Pseudomonas capable of preventing and controlling citrus canker and promoting plant growth and application thereof

Abstract

The invention discloses a pseudomonas capable of preventing and controlling citrus canker and promoting plant growth and application of the pseudomonas. The pseudomonas sp. CR1201 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on January 17, 2024, and the preservation number of the pseudomonas sp. CR1201 is GDMCC No: 64290. The pseudomonas sp. CR1201 is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on January 17, 2024. The pseudomonas disclosed by the invention is a new species of the genus, can effectively antagonize citrus canker pathogen Xcc, and can promote plant growth by generating siderophores and plant growth hormone IAA. Therefore, the pseudomonas CR1201 disclosed by the invention can be used for preparing a microbial preparation product for preventing and treating the citrus canker pathogen, and has important application potential in the aspects of microbial agents, biological fertilizers and the like for promoting the growth and development of plants.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and specifically relates to a Pseudomonas strain capable of preventing and controlling citrus canker and promoting plant growth, and an application thereof. Background Art

[0002] Citrus is a plant of the Rutaceae family and is one of the fruits with the largest planting area and the highest yield in the world. Citrus fruits are rich in nutrients such as vitamin C, trace elements, soluble sugars and folic acid, and are deeply loved by consumers. At the same time, citrus fruits also have anti-cancer and anti-inflammatory properties and are of high value. For many regions, the citrus industry also plays a very important role.

[0003] However, with the high incidence of various diseases, the citrus industry is also facing severe challenges. At present, the diseases that affect citrus production and quality mainly include citrus canker, Huanglongbing, anthracnose, gray mold, white scab, sand bark, green mold and penicillium, while insect pests mainly include psyllids, scale insects and red spiders. Among them, citrus canker is one of the most destructive bacterial diseases in the citrus industry. Citrus canker is a devastating bacterial disease caused by Xanthomonas citrisubsp.citri (Xcc), which has caused great harm to the citrus industry.

[0004] The current prevention and control measures for citrus canker mainly include planting citrus varieties that are resistant or insensitive to Xcc, establishing windbreaks, regularly spraying copper-based fungicides and controlling leaf miners, etc. However, there are few disease-resistant varieties available, and chemical control is very likely to cause environmental pollution, seriously threatening the safety of citrus consumption and public health. Using growth-promoting and disease-resistant microorganisms to control citrus canker is a green and potential alternative to chemical control methods, but the current available biocontrol bacteria resources are scarce. Continuously exploring new resources of growth-promoting and disease-resistant microorganisms from citrus, and developing related microbial agents, microbial fertilizers and other products are of great significance to promoting the green development of the citrus industry. Summary of the invention

[0005] The first object of the present invention is to provide a strain of Pseudomonas sp. CR1201, whose deposit number is: GDMCC No: 64290.

[0006] Pseudomonas sp. CR1201 is a new species of bacteria of the genus Pseudomonas that can prevent and control citrus canker bacteria and promote plant growth. The strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on January 17, 2024. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its collection number is: GDMCC No: 64290.

[0007] The second object of the present invention is to provide the use of the above-mentioned Pseudomonas CR1201 in producing siderophores or IAA.

[0008] The third object of the present invention is to provide a preparation containing the above-mentioned Pseudomonas CR1201, a culture thereof or a fermentation broth thereof as an active ingredient.

[0009] Preferably, the preparation is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned Pseudomonas CR1201 or the above-mentioned preparation in preventing and controlling citrus bacterial diseases.

[0011] Preferably, the invention is used for inhibiting citrus canker bacteria.

[0012] Preferably, the Xenopus citri sanguinis is Xenopus citri sanguinis Xcc.

[0013] The fifth object of the present invention is to provide the use of the above-mentioned Pseudomonas CR1201 or the above-mentioned preparation in the preparation of drugs for preventing and controlling citrus bacterial diseases.

[0014] The sixth object of the present invention is to provide a method for preventing and controlling citrus bacterial diseases, which utilizes the above-mentioned Pseudomonas CR1201 or the above-mentioned preparation to prevent and control citrus bacterial diseases.

[0015] The seventh object of the present invention is to provide the use of the above-mentioned Pseudomonas CR1201 or the above-mentioned preparation in promoting plant growth by producing iron carriers and plant growth hormone IAA.

[0016] A method for promoting plant growth comprises treating plants with the above-mentioned Pseudomonas CR1201 or the above-mentioned preparation, thereby promoting plant growth by producing iron carriers and plant growth hormone IAA.

[0017] The present invention has the following advantages over the prior art:

[0018] 1) The Pseudomonas CR1201 provided by the present invention is a new species different from other Pseudomonas strains;

[0019] 2) Pseudomonas CR1201 can not only antagonize citrus canker bacteria, but also promote plant growth by producing iron carriers and plant growth hormone IAA, and has the function of promoting growth and resisting diseases. Therefore, the Pseudomonas CR1201 disclosed in the present invention has broad application prospects in the preparation of microbial agents, organic biofertilizers and promoting the growth of crops.

[0020] Pseudomonas sp. CR1201 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 17, 2024, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is: GDMCC No: 64290. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphology of Pseudomonas CR1201 of the present invention on the R2A plate, the scale of Figure A is 2 mm, and that of Figure B is 200 μm.

[0022] Figure 2 This is a transmission electron microscope observation picture of the cells of Pseudomonas CR1201 of the present invention, and the scale bar is 1 μm.

[0023] Figure 3 16S rRNA gene phylogenetic tree of Pseudomonas sp. CR1201 of the present invention (NJ).

[0024] Figure 4 The figure is a phylogenetic tree of the genome of Pseudomonas CR1201 obtained in the present invention.

[0025] Figure 5 The results are the results of the antagonistic activity test of Pseudomonas sp. CR1201 against citrus canker pathogen Xcc.

[0026] Figure 6 This is the qualitative determination result of the siderophore production by Pseudomonas CR1201 in the present invention.

[0027] Figure 7 This is the quantitative determination result of the siderophore and plant growth hormone IAA produced by Pseudomonas CR1201 in the present invention. DETAILED DESCRIPTION

[0028] The following examples are further explanations of the present invention. It should be noted that these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Modifications and substitutions made to the details and forms of the embodiments under the concept and scope of the present invention all fall within the scope of protection of the present invention.

[0029] The present invention is further described below in conjunction with the embodiments. The experimental methods in the following embodiments are conventional experimental methods unless otherwise specified. The experimental reagents and consumables described in the following embodiments are from conventional biochemical reagent companies unless otherwise specified.

[0030] Example 1: Isolation and purification of Pseudomonas CR1201

[0031] 1. Isolation culture medium

[0032] R2A agar medium (g / L Qingdao Haibo Biological HB0167-5): 0.5g yeast extract powder, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, 15g agar. When using, weigh 15.2g of the above culture medium product, heat and dissolve it in 1L distilled water, sterilize it with high-pressure steam at 121℃ for 15min, and set aside.

[0033] 2. Experimental steps

[0034] (1) Sample collection: Soil samples around the rhizosphere of Gonggan were collected from the Gonggan orchard in Yongfeng Town, Deqing County, Zhaoqing City, Guangdong Province (23°17′21.41'N, 112°12′32.40'E), placed in sampling bags and brought back to the laboratory for natural air drying at room temperature.

[0035] (2) Preparation of soil gradient dilution: Weigh 10 g of soil and add it to a sterile Erlenmeyer flask with glass beads and 90 mL of sterile water. Incubate at 200 rpm on a shaker at 30°C for 30 min. After standing for 5 min, take 1 mL of the supernatant and add it to a test tube with 9 mL of sterile water. Then perform gradient dilutions to 10. -2 -10 -7 g / mL soil suspension for later use.

[0036] (3) Isolation of culturable bacteria: Take 100 μL of the diluted soil bacterial suspension (10 -2 -10 -7 g / mL), and evenly spread it on the R2A medium plate with a disposable plastic coating rod. Three replicates were set for each concentration. After coating, the plate was inverted and placed in a 30°C constant temperature incubator for culture. The growth of the colonies on the plate was observed every other day, and strains of different forms that produced transparent circles were selected and streaked on the R2A solid plate. The strains were purified, and finally strain CR1201 was isolated.

[0037] Example 2: Classification and Identification of Strain CR1201

[0038] The isolated strains were identified with reference to the Manual of Identification of Common Bacterial Systems (Edited by Dong Xiuzhu, Science Press).

[0039] (1) Phenotypic characteristics:

[0040] The strain CR1201 was inoculated on the R2A medium plate by the three-zone streak method, and its colony morphology was observed after culturing at 30°C for 3 days. The cultured colonies were selected for Gram staining and morphological observation using an optical microscope, and samples for transmission electron microscopy were prepared, and the cell morphology of the strain was observed using a Hitachi H7650 transmission electron microscope.

[0041] The strain CR1201 was Gram-negative, and was milky white on R2A medium. The single colony was round, smooth, with neat edges and no fluidity ( Figure 1 ); Under transmission electron microscopy, the cells appear short rod-shaped and have flagella ( Figure 2 ).

[0042] (2) System evolution analysis:

[0043] The DNA of strain CR1201 was extracted by boiling method, and the 16S rRNA gene sequence of CR1201 was amplified using bacterial 16S rRNA specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAATCGC-3′) and Taq enzyme. The amplified product was analyzed by electrophoresis to produce a band of about 1500b. After gel cutting and recovery, the band was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for Sanger sequencing. The sequence obtained by sequencing was spliced ​​by DNAMAN software to obtain the 16S rRNA sequence. Its nucleotide sequence is shown in SEQ ID NO.1 and submitted to the EzBioClode website (https: / / www.ezbiocloud.net / ) for identification. The NeighborJoining phylogenetic tree based on the 16S rRNA sequence was constructed using MEGA 10 software, and the calculation model was Kimura 2-paremeter. The full length of the 16S rRNA gene of strain CR1201 obtained by Sanger sequencing is 1412bp (SEQ ID NO.1). Sequence comparison revealed that the similarity between strain CR1201 and the published model bacteria sequence was lower than the critical value of 98.65% for species differentiation (Table 1), among which the model bacteria with the highest similarity was Pseudomonas lalkuanensis PE08. T The phylogenetic tree constructed based on the 16S rRNA gene sequence and the genome sequence showed that strain CR1201 belonged to the genus Pseudomonas and was closely related to Pseudomonas lalku anensis PE08. T, Pseudomonas furukawaii KF707 T , Pseudomonas resinovorans LMG2274 T and P. seudomonasboanensis DB1 T The closest relatives of the model bacteria are Figure 3 In summary, based on the 16S rRNA gene sequence and morphological characteristics, strain CR1201 was identified as a potential new species of the genus Pseudomonas.

[0044] (3) Comparative genomics analysis:

[0045] The genomic DNA of strain CR1201 was extracted using a bacterial genomic DNA extraction kit (Magen), and then the genome sequencing was completed by Shanghai Meiji Biotechnology Co., Ltd. after verification by 16S rRNA gene amplification. The construction of double-end libraries and Illumina Hiseq 2500 sequencing were commissioned to this company. The off-machine data were assembled using SPAdes (3.13.0) software for genome sequence splicing. The genome sequences of the published model species intended for comparative analysis were downloaded from the NCBI database, and the average nucleotide identity (ANI) of each strain was calculated using FastANI based on the genome sequence. The DNA-DNA hybridization value (dDDH) based on the genome sequence was calculated using the online tool GGDC 2.1 (http: / / hhdc.dsmz.de / home.php). The genome ML phylogenetic tree was constructed using UBCG. The genome size of strain CR1201 is about 5.5Mb, and the GC content is 64.5mol%. The results of genome evolution analysis are consistent with those of 16s rRNA gene sequence evolution analysis, showing that strain CR1201 belongs to the genus Pseudomonas ( Figure 3 , Figure 4 ). The results of comparative analysis based on the genome sequence are shown in Table 1. The ANI values ​​and dDDH between strain CR1201 and the closest published model strains are 80.23% to 92.76% and 21.3% to 49.8%, respectively, which are significantly lower than the currently recognized thresholds of 95% to 96% ANI and 70% dDDH as species boundaries. These results indicate that strain CR1201 represents a different new species of the genus Pseudomonas. The present invention names strain CR1201 as Pseudomonas sp. CR1201.

[0046] Pseudomonas sp. CR1201 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 17, 2024, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510070, and its deposit number is: GDMCC No: 64290.

[0047] Table 1 Comparative genome analysis of strain CR1201 and 10 published closely related model species

[0048]

[0049] Example 3: Evaluation of the antibacterial activity of Pseudomonas CR1201 against citrus canker bacteria

[0050] The Oxford cup dual culture method was used to determine the antagonistic activity of Pseudomonas CR1201 against citrus canker pathogen Xcc. The specific method is as follows: (1) A single colony of citrus canker pathogen Xcc was selected and inoculated into 5 mL of NB liquid culture medium, and then placed in a horizontal shaker at 30°C and shaken at 200 rpm for about 30 h until the OD 600 =1 for standby; (2) Use an Oxford cup to prepare a NA solid plate with holes containing 1% (v / v) pathogens, that is, after the NA solid culture medium is cooled to about 50°C, add the citrus canker pathogen Xcc culture solution cultured in step (1) at a volume ratio of 1%, mix well and pour the plate; (3) Add 80 μL of Pseudomonas CR1201 cultured in NB liquid culture medium for 2 days to each well (the preparation method of Pseudomonas CR1201 culture solution is: activate strain CR1201 on R2A solid plate, culture at 30°C for 2 days, pick a single colony and inoculate it into a test tube containing 5mL NB liquid culture medium, and then place it at 30°C, 200rpm and shake on a horizontal shaker for 2 days). Carefully place the plate in a 30°C incubator and incubate for 48 hours. Evaluate the antagonistic activity based on the size of the inhibition zone. Repeat the plate experiment three times.

[0051] The results of the antagonistic effect of Pseudomonas CR1201 on citrus canker pathogens are shown in Figure 5 As shown, it can be seen that after the CR1201 bacterial solution was inoculated into a well on the NA solid plate containing citrus canker pathogen Xcc, an obvious inhibition zone appeared around the well, indicating that Pseudomonas CR1201 can effectively antagonize citrus canker pathogen Xcc.

[0052] NA (nutrient broth) solid culture medium: peptone 10.0 g / L, beef extract powder 3.0 g / L, sodium chloride 5.0 g / L, agar 15 g / L, pH 7.0; the preparation method is to weigh the above culture medium components, dissolve them in distilled water and mix them evenly, adjust the pH value, sterilize them with high-pressure steam at 121°C for 15 minutes, and set them aside.

[0053] NA solid medium without adding agar is NB liquid medium.

[0054] Example 4: Determination of the ability of Pseudomonas CR1201 to produce siderophore

[0055] The qualitative determination experiment of iron carrier production was carried out by using the CAS plate covering method. Pseudomonas CR1201 was activated on the R2A solid plate, cultured at 30℃ for 2 days, and the bacteria were scraped and inoculated on a new R2A plate (four points were inoculated on each plate). After culturing at 30℃ for 2 days, a layer of CAS detection solution containing 1% agarose was covered on the plate (heated to dissolve, and after cooling, CAS was poured on the plate where the test bacteria had grown), and the plate was placed in a 30℃ incubator for incubation, and the color change was observed at any time.

[0056] The quantitative determination method of iron carrier production is to pick a single colony of strain CR1201 and inoculate it into a test tube containing 5mL R2A liquid culture medium (R2A agar culture medium does not contain agar components), then place it on a horizontal shaker at 30°C and 200rpm for shaking culture for 2d, take 1mL of the cultured bacterial liquid in a 1.5mL EP tube, centrifuge it at 12000rpm for 5min, take 100μL of the supernatant in a 96-well plate, add an equal volume of CAS detection solution and mix evenly, let it stand for 1h, and use an enzyme marker to measure the absorbance value As at 630nm wavelength, and adjust it to 0 by adding ddH2O as a control, and take an equal volume of uninoculated sterilized R2A culture medium and mix it with an equal volume of CAS detection solution, and the measured absorbance value is the reference value Ar. Finally, the amount of iron carrier produced is calculated according to the formula. The amount of iron carrier produced = (Ar-As) / Ar×100% iron carrier amount unit. If the calculated result is less than 10%, it is considered that the iron carrier secretion is negative.

[0057] The qualitative determination results of the siderophore produced by Pseudomonas CR1201 are shown in Figure 6 , it can be seen that an obvious yellow halo appeared around the inoculated strain, indicating that Pseudomonas CR1201 can produce siderophores; quantitative determination results showed that after 2 days of inoculation, the amount of siderophores produced by Pseudomonas CR1201 was 21.42±0.48% ( Figure 7 ).

[0058] CAS detection solution (1 L): Chrome azurol S (chrome azurol) 60.5 mg, CTAB 72.9 mg, PIPES (piperazine-1,4-diethanesulfonic acid) 30.24 g, FeCl3·6H2O 1 mM, HCl 10 mM, agarose 0.9% (w / v).

[0059] Example 5: Determination of iron-producing plant growth hormone IAA by Pseudomonas CR1201

[0060] The culture method of the strain is the same as that of the iron carrier determination in Example 4 above. Take 1 mL of the cultured bacterial solution and centrifuge it at 10000 rpm in an EP tube for 5 minutes. Take 50 μL of the supernatant in a 96-well plate and add 50 μL of Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5M FeCl3). Add 50 μL of 50 mg / L IAA colorimetric solution as a positive control. Place the 96-well plate in the dark at room temperature for 30 minutes and measure the OD with an ELISA reader. 530 The absorbance value was calculated and a standard curve was prepared using gradiently diluted 50 mg / L IAA as the standard. Finally, the IAA content of the strain was calculated according to the standard curve equation.

[0061] The results of the determination of IAA production by Pseudomonas CR1201 are shown in Figure 7 After 2 days of inoculation and culture, the amount of IAA produced was 31.28±0.81 mg / L.

[0062] SEQ ID NO.1

[0063] GCTACCATGCAGTCGAGCGGCAGCGGGTCCTTCGGGATGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGAGGTGAAATCCCCGGGCTCAACCTGGGAACTGCCTCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAACCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCGCAAGGGGGACGGTACCA

Claims

1. Pseudomonas sp. CR1201, characterized in that The deposit number is: GDMCC No: 64290.

2. Use of the Pseudomonas CR1201 according to claim 1 in producing siderophores or IAA.

3. A preparation, characterized in that Contains the Pseudomonas CR1201, its culture or its fermentation liquid as claimed in claim 1 as an active ingredient.

4. The preparation according to claim 3, characterized in that The preparation is a microbial agent, a microbial fertilizer, a microbial pesticide or a microbial additive.

5. Use of the Pseudomonas CR1201 according to claim 1 or the preparation according to claim 3 in preventing and controlling citrus bacterial diseases.

6. The use according to claim 5, characterized in that: It is used in inhibiting citrus canker bacteria.

7. The use according to claim 6, characterized in that: The citrus canker bacteria is citrus canker bacteria Xcc.

8. Use of the Pseudomonas CR1201 according to claim 1 or the preparation according to claim 3 in the preparation of drugs for preventing and controlling citrus bacterial diseases.

9. A method for preventing and controlling citrus bacterial diseases, characterized in that: The method comprises using the Pseudomonas CR1201 described in claim 1 or the preparation described in claim 3 to prevent and control citrus bacterial diseases.

10. Use of the Pseudomonas CR1201 according to claim 1 or the preparation according to claim 3 in promoting plant growth by producing siderophores and plant growth hormone IAA.

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