Pseudomonas palleronii strain jct-2 and uses thereof

Abstract

The application discloses a Palleronii Pseudomonas strain Jct-2 and application thereof, relates to the field of microorganisms, and particularly relates to a Palleronii Pseudomonas strain (Pseudomonas palleronii) Pseudomonas palleroniana ) Jct-2 and a prevention and treatment effect on leaf spot disease. The Palleronii Pseudomonas strain (Pseudomonas palleronii) Pseudomonas palleroniana ) Jct-2 has a preservation number of CCTCC NO: M 20251022. Experiment proves that the Palleronii Pseudomonas strain Jct-2 is used for preparing products for inhibiting or preventing and treating leaf spot disease and a pathogenic bacterium thereof. Compared with prevention and treatment by using a chemical synthetic pesticide, the Palleronii Pseudomonas strain Jct-2 has advantages of high efficiency, low toxicity, no pollution to the environment and no drug resistance, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of *Pseudomonas pallens* (…). Pseudomonas palleroniana Jct-2 and its applications. Background Technology

[0002] Leaf spot is a widespread plant disease that can affect a variety of plant species, severely impacting their growth, development, and yield. It is typically caused by a variety of pathogens. The main pathogens are fungi, such as *Cercospora*, *Helminthosporium*, *Septoria*, *Phyllosticta*, and *Alternaria*. In addition, bacteria of the genera *Xanthomonas* and *Pseudomonas* can also cause leaf spot.

[0003] Both lemongrass and basil are plants of significant economic value and diverse uses. Lemongrass is commonly used in cooking, spice extraction, and has numerous applications in traditional medicine; basil, on the other hand, is a common herb, highly favored in cooking and aromatherapy. Basil, belonging to the genus *Ocimum* of the Lamiaceae family, is an annual herb native to tropical Asia and now widely distributed worldwide. Its plants are rich in various bioactive components, and its unique aroma, flavor, and pharmacological effects have made it an important part of human production and daily life, becoming a research hotspot for cross-disciplinary applications. For example, basil leaves are rich in vitamins (such as vitamin C, vitamin K, and vitamin A), minerals (such as calcium, iron, and magnesium), and antioxidants (such as flavonoids and phenolic compounds), making basil a potentially health-promoting edible and medicinal plant. 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. Meanwhile, basil essential oil can also be added to food and beverages as a natural flavoring 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 *Helicobacter pylori* (…). Bipolaris secalis This pathogen causes lemongrass leaves to turn yellow and curl, with brown or purple, irregularly shaped lesions. In severe cases, multiple lesions can merge, leading to extensive leaf tissue necrosis, leaf drying, and curling, significantly impacting the photosynthetic efficiency of lemongrass and thus reducing its growth vitality and yield. This not only affects the plant's appearance but also its edible value and aroma content. Basil leaf spot disease is caused by the pathogen Alternaria alternifolia (…). Alternariaalternata This disease is caused by infection. Symptoms of basil leaf spot are clearly visible on the leaves. In the early stages, small brown spots appear on the leaf surface, usually round or nearly round, and lighter in color, with slight yellowing of the surrounding tissue. In the middle stage of the disease, the spots gradually enlarge and turn dark brown, with clear edges. Multiple spots can merge to form large, irregularly shaped necrotic areas. The leaf tissue around the spots shows obvious yellowing or drying. In severe cases, the spots cover the entire leaf, causing the leaf to wither and fall off.

[0004] In current agricultural production practices, chemical control methods still dominate crop pest and disease control due to their advantages such as rapid effectiveness, ease of operation, and high economic benefits. However, the "3R" problems of chemical control—pesticide residues, resurgence of harmful organisms, and increased pathogen resistance—have become global challenges. In light of this, biological control, as a non-toxic, harmless, and pollution-free control strategy that is less likely to induce resistance, has gradually gained widespread attention, and related research and applications are continuously deepening. In recent years, biological control has become a hot research area in leaf spot disease control.

[0005] Pseudomonas pallensii ( P. palleroniana *Pseudomonas pallens* is an important species in the genus *Pseudomonas*, and is a Gram-negative bacterium. Under a microscope, it typically appears as a straight or slightly curved rod-shaped organism, generally ranging in size from 0.5 to 1.0 × 1.5 to 5.0 μm. A search revealed that *Pseudomonas pallens* (*Pseudomonas pallens*) is a Gram-negative bacterium. P. palleroniana Reported studies have focused on its control of damping-off in cucurbit seedlings, black shank in tobacco, blight in peppers, and anthracnose in tea. There are currently no reports on the control of leaf spot by *Pseudomonas pallens*. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain of Pseudomonas pallensii and its applications.

[0007] This invention provides a strain of *Pseudomonas pallens* ( Pseudomonas palleroniana Jct-2, the *Pseudomonas pallens* strain ( Pseudomonas palleroniana Jct-2 was deposited on May 12, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20251022, located at Wuhan University, Wuhan, China.

[0008] The colonies of the strain are slightly yellow, mostly round, with neat edges, smooth and glossy surfaces, a sticky and moist texture, and are semi-transparent. The colonies are relatively soft, with a diameter of 1-2 mm. The *Pseudomonas pallensii* Jct-2 was prepared by culturing in a culture medium containing a carbon source, a nitrogen source, water, and inorganic salts.

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

[0010] The present invention also provides a biocontrol agent prepared from the aforementioned Pseudomonas pallensii strain Jct-2 or its volatile substances.

[0011] This invention also protects the use of biocontrol agents in the preparation of products that inhibit leaf spot disease or the pathogens causing leaf spot disease.

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

[0013] Furthermore, the pathogen of the leaf spot disease is *Helicobacter ryegrass* (…). Bipolaris secalis ) or Alternaria alternifolia ( Alternaria alternata ).

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention isolates a strain of *Pseudomonas pallens*, which has a significant antibacterial effect, proving its potential as a biocontrol agent. The present invention combines *Pseudomonas pallens* with the pathogens of black spot disease in basil and sparse-haired basil (… A. alternata In a confrontation culture, the results showed that the biocontrol bacterium significantly inhibited the growth of *Alternaria alternata*, with an inhibition rate of 47.11 ± 6.39%. This biocontrol bacterium also showed resistance to *Helicobacter ryegrass*, the pathogen of lemongrass leaf spot disease. B. secalis In a confrontation culture experiment, the results showed that the biocontrol bacterium significantly inhibited the growth of *Alternaria alternata*, with an inhibition rate of 51.44±5.59%. In inoculation trials, the bacterium demonstrated control efficacy against black spot disease in basil and sparse-haired basil of 70.57±35.46% and 68.44±36.82%, respectively. This indicates that the biocontrol bacterium has a good inhibitory effect on *Alternaria alternata* and *Alternaria alternata*.

[0015] This invention is the first report of *Pseudomonas pallens* ( P. palleroniana Jct-2 exhibits excellent inhibitory effects against the plant pathogen Alternaria alternifolia, and also demonstrates good biocontrol efficacy against black spot disease in basil and basil sparse hairs caused by Alternaria alternifolia. This strain produces volatile compounds, which are the main components responsible for its antibacterial effect and control of plant diseases. The first discovery of this strain and the application of its novel functions enrich my country's resources of disease-resistant microorganisms. Attached Figure Description

[0016] Figure 1 The colony morphology of Pseudomonas pallensii Jct-2 on LB agar plates.

[0017] Figure 2The phylogenetic tree of Pseudomonas palleroniana Jct-2 strain constructed based on 16S rDNA gene.

[0018] Figure 3 Antagonistic effect of Pseudomonas palleroniana Jct-2 on Alternaria alternata, the pathogen of black spot disease of basil. Note: CK: control group; A: treatment group; *** between groups indicates P<0.001, significant difference.

[0019] Figure 4 Antagonistic effect of Pseudomonas palleroniana Jct-2 on B. graminum, the pathogen of leaf spot disease of lemongrass. Note: CK: control group; A: treatment group; *** between groups indicates P<0.001, significant difference.

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

[0021] Figure 6 Inhibitory effect of volatile compounds of Pseudomonas palleroniana Jct-2 on mycelial growth of B. graminum, the pathogen of leaf spot disease of lemongrass. Note: CK: control group; A: treatment group; *** between groups indicates P<0.001, significant difference.

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

[0023] Figure 8 Preventive effect of volatiles of Pseudomonas palleroniana Jct-2 on black spot disease of loose-hair basil. Note: CK: control group; A: treatment group; ** between groups indicates P<0.01, significant difference. DETAILED DESCRIPTION

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

[0025] Example 1 Isolation and preservation of Pseudomonas palleroniana (Palleronia) P. palleroniana

[0026] ​The strain Jct-2 obtained by purification from the healthy part of the Ocimum sanctum L. plant tissue was cultured on LB solid medium at 28°C for 3 days, and the colony morphology of the Jct-2 strain was observed. The colony of the Jct-2 strain was slightly yellow, most of the colonies were round, the edges were neat, the surface was smooth, and there was a certain sense of luster, the texture was sticky and moist, and the colony was translucent. The colony texture was relatively soft, and the colony diameter was about 1-2 mm, like Figure 1 The purified strain Jct-2 was inoculated into LB liquid medium and cultured at 28°C and 180 rpm until the logarithmic growth phase. The bacterial solution was mixed with 50% glycerol at a volume ratio of 1:1, and then was divided into sterile cryotubes and stored in a -80°C ultra-low temperature refrigerator.

[0027] Example 2 Identification of Pseudomonas palleroniana Jct-2

[0028] The antagonistic bacteria Jct-2 was inoculated into LB solid medium and cultured at 37°C for 24 h, and the morphological characteristics of the colony were observed and recorded. The 16S rRNA gene sequence of the strain Jct-2 was amplified using the universal primer 27F / 1492R, i.e., 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), using the bacterial 16S rRNA gene solution as the DNA template. The sequences of the universal primers are shown as SEQ ID NO:1-2. The total volume of the PCR reaction system was 25 μL, including 2 μL of the upstream primer, 2 μL of the downstream primer, 2 μL of the DNA template, 6.5 μL of ddH2O, and 12.5 μL of 2×PCR Master Mix. After amplification, a suitable amount of PCR product was subjected to gel electrophoresis detection. In the case where the target band was determined to exist, the remaining PCR product was sequenced, and the sequence is shown as SEQ ID NO:3, with a length of 1397 bp.

[0029] The 16S rRNA gene sequence of the strain Jct-2 was submitted to the NCBI database for sequence homology comparison. The comparison results showed that the 16S rRNA gene sequence of the strain Jct-2 had the highest similarity of 100% with the 16S rRNA gene sequence (accession number PP086517.1) of the strain 18ksbl17sm1. Pseudomonas palleroniana The 16S rRNA gene similarity analysis showed that the strain Jct-2 had the closest phylogenetic relationship with P. palleroniana . Palleroniana, and the strain Jct-2 was named P. palleroniana) Jct-2. The sequence of the obtained strain Jct-2 was used to construct a phylogenetic tree with the gene fragments of the closely related strains of the sequences with high homology and similarity in GenBank using the neighbor-joining (NJ) method. Strain Jct-2 was clustered with Pseudomonas palleroniana with a support of 96%, as shown in Figure 2 . Therefore, strain Jct-2 was identified as Pseudomonas palleroniana.

[0030] The inventors deposited the strain in the China Center for Type Culture Collection on May 12, 2025, with the accession number CCTCC NO: M 20251022, and the address of the depositary is Wuhan, China, Wuhan University.

[0031] Example 3 Antagonistic effect of Pseudomonas palleroniana Jct-2 on Alternaria alternata (Aa) A. alternata and Bipolaris sorokiniana (Bs) B. secalis

[0032] The plate streaking method was used to inoculate a 7-mm-diameter pathogenic fungus cake in the center of a PDA plate, and the endophytic bacterial liquid was inoculated at a distance of about 45 mm on one side. The experimental group and the control group were set up, and each treatment had 3 replicates. The plates were placed in a 28°C constant temperature incubator for culture, and the inhibition was observed.

[0033] The antagonistic effect of strain Jct-2 on the pathogenic fungus Alternaria alternata (Aa) A. alternata was counted, and the width of the inhibition zone was measured after 7 days. The inhibition of Aa by strain Jct-2 is shown in Figure 3 . The t-test was used for significant analysis of the pathogenic mycelium diameter data of the control group and the treatment group, and the visualization was presented by a column chart. It can be found that there is a significant difference between the mycelium diameter of the control group and the mycelium diameter after treatment with Jct-2. The mycelium diameters of the control group and the treatment group were 45.05 ± 0.35 mm and 23.21 ± 0.42 mm, respectively. The inhibition rate of strain Jct-2 on Aa A. alternata was 48.48 ± 1.33%.

[0034] The antagonistic effect of strain Jct-2 on the pathogenic fungus Bipolaris sorokiniana (Bs) B. secalis of lemon grass leaf spot was counted, and the width of the inhibition zone was measured after 7 days. The inhibition of Bs by strain Jct-2 is shown in Figure 4 ​As shown, the pathogen mycelium diameter data of the control group and the treatment group were subjected to significance analysis by t-test, and visualized by column chart. It can be found that there was a significant difference between the mycelium diameter of the control group and the mycelium diameter after Jct-2 treatment. The mycelium diameters of the control group and the treatment group were 55.60±0.27 mm and 22.28±0.74 mm, respectively. The inhibition rate of strain Jct-2 on B. secalis was 59.93±1.53%.

[0035] Example 4 Biocontrol effect of Pseudomonas plecogoniicola Jct-2 volatile

[0036] The antagonistic effect of the volatile of strain Jct-2 on Alternaria alternata and Helminthosporium catenatum, the pathogens of basil black spot and lemon grass leaf spot, respectively, was investigated by dual-plate cross-coupling. The selected antagonistic strain was added into a culture bottle containing LB liquid medium, and placed in a shaker at 28°C and 180 rpm for 72 h until the OD600 value of the culture solution was between 0.6 and 1. Thereafter, 100 μL of the antagonistic bacterial solution was evenly spread on a solid LB plate. A 6 mm diameter pathogen plug was placed in the center of a solid PDA plate. The above LB plate coated with the antagonistic bacterial solution was inverted and covered on the PDA plate containing the pathogen plug, and sealed with a sealing film to prevent the loss of volatile. As a control group, the solid LB plate coated with 100 μL of sterile water was inverted and covered on the PDA plate containing the pathogen plug and sealed. The control group and the experimental group were each set in triplicate, and the plates were incubated at 28°C for 72 h. The diameter of the pathogen mycelium was measured and the inhibition rate of the bacterial volatile was calculated.

[0037] The inhibition of the volatile of strain Jct-2 on A. alternata was as shown in Figure 5 The pathogen diameter data of the control group and the Jct-2 volatile treatment group were subjected to significance analysis by t-test, and visualized by column chart. The results showed that there was a significant difference in the pathogen mycelium diameter between the control group and the Jct-2 volatile treatment group. The pathogen diameters of the control group and the treatment group were 71.64±0.43 mm and 37.89±4.35 mm, respectively. The inhibition rate of the volatile of strain Jct-2 on A. alternata was 47.11±6.39%.

[0038] The inhibition of the volatile of strain Jct-2 on H. catenatum was as shown in Figure 6, the diameter of the pathogen was measured using the cross method. The results are shown in FIG. 6. The diameter of the pathogen in the control group and the Jct-2 volatile-treated group was 70.59 ± 0.38 mm and 34.28 ± 3.76 mm, respectively. The volatiles of the strain Jct-2 had a significant effect on the pathogenicity of the pathogen in the leaves of the Ocimum basilicum. A. alternata The inhibition rate was 51.44 ± 5.59%.

[0039] Example 5 Pseudomonas palleroniana P. palleroniana Inhibition of Pseudomonas palleroniana by Jct-2

[0040] The leaves of the aromatic plant inoculated with the pathogen were placed on the other side as the experimental group. The control group was coated with 100 μL of sterile water on one side of the dual-zone medium containing solid LB, and the leaves of the aromatic plant inoculated with the pathogen were placed on the other side. Both the control group and the experimental group were sealed with sealing film to prevent the loss of volatiles. Each group had 3 replicates. After 2-3 days, the size of the lesion was measured using the cross method.

[0041] The results of the effect of the volatiles of the antagonistic bacteria on the pathogenicity of the pathogen in the leaves of O. basilicum are shown in FIG. 7. The diameter of the lesion in the control group and the Jct-2 volatile-treated group was 3.77 ± 1.85 mm and 1.19 ± 0.47 mm, respectively. Compared with the control group, the size of the lesion in the leaves of the treatment group was significantly reduced, and the inhibition rate was 68.44 ± 36.82%. The volatiles of the strain Jct-2 had a significant effect on the occurrence of the leaf spot disease of O. basilicum. Figure 7

[0042] The results of the effect of the volatiles of the antagonistic bacteria on the pathogenicity of the pathogen in the leaves of O. basilicum are shown in FIG. 7. The diameter of the lesion in the control group and the Jct-2 volatile-treated group was 3.77 ± 1.85 mm and 1.19 ± 0.47 mm, respectively. Compared with the control group, the size of the lesion in the leaves of the treatment group was significantly reduced, and the inhibition rate was 68.44 ± 36.82%. The volatiles of the strain Jct-2 had a significant effect on the occurrence of the leaf spot disease of O. basilicum. Figure 8 The results of the effect of the volatiles of the antagonistic bacteria on the pathogenicity of the pathogen in the leaves of O. basilicum are shown in FIG. 7. The diameter of the lesion in the control group and the Jct-2 volatile-treated group was 3.77 ± 1.85 mm and 1.19 ± 0.47 mm, respectively. Compared with the control group, the size of the lesion in the leaves of the treatment group was significantly reduced, and the inhibition rate was 68.44 ± 36.82%. The volatiles of the strain Jct-2 had a significant effect on the occurrence of the leaf spot disease of O. basilicum. ​

[0043] The above results show that Pseudomonas pelioli strain Jct-2 is a high-efficiency biocontrol strain with significant inhibition of Alternaria alternata, the pathogen of basil black spot disease, and the volatile compounds produced by the strain can be used for the prevention and control of basil black spot disease and O. tenuissima black spot disease, or for the production of microbial preparations, microbial fertilizers, etc., and further applied to the prevention and control of basil black spot disease and O. tenuissima black spot disease.

Claims

1. Pseudomonas palleroniana strain Jct-2, characterized in that, The Palleronii Pseudomonas strain Pseudomonas palleroniana Jct-2 was preserved in China Center for Type Culture Collection on May 12, 2025, with a preservation number of CCTCC NO: M 20251022, and a preservation address of Wuhan, China, Wuhan University.

2. The Pseudomonas paucimobilis strain Jct-2 according to claim 1, characterized by: The colony of the strain is slightly yellow, mostly round, with neat edges, smooth surface, glossy, sticky and moist texture, translucent, soft colony texture, and colony diameter of 1-2 mm.

3. The Pseudomonas paucimobilis strain Jct-2 according to claim 1, characterized by: The Palleroniana Pseudomonas Jct-2 is obtained by culturing in LB medium.

4. The Palleronii Pseudomonas strain Jct-2 of claim 3, characterized by: The LB 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 Palleroniana Pseudomonas strain Jct-2 according to any one of claims 1-4.

6. Use of Pseudomonas palleroniana strain Jct-2 according to any one of claims 1 to 4 or of the biocontrol agent according to claim 5 for the manufacture of a product for inhibiting leaf spot or the pathogen of leaf spot, said leaf spot being Cymbopogon citratus leaf spot or Ocimum basilicum black spot, said pathogen of leaf spot being Pythium aphanidermatum (Drechsler 1916) Fitzpatrick 1923 or Alternaria alternata (Fr.) Keissler 1912. Bipolaris secalis .

7. Use of Pseudomonas palleroniana strain Jct-2 according to any one of claims 1 to 4 or of the biocontrol agent according to claim 5 for the manufacture of a product for inhibiting leaf spot or the pathogen of leaf spot, said leaf spot being Cymbopogon citratus leaf spot or Ocimum basilicum black spot, said pathogen of leaf spot being Pythium aphanidermatum (Drechsler 1916) Fitzpatrick 1923 or Alternaria alternata (Fr.) Keissler 1912. Alternaria alternata .

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