Pseudomonas chlororaphis strain LQ29 and application thereof

Abstract

This invention discloses a strain of Pseudomonas aeruginosa, LQ29, and its applications. LQ29 can produce volatile substances such as tert-butanol, N-methylthioformamide, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane, which can inhibit Rhizoctonia graminearum and Phytophthora cirrhosa, and can be used for the biological control of tobacco diseases such as tobacco black shank.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Pseudomonas aeruginosa, LQ29, and its applications. Background Technology

[0002] Tobacco black shank is one of the most devastating diseases in tobacco production. It is an important soil-borne oomycete disease caused by Phytophthora infestans, which mainly infects the roots and base of the stem of tobacco plants. When the disease occurs in mature plants, it often forms large, sunken black lesions on the stem, hence the name "black shank".

[0003] The pathogen of tobacco black shank is Phytophthora parasitica var. nicotianae (Bredadehean) Tuker, which belongs to the kingdom Algae, class Oomycetes, order Peronomycetes, and genus Phytophthora.

[0004] Tobacco black shank is prevalent worldwide, particularly in temperate, subtropical, and tropical regions, affecting nearly 40 countries and regions. In China, it is widely distributed, with the most severe outbreaks in Henan, Shandong, and Anhui provinces, and also relatively common in Hunan, Hubei, Fujian, Guangdong, Guangxi, Yunnan, Guizhou, and Sichuan provinces.

[0005] Currently, there is a lack of effective measures for the prevention and control of tobacco black shank, especially after the disease occurs. In production, it is still necessary to adhere to the control strategy of "prevention first, comprehensive control," emphasizing prevention and mastering the methods of pesticide application. With the development of pollution-free tobacco production, biological control of black shank, as a major disease in tobacco production, has gradually gained attention and become one of the important measures for its control. Summary of the Invention

[0006] To address the above-mentioned technical shortcomings, this invention provides a *Pseudomonas aeruginosa* strain LQ29 and its applications.

[0007] This invention is achieved through the following technical solution:

[0008] Rhizosphere soil samples were collected from healthy tobacco plants in tobacco fields with severe black shank disease. Bacteria were isolated from the soil samples using the plate dilution method, and Pseudomonas chlororaphis strain LQ29 with preservation number CGMCC No. 27828 was obtained.

[0009] The aforementioned *Pseudomonas aeruginosa* can produce volatile substances such as tert-butanol, N-methylthioformamide, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane.

[0010] The highest concentration of the volatile substance produced was N-methylthioformamide, at 1.22 mg / L, followed by hexamethylcyclotrisiloxane at 0.72 mg / L.

[0011] The present invention provides a strain of Pseudomonas aeruginosa, LQ29, which can be used in the biological control of plant diseases.

[0012] The *Pseudomonas aeruginosa* strain LQ29 provided by this invention can be used in the prevention and control of tobacco black shank disease.

[0013] The *Pseudomonas aeruginosa* strain LQ29 provided by this invention can be used to inhibit *Phytophthora indicum*.

[0014] This invention demonstrates that the volatile substances produced by the *Pseudomonas aeruginosa* strain LQ29 can inhibit *Rhizoctonia graminearum*, and the *Pseudomonas aeruginosa* strain LQ29 provided by this invention can be used to inhibit *Rhizoctonia graminearum*.

[0015] This invention demonstrates that the volatile substances produced by the *Pseudomonas aeruginosa* strain LQ29 can promote the growth of *Arabidopsis thaliana*, and the *Pseudomonas aeruginosa* strain LQ29 provided by this invention can be used to promote the growth of *Arabidopsis thaliana*.

[0016] The beneficial effects of this invention are:

[0017] The *Pseudomonas aeruginosa* strain LQ29 provided by this invention can produce volatile substances such as tert-butanol, N-methylthioformamide, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane, which can inhibit *Rhizoctonia graminearum* and *Phytophthora indicum*, and can be used for the biological control of diseases such as tobacco black shank.

[0018] The biological sample preservation information is as follows: Pseudomonas chlororaphis strain LQ29, preservation number CGMCCNo.27828, preservation date July 7, 2023, depositary address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. A certificate of viability has been issued by the depositary. Attached Figure Description

[0019] Figure 1 The inhibitory effect of strain LQ29 on Phytophthora indica is shown, where a represents the control (CK) and b represents LQ29.

[0020] Figure 2 These are the morphological characteristics of strain LQ29.

[0021] Figure 3 The bacterial cell concentration is 1×10 8 Photographs showing the inhibitory effect of volatile substances produced by strain LQ29 on Phytophthora indicum, where a represents 100 μL; b represents 50 μL; c represents 30 μL; and d represents the control (CK).

[0022] Figure 4 The bar chart shows the inhibitory effect of volatile substances from strain LQ29 on Phytophthora indica. Different letters in the chart indicate significant differences (p < 0.05).

[0023] Figure 5 This is a chromatogram of volatile substances from strain LQ29 analyzed by HS-SPME / GC-MS.

[0024] Figure 6 These images show the inhibitory effects of three compounds (pure) on *Phytophthora indicum*, based on the volatile substances produced by strain LQ29. In the images, a represents methanol (CK1); b represents LQ29 bacterial culture (CK2); c represents a 100 μL mixture of the three substances; df represents 30 μL, 50 μL, and 100 μL of tert-butanol; gi represents 30 μL, 50 μL, and 100 μL of hexamethylcyclotrisiloxane; and jl represents 30 μL, 50 μL, and 100 μL of N-methylthioformamide.

[0025] Figure 7 These are photographs showing the inhibitory effects of three compounds on other pathogenic fungi, where a is methanol (CK); b is tert-butanol; c is hexamethylcyclotrisiloxane; d is N-methylthioformamide; and e is a mixture of the three.

[0026] Figure 8 These are photographs showing the effects of three compounds on Arabidopsis thaliana growth, where a is methanol (CK1); b is water (CK2); c is tert-butanol; d is hexamethylcyclotrisiloxane; e is N-methylthioformamide; and f is a mixture of the three compounds.

[0027] Figure 9 These are photos showing the effects of three compounds on the prevention and treatment of tobacco black shank, where a is water (CK); b is tert-butanol; c is hexamethylcyclotrisiloxane; d is N-methylthioformamide; and e is a mixture of the three. Detailed Implementation

[0028] Example 1: Isolation and identification of strain LQ29.

[0029] Rhizosphere soil samples were collected from healthy tobacco plants in a tobacco field in Weifang, Shandong Province, where black shank disease was severe. Bacterial isolation was performed on the soil samples using the plate dilution method. 10g of soil sample was weighed and added to an Erlenmeyer flask containing 90mL of sterile water to prepare a soil suspension. The soil suspension was then serially diluted 10-fold with sterile water to prepare 10... -2 —10 -5Gradient soil dilution suspensions were prepared by pipetting 100 μL of each dilution onto NA solid medium. Each dilution was repeated three times. The plates were incubated at 28°C for 5 days. Single colonies that grew on the plates were streaked for purification and then inoculated onto NA medium plates for storage.

[0030] The antibacterial activity of each isolated strain was determined using a plate confrontation culture method. Preserved *Phytophthora nicotine* strains were activated and cultured on PDA medium. After the mycelium had fully colonized the entire plate, a 5 mm diameter mycelial cake was collected. 10 μL LOD was added to the center of each PDA medium plate. 600 A 0.5 μL bacterial suspension was inoculated with *Phytophthora indicum* bacterial pellets at 2.5 cm distance from the center on both sides. The pellets were incubated at 28°C for 5 days. A culture plate containing only *Phytophthora indicum* served as a control. Each strain was replicated three times. The antagonistic effect of each bacterial strain against *Phytophthora indicum* was determined by measuring the radius of the pathogen, and the inhibition rate was calculated. The bacterial strains with the strongest inhibitory effect were selected. Inhibition rate = (Diameter of pathogen in control group - Diameter of pathogen in treatment group) / Diameter of pathogen in control group × 100%.

[0031] According to the *Handbook of Systematic Identification of Common Bacteria*, strains with strong antagonistic ability were selected and plated on NA solid medium plates. After single colonies grew, their morphology was observed, including size, color, and surface shape, and further confirmation was performed using molecular identification. DNA was extracted from the bacterial strains using a DNA kit (Nanjing Vazyme Biotech Co., Ltd., China). The 16S rDNA gene sequence of the strains was amplified by PCR using universal primers 27F (5'AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'GGTTACGTTACGACTT-3'). The PCR reaction conditions were: 94℃, 5 min; (94℃, 40 s; 55℃, 40 s; 72℃, 1 min) 30 cycles, 72℃, 1 min, 4℃, and then stored. The PCR products were detected by agarose gel electrophoresis and sequenced by Shanghai Sangon Biotech Co., Ltd. The determined base sequence results were analyzed for sequence homology using the BLAST program.

[0032] One bacterial strain with strong antibacterial activity against Phytophthora tobaccois was screened from 71 bacterial strains isolated from tobacco rhizosphere soil and named LQ29. Figure 1 In the plate confrontation experiment, this strain had a strong inhibitory effect on Phytophthora nicotineis, with an inhibition rate of 82.81% (Table 1).

[0033] Table 1. Inhibitory effect of LQ29 on Phytophthora indicata.

[0034] CK 2.50±0.14a / LQ29 0.43±0.04c 82.81±0.23a

[0035] Observe the bacterial colony morphology on NA solid medium. Figure 2 The cells of strain LQ29 are reddish-orange in color, with a smooth, slightly raised surface and regular edges. Sequencing analysis of the PCR amplification product revealed that the full-length 16S rDNA sequence of strain LQ29 consists of 1434 bases, and its accession number in GenBank is ON860634. Homology analysis showed that LQ29 exhibits high similarity (99%) to *Pseudomonas aeruginosa*. Therefore, strain LQ29 was identified as *Pseudomonas aeruginosa*.

[0036] Strain LQ29 has been deposited at the China General Microbiological Culture Collection Center of the China Microbiological Culture Collection Committee.

[0037] Example 2: Inhibitory effect of volatile substances from strain LQ29 on Phytophthora indicans

[0038] The inhibitory effect of volatile substances from strain LQ29 on *Phytophthora indicum* was determined using a petri dish inverting method. Strain LQ29 was inoculated into NB medium and cultured in shake flasks for 48 hours. Its OD value was then adjusted... 600 =1. Take 30 μL, 50 μL, and 100 μL of bacterial suspension respectively and transfer them to NA plates, spreading them evenly using a sterile spreader. Take a 5 mm diameter mycelial disc from a plate contaminated with *Phytophthora nicotine* and inoculate it into the center of a new PDA plate. Invert the evenly spread NA plate onto the PDA plate containing *Phytophthora nicotine* and seal it, with the PDA plate on top. Repeat each treatment three times. Spread sterile water on the control plate. Incubate the tightly sealed plates in a 28°C dark incubator for 7 days. Observe and measure the diameter of *Phytophthora nicotine* in the treatment and control groups, and calculate the inhibition rate of LQ29 against *Phytophthora nicotine*. Inhibition rate = (Diameter of pathogen in control group - Diameter of pathogen in treatment group) / Diameter of pathogen in control group × 100%.

[0039] The test results of the petri dish inverted are as follows Figure 3 , Figure 4 As shown, the growth of *Phytophthora indicum* was significantly inhibited after treatment with the volatile substances of LQ29, showing a marked difference from the control. The treatment with 100 μL of bacterial suspension exhibited the strongest inhibitory effect on *Phytophthora indicum*, with an inhibition rate of 84.46%. Treatments with 50 μL and 30 μL of suspension showed inhibition rates of 74.7% and 69.51%, respectively. These results indicate that the volatile substances produced by strain LQ29 can effectively inhibit the growth of *Phytophthora indicum*, and the inhibitory effect is directly proportional to the amount of bacterial suspension used.

[0040] Example 3: Analysis of volatile components of strain LQ29

[0041] Volatile substances released by strain LQ29 were analyzed using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Strain LQ29 was inoculated into headspace vials containing NA medium and incubated at 28°C in the dark for 5 days. Volatile substances were then collected. A fiber extraction tip was placed in the GC-MS inlet, inserted into the headspace vial, and then withdrawn. Extraction was performed using ethanol at a 1:1 mass ratio. Ethanol was added, and the extraction was carried out ultrasonically at 60°C for 2 hours. The ethanol solution was then used for testing. Headspace vials containing NA medium but not inoculated with LQ29 served as blank controls. GC-MS analysis was performed using an Agilent 7890A gas chromatography system and mass spectrometer. The system used an Agilent DM-WAX2 column (30m × 250μm × 0.25μm) in splitless mode with an injection port temperature of 250℃. Ammonia was used as the carrier gas, the septum purge flow rate at the inlet was 3 mL / min, the total flow rate was 24 mL / min, and the split ratio was 20:1. The column oven temperature was maintained at 40℃ for 5 min, then increased to 240℃ at a rate of 10℃ / min and held for 3 min. The pre-injection, transfer line, quadrupole, and ion source temperatures were 260℃, 250℃, 150℃, and 250℃, respectively. The energy in electron collision mode was 70 eV. Mass spectrometry data were acquired in full scan mode, with an m / z range of 20–400. Database matching was used to select components with a relative peak area greater than 1% and both RSI and SI greater than 800 for dynamic component analysis.

[0042] Table 2. Analysis of volatile components in strain LQ29

[0043]

[0044] The volatile substances of strain LQ29 collected in headspace vials were adsorbed using headspace solid-phase microextraction (HS-SPME) and then analyzed by gas chromatography-mass spectrometry (GC-MS). Volatile substances with a relative peak area >1% were selected. The results are as follows: Figure 5 As shown in Table 2, strain LQ29 produced a total of 5 volatile substances, namely tert-butanol, N-methylthioformamide, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane. Among them, N-methylthioformamide had the highest concentration at 1.22 mg / L, followed by hexamethylcyclotrisiloxane at 0.72 mg / L.

[0045] Example 4: Determination of the inhibitory effect of some volatile substances (compounds) on Phytophthora indicans.

[0046] Based on the concentrations of various volatiles produced by strain LQ29, the inhibitory effects of single substances and mixtures of tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane on the mycelial growth of *Phytophthora nicotine* were determined using bipartite culture dishes. The test compounds were dissolved and diluted with methanol at the concentrations listed in Table 2. One half of the bipartite culture dish was filled with PDA medium, and the other half was placed with a 6 cm long and 1 cm wide sterile filter paper strip. *Phytophthora nicotine* mycelial cakes with a diameter of approximately 5 mm were inoculated onto the side of the bipartite culture dish containing PDA medium. The side containing the sterile filter paper strip was infiltrated with 30 μL, 50 μL, and 100 μL of the single compound solution, respectively; the volume of the mixed solution of the three compounds was 100 μL. A sterile filter paper strip infiltrated with 100 μL of LQ29 bacterial solution served as a positive control (Control 2), and a sterile filter paper strip infiltrated with 100 μL of methanol served as a negative control (Control 1). Each treatment was repeated three times. All plates used in the experiment were sealed and placed in a dark incubator at 28°C for 7 days. The antibacterial rate was then determined using the formula described above.

[0047] Table 3. Inhibitory effects of three compounds and their mixtures on Phytophthora indicum.

[0048]

[0049] The inhibitory effects of single compounds and mixtures on Phytophthora tobaccora were determined using bipartite culture dishes, as follows: Figure 6 As shown in Table 3, compared with Control 1, the three selected compounds and their mixture all showed significant antibacterial activity against *Phytophthora indicum*. N-methylthioformamide showed the best effect, with an inhibition rate of 73.17% at the maximum dosage. Tert-butanol and hexamethylcyclotrisiloxane showed inhibition rates of 66.22% and 69.27% ​​at the maximum dosage, respectively, with the inhibition rate directly proportional to the dosage. However, compared with the single substance and the positive control, the mixture of the three substances showed stronger antibacterial activity, with an inhibition rate of 82.32%. Therefore, this indicates that these three compounds all have good antibacterial effects against *Phytophthora indicum*, playing a significant inhibitory role in the volatile substances produced by strain LQ29, and the mixture of the three compounds achieves a better inhibitory effect against *Phytophthora indicum*.

[0050] Example 5: Determination of the antifungal activity of three compounds and their mixtures against other pathogenic fungi.

[0051] The inhibitory effects of single substances and mixtures of tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane on other common pathogenic fungi were determined using bipartite culture dishes. One half of the bipartite culture dish was filled with PDA medium, and the other half held sterile filter paper strips measuring 6 cm in length and 1 cm in width. *Rhizoctonia graminearum*, *Fusarium verticillata*, *Fusarium oxysporum*, *Fusarium graminearum*, and *Leuconostoc mesenteroides* (approximately 5 mm in diameter) were inoculated onto one side of the bipartite culture dish containing PDA medium. The side containing the sterile filter paper strip was infiltrated with 100 μL of the single compound or its mixture, and the inhibitory effects on the five common pathogenic fungi were determined. All substances were dissolved and diluted with methanol at the same concentration as above. Sterile filter paper strips infiltrated with an equal volume of methanol served as controls. Each treatment was repeated three times. All plates were sealed and incubated in a dark incubator at 28°C for 7 days. The inhibition rate was calculated using the same formula as above.

[0052] Table 4. Inhibitory effects of the three compounds and their combinations on other pathogenic fungi.

[0053]

[0054]

[0055] Table 4 shows the inhibitory effects of the three compounds and their mixtures on other root and stem pathogens, as determined using bipartite culture dishes. The results indicate that the three compounds and their mixtures had no significant inhibitory effect on *Leuconostoc mesenteroides*, *Fusarium graminearum*, *Fusarium verticillatum*, and *Fusarium oxysporum*, but exhibited good inhibitory effects on *Rhizoctonia graminearum*. Figure 7 Among the various antibacterial agents, the mixture of three substances showed the strongest antibacterial activity, with an inhibition rate of 41.67%. The antibacterial rates of the other substances were 37.38% for tert-butanol, 34.88% for hexamethylcyclotrisiloxane, and 27.98% for N-methylthioformamide. Further research is needed to investigate the antibacterial mechanism of these volatile substances against pathogens.

[0056] Example 6: Determination of the effects of three compounds and their mixtures on Arabidopsis thaliana growth.

[0057] Arabidopsis seeds were placed in sterile Petri dishes and soaked in 75% alcohol for 1 minute. The alcohol was then removed using a pipette, and the seeds were soaked in 3% sodium hypochlorite solution for 10 minutes. After 10 minutes, the sodium hypochlorite solution was removed using a pipette, and the seeds were rinsed 3-4 times with sterile water. Ten seeds were then inoculated into each half of a bipartite Petri dish containing MS medium. A 6cm long and 1cm wide sterile filter paper strip was placed in the other half of the dish. The Petri dishes were sealed and placed in a 4°C refrigerator for 2 days to vernalize the Arabidopsis seeds. After 2 days, the vernalized seed plates were placed in a 26°C light incubator for alternating light and dark incubation for 2 weeks. After Arabidopsis seeds germinated, 100 μL of individual solutions of tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane, or their mixtures, were separately applied to sterile filter paper strips in the other half of a two-compartment culture dish. The concentrations used were the same as above. Controls were prepared by applying equal volumes of methanol and water. Each treatment was repeated three times. The culture dishes were then sealed tightly and returned to an incubator under light for three weeks of further cultivation. Changes in Arabidopsis growth were then observed.

[0058] Table 5. Growth-promoting effects of three compounds and their mixtures on Arabidopsis thaliana.

[0059] Clear water 9.05±0.78d / methanol 9.17±0.42d / tert-Butanol 23.85±1.48b 2.64 Hexamethylcyclotrisiloxane 28.50±1.27a 3.15 N-methylthioformamide 25.60±0.99b 2.83 mix 19.2±0.85c 2.12

[0060] The effects of each compound on Arabidopsis growth were determined using bipartite culture dishes. The results showed that all three substances, individually and in combination, significantly promoted the growth of Arabidopsis, such as... Figure 8 As shown in Table 5, observations revealed that the number of leaves and leaf area of ​​Arabidopsis treated with each substance increased, and the fresh weight differed significantly from the control. The statistical results showed that the fresh weight of Arabidopsis treated with water and methanol was 9.05 mg and 9.17 mg, respectively, while the fresh weights of Arabidopsis treated with tert-butanol, hexamethylcyclotrisiloxane, N-methylthioformamide, and mixtures reached 23.85 mg, 28.50 mg, 25.60 mg, and 19.2 mg, respectively, all significantly promoting Arabidopsis growth. Hexamethylcyclotrisiloxane showed the best growth-promoting effect.

[0061] Example 7: Effects of three compounds and their mixture on the prevention and treatment of tobacco black shank.

[0062] Tobacco seedlings were raised using a floating method and transplanted when they reached the 4-5 leaf stage. The ratio of substrate to sterilized soil used for transplanting was 2:1. Two weeks after the seedlings had recovered, treatment was administered. The prepared substances and their mixtures were applied to the roots of each seedling using 100 mL of the same concentration as above. The pots were covered with plastic wrap. A control group was treated with plain water. Each treatment consisted of 10 seedlings, and treatments were repeated every 7 days for a total of 3 treatments. Three days after the first treatment, the plastic wrap was removed, and each seedling was watered with 100 mL of a 10% concentration solution. 8A suspension of *Phytophthora indicum* spores (cfu / mL) was applied, and the tobacco was continuously irrigated with an equal amount of water to maintain humidity. After three weeks of growth, the disease incidence was observed and recorded. The disease index and control effect were calculated according to the tobacco black shank disease grading standard. Disease index = (∑(number of diseased plants at each level × representative value at each level) × 100) / (total number of plants in the treatment × highest representative level); Control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0063] Table 6. The effects of three compounds and their mixtures on the prevention and treatment of tobacco black shank.

[0064] CK (Shimizu) 88.89 / tert-Butanol 34.38 61.32 Hexamethylcyclotrisiloxane 38.67 56.50 N-methylthioformamide 33.34 62.49 mix 28.19 68.29

[0065] The effects of various compounds on the control of tobacco black shank disease were analyzed through pot experiments. The results are shown in […]. Figure 9 See Table 6. It is evident that both individual substances and mixtures exhibit significant control effects against tobacco black shank. Compared to the control, the disease index of treated tobacco plants was significantly reduced. The control effects of tert-butanol, hexamethylcyclotrisiloxane, and N-methylthioformamide on tobacco black shank reached 61.32%, 56.50%, and 62.49%, respectively. The mixture of the three substances achieved a control effect of 68.29%. Pot experiment results indicate that the three compounds significantly inhibited the infection of tobacco plants by Phytophthora infestans and demonstrated good control effects against tobacco black shank.

[0066] The above description is only a preferred embodiment of this patent. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of this patent, and these improvements and substitutions should also be considered within the scope of protection of this patent.

Claims

1. A type of Pseudomonas aeruginosa ( Pseudomonas chlororaphis LQ29, characterized in that: The green needle-like Pseudomonas ( Pseudomonas chlororaphis LQ29 has the accession number CGMCCNo.27828, the accession date is July 7, 2023, the depositary institution is the China General Microbiological Culture Collection Center, and the depositary address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

2. A *Pseudomonas aeruginosa* strain according to claim 1 (… Pseudomonas chlororaphis LQ29, characterized in that: The aforementioned Pseudomonas aeruginosa ( Pseudomonas chlororaphis LQ29 can produce volatile substances such as tert-butanol, N-methylthioformamide, hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, and decamethylcyclopentasiloxane.

3. A *Pseudomonas aeruginosa* strain according to claim 2 ( Pseudomonas chlororaphis LQ29, characterized in that: The highest concentration of the volatile substance N-methylthioformamide was 1.22 mg / L, followed by hexamethylcyclotrisiloxane at 0.72 mg / L.

4. A *Pseudomonas aeruginosa* strain according to claim 1 ( Pseudomonas chlororaphis The application of LQ29 is characterized by: The aforementioned Pseudomonas aeruginosa ( Pseudomonas chlororaphis Application of volatile substances tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane and their mixtures produced by LQ29 in the prevention and treatment of tobacco black shank.

5. A *Pseudomonas aeruginosa* strain according to claim 4 ( Pseudomonas chlororaphis The application of LQ29 is characterized by: The aforementioned Pseudomonas aeruginosa ( Pseudomonas chlororaphis Application of volatile substances tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane and their mixtures produced by LQ29 in the inhibition of Phytophthora intoxin.

6. A *Pseudomonas aeruginosa* strain according to claim 4 ( Pseudomonas chlororaphis The application of LQ29 is characterized by: The aforementioned Pseudomonas aeruginosa ( Pseudomonas chlororaphis Application of volatile substances tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane and their mixtures produced by LQ29 in the inhibition of Rhizoctonia gracilis.

7. A *Pseudomonas aeruginosa* strain according to any one of claims 1-3 ( Pseudomonas chlororaphis Application of volatile substances tert-butanol, N-methylthioformamide, and hexamethylcyclotrisiloxane and their mixtures produced by LQ29 in promoting Arabidopsis thaliana growth.

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