Low-temperature-resistant pseudomonas with biocontrol effect and application of low-temperature-resistant pseudomonas

Abstract

The invention discloses a low-temperature-resistant pseudomonas with a biocontrol effect and application thereof, relates to the field of microorganisms, and aims to solve the problem that the pathogen antagonism capability is reduced due to the fact that the activity of the existing biocontrol strain is inhibited in a low-temperature environment. The pseudomonas is pseudomonas koreana B12, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is February 17, 2025, and the preservation number is CGMCC No.33538. The pseudomonas koreana B12 can be applied to the field of biological engineering. The low-temperature-resistant pseudomonas disclosed by the invention is used for inhibiting phytopathogens at a low temperature of 5-15 DEG C and is used for decomposing organic phosphorus, glucan, starch and cellulose.

Description

Technical Field

[0001] The present invention relates to the field of biological control of agricultural diseases, and in particular to a cold-resistant Pseudomonas bacterium with biological control function and application thereof. Background Art

[0002] Crop diseases are one of the main factors affecting crop yield and quality. Traditional disease control relies primarily on chemical pesticides. However, overreliance on chemical pesticides not only leads to increased resistance among pathogens, but also causes soil degradation and agricultural product safety risks, which is contrary to the national policy goals of green agricultural development and the reduction of chemical fertilizers and pesticides. Biological control is a green, safe, and environmentally friendly means of managing agricultural diseases. However, due to the living nature of biocontrol agents, their activity is affected by many factors, among which low temperatures are one of the main reasons for inhibiting the activity of biological agents. In cold regions, the long duration of low temperatures in winter makes it difficult for traditional agricultural microbial agents to colonize in the soil and significantly reduces their ability to antagonize pathogens. This directly leads to problems such as frequent soil-borne diseases, slow straw decomposition, and reduced fertilizer utilization. Summary of the Invention

[0003] The present invention aims to solve the problem that the activity of existing biocontrol strains is inhibited in a low-temperature environment, resulting in a decrease in the ability to antagonize pathogens, and to provide a low-temperature resistant Pseudomonas strain with biocontrol effects and its application.

[0004] The present invention provides a cold-resistant Pseudomonas with biocontrol function, which is Pseudomonas koreensis B12. It has been deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 17, 2025, and the deposit number is CGMCC No. 33538.

[0005] The cold-resistant Pseudomonas bacteria of the present invention have regular round, opaque colonies with a moist and smooth surface, a uniform, slightly transparent, light yellow texture, and a uniform color on both sides. The bacteria are short rod-shaped with blunt ends, and some of the bacteria are slightly curved.

[0006] The Gram staining result of the cold-resistant Pseudomonas sp. of the present invention is negative. Among 21 typical biochemical experiments, 5 reactions are positive, specifically β-galactosidase activity test, arginine, lysine, ornithine, and oxidase reaction are positive, and the remaining 16 reactions are negative.

[0007] The 16S rDNA sequence alignment analysis of the present invention clustered with Pseudomonas koreensis (CP111114) into a highly reliable clade (99%), demonstrating a strong genetic relationship between the two. Combining bacterial morphology, growth conditions, and physiological and biochemical characterization results, the present invention's psychrotolerant Pseudomonas was confirmed to be Pseudomonas koreensis.

[0008] The low-temperature-resistant pseudomonas of the present invention is used for inhibiting plant pathogens at low temperatures.

[0009] Furthermore, the low temperature is 5-15°C.

[0010] Furthermore, the plant pathogens are corn stalk rot, beans anthracnose, rice seedling bakanae, tomato gray mold and Rhizoctonia solani.

[0011] The cold-resistant pseudomonas of the present invention is used for decomposing organic phosphorus, glucan, starch and cellulose.

[0012] Beneficial effects of the present invention:

[0013] The present invention screened a functional strain with low-temperature adaptability from soil environments in high-altitude cold regions. This strain, Pseudomonas Koreanus, can stably exert its biological control effects at temperatures between 5 and 15°C. It exhibits broad-spectrum antibacterial properties, inhibiting corn stalk rot, bean anthracnose, rice seedling bakanae, tomato gray mold, and Rhizoctonia solani to varying degrees. The Pseudomonas Koreanus strain also has the ability to decompose organic phosphorus, glucans, starch, and cellulose.

[0014] This strain breaks through the low temperature limitations of traditional microbial agents and provides a green control solution for agriculture in northern cold regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the Gram staining result of Pseudomonas Koreana B12;

[0016] Figure 2 This is the phylogenetic tree of Pseudomonas koreacens B12;

[0017] Figure 3 A plate confrontation of multiple pathogenic fungi for Pseudomonas koreacens B12;

[0018] Figure 4 These are the results of the metabolite production capacity test of Pseudomonas koreacens B12. DETAILED DESCRIPTION

[0019] The following embodiments of the present invention are described in detail. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation plans and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0020] Example 1:

[0021] The cold-resistant Pseudomonas with biocontrol activity in this embodiment is Pseudomonas koreana B12, which has been deposited at the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on February 17, 2025, and the deposit number is CGMCC No. 33538.

[0022] The isolation method of the cold-resistant Pseudomonas in this embodiment is as follows:

[0023] The strain was isolated from soil samples 0-10 cm around the tubers of continuously planted potatoes (mature stage) in the Daxing'anling region. Take 1 g of soil sample and add it to a 250 mL Erlenmeyer flask containing 99 mL of sterile distilled water and several glass beads (diluted 100 times). Shake well and shake in a low-temperature constant temperature shaker at 15°C and 150 rpm for 20 minutes to fully disperse the soil sample in the water. Take 1 mL of the suspension and mix it with 9 mL of sterile water to make 10 -3 Repeat this process to obtain 10 -4 , 10 -5 , 10 -6 Dilution: Take 100 μL of different gradient dilutions and evenly spread them on the surface of beef extract peptone solid culture medium plate, and invert it in a constant temperature incubator at 15°C for culture.

[0024] Rice blast pathogens were activated two to three times on PDA solid medium plates. A hole punch was used to evenly punch holes in the activated plates. A bacterial cake was placed in the center of the PDA plate. The selected strain was streaked 3 cm from the cake and incubated in a 20°C incubator for 7 days. After the blank control plate had completely covered the entire plate, the plate was removed and plates with a clear inhibition zone between the antagonist and pathogen colonies were selected. The pathogen colony area was measured using ImageJ software, and the inhibition rate of the antagonist was calculated. Inhibition rate = (blank control pathogen colony area - experimental control pathogen colony area) / blank control pathogen colony area × 100%. Strain B12 was screened and found to have an inhibition rate of 63.75% against the rice blast pathogen at 15°C.

[0025] Example 2: Identification of strain B12

[0026] 1. Morphological observation of strains:

[0027] After culturing strain B12 on PDA solid medium at 20°C for 24 hours, the colonies were observed to be regular, round, opaque, with a moist and smooth surface, a uniform, slightly transparent, light yellow texture, and the color of the front and back of the colonies was consistent. Gram staining was negative (e.g. Figure 1 ), the bacteria are short rod-shaped with blunt ends, and some bacteria are slightly curved.

[0028] 1. Physiological and biochemical identification of strains:

[0029] Strain B12 was subjected to physiological and biochemical identification using the Bojian Gram-negative bacteria identification system. The results are shown in Table 1. This system includes 21 typical biochemical tests. The color results of each reaction were converted to an octal number. Using the standard database query system provided by Qingdao Hi-Tech Park Haibo Biotechnology, the relative probability of antagonistic strain identification, T value, and R value were obtained, providing a certain degree of identification of the strain. Of the 21 typical biochemical tests, five reactions, including β-galactosidase activity, arginine, lysine, ornithine, and oxidase reactions, were positive, while the remaining 16 were negative. After converting the test results into octal numbers and entering them into the standard database query system, the identification result indicated that strain B12 belonged to the genus Pseudomonas.

[0030] Table 1

[0031]

[0032] 2. Molecular biological identification of strains:

[0033] The genome of strain B12 was extracted and sequenced for 16S rDNA identification. The universal primers 27F / 1492R for bacterial identification were used to amplify the gene fragment of 1391 bp. The sequence is shown in SEQ ID NO: 1 in the sequence list. The sequence was Blast aligned in NCBI. The 16S rDNA sequences of adjacent species were selected and a phylogenetic tree was constructed using Mega7.0. Figure 2 The results showed that strain B12 and Pseudomonas koreensis (CP111114) clustered into a highly reliable branch (99% confidence), indicating that the two strains were highly related.

[0034] According to the Bergey's Manual of Bacterial Identification and the Manual of Common Bacterial Identification, strain B12 was identified as Pseudomonas koreensis based on the strain's morphology, physiological and biochemical characteristics and 16SrDNA sequence analysis.

[0035] Example 3: Determination of broad-spectrum antibacterial performance

[0036] Pseudomonas koreensis B12 was plated against each of the pathogens Fusarium graminearum, Colletotrichum gloeosporioides, Fusarium moniliforme, Botrytis cinerea, and Rhizoctonia solani. Figure 3 As shown in Table 2, Pseudomonas koraiensis B12 has different degrees of inhibitory effect on pathogenic bacteria. The inhibition rate is shown in Table 2. It has broad-spectrum antibacterial properties and has the potential to play a biocontrol role against a variety of fungal crop diseases.

[0037] Table 2 Inhibition rate of strain B12 against various pathogenic fungi

[0038] serial number pathogens Antibacterial rate 1 Corn stalk rot (Fusarium graminearum) 60.83±1.18 2 Colletotrichum gloeosporioides 60.92±1.12% 3 Fusarium moniliforme 55.25±0.31% 4 Tomato gray mold (Botrytis cinerea) 59.83±0.31% 5 Rhizoctonia solani 51.25±0.74%

[0039] Example 4: Test of bacterial strain metabolic capacity

[0040] Determination of organophosphate degradability: Pseudomonas koreanicus B12 was tested using Montana organophosphate medium to determine its organophosphate degradability. Strain B12 was inoculated in the center of the culture medium and incubated at 15°C for 48 hours. The B12 colonies were observed for the formation of a clearing zone around them. Results showed a 0.5 cm clearing zone around the Pseudomonas koreanicus B12 colony, indicating that the strain possessed a moderate organophosphate degradability.

[0041] Dextranase production capacity was determined using a dextran-Congo red medium to identify the ability of Pseudomonas koreana B12 to produce glucanase. Strain B12 was inoculated in the center of the identification medium and incubated at 15°C for 48 hours. After incubation, the B12 colonies were observed for the formation of a clearing zone around them. Results showed a 2.6 cm clearing zone around the Pseudomonas koreana B12 colony, indicating that the strain had strong glucanase production capacity.

[0042] Cellulase production capacity was determined using carboxymethyl cellulose culture medium to identify the cellulase-producing ability of Pseudomonas koreana B12. Strain B12 was inoculated in the center of the culture medium and incubated at 15°C for 48 hours. After incubation, the plate was stained with Congo red (1 mg / mL) for 30 minutes. The plate was then immersed in NaCl (1 mol / L) for 20 minutes. After rinsing with sterile water, the plate was observed for the formation of a clearing zone around the B12 colonies. The results showed that a 2.10 cm clearing zone formed around the Pseudomonas koreana B12 colonies, indicating that the strain had strong cellulase production capacity.

[0043] Amylase production capacity was determined using amylase culture medium to identify the amylase-producing ability of Pseudomonas koreana B12. Strain B12 was inoculated in the center of the identification medium and incubated at 15°C for 48 hours. After incubation, the surface of the medium was thoroughly covered with iodine solution (1 mg / mL). Color was developed for 60 minutes, the iodine solution was removed, and the formation of a clearing zone around the B12 colony was observed. Results showed that a 1.4 cm clearing zone formed around the Pseudomonas koreana B12 colony, indicating that the strain had strong amylase production capacity.

[0044] Through the above metabolite production capacity determination, it was found that Pseudomonas koreanicus B12 could form transparent circles on the surface of identification culture medium when decomposing organophosphorus, glucan, starch and cellulose. Figure 4 As shown. It has the ability to decompose organic phosphorus, glucan, starch and cellulose.

[0045] Example 5: Low temperature stability test of strains

[0046] Single colonies of Pseudomonas koronicum B12 were streaked onto multiple LB medium plates and incubated at 15°C, 10°C, and 5°C, with three replicates at each temperature. Cultivation revealed that cultivation at 15°C required 1-2 days, 3-4 days at 10°C, and 6-8 days at 5°C. Pseudomonas koronicum B12 grew stably within the low-temperature range of 5-15°C, and its growth rate gradually slowed with decreasing temperature (15°C → 10°C → 5°C), with the time it took for colonies to appear increasing from 1-2 days to 5-6 days. This indicates that Pseudomonas koronicum B12 possesses significant cold adaptability and metabolic stability, making it a typical psychrotrophic bacterium.

[0047] The experimental results showed that Pseudomonas koreensis B12 can grow stably at 5-15℃ and play a biocontrol role.

[0048] Example 6: Strain safety test

[0049] Hemolysis test: prepare 1.0×10 8 A suspension of strain B12 at 10 CFU / mL was streaked onto the surface of a blood agar plate using a sterile disposable inoculating loop. The plate was then incubated at 28°C for 24 hours and observed for the formation of hemolytic rings. Staphylococcus aureus, a known hemolytic-positive strain, was used as a control. While clear hemolytic rings formed around the positive S. aureus colonies in the control group, no clear hemolytic rings formed around the B12 colonies, indicating that strain B12 is not hemolytic.

[0050] Antimicrobial susceptibility test: Take 100 μL of strain B12 suspension (1.0×10 8CFU / mL) were inoculated onto the surface of a MH agar plate. Spread evenly with a disposable sterile applicator, cover the plate, and allow to air dry at room temperature for 5 minutes. Escherichia coli was used as a quality control strain. Sterile antimicrobial susceptibility paper strips were applied to the surfaces of the B12 and E. coli quality control plates, with four strips placed on each plate. The plates were then incubated at 28°C for 24 hours, and the formation of inhibition zones around the antimicrobial susceptibility paper strips was observed. The results are shown in Table 3. The results showed that the Escherichia coli quality control strain was extremely sensitive to all drugs, indicating that the activity of the drug sensitivity paper was qualified and the operation was correct; Korean Pseudomonas B12 was highly sensitive to carbapenems (meropenem), cephalosporins (ceftazidime) and aminoglycosides (amikacin); it was lowly sensitive to β-lactams (piperacillin) and fluoroquinolones (ciprofloxacin), so strain B12 may show a certain degree of resistance to these two classes of drugs.

[0051] Table 3

[0052]

[0053] Drug sensitivity determination criteria: inhibition zone diameter >20mm, extremely sensitive; 15-20mm, highly sensitive; 10-14mm, moderately sensitive; <10mm, low sensitive; 0mm, insensitive.

Claims

1. A cold-resistant Pseudomonas strain with biocontrol properties, characterized in that: The Pseudomonas is Pseudomonas koreensis B12, which has been deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is February 17, 2025, and the deposit number is CGMCC No. 33538.

2. Use of the cold-resistant Pseudomonas as claimed in claim 1 in inhibiting plant pathogens at low temperatures.

3. The use according to claim 1, characterized in that The low temperature is 5-15°C.

4. The use according to claim 1, characterized in that The plant pathogens are corn stalk rot fungus, bean anthracnose fungus, rice seedling bakanae fungus, tomato gray mold fungus and Rhizoctonia solani. The psychrotolerant Pseudomonas according to claim 1 is used for decomposing organophosphorus, glucan, starch and cellulose.

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