Paenibacillus polymyxa ZR-4 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to paenibacillus polymyxa ZR-4 and application of the paenibacillus polymyxa ZR-4, the paenibacillus polymyxa ZR-4 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, West Beichen Road, Chaoyang District, Beijing, the preservation date is May 17, 2024, and the preservation number is CGMCC No.30589. The paenibacillus polymyxa ZR-4 is named as Paenibacillus polymyxa ZR-4. The paenibacillus polymyxa ZR-4 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 Paenibacillus polymyxa ZR-4 and applications thereof. Background Art

[0002] In the past few decades, in order to ensure crop yields and solve the problem of food and clothing for mankind, chemical pesticides and chemical fertilizers have been used in agriculture. However, chemical fertilizers and chemical pesticides are not used scientifically during use, which not only causes serious pollution of water, air and soil, but also enters the human body through crop residues or food chains, endangering human health. In addition, while chemical pesticides 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 drugs. Biofertilizers and biopesticides not only have good crop quality and yield improvement effects, but also do not pollute the environment. They can also establish a balanced relationship between new biological populations and have a sustainable prevention and 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] At present, many strains of Paenibacillus polymyxa with high application potential that can be used as PGPR have been discovered, and the antagonistic effects of these Paenibacillus polymyxa on common plant disease microorganisms, the biological control effects of common plant diseases, and the effects on the phenotypes or yields of some plants have been studied. Regarding the growth-promoting effect of Paenibacillus polymyxa on strawberry and corn plants, only the increase in yield after the prevention and control of plant diseases has been studied, and the effect of Paenibacillus polymyxa directly promoting the growth of strawberry and corn plants has not been studied. In addition, although some people have begun to study the improvement of the phenotype of rice plants by Paenibacillus polymyxa, the existing Paenibacillus polymyxa does not significantly improve the root length and plant height of rice plants.

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

[0006] The problem that the present application aims to solve is to provide Bacillus polymyxa ZR-4, which can not only promote 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 excellent antagonistic effects on pathogens of various plant diseases, and has broad application prospects and development space.

[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 Paenibacillus polymyxa ZR-4, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen Road West, Chaoyang District, Beijing, the deposit date is May 17, 2024, and the deposit number is CGMCC No.30589.

[0009] The source of the Paenibacillus polymyxa ZR-4 is from the rhizosphere soil of Taxus × media, separated and screened by the dilution spread plate method. Sampling site: Emeishan Nature Reserve, Leshan City, Sichuan Province.

[0010] This application identifies Paenibacillus polymyxa ZR-4 from the perspective of morphology and molecular biology:

[0011] Morphological identification: The colonies of Paenibacillus polymyxa ZR-4 were white, round, translucent, with a smooth and moist surface, and Gram-stained blue, indicating that they were Gram-positive bacteria.

[0012] Molecular biological identification: The fragment size of 16S rDNA of Paenibacillus polymyxa ZR-4 is about 1500bp, which is most closely related to Paenibacillus polymyxa. It is clustered together with P. polymyxa (NR-112641.1), P. polymyxa (NR-114810.1) and P. polymyxa (NR-112117.1), and its bootstrap support rate is 95%.

[0013] On the one hand, the present application provides the use of the above-mentioned Bacillus polymyxa ZR-4 in at least one of solubilizing phosphate, secreting siderophores, producing indoleacetic acid, and producing coumarin compounds.

[0014] On the one hand, the present application provides the application of a preparation having the above-mentioned Paenibacillus polymyxa ZR-4 as an active ingredient in agriculture.

[0015] In one aspect, the present application provides a bacterial agent containing Paenibacillus polymyxa ZR-4.

[0016] On the one hand, the present application provides the use of the above-mentioned Bacillus polymyxa ZR-4 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 Paenibacillus polymyxa ZR-4 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 Paenibacillus polymyxa T4 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 polymyxa bacillus ZR-4 provided in the present application has the ability to solubilize phosphate, secrete siderophores, and secrete IAA, which can not only improve the plant's absorption of elements such as phosphorus and iron, regulate plant growth and development, promote plant wound healing, but also improve the environment and reduce environmental pollution.

[0024] (2) The polymyxa bacillus ZR-4 provided in the present 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. It is a plant rhizosphere growth-promoting bacterium with strong application value.

[0025] (3) The polymyxa bacillus ZR-4 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., Polytrichomoniasis spp., Colletotrichum gloeosporioides, Fusarium graminearum, Alternaria solani, Alternaria alternata, Fusarium solani and Colletotrichum boninensis, and is a bacterium with research value and development prospects.

[0026] (4) The polymyxa Bacillus ZR-4 provided in the present application is the first polymyxa Bacillus discovered to be able to secrete coumarin, coumarin-3-carboxylic acid and 6-hydroxy-4-methylcoumarin, providing a new technical route for the biosynthesis of coumarin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 The morphological characteristics of Paenibacillus polymyxa ZR-4 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);

[0029] Figure 2 This is the electrophoresis detection result of the PCR amplification product of Paenibacillus polymyxa ZR-4 (lane M is 2000bpMaker);

[0030] Figure 3 This is the phylogenetic tree of the T-416S rDNA gene of Paenibacillus polymyxa;

[0031] Figure 4 The growth diagram of Paenibacillus polymyxa ZR-4 on different culture media (a. PVK medium, b. calcium phytate medium, c. Ashby medium, d. CAS medium);

[0032] Figure 5 The photos show the effect of Paenibacillus polymyxa ZR-4 on the disease of detached strawberry leaves infected with Botrytis cinerea.

[0033] Figure 6 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the lesion area of ​​detached strawberry leaves infected with Botrytis cinerea;

[0034] Figure 7 The photos show the effects of Paenibacillus polymyxa ZR-4 on the disease of living strawberry leaves infected with Botrytis cinerea.

[0035] Figure 8 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the disease index of living strawberry leaves infected with Botrytis cinerea;

[0036] Fig. 9 This is a statistical bar chart of the control effect of Paenibacillus polymyxa ZR-4 on the disease of living strawberry leaves infected with Botrytis cinerea;

[0037] Fig.10The photos show the effects of Paenibacillus polymyxa ZR-4 on the disease of detached corn leaves infected with Helicoverpa maydis (a. blank control group, b. experimental group, c. positive control group);

[0038] Fig.11 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the lesion area of ​​detached corn leaves infected with Helicoverpa maydis.

[0039] Fig.12 The photos show the effects of Paenibacillus polymyxa ZR-4 on the disease of living corn leaves infected with Helicoverpa maydis (a. blank control group, b. experimental group, c. positive control group);

[0040] Fig.13 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the disease index of living corn leaves infected with Helicoverpa maydis.

[0041] Fig.14 This is a statistical bar graph of the disease prevention and control effect of Paenibacillus polymyxa ZR-4 on living corn leaves infected with Helicoverpa maydis;

[0042] Fig.15 The photos show the phenotypic effects of Paenibacillus polymyxa ZR-4 on strawberry seedlings.

[0043] Fig.16 This is a statistical bar graph showing the effects of Paenibacillus polymyxa ZR-4 treatment on some agronomic traits of strawberry seedlings (a. plant height, b. maximum leaf area, c. fresh weight, d. dry weight);

[0044] Fig.17 This is a photo of the phenotypic effects of Paenibacillus polymyxa ZR-4 treatment on potted corn seeds (the pots were removed when the photos were taken);

[0045] Fig.18 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the plant height of potted corn seeds;

[0046] Fig.19 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the root length of potted corn seeds;

[0047] Fig. 20 The photos show the phenotypic effects of Paenibacillus polymyxa ZR-4 treatment on corn seedlings in the field.

[0048] Fig.21 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the plant height of corn seedlings in the field;

[0049] Fig. 22 The photos show the phenotypic effects of Paenibacillus polymyxa ZR-4 treatment on rice seedlings.

[0050] Fig.23 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the plant height of rice seedlings;

[0051] Fig.24 This is a statistical bar graph showing the effect of Paenibacillus polymyxa ZR-4 treatment on the root length of rice seedlings;

[0052] Fig.25 Some secondary metabolite components and their relative contents in the fermentation broth of Paenibacillus polymyxa ZR-4;

[0053] Fig.26 This is the standard curve of coumarin content in the fermentation broth of Bacillus polymyxa ZR-4. DETAILED DESCRIPTION

[0054] 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.

[0055] Example 1: Screening of strain ZR-4

[0056] 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.

[0057] 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;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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 mixture 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 and control groups were cultured for 7 days to screen the strain with antagonistic effect, which was named ZR-4.

[0063] Example 2: Identification of antagonistic strain ZR-4

[0064] The colonies of the antagonistic strain ZR-4 were white, round, translucent, smooth and moist, and Gram-stained blue, belonging to Gram-positive bacteria ( Figure 1 );

[0065] The 16S rDNA fragment of the antagonistic strain ZR-4 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 ZR-4 was about 1500 bp ( Figure 2 );

[0066] The sequencing results of the 16S rDNA fragment amplification products of the antagonistic strain ZR-4 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 ( Figure 3 ), in the homology comparison results of NCBI, the antagonistic strain ZR-4 was most closely related to Paenibacillus polymyxa, and clustered with P.polymyxa (NR-112641.1), P.polymyxa (NR-114810.1) and P.polymyxa (NR-112117.1), with a bootstrap support rate of 95%;

[0067] Combined with the morphological characteristics and molecular biological identification results of the antagonistic strain ZR-4, it can be determined that the antagonistic strain ZR-4 is Paenibacillus polymyxa.

[0068] Example 3: Preparation of bacterial fermentation broth and sterile fermentation broth of Paenibacillus polymyxa ZR-4

[0069] 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;

[0070] 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.

[0071] Example 4: Antibacterial effect test of Paenibacillus polymyxa ZR-4

[0072] The plate confrontation method was used to determine the broad-spectrum antibacterial activity of Bacillus polymyxa ZR-4 against plant disease microorganisms such as Helminthosporium of mays, Botrytis cinerea, Aspergillus flavus, Rhizoctonia solani, Fusarium oxysporum, Fusarium laminarum, Curvularia lunulata, Verticillium dahliae, Colletotrichum spp., Pseudomonas aeruginosa, Colletotrichum gloeosporioides, Fusarium graminearum, Alternaria solani, Alternaria alternata, Fusarium solani and Colletotrichum boninensis. The results are shown in Table 1.

[0073] Table 1 Determination of broad-spectrum antibacterial activity of Paenibacillus polymyxa ZR-4

[0074] Serial number Pathogen Average antibacterial rate% 1 B.maydis 78.26％ 2 Botrytis cinerea 80.00％ 3 Aspergillus flavus 57.33％ 4 Rhizoctonia solani 62.20％ 5 Fusarium oxysporum 62.54％ 6 F. proliferatum 63.41％ 7 C.lunata 77.94％ 8 Verticillium dahliae 78.45％ 9 C.fructicola 84.14％ 10 P.theae 84.14％ 11 Colletotrichum coccodes 74.29％ 12 Fusarium graminearum 73.11％ 13 Alternaria solani 65.33％ 14 A. alternata 77.23％ 15 Fusarium solanni 50.12％ 16 C. boninense 75.60％

[0075] Among the existing polymyxa bacillus, the antibacterial rates of ZF129 against Botrytis cinerea, Rhizoctonia solani, Fusarium oxysporum, Verticillium dahliae and Alternaria solani were 63.33%, 55.29%, 60.00%, 72.16% and 57.65%, respectively; the antibacterial rates of KDB against Curvularia lunula and Fusarium graminearum were 83.52% and 60.36%, respectively; the antibacterial rate of DQ-3 against Helicoverpa maydis was 72.96%; the antibacterial rate of Pp146 against Fusarium solani was 48.21%, and there was no obvious antibacterial effect on Fusarium solani; the antibacterial rate of BST292 against Alternaria alternata was 71.72%; there is no research on the antibacterial rate against Aspergillus flavus, Colletotrichum spp., Polytrichomoniasis spp., Colletotrichum sphaerocephala and Colletotrichum boninensis.

[0076] According to the above data, the polymyxa bacillus ZR-4 of the present application has a good antagonistic effect on a variety of common leaf disease pathogens, among which the antagonistic effect on corn flat umbilical spores, Botrytis cinerea, Crescent spores, dahlia verticillium, fruit spiny disc spores, tea pseudodisk multihistospores, alternaria and bonin anthracnose is more significant, with an inhibition rate of more than 75%.

[0077] Example 5: Evaluation of the Growth-Promoting Potential of Paenibacillus polymyxa ZR-4

[0078] Paenibacillus polymyxa ZR-4 was inoculated into different culture media for cultivation. The growth of the colonies on each culture medium was analyzed. Figure 4 ) to determine its ability to dissolve phosphate, fix nitrogen, and secrete iron carriers; add a colorimetric reagent to the fermentation broth of Bacillus polymyxa ZR-4 and an IAA standard solution, and determine its ability to secrete IAA based on the color development of the reagent.

[0079] Phosphorus solubilization ability test: 10 μL of the bacterial fermentation broth prepared in Example 3 was inoculated onto a PVK solid culture medium. After culturing at a constant temperature of 28°C for 4 days, it was observed that a transparent halo was formed around the colonies, indicating that Bacillus polymyxa ZR-4 has the ability to dissolve inorganic phosphorus; 10 μL of the bacterial fermentation broth prepared in Example 3 was inoculated onto a calcium phytate solid culture medium. After culturing at a constant temperature of 28°C for 4 days, colony growth was observed, indicating that Bacillus polymyxa ZR-4 has the ability to dissolve organic phosphorus.

[0080] 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 no transparent circle was formed around the strain, indicating that Paenibacillus polymyxa ZR-4 did not have the ability to fix nitrogen.

[0081] Siderophore secretion ability test: 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 Paenibacillus polymyxa ZR-4 had the ability to secrete siderophores.

[0082] 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) in a Salkowski colorimetric reagent. The reagent turned red, indicating that Bacillus polymyxa ZR-4 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 colorimetric 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 Bacillus polymyxa ZR-4 in the LB liquid culture medium containing tryptophan is 0.918 μg / L.

[0083] Example 6: Biocontrol assay of Paenibacillus polymyxa ZR-4 against strawberry gray mold

[0084] 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 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 situation ( Figure 5 ), and the lesion area was counted ( Figure 6 ).

[0085] The results showed that the lesion area of ​​the blank control group was 3.68 cm 2The lesion area of ​​the experimental group was reduced by 0.94 times compared with that of the control group, which was significantly smaller than that of the blank control group. No lesions appeared on the leaves of the positive control group. It can be seen that the sterile fermentation liquid of Bacillus polymyxa ZR-4 has a good inhibitory effect on the pathogenicity of Botrytis cinerea on detached strawberry leaves.

[0086] 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 strawberry seedlings are cultured alternately in a light-dark environment of 14:10 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 ( Figure 7 ) and calculate the disease index of strawberry seedlings ( Figure 8 ) and control effect ( Fig. 9 ), please refer to Table 2 for the classification of disease levels.

[0087] Table 2 Grades of gray mold on strawberry leaves

[0088] 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%

[0089] Disease index = ∑ (disease level × total number of leaves at that level) / (highest disease level × total number of leaves) × 100%

[0090] Preventive and therapeutic effect = (disease index of blank control group - disease index of treatment group) / disease index of blank control group × 100%

[0091] 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 19.94%, and a small number of diseased leaves appeared at the base of the strawberry seedlings, and the control effect reached 73.19%; the disease index of the positive control group was 14.22%. It can be seen that the sterile fermentation liquid of Bacillus polymyxa ZR-4 has an excellent biological control effect on Botrytis cinerea on living strawberry leaves, which is equivalent to the control effect of the chemical agent procymidone.

[0092] Example 7: Biocontrol assay of Paenibacillus polymyxa ZR-4 against corn leaf blight

[0093] In vitro corn leaf control experiment: Select healthy corn seedling leaves with similar growth conditions, and after surface disinfection, spray the same amount of sterile fermentation liquid, LB liquid culture medium and chemical agent carbendazim prepared in Example 3 on the surface of each leaf. Three leaves are set for each treatment group, 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 for 12 hours, the corn seedling leaves of each treatment group are pierced with an inoculation needle and inoculated with Zea mays flat navel helminth spores. The inoculated leaves are placed in a culture dish containing moist sterile filter paper, and cultured for 7 days at 14:10 light-dark alternation at 28°C and 70% humidity. The above test is carried out 3 times in total to observe the disease condition of the leaves ( Fig.10 ), and the lesion area was counted ( Fig.11 ).

[0094] 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 0.13 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 Bacillus polymyxa ZR-4 had a good inhibitory effect on the pathogenicity of Helicoverpa maydis on detached corn leaves.

[0095] Live corn leaf control experiment: The experimental field was divided into several areas of 1m 2 The test 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 trumpet stage with the same 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. The corn seedlings were cultivated for 30 d under natural conditions, and the incidence of corn leaves in all treatment group plots was observed and recorded ( Fig.12 ) and calculate the disease index of corn seedlings ( Fig.13 ) and control effect ( Fig.14 ), please refer to Table 3 for the classification of disease levels.

[0096] Table 3 Incidence grade of corn leaf spot

[0097]

[0098] Disease index = ∑ (disease level × total number of plants at that level) / (highest disease level × total number of plants) × 100%

[0099] Preventive and therapeutic effect = (blank control group disease index - treatment disease index) / blank control group disease index × 100%

[0100] 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 27.56%, and a small amount of brown spots appeared on the base leaves of the corn plants, and the control effect reached 68.36%; 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 Bacillus polymyxa ZR-4 has an excellent biological control effect on Zea maydis on living corn leaves, which is close to the control effect of the chemical agent carbendazim.

[0101] Example 8: Growth promotion assay of Paenibacillus polymyxa ZR-4 on strawberry seedlings

[0102] 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 are observed and recorded ( Fig.15 ) and counted various growth indicators of strawberry seedlings ( Fig.16 ).

[0103] The results showed that after the strawberry seedlings were treated with the fermented liquid of Paenibacillus polymyxa ZR-4 and cultured for 21 days, the aboveground phenotypes were significantly different compared with the blank control group, including an increase of 88.12% in plant height, 116% in maximum leaf area, 55.16% in fresh weight, and 50.42% in dry weight compared with the blank control group. It can be seen that the fermented liquid of Paenibacillus polymyxa ZR-4 can promote the growth of strawberry seedlings.

[0104] Example 9: Growth promotion assay of corn by Paenibacillus polymyxa ZR-4

[0105] 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 was observed and recorded ( Fig.17 ), and measure and count the plant height growth index of corn seedlings ( Fig.18 ) and root length growth index ( Fig.19 ).

[0106] The results showed that compared with the blank control group, the plant height and root length of the experimental group were significantly different. The plant height increased by 30.71% and the root length increased by 39.93% compared with the blank control group. In addition, compared with the blank control group, the number and length of lateral roots of corn seedlings increased significantly after being treated with bacterial fermentation liquid, which allowed the plants to absorb more nutrients, thereby promoting the growth of the aboveground part.

[0107] The field growth promotion effect on corn seedlings: The experimental field was divided into several areas of 1m 2 The 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. 20 ), and measure and count the plant height growth index of corn seedlings ( Fig.21 ).

[0108] The results showed that after field root irrigation, the plant height of the experimental group increased by 38.71% compared with the blank control group.

[0109] It can be seen that the fermentation broth of Bacillus polymyxa ZR-4 can promote the growth of corn seedlings.

[0110] Example 10: Growth promotion assay of rice seeds by Paenibacillus polymyxa ZR-4

[0111] Rice seeds with similar germination degree after germination were selected and sown evenly in hydroponic boxes. 20 mL of 1×105 CFU / mL 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 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 ( Fig. 22 ), and measure and count the plant height growth index of rice seedlings ( Fig.23 ) and root length growth index ( Fig.24 ).

[0112] The results showed that after the rice seeds in the hydroponic box were treated with the fermented liquid of Paenibacillus polymyxa ZR-4 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 43.41% and the root length increased by 39.33% compared with the blank control group. The existing Paenibacillus polymyxa X-11 increased the plant height of rice seedlings by 15.4% and the root length by 6.8%. It can be seen that the fermented liquid of Paenibacillus polymyxa ZR-4 has a better promoting effect on the growth of rice seedlings.

[0113] Example 11: Analysis of major secondary metabolites in the fermentation broth of Paenibacillus polymyxa ZR-4

[0114] The sterile fermentation broth prepared in Example 3 was extracted with ethyl acetate to obtain a crude extract, which was then analyzed and determined to obtain some secondary metabolite components and their relative contents ( Fig.25 ), and from the analysis results it was found that Bacillus polymyxa ZR-4 has the ability to secrete coumarin compounds.

[0115] The coumarin standard was prepared into 5, 10, 20, 40, and 80 ng / mL standard solutions, the OD450 value at each standard concentration was measured, and a coumarin content standard curve was drawn ( Fig.26 ), its linear equation is Y = 0.0285x + 0.9908, R 2 =0.9199. According to the instruction of the total coumarin ELISA kit of Wuhan Kailai Enzyme Biotechnology Co., Ltd. and the standard curve, after the fermentation of Bacillus polymyxa ZR-4 at 28°C and 200 r / min for 30 hours, the coumarin content in the fermentation broth was 236.25 ng / L.

[0116] In summary, the polymyxa bacillus ZR-4 provided by the present application has the ability to dissolve inorganic phosphorus and organic phosphorus, 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 prevention and control and plant growth promotion for agricultural production. In addition, coumarin compounds, as a class of compounds with antibacterial ability, are currently mainly isolated from plants, and the present application has discovered for the first time a polymyxa bacillus capable of producing coumarin, coumarin-3-carboxylic acid and 6-hydroxy-4-methylcoumarin. Through in-depth research and application development, polymyxa bacillus ZR-4 is not only expected to provide more environmentally friendly and efficient biological pesticides and plant growth promoters for agricultural production, promote the sustainable development of agriculture, but also provide a new technical route for the biosynthesis of coumarin compounds.

[0117] 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. Paenibacillus polymyxa ZR-4, characterized in that The polymyxa bacillus ZR-4 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen Road West, Chaoyang District, Beijing, the deposit date is May 17, 2024, and the deposit number is CGMCC No.30589.

2. Use of the Bacillus polymyxa ZR-4 according to claim 1 in at least one of phosphate solubilization, siderophore secretion, indoleacetic acid production and coumarin compound production.

3. Use of a preparation containing the polymyxa bacillus ZR-4 as claimed in claim 1 as an active ingredient in agriculture.

4. A bacterial agent containing the Paenibacillus polymyxa ZR-4 according to claim 1.

5. Use of the polymyxa bacillus ZR-4 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 containing the Paenibacillus polymyxa ZR-4 according to claim 1 or the bacterial agent according to claim 4, characterized in that: The plants include Rosaceae and Poaceae.

7. A medicine containing the Paenibacillus polymyxa ZR-4 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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