Pseudomonas sp. A-3 and its application in degradation of fungicide difenoconazole

Abstract

The application discloses a Pseudomonas sp. A-3, which is preserved in the Guangdong Microbial Culture Collection Center with a preservation number of GDMCC NO:63819; the strain is applied in degrading diafenthiuron, and experimental results show that the strain can effectively degrade diafenthiuron in a wide pH and temperature range, and can be applied in bioremediation of polluted environments such as water bodies or soil as an excellent pesticide degrading strain, the application provides a new way for treating pesticide residues, enriches a germplasm resource bank of pesticide degrading strains, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation technology, specifically relating to a strain of Pseudomonas sp. A-3 and its application in the degradation of difenoconazole. Background Technology

[0002] Difenoconazole belongs to the triazole systemic fungicide class. Due to its high efficiency and broad spectrum, it is widely used to effectively control fungal diseases caused by basidiomycetes and ascomycetes. It has been extensively researched and applied over the past 40 years, and is widely used for disease control in various crops, providing long-lasting protection, extending crop lifespan, and increasing yield, such as in rice, wheat, corn, and vegetables. It can inhibit many common crop pathogenic fungi, such as wheat scab, wheat powdery mildew, vegetable blight, rice sheath blight, rust, leaf spot, brown spot, and anthracnose. In addition, difenoconazole is also used to control fungal infections in fruit trees and turfgrass.

[0003] Due to its high stability and persistence, difenoconazole in soil migrates into the environmental media through rainwater runoff and surface runoff, posing a long-term risk to aquatic and soil ecosystems. It is difficult to degrade in soils in various regions within a short period, resulting in long-term environmental pollution and entry into the food chain, harming humans and other non-target organisms. Currently, various toxicological effects of difenoconazole on different organisms and its adverse effects throughout the entire life cycle have been reported. For example, difenoconazole can induce developmental toxicity, thyroid endocrine disorders, immune system damage, metabolic disorders, and reproductive toxicity; DNA damage and mitochondrial-mediated apoptosis; hepatotoxicity in zebrafish larvae and adults; and severe tissue lesions in spleen tissue. Furthermore, some studies have indicated that difenoconazole residues can enter water bodies through runoff and washout, potentially producing toxic effects on aquatic organisms and non-target species, such as inhibiting the growth and development of fish and algae, and also having a certain toxic effect on bees. In addition, it reduces soil bacterial community diversity and disrupts soil ecological balance. This demonstrates that difenoconazole poses a serious threat to ecosystems and human health.

[0004] Research on microbial degradation of pesticides began in the late 1940s. Among existing methods for treating pesticide residues, microbial degradation has attracted considerable attention from researchers both domestically and internationally due to its advantages of safety, low cost, ease of operation, and lack of secondary pollution. However, microbial degradation exhibits significant specificity, thus requiring the screening of highly efficient degrading strains. Currently, research on difenoconazole-degrading bacteria is very limited, and there are no reports on the bioremediation of difenoconazole. Summary of the Invention

[0005] To address the shortcomings of pesticide residue degradation and remediation technologies for difenoconazole fungicide, this invention provides a strain of Pseudomonas sp. A-3, which was deposited on September 18, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC NO:63819), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Another objective of this invention is to apply the aforementioned Pseudomonas sp. A-3 in the degradation of difenoconazole.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] 1. Soil was collected from the planting area of ​​Jijie Town, Xundian Hui and Yi Autonomous County, Kunming City, Yunnan Province. The soil was added to MSM liquid medium containing difenoconazole. After continuous enrichment culture, multiple strains with the ability to degrade difenoconazole were obtained. The degradation ability of the strains to difenoconazole was detected by high performance liquid chromatography (HPLC). Finally, the strain A-3 with the strongest degradation ability was selected for subsequent experiments.

[0009] 2. Identification of strain A-3

[0010] (1) Morphological characteristics of strain A-3: On LB medium, the colonies are round, slightly raised, milky yellow, moist, opaque, with neat edges and smooth surface. Under scanning electron microscopy, the bacterial cells can be observed to be rod-shaped.

[0011] (2) Molecular identification

[0012] Genomic DNA was extracted from strain A-3 using a bacterial genomic DNA extraction kit. Using the extracted genome as a template, PCR amplification was performed using 16S rDNA bacterial universal primers. After sequencing the PCR products, the sequencing results were compared with the sequences on NCBI. Based on morphological characteristics and molecular identification results, the strain was finally identified as Pseudomonas sp.

[0013] 3. When Pseudomonas aeruginosa A-3 was applied to degrade difenoconazole, the experimental results showed that the degradation rate of difenoconazole by this strain reached 62.6% after 7 days. The optimal degradation conditions for strain A-3 were pH 5 and an initial difenoconazole concentration of 200 mg·L⁻¹. -1 The temperature is 30℃.

[0014] The present invention has the following beneficial effects:

[0015] The Pseudomonas A-3 strain obtained by screening in this invention can be used to remediate natural environments such as water and soil contaminated with difenoconazole. The Pseudomonas A-3 strain provided by this invention enriches the germplasm resource bank of pesticide-degrading bacteria and has significant application value in the bioremediation of water and soil contaminated with difenoconazole residues. It provides a new development approach to break through the existing bottlenecks in the treatment of pesticide residue pollution. Attached Figure Description

[0016] Figure 1 The colony morphology characteristics of strain A-3 after 1 day of culture on LB solid medium;

[0017] Figure 2 This is a scanning electron microscope image of strain A-3;

[0018] Figure 3 Phylogenetic tree of 16S rDNA of strain A-3;

[0019] Figure 4 For OD 600 Fitting curves with corresponding colony counts;

[0020] Figure 5 This is a standard curve for the HPLC detection of difenoconazole;

[0021] Figure 6 The dynamic relationship between the growth and degradation of difenoconazole by strain A-3;

[0022] Figure 7 The degradation effect of strain A-3 on difenoconazole under different pH conditions;

[0023] Figure 8 The degradation effect of strain A-3 on difenoconazole at different temperatures;

[0024] Figure 9 The degradation effect of strain A-3 on different concentrations of difenoconazole; Detailed Implementation

[0025] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Those skilled in the art can implement the invention by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention;

[0026] The culture medium formulations in the following examples are as follows:

[0027] MSM liquid medium (1L): (NH4)2SO4 2.0g, CaCl2·2H2O 0.01g, FeSO4·7H2O 0.001g, Na2HPO4·12H2O 1.5g, MgSO4·7H2O 0.2g, KH2PO4 1.5g, pH 8.0; MSM solid medium is prepared by adding 17-20g of agar powder to each 1L of liquid medium.

[0028] LB liquid medium: 5.0g yeast extract, 10.0g peptone, 10.0g sodium chloride, 1000mL deionized water, pH 7.2, sterilized at 121℃ for 20min; LB solid medium: 17-20g agar powder added to every 1L of liquid medium.

[0029] Example 1: Isolation and Identification of Strain A-3

[0030] 1. Screening and isolation of difenoconazole-degrading strains:

[0031] Soil samples were collected from the planting area in Jijie Town, Xundian Hui and Yi Autonomous County, Kunming City, Yunnan Province. 5g of soil sample was added to 50mL of MSM liquid medium containing difenoconazole (50mg / L). After incubation at 30℃ and 200rpm for 3 days, 20% of the culture solution was inoculated into MSM liquid medium containing 100mg / L difenoconazole and incubated for 3 days. This process was repeated, with the concentration of difenoconazole in the MSM liquid medium being sequentially increased to 200mg / L, 400mg / L, 800mg / L, and 1000mg / L. The final concentration of the culture solution was then serially diluted 10-fold to 1×10⁻⁶. -7 The culture was then spread on MSM solid medium containing 50 mg / L difenoconazole and incubated upside down at 30°C for 2 days. After single colonies grew on the plates, single colonies were picked and streaked multiple times for purification to obtain purified strains A-1, A-2, and A-3.

[0032] Three bacterial strains were expanded in LB liquid medium and then inoculated at a 20% inoculum into 50 mL of MSM liquid medium containing difenoconazole (50 mg / L). After 7 days of culture, the concentration of difenoconazole in the culture medium was detected by high performance liquid chromatography (HPLC). The concentration of difenoconazole was determined based on the size of the HPLC peak area. A control group without inoculation was also included. The results showed that the degradation rate of strain A-1 was 52.33%, strain A-2 was 52.20%, strain A-3 was 65.59%, and the degradation rate of the blank control was 33.94%. The results indicated that strain A-3 had the strongest degradation ability for difenoconazole, and strain A-3 was selected for subsequent experiments.

[0033] 2. Identification of strain A-3

[0034] (1) Morphological identification

[0035] Strain strain A-3 was inoculated onto LB agar plates and incubated upside down at 30°C for 1 day. Colony morphology was then observed. Colonies cultured on LB plates for 1 day were round, slightly raised, milky yellow, moist, opaque, with neat edges and a smooth surface (see...). Figure 1 Under a scanning electron microscope, the bacterial cells can be observed to be rod-shaped (see...). Figure 2 );

[0036] (2) Molecular biological identification of 16S rDNA

[0037] Genomic DNA was extracted from strain A-3 using a bacterial genomic DNA extraction kit (Solepro). Following the kit's instructions, genomic DNA was extracted from strain A-3. Using the extracted genome as a template, PCR amplification was performed using universal 16S rDNA primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The obtained 16S rDNA sequences were aligned to the GenBank database using BLAST. Phylogenetic trees were constructed using MAGE 5.0 software to analyze evolutionary relationships, and the results are shown below. Figure 3 The 16S rDNA sequence of strain A-3 isolated and purified by this invention has 99% homology with Pseudomonas putida strain MnS2201005, and is the closest in evolutionary distance; its morphological characteristics and scanning electron microscopy observation characteristics are also most similar to those of Pseudomonas, therefore strain A-3 is identified as Pseudomonas sp.

[0038] Example 2: Degradation experiment of p-difenoconazole by strain A-3

[0039] (1) Seed liquid preparation

[0040] The purified strain A-3 was inoculated into 100 mL of LB liquid medium and activated overnight at 30 °C and 150 rpm until the logarithmic phase. 30 mL of the culture was transferred into a 50 mL sterile centrifuge tube and centrifuged at 4 °C and 4000 rpm for 10 min. The bacterial cells were collected, rinsed with sterile water, and then 30 mL of sterile water was added to prepare the seed culture.

[0041] (2) Degradation performance determination

[0042] Seed culture was inoculated into 50 mL of liquid medium containing 50 mg / L difenoconazole MSM at an inoculation rate of 20%. A control group without inoculation was also included. Each group was divided into three replicates. The culture was incubated at 30°C and 150 rpm in a shaker for 7 days. Samples were taken daily, and the OD of the culture medium was measured using a spectrophotometer. 600 The amount of difenoconazole in MSM liquid culture medium was determined by HPLC each day.

[0043] Chromatographic conditions: An Agilent 1260 Infinity II high-performance liquid chromatograph was used; the chromatographic column was Elite C10. 18 The reversed-phase column (Supersil AQ-C18, 250nm×4.60mm, 5μm) was used. The injection temperature was 25℃, the injection volume was 20μL, the flow rate was 1.000mL / min, and the mobile phase was V(acetonitrile)∶V(water, pH adjusted to 3.0 with acetic acid)=70∶30. The detection wavelength was 230nm.

[0044] Dilute bacterial suspensions of different concentrations to 1×10⁻⁶. -7 Subsequently, the bacterial culture was spread onto LB solid medium and the colony count was recorded. Simultaneously, the OD600 values ​​of different concentrations of bacterial solutions were measured using a spectrophotometer. Finally, a curve was fitted using the colony counts corresponding to different OD values, with three replicates for each concentration. The results are shown in [Figure number missing]. Figure 4 ;

[0045] Quality control: Standard curves were prepared using the external standard method to calibrate standard reference materials according to conventional methods. Figure 5 );

[0046] according to Figure 4 Calculate the number of bacterial strains in the 7-day sample, and simultaneously calculate the degradation rate of difenoconazole in the 7-day sample according to the degradation rate formula. Difenoconazole degradation rate (%) = [1-(A1 / A0)]×100%, where A1 is the concentration of difenoconazole in the culture medium after 7 days of treatment with strain A-3, and A0 is the initial concentration of difenoconazole in the culture medium;

[0047] See results Figure 6Strain A-3 rapidly degraded difenoconazole in the early stages of cultivation. With prolonged cultivation, the degradation of difenoconazole pesticide was positively correlated with the growth of strain A-3. Under difenoconazole as the sole carbon source, strain A-3 exhibited no growth or degradation lag phase. The degradation rates reached 51.9% and 60.52% after 1 and 2 days of cultivation, respectively. The degradation rate slowed after 3 days, reaching 62.6% by 7 days. Furthermore, the bacterial population growth slowed after 6 days, with the bacterial count almost matching that of 7 days. Data from 0 to 2 days showed that the degradation rate of difenoconazole increased with the continuous increase in bacterial density. On 6 days of cultivation, strain A-3 entered a stationary phase, at which point 61.7% of the difenoconazole was degraded. After 7 days, the strain began to decline, and the bacterial density decreased. On 7 days, the degradation rate of difenoconazole by strain A-3 was measured at 62.6%. The control group (7-day natural degradation rate) was only 21.44%.

[0048] The results showed that strain A-3 could use difenoconazole as the sole carbon and energy source for growth and reproduction. At a difenoconazole concentration of 50 mg / L, the degradation rate was 51.9% after 1 day of cultivation and 62.6% after 7 days of cultivation, indicating that the strain has the ability to efficiently and rapidly degrade difenoconazole.

[0049] Example 3: Study on the degradation characteristics of p-difenoconazole by strain A-3

[0050] (1) Effect of pH on the degradation of difenoconazole by strain A-3

[0051] Seed culture was inoculated into 50 mL of MSM liquid medium at different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) at a 20% inoculation rate, and difenoconazole was added to achieve a final concentration of 50 mg / L. Each group was replicated three times. After incubation at 30℃ and 150 rpm in a constant temperature shaker for 5 days, the degradation of difenoconazole by strain A-3 under different pH conditions was determined by HPLC.

[0052] See results Figure 7 As shown in the figure, this bacterium can degrade difenoconazole within a pH range of 5.0 to 9.0, with the best degradation effect at pH 5.0. The degradation efficiency is better under acidic or alkaline conditions than under neutral conditions. This may be because, during the microbial degradation of difenoconazole, some enzymes are more able to catalyze the demethylation reaction of difenoconazole under acidic or alkaline environments, ultimately degrading difenoconazole into non-toxic or less toxic compounds.

[0053] (2) Effect of temperature on the degradation of difenoconazole by strain A-3

[0054] Seed culture was inoculated into 50 mL of MSM medium at a 20% inoculation rate, and difenoconazole was added to achieve a final concentration of 50 mg / L. The culture was then placed in a constant temperature shaker at 150 rpm at 20℃, 25℃, 30℃, 35℃, and 40℃, with three replicates per group. After 5 days of culture, the degradation of difenoconazole by strain A-3 under different culture temperature conditions was determined by HPLC.

[0055] See results Figure 8 As shown in the figure, strain A-3 can degrade difenoconazole within the temperature range of 20–40℃, with the highest degradation rate (approximately 76.89%) at 30℃. The degradation ability decreases above 30℃, reaching approximately 63.54% at 35℃ and approximately 45.47% at 40℃.

[0056] (3) Effect of initial concentration of difenoconazole on degradation performance of strain A-3

[0057] Seed culture was inoculated into 50 mL of MSM liquid medium containing different initial concentrations of difenoconazole (20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 400 mg / L) at a 20% inoculation rate. Each group was inoculated in triplicate. After culturing at 30°C and 150 rpm for 5 days, the degradation of difenoconazole by the strain at different initial concentrations was determined by HPLC.

[0058] See results Figure 9 As shown in the figure, this strain can degrade difenoconazole in the range of initial concentrations of 20–400 mg / L. When the initial concentration of difenoconazole is 200 mg / L, strain A-3 has the best degradation performance. When the initial concentration is increased to 400 mg / L, the degradation rate decreases rapidly.

Claims

1. A strain of Pseudomonas ( Pseudomonas Application of sp.) A-3 in the degradation of difenoconazole; The accession number of Pseudomonas A-3 at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC NO:63819.

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