CN110144305B - Pseudomonas putida, and microbial inoculum and application thereof - Google Patents

Abstract

The invention relates to Pseudomonas putida, which is Pseudomonas putida WH-B3(Pseudomonas putida WH-B3) and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2019048. The invention provides a preparation method of a microorganism bacterium and a microorganism bacterium agent containing the pseudomonas putida, and provides an application method of the microorganism bacterium agent in degradation of benzoic acid and alleviation of continuous cropping obstacles of peaches. The Pseudomonas putida Putida WH-B3 has extremely strong degradation capability on benzoic acid, and can degrade 500mg kg of soil within 24 hours^‑1The degradation efficiency of the benzoic acid can reach more than 99 percent, and the degradation product is nontoxic and has good development and application prospects.

Description

Pseudomonas putida, and microbial inoculum and application thereof

Technical Field

The invention relates to the field of plant protection, in particular to pseudomonas putida, a microbial inoculum thereof and application thereof in degrading benzoic acid and relieving continuous cropping obstacle of peaches.

Background

Peach is one of four fruits in China, has wide cultivation area in each province and each region of China, and is also planted in all the world. The peach has beautiful appearance, delicious taste and rich nutritive value, and is deeply welcomed by the consumers. In recent years, the peach yield and the consumption of China are at the top of the world, but with the increase of peach demand, continuous cropping obstacles are commonly generated in some orchards due to the fact that the land is tight, the management system is deficient, the soil remediation technology is inappropriate and the like, and the peach continuous cropping obstacles become important factors for restricting the healthy and continuous development of the peach industry. The continuous cropping obstacle of the fruit tree is the phenomenon that when a new tree is planted after old trees are planed in an orchard, the root system of a young tree is dysplastic, the growth is slow, the plant is short, the resistance is reduced, and even the whole plant dies (Hoestra 1988). The phenomenon of continuous cropping obstacle is found in apple, peach, plum, apricot, orange, strawberry, cherry, grape, pear, banana and other fruit trees, and the phenomenon is widely distributed and occurs in different fruit trees in orchards in various countries and regions such as Asia, America, Africa, Europe, and Atlanta (Mai and Abawi 1981; Hoestra 1968), which is a global problem. The discovery of the continuous cropping obstacle of the peach tree has been over two hundred years old, the peach tree is ubiquitous in all peach cultivation areas, and students begin to research the continuous cropping obstacle of the peach tree in the last 40 th century, but so far, the research on the continuous cropping obstacle of the peach tree is still less at home and abroad. The reason for the continuous cropping obstacle is considered as follows: (1) deterioration of physical and chemical properties of soil, (2) change of soil microbial community structure, and (3) accumulation of autotoxic substances.

At present, continuous cropping obstacle relieving measures mainly comprise the following aspects of fertilizer application, breeding of disease-resistant varieties of crops, crop rotation of other crops, soil replacement, soil disinfection and the like. For peach trees, because the research on genetic transformation is less, the system is still not perfect enough, and the breeding of resistant varieties is difficult; soil replacement is only suitable for small-area operation, and chemical drugs can cause soil residue and environmental pollution; other traditional methods are possibly more convenient on herbaceous plants, but are time-consuming and labor-consuming for woody fruit trees, the solution is not strong in pertinence, the soil physicochemical properties and microbial communities can be effectively improved, but the influence of self-toxic substances cannot be completely removed. Therefore, removing autotoxic substances from the continuous cropping soil by some methods may be more effective in resolving the continuous cropping obstacles. In recent years, studies for alleviating continuous cropping obstacles by using autotoxic substance-degrading bacteria have been receiving attention.

Autotoxic substances are secondary metabolites released by plants into the environment, and can enter the environment through volatilization, leaching, root secretion, stubble degradation and other ways to further act on surrounding receptors, so that the autotoxic substances are gradually found to be the main cause of continuous cropping obstacles. Research results show that some secondary metabolites generated by the acetic acid pathway, the shikimic acid pathway or the combination of the two pathways in plants or phenolic substances generated by the decay of plant residues in soil are the most reported autotoxic substances, and mainly comprise phlorizin, phloretin, phloroglucinol, chlorogenic acid, benzoic acid, cinnamic acid, caffeic acid, syringic acid, ferulic acid, p-hydroxybenzoic acid, phthalic acid, salicylic acid, gallic acid, vanillin and the like. Peach trees generate a large amount of autotoxic substances including mandelonitrile, benzoic acid, benzaldehyde, hydrocyanic acid, palmitic acid and the like mainly through hydrolysis of cyanogenic glycoside rich in root systems and root secretion, and the substances influence membrane systems, photosynthesis, enzyme activity, soil microbial communities, physicochemical properties and the like of plants to further inhibit the growth of plant seedlings. Therefore, proper methods for removing autotoxic substances in the soil of the root system of the crops can reduce the adverse effects on the growth of the crops. Research in this regard has become one of the hotspots and focuses in the environmental and agricultural fields in recent years. Some microorganisms have a certain ability to decompose autotoxic substances, and the microorganisms have been found to include plant rhizobacteria of more than 20 genera, such as Phanerochaete chrysosporium, Acinetobacter calcoaceticus, Bacillus, Burkholderia, and the like. However, the research of the microbial degradation of autotoxic substances by the predecessors mainly focuses on herb crops such as strawberries and cucumbers, the research of woody fruit trees is more with apples, and the related research of peaches is rarely reported. Meanwhile, the types of autotoxic substances of different plants and the content of the autotoxic substances in rhizosphere soil are different, so that the degrading bacteria screened by other crops are not necessarily suitable for the autotoxic substances of peach trees. On the other hand, most strains in the microbial fertilizer belong to exogenous microorganisms, so that the original ecological environment of soil can be possibly influenced. Therefore, the microorganism capable of degrading the autotoxic substances directly separated from the continuous cropping soil of the peaches has greater ecological safety advantage and wider practical application value.

Disclosure of Invention

The invention provides pseudomonas putida, a microbial inoculum thereof and application of the microbial inoculum in degrading benzoic acid and relieving continuous cropping obstacle of peaches to solve the problems.

The technical scheme for solving the technical problems is as follows: the Pseudomonas putida is pseudomonad, namely Pseudomonas putida WH-B3, is obtained by screening from peach rhizosphere soil, is identified as the Pseudomonas putida by combining morphological characteristics and molecular characteristics, is named as Pseudomonas putida WH-B3, is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 1 and 15 days, has the address of Wuchang Lojiashan mountain in Wuhan city, Hubei province, and has the preservation number of CCTCC M2019048.

Experiments prove that the pseudomonas putida can degrade benzoic acid, and the invention provides the application of the pseudomonas putida in the degradation of benzoic acid.

The experiment proves that the pseudomonas putida can relieve the continuous cropping obstacle of the peach, and the invention provides the application of the pseudomonas putida in relieving the continuous cropping obstacle of the peach.

The invention also provides a microbial agent which comprises the pseudomonas putida.

The invention also provides a preparation method of the microbial agent, and the microbial agent is obtained by inoculating the pseudomonas putida in a culture medium.

Further, the culture medium is LB culture medium or inorganic salt culture medium with benzoic acid as the only carbon source. The microbial agent has the advantages of simple preparation method, high growth speed, short culture time and low production cost.

The invention also provides application of the microbial agent in degradation of benzoic acid.

The invention also provides application of the microbial agent in relieving continuous cropping obstacles of peaches.

Compared with the prior art, the invention has the following advantages and effects:

1. the Pseudomonas putida WH-B3 is separated from the peach tree rhizosphere soil, and has stable ecological environment.

2. The Pseudomonas putida Putida WH-B3 has extremely strong degradation capability on benzoic acid, and can degrade 500mg kg of soil within 24 hours^-1The degradation efficiency of the benzoic acid can reach more than 99 percent, and the degradation product is nontoxic and has good development and application prospects.

3. The Pseudomonas putida WH-B3 can relieve the continuous cropping obstacle of the peach tree.

Drawings

FIG. 1 is a photograph showing the morphology of Pseudomonas putida WH-B3 of the present invention, wherein FIG. 1A is the colony morphology and FIG. 1B is the single bacterial morphology;

FIG. 2 is a graph showing the growth of Pseudomonas putida WH-B3 and the degradation curve of benzoic acid in example 2 of the present invention, wherein FIG. 2A is a graph showing the growth curve of WH-B3 in a liquid medium, FIG. 2B is a graph showing the degradation curve of benzoic acid in a liquid medium, FIG. 2C is a graph showing the growth curve in soil, and FIG. 2D is a graph showing the degradation curve in soil;

FIG. 3 shows the germination status of lettuce seeds at day 3 in example 3 of the present invention, wherein the control group is control 1, the BA group is control 2, and the degradation product group is the experimental group;

FIG. 4 shows the effect of soil inoculation with Pseudomonas putida WH-B3 of Pseudomonas putida in example 4 of the present invention on the growth of peach seedlings, wherein FIG. 4A shows the plant heights of different groups of peach seedlings, FIG. 4B shows the stem dry weight of different groups of peach seedlings, FIG. 4C shows the stem dry weight of different groups of peach seedlings, and FIG. 4D shows the root dry weight of different groups of peach seedlings.

Detailed Description

The principles and features of this invention are described in connection with the drawings and the detailed description of the invention, which are set forth below as examples to illustrate the invention and not to limit the scope of the invention.

Experimental Material

The soil separated by the benzoic acid degrading bacteria is taken from rhizosphere soil of 9-year-old peach trees in fruit tree teaching experiment base of university of agriculture in Huazhong. The soil used for the pot experiment comprises continuous cropping soil and leisure soil, wherein the continuous cropping soil is taken from soil in the 9-year peach orchard, and the leisure soil is taken from soil which is not planted with peach trees and other crops nearby the peach orchard. And collecting soil by adopting a five-point sampling method, then air-drying the soil, uniformly mixing, removing root systems and broken stones, and sieving by using a 1cm sieve for later use. Selecting wild peach young seeds which grow consistently and are about 10cm to perform pot culture tests in plastic pots which are 25cm multiplied by 18 cm.

Experimental reagent and instrument

Benzoic acid (national drug group chemical reagent limited), a DNA extraction kit (beijing edlely biotechnology limited), a gram stain (south kyo established technology limited), an HP400G-C incubator (wuhan ruihua instruments limited), a UV-2450 UV-visible spectrophotometer (Shimadzu, japan), and a high performance liquid chromatograph (Agilent 1200, usa).

Culture medium

(1) Enrichment culture medium: 10g of peptone, 1g of glucose, 1g of NaCl and KH₂PO₄1g, adding carbon source according to the need, adding distilled water to 1000mL, and adjusting pH to 7.0-7.2.

(2) Inorganic salt medium (MSM): (NH4)₂SO₄(2.0g)，MgSO₄·7H₂O(0.2g)，KH₂PO₄(1.5g)，Na₂HPO₄·12H₂O (1.5g) and benzoic acid as the only carbon source were added as needed, distilled water was made to 1000mL, pH was adjusted to 7.0-7.2, and lmL sterilized trace element solution was added before inoculation. 15g of agar was added to the solid medium.

(3) Solution of trace elements: CaCl₂·2H₂O(0.05g)，FeSO₄·7H₂O(0.004g)，ZnSO₄(0.1g)，MnSO₄·H₂O(0.008g)，CuCl₂·2H₂O(0.05g)，H₃BO₃(0.1g)，CoCl₂(0.05g)，Na₂MoO₄·2H₂O (0.1g) and HCl (0.1M), distilled water to 1000 mL.

(4) LB culture medium: yeast extract (5.0g), tryptone (10.0g), NaCl (10.0g), distilled water to 1000mL, pH 7.0-7.2. Agar 15g was added to the solid LB medium.

Example 1 screening and identification of benzoic acid degrading bacteria

1. Separation and purification of benzoic acid degrading bacteria

Weighing 10g of soil, adding the soil into 100mL of sterile water, placing the mixture on a magnetic stirrer, stirring the mixture for 30min, and standing the mixture, wherein the supernatant is the soil leaching solution. Placing 5mL of soil leaching solution into 45mL of enrichment medium, and performing shake culture in a 150mL conical flask at 30 ℃ and 150r/min for 3 d. Subsequently, 45mL of new enrichment medium (containing 100mg/L benzoic acid) and 5mL of previous enrichment medium are added into a 150mL conical flask, the conical flask is placed at 30 ℃ and subjected to shaking culture at 150r/min for 3d to form an acclimatization period, the conical flask is inoculated into fresh enrichment medium in an inoculation amount of 10% after each period is finished, and the concentration of the benzoic acid is gradually increased from 100mg/L and 200mg/L to 500 mg/L. Then, an MSM culture medium containing benzoic acid of 100mg/L is used to replace an enrichment culture medium, the domestication is continued according to the concentration increasing mode, finally, a culture solution with the highest turbidity is selected, after proper dilution, the culture solution is coated on a solid MSM culture medium (containing benzoic acid of 500mg/L) with benzoic acid as a unique carbon source by a coating method, and the culture is carried out at 30 ℃. After the colonies grow out, selecting a single colony, repeatedly streaking and purifying the single colony on an MSM solid culture medium for at least 3 times, inoculating a strain WH-B3 obtained by final purification on an LB solid inclined plane, and storing the strain in a refrigerator at 4 ℃.

2. Identification of benzoic acid degrading Strain WH-B3

a. Morphological characteristics of benzoic acid degrading Strain WH-B3

The WH-B3 obtained by screening is cultured on an LB solid medium for 24h, the colony morphology is shown in figure 1A, the colony is circular, the diameter of the colony is 3-5mm, and the colony is light yellow, opaque and smooth in edge. The single bacterium is shown in FIG. 1B, and the single bacterium is a Brevibacterium, about 0.2-0.8X 0.7-1.5 μm in size, has flagella at one end, and is a gram-negative bacterium.

b. Molecular identification of benzoic acid degrading strain WH-B3

1mL of fresh bacterial liquid is taken, after centrifugation, thalli are collected, then DNA is extracted by using a bacterial genome kit, and universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGTACCTTGTTACGACTT-3') are selected to carry out PCR amplification on the 16SrRNA gene. The PCR amplification system comprises 12.5 mu L of Mix, 2 mu L of double distilled water 8.5 mu L, DNA template and 1 mu L of each primer. PCR reaction procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, and final extension at 72 ℃ for 5 min. The amplified product is subjected to sequencing by Wuhan Strongzidae biotechnology limited company, and the 16S rDNA sequence of the amplified product is shown as SEQ ID NO: 1:

AACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCACGGTTGATTACTG

the sequence is 1426bp in total, BLAST analysis is carried out on the sequence shown in SEQ ID NO:1 and the sequence in a GenBank database, a phylogenetic tree is constructed by using MEGA 6, the sequence homology of the strain WH-B3 and Pseudomonas putida (Pseudomonas putida KT2440) is more than 99 percent, and the strain can be considered to belong to the same species.

The strain WH-B3 was judged to belong to Pseudomonas putida, and was named Pseudomonas putida WH-B3(Pseudomonas putida WH-B3), in combination with the morphological characteristics and molecular biological identification described above.

Pseudomonas putida WH-B3 was preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 1 month and 15 days, with the address of Wuhan city, Lojia mountain, Hubei province, and the preservation number of CCTCC M2019048.

Example 2 degradation Properties of Pseudomonas putida WH-B3(Pseudomonas putida WH-B3)

a. Liquid culture degradation test: inoculating bacteria into 50mL MSM culture medium containing benzoic acid 500mg/L, shake culturing at 30 deg.C and 150r/min to obtain the position without bacteriaThe principle is used as a control. The OD value of the bacterial suspension was measured every 4 hours at a wavelength of 600nm with a spectrophotometer, and a growth curve was plotted, the result being shown in FIG. 2A. Meanwhile, the concentration of benzoic acid was measured every 4 hours by high performance liquid chromatography, and a degradation curve of benzoic acid was plotted, and the result is shown in fig. 2B. 5mL of bacterial liquid is taken each time, centrifuged at 2000rpm for 10min, the supernatant is extracted with 10mL of ether for 2 times, then evaporated to dryness by a vacuum concentrator, and 5mL of methanol is added to dissolve solid substances for measuring the content of benzoic acid. The HPLC column is ZORBAXC18(4.6mm × 250mm, 5 μm), the mobile phase is 95% water (pH adjusted to 4.2 with glacial acetic acid) and 5% acetonitrile, and methanol is used as cleaning solution. The detector is a diode array detector, the flow rate is 1.0mL/min, the column temperature is 25 ℃, the sample injection volume is 5 mu L, and the light absorption value at 228nm is detected. Drawing a benzoic acid standard curve: respectively taking 0, 2, 4, 6, 8 and 10mL of 1.00g/L benzoic acid standard solution, using methanol to make the solution be dissolved in a 100mL volumetric flask to obtain the final benzoic acid solution whose concentration gradient is 0, 20, 40, 60, 80 and 100mg/L, using the obtained benzoic acid solution to make standard curve determination, after WH-B3 is cultured for 12h, the maximum growth quantity can be reached to 4X 10⁸CFU/mL, and 92.5% of benzoic acid was degraded.

b. Soil culture degradation test: the test soil is leisure soil, and is sterilized for 1h by high-pressure steam at 121 ℃ for 3 times. Then 100g of sterilized soil is added into 20mL of 500mg/L benzoic acid solution to ensure that the concentration of benzoic acid in the soil is 100mg/kg, after bacteria are cultured to a logarithmic phase, a proper amount of bacterial liquid is taken out for centrifugation and supernatant is removed, then 0.85% of NaCl solution with the same amount is added for resuspension once, and the final bacterial liquid is evenly added into the sterilized soil to ensure that the content of bacteria in the soil is about 4 multiplied by 10⁷CFU/g. Under the same conditions, the same amount of 0.85% NaCl treatment was used as a control. The water content in the soil was kept around 50% and all samples were placed in a 30 ℃ incubator. Taking 5g of soil sample every 4h, extracting with 5mL of methanol for 30min, extracting twice to obtain total extractive solutions, filtering with 0.22 μm nylon filter, and determining benzoic acid content. Then another 5g of soil sample is taken and mixed with 100mL of sterile water, and diluted by 10 percent⁵Taking 100 mul of the diluted solution, evenly coating the diluted solution on an MSM solid plate containing benzoic acid, recording the colony number after 3 days, and culturing in soilWH-B3 reached a maximum bacterial count of 1.39X 10 within 16h⁸CFU/g (shown in FIG. 2C), and 99.0% of benzoic acid was degraded (shown in FIG. 2D).

Example 3 toxicity analysis of P.putida WH-B3 on degradation products of benzoic acid

The test was carried out using the internationally common chemosensory model plant lettuce as the test receptor. Three filter papers are soaked in 0.2 percent oxalic acid in advance, and then the air-dried filter papers are washed by distilled water and put into a culture dish with the diameter of 9cm and sterilized at high temperature for standby. Culturing degrading bacteria with MSM containing 500mg/L benzoic acid for 12h (benzoic acid degradation is basically completed), centrifuging 10mL of bacteria liquid at 2000rpm for 10min, extracting the supernatant with 20mL of diethyl ether for 2 times, concentrating the supernatant to dryness with a vacuum concentrator, adding 5mL of methanol to completely dissolve solid residues, adding the solid residues into a culture dish with filter paper, and adding 10mL of sterilized distilled water after the methanol is completely volatilized. Lettuce seeds were sterilized with 1% sodium hypochlorite solution for 10min and then washed several times with distilled water. Selecting healthy and plump seeds with consistent sizes, soaking the seeds in distilled water for 30min, sucking water on the surfaces of the seeds by using filter paper, uniformly placing 30 seeds in each dish, treating the seeds with the same amount of distilled water as a control 1, treating the seeds with 500mg/L benzoic acid as a control 2, and repeating the treatment for 3 times. All dishes were then placed in a 25 ℃ light incubator under 5000lx for 12h and dark at 16 ℃ for 12 h. The humidity of the dishes was maintained during the cultivation period, the number of germination of the seeds was recorded every day for the first three days, and fig. 3 shows the germination of the seeds for the third day, and the height and fresh weight of lettuce seedlings in each dish were measured on the 7 th day. According to the determination indexes and the method, according to the international seed germination regulation, half of the length of the protruded seed coat of the radicle is the germinated seed, and the related indexes of the lettuce seedling are determined on the 7 th day. Wherein the germination rate calculation formula is as follows:

after the lettuce seeds are treated by 500mg/L of benzoic acid, the germination of the seeds is strongly inhibited, the germination rate within 3d is 0, 3-4 seeds germinate after 7d, but the growth of the germinated seedlings is also severely inhibited. The germination rate of the lettuce seeds treated by the WH-B3 degradation products reaches 96%, the growth condition of seedlings is not obviously different from that of clear water control treatment, and the lettuce seeds are not inhibited basically.

Example 4 Effect of inoculation of WH-B3 on soil benzoic acid and peach shoot growth in peach shoot potting test

The peach seedling pot experiment is carried out in a four-side ventilated greenhouse in a fruit tree teaching experiment base of Huazhong university of agriculture under the conditions of natural illumination and humidity. The test is provided with 5 soil treatments, each treatment is used for planting 30 peach seedlings, and the specific soil design is shown in table 1. The test started on 1 day of 4 months and continued for 2 months, during which time water was applied every other week or so. And 6, measuring related indexes on 1 day in 6 months. The plant height is measured by using a measuring tape, and the trunk diameter is measured at the same position of the trunk of the peach seedling by using a vernier caliper. Separating stem and root of peach seedling, oven drying in oven at 105 deg.C for 15min, oven drying at 75 deg.C to constant weight, and measuring dry weight.

TABLE 1 exogenous benzoic acid test

The results are shown in fig. 4, the addition of 100mg/kg of exogenous benzoic acid to the leisure soil significantly inhibited the growth of peach seedlings, and the plant height, the stem diameter, the stem weight and the root dry weight of the peach seedlings were respectively reduced by 28.3%, 17.1%, 50.1% and 52.2% compared with the control. And by inoculating WH-B3 and the benzoic acid, the inhibitory action of the benzoic acid is basically disappeared, the peach seedling growth is not obviously different from that of a control, and the benzoic acid in the soil is degraded by 94.4%.

The growth of the peach seedlings planted in the continuous cropping soil is obviously inhibited, and compared with the control treatment, the plant height, the stem diameter, the stem weight and the root dry weight of the peach seedlings are respectively reduced by 20.6%, 16.1%, 50.0% and 37.2%. After continuous cropping soil inoculation with WH-B3, the inhibition effect is remarkably relieved and is reduced by only 5.2%, 9.7%, 27.3% and 17.9% compared with the control. The initial content of benzoic acid in the continuous cropping soil was 44.9mg/kg, while no benzoic acid was detected initially in the fallow soil. After continuous cropping soil inoculation WH-B3, the benzoic acid in the soil was degraded by 65.0%.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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<120> pseudomonas putida, and microbial inoculum and application thereof

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Claims (8)

1. The pseudomonas putida is characterized by being pseudomonas putida WH-B3(Pseudomonas putida WH-B3) which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2019048.

2. Use of the pseudomonas putida of claim 1 for degrading benzoic acid.

3. Use of pseudomonas putida according to claim 1 for alleviating continuous cropping disorders of peach.

4. A microbial inoculant comprising the pseudomonas putida of claim 1.

5. A method for preparing the microbial agent according to claim 4, wherein said Pseudomonas putida is inoculated in a culture medium to obtain said microbial agent.

6. The method for preparing a microbial inoculant according to claim 5, wherein the culture medium is LB medium or inorganic salt medium with benzoic acid as the sole carbon source.

7. Use of the microbial inoculant of claim 4 for degrading benzoic acid.

8. Use of the microbial inoculant of claim 4 for alleviating continuous cropping disorders of peaches.

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