Pseudomonas geissii PG-9 and its application

Abstract

The present invention discloses Pseudomonas gessardii PG-9, which relates to the field of microbial technology. The strain PG-9 was isolated from apple roots in Xingtai City, Hebei Province, and deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with a deposit date of 2022.10.10, a deposit number of CGMCC No.25887, and a classification name of Pseudomonas gessardii. The Pseudomonas gessardii PG-9 strain provided by the present invention has ACC deaminase activity and a strong ability to dissolve inorganic phosphorus. Watering the roots of apples with a liquid bacterial suspension prepared with the Pseudomonas gessardii PG-9 strain can increase apple yield and improve apple quality.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a strain of Pseudomonas Geisleri PG-9 and applications thereof. Background Art

[0002] Apple yield and quality directly impact farmers' economic returns. While long-term, heavy application of chemical fertilizers increases yields, it also threatens soil health, weakening trees and severely impacting both yield and quality. Therefore, reducing chemical fertilizer use and finding effective fertilization strategies are crucial.

[0003] Research has found that some beneficial endophytic bacteria improve soil fertility and promote soil nutrient conversion, stimulating plant growth and improving yield and quality. These bacteria participate in various plant life activities and have functions such as regulating plant hormone levels and metabolism, solubilizing phosphate, and inhibiting bacteria. Therefore, providing more strains that can alleviate apple tree decline, enhance plant resistance, and promote apple growth is an urgent need for those skilled in the art. Summary of the Invention

[0004] In view of this, the present invention provides a strain of Pseudomonas Gaertneri PG-9 and applications thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A strain of Pseudomonas gessardii PG-9, the Pseudomonas gessardii PG-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 10, 2022, with the deposit number CGMCC No. 25887, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Pseudomonas gessardii.

[0007] Preferably, the Pseudomonas gaeae PG-9 has the ability to produce ACC deaminase and the ability to solubilize inorganic phosphorus.

[0008] Pseudomonas gaesii PG-9 has strong colonization and rapid reproduction, enabling beneficial symbiosis with host plants. ACC deaminase breaks down ACC, a precursor for ethylene synthesis, into α-ketobutyric acid and ammonia, regulating ethylene levels and promoting plant growth and development.

[0009] As an inventive concept identical to the above technical solution, the present invention further seeks protection for an engineered strain, which is constructed using the above-mentioned Pseudomonas Gaesii PG-9 as a starting strain.

[0010] As an inventive concept identical to the above technical solution, the present invention also seeks protection for a microbial agent comprising the above strain.

[0011] Preferably, the bacterial agent is in the form of a bacterial suspension, powder or tablet, preferably a bacterial suspension.

[0012] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the application of the above-mentioned Pseudomonas Gaesii PG-9 or the above-mentioned engineered strain or the above-mentioned microbial agent.

[0013] Preferably, the application is to promote plant growth.

[0014] Preferably, the application is to increase apple yield.

[0015] Preferably, the application is to regulate the available phosphorus content in soil.

[0016] More preferably, the application is to apply the Pseudomonas Gaesii PG-9 or the engineered strain or the microbial agent to the soil around the roots of plants.

[0017] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses Pseudomonas gaesii PG-9 and its application, which have the following beneficial effects:

[0018] 1. The Pseudomonas gaeae PG-9 strain provided by the present invention has the ability to produce ACC deaminase, with an ACC deaminase activity ranging from 12.40 U / mg to 13.22 U / mg, which helps to reduce ethylene stress, improve plant resistance, and promote plant growth.

[0019] 2. The Pseudomonas gaeae PG-9 provided by the present invention has the ability to dissolve inorganic phosphorus. 600 = 0.5 concentration, the inoculum was inoculated into NBRIP liquid medium at an inoculum volume of 1% of the inoculation medium volume. After shaking for 7 days, the phosphate solubilization capacity was between 506.67 mg·L -1 ~539.13mg·L -1 , regulate the content of available phosphorus in the soil, help plants absorb nutrients, promote plant growth and other advantages.

[0020] 3. The Pseudomonas gaeae PG-9 strain provided by the present invention can increase apple yield and improve apple quality. The PG-9 strain can produce 62.67t / hm2 of apples. 2 ~68.54t / hm 2 The apple soluble sugar is 10.50% to 10.94%, and the apple soluble acid is 0.16% to 0.18%, which has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 This is a colony morphology diagram of Pseudomonas Galeae PG-9 on LB medium of the present invention;

[0023] Figure 2 This is the phylogenetic tree of Pseudomonas Gaesii PG-9 of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1 Isolation, identification and preservation of Pseudomonas gaeae PG-9

[0026] (1) Separation:

[0027] Pseudomonas gaesii PG-9 was isolated from apple roots on a farm in Xingtai City, Hebei Province. The apple tree root tissue was surface-disinfected, minced, and diluted with sterile water to create different bacterial suspensions. The suspensions were then spread onto KB solid medium and incubated at 30°C for 2 days. Bacteria that fluoresced under UV light were selected and purified. The isolated strain was inoculated into 50 mL of PFA liquid medium and incubated with shaking at 25°C and 200 rpm for 24 hours. One mL of the suspension was transferred to another 50 mL of PFA liquid medium and incubated once under the same conditions to enrich the bacteria in the medium. After the incubation period, one mL of the suspension was transferred to 50 mL of DF liquid medium and incubated with shaking at 25°C and 200 rpm for another 24 hours. Finally, another 1 mL of the suspension was transferred to 50 mL of ADF medium (using 3 mmol / L ACC reagent instead of (NH₄)₂SO₄ as the nitrogen source) and incubated with shaking at 25°C and 200 rpm for 24 hours. Pipette 100 μL of bacterial suspension and spread it evenly on ADF solid medium, culture it at 25℃ for 3 days, and transfer it to ADF solid medium again after colonies grow out for streak purification. Select the strains that can still grow, store them on TSB solid medium and place them in a 4℃ refrigerator for later use.

[0028] (2) Screening

[0029] ACC deaminase activity screening: The isolated strain was inoculated into LB liquid medium and cultured overnight at 28°C and 200 rpm with shaking for 12 hours. The cells were then collected by centrifugation for 10 minutes (4°C and 10,000 rpm). The suspension was washed twice with 7.5 mL of DF liquid medium without (NH4)2SO4. 45 μL of 0.5 mol / L ACC solution was added to the suspension through a 0.22 μm sterilizing filter and cultured with shaking at 28°C and 200 rpm for 24 hours. The suspension was centrifuged for 10 minutes (4°C and 10,000 rpm) to collect the cells, weigh them, and then centrifuged (4°C and 10,000 rpm) with 5 mL of Tris-HCl buffer (0.1 mol / L, pH = 7.6). The cells were washed twice to remove the DF liquid medium and divided equally into three 1.5 mL EP tubes. The cells were then resuspended in 600 μL of Tris-HCl buffer (0.1 mol / L, pH 8.5), and 30 μL of toluene was added and shaken on a shaker for 30 s to disrupt the cells. 100 μL of the cell extract was taken and the protein content was determined using the Coomassie Brilliant Blue G-250 method. 200 μL of the cell extract was aspirated and added to 20 μL of ACC solution (0.5 mol / L). The mixture was incubated at 30°C for 15 min, followed by the addition of 1 mL of HCl (0.56 mol / L) and centrifugation for 8 min (10,000 rpm). 1 mL of the supernatant was then added to 800 μL of HCl (0.56 mol / L) and 300 μL of 2,4-dinitrophenylhydrazine (0.2% by mass, dissolved in HCl (2 mol / L)). The mixture was incubated at 30°C for 30 min, followed by the addition of 2 mL of NaOH (2 mol / L) for colorimetry at 540 nm. Sterile water was used as a blank. Three replicates were performed for each treatment. ACC deaminase activity was calculated based on protein determination and α-ketobutyric acid standard curve.

[0030] After calculation, the ACC deaminase activities of the PG-9 strain were 13.22U / mg, 12.40U / mg and 12.40U / mg, respectively, and the ACC deaminase activities of another control Pseudomonas Galea strain were 8.75U / mg, 8.38U / mg and 7.68U / mg, respectively. The results showed that the ACC deaminase activity of the PG-9 strain ranged from 12.40U / mg to 13.22U / mg, and the activity of the control strain ranged from 7.68U / mg to 8.75U / mg.

[0031] Screening of the ability to dissolve inorganic phosphorus: The isolated strains were inoculated into 4 areas of NBRIP solid culture medium, repeated 3 times, and cultured at 28°C for 8 days. The colony diameter (d) and the diameter of the phosphorus solubilization zone (D) were measured, and the ratio of D / d was calculated to preliminarily determine the size of the phosphorus solubilization ability. Rescreening was performed by liquid shake flask test. Different isolated strains were inoculated into LB liquid culture medium and cultured at 28°C and 180 rpm for 24 hours. After centrifugation for 10 minutes (4°C, 10000 rpm), the bacteria were resuspended in sterile water to prepare a bacterial suspension (OD 600 =0.5). The bacterial suspension was inoculated into NBRIP liquid medium at an inoculum volume of 1% of the volume of the inoculation medium. NBRIP liquid medium without bacterial suspension was set as a blank control. Each treatment was repeated three times. The culture was shaken at 28°C and 150 rpm for 7 days. The culture solution was centrifuged for 10 minutes (4°C, 10,000 rpm), and 1 mL of the supernatant was used to determine the available phosphorus content using the molybdenum antimony colorimetric method.

[0032] The results showed that the PG-9 strain had a significant phosphate solubilization capacity on NBRIP medium, with the concentrations of 539.13 mg·L -1 , 535.65mg·L -1 and 506.67 mg·L -1 The inorganic phosphorus solubility capacity of the other control strain Pseudomonas gaesii was 280.00 mg·L -1 , 290.43mg·L -1 and 277.10 mg·L -1 Therefore, the PG-9 strain has the highest phosphate solubilizing ability, and its phosphate solubilizing ability is quantitatively determined to be between 506.67 mg·L -1 ~539.13mg·L -1 higher than the control.

[0033] (3) Identification

[0034] Morphological identification: PG-9 strain was streaked on LB solid medium and cultured at 28°C for 2 days. The strain morphology was observed and its morphological characteristics were as follows: the colonies were round, moist, with neat edges and opaque light yellow color (see Appendix). Figure 1 The strain was Gram-stained and observed under an optical microscope. The results showed that the strain was Gram-negative and the cell morphology was long rod-shaped.

[0035] Molecular identification: The total DNA of the strain was extracted using a bacterial genomic DNA extraction kit (Omega Bio-Tek, USA). PCR amplification was performed using Pseudomonas-specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.1) and 1495R (5′-ACGGCTACCTTGTTACGACT-3′, SEQ ID NO.2), and sequencing was commissioned to Beijing Liuhe BGI Genomics Technology Co., Ltd. The result was a 1399 bp sequence. After sequencing analysis and comparison with the GenBank database, it was found that PG-9 had a similarity of 99.93% with Pseudomonas gessardii (MK883134.1). The sequencing results were further used to construct a phylogenetic tree (see Appendix Figure 2 ), and it was found that the PG-9 strain and Pseudomonas gessardii clustered into one branch.

[0036] The 16S rDNA sequence is as follows:

[0037]

[0038]

[0039] (4) Preservation

[0040] Pseudomonas gessardii PG-9 was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC No. 25887, the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification name being Pseudomonas gessardii. The deposit date is October 10, 2022.

[0041] Example 2 Preparation of Pseudomonas Gaertneri PG-9 bacterial suspension (bacteria)

[0042] Pseudomonas gaeae PG-9 strain was inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 24 h. The culture was then centrifuged at 4°C and 10,000 rpm for 10 min. The cells were resuspended in sterile water to a viable count of 2 × 10 9 cfu·mL -1 .

[0043] Example 3 Pseudomonas gaeae PG-9 regulates soil available phosphorus content

[0044] The bacterial suspension obtained in Example 2 was tested for soil phosphorus content:

[0045] Application method: The experiment adopted a randomized block design and selected apple trees with basically the same growth. On July 11, 2021, the final concentration of 2×10 7 cfu·mL -1 The PG-9 strain bacterial suspension was evenly poured into the tree tray with a radius of 1.5m from the rhizosphere of each apple tree, and an average of 2L of bacterial agent was poured into each tree. On November 10, 2021, the apple rhizosphere soil was collected at a distance of 50cm from the tree trunk. The soil collected from 0-40cm was air-dried and sieved for the determination of soil phosphorus content. The results showed that the total phosphorus contents of the rhizosphere soil of apples treated with PG-9 strains were 1.51g / kg, 1.49g / kg, and 1.44g / kg, respectively; the total phosphorus contents of the rhizosphere soil treated with another control Pseudomonas Gaertnsis strain were 1.83g / kg, 1.68g / kg, and 1.61g / kg, respectively; the total phosphorus content in the soil treated with PG-9 strains was 1.44g / kg~1.51g / kg, and that of the control bacteria was 1.61g / kg~1.83g / kg. The available phosphorus contents in the rhizosphere soil of the PG-9 strain were 226.80 mg / kg, 237.34 mg / kg, and 260.71 mg / kg, respectively, while those in the rhizosphere soil treated with the control strain, Pseudomonas galli, were 239.46 mg / kg, 229.74 mg / kg, and 197.53 mg / kg, respectively. The available phosphorus content in the soil treated with the PG-9 strain ranged from 226.80 mg / kg to 260.71 mg / kg, while that in the control strain ranged from 197.53 mg / kg to 239.46 mg / kg, indicating that the PG-9 strain has a stronger ability to transform insoluble phosphorus in the soil.

[0046] Example 4 Application of Pseudomonas geissii PG-9 in apple cultivation

[0047] The effect of the bacterial suspension obtained in Test Example 2 on apples: A 16-year-old "Red Fuji" orchard was selected for testing on July 15, 2021. The test method was the same as in Example 3, with 3 plants treated each time and repeated 3 times.

[0048] During the apple harvest period (October 25-30, 2021), the number of fruits per tree per treatment and per replicate was recorded, and 50 fruits were randomly selected from different positions of each apple tree, weighed one by one, and the mass of each apple (g) was calculated; the calculation was converted to 667m 2 Area yield (kg); 10 fruits were randomly selected from different positions of apple trees in each treatment and replicate to analyze the effect of PG-9 strain on apple quality.

[0049] The results showed that the apple yields of the PG-9 strain treatment were 68.54t / hm 2 、62.67t / hm 2 、63.48t / hm 2and the other control strain Pseudomonas gaeae strain treated apple yields of 58.67t / hm 2 、58.65t / hm 2 、59.53t / hm 2 PG-9 strain treatment apple yield 62.67t / hm 2 ~68.54t / hm 2 , the control bacteria was 58.65t / hm 2 ~59.53t / hm 2 The PG-9 strain increased soluble sugars in apples by 10.91%, 10.94%, and 10.50%, respectively; the control strain, Pseudomonas gaeae strain, increased soluble sugars by 9.81%, 9.11%, and 9.14%, respectively. The PG-9 strain increased soluble sugars by 10.50% to 10.94%, while the control strain increased soluble sugars by 9.11% to 9.18%. The PG-9 strain increased soluble acids in apples by 0.17%, 0.16%, and 0.18%, respectively; the control strain, Pseudomonas gaeae strain, increased soluble acids by 0.27%, 0.23%, and 0.22%, respectively. The G-9 strain increased soluble acids in apples by 0.16% to 0.18%, while the control strain increased soluble acids by 0.22% to 0.27%. This suggests that the PG-9 strain can promote fruit growth and significantly increase yield by converting soluble acids into soluble sugars, improving fruit quality and the percentage of high-quality fruit. It has promising development prospects.

[0050] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Pseudomonas gaesii ( Pseudomonas gessardii ) PG-9, characterized in that The Pseudomonas gaesii PG-9 was deposited in the General Microbiology Center of the China Culture Collection Administration on October 10, 2022, with the deposit number CGMCC No. 25887, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and is classified as Pseudomonas gaesii. Pseudomonas gessardii .

2. A microbial agent, characterized in that: The invention comprises the Pseudomonas Galeae PG-9 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The bacterial agent dosage form is bacterial suspension, powder or tablet.

4. The microbial agent according to claim 3, characterized in that The bacterial agent dosage form is a bacterial suspension.

5. Use of the Pseudomonas geissii PG-9 according to claim 1 or the microbial agent according to any one of claims 2 to 4, characterized in that: The application is any of the following: A. Producing ACC deaminase; B. Regulate the available phosphorus content in the soil; C. Promote apple growth; D. Increase apple production; E. Improve apple quality.

6. The application according to claim 5, characterized in that: Apply the Pseudomonas Gaesii PG-9 described in claim 1 or the microbial agent described in any one of claims 2 to 4 to the soil around the roots of the plants.

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