CN111575216B - Pseudomonas putida capable of degrading phenolic acid autotoxic substances and application thereof - Google Patents
Pseudomonas putida capable of degrading phenolic acid autotoxic substances and application thereof Download PDFInfo
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Abstract
The invention discloses pseudomonas putida capable of degrading phenolic acid autotoxic substances and application thereof. The degrading bacterium is Pseudomonas putida (Pseudomonas putida) H1CGMCC NO. 19841. The strain can efficiently degrade phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid, and can prevent and treat continuous cropping obstacles caused by various phenolic acid substances.
Description
Technical Field
The invention relates to a bacterium capable of efficiently degrading a plurality of phenolic acid autotoxic substances of plants and application thereof, in particular to a phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid degrading bacterium with a growth promoting effect and application thereof in preventing and treating continuous cropping obstacles of strawberries, lilies, watermelons and poplar.
Background
Allelopathy (allelophy) was first proposed by german scientist Molish in 1937, and Rice defined in 1984 as a chemo-ecological phenomenon in which plants (or microorganisms) release chemical substances to the surrounding environment, thereby affecting the growth and development of adjacent plants (or microorganisms), and is a very complex interaction result of the plants, the microorganisms and soil. Autotoxic effects are a major type of allelochemical effects in plants, which means that allelochemical substances of plants are released into the surrounding environment and exert an inhibitory effect on the growth of the same plants. Autotoxic effects are prevalent in most plants. The autotoxic substances can be released into the soil environment through the ways of leaching on the overground part, root secretion, plant stubble decay and the like. Along with the growth and development process of plants, autotoxic substances in soil are continuously accumulated, and the microenvironment around the rhizosphere is changed, so that the growth of the plants is not facilitated. Meanwhile, autotoxic substances can also stimulate the growth of rhizosphere pathogenic bacteria and inhibit the growth of beneficial microorganisms, so that the rhizosphere microbial community structure is unbalanced, the microbial diversity level is changed, the proportion of the pathogenic bacteria is increased, and the incidence of soil-borne diseases is aggravated. The accumulation of autotoxic substances in soil is one of the important causes of continuous cropping obstacles of plants.
The previous research shows that phenolic acids, glycosides and flavonoids are main autotoxic substances. The phenolic acid is the main component of self-toxic substances of soybean, rice, cowpea, peanut, watermelon, melon, hot pepper, asparagus, American ginseng, liquorice, crabapple, peach, poplar, pseudo-ginseng, strawberry, lily and other plants. The phenolic acid substance takes a benzene ring with active carboxyl as a molecular skeleton, and various substituent groups and substitution sites on the benzene ring form the diversity of molecular structures and properties, and can generate toxic action on the growth and development of plants by influencing a membrane system, photosynthesis, enzyme activity, soil microbial activity, soil physicochemical properties and the like of the plants. The phenolic acid autotoxic substances reported at present are phthalic acid, p-hydroxybenzoic acid, cinnamic acid, ferulic acid, salicylic acid, syringic acid, p-hydroxybenzoic acid, vanillic acid, coumaric acid, benzoic acid, coumarin and the like. In nature, usually one plant can produce several to more than ten kinds of phenolic acid autotoxicants simultaneously, and the main phenolic acid autotoxicants produced by different plants are different.
The measures for relieving the autotoxicity include resistant varieties, crop rotation, interplanting, biological carbon adsorption, degradation by beneficial microorganisms and the like. Wherein the microbial degradation has the characteristics of economy, high efficiency, no secondary pollution and the like, and has wide application prospect.
Disclosure of Invention
The invention aims to provide a degrading bacterium capable of efficiently degrading phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid and application thereof in preventing and treating plant continuous cropping obstacles.
The degrading bacteria provided by the invention are Pseudomonas putida (Pseudomonas putida) H1. The Pseudomonas putida (Pseudomonas putida) H1 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 Siro. 1 Hospital, Chaoyang, China) at 19.5.2020 with the deposition number of No. 19841. The Pseudomonas putida (Pseudomonas putida) H1 was isolated from Xiangshan soil and was a gram-negative bacterium. The thalli can form milky colonies on an LB culture medium, the edges of the colonies are neat, the surface is smooth, and the colonies have slight viscosity. The bacterial colony is milky white at the initial stage, then becomes a faint yellow purulent colony with regular circular edge, smooth, flat and opaque, and moist surface, and can grow on an MSM culture medium taking phthalic acid as a unique carbon source.
The invention also protects the application of the Pseudomonas putida (Pseudomonas putida) H1CGMCC NO.19841 in degrading phthalic acid, syringic acid, p-hydroxybenzoic acid and/or ferulic acid.
The application of Pseudomonas putida (Pseudomonas putida) H1 in preventing and treating plant continuous cropping obstacles also belongs to the protection scope of the invention. Wherein the plant can be strawberry, lily, watermelon and/or poplar.
The biological agent containing the Pseudomonas putida (Pseudomonas putida) H1 as an active ingredient also belongs to the protection scope of the invention.
The strain can prevent and treat continuous cropping obstacles caused by various phenolic acid substances, and can efficiently degrade phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid in MSM culture medium and soil. The Pseudomonas putida (Pseudomonas putida) H1 is a strain with good degradation of phenolic acid autotoxicants.
Experiments prove that the Pseudomonas putida (Pseudomonas putida) H1 which has the degradation effect on phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid can be efficiently degraded, and the better prevention and treatment effect on the continuous cropping obstacle caused by the substances can be realized.
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FIG. 1 shows the effect of Pseudomonas putida (Pseudomonas putida) H1 in reducing the autotoxicity of phenolic acids on strawberry, watermelon and poplar.
FIG. 2 shows the effect of Pseudomonas putida (Pseudomonas putida) H1 in reducing the effect of phenolic acid on lily autotoxicity.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
The percentages in the following examples are by mass unless otherwise specified.
Example 1 obtaining of Pseudomonas putida (Pseudomonas putida) H1, a phenolic acid degrading bacterium of the present invention
Isolation of Pseudomonas putida (Pseudomonas putida) H1
The strain is separated from the soil of the Yingpo mountain of Beijing, and the specific method comprises the following steps: weighing 5g of rhizosphere soil, putting the rhizosphere soil into a 300mL triangular flask (added with 0.2g/L of phthalic acid) filled with 100mL of MSM liquid culture medium (5.8 g of dipotassium phosphate, 4.5g of monopotassium phosphate, 2.0g of ammonium sulfate, 0.16g of magnesium chloride, 0.02g of calcium chloride, 0.2g of sodium chloride, 0.0015g of manganese chloride dihydrate, 0.0018g of ferric chloride, 1000mL of distilled water and pH7.0), culturing at 30 ℃ in a dark place at 170rpm and for 7 days by shaking; transferring 2mL of the enriched culture of the soil suspension into a 300mL triangular flask (0.3 g/L of phthalic acid is added) filled with 100mL of liquid MSM culture medium, and continuously culturing for 7 days under the same culture conditions; transferring 2mL of the enrichment culture into a 300mL triangular flask (added with 0.4g/L phthalic acid) filled with 100mL MSM liquid culture medium, and continuously culturing for 7 days under the same culture conditions; 2mL of the enriched culture was transferred to a 300mL Erlenmeyer flask containing 100mL of MSM liquid medium (0.5 g/L phthalic acid was added) and cultured for 7 days under the same conditions. 2mL,1mL,0.5mL, and 0.1mL of the final culture suspension were uniformly applied to an MSM plate (dipotassium hydrogenphosphate.8 g, potassium dihydrogenphosphate 4.5g, ammonium sulfate 2.0g, magnesium chloride 0.16g, calcium chloride 0.02g, sodium chloride 0.2g, manganese chloride dihydrate 0.0015g, ferric chloride 0.0018g, agar 18g, distilled water 1000mL, pH 7.0; 0.5mg of phthalic acid was added). And culturing the coated MSM plate at 30 ℃, continuously observing for 7d, randomly picking 100 single colonies, and streaking, purifying and storing for later use. And (3) re-screening the separated and purified phthalic acid degrading bacteria on an MSM (metal-organic-metal-semiconductor) plate (0.5 mg of syringic acid, p-hydroxybenzoic acid and ferulic acid are respectively added), and screening out 6 degrading bacteria capable of simultaneously degrading phthalic acid, syringic acid, p-hydroxybenzoic acid and ferulic acid, wherein one degrading bacteria is named as H1.
Example 2 identification of Pseudomonas putida (Pseudomonas putida) strain H1 genus
Example 1 the physiological, biochemical and basic biological properties of the strain H1 obtained by separation and screening are shown in Table 1.
TABLE 1 basic biological characteristics of H1
Note: "+" indicates that a carbon source (or a nitrogen source) can be utilized, and "-" indicates that a carbon source (or a nitrogen source) cannot be utilized
According to the handbook of identification of common bacteria, the above physiological and biochemical characteristics of the H1 strain are the same as those of Pseudomonas, and the strain is named as Pseudomonas (Pseudomonas sp.) H1.
The H1 strain was identified by conventional physiological and biochemical methods and 16S rDNA sequence analysis, and it was confirmed that the strain was Pseudomonas putida (Pseudomonas putida).
Using genome DNA of Pseudomonas sp H1 as a template and a bacterial 16SrDNA sequence universal primer as a primer (27F: 5 '-AGAGTITGATCCTG GCTCAG-3'; reverse primer 1492R: 5'-TACGGGTACCTTGTTACGACTT-3'), a product fragment of about 1.5kb was PCR-amplified, and the sequence was determined with 27F and 1492R primers to obtain 1443 nucleotides (sequence 1 in the sequence listing). The sequence was blast aligned at NCBI and showed that the 16SrDNA sequence of H1 has the highest homology with Pseudomonas putida strain W30 (accession number: GQ303714.1) up to 99.72%.
The Pseudomonas putida (Pseudomonas putida) H1 strain has been deposited in China general microbiological culture Collection center (CGMCC, No. 3 of the institute of microbiology of China academy of sciences, Naja, Chao, China) at 19.5.2020, with a collection number of CGMCC NO. 19841.
Example 3 determination of the ability of Pseudomonas putida (Pseudomonas putida) H1 to degrade phenolic acids
1. Degradation of phenolic acids by Pseudomonas putida (Pseudomonas putida) H1 in MSM liquid medium
1.1, preparing a standard solution and preparing a standard curve, namely preparing a gradient standard curve solution: preparing 1mg/mL phenolic acid substance (phthalic acid, syringic acid, p-hydroxybenzoic acid or ferulic acid) solution with 50% methanol solution as solvent, and preparing 2.5, 5.0, 10.0, 20.0, 50.0 μ g/mL standard solutions respectively according to gradient dilution method.
1.2 determination of the content of phenolic acids by HPLC Pseudomonas putida (Pseudomonas putida) H1 was picked and added to liquid LB medium and cultured overnight at 28 ℃ and 170 rpm. And (3) centrifuging the overnight-cultured bacterial liquid at 4000rpm for 5min, discarding the supernatant, and then resuspending the supernatant by using sterile water to prepare bacterial suspension with the OD600 value of 1.0. Adding 120 μ L of the above bacterial suspension into 12mL of MSM liquid culture medium (respectively adding phthalic acid, syringic acid, p-hydroxybenzoic acid or ferulic acid to make the final concentration of phthalic acid, syringic acid, p-hydroxybenzoic acid or ferulic acid 0.5g/L), and culturing at 28 deg.C for 36h in a shaking incubator at 170 rpm. Sterile water was used as a control. The culture broth was centrifuged at 12000rpm for 5min, and the supernatant was mixed with methanol at a ratio of 1: 1. After mixing, the mixture is diluted by 50% methanol solution. Taking 2mL of diluted solution, filtering and sterilizing the diluted solution by using a 0.22 mu m organic injection filter, and detecting the residual quantity of four phenolic acid substances by using high performance liquid chromatography. A chromatographic column: XDB-C18 column (4.6mm X250 mm), column temperature room temperature. The mobile phase solution was formulated from 0.1% HCOOH + 2% meoh (a) and acetonitrile (B). The gradient elution composition was as follows: 0min → 96% A + 4% B → 10 min; 10% A + 90% B → 16 min; 96% A + 4% B → 30min, flow rate 1.0 mL. min-1, test wavelength 234nm, sample size 10. mu.L. And (4) recording chromatographic data, and obtaining the concentration of the phenolic acid substances in the soil sample according to the used peak area by an external standard method. The degradation rate of phthalic acid was obtained by calculation using the following formula. The degradation rate (%) [1- (treatment group-remaining amount/control group-remaining amount) ] × 100%.
1.3 results
As shown in Table 2, the degradation rate of P.putida (Pseudomonas putida) H1 in MSM broth after 36 hours treatment was 99% or more for both o-phthalic acid and p-hydroxybenzoic acid. The degradation rates of ferulic acid and syringic acid are 87.7% and 64.1%, respectively.
TABLE 2 degradation of phenolic acids by H1 in MSM broth
2. Degradation of phenolic acids in soil by Pseudomonas putida (Pseudomonas putida) H1
2.1 soil treatment: the soil samples were air-dried at room temperature. And screening the air-dried soil by using a 2mm sieve. Pseudomonas putida (Pseudomonas putida) H1 strain was cultured overnight at 200rpm in LB liquid medium at 28 ℃. Centrifuging the bacterial solution at 4000rpm for 5min, removing supernatant, re-suspending with sterile water, and adjusting OD with spectrophotometer6001.0. 200g of air-dried soil is put into a flowerpot, 10mL of phthalic acid, syringic acid, ferulic acid or p-hydroxybenzoic acid solution is respectively added, so that the contents of the phthalic acid, the syringic acid, the ferulic acid or the p-hydroxybenzoic acid in the soil are respectively 30 mu g/g, 35 mu g/g,100 mu g/g and 250 mu g/g, and each group is repeated for 3 times. 20mL of experimental group inoculum suspension (theoretical concentration of degrading bacteria in soil is 10)7cfu/g), 3 replicates per group. The cells were placed in a light incubator (25 ℃ C./20 ℃ C., light/dark 16/8h), 20mL of sterile water was added to the control group, and samples were taken every 72h during the incubation period.
2.2 extracting phenolic acid substances in soil: the soil samples were air dried and 5g of the sample was weighed and extracted by adding 8ml of 1mol/LNaOH (15ml centrifuge tube) and shaking for 12h at 200rpm on a shaker at room temperature. The supernatant was aspirated and the pH was adjusted to 2.5 with 3mol/L HCl. Adding 5ml ethyl acetate, ultrasonic extracting for 10min, extracting phenolic substances from the acidified solution, and collecting the extract. 5ml ethyl acetate was added to the acidified solution and the above steps were repeated to extract 3 times. The combined extracts were evaporated to dryness in a rotary evaporator at 35 ℃ under vacuum, the dried extract was redissolved in 2ml of methanol and filtered through a 0.22 μm pore filter and transferred to a chromatography flask.
2.3HPLC method for measuring the degradation of phenolic acid substances in soil:
a chromatographic column: XDB-C18 column (4.6mm X250 mm), column temperature room temperature. The mobile phase solution was prepared from acetonitrile (a) and phosphoric acid water (pH 3.00) (B). The gradient elution composition was as follows: 0min → 10% a + 90% B → 20 min; 90% A + 10% B → 25 min; 100% A + 0% B → 30min, flow rate 1.0 mL. min-1, sample size 5. mu.l, column temperature 30 ℃. And (4) recording chromatographic data, and obtaining the concentration of the phenolic acid substances in the soil sample according to the used peak area by an external standard method. The degradation rate of the Phenolic Acid (PA) is calculated by the following formula. The PA degradation rate (%) × 100% for [1- (treatment group PA-remaining amount/control group PA-remaining amount) ].
2.4 results
As a result, as shown in Table 3, the degradation rates of syringic acid, phthalic acid, ferulic acid and p-hydroxybenzoic acid in the soil were 99.23%, 98.94%, 82.07% and 98.94% respectively for H1 after 9H of treatment.
TABLE 3 degradation of phenolic acids in soil by H1
Example 4 testing of the Effect of H1 on reducing the autotoxicity of plants
1. The method for soil treatment and bacterial suspension preparation in this experiment was the same as the method for degrading phenolic acids in soil by Pseudomonas putida (Pseudomonas putida) H1 shown in step 2 of example 3. The test plant species and corresponding phenolic acids were selected according to literature reports of plant autotoxic substances. The effects of the H1 strain on alleviating the self-toxicity of p-hydroxybenzoic acid on strawberry, ferulic acid on poplar, syringic acid on watermelon and phthalic acid on lily were determined respectively. After planting, the plants are cultured in an illumination incubator (light at 25 ℃/20 ℃ and light/dark 16/8h), and after 45d, a series of growth indexes such as plant height, stem thickness, leaf number, fresh weight of the ground and underground parts and the like of the plants are observed and recorded. Three replicates per treatment, 3 plants per replicate, were treated with sterile water as control and soil without added phenolic acid as mock.
The results are shown in tables 4-7 and fig. 1 and 2, the growth amounts of strawberry, poplar, watermelon and lily treated by Pseudomonas putida (Pseudomonas putida) H1 are obviously better than those of the clear water control, and the H1 strain is proved to be capable of effectively reducing the self-toxicity of p-hydroxybenzoic acid to strawberry, ferulic acid to poplar, syringic acid to watermelon and phthalic acid to lily. And the growth indexes of the strawberries, the poplars and the watermelons treated by the H1 are obviously superior to those of mock without adding phenolic acid substances, which shows that the H1 strain has obvious promotion effects on the three plants.
TABLE 4 Effect of Pseudomonas putida (Pseudomonas putida) H1 in reducing the autotoxicity of p-hydroxybenzoic acid on strawberry
*P<0.05
TABLE 5 Effect of Pseudomonas putida (Pseudomonas putida) H1 in alleviating the autotoxic effect of ferulic acid on poplar
*P<0.05
TABLE 6 Effect of Pseudomonas putida (Pseudomonas putida) H1 in alleviating the autotoxic effect of syringic acid on watermelon
#1-20 fibrous roots per plant + +,21-40 fibrous roots per plant; over 40 fibrous roots per plant ++, in
*P<0.05
TABLE 7 Effect of Pseudomonas putida (Pseudomonas putida) H1 on reducing the effect of phthalic acid on lily autotoxicity
*P<0.05
The foregoing description is intended to be illustrative rather than limiting, and it will be appreciated by those skilled in the art that many modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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Claims (6)
1. Pseudomonas putida (b)Pseudomonas putida) Characterized in that the name is pseudomonas putida (f)Pseudomonas putida) H1, Committee for China Collection of microorganismsThe preservation number of the general microorganism center of the member is CGMCC No. 19841.
2. Pseudomonas putida (B)Pseudomonas putida) H1 is used for preventing and treating plant continuous cropping obstacles; the autotoxic substances in the plant continuous cropping obstacle are phthalic acid, syringic acid, p-hydroxybenzoic acid and/or ferulic acid; wherein, Pseudomonas putida (B)Pseudomonas putida) The preservation number of H1 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC number 19841.
3. Use according to claim 2, wherein the plant is strawberry, lily, watermelon and/or poplar.
4. Pseudomonas putida (B)Pseudomonas putida) H1 application in preparing biological agent for preventing and treating plant continuous cropping obstacle; pseudomonas putida (B)Pseudomonas putida) The preservation number of H1 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC number 19841.
5. The biological agent for preventing and treating plant continuous cropping obstacle is characterized in that the active ingredient of the biological agent is pseudomonas putida (pseudomonas putida)Pseudomonas putida）H1 CGMCC No. 19841。
6. Pseudomonas putida (B)Pseudomonas putida) Application of H1CGMCC number 19841 in degrading phthalic acid, syringic acid, p-hydroxybenzoic acid and/or ferulic acid.
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| CN110144305A (en) * | 2019-04-05 | 2019-08-20 | 华中农业大学 | A kind of pseudomonas putida and its microbial inoculum and application |
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