CN117264838B

CN117264838B - Pseudomonas with growth promoting function on plant lead stress and application thereof

Info

Publication number: CN117264838B
Application number: CN202311298152.XA
Authority: CN
Inventors: 殷秀杰; 陈子瑞; 王孜成; 燕昌江; 吴禹辰; 穆美琪; 马泽旺; 聂婉婷; 赵思文; 崔国文
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2023-10-09
Filing date: 2023-10-09
Publication date: 2024-04-19
Anticipated expiration: 2043-10-09
Also published as: CN117264838A

Abstract

The invention discloses Pseudomonas sp.Y22 with a growth promoting function on plant lead stress and application thereof, belonging to the technical field of microorganisms. The strain is preserved in China center for type culture Collection, and the preservation address is as follows: eight paths of 299-grade university of Wuhan in Wuchang district of Wuhan, hubei province have a preservation date of 2023, 07 month and 26 days, a preservation number of CCTCC No. M20231339, and are classified and named as pseudoomonas sp.Y22. The strain has no obvious change of growth trend under the conditions of lead stress concentration of 0, 1000 and 5000mg/L, has obviously higher lead tolerance than other existing pseudomonas, has the functions of producing auxin, secreting protease, decomposing inorganic phosphorus and producing ACC deaminase, can be used for phytoremediation of lead-polluted soil, and has the advantages of low cost and high efficiency.

Description

Pseudomonas with growth promoting function on plant lead stress and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to Pseudomonas sp.Y22 with a growth promoting function on plant lead stress and application thereof.

Background

Soil is an important material basis for agricultural production, and heavy metal pollution of soil is a problem which is not negligible at present, wherein the proportion of lead pollution is relatively high. The lead element not only can reduce the yield and quality of crops in the environment, but also can accumulate on the ecological chain through the food chain to influence the health of animals and human beings.

The soil lead pollution remediation technology realizes the remediation of the soil environment by preventing the migration of pollutants, reducing the total concentration of lead pollution, reducing the enrichment effect of organisms on lead and the like, and can be divided into physical, chemical, biological and combined remediation modes, but the traditional remediation method has the defects of high cost, long time, large engineering quantity and lower soil utilization rate. Some bacteria in the soil can form a bacteria-plant system with the repairing plants, so that the absorption of the plants to soil nutrients is improved, the toxicity of lead ions in the soil is reduced, the plant resistance is enhanced by promoting the enzyme activity of the soil and generating plant hormones and the like, the plant growth is promoted, and finally the lead repairing efficiency of the plants is improved, and therefore, a lead-resistant plant rhizosphere growth-promoting bacteria is needed.

Disclosure of Invention

Based on the defects, the invention aims to provide Pseudomonas sp.Y22 with a growth promoting function on plant lead stress, wherein the bacterial colony is in a long rod shape, white, opaque and smooth in surface, and flocculent in the middle of the bacterial colony, and the bacterial colony is flat, shows stronger lead tolerance and has a positive effect on plant growth, can be used for bioremediation of lead contaminated soil, and has a good promoting capability on plant growth under the lead stress.

The technical scheme adopted by the invention is as follows: pseudomonas sp.Y22, which has growth promoting function on plant lead stress, is deposited in China center for type culture Collection, accession number: eight paths of No. 299 Wuhan university in Wuchang district of Wuhan, hubei province have a preservation date of 2023, 07 month and 19 days, a preservation number of CCTCC No. M20231339, and are classified and named as Pseudomonas sp.Y22.

The invention also provides a method for culturing pseudomonas with growth promoting function on plant lead stress, which comprises the following steps: taking OD ₆₀₀ =1.0 seed solution, adding 0.1% of the seed solution into the culture medium according to the volume ratio, and carrying out shaking culture for 24 hours at the temperature of 28 ℃ and under the condition of 180r/min and pH=7.0.

Further, as described above, the culture medium adopts LB liquid culture medium, and the formula is: 10g/L of sodium chloride, 5g/L of yeast extract, 10g/L of tryptone, 15g/L of agar and pH=7.

It is another object of the present invention to provide the use of a Pseudomonas having a growth promoting function on lead stress in plants as described above as a microbial product for promoting growth of plant rhizosphere under lead stress.

It is another object of the present invention to provide a plant rhizosphere promoter for plants under lead stress, comprising a Pseudomonas bacterium having a promoting function on lead stress of plants as described above.

Further, the bacterial liquid of pseudomonas was adjusted to OD ₆₀₀ =0.50±0.02, and the bacterial liquid was irrigated to the root of the plant every 5 days, 50mL each time.

Further, the plant as described above is alfalfa.

The invention has the advantages and beneficial effects that: the strain has no obvious change on the growth trend under the conditions that the concentration of lead stress is 0, 1000 and 5000mg/L, has obviously higher tolerance to lead than other existing pseudomonas, has the functions of generating auxin, secreting protease, decomposing inorganic phosphorus and generating ACC deaminase, has better promotion capability on the growth of plants under the stress of lead, can be used for repairing lead-polluted soil by plants, and has the advantages of low cost and high efficiency.

Drawings

FIG. 1 is a diagram of a bacterial morphology taken by an electron microscope;

FIG. 2 is a phylogenetic tree analysis chart of the strain;

FIG. 3 is a standard graph of the α -butanoic acid content;

FIG. 4 is an IAA standard graph;

FIG. 5 is a graph showing the growth of the strain under different lead stresses;

FIG. 6 is a phenotype diagram of the strain on plant germination under lead stress;

FIG. 7 is a phenotype diagram of the strain on plant germination under low temperature stress;

FIG. 8 is a bar graph showing the effect of the strain on chlorophyll a content in plants under lead stress;

FIG. 9 is a bar graph showing the effect of the strain on plant chlorophyll b content under lead stress;

FIG. 10 is a bar graph showing the effect of the strain on plant carotenoid content under lead stress;

FIG. 11 is a bar graph showing the effect of the strain on total chlorophyll content in plants under lead stress;

FIG. 12 is a bar graph showing the effect of the strain on plant Malondialdehyde (MDA) content under lead stress;

FIG. 13 is a bar graph showing the effect of the strain on plant superoxide dismutase (SOD) content under lead stress;

FIG. 14 is a bar graph showing the effect of the strain on plant Peroxidase (POD) content under lead stress;

FIG. 15 is a bar graph showing the effect of this strain on plant Catalase (CAT) content under lead stress.

Detailed Description

The following examples illustrate the invention in further detail, but it should be understood that the detailed description is merely illustrative and explanatory of the invention, as well as of the technical aspects and advantages thereof, and is not intended to limit the invention in any way.

Example 1 isolation, screening and identification of strains

The lead-resistant plant rhizosphere pseudomonads (Pseudomonas sp.y22) provided in this example was isolated from the rhizosphere soil of red clover plants in the university campus of northeast agriculture in hardb, the province of black longjiang. The lead content of the soil is 4.5g/kg. 5g of red clover rhizosphere soil was placed in 45mL of sterile water and shaken for 30min without interruption. At this time, the concentration of the bacterial suspension is 10 ^-1, and then 10 times of gradient dilution is carried out for 5 times until the concentration reaches 10 ^-5. Extracting 100 μl of each bacterial suspension from the dilution of 10 ^-3、10^-4、10^-5, uniformly coating the bacterial suspensions on LB culture medium (sodium chloride 10g, yeast extract 5g, tryptone 10g, agar 15 g) plates by using a sterilized coater, sealing a culture dish, inverting the culture dish, culturing for 48h in a constant temperature incubator at 28 ℃, observing the growth state of the bacterial colonies, performing preliminary screening according to the form, size, color and the like of the bacterial colonies after the bacterial colonies grow out, picking single bacterial colonies with different types, carrying out streak purification on the bacterial colonies by using a plurality of plates until single purified bacterial strains are obtained, and storing the purified bacterial strains in a refrigerator at-80 ℃. And (3) carrying out growth promotion capability measurement on the separated and purified strain, including but not limited to protease and ACC deaminase production, IAA production and inorganic phosphate leaching capability identification, selecting the strain with the largest growth promotion capability as a target strain, and carrying out strain identification. The plant rhizosphere growth promoter with lead resistance obtained by screening is Pseudomonas sp.

The strain is cultured on an LB plate at 28 ℃ for 24 hours, the colony form of the strain is white, opaque and smooth in surface, the middle of the colony is flocculent and flat, and the strain is in a long rod shape (figure 1) through microscopic observation, and various physiological and biochemical tests are carried out on the strain, and the characteristics are as follows (table 1):

Table 1: physiological and biochemical characteristics of strain

Note that: "+" indicates positive and "-" indicates negative

Example 2 identification of 16SrDNA of bacteria:

1ml of bacterial liquid with OD ₆₀₀ =1-1.5 was collected as bacterial culture by a 2ml centrifuge tube, centrifuged for 30s at 12000 Xg, the supernatant was discarded, the strain DNA was extracted by using a kit, and the bacterial genome PCR amplification was performed using 27F ("5-AGTTTGATCCTGGGCTCAG-3") and 1492R ("5-CTACGGCTACCTTGTTACGA-3"), and the sample was sent to the Sharphead Homekino Biotech Co., ltd for bacterial 16Sr DNA sequencing, the sequence of which is shown in SEQ ID No. 1. Sequence alignment analysis was performed in the BLAST program in NCBI website based on the sequencing result, and phylogenetic tree was constructed using MEGA7.0 (fig. 2), and the result showed that the bacterium was Pseudomonas sp.

EXAMPLE 3 protease production ability of Strain

The strain obtained by screening was inoculated into LB liquid medium (sodium chloride 10g/L, yeast extract 5g/L, tryptone 10g/L, agar 15g/L, pH=7) with an inoculating loop, and shake-cultured at 28℃for 24 hours at 180r/min under pH=7.0 to obtain a seed solution. The seed solution was diluted with sterile water in an ultra clean bench to OD ₆₀₀ = 0.5 for use. Inoculating the activated strain to protease screening culture medium with sterilized fungus receiving ring in ultra clean bench, sealing culture dish, inverting at 28deg.C constant temperature incubator, culturing for 48 hr, observing whether the outer ring of colony generates transparent aperture, which is representative of the strain with protease production capability, measuring maximum diameter (D) of the transparent aperture and colony diameter (D), and calculating (D/D) value. The results are shown in Table 2.

TABLE 2D/D values of strains on protease Medium

Example 4 ACC deaminase production ability of Strain

Bacterial solutions were prepared in the same manner as described above. Inoculating the sterilized strain-inoculating loop to ACC deaminase screening culture medium (ADF) in an ultra-clean workbench, sealing a culture dish, inverting the culture dish, culturing in a constant temperature incubator at 28 ℃ for 48 hours, and observing whether bacteria can grow or not, wherein the bacteria can grow, namely the strain has ACC deaminase activity.

And (3) taking the alpha-butanoic acid as a standard sample, measuring an OD ₄₅₀ value by an ultraviolet spectrophotometer, and comparing the alpha-butanoic acid standard curve with a bovine serum albumin measurement standard curve to calculate the ACC deaminase activity of the strain. From FIG. 3 and Table 3, it can be seen that the strain has ACC deaminase-producing ability.

TABLE3 ACC deaminase Activity by strains

Example 5 IAA Producing ability of Strain

Bacterial solutions were prepared in the same manner as described above. The seed solution was diluted with sterile water in an ultra clean bench to OD ₆₀₀ = 0.5 for use. The purified target strains are respectively inoculated into LB liquid medium containing 300 mug/mL tryptophan and placed at 180r/min for shake enrichment culture at 28 ℃ for 24 hours. 1mL of the bacterial suspension is taken to be placed in a washed test tube, an equivalent amount Salkowski of chromogenic liquid is added to carry out chromogenic reaction, and an equivalent amount of IAA standard liquid is added to be used as positive control, and the test tube is placed at room temperature for color development for 30min under the condition of light shading, so that the IAA can be produced when the color becomes red.

IAA standard solutions with concentrations of 0, 10, 20, 30, 40 and 50 mug/mL were prepared, mixed with Salkowski colorimetric solutions according to a volume ratio of 1:1, left at room temperature in the dark for 30min, and then OD ₅₃₀ at each concentration was measured separately (a 1:1 mixed solution of distilled water and Salkowski colorimetric solution was used as a blank). And finally, drawing by taking IAA concentration as an abscissa and OD ₅₃₀ as an ordinate to obtain an IAA standard curve. The corresponding IAA concentration values were calculated by standard curves of OD ₅₃₀ and IAA concentration. It can be seen from FIG. 4 and Table 4 that the strain has IAA-producing ability.

TABLE 4 IAA concentration production by strains

Example 6 inorganic phosphate leaching Capacity of Strain

Bacterial solutions were prepared in the same manner as described above. The seed solution was diluted with sterile water in an ultra clean bench to OD ₆₀₀ = 0.5 for use. Inoculating the activated target strain to inorganic phosphorus culture medium (NPA) with sterilized fungus receiving rings in an ultra-clean workbench, sealing a culture dish, inverting the culture dish, culturing in a constant temperature incubator at 28 ℃ for one week, observing whether a transparent aperture is generated on the outer ring of a colony, and measuring the maximum diameter (D) of the generated transparent aperture and the diameter (D) of the colony to calculate the value (D/D). The results are shown in Table 5.

TABLE 5D/D values of strains on inorganic phosphate-solubilizing Medium (NPA)

Example 7 growth curves of strains under lead stress

The bacterial liquid was prepared in the same manner as above, and the seed liquid was diluted OD ₆₀₀ =0.5±0.02 in a sterile water super clean bench for use. Respectively preparing LB liquid culture media containing 0, 250, 500, 1000 and 5000mg/L, sucking 1mL of target bacterial liquid, pouring the prepared LB culture media containing lead, placing the culture media in a shaking table at 180r/min and 28 ℃ for shake culture, measuring OD ₆₀₀ every 3 hours, drawing a growth curve, and obtaining that the strain is less influenced by lead stress, has no obvious change of growth trend under the lead stress of different concentrations and has stronger lead stress resistance.

Example 8 Effect of strains on germination of alfalfa under lead stress

The test sets 6 lead stress concentrations (0 mg/L,250mg/L,500mg/L,1000mg/L,2500mg/L,5000 mg/L), the control group (0 mg/L) uses deionized water as a treatment liquid, the bacterial liquid is regulated to OD ₆₀₀ = 0.50 plus or minus 0.02, and the prepared bacterial agent is used for seed soaking treatment according to the proportion of 10ml/100 alfalfa seeds, the time is 48 hours, the temperature is 28 ℃, and the pH is = 7.0. Taking out the seeds, culturing at constant temperature for 4 days, and measuring germination vigor; after 7 days, germination rate and embryo length and radicle length were measured, and 3 replicates were set per 40 seeds in the petri dish. As can be seen from tables 6, 7, 8, 9 and fig. 6, the strain significantly improved germination rate, germination vigor, radicle length and embryo length of alfalfa seeds under Pb stress as compared with the control group, and the higher stress concentration was more significant.

TABLE 6 influence of the strains on germination of alfalfa seeds under different lead concentrations

Note that: the capital letters in the table are the significance of differences (P < 0.05) under different concentrations of lead stress in the vaccinated bacteria treatment group; the lower case letters are the significance of the differences (P < 0.05) between the inoculated bacteria and the control group in the same concentration lead stress treatment, as follows.

TABLE 7 influence of strains on germination vigor of alfalfa seeds under different concentrations of lead stress

TABLE 8 influence of strains on alfalfa seed radicle length under different concentrations of lead stress

TABLE 9 influence of strains on alfalfa seed embryo length under different concentrations of lead stress

Example 9 Effect of strains on alfalfa germination under Low temperature stress

The experiment was performed with 4 temperature gradients (5 ℃, 10 ℃, 15 ℃ for low temperature stress treatment and 25 ℃ for control), and alfalfa seeds were treated in the same manner as described above. After seed inoculation is completed, culturing for 4 days at constant temperature, and measuring germination vigor; after 7 days, germination rate and embryo length and radicle length were measured, and 3 replicates were set per 40 seeds in the petri dish. From tables 10, 11, 12, 13 and fig. 7, it can be seen that the strain can significantly improve germination rate, germination vigor radicle length and germ length of alfalfa seeds under low temperature stress, and the effect is more significant at lower stress temperature.

TABLE 10 influence of the strains on germination of alfalfa seeds under Low temperature stress

TABLE 11 influence of the strains on the germination vigor of alfalfa seeds under Low temperature stress

TABLE 12 Effect of strains on alfalfa seed radicle growth under Low temperature stress

TABLE 13 influence of strains on the growth of alfalfa seed embryos under low temperature stress

Example 10 Effect of strains on the photosynthetic pigment content of alfalfa under lead stress

3 Kinds of lead stress treatments with different concentrations of 0mg/kg,1000mg/kg and 5000mg/kg are set, alfalfa seeds with proper sizes and full grains are selected, and sterilization treatment is carried out on the alfalfa seeds. Placing seeds in a culture dish with sterile filter paper, inoculating 10mL of target bacterial liquid into each dish, and soaking the seeds for 48 hours; and then the seeds are moved to a culture dish for germination, after the seeds germinate, the germinated seeds with similar growth vigor and good vigor are selected and transplanted to a flowerpot of passivated and sterilized gardening soil, 8 alfalfa seeds are sown in each pot, and after the seedlings grow to a three leaf one-heart period, bacterial liquid treatment is carried out, and each treatment is repeated three times. Watering every 3 days, 100mL each time, to maintain proper water content in the soil; regulating the bacterial liquid to OD ₆₀₀ = 0.50 plus or minus 0.02, and irrigating the bacterial liquid to the root of the plant every 5 days for 50mL each time so as to maintain stable abundance of bacterial groups in soil; and (5) after the alfalfa grows for 30 days, measuring phenotype and various physiological indexes of the alfalfa.

Collecting fresh plant leaf samples, weighing 0.1g, shearing the leaves, adding the leaf samples into a centrifuge tube, mixing the leaf samples with chlorophyll leaching solution, hermetically placing the mixture under the conditions of room temperature and darkness for leaching for 24 hours, taking blank leaching solution as blank for zeroing after the material turns white, respectively measuring the light absorption values at the positions of 663nm,645nm and 470nm on a spectrophotometer, recording, repeating each treatment for three times, and then calculating chlorophyll a, chlorophyll b, total chlorophyll content and carotenoid content, wherein the addition of the strain can be seen from figures 8, 9, 10 and 11 to obviously improve the chlorophyll and carotenoid content of plants under lead stress.

Example 11 Effect of strains on malondialdehyde content of alfalfa under lead stress

Grinding 0.1g of fresh plant sample, adding 10%1.5ml of trichloroacetic acid (TCA), grinding for 1min in an ice bath, centrifuging to obtain a supernatant, centrifuging at 4 ℃ for 4000r/min for 10min, collecting 1ml of supernatant which is a Malondialdehyde (MDA) -containing supernatant, mixing with 1ml of 0.67% thiobarbituric acid solution (TBA), treating in a boiling water bath for 15min, taking out the reaction solution, rapidly cooling, centrifuging at 4000r/min for 15min, collecting the supernatant, and measuring the absorbance at 532nm, 600nm and 450nm by using an enzyme-labeled instrument and recording. From fig. 12, it can be seen that the strain can significantly reduce the malondialdehyde content of the plant, indicating that the stress response of the plant is reduced.

Example 12 Effect of strains on antioxidant capacity of alfalfa under lead stress

Superoxide dismutase (SOD): measured by adopting a Nitrogen Blue Tetrazolium (NBT) photoreduction method. 0.1g of fresh plant sample is weighed, added with an extraction medium (pre-cooling is needed), ground and centrifuged at 4 ℃ for 10min at 10000r/min, and the obtained supernatant is the enzyme extract. The same test tube with good transparency was used as 3 control, 3 as measurement and 1 as dark control (for zeroing). Absorbing 0.1mL of the extracting solution into test tubes, adding 1.5mL of 50mmol/L phosphoric acid buffer solution (pH 7.0) +0.5mL of distilled water +0.3mL Met+0.3mL NBT+0.3mL EDTA-Na2+0.3mL of riboflavin into each test tube, replacing the dark control test tube extracting solution with distilled water, immediately sleeving a black paper sleeve after adding the riboflavin for shading treatment, shaking uniformly after adding the reagent, placing in an illumination incubator, performing a color development reaction for 30min (after the color is changed) at the temperature of 4000lx, and covering with shading cloth to terminate the reaction. The reaction solution absorbance in the test tube was measured and recorded on a spectrophotometer at a wavelength of 560nm with a blank (zeroing) in the dark control tube.

Peroxidase (POD): the guaiacol method is adopted for measurement; 0.1g of fresh plant sample is weighed, an extraction medium (pre-cooling is needed) is added, grinding and centrifugation are carried out at a low temperature of 4 ℃, the centrifugation procedure is 10000r/min and 10min, and the obtained supernatant is the enzyme extract. The same test tube with good transparency was used, 1 control (zeroing) and 3 test tubes. Each test tube was first added with 3mL of the reaction mixture (28. Mu.L of guaiacol in 50mL of 100mmol/L phosphate buffer (pH 6.0), placed on a magnetic stirrer, heated and stirred until dissolved, after the solution cooled, 19. Mu.L of 30% hydrogen peroxide was added, mixed well, stored at low temperature for later use), 0.1mL of enzyme extract was added to the post-measurement tube, 0.1mL of 50mmol/L phosphate buffer (pH 7.0) was added to the control tube in place of the enzyme extract, zeroed with the control tube, stop watch counting was started immediately after each enzyme extract was added to the measurement tube, absorbance was measured at a wavelength of 470nm on a spectrophotometer, and readings were taken every 1min and recorded.

Catalase (CAT): measuring by ultraviolet absorption method; weighing 0.1g of fresh plant sample, adding extraction medium (pre-cooling in advance), grinding at low temperature of 4deg.C, centrifuging for 10min at 10000r/min, and collecting supernatant as enzyme extractive solution. The same test tube with good transparency was used, 1 control (zeroing) and3 test tubes. 50mmol/L phosphate buffer (pH 7.0) 1 mL+enzyme extract 0.1 mL+distilled water 1mL was added to the test tube, the control tube was added with 0.1mL of enzyme extract after heating, inactivating and cooling in advance, the test tube was subjected to water bath preheating treatment, water bath program 25 ℃ for 3min, then 0.2mL 200mmol/L H ₂O₂ solution was added to each tube, each tube was immediately measured on a spectrophotometer at 240nm wavelength (distilled water zeroing) and read every 1min for 4 times and recorded.

From FIGS. 13, 14 and 15, it can be seen that the strain can significantly improve the activity of plant antioxidant enzymes (SOD, POD and CAT), which indicates that the antioxidant capacity of plants is increased.

Claims

1. Pseudomonas with growth promoting function for plant lead stress is preserved in China center for type culture Collection, with a preservation address: the preservation date is 2023, 07 and 19 days, the preservation number is CCTCC No. M20231339, and the classification name is Pseudomonas sp.Y22.

2. The method for culturing pseudomonas with growth promoting function for lead stress of plant according to claim 1, wherein the method comprises the following steps: taking OD ₆₀₀ =1.0 bacterial liquid according to the inoculum size of 0.1% of volume ratio into a culture medium, and shake culturing for 24 hours at the temperature of 28 ℃ under the conditions of 180 r/min and pH=7.0.

3. The use of a pseudomonas with a growth promoting function against plant lead stress according to claim 1 as a microbial product for enhancing germination rate, germination vigor, radicle length and germ length of alfalfa seeds under lead stress.

4. A microbial product for alfalfa under lead stress, characterized in that: comprising a Pseudomonas bacterium having a growth-promoting function against lead stress in plants as claimed in claim 1.