Pseudomonas putida qL9 strain and application thereof

Abstract

The application provides a Pseudomonas putida QL9 strain and application thereof. The Pseudomonas putida QL9 strain is preserved in the Guangdong Microbial Culture Collection Center on October 13, 2023, has a preservation number of GDMCC No: 63881, and a preservation address of No. 59 Building, 5th Floor, Guangzhou, Guangdong, China. The Pseudomonas putida QL9 strain is a new species of Pseudomonas, can degrade 3-hydroxy palmitic acid methyl ester signal molecules produced by Ralstonia solanacearum, namely adopts a quorum quenching mode to interfere with normal expression of pathogenic factors of Ralstonia solanacearum, block information exchange between Ralstonia solanacearum, and then specifically inhibit pathogenicity of Ralstonia solanacearum. Moreover, the quorum quenching mode adopted by the Pseudomonas putida QL9 strain does not cause excessive survival pressure on Ralstonia solanacearum to form super bacteria. Therefore, the Pseudomonas putida QL9 strain is suitable for preparing products for preventing and / or treating plant Ralstonia solanacearum diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological control, and more particularly relates to a Pseudomonas polia sp.nov. QL9 strain and application thereof. BACKGROUND

[0002] Ralstonia solanacearum is a gram-negative plant pathogen, which is widely distributed in tropical, subtropical and temperate regions, and can infect more than 450 species of plants in more than 50 families, and can occur in tobacco, eggplant, tomato, peanut, Casuarina equisetifolia and other plants, and is the pathogenic bacterium causing plant bacterial wilt.

[0003] The current prevention methods of bacterial wilt mainly include cultivating disease-resistant varieties, chemical control, biological control and the like, which can reduce the occurrence of plant bacterial wilt to a certain extent, but all have great defects, for example: (1) cultivating disease-resistant varieties: there are few disease-resistant varieties at present, the breeding process is complex, and it is difficult to breed, and has defects such as poor resistance effect and easy disappearance of resistance; (2) chemical control: chemical reagents are easy to remain, which can cause environmental safety problems; (3) biological control: the existing antagonistic bacteria of bacterial wilt generally play antagonistic effect by killing pathogenic bacteria, but this method is easy to form super bacteria, which leads to limited control effect.

[0004] Quorum quenching (QQ) can inhibit or interfere with the quorum sensing system between pathogenic bacteria, interfere with the normal expression of pathogenic factors of pathogenic bacteria, block the 'information exchange' between pathogenic bacteria, and then specifically inhibit the pathogenicity of pathogenic bacteria, and the quorum quenching will not cause too much survival pressure to pathogenic bacteria, that is, will not lead to drug resistance of pathogenic bacteria, and will not form super bacteria, which is a practical and effective biological prevention and control method, but there are still few microorganisms that can prevent and control bacterial wilt by quorum quenching at present. SUMMARY

[0005] The present application aims at the deficiencies in the prior art, and provides a Pseudomonas polia sp.nov. QL9 strain, which can interfere with the normal expression of pathogenic factors of Ralstonia solanacearum by quorum quenching, block the 'information exchange' between Ralstonia solanacearum, and then specifically inhibit the pathogenicity of Ralstonia solanacearum.

[0006] The first object of the present application is to provide a Pseudomonas polia sp.nov. QL9 strain.

[0007] The second object of the present application is to provide application of the above-mentioned Pseudomonas polia sp.nov. QL9 strain in preparation of products for preventing and / or treating plant bacterial wilt.

[0008] A third object of the present application is to provide a method for preventing and / or treating plant bacterial wilt.

[0009] A fourth object of the present application is to provide a product for preventing and / or treating plant bacterial wilt.

[0010] The above objects of the present application are achieved by the following technical solutions:

[0011] The present application provides a Pseudomonas polia sp. nov. QL9 strain, which was deposited in the Guangdong Microbial Culture Collection Center on October 13, 2023, with the accession number GDMCC No: 63881. It is a new species of Pseudomonas sp. and is deposited at No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard.

[0012] The Pseudomonas polia sp. nov. QL9 strain of the present application can inhibit the pathogenicity of Ralstonia solanacearum by degrading the quorum sensing signal produced by Ralstonia solanacearum, i.e. 3-hydroxy palmitate methyl ester 3-OH PAME, thereby effectively preventing and / or treating plant bacterial wilt. Therefore, the application of the above-mentioned Pseudomonas polia sp. nov. QL9 strain in the preparation of preparations for preventing and / or treating plant bacterial wilt should be within the scope of protection of the present application.

[0013] Preferably, the plant is one or more of Casuarina equisetifolia, peanut, and tomato.

[0014] Preferably, the bacterial wilt is a plant disease caused by Ralstonia solanacearum.

[0015] Preferably, the prevention and / or treatment of plant bacterial wilt is to inhibit the pathogenicity of Ralstonia solanacearum.

[0016] Further preferably, the inhibition of the pathogenicity of Ralstonia solanacearum is to degrade the quorum sensing signal produced by Ralstonia solanacearum.

[0017] Further preferably, the quorum sensing signal is 3-hydroxy palmitate methyl ester.

[0018] The present application also provides a method for preventing and / or treating plant bacterial wilt, i.e. inoculating the above-mentioned Pseudomonas polia QL9 strain on plants, such as inoculating Casuarina equisetifolia and tomato by root injury and bacteria inoculation method, or inoculating peanut by injection method.

[0019] In addition, the present application also provides a product for preventing and / or treating plant bacterial wilt, which comprises the above-mentioned Pseudomonas polia QL9 strain.

[0020] Preferably, the product is one or more of a bacterial solution, a fermentation solution, a medicine.

[0021] Further preferably, the concentration of the Pseudomonas polia QL9 strain in the medicine is OD 600 1.0-1.2.

[0022] Preferably, the plant is one or more of Casuarina equisetifolia, peanut, tomato.

[0023] Preferably, the inoculation is root wound irrigation inoculation or injection inoculation.

[0024] The present application has the following beneficial effects:

[0025] (1) The Pseudomonas polia sp. nov. QL9 strain is a new species of the genus Pseudomonas, which can interfere with the normal expression of the pathogenic factor of Pseudomonas solanacearum by degrading the 3-hydroxy palmitate signal molecule produced by Pseudomonas solanacearum, i.e. by adopting a quenching method to block the "information exchange" between Pseudomonas solanacearum, thereby specifically inhibiting the pathogenicity of Pseudomonas solanacearum. Moreover, the quenching method adopted by the Pseudomonas polia QL9 strain will not make Pseudomonas solanacearum form super bacteria.

[0026] (2) The Pseudomonas polia sp. nov. QL9 strain has simple culture conditions and can be easily produced industrially, and can be used as an excellent plant Pseudomonas solanacearum disease control agent. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is the colony morphology of the Pseudomonas polia QL9 strain cultured on LB medium for 24 h.

[0028] Figure 2 It is the circular chromosome of the Pseudomonas polia QL9 strain.

[0029] Figure 3 It is the growth condition of the plants in Example 2, wherein, Figure 3 A in the above table is the growth condition of Casuarina equisetifolia, Figure 3 B in the above table is the growth condition of peanut, Figure 3 C in the above table is the growth condition of tomato.

[0030] Figure 4 It is the LC-MS spectrum of 3-hydroxy palmitate in the blank group.

[0031] Figure 5 It is the LC-MS spectrum of 3-hydroxy palmitate in the sample group.

[0032] Figure 6Statistical results of the degradation rate of methyl 3-hydroxypalmitate for the sample groups. DETAILED DESCRIPTION

[0033] The application will be further described in conjunction with the drawings and specific examples of the application, which are presented herein for the purpose of illustration only and are not intended to limit the scope of the application. The experimental methods used in the following examples are conventional unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified.

[0034] In the following examples, the methyl 3-hydroxypalmitate used is methyl 3-hydroxypalmitate dissolved in methanol, and the concentration of methyl 3-hydroxypalmitate in methanol is 1 nM.

[0035] Example 1 Screening, isolation, identification and preservation of Pseudomonas monteilii QL9 strain

[0036] (1) Screening and isolation of Pseudomonas monteilii QL9 strain

[0037] The 20 g of soil of 5-10 cm in the rhizosphere of pepper was collected in Qilinbei farm of South China Agricultural University in Tianhe District of Guangzhou, Guangdong Province, and the collected soil sample was thoroughly mixed after removing visible roots and other impurities, and was bagged and stored for bringing back to the laboratory for isolation.

[0038] 5.0 g of soil sample was weighed with a balance and was put into 200 mL of MSM medium added with methyl 3-hydroxypalmitate (the final concentration of methyl 3-hydroxypalmitate was 1 μM). After the soil sample was shaken, it was placed in a 28℃, 200 rpm shaker for 7 days of shaking culture. After 7 days, the transfer was performed on a clean bench, 200 μL of the mixed soil suspension was sucked with a pipette and was put into 200 mL of new MSM medium added with methyl 3-hydroxypalmitate (the final concentration of methyl 3-hydroxypalmitate was 1 μM), which was placed in a 28℃, 200 rpm shaker for 7 days of shaking culture again. After 7 days, 100 μL of the mixed soil suspension was again sucked with a pipette on a clean bench, and was diluted 10 times, 10 times, 10 times, 10 times and 10 times with sterilized water respectively, and 150 μL of the 10 times, 10 times, 10 times, 10 times and 10 times diluted soil suspension was respectively sucked. 1 ,10 2 ,10 3 ,10 4 ,10 0 ,10 1 ,10 2 ,10 3 ,10 4The dilution solution was diluted by 2 times and evenly coated on the surface of LB plate, and then dried at 25℃. After sealing the plate, it was cultured in a 28℃ incubator for 24h. After the colonies grew, the size, color, dryness, smoothness and halo were observed, and a single colony was picked and inoculated on a new LB medium for streaking and purification. The purified strain was numbered as QL9.

[0039] (2) Identification of Pseudomonas polia sp. nov. QL9 strain

[0040] The strain QL9 was Gram-negative. The colony morphology cultured in LB medium for 24h was shown in Figure 1 It could be seen that the colony of strain QL9 was wet, round, 2-3mm in diameter, gray in color and smooth in surface in LB medium. The colony edge was diffuse in the late stage of culture.

[0041] To further clarify the taxonomic status of strain QL9, we extracted the whole genome DNA of QL9 and sent it to Baimaikesi Biological Technology Co., Ltd. for whole genome sequencing. The sequencing results were assembled to obtain a complete circular chromosome (see Figure 2 ), and homologous comparison was performed in the NCBI database. It was found that QL9 was most closely related to Pseudomonas nicosulfuronedens LAM1902 strain in the database, with ANI of 92.8145% and coverage of 83.0477%, which was significantly lower than the threshold of the same species (95%), indicating that QL9 was a new species. We named QL9 as Pseudomonas polia sp. nov. according to the colony characteristics.

[0042] (3) Preservation of Pseudomonas polia sp. nov. QL9 strain

[0043] Pseudomonas polia sp. nov. QL9 strain was preserved in Guangdong Microbial Culture Collection Center on October 13, 2023, with the preservation number of GDMCC No: 63881 and the preservation address of No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard.

[0044] Example 2: Pseudomonas polia sp. nov. QL9 strain can effectively prevent and / or treat plant bacterial wilt

[0045] (1) Test method

[0046] In the clean bench, the single colony of Ralstonia solanacearum NS25 on TTC plate was picked up with a inoculating loop into 10 mL TTC liquid medium, and shaken for 24 h to obtain NS25 seed liquid. Then, the NS25 seed liquid was inoculated into 110 mL TTC liquid medium, so that the volume ratio of NS25 seed liquid to TTC liquid medium was 1:100, and was placed in a shaking bed at 28℃ and 200 rpm to shake and culture until OD 600 was 1.2, to obtain NS25 bacterial liquid.

[0047] In the clean bench, QL9 on LB plate was picked up with a inoculating loop into 10 mL LB liquid medium, and shaken for 24 h to obtain QL9 seed liquid. Then, the QL9 seed liquid was inoculated into 55 mL LB liquid medium, so that the volume ratio of QL9 seed liquid to LB liquid medium was 1:100, and was placed in a shaking bed at 28℃ and 200 rpm to shake and culture until OD 600 was 1.2, to obtain QL9 bacterial liquid.

[0048] Three groups were set up in the test:

[0049] ① NS25+QL9 group: 5 mL QL9 bacterial liquid was mixed with 5 mL NS25 bacterial liquid to obtain treatment liquid;

[0050] ② NS25 group: 5 mL NS25 bacterial liquid was mixed with 5 mL LB liquid medium to obtain treatment liquid;

[0051] ③ CK group: 5 mL LB liquid medium was mixed with 5 mL TTC liquid medium to obtain treatment liquid;

[0052] NS25+QL9 group, NS25 group and CK group were respectively set up with 4 parallel samples, and the following test was carried out:

[0053] ① Casuarina equisetifolia (wound root irrigation method):

[0054] The normal root system of Casuarina equisetifolia was trimmed with scissors after alcohol disinfection, and a wound was created (attention should be paid to not cutting the main root). Then, Casuarina equisetifolia was planted in a small flowerpot, and 10 mL of the above treatment liquid was irrigated on the root of Casuarina equisetifolia. The growth condition of Casuarina equisetifolia was observed every day, and the record was made. The photo of Casuarina equisetifolia on the 46th day after irrigation was taken.

[0055] ② Peanut (injection method):

[0056] 200 μL of the above treatment liquid was sucked into a 1 mL syringe, and was injected into the stem of peanut. The growth condition of peanut was observed every day, and the record was made. The photo of peanut on the 8th day after injection was taken.

[0057] ③ Tomato (wound root irrigation method):

[0058] The normal root system of the tomato is trimmed using an alcohol-disinfected scissors to create a wound (be careful not to cut the main root), the tomato is planted in a small flowerpot, and then 10 mL of the above treatment solution is irrigated on the root of the tomato. The growth condition of the tomato is observed every day, and the record is made. The photo of the tomato on the 9th day after irrigation is taken.

[0059] (2) Test results

[0060] The results are shown in Figure 3 wherein, Figure 3 A in the table is the growth condition of Casuarina equisetifolia, Figure 3 B in the table is the growth condition of Arachis hypogaea, Figure 3 C in the table is the growth condition of Lycopersicon esculentum.

[0061] It can be seen that:

[0062] ① The NS25 group of Casuarina equisetifolia began to be diseased after 8 days of inoculation, and the NS25+QL9 group began to be diseased after 9 days of inoculation. After 46 days of inoculation, the growth condition of Casuarina equisetifolia in the NS25+QL9 group was significantly better than that in the NS25 group, indicating that the Pseudomonas polia sp. nov. QL9 strain can effectively prevent and / or treat the Casuarina equisetifolia bacterial wilt.

[0063] ② The NS25 group of Arachis hypogaea began to be diseased after 6 days of inoculation, and the NS25+QL9 group began to be diseased after 8 days of inoculation. After 8 days of inoculation, all the Arachis hypogaea in the NS25 group were close to death, while only one Arachis hypogaea in the NS25+QL9 group was diseased and the degree was lighter, i.e. the growth condition of Arachis hypogaea in the NS25+QL9 group was significantly better than that in the NS25 group, indicating that the Pseudomonas polia sp. nov. QL9 strain can effectively slow down the occurrence of the Arachis hypogaea bacterial wilt.

[0064] ③ The NS25 group of Lycopersicon esculentum began to be diseased after 7 days of inoculation, and the NS25+QL9 group began to be diseased after 8 days of inoculation. After 9 days of inoculation, all the Lycopersicon esculentum in the NS25 group were dead, while only one Lycopersicon esculentum in the NS25+QL9 group was diseased and dead, and the other plants grew well, i.e. the growth condition of Lycopersicon esculentum in the NS25+QL9 group was significantly better than that in the NS25 group, indicating that the Pseudomonas polia sp. nov. QL9 strain can effectively slow down the occurrence of the Lycopersicon esculentum bacterial wilt.

[0065] In summary, the Pseudomonas polia sp. nov. QL9 is a biocontrol strain with the function of slowing down the occurrence of plant bacterial wilt, which can effectively treat the Casuarina equisetifolia bacterial wilt, the Arachis hypogaea bacterial wilt and the Lycopersicon esculentum bacterial wilt through the root injury irrigation method or the injection method.

[0066] Example 3 The Pseudomonas polia sp. nov. QL9 strain can efficiently degrade 3-hydroxy palmitic acid methyl ester

[0067] (1) Test method

[0068] QL9 strain was activated on LB solid medium plate, and a single colony of QL9 was picked from the plate with an inoculation loop into a 50 mL centrifuge tube containing 10 mL of LB liquid medium, and the centrifuge tube was placed in a 28°C shaker for culture until the OD 600 was 1.2, and QL9 bacterial liquid was obtained.

[0069] The experiment was divided into two groups:

[0070] ① Sample group: 10 mL of MSM medium was taken in a new 50 mL centrifuge tube in a dark clean bench, and 3-hydroxypalmitic acid methyl ester (the final concentration of 3-hydroxypalmitic acid methyl ester in the MSM medium was 1 μM) and 100 μL of QL9 bacterial liquid with OD 600 1.2 were added.

[0071] ② Blank group: 10 mL of MSM medium was taken in a new 50 mL centrifuge tube in a dark clean bench, and 3-hydroxypalmitic acid methyl ester (the final concentration of 3-hydroxypalmitic acid methyl ester in the MSM medium was 1 μM) was added, and no QL9 bacterial liquid was added.

[0072] The sample group and the blank group were each prepared in four parallel samples, and the following experiments were performed:

[0073] After the sample group and the blank group were placed in a 28°C, 200 rpm shaker for 7 days of dark shaking culture, the obtained liquid was poured into a 60 mL separatory funnel, 10 mL of dichloromethane was added for extraction, and 50 mL of a conical flask was used to collect the lower organic phase, and the upper aqueous phase was extracted three times. The collected organic phase was rotary evaporated to completely evaporate the dichloromethane, 1 mL of chromatographic methanol was taken with a syringe to elute the substances in the conical flask, a disposable plastic dropper was used to suck the eluent into a 1.5 mL centrifuge tube, and then 150 μL was taken with a syringe and placed in a sample bottle. Finally, the degradation rate of 3-hydroxypalmitic acid methyl ester by strain QL9 was determined by LC-MS.

[0074] The determination conditions of LC-MS were as follows:

[0075] LC-MS model: Q Exactive Focus liquid chromatograph-mass spectrometer of Thermo Fisher Company;

[0076] Chromatographic column: waters chromatographic column (SKU: 186003539, ACQUITY UPLC HSS T3 Column, 1.8 μm, 2.1 mm X 100 mm, 1 / pk);

[0077] Flow rate: 0.3 mL / min;

[0078] Column temperature: 40℃;

[0079] Mobile phase: Methanol: Water = 80:20 (v / v);

[0080] Characteristic ion: Na + ;

[0081] Molecular weight: 309;

[0082] Peak emission time: 6.0–6.5 min;

[0083] Injection volume: 10 μL.

[0084] (2) Test Results

[0085] The results are as follows Figures 4-6 As shown, where, Figure 4 This is the LC-MS spectrum of methyl 3-hydroxypalmitate in the blank group. Figure 5 The LC-MS spectrum of methyl 3-hydroxypalmitate in the sample group is shown below. Figure 6 The results show the statistical results of the degradation rate of methyl 3-hydroxypalmitate in the sample groups.

[0086] from Figures 4-6 The degradation rate of methyl 3-hydroxypalmitate in the sample group was 80.20%, indicating that Pseudomonas glaucusiformis strain QL9 can effectively degrade methyl 3-hydroxypalmitate, a quorum sensing signal produced by Ralstonia solanacearum, and can effectively prevent and / or treat bacterial wilt of plants.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Pseudomonas polia sp. nov. QL9 strain, characterized in that, The Pseudomonas putida QL9 strain is preserved in the Guangdong Microbial Culture Collection Center on October 13, 2023, and the preservation number is GDMCC No: 63881.

2. The use of the Pseudomonas putida QL9 strain of claim 1 in the preparation of a product for preventing and / or treating plant bacterial wilt.

3. Use according to claim 2, characterized in that, The plant is one or more of Casuarina equisetifolia, peanut, and tomato.

4. Use according to claim 2, characterized in that, The prevention and / or treatment of plant bacterial wilt is to inhibit the pathogenicity of Ralstonia solanacearum.

5. Use according to claim 4, characterized in that, The inhibition of the pathogenicity of Ralstonia solanacearum is to degrade the quorum sensing signal produced by Ralstonia solanacearum.

6. Use according to claim 5, characterized in that, The quorum sensing signal is 3-hydroxy palmitate methyl ester.

7. A method for preventing and / or treating bacterial wilt of plants, characterized by, The Pseudomonas putida QL9 strain of claim 1 is inoculated on the plant.

8. The method of claim 7, wherein, The inoculation is root wound bacteria inoculation or injection inoculation.

9. The method of claim 7, wherein, The plant is one or more of Casuarina equisetifolia, peanut, and tomato.

10. A product for preventing and / or treating bacterial wilt of plants, characterized in that, It comprises the Pseudomonas putida QL9 strain of claim 1.

Images