be aligned to be compared with the bacterial strain. The results are shown in fig. 5 and 6.
EXAMPLE 2 Pseudomonas B1207 enhanced plant immunity test
(1) Plant pretreatment
Selecting strains (B0861, B0927 and B1207) with good control rate of aligned sclerotium rolfsii in a re-screening, inoculating the strains into a TSB liquid culture medium of 2 mL according to the proportion of 0.5 percent for resuscitation, inoculating the strains into a TAB culture medium of 50mL according to the proportion of 2 percent, culturing the strains to a logarithmic growth phase (OD 600 value of 0.8) under the conditions of 30 ℃ and 120 rpm, centrifuging the strains, collecting thalli, re-suspending the thalli by using a 10 mmol/L sterile MgCl 2 solution, diluting the thalli to an OD 600 value of 0.5, selecting sterile seedlings with uniform morphology and size, transplanting the sterile seedlings into a circular glass flowerpot of 18X 35 cm, sealing a bottle mouth by using a filter membrane of 0.45 mu m, growing the sterile seedlings in a greenhouse with the diurnal mode of 16 h/8 h, the temperature of 30 ℃/25 ℃ and the humidity of more than 90 percent and the light quantum flux of 200 mu mol.m -2·s-1 for 3 days, and repeating the treatments at the root.
(2) Plant leaf ROS test
After 7 days of inoculation, a leaf with the best growth vigor is selected, living tissue is cut off by a puncher of 5 mm, the leaf is placed in a 96-well plate containing 100 mu L of sterile water in a right-side up mode, the leaf is incubated overnight under 22 ℃ continuous illumination, stress response is reduced, and peanut leaf oxidative stress response is measured.
Sterile water was removed with a pipette and 100. Mu.L of an immunoassay solution containing 200. Mu.M luminol (3-amino-phthalhydrazide) and 100. Mu.g/mL Horseradish Peroxidase (horseradish peroxidase) and 500. Mu.g/mL chitin was added, and the plate was immediately placed in a microplate reader and the luminescence intensity was measured once per minute in 470 nm (chemiluminescent assay mode) for more than 80 minutes until the luminescence intensity fell to the control level. A blank control was set up for each test, including a negative blank without leaf blades and with saline instead of bacterial fluid. The integral data of the ROS kinetic curve is calculated by adopting R software Bolstad package SINTEGRAL command, and the result is shown in fig. 7, so that the living tissue of the blade can generate a large amount of ROS, and the immunity of the plant is effectively enhanced.
(3) Plant root system callose deposition detection
Taking out the plant from the flowerpot after 7 days of inoculation, flushing the root system with sterile water to ensure the integrity of the root system as much as possible, intercepting a main root section which is about 2 cm-5 cm away from the root tip, directly immersing in Carnoy fixing solution (V acetic acid: V ethanol=1:3) for fixing for 24 hours, immersing and washing twice with absolute ethanol for 1 minute each time, transferring into 100% ethanol with the volume of at least 10 times for preservation, taking out the tissue before dyeing, immersing in 50% ethanol for balancing 30min, taking out and draining slightly, immersing in 1 XPBS for balancing 30min, taking out and draining slightly, preparing dyeing working solution before use, immersing the root system tissue in callose dyeing working solution, and dyeing for 1 hour at room temperature and in a dark place. 25 μl of aqueous gelatin-coated slide was added dropwise to the slide, and the stained tissue was transferred to the slide. The images were taken with a fluorescence microscope (Leika DM 6B) (excitation light 370 nm, emission light 590 nm) at 5, 10 and 20 times objective, respectively, with a resolution of 1, 300X 1,030, and exposure times of 700 ms, 350 ms, 150 ms, respectively. As shown in figure 8, the root system has a large amount of callose deposition, so that the immunity of the plant is effectively enhanced.
Example 3 Pseudomonas B1207 identification
B1207 bacterial genome DNA is extracted by using a bacterial fungus genome extraction kit. The 16S rDNA sequence amplification primer is 27F 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1), 1495R 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2). PCR amplification conditions were 96℃for 5min, 96℃for 30 s, 56℃for 30 s, 72℃for 1min, total 30 cycles, 72℃for 10 min, and 4℃for storage, and PCR products were detected by 1% agarose gel electrophoresis and labeled with 100~5000 bp marker, as shown in FIG. 9, and purified and sequenced by Shenzhen Hua megagene Co., ltd. DNA sequences obtained by sequencing the strain were aligned for homology using Basic BLAST on NCBI website, and phylogenetic tree was constructed using N-J method using MEGA 7.0 software, as shown in FIG. 2. Strain B1207 is in the same branch of the evolutionary tree as Pseudomonas sp, identified as Pseudomonas and designated Pseudomonas sp (Pseudomonas sp.) B1207.
Example 4 Pseudomonas B1207 evaluation of the Protoffee
Pseudomonas B1207 preserved strains are inoculated into a TSB liquid culture medium of 2mL according to the proportion of 0.5 percent for resuscitation, then are inoculated into a TAB culture medium of 50 mL according to the proportion of 2 percent, are cultured to a logarithmic growth phase (OD 600 value of 0.8) under the conditions of 30 ℃ and 120 rpm, are collected after centrifugation, are diluted to an OD 600 value of 0.2 by using a bacterial solution of 10 mmol/L sterile MgCl 2, sterile peanut seedlings with uniform morphology size are selected, 10 plants are treated each, root systems are soaked in a rescreening bacterial solution for 12 h (the control is soaked in 10 mmol/L sterile MgCl 2), are transplanted into a circular flowerpot of 10 multiplied by 15 cm, are cultivated in a greenhouse with the light quantum flux of about 200 mu mol.m -2·s-1 at the temperature of 30 ℃ for potting, and the plant growth condition is evaluated after 30 days.
The results in FIG. 10 show that the strain can effectively promote plant growth and development under the condition of no pathogenic bacteria stress, and after 30 d times of inoculation, the average plant height of a control is 7.5 cm, the average plant height of B1207 is 10.57: 10.57 cm, and the growth rate of the strain is 40.9 percent compared with that of the control.
EXAMPLE 5 Pseudomonas B1207 growth promoting function Studies
1. Testing the capacity of the siderophore, namely inoculating an activated pseudomonas B1207 single colony on a universal CAS culture medium (chrome azure 60.5 mg, cetyl trimethyl ammonium bromide 72.9 mg, feCl 3·6H2 O2.645 mg, peptone 4.5 g, glucose 9g, beef extract powder 2.7 g, naCl 4.5 g, agar 20g and distilled water 1000 mL) for detecting the siderophore, culturing 5D at 30 ℃, determining the diameter D (cm) of the transparent ring and the diameter D (cm) of the colony if the transparent ring appears around the colony, calculating the D/D value, and setting 3 repeated parallel experiments when the ratio is larger to show that the activity of the siderophore is larger.
The results in FIG. 11 show a clear transparent circle around the Pseudomonas B1207 strain, with a D/D value of 1.80, indicating that the strain has siderophore-producing ability.
2. And (3) carrying out organophosphorus dissolving capability test, namely picking the single bacterial drop point of the activated pseudomonas B1207, connecting the single bacterial drop point to a Meng Jinna organophosphorus culture medium (formula (NH 4)2SO4, 0.5, g, naCl, 0.3, g, KCl, 0.3, g, glucose, 10 g、FeSO4·7H2O 0.03 g、MnSO40.03 g、CaCO3 5 g、MgSO4·7H2O 0.3 g、, lecithin, 0.2, g, agar, 20, g and distilled water 1000, mL) plate, culturing at 30 ℃ for 5, culturing for 5, and culturing for D, wherein a transparent ring appears around a colony to be positive, measuring the diameter D (cm) of the transparent ring and the diameter D (cm) of the colony, calculating the D/D value, wherein the larger the ratio is to show that the organophosphorus dissolving capability is stronger, and setting 3 repeated parallel experiments.
The results in FIG. 12 show a clear transparent ring around the Pseudomonas B1207 strain, with a D/D value of 2.20, indicating that the strain has an ability to solubilize organic phosphorus.
3. And (3) testing the inorganic phosphorus dissolving capability, namely picking the single bacterial drop point of the activated pseudomonas B1207 to be connected to a Pikovasky's phosphorus dissolving culture medium （NaCl 0.3 g、MgSO4·7H2O 0.3 g、MnSO40.03 g、KCl 0.3 g、(NH4)2SO40.5 g、FeSO4·7H2O 0.03 g、Ca3(PO4)25 g、 glucose 10 g, agar 20 g and distilled water 1000 mL) flat plate, culturing at 30 ℃ for 5 d, and indicating that the functional strain has the inorganic phosphorus dissolving capability if a transparent ring appears around the bacterial colony. The diameter of the transparent ring, D (cm), and the diameter of the colony, D (cm), were measured, and D/D values were calculated, with a larger ratio indicating greater inorganic phosphorus dissolving capacity, each experiment being repeated 3 times.
The results in FIG. 13 show a clear transparent ring around the Pseudomonas B1207 strain, with a D/D value of 2.33, indicating that the strain has the ability to dissolve inorganic phosphorus.
4. The amylase production capability test comprises the steps of picking an activated pseudomonas B1207 single colony strain, inoculating the strain on a screening culture medium [ the screening culture medium formula (g/L): beef extract 0.5, yeast powder 10.0, KH 2PO420.0,MgSO4·7H2 O0.5, soluble starch 10.0, agar 20.0 and distilled water 1000 mL ] plate, and culturing at 30 ℃ for 2 d. After the Lugol's iodine solution is dripped into the screening culture medium, the screening culture medium is washed by deionized water, and transparent circles appear on the flat plate to show that the strain has the capability of producing amylase. The diameter of the transparent ring (D) was measured and each experiment was repeated 3 times.
The results in FIG. 14 show a clear transparent ring around the Pseudomonas B1207 strain, D-value of 0.7, indicating that the strain has starch producing ability.
The preparation method of Lugol's iodine solution comprises adding 5.5 g potassium iodide into 20 mL pure water, dissolving with stirring rod, adding 2.5. 2.5 g iodine, stirring for a period of time to dissolve completely, and fixing volume to 50 mL.
5. Hydrogen peroxidase production test: picking a colony on a solid culture medium, picking a single colony by using a toothpick, placing the single colony in a clean test tube, dripping 3% hydrogen peroxide solution 2 mL, observing the result, wherein the person with bubbles in half a minute is positive, and the person without bubbles is negative.
The results in FIG. 15 show that after 3% hydrogen peroxide was added to the tube, the Lima produced a large amount of air bubbles, indicating that the strain had the ability to produce H 2O2 enzyme.
6. Bacterial biofilm test (1) bacterial culture, namely, picking single colony on a solid culture medium, inoculating the single colony into a TSB liquid culture medium, shake-culturing the single colony in a shaking table at 30 ℃ until the single colony reaches a logarithmic growth phase, (2) biofilm formation, namely, adding 4 mu L of bacterial liquid (diluted by 1:500) into a high-boron-silicon test tube containing 2mL TSB culture medium, placing the test tube into a30 ℃ constant-temperature incubator for static culture 96 h, (3) biofilm visualization, namely, collecting a biofilm sample after 72 h, gently sucking out suspended bacteria by a pipetting gun, washing the suspended bacteria with sterile water for 3 times (operation should be eased, avoiding damaging the biofilm), staining the formed biofilm with 2.5 mL of 0.1% crystal violet for 20 minutes without shaking, sucking dye out the dye by the pipetting gun, washing the unbound dye by sterile water, repeating for 3 times, adding 2mL of 100% ethanol into the test tube after air drying, and judging the biofilm formation capacity according to color depth.
The results in FIG. 16 show that after 72 h of stationary culture in a test tube, a thin film was visible on the liquid surface, and the solution was dark purple after dye redissolution, indicating that the strain had the ability to form a biofilm.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
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