Pseudomonas fulva SY2-8 and its applications

Abstract

This invention belongs to the field of saline-alkali land improvement technology. This invention discloses a strain of *Pseudomonas aeruginosa* (…). Pseudomonas fulva )SY2-8, its preservation number is CGMCC NO.25613. The *Pseudomonas aeruginosa* (*Pseudomonas chrysogenum*) provided by this invention... Pseudomonas fulva SY2-8 is easy to cultivate, has a short cycle, is non-ecotoxic, is salt-tolerant, and has a rich variety of available carbon sources, which can effectively promote the growth of crops such as cabbage and corn.

Description

Technical Field

[0001] This invention belongs to the field of saline-alkali land improvement technology. Background Technology

[0002] Antagonistic bacteria in the plant rhizosphere achieve biocontrol through competition for space and nutrients, antimicrobial activity, and the induction of resistance. They form hyphal structures on the root surface, creating a physical defense system against pathogens, while simultaneously competing directly with pathogens for nutrients and space. The secretions of antagonistic bacteria contain phytoalexins, which inhibit pathogen growth, alter the microbial environment around the roots, induce the synthesis of resistance-related substances, and enhance plant disease resistance. In agricultural production, utilizing antagonistic bacteria for biological control of crop diseases aligns with the strategic policy of sustainable agricultural development and is gaining increasing attention. Traditional pesticide control of various pests and diseases is widespread, but the environmental pollution caused by excessive pesticide use is also worsening. To achieve high-yield, high-quality agricultural products, reducing pesticide use and using beneficial microorganisms to antagonize pathogens is crucial. Summary of the Invention

[0003] In view of this, the first objective of the present invention is to provide a strain of *Pseudomonas aeruginosa* (…). Pseudomonas fulva )SY2-8, its accession number is CGMCC NO. 25613.

[0004] The *Pseudomonas aeruginosa* provided by this invention ( Pseudomonas fulva SY2-8 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25613, on August 30, 2022. The depositary institution is referred to as CGMCC and its address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0005] A second objective of this invention is to provide *Pseudomonas aeruginosa* (…). Pseudomonas fulva Application of SY2-8 in saline-alkali land improvement microbial agents.

[0006] The present invention provides *Pseudomonas aeruginosa* ( Pseudomonas fulva SY2-8 is easy to cultivate, has a short cycle, is non-ecologically toxic, and has a rich variety of available carbon sources. It can effectively promote plant growth and improve the growth of corn, cabbage, and potatoes. Attached Figure Description

[0007] Figure 1 It is Pseudomonas aeruginosa ( Pseudomonas fulva Tolerance diagram of SY2-8 to mancozeb, chlorothalonil and carbendazim.

[0008] Figure 2It is Pseudomonas aeruginosa ( Pseudomonas fulva ) Effect of SY2-8 on maize growth.

[0009] Figure 3 It is Pseudomonas aeruginosa ( Pseudomonas fulva The effect of SY2-8 on the growth of Chinese cabbage.

[0010] Figure 4 It is Pseudomonas aeruginosa ( Pseudomonas fulva The effect of SY2-8 on potato growth. Detailed Implementation

[0011] Unless otherwise specified, the strains tested in the following examples all refer to *Pseudomonas aeruginosa* (…). Pseudomonas fulva )SY2-8.

[0012] Unless otherwise specified, the activation in the following examples refers to *Pseudomonas aeruginosa* (…). Pseudomonas fulva SY2-8 was cultured three times on potato solid medium.

[0013] Example 1 Isolation and identification of bacterial strains 1. Culture medium: Potato Dextrose agar (PDA) medium (1L): 200g potato, 20g glucose, 15g agar, 1000mL distilled water, pH natural.

[0014] 2. Source of strain: Pseudomonas aeruginosa ( Pseudomonas fulva SY2-8 was invented by the inventor in November 2021 in Sanya City, Hainan Province.

[0015] Take 1g of soil sample and put it into a 50ml centrifuge tube. Add 10ml of sterile water and shake well. Shake on a shaker at 37℃ and 200 rpm for 30 minutes. Let it stand until clear to obtain a soil suspension.

[0016] The prepared soil suspension was serially diluted in a clean bench, and 100 µL of each solution was taken. -3 10 -4 10 -5 The diluted solution was spread onto LB agar plates three times, and incubated at 37 °C for 24 h. After 24 h, colonies with different morphologies were picked and streaked for purification in a laminar flow hood, and incubated at 37 °C for 24 h. This purification process was repeated three times. The resulting pure single colonies were transferred to test tube slant agar and stored at 4 °C for later use.

[0017] 3. Screening Results: The isolated strain was named strain SY2-8. It is Gram-positive, rod-shaped, and reproduces by binary fission. The colonies are yellowish-white, round, with regular, flat, smooth, glossy, and viscous. It produces spores and decomposes glucose, maltose, and other sugars.

[0018] 4. Methods for identifying bacterial strains: Genomic DNA was extracted using the TIANGEN bacterial genomic DNA extraction kit. Using the genomic DNA of the test strain as a template, the target fragment was amplified using the universal primers 27F / 1492R for the bacterial 16S rRNA gene. The PCR product was detected by 1% agarose gel electrophoresis, and the PCR stock solution was sent to Bomeide Biotechnology Co., Ltd. for sequencing. 16S rRNA gene sequence analysis, after sequence comparison and identification, confirmed strain SY2-8 as *Pseudomonas aeruginosa* using MK757660.1 (NCBI) as the standard. Pseudomonas fulva The sequencing sequence is as described in SEQ ID NO.1 in the sequence listing.

[0019] Pseudomonas aeruginosa ( Pseudomonas fulva SY2-8 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25613, on August 30, 2022. The depositary institution is referred to as CGMCC and its address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] Example 2 Physiological and biochemical analysis of strains

[0021] The physiological and biochemical parameters of strain SY2-8 were tested using the bioMérieux API 20NE non-fermenting Gram-negative bacillus identification kit. *Pseudomonas aeruginosa* (… Pseudomonas fulva SY2-8 can utilize pyruvate, glucose, maltose, Kohn gelatin, honey, arabinose, etc.

[0022] The detailed results are shown in the table below: Table 1. Analysis of physiological and biochemical properties

[0023] Example 3 Salt tolerance test of strains The basal medium was LB liquid medium. NaCl was added to the LB solid medium to adjust the salt concentration to 1%, 3%, 5%, 9%, and 11%, and the pH was adjusted to 7.0. The medium was then autoclaved at 121°C for 20 minutes.

[0024] The activated test strain SY2-8 was inoculated onto five different salt concentrations of LB liquid medium, with uninoculated medium serving as a blank control. The media were incubated at 37°C for 24 hours to observe the growth of strain SY2-8. The OD value was measured.

[0025] As the concentration of NaCl increased, the concentration of strain SY2-8 gradually decreased. However, it could still grow when the NaCl concentration reached 11%, although growth was significantly inhibited. This demonstrates that this strain can be used in the improvement of saline-alkali land, exhibits salt tolerance, and is suitable as a microbial agent for improving saline-alkali land.

[0026] Table 2. Growth of SY2-8 at different salt concentrations

[0027] Example 4 Determination of strain sensitivity to different pH values LB broth was used as the basal medium, with pH values ​​adjusted to 3, 5, 6, 7, 8, 9, and 11. The activated test strain SY2-8 was inoculated onto the six pH-sensitive media and incubated at 37°C for 24 hours. The growth of strain SY2-8 was observed. Normal growth indicated that strain SY2-8 could tolerate the specified pH; conversely, abnormal growth indicated that strain SY2-8 could not tolerate the specified pH. The concentration OD value was measured. Growth of strain SY2-8 was significantly inhibited at pH values ​​below 3 and above 9. Growth continued at pH 9, but was somewhat inhibited. This demonstrates that the strain can survive and reproduce in environments with pH values ​​from 1 to 11, indicating its applicability in saline-alkali land improvement. It exhibits acid and alkali tolerance and is suitable as a microbial agent for saline-alkali land improvement.

[0028] Table 3. Growth of SY2-8 at different pH values

[0029] Example 5 Strain tolerance to different fungicides Take the activated strain SY2-8 into 50 ml of potato broth and incubate it in a shaker at 37°C for 2-5 days. After centrifuging the fermentation broth at 12,000 r / min for 1 min, discard the supernatant and suspend the precipitate in an equal volume of sterile water to prepare a bacterial suspension.

[0030] Three commonly used pesticides, namely mancozeb, chlorothalonil, and carbendazim, were purchased from Beijing Zhongbao Lvnong Technology Group Co., Ltd., and were diluted to the recommended concentrations for later use.

[0031] The activated test strain SY2-8 was spread onto LB solid medium. 10 μl of the prepared pesticide was dropped into the center of the plate. A blank control (the plated culture medium without pesticide) was used as a blank control. The plates were incubated at 37°C for 24 hours to observe the growth of strain SY2-8. Figure 1 As shown in the results, strain SY2-8 has a higher tolerance to mancozeb than chlorothalonil and carbendazim. In the production process, strain SY2-8 can be combined with mancozeb to improve the disease control effect while promoting crop growth.

[0032] Example 6 The effects of bacterial strains on the growth of cabbage, corn, and potatoes After soaking cabbage, potato, and corn seeds at room temperature for 24 hours, they were transplanted into vermiculite pots (17 cm in diameter). After 10 days of growth, seedlings with uniform growth were selected and inoculated with 100 Ml of SY2-8 spore suspension (1×10⁻⁶) using the root drenching method. 6 CFU / mL was added to the flowerpot; chlorophyll content of cabbage and potatoes was measured after 20 days of growth, and chlorophyll content and plant height of corn were measured after 30 days of growth.

[0033] Table 4. Plant height data of maize after 30 days of growth.

[0034] Table 5. Chlorophyll data measured after 30 days of maize growth.

[0035] Table 6. Chlorophyll data measured after 20 days of growth in Chinese cabbage

[0036] Table 7. Chlorophyll data measured after 20 days of potato growth

[0037]

[0038] The results showed that strain SY2-8 had a certain degree of growth-promoting effect on cabbage, corn, and potato, and is suitable for development and application as a crop growth-promoting substance.

[0039] This invention produces a strain of *Pseudomonas aeruginosa* with salt and alkali tolerance, which can effectively improve the growth rate of corn, cabbage, and potatoes. To ensure high-quality crop production, it can reduce the amount of pesticides and fertilizers used. This strain can replace part of the pesticides and fertilizers used, thereby promoting plant growth and improving the soil ecological environment.

Claims

1. Pseudomonas aeruginosa ( Pseudomonas fulva SY2-8, with accession number CGMCC NO. 25613.

2. The *Pseudomonas aeruginosa* as described in claim 1 (… Pseudomonas fulva Application of SY2-8 in the preparation of saline-alkali land improvement inoculants; The saline-alkali land improvement microbial agent has a salt tolerance of no more than 9% NaCl mass concentration; The alkali resistance of the saline-alkali land improvement microbial agent is a pH value not greater than 11.

3. The *Pseudomonas aeruginosa* as described in claim 1 ( Pseudomonas fulva Application of SY2-8 in the preparation of maize growth promoters.

4. The *Pseudomonas aeruginosa* as described in claim 1 (… Pseudomonas fulva Application of SY2-8 in increasing chlorophyll content in corn, cabbage or potatoes.

Images