Paenibacillus sp. WF1 strain having antifungal effect against plant pathogenic fungi and improving crop growth, and use of thereof

Abstract

The present invention relates to a novel strain of the genus Paenibacillus, Paenibacillus sp. WF1 (KCTC 1525BP), which promotes plant growth and has antibacterial activity against plant pathogenic fungi, and to increasing plant growth and yield by applying the same to plant cultivation.

Description

Technology Field

[0001] The present invention relates to a novel strain of the genus Paenibacillus that has antibacterial activity against plant pathogenic fungi and promotes plant growth by decomposing organic matter in the soil, and to the use thereof. Background Technology

[0002] Climate change caused by human activity is currently manifesting itself in various parts of the world through extreme weather events and consequent declines in crop yields. Consequently, conventional farming methods, such as chemical-based fertilization and pest control, are gradually shifting toward alternatives, and microorganisms beneficial to plant growth are being utilized as the most promising alternative. Bacteria in the soil that are beneficial to plants are called plant growth-promoting rhizobacteria (PGPRs); these bacteria aid plant growth by decomposing organic matter in the soil and converting it into nutrients available to plants, or by exhibiting antimicrobial activity against plant pathogens (Olanrewaju et al. (2017) World J Microbiol. Biotechnol. 33:197).

[0003] Accordingly, several soil bacteria have been developed for agricultural use and are being utilized in agriculture. However, the types are Bacillus , Pseudomonas , BurkholderiaIt is limited to a very small number of genera. It has been reported that the treatment effects (of organic fertilizers or microorganisms) in the field sometimes fall short of expectations (Sharpe et al. (2020) PLoS ONE. 15(1):e0227429). To enhance the treatment effects of microorganisms in actual application, it is necessary to develop diverse strains that can be utilized in various field environments. Although the potential for use of bacteria of the genus Paenibacillus has been reported as they aid plant growth and exhibit various antimicrobial activities (Grady et al. (2016) Microbial Cell Factories. 15:203), there are currently no strains in Korea that have been commercially developed or commercialized for agriculture. Prior art literature

[0004] Korean Registered Patent No. 10-1183538 (Novel Paenibacillus polymixa and microbial preparation for preventing plant blight containing the same) Korean Registered Patent No. 10-1498155 (Method for promoting potato production using a novel Paenibacillus genus strain)

[0005] Grady et al. (2016) Current knowledge and perspectives of Paenibacillus: a review. Microbial Cell Factories. 15:203 The problem to be solved

[0006] The types of agricultural microorganisms with antifungal activity currently on the market are extremely limited. In particular, products containing strains isolated and developed overseas often fail to produce the expected effects when applied by domestic farms. Therefore, in order to expand the scope of application and increase the efficiency of microorganisms in actual agricultural fields, it is necessary to secure and develop diverse microbial strains derived from the various environments of our soil (Sa Dong-min et al. 2011. (Project Report) Research on the Development of Technology to Enhance Field Utilization of Microbial Fertilizers). Furthermore, it is necessary to secure a variety of materials that can be directly applied to the agricultural industry by verifying the plant protective functions and plant growth-promoting effects of these strains. means of solving the problem

[0007] The present invention relates to a species of Paenibacillus (deposited under accession number KCTC15125BP) isolated from the plant rhizosphere in a barren environment Paenibacillus sp.) WF1 strain (hereinafter WF1 strain) is provided.

[0008] The present invention relates to a plant cultivation experiment in which plant growth increased when the culture medium was treated with the WF1 strain, and lettuce sclerotinia pathogen ( Sclerotinia sclerotiorum The study was completed by confirming that the incidence of sclerotinia disease was reduced compared to the control group when a WF1 strain culture solution was sprayed on plants inoculated with the strain. The strain exhibited organic matter decomposition activity and various enzyme activities, as well as antimicrobial activity against plant pathogenic fungi.

[0009] Considering the foregoing, the composition of the present invention can be understood as a microbial agent that promotes plant growth in one aspect, and as a composition for controlling plant pathogenic fungi in another aspect.

[0010] In this invention, "microbial preparation" refers to an agricultural material that utilizes a biological method among physical, chemical, and biological methods for improving crop productivity.

[0011] The above microbial preparation may use a culture medium containing or from which the WF1 strain has been removed, but is not limited thereto.

[0012] In addition, the microbial preparation of the present invention may include excipients, stabilizers, and additives depending on the formulation or intended use, but is not limited to specific ones, and the components and amounts added may be selected within a range that does not impair the effect of the microbial preparation.

[0013] The above "plant pathogenic fungus" is pepper blight ( Phytophthora capsisi ), sheath blight ( Rhyzoctonia solani ), gray mold disease( Botrytis cinerea ), lettuce sclerotinia disease( Sclerotinia sclerotiorum It is preferable to select one of the groups consisting of ), but is not limited thereto.

[0014] For the control of plant pathogenic fungi, infection can be suppressed by spraying or misting the culture solution of the strain of the present invention onto the leaves of plants exposed to the fungus, and when using a culture solution containing live bacteria, the concentration of WF1 bacteria in the culture solution is the optical density (OD) at 600 nm 600 ) 0.1 to 1.0 is preferable, but the treatment method and concentration of the strain are not limited to this.

[0015] In addition, the microbial preparation of the present invention exhibits a synergistic effect when mixed with organic fertilizer, preferably chicken manure compost, in barren soil where plant growth is unfavorable, such as construction waste, as shown in the following examples, but is not limited to the type of soil, the type of organic fertilizer mixed, or the amount of fertilizer applied. Effects of the invention

[0017] The present invention PaenibacillusThe use of the sp. WF1 strain in plant cultivation can lead to rapid growth and increased yields. In particular, this strain exhibits organic matter decomposition activity and various enzymatic activities, enabling synergistic effects when used in combination with organic fertilizers. The growth inhibitory effect of this strain against plant pathogenic fungi indicates its potential as a plant disease control agent and suggests that it can be utilized as a biopesticide material to replace chemical pesticides. Brief explanation of the drawing

[0018] Figure 1 is a phylogenetic tree showing the 16S rDNA sequences of the WF1 strain and other species of Paenibacillus. Figure 2 shows the organic matter decomposition activity of the WF1 strain. Figure 3 shows the various enzyme activities of the WF strain. Figure 4 shows the ammonia activity of the WF1 strain, confirmed using Nessler's reagent. Figure 5 shows the antimicrobial activity of the WF1 strain against plant pathogenic fungi. Figure 6 shows the inhibitory effect of the WF1 strain on the development of lettuce sclerotinia disease. Specific details for implementing the invention

[0019] The present invention will be described in detail below through examples. However, these examples are described for illustrative purposes to specifically explain the process of the present invention, and the scope of the present invention is not limited to these examples; it is obvious that variations and modifications can be made by those skilled in the art within the concept and scope of the present invention.

[0020] <Example 1> Paenibacillus Isolation and identification of sp. WF1 strain

[0021] To isolate soil microorganisms that aid plant growth in the rhizosphere of plants in environments where it is difficult for plants to establish themselves, soil surrounding weed roots was collected from the soil of a sidewalk frequently visited by people and exposed to the hot heat and sunlight of a dry midsummer. Sterile water (about 10 mL), approximately twice the volume of the collected soil (about 5 mL), was added to the soil and left at room temperature for about 30 minutes. After the soil settled, the supernatant was diluted again in sterile water by 1 / 100 and 1 / 500. 50 μL of each diluted solution was spread onto a plate containing 1 g / L each of tryptone, ammonium sulfate ((NH4)2SO4), and sodium nitrate (NaNO3), and 1% soil extract (hereinafter referred to as soil bacteria medium A), and incubated overnight at room temperature. The generated strains were each cultured in new soil bacterial medium A, and then requested to Bionics Co., Ltd. for 16S rDNA gene sequencing analysis for identification. The 16S rDNA was amplified and analyzed using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the resulting sequences were analyzed by searching for species with similar sequences using the blastn algorithm in the NCBI (National Center for Biotechnology Information) database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), thereby identifying Paenibacillus ( Paenibacillus It was identified as a bacterium of the genus ).

[0022] The 16S rDNA sequence of the WF1 strain is in the database Paenibacillus It was most similar to bacteria of the genus, and the most similar Phenibacillus bovis ( P. bovis It was found to match up to 99% with ). Collected from the database PaenibacillusThe phylogenetic tree is shown in Figure 1 by comparing the 16S rDNA sequences of bacteria and Bacillus with the sequence of the WF1 strain. The sequences compared in Figure 1 are as shown in Table 1 below.

[0023] strain name GenBank® ID Paenibacillus sp. WF1 (Strain of the present invention) Paenibacillus bovis strain BD3526 NR_148888.1 Paenibacillus shenyangensis strain A9 NR_178643.1 Paenibacillus dauci strain H9 NR_136852.1 Paenibacillus sp. FZW.41 KT427400.1 Bacillus sp. strain FJAT-29882 MF948342.1 Paenibacillus elgii JCK 1400 MK801237.1 Paenibacillus kribbensis T-9 KF019740.1 Paenibacillus xylanexedens S210B16 KM507159.1

[0025] <Example 2> Paenibacillus Organic matter decomposition activity of sp. WF1 strain

[0026] To test the protein, starch, and cellulose degradation activities of the above strain, soil bacterial medium A was used, supplemented with 1 g / L of skim milk for protein degradation activity, 2 g / L of corn starch for starch degradation activity, and 2 g / L of carboxymethyl cellulose for cellulose degradation activity. 1, 2, and 4 μL of liquid-cultured WF1 suspensions, and 1, 2, and 4 μL of culture suspensions of a control strain (D12) with no organic matter degradation activity were dropped onto the above agar plates, respectively, and incubated overnight at room temperature. Protein degradation activity was confirmed by the formation of a clear halo around the colony where the strain grew, while starch and cellulose degradation activities were confirmed by the presence of a clear halo after pouring Lugol's solution diluted to 1 / 5 onto the agar plate where the strain grew, staining for about 5 minutes, and then repeatedly rinsing with distilled water until the stain was removed to some extent. These organic matter analysis methods were carried out by referring to and modifying the following literature.

[0027] Jeong, Jeong-yong et al. (2011) Isolation and selection of functional microorganisms from livestock manure compost for eco-friendly turf management. Weed & Turf grass Science (Korean Journal of Weed & Turfgrass Science) 6(2): 158-164

[0028] Chand et al. (2008) A rapid and easy method for the detection of microbial cellulases on agar plates using Gram's iodine. Curr Microbiol 57: 503-507

[0029] Hossain et al. (2020) Hydrolytic exoenzymes produced by bacteria isolated and identified from the gastrointestinal tract of bombay duck. Frontiers in Microbiology. doi: 10.3389 / fmicb.2020.02097

[0031] Test results showed that the WF1 strain exhibited high degradation activity against all four organic substances: proteins, carbohydrates, lipids, and cellulose. Fig. 2 ).

[0033] <Example 3> Paenibacillus Enzyme activity of sp. WF1 strain

[0034] The API® ZYM kit (Biomerieux 25 200) is an experimental set (kit) for testing the enzymatic activities (19 types) of microorganisms, and it was used to determine the enzymatic activity of WF1. 65 μL of bacterial culture adjusted to a McFarland turbidity of 5 was inoculated into each well of the kit strip and incubated at 30°C for approximately 4 hours. After incubation, one drop each of ZYM A and ZYM B reagents was added to each well, and the enzymatic activity was determined by observing the intensity of color development after leaving the samples under strong light at room temperature for about 1 minute. The WF1 strain exhibited strong activity for esterase, esterase lipase, and naphtol-AS-BI-phosphohydrolase, and weak activity for alkaline phosphatase, leucin arylamidase, valine arylamidase, crystine arylamidase, and acid phosphatase (Fig. 3).

[0035] <Example 4> Paenibacillus Ammonification activity of sp. WF1 strain

[0036] 1 mL of the suspension in which the strain of the present invention was grown was centrifuged, the supernatant was discarded, and only the cells (bacteria) were resuspended in peptone water (0.1% peptone) and incubated at 28°C for approximately 2 hours. After incubation, the solution was centrifuged again, and the supernatant excluding cells was transferred to a new tube. As a control, only peptone water without cell culture was added. 50 μL of Nessler's reagent was added to this solution, and by observing that the solution containing the cells turned yellow, it was confirmed that the cells metabolized the organic nitrogen contained in the peptone water and converted it into ammonia. Fig. 4 ).

[0038] <Example 5> Paenibacillus Antifungal activity of sp. WF1 strain

[0039] The strain of the present invention and a control strain without antimicrobial activity were streaked parallel to each other in transverse lines on soil bacterial medium A, supplemented with 2 g / L of sucrose. Then, an agar piece with hyphae of a plant pathogenic fungus was placed in the center and cultured together at room temperature. The plant pathogenic fungus was obtained from the National Institute of Agricultural Sciences Microbial Bank (Korean Agricultural Culture Collection, KACC), and detailed information is as follows.

[0040] Phytophthora capsici (KACC 40158) Pepper blight pathogen

[0041] Botritys cinerea (KACC 43528) Gray mold

[0042] Rhizoctonia solani AG2-2 (IV) (KACC 40152) Sheath blight

[0043] Sclerotinia sclerotiorum (KACC 40457) Lettuce Sclerotinia

[0044] As a result, unlike the control strain which had no antibacterial activity, it was confirmed that fungal hyphae could not grow around the WF1 strain (Fig. 5).

[0046] <Example 6> Paenibacillus Plant growth-promoting effect of sp. WF1 strain

[0047] Two seedlings each of kale and lettuce (Asia Seed), each having developed one true leaf, were transplanted into pots with a capacity of approximately 3L. For the growing medium, a mixture of soil from construction waste treatment and commercially available potting soil was used in a ratio of approximately 9:1. To observe the effect of treatment with organic fertilizer, 5g of chicken manure compost (compressed dry chicken manure, Hansung Industry) was added to the soil in each pot before planting, submerging it about halfway. A liquid-cultured bacterial suspension was poured onto the growing medium at a rate of 25mL at a time, approximately four times over the first two weeks of plant cultivation. The growth status of the plants after about eight weeks and the weight of leaves harvested a total of three times are shown in Figure 6.

[0048] As a result of the measurements, the WF1 culture solution treatment group showed superior growth compared to the control group, and the yield increased in all three trials. In particular, when chicken manure compost and WF1 were treated together, a higher yield was observed compared to plants treated with only chicken manure compost.

[0050] <Example 7> Paenibacillus Inhibitory effect of sp. WF1 strain on plant pathogenic fungal infection

[0051] As in Example 6, lettuce leaves grown by germination and transplantation were evenly sprayed with lettuce sclerotinia fungus cultured in potato dextrose browth medium. About 1 hour after fungal treatment, the WF1 strain cultured in liquid medium A, a soil bacterial medium without added agar, was sprayed onto the WF1 treatment group. After repeating the treatment three times at two-day intervals, as shown in Figure 7, it was confirmed that the WF1 treatment group had less infection by lettuce sclerotinia fungus and continued growth compared to the control group.

[0053] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15125BP Date of Deposit: 2022-10-06

Claims

Claim 1 Novel Paenibacillus WF1 strain deposited under accession number KCTC 15125BP ( Paenibacillus sp. WF1) Claim 2 In paragraph 1, the Paenibacillus WF1 strain deposited under accession number KCTC 15125BP ( Paenibacillus sp. WF1) is a strain characterized by having the rDNA base sequence of SEQ ID NO.

1. Claim 3 A composition for promoting the growth of lettuce and kale comprising the strain of claim 1 or a culture solution thereof as an active ingredient. Claim 4 An antifungal composition for one or more fungi selected from the group consisting of lettuce sclerotinia pathogen, sheath blight pathogen, pepper blight pathogen, and gray mold pathogen, comprising the strain of claim 1 or a culture solution thereof as an active ingredient.

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