Pseudomonas S11 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to Pseudomonas S11 and application thereof, the Pseudomonas S11 is preserved in China Center for Type Culture Collection, the preservation address is No. 299, eight road, Wuchang District, Wuhan City, Hubei Province, the preservation date is March 21, 2022, and the preservation number is CCTCC NO: M2022294. The pseudomonas S11 provided by the invention not only can promote the growth of plants such as strawberries, corns and rice, but also has a biological control effect on agricultural diseases such as strawberry gray mold and corn southern leaf blight, has an excellent antagonistic effect on pathogenic bacteria of various plant diseases, and has a wide application prospect and development space.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to Pseudomonas S11 and applications thereof. Background Art

[0002] While chemical pesticides can prevent and control crop diseases, insects, weeds, rats and other harmful organisms, they may also have toxic effects on other beneficial organisms, destroy the balance and biodiversity of the ecosystem, and make many harmful organisms resistant to pesticides. However, biological fertilizers and biological pesticides not only have good crop quality and yield-enhancing effects, but also do not pollute the environment. They can also establish a new balance between biological populations and have a sustainable control effect.

[0003] In the natural environment, there are many fungi that are beneficial to plants. They can have beneficial effects on plants in various ways. However, most beneficial bacteria cannot have the ability to promote growth and biocontrol at the same time, and often need to be used in combination with fungi with another ability. In recent years, plant growth-promoting rhizobacteria (PGPR), which can promote plant growth and its absorption and utilization of mineral nutrients while inhibiting harmful organisms, have become a research hotspot in fields such as microbiology and plant pathology.

[0004] Pseudomonas is a rod-shaped, motile Gram-negative bacterium that is widely present in various environments. It is a very potential plant growth-promoting bacterium. Pseudomonas, due to its denitrification ability, is currently mainly used for water treatment of industrial wastewater or polluted rivers and ponds and soil remediation. As in the prior art, ZL201210272041.7 and disclose that Pseudomonas flavus can be used for soil or water pollution remediation, and ZL202010070032.4 discloses that a microbial composite bacterial agent containing Pseudomonas flavus can be used for petroleum contaminated soil remediation. In addition, CN115948299A also discloses that Pseudomonas flavus can be used to prevent and treat citrus canker. However, there is currently no report on the promotion of growth of Pseudomonas in strawberry, corn and rice and the biological control of strawberry gray mold and corn leaf spot, nor is there a report on Pseudomonas in broad-spectrum antibacterial.

[0005] Therefore, it is necessary to strengthen the selection and breeding of Pseudomonas strains to solve the current problems. Summary of the invention

[0006] The problem that this application wants to solve is to provide Pseudomonas S11, which not only promotes the growth of plants such as strawberry, corn and rice, but also has a biological control effect on agricultural diseases such as strawberry gray mold and corn leaf spot, and at the same time has an excellent antagonistic effect on pathogens of various plant diseases.

[0007] In order to solve the above technical problems, this application adopts the following technical solutions:

[0008] On the one hand, the present application provides Pseudomonas sp. S11, which is a Pseudomonas fulva. The Pseudomonas S11 is deposited in the China Center for Type Culture Collection, the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date is March 21, 2022, and the preservation number is CCTCCNO: M2022294.

[0009] The Pseudomonas S11 is derived from the rhizosphere soil of Taxus × media and is isolated and screened by the dilution spread plate method. Sampling site: Emeishan Nature Reserve, Leshan City, Sichuan Province.

[0010] This application identified Pseudomonas S11 from the perspective of morphology and molecular biology:

[0011] Morphological identification: Pseudomonas S11 colonies on LB medium are small yellowish circles with neat edges and a bulge in the middle. The surface is smooth and easy to pick up. Gram staining is red, indicating that it is a Gram-negative bacterium.

[0012] Molecular biological identification: The fragment size of 16S rDNA of Pseudomonas S11 is about 1800bp, and its homology with Pseudomonas fulva in the genus Pseudomonas is 99%. It is closely related to strains Pseudomonas fulva (NR-113857.1) and Pseudomonas fulva (NR-104280.1), and is clustered together with Pseudomonas fulva (NR-104280.1).

[0013] On the one hand, the present application provides the use of the above-mentioned Pseudomonas S11 in at least one of phosphate solubilization, nitrogen fixation, siderophore secretion and indoleacetic acid production.

[0014] On the one hand, the present application provides the application of the preparation containing the above-mentioned Pseudomonas S11 as an active ingredient in agriculture.

[0015] In one aspect, the present application provides a bacterial agent containing the above-mentioned Pseudomonas S11.

[0016] On the one hand, the present application provides the use of the above-mentioned Pseudomonas S11 or the above-mentioned bacterial agent in promoting plant growth.

[0017] In some embodiments, the plants include Rosaceae and Poaceae.

[0018] In one aspect, the present application provides a plant growth agent for promoting plant growth containing the above-mentioned Pseudomonas S11 or the above-mentioned bacterial agent.

[0019] In some embodiments, the plants include Rosaceae and Poaceae.

[0020] In one aspect, the present application provides a medicine containing the above-mentioned Pseudomonas S11 or the above-mentioned bacterial agent.

[0021] In some embodiments, the drug can be used for biological control of strawberry gray mold and corn leaf spot.

[0022] This application has the following beneficial effects:

[0023] (1) The Pseudomonas S11 provided in the present application has the ability to solubilize phosphate, fix nitrogen, secrete iron carriers and secrete IAA, which can enhance the plant's absorption of elements such as phosphorus, nitrogen and iron, regulate plant growth and development, and promote plant wound healing.

[0024] (2) The Pseudomonas S11 provided in this application has an excellent growth-promoting effect on plants such as strawberry, corn and rice, and has a significant biological control effect on strawberry gray mold and corn leaf spot, and has a strong application value.

[0025] (3) The Pseudomonas S11 provided in the present application has a good antagonistic effect on plant disease microorganisms such as Zea mays, Botrytis cinerea, Aspergillus flavus, Rhizoctonia solani, Fusarium oxysporum, Fusarium solani, Curvularia lunulata, Verticillium dahliae, Sporangium spp., Pseudomonas tea, Colletotrichum gloeosporioides, Fusarium graminearum, Alternaria solani, Alternaria alternata, Fusarium solani and Colletotrichum boninensis, and is a strain with great research potential and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present disclosure, the following briefly introduces the drawings required to be used in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams, and are not limitations on the actual process of the method involved in the embodiments of the present disclosure.

[0027] Figure 1 The morphological characteristics of Pseudomonas S11 are shown in Figure 1 (a. Colony plate growth diagram, scale bar is 1 cm; b. Single colony morphology diagram, scale bar is 100 μm; c. Gram staining diagram, scale bar is 10 μm; d. 3000x scanning electron microscope diagram, scale bar is 500 nm);

[0028] Figure 2 This is the electrophoresis detection result of the PCR amplification product of Pseudomonas S11 (lane M is 2000bp Maker);

[0029] Figure 3 This is the phylogenetic tree of Pseudomonas S1116S rDNA gene;

[0030] Figure 4 The antagonistic effect of Pseudomonas S11, Botrytis cinerea and Helminthosporium maydis in a dish (a: Botrytis cinerea group; b: Helminthosporium maydis group; Mock: blank control group);

[0031] Figure 5 The antagonistic effects of volatile substances produced by Pseudomonas S11 on Botrytis cinerea and Helminthosporium maydis (a: Botrytis cinerea group; b: Helminthosporium maydis group);

[0032] Figure 6 The growth diagram of Pseudomonas S11 on different culture media (a. PVK medium, b. calcium phytate medium, c. Ashby medium, d. CAS medium);

[0033] Figure 7 The photos show the effect of Pseudomonas S11 on the disease of detached strawberry leaves infected with Botrytis cinerea.

[0034] Figure 8 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the lesion area of ​​detached strawberry leaves infected with Botrytis cinerea;

[0035] Fig. 9 The photos show the effects of Pseudomonas S11 on the disease of living strawberry leaves infected with Botrytis cinerea.

[0036] Fig.10 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the disease index of living strawberry leaves infected with Botrytis cinerea;

[0037] Fig.11 This is a statistical bar chart of the control effect of Pseudomonas S11 on the disease of living strawberry leaves infected with Botrytis cinerea;

[0038] Fig.12 Photos showing the effect of Pseudomonas S11 on the disease of detached corn leaves infected with Helicoverpa maydis (a. blank control group, b. experimental group, c. positive control group);

[0039] Fig.13 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the lesion area of ​​detached corn leaves infected with Helicoverpa maydis.

[0040] Fig.14 Photos showing the effects of Pseudomonas S11 on the disease of living corn leaves infected with Helicoverpa maydis (a. blank control group, b. experimental group, c. positive control group);

[0041] Fig.15 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the disease index of living corn leaves infected with Helicoverpa maydis.

[0042] Fig.16 This is a statistical bar graph of the control effect of Pseudomonas S11 on the disease of living corn leaves infected with Helicoverpa maydis.

[0043] Fig.17 The photos show the phenotypic effects of Pseudomonas S11 treatment on strawberry seedlings.

[0044] Fig.18 This is a statistical bar graph showing the effects of Pseudomonas S11 treatment on some agronomic traits of strawberry seedlings (a. plant height, b. maximum leaf area, c. fresh weight, d. dry weight);

[0045] Fig.19 This is a photo of the phenotypic effects of Pseudomonas S11 treatment on potted corn seedlings (the pots were removed when the photo was taken);

[0046] Fig. 20 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the plant height of potted corn seedlings;

[0047] Fig.21 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the root length of potted corn seedlings;

[0048] Fig. 22 The photos show the phenotypic effects of Pseudomonas S11 treatment on corn seedlings in the field;

[0049] Fig.23 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the plant height of corn seedlings in the field;

[0050] Fig.24 The photos show the phenotypic effects of Pseudomonas S11 treatment on rice seedlings;

[0051] Fig.25 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the plant height of rice seedlings;

[0052] Fig.26 This is a statistical bar graph showing the effect of Pseudomonas S11 treatment on the root length of rice seedlings. DETAILED DESCRIPTION

[0053] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0054] Example 1: Screening of strain S11

[0055] S1. Weigh 5 g of the shade-dried rhizosphere soil sample of Taxus chinensis and put it into 100 ml of a sterile aqueous solution containing glass beads. After shaking at 180 r / min at 4°C for 30 min, a rhizosphere soil suspension was obtained.

[0056] S2. The rhizosphere soil suspension was allowed to stand for 5 minutes, and the supernatant was diluted to 10 by 10-fold dilution method. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 5 dilution gradient solutions, each dilution gradient solution is 10mL;

[0057] S3. Take 100 μL of each dilution gradient solution and evenly apply it on the LB solid plate, and apply 3 plates for each gradient dilution solution;

[0058] S4. Place each of the plates upside down in a constant temperature incubator, culture at 28° C. for 2 to 3 days, and collect single colonies with different morphologies and colors;

[0059] S5. Inoculate each of the single colonies into a flask containing 50 mL of LB liquid culture medium, shake and culture at 28°C and 200 r / min for 24 h to obtain a number of activated bacterial solutions;

[0060] S6. Inoculate the pathogenic fungus as the target fungus on a PDA solid plate, and after constant temperature culture at 28°C for 3 to 5 days, use a 6 mm diameter puncher to punch holes in the cultured target fungus culture medium to obtain a bacterial cake, and inoculate the bacterial cake to the center of a new PDA solid culture medium. Each pathogenic fungus is inoculated with 3 new PDA solid culture media;

[0061] S7. 10 μL of each activated bacterial solution was spot-spread at a distance of 2 cm from the center of each new PDA solid culture medium, and the culture was evenly streaked as the experimental group. The new PDA solid culture medium inoculated with only bacterial cake was used as the control group. The experimental group and the control group were cultured for 7 days, and the strain with antagonistic effect was screened and named S11.

[0062] Example 2: Identification of antagonistic strain S11

[0063] The colony morphology of the antagonistic strain S11 on LB medium is light yellow, small and round, with neat edges, a bulge in the middle, a smooth surface that is easy to pick up, and a slightly reddish Gram stain, which belongs to Gram-negative bacteria (such as Figure 1 shown);

[0064] The 16S rDNA fragment of the antagonistic strain S11 was amplified, and the amplified product was subjected to gene sequencing and agarose gel electrophoresis. The results of agarose gel electrophoresis showed that the size of the 16S rDNA fragment of the antagonistic strain S11 was about 1800 bp (e.g. Figure 2 shown);

[0065] The sequencing results of the 16S rDNA fragment amplification products of the antagonistic strain S11 were compared with the known sequences in GenBank using BLAST, and the sequences of the strains with the closest relationship were downloaded. The phylogenetic tree was established using the Neighbor-Join method using MEGA7.0 software (e.g. Figure 3 As shown in the homology comparison results of NCBI, the homology between the antagonistic strain S11 and Pseudomonas fulva in the genus Pseudomonas reached 99%, and it was closely related to the strains Pseudomonas fulva (NR-113857.1) and Pseudomonas fulva (NR-104280.1). At the same time, it was clustered with Pseudomonas fulva (NR-104280.1), and its bootstrap support rate was 88%.

[0066] Combined with the morphological characteristics and molecular biological identification results of the antagonistic strain S11, it can be determined that the antagonistic strain S11 is Pseudomonas fulva.

[0067] Example 3: Preparation of bacterial fermentation broth and aseptic fermentation broth of Pseudomonas S11

[0068] S21. Inoculate Paenibacillus polymyxa ZR-4 into LB liquid culture medium, place it at 28°C for constant temperature culture and activation for 24 hours, take 100 μL of the activated bacterial solution and apply it on LB solid culture medium, culture it at 28°C for 24 hours, pick a single colony and inoculate it on LB liquid culture medium, culture it at 28°C for 24 hours, and then shake it at 28°C at a constant temperature of 200 r / min for 30 hours to obtain the bacterial fermentation liquid;

[0069] S22. Centrifuge part of the bacterial fermentation liquid at 6000 r / min in a high-speed centrifuge for 5 min, aspirate the supernatant, and filter the bacteria using a 0.22 μm aqueous phase filter to obtain a sterile fermentation liquid.

[0070] Example 4: Antibacterial effect test of Pseudomonas S11 and volatile substances produced by it

[0071] Antibacterial effect of Pseudomonas S11: The streak plate confrontation method was used to determine the in-plate antagonistic effect of Pseudomonas S11 on two pathogens, Botrytis cinerea and B. maydis. Figure 4 According to the antagonistic results, the inhibition rate of Pseudomonas S11 against Botrytis cinerea was 71.23%, and the inhibition rate against B. maydis was 70.45%. The streak plate confrontation method was also used to determine the broad-spectrum antibacterial activity of Pseudomonas S11 against plant disease microorganisms such as Aspergillus flavus, Rhizoctonia solani, Fusarium oxysporum, Fusarium lamina, Curvularia lunulata, Verticillium dahliae, Colletotrichum spp., Pseudomonas graminearum, Alternaria solani, Alternaria alternata, Fusarium solani, and Colletotrichum boninensis. The results are shown in Table 1.

[0072] Table 1 Determination of broad-spectrum antibacterial activity of Pseudomonas S11

[0073] Serial number Pathogen Average antibacterial rate% 1 Aspergillus flavus 53.56％ 2 Rhizoctonia solani 45.02％ 3 Fusarium oxysporum 54.32％ 4 F. proliferatum 69.72％ 5 C.lunata 58.56％ 6 Verticillium dahliae 44.43％ 7 C.fructicola 59.40％ 8 P.theae 59.40％ 9 Colletotrichum coccodes 60.62％ 10 Fusarium graminearum 70.11％ 11 Alternaria solani 59.76％ 12 A. alternata 59.11％ 13 Fusarium solanni 53.14％ 14 C. boninense 61.31％

[0074] According to the above data, the Pseudomonas S11 of the present application has a good antagonistic effect on a variety of common leaf disease pathogens, among which the antagonistic effect on Helicoverpa maydis, Botrytis cinerea and Fusarium graminearum is more significant, with an inhibition rate of more than 70%.

[0075] Antibacterial effect of volatile substances produced by Pseudomonas S11:

[0076] The antagonistic effect of volatile substances produced by Pseudomonas S11 on Botrytis cinerea and Helminthosporium maydis was determined by the two-compartment confrontation culture method. After 7 days of confrontation treatment, the mycelial distribution area on the side inoculated with pathogens in the blank group was significantly larger than that in the treatment group (such as Figure 5 As shown in the figure, after calculation, the volatile substances produced by Pseudomonas S11 had an antibacterial rate of 50.12% against Botrytis cinerea and 45.21% against Helicoverpa maydis.

[0077] According to the above data, the volatile substances produced by Pseudomonas S11 also have a good inhibitory effect on Botrytis cinerea and Helicoverpa maydis.

[0078] Example 5: Evaluation of the Growth-Promoting Potential of Pseudomonas S11

[0079] Pseudomonas S11 was inoculated into different culture media and cultured according to the growth of colonies on each culture medium (e.g. Figure 6The ability of phosphate solubilization, nitrogen fixation and iron carrier secretion was determined by adding a colorimetric reagent to the fermentation broth of Pseudomonas S11 and an IAA standard solution, and the ability of IAA secretion was determined according to the color development of the reagent.

[0080] Phosphorus solubilization ability test: 10 μL of the fermented liquid prepared in Example 3 was inoculated onto a PVK solid culture medium. After culturing at 28°C for 4 days, it was observed that a transparent halo was formed around the colonies, indicating that Pseudomonas S11 has the ability to dissolve inorganic phosphorus; 10 μL of the fermented liquid prepared in Example 3 was inoculated onto a calcium phytate solid culture medium. After culturing at 28°C for 4 days, colony growth was observed, indicating that Pseudomonas S11 has the ability to dissolve organic phosphorus.

[0081] Nitrogen fixation ability test: 10 μL of the fermentation liquid prepared in Example 3 was inoculated onto Ashby solid culture medium. After culturing at 28° C. for 4 days, it was observed that the strain could grow normally and a transparent halo was generated around the colony, indicating that Pseudomonas S11 had the ability to fix nitrogen.

[0082] Test for the ability to secrete siderophore: 10 μL of the fermentation broth prepared in Example 3 was inoculated into the CAS test medium. After culturing at 28° C. for 4 days, an orange halo was observed to form around the colony, indicating that Pseudomonas S11 had the ability to secrete siderophore.

[0083] The ability to secrete indoleacetic acid (IAA) was tested: 200 μL of the fermented liquid obtained in Example 3 was added to a liquid LB medium containing L-tryptophan (0.5 g / L), and cultured in a shaker at 28°C and 200 r / min for 2 days. 2 ml of the cultured bacterial liquid was taken into a centrifuge tube, and centrifuged at 8000 r / min for 10 min. The supernatant was collected, and 1 mL of the collected supernatant was mixed with 1 mL of Salkowski colorimetric reagent, and reacted at room temperature in the dark for 30 min. The results showed that the supernatant could secrete indoleacetic acid (IAA). The color reagent turned red, indicating that Pseudomonas S11 had the ability to secrete IAA; the IAA standard was prepared into standard solutions of 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 μg / L, and 3 standard solutions were set for each concentration. Each standard solution was reacted with the Salkowski color reagent for 30 minutes. After the reaction was completed, 200 μL of the reaction solution was drawn and added to the ELISA plate. The OD530 value of the standard solution at each concentration was determined, and the IAA standard curve was drawn. Its linear equation was Y=0.0589X+0.0863, R 2 =0.9955. According to the standard curve, the content of IAA secreted by Pseudomonas S11 in LB liquid culture medium containing tryptophan is 1.338 μg / L.

[0084] Example 6: Biocontrol assay of Pseudomonas S11 against strawberry gray mold

[0085] In vitro strawberry leaf control test: Select healthy strawberry seedling leaves with similar growth conditions. After surface disinfection, spray the sterile fermentation liquid, LB liquid culture medium and chemical agent Procymidone prepared in Example 3 in equal amounts on the surface of each leaf. Each treatment group is provided with 3 leaves, wherein the sterile fermentation liquid treatment group is used as the experimental group, the LB liquid culture medium treatment group is used as the blank control group, and the chemical agent Procymidone treatment group is used as the positive control group. After spraying for 12 hours, the strawberry seedling leaves of each treatment group are pierced with an inoculation needle and inoculated with Botrytis cinerea. The inoculated leaves are placed in a culture dish containing moist sterile filter paper, and cultured in an environment of 28°C and 70% humidity for 7 days with a 14:10 light-dark alternation. The above test is carried out 3 times in total to observe the leaf disease (such as Figure 7 ), and the lesion area was counted (as shown in Figure 8 shown).

[0086] The results showed that the lesion area of ​​the experimental group was significantly smaller than that of the blank control group, and no lesions appeared on the leaves of the positive control group. This shows that the sterile fermentation liquid of Pseudomonas S11 has a good inhibitory effect on the pathogenicity of Botrytis cinerea on detached strawberry leaves.

[0087] In vivo strawberry leaf control experiment: Select strawberry seedlings with similar growth, disinfect the leaf surface, spray the sterile fermentation liquid, LB liquid culture medium and chemical agent Procymidone obtained in Example 3 on the surface of each leaf, and each treatment group is provided with 3 leaves, wherein the sterile fermentation liquid treatment group is used as the experimental group, the LB liquid culture medium treatment group is used as the blank control group, and the chemical agent Procymidone treatment group is used as the positive control group. After spraying for 24 hours, a spore suspension of Botrytis cinerea is evenly sprayed on the surface of the strawberry seedling leaves of each treatment group using a spray bottle, and the leaves are cultured alternately in 14:10 light-darkness at 28°C and 70% humidity for 14 days. The above test is carried out 3 times to observe the incidence of the strawberry seedling leaves (such as Fig. 9 As shown), and the disease index of strawberry seedlings was calculated (as shown Fig.10 shown) and control effects (as shown Fig.11 For classification of disease severity, refer to Table 2.

[0088] Table 2 Grades of gray mold on strawberry leaves

[0089] Disease grade The proportion of lesion area to leaf area Level 0 No disease spots; Level 1 The lesion area is less than 5% of the total area; Level 2 5% ≤ lesion area < 20% of the total area Level 3 20% ≤ lesion area < total area 35% Level 4 35% ≤ lesion area < 50% of total area Level 5 Lesion area ≤ 50%

[0090] Disease index = ∑ (disease level × total number of leaves at that level) / (highest disease level × total number of leaves) × 100%

[0091] Preventive and therapeutic effect = (disease index of blank control group - disease index of treatment group) / disease index of blank control group × 100%

[0092] The results showed that after spraying the spore suspension of Botrytis cinerea, the disease index of the blank control group was 95.23%. In the early stage of pathogen infection, water-soaked spots or inverted "V"-shaped spots appeared on the base leaves. In the late stage of the disease, all the leaves died, and gray-white mold-like substances were generated on the spots of leaves with high humidity; the disease index of the experimental group was 68.73%, and a small number of diseased leaves appeared at the base of the strawberry seedlings, and the control effect reached 34.21%; the disease index of the positive control group was 14.22%. It can be seen that the sterile fermentation liquid of Pseudomonas S11 has a good biological control effect on Botrytis cinerea on living strawberry leaves.

[0093] Example 7: Biocontrol assay of Pseudomonas sp. S11 against corn leaf blight

[0094] In vitro corn leaf control experiment: Select healthy corn seedling leaves with similar growth conditions, and after surface disinfection, spray the surface of each leaf with equal amounts of the sterile fermentation liquid prepared in Example 3, LB liquid culture medium and chemical agent carbendazim. Each treatment group is provided with 3 leaves, wherein the sterile fermentation liquid treatment group is used as the experimental group, the LB liquid culture medium treatment group is used as the blank control group, and the chemical agent carbendazim treatment group is used as the positive control group. After spraying treatment for 12 hours, the corn seedling leaves of each treatment group are pierced with an inoculation needle and inoculated with Zea mays flat navel helminthespores. The inoculated leaves are placed in a culture dish containing moist sterile filter paper, and cultured in an environment of 28°C and 70% humidity for 7 days with a 14:10 light-dark alternation. The above test is carried out 3 times in total, and the disease condition of the leaves is observed (such as Fig.12 ), and the lesion area was counted (as shown in Fig.13 shown).

[0095] The results showed that the lesion area of ​​the blank control group was 1.22 cm 2 The lesion area of ​​the experimental group was 0.43 cm 2 , which was significantly lower than that of the blank control group. No lesions appeared on the leaves of the positive control group. The experimental results showed that the sterile fermentation liquid of Pseudomonas S11 had a significant inhibitory effect on the pathogenicity of Helicoverpa maydis on detached corn leaves.

[0096] In vivo corn leaf control experiment: The experimental field was divided into two sections with an area of ​​1m 2The test plots were 50 cm apart, and when the corn seedlings in each plot were in the trumpet stage with consistent growth, the sterile fermentation liquid, LB liquid culture medium and chemical agent carbendazim prepared in Example 3 were sprayed on the surface of the leaves of the corn seedlings in each plot using a spray bottle. A randomized block design was adopted, and three plots were set for each treatment group, wherein the sterile fermentation liquid treatment group plot was used as the experimental group, the LB liquid culture medium treatment group plot was used as the blank control group, and the chemical agent carbendazim treatment group plot was used as the positive control group. After spraying for 24 h, a spore suspension of Zea mays was evenly sprayed on the surface of the leaves of the corn seedlings in each treatment group plot using a spray bottle, and the corn seedlings were cultivated for 30 d under natural conditions. The incidence of corn leaves in all treatment group plots was observed and recorded (such as Fig.14 ), and calculate the disease index of corn seedlings (as shown Fig.15 shown) and control effects (as shown Fig.16 For classification of disease severity, refer to Table 3.

[0097] Table 3 Incidence grade of corn leaf spot

[0098]

[0099] Disease index = ∑ (disease level × total number of plants at that level) / (highest disease level × total number of plants) × 100%

[0100] Preventive and therapeutic effect = (blank control group disease index - treatment disease index) / blank control group disease index × 100%

[0101] The results showed that after the corn seedlings in the blank control group were sprayed with the spore suspension of Zea maydis, the final statistical disease index was 88.17%. In the early stage of pathogen infection, small brown spots appeared on the base leaves, and the spots gradually connected into long ovals and spread upward. In the late stage of the disease, the top leaves of the corn plants were also infected, and the elliptical or circular spots were densely connected into pieces, and the whole plant was infected; the disease index of the experimental group was 43.33%, and a small number of brown spots appeared on the base leaves of the corn plants, and the control effect reached 50.11%; the disease index of the positive control group was 16.98%, and the control effect was 75.32%. It can be seen that the sterile fermentation liquid of Pseudomonas S11 has a good biological control effect on Zea maydis on living corn leaves.

[0102] Example 8: Growth promotion assay of strawberry seedlings by Pseudomonas S11

[0103] Select the aseptic soil potted strawberry seedlings with similar growth, and each strawberry seedling is root-irrigated with 20 mL 1 × 105 CFU / mL of the bacterial fermentation liquid prepared in Example 3 and 20 mL LB liquid medium. Each treatment group is provided with 3 potted plants, wherein the bacterial fermentation liquid treatment group is used as the experimental group, and the LB liquid medium treatment group is used as the blank control group. After root irrigation treatment, the potted plants of each treatment group are placed in a greenhouse for 21 days. The above test is carried out 3 times in total, and the phenotypes of the strawberry seedlings (such as Fig.17 As shown), and statistics of various growth indicators of strawberry seedlings (such as Fig.18 shown).

[0104] The results showed that after the strawberry seedlings were treated with the fermented liquid of Pseudomonas S11 and cultured for 21 days, the aboveground phenotypes were significantly different compared with the blank control group, including a 110% increase in plant height, a 128% increase in maximum leaf area, a 59% increase in fresh weight, and a 56.41% increase in dry weight compared with the blank control group. It can be seen that the fermented liquid of Pseudomonas S11 has a significant effect on promoting the growth of strawberry seedlings.

[0105] Example 9: Pseudomonas S11 growth promotion assay for corn

[0106] Potted growth promotion assay for corn seeds: corn seeds with similar germination degree after germination were selected and sown in sterile soil pots. Each corn seed was root-irrigated with 20 mL of 1×105 CFU / mL bacterial fermentation liquid prepared in Example 3 and 20 mL of LB liquid culture medium. Five pots were set for each treatment group, among which the bacterial fermentation liquid treatment group was used as the experimental group and the LB liquid culture medium treatment group was used as the blank control group. After root irrigation treatment, each treatment group was placed in a greenhouse for 14 days. The above test was carried out 3 times in total, and the phenotype of the corn seedlings (such as Fig.19 ), and measure and count the plant height growth index of corn seedlings (as shown in Fig. 20 ) and root length growth indicators (such as Fig.21 shown).

[0107] The results showed that compared with the blank control group, the plant height and root length of the experimental group were significantly different, among which the plant height increased by 47.78% and the root length increased by 39.72% compared with the blank control group. In addition, compared with the blank control group, after treatment with the bacterial fermentation liquid, the number and length of the lateral roots of the corn seedlings increased significantly, which allowed the plants to absorb more nutrients, thereby promoting the growth of the aboveground part.

[0108] The field growth promotion effect on corn seedlings: The experimental field was divided into several areas of 1m 2The experimental plots were 50 cm apart, and 9 corn seedlings with the same growth were transplanted in each plot. When the corn seedlings in each plot were in the three-leaf stage with the same growth, the fermented liquid and LB liquid culture medium prepared in Example 3 were respectively irrigated at the root of the corn seedlings. A random block design was adopted, and 3 plots were set for each treatment group, among which the fermented liquid treatment group was used as the experimental group, and the LB liquid culture medium treatment group was used as the blank control group. After 30 days of root canal treatment, corn plants were randomly selected and transplanted into pots, and the phenotypes of the corn seedlings were observed and recorded ( Fig. 22 ), and measure and count the plant height growth index of corn seedlings ( Fig.23 ).

[0109] The results showed that after field root irrigation, the plant height of the experimental group increased by 40.32% compared with the blank control group.

[0110] It can be seen that the fermentation broth of Pseudomonas S11 can promote the growth of corn seedlings.

[0111] Example 10: Growth promotion assay for rice seedlings

[0112] Rice seeds with similar germination degree after germination were selected and sown evenly in hydroponic boxes. 20 mL of 1×105 CFU / mL bacterial fermentation liquid prepared in Example 3 and 20 mL of LB liquid culture medium were added to each hydroponic box. Three hydroponic boxes were set for each treatment group, among which the bacterial fermentation liquid treatment group was used as the experimental group and the LB liquid culture medium treatment group was used as the blank control group. The seeds were placed in the greenhouse for 14 days, and the phenotypes of the rice seedlings were observed and recorded (such as Fig.24 ), and measure and count the plant height growth index of rice seedlings (as shown in Fig.25 ) and root length growth indicators (such as Fig.26 shown).

[0113] The results showed that after the rice seeds in the hydroponic box were treated with the bacterial fermentation liquid of Pseudomonas S11 and cultivated for 14 days, the plant height and root length were significantly different from those of the blank control group. The plant height increased by 47.78% and the root length increased by 39.72% compared with the blank control group. It can be seen that the bacterial fermentation liquid of Pseudomonas S11 has a good promoting effect on the growth of rice seedlings.

[0114] In summary, the Pseudomonas S11 provided in this application has the ability to dissolve inorganic phosphorus and organic phosphorus and fix nitrogen, can secrete siderophores and IAA, has excellent growth-promoting effects on plants such as strawberry, corn and rice, and also has significant biological control effects on strawberry gray mold and corn leaf spot, and has good antagonistic effects on a variety of plant pathogenic microorganisms, providing a new biological solution for plant disease control and plant growth promotion for agricultural production. Through in-depth research and application development, Pseudomonas S11 is expected to provide more environmentally friendly and efficient biological pesticides and plant growth promoters for agricultural production, and promote the sustainable development of agriculture.

[0115] The above is only a preferred embodiment of the present disclosure. It should be understood that the present disclosure is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present disclosure, and should be within the scope of protection of the claims attached to the present disclosure.

Claims

1. Pseudomonas sp. S11, characterized in that The Pseudomonas S11 is a fulva Pseudomonas (Pseudomonas fulva), and the Pseudomonas S11 is preserved in the China Center for Type Culture Collection, the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation date is March 21, 2022, and the preservation number is CCTCC NO: M2022294.

2. Use of the Pseudomonas S11 according to claim 1 in at least one of phosphate solubilization, nitrogen fixation, siderophore secretion and indoleacetic acid production.

3. Use of the preparation containing the Pseudomonas S11 as claimed in claim 1 as an active ingredient in agriculture.

4. A bacterial agent containing the Pseudomonas S11 according to claim 1.

5. Use of the Pseudomonas S11 according to claim 1 or the bacterial agent according to claim 4 in promoting plant growth, characterized in that: The plants include Rosaceae and Poaceae.

6. A plant growth agent for promoting plant growth comprising the Pseudomonas S11 according to claim 1 or the bacterial agent according to claim 4, characterized in that: The plants include Rosaceae and Poaceae.

7. A medicine comprising the Pseudomonas S11 according to claim 1 or the bacterial agent according to claim 4.

8. The drug according to claim 7, characterized in that The drug can be used for biological control of strawberry gray mold and corn leaf spot.

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