Pseudomonas pakerneli strain Jct-2 and application of pseudomonas pakerneli strain Jct-2

Abstract

The invention discloses a Pseudomonas paunoniana strain Jct-2 and application thereof, relates to the field of microorganisms, and in particular relates to a Pseudomonas paunoniana strain Jct-2 and a prevention and treatment effect of the Pseudomonas paunoniana strain Jct-2 on a leaf spot disease. The preservation number of the Pseudomonas palustris Jct-2 is CCTCC (China Center for Type Culture Collection) NO: M 20251022, and the preservation number of the Pseudomonas palustris Jct-2 is CCTCC NO: M 20251022. Experiments prove that the Pseudomonas pakerneli strain Jct-2 is applied to preparation of products for inhibiting or preventing and treating the leaf spot disease and pathogenic bacteria of the leaf spot disease. Compared with the prevention and control by using chemically synthesized pesticides, the compound is from the natural environment, has the advantages of high efficiency, low toxicity, no environmental pollution, low possibility of generating drug resistance and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a strain of Pseudomonas paleronis ( Pseudomonas palleroniana )Jct-2 and its applications. Background Art

[0002] Leaf spot is a widespread plant disease that can infect a wide range of plant species, severely negatively impacting growth, development, and yield. Leaf spot is typically caused by a variety of pathogens. These pathogens are primarily fungi, such as Cercospora, Helminthosporium, Septoria, Phyllosticta, and Alternaria. Bacteria from the genera Xanthomonas and Pseudomonas can also cause leaf spot.

[0003] Both lemongrass and basil are economically valuable plants with diverse uses. Lemongrass is commonly used in cooking, spice extraction, and traditional medicine; basil, on the other hand, is a common herb favored in cooking and aromatherapy. Basil, an annual herb in the genus Ocimum, belongs to the Lamiaceae family. It is native to tropical Asia and is now widely distributed worldwide. Rich in a variety of bioactive compounds, its unique aroma, flavor, and pharmacological properties have made it a vital component of human life and a research hotspot for cross-disciplinary applications. For example, basil leaves are rich in vitamins (such as C, K, and A), minerals (such as calcium, iron, and magnesium), and antioxidants (such as flavonoids and phenolic compounds), making it a promising edible and medicinal plant with potential health benefits. The unique aroma of basil essential oil makes it highly sought after in the fragrance industry. High-purity basil essential oil can be obtained through extraction processes such as distillation and pressing, and is widely used in perfumes, cosmetics, detergents, and other daily chemical products. At the same time, basil essential oil can also be added as a natural flavor to food and beverages to develop new flavor products and meet consumers' demand for natural and healthy food additives. However, leaf spot disease has a significant impact on their growth and quality. Lemongrass leaf spot disease is caused by Helicoverpa truncatula ( Bipolaris secalis ). After this pathogen infects lemongrass, the leaves of the lemongrass plant turn yellow and curl, and the lesions are brown or purple with irregular shapes. In severe cases, multiple lesions will merge with each other, causing large areas of leaf tissue to necrotize, and the leaves to dry and curl, greatly affecting the photosynthesis efficiency of the lemongrass, thereby reducing its growth vitality and yield. This not only affects the appearance of the plant, but also affects the edible value and aroma content of its leaves. Basil leaf spot disease is caused by the pathogen Alternaria alternata ( Alternaria alternata ) infection. Basil leaf spot symptoms are noticeable on the leaves. In the early stages of the disease, tiny brown lesions appear on the leaf surface. These are usually circular or nearly circular, light in color, and the surrounding tissue turns slightly yellow. In the middle stages of the disease, the lesions gradually enlarge and turn dark brown with distinct edges. Multiple lesions may fuse, forming large, irregular necrotic areas. The mesophyll tissue surrounding the lesions becomes noticeably yellow or withers. In severe cases, the lesions can cover the entire leaf, causing it to wither and fall.

[0004] In current agricultural production practices, chemical control methods still dominate crop pest and disease control due to their rapid effectiveness, ease of use, and high economic returns. However, the "3R" issues associated with chemical control—pesticide residues, the resurgence of pests, and the development of pathogen resistance—have become a global challenge. Consequently, biological control, as a non-toxic, harmless, pollution-free control strategy that is less susceptible to developing resistance, has gradually garnered widespread attention, with research and application continuing to deepen. In recent years, biological control has become a hot topic in research on leaf spot control.

[0005] Pseudomonas paleronii ( P. palleroniana ) is an important species of Pseudomonas, a Gram-negative bacterium. Observed under a microscope, it usually appears as a straight or slightly curved rod, with a size generally ranging from 0.5 to 1.0 × 1.5 to 5.0 μm. P. palleroniana ), reported studies have focused on its use against damping-off disease in melon seedlings, tobacco black shank, pepper blight, and tea anthracnose. Currently, there are no reports on the use of Pseudomonas paleronis in the control of leaf spot. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a strain of Pseudomonas paleroni and applications thereof.

[0007] The present invention provides a strain of Pseudomonas paleronii ( Pseudomonas palleroniana ) Jct-2, the Pseudomonas paleronii strain ( Pseudomonas palleroniana ) Jct-2 was deposited in the China Center for Type Culture Collection on May 12, 2025, with the deposit number CCTCC NO: M 20251022, and the deposit address is Wuhan University, Wuhan, China.

[0008] The strain produces slightly yellowish colonies that are mostly round, have neat edges, a smooth, shiny surface, a sticky, moist texture, and are translucent. The colonies are relatively soft and have a post-colonial diameter of 1 to 2 mm. The Pseudomonas paleronisi Jct-2 is cultured in a culture medium comprising a carbon source, a nitrogen source, water, and inorganic salts.

[0009] Furthermore, the culture medium is LB culture medium.

[0010] The present invention also provides a biocontrol agent, which is prepared from the Pseudomonas paleroni strain Jct-2 or a volatile substance thereof.

[0011] The present invention also protects the use of the biocontrol agent in preparing a product for inhibiting leaf spot disease or the pathogenic bacteria of leaf spot disease.

[0012] Furthermore, the leaf spot disease is citronella leaf spot disease or basil black spot disease.

[0013] Furthermore, the pathogen of the leaf spot disease is Helicoverpa truncatula ( Bipolaris secalis ) or Alternaria interspersata ( Alternaria alternata ).

[0014] Compared with the prior art, the present invention has the following beneficial effects: the present invention isolates a strain of Pseudomonas paleronis, which has a significant antibacterial effect, proving its potential as a biocontrol agent. The present invention isolates Pseudomonas paleronis and the pathogen of black spot disease of basil and basil pubescent ( A. alternata ) were cultured in a confrontation culture. The results showed that the biocontrol bacteria had a significant inhibitory effect on the growth of Alternaria alternata, with an inhibition rate of 47.11±6.39%; ... the pathogen of lemongrass leaf spot disease, Helicoverpa truncatula ( B. secalis ) in a standoff culture. The results showed that the biocontrol agent significantly inhibited the growth of Helminthosporium tiliaceum, with an inhibition rate of 51.44±5.59%. Inoculation experiments, the agent demonstrated efficacy against black spot disease in basil and basil of pubescent cultivars of 70.57±35.46% and 68.44±36.82%, respectively. This suggests that the biocontrol agent has a strong inhibitory effect against Alternaria alternata and Helminthosporium tiliaceum.

[0015] The present invention reports for the first time that Pseudomonas paleronii ( P. palleroniana Jct-2 has a strong inhibitory effect against the plant pathogen Alternaria alternata and demonstrates excellent biocontrol effectiveness against black spot disease of basil and basil caused by Alternaria alternata. This strain produces volatile compounds, which are the primary components for antibacterial activity and plant disease control. The discovery of this strain and its application of new functions have enriched my country's resources for disease-resistant microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colony morphology of Pseudomonas paleroni Jct-2 on LB plate.

[0017] Figure 2This is the phylogenetic tree of Pseudomonas paleroni Jct-2 strain constructed based on the 16S rDNA gene.

[0018] Figure 3 The antagonistic effect of Pseudomonas paleroni Jct-2 against Alternaria alternata, the pathogen of basil black spot. Note: CK: control group; A: treatment group; *** indicates significant difference between groups (P < 0.001).

[0019] Figure 4 The antagonistic effect of Pseudomonas paleroni Jct-2 against Helminthosporium tiliaceum, the pathogen of citronella leaf spot. Note: CK: control group; A: treatment group; *** indicates significant difference between groups (P < 0.001).

[0020] Figure 5 The inhibitory effect of volatile compounds from Pseudomonas paleroni Jct-2 on the mycelial growth of Alternaria alternata, the pathogen of basil black spot. Note: CK: control group; A: treatment group; *** indicates significant difference between groups (P < 0.001).

[0021] Figure 6 The inhibitory effect of volatile compounds from Pseudomonas paleroni Jct-2 on the mycelial growth of Helminthosporium teganophyllum, the pathogen of citronella leaf spot. Note: CK: control group; A: treatment group; *** indicates significant difference between groups (P < 0.001).

[0022] Figure 7 The control effect of volatiles of Pseudomonas paleroni Jct-2 on black spot of basil. Note: CK: control group; A: treatment group; ** indicates significant difference between groups (P < 0.01).

[0023] Figure 8 The control effect of volatiles of Pseudomonas paleroni Jct-2 on black spot of Ocimum pubescens. Note: CK: control group; A: treatment group; ** indicates significant difference between groups (P < 0.01). DETAILED DESCRIPTION

[0024] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0025] Example 1 Pseudomonas paleronii ( P. palleroniana ) separation and preservation The strain Jct-2 isolated and purified from the healthy part of Ocimum pubescens plant tissue was cultured in LB solid medium at 28°C for 3 days. The colony morphology of the Jct-2 strain was observed. The colonies were slightly yellow, mostly round, with neat edges, smooth surface, a certain gloss, sticky and moist texture, translucent, and relatively soft. The diameter of the colonies was about 1-2 mm. Figure 1 The purified strain Jct-2 was inoculated into LB liquid medium and cultured at 28°C and 180 rpm with shaking until the logarithmic growth phase. The bacterial solution was mixed with 50% glycerol at a volume ratio of 1:1, dispensed into sterile cryovials, and stored in a -80°C ultra-low temperature freezer.

[0026] Example 2 Identification of Pseudomonas paleroni Jct-2 The antagonistic bacterium Jct-2 was inoculated into LB solid medium and cultured at 37°C for 24 h. The morphological characteristics of its colonies were observed and recorded. The antagonistic bacterium liquid was used as a DNA template, and the 16S rRNA gene sequence of strain Jct-2 was amplified using the bacterial 16S rRNA gene amplification universal primers 27F / 1492R, i.e., 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The sequences of the universal primers are shown in SEQ ID NO:1-2. The total volume of the PCR reaction system was 25 μL, including 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of DNA template, 6.5 μL of ddH2O, and 12.5 μL of 2×PCR Master Mix. After amplification, an appropriate amount of PCR product was taken for gel electrophoresis detection. When the target band was confirmed, the remaining PCR product was sequenced. The sequence is shown in SEQ ID NO: 3, with a length of 1397 bp.

[0027] The 16S rRNA gene sequence of strain Jct-2 was submitted to the NCBI database for sequence homology comparison. Pseudomonas palleroniana The 16S rRNA gene sequence of strain 18ksbl17sm1 (accession number PP086517.1) had the highest similarity, reaching 100%. 16S rRNA gene similarity analysis showed that strain Jct-2 had the highest similarity with P. palleroniana The strain Jct-2 was named Pseudomonas paleroni (P. palleroniana) Jct-2. The obtained strain Jct-2 sequence was compared with the gene fragments of closely related strains with high homology and similarity in GenBank using the nearest neighbor method (NJ) to construct a phylogenetic tree. Strain Jct-2 was clustered with Pseudomonas paleroni with a support of 96%, as shown in the following figure. Figure 2 Therefore, strain Jct-2 was identified as Pseudomonas paleroni.

[0028] The inventor deposited the strain in the China Center for Type Culture Collection on May 12, 2025, with the deposit number CCTCC NO: M 20251022, and the deposit address is Wuhan University, Wuhan, China.

[0029] Example 3 Effect of Pseudomonas paleroni Jct-2 on the pathogen Alternaria alternata ( A. alternata ) and Helminthosporium tiliaceum ( B. secalis ) antagonistic effect Using the streak method, a 7 mm diameter pathogen cake was inoculated in the center of a PDA plate. Endophytic bacterial liquid was streaked approximately 45 mm from one side. Experimental and control groups were set up, with three replicates for each treatment. The plates were incubated at 28°C, and the inhibition was observed.

[0030] The statistical analysis of the strain Jct-2 against the pathogen of basil black spot disease, Alternaria alternata ( A. alternata The antagonistic effect of strain Jct-2 on Alternaria alternata was measured after 7 days. Figure 3 As shown in the figure, the t-test was used to analyze the significance of the mycelial diameter data of the control group and the treatment group, and the data were visualized by a bar graph. It can be found that there is a significant difference between the mycelial diameter of the control group and the mycelial diameter after Jct-2 treatment. The mycelial diameters of the control group and the treatment group are 45.05±0.35 mm and 23.21±0.42 mm, respectively. A. alternata The inhibition rate was 48.48±1.33%.

[0031] The statistical analysis of the strain Jct-2 against the pathogen of lemongrass leaf spot ( B. secalis The antagonistic effect of strain Jct-2 on Helminthosporium ryegrass was measured after 7 days. Figure 4 As shown in the figure, the t-test was used to analyze the significance of the mycelial diameter data of the control group and the treatment group, and the data were visualized by a bar graph. It can be found that there is a significant difference between the mycelial diameter of the control group and the mycelial diameter after Jct-2 treatment. The mycelial diameters of the control group and the treatment group are 55.60±0.27 mm and 22.28±0.74 mm, respectively. B. secalis The inhibition rate was 59.93±1.53%.

[0032] Example 4 Biocontrol Effects of Volatiles from Pseudomonas paleroni Jct-2 The antagonistic effects of volatiles from strain Jct-2 against the pathogens of basil black spot, Alternaria alternata, and Citronella tiliacea, the leaf spot pathogen of citronella, were investigated using a double-plate method. Two μL of the selected antagonistic strains were added to a flask containing LB liquid medium. The culture was incubated in a shaker at 28°C and 180 rpm for 72 hours until the OD600 value of the culture reached between 0.6 and 1. Subsequently, 100 μL of the antagonistic bacterial solution was evenly spread onto a solid LB plate. A 6 mm diameter pathogen cell was placed in the center of a solid PDA plate. The LB plate coated with the antagonistic bacteria was inverted and placed on the PDA plate containing the pathogen cell. The plate was sealed with parafilm to prevent loss of volatiles. As a control group, a solid LB plate coated with 100 μL of sterile water and a PDA plate containing pathogenic bacteria were inverted and sealed. Three replicates were set for both the control and experimental groups. The plates were incubated at 28°C for 72 h. The diameter of the pathogen hyphae was measured, and the inhibition rate of the bacterial volatiles was calculated.

[0033] The inhibitory effect of the volatiles of strain Jct-2 on Alternaria alternata is shown in the figure. Figure 5 The t-test was used to analyze the significance of the pathogen diameter data of the control group and the Jct-2 volatiles treatment group, and the data were visualized by bar graphs. The results showed that there was a significant difference in the diameter of the pathogen hyphae between the control group and the group treated with Jct-2 volatiles. The diameters of the pathogens in the control group and the treatment group were 71.64±0.43 mm and 37.89±4.35 mm, respectively. A. alternata The inhibition rate was 47.11±6.39%.

[0034] The inhibitory effect of the volatiles of strain Jct-2 on Helminthosporium tiliaceum is shown in the figure. Figure 6 The t-test was used to analyze the significance of the pathogen diameter data of the control group and the Jct-2 volatiles treatment group, and the data were visualized by bar graphs. The results showed that there was a significant difference in the diameter of the pathogen hyphae between the control group and the group treated with Jct-2 volatiles. The diameters of the pathogens in the control group and the treatment group were 70.59±0.38 mm and 34.28±3.76 mm, respectively. A. alternata The inhibition rate was 51.44±5.59%.

[0035] Example 5 Pseudomonas paleroni P. palleroniana Inhibitory effect of Jct-2 on black spot disease of Ocimum basilicum and Ocimum pubescens 100 μL of the antagonistic bacterial solution was evenly applied to one side of a two-zone medium containing solid LB, and the spice plant leaves inoculated with the pathogen were placed on the other side as the experimental group. For the control group, 100 μL of sterile water was applied to one side of the two-zone medium containing solid LB, and the spice plant leaves inoculated with the pathogen were placed on the other side. Both zones were sealed with parafilm to prevent the loss of volatiles. Three replicates were set up for each control and experimental group. Lesion size was measured 2–3 days later using the cross-hatch method.

[0036] The results of the effects of volatiles from antagonistic bacteria on the pathogenicity of Ocimum pubescens leaves infected by pathogens are as follows: Figure 7 The lesion diameter data for the control and Jct-2 volatiles-treated groups were analyzed using a t-test for significance and visualized using a bar graph. The results showed significant differences between the control and Jct-2 volatiles-treated groups. The lesion diameters of the control and Jct-2 volatiles-treated groups were 3.77 ± 1.85 mm and 1.19 ± 0.47 mm, respectively. Compared with the control group, the leaf lesion size in the treated group was significantly reduced, with an inhibition rate of 68.44 ± 36.82%. Volatiles from strain Jct-2 are significantly effective in preventing and controlling black spot disease of Ocimum pubescens.

[0037] The results of the effects of antagonistic bacterial volatiles on the pathogenicity of basil leaves infected by pathogens are as follows: Figure 8 The lesion diameter data for the control and Jct-2 volatiles-treated groups were analyzed using a t-test for significance and visualized using a bar graph. The results showed significant differences between the control and Jct-2 volatiles-treated groups. The lesion diameters of the control and Jct-2 volatiles-treated groups were 10.26±4.56 mm and 3.02±1.58 mm, respectively. Compared with the control group, the leaf lesion size in the treated group was significantly reduced, with an inhibition rate of 70.57±35.46%. Volatiles from strain Jct-2 are significantly effective in preventing and controlling black spot disease in basil.

[0038] The above results show that Pseudomonas paleronisi strain Jct-2 is a highly effective biocontrol strain that can significantly inhibit the pathogen Alternaria alternata of basil black spot disease. The volatile compounds it produces can be used to prevent and control basil black spot disease and basil black spot disease, or used in the production of microbial preparations, microbial fertilizers, etc., and then used in the prevention and control of basil black spot disease and basil black spot disease.

Claims

1. Pseudomonas paleroni strain Jct-2, characterized in that The Pseudomonas paleronii strain ( Pseudomonas palleroniana ) Jct-2 was deposited in the China Center for Type Culture Collection on May 12, 2025, with the deposit number CCTCC NO: M 20251022, and the deposit address is Wuhan University, Wuhan, China.

2. The Pseudomonas paleronisi strain Jct-2 according to claim 1, characterized in that: The colonies of the strain are slightly yellow, mostly round, with neat edges, smooth and shiny surfaces, sticky and moist textures, and are translucent. The colonies are relatively soft and have a diameter of 1 to 2 mm after colonization.

3. The Pseudomonas paleronisi strain Jct-2 according to claim 1, characterized in that: The Pseudomonas paleroni Jct-2 is cultured in LB medium.

4. The Pseudomonas paleronisi strain Jct-2 according to claim 3, characterized in that: The LB culture medium comprises a carbon source, a nitrogen source, water and inorganic salts.

5. A biocontrol agent, characterized in that The biocontrol agent is prepared from the Pseudomonas paleroni strain Jct-2 or its volatile substances according to claim 3.

6. Use of the Pseudomonas paleronisi strain Jct-2 according to claim 1 or 2 or the biocontrol agent according to claim 5 in the preparation of a product for inhibiting leaf spot disease or the pathogen of leaf spot disease.

7. The method according to claim 6, wherein: The leaf spot disease is citronella leaf spot disease or basil black spot disease.

8. The method according to claim 6, wherein: The pathogen of the leaf spot disease is Helicoverpa truncatula ( Bipolaris secalis ) or Alternaria interspersata ( Alternaria alternata ).

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