KR20210055179A - Burkholderia spp. strain having antifungal activity against red pepper anthracnose and composition for controlling red pepper anthracnose - Google Patents

Abstract

The present invention relates to a novel Burkholderia sp. KF1 strain and a composition for controlling pepper anthrax including the same. The strain inhibits mycelial growth and spore germination of pathogens against Colletotrichum species that cause pepper anthrax, so the control of red pepper anthrax using the above strain reduces harmful effects such as ecological pollution caused by the use of chemical pesticides and effectively controls diseases to reduce yield loss due to anthrax, thereby reducing damage to farmers, further securing the stability of food, and expecting ecosystem conservation.

Description

The composition for controlling capsicum anthrax comprising the strain of the genus Burkholderia having antifungal activity against capsicum anthrax bacteria {Burkholderia spp. strain having antifungal activity against red pepper anthracnose and composition for controlling red pepper anthracnose}

The present invention relates to a strain of Burkholderia sp. having antifungal activity against capsicum anthrax bacteria, and more particularly, hyphae growth of pathogens against Colletotrichum species causing capsicum anthrax And a novel isolated strain of the genus Buckholderia having an effect of inhibiting spore germination and a composition for controlling pepper anthrax using the same.

Red pepper (Capsicum annuum L.) is one of the major economic crops with a domestic cultivation area of 28,824 ha and production capacity of 71,509 tons (KOSTAT, 2018). It is high in vitamins A and C and is an important ingredient in Korean food. Capsicin contained in red pepper stimulates appetite and promotes metabolism, preventing fat accumulation in the body, and enhancing the body's immunity. To help. It is also used as an anti-inflammatory agent because of its inflammation-relieving effect, and is known to improve blood circulation and have anti-cancer activity (Kwon et al., 2014; Saxena et al., 2016).

According to the National Agricultural Crop Pest Management System (RDA, http://ncpms .rda.go.kr), major fungal diseases occurring in peppers are brown spotting disease caused by Cercospora capsici, black fungal disease caused by Alternaria spp., and Phytophthora capsici. 14 diseases, including plague, have been reported. Capsicum anthrax caused by Colletotrichum spp. is well known as a major disease that seriously damages not only the yield but also the marketability of fruits (Kim et al., 2015). Capsicum anthrax is caused by the genus Colletotrichum, and the characteristic of capsicum anthrax is a circular brown lesion that is hollow in the infected area of the fruit, and as the disease progresses, orange conidia mucous masses form in the lesion, and the lesion gradually expands. And eventually wraps up the whole fruit. Spores of pathogens are spread by rain and wind, heavy rain, etc., and winter in the form of ascaris and hyphae in infected plant debris or soil. The optimum temperature for infection of pathogens is 20~24℃ and occurs in a humid environment with a relative humidity of about 80% (Pernezny K et al., 2003; Oo & Oh, 2016).

In particular, in the open field cultivation of peppers in summer, the spread of spores is active due to frequent rainfall, and anthrax is prone to occurrence due to high temperature and humidity. Therefore, in order to control anthrax, more than 10 frequent treatments are required until harvest. However, chemical pesticides not only negatively affect the ecosystem, but also the harmfulness of chemical substances remaining in food materials is a problem that must be solved. In order to solve this problem, various environmentally friendly materials or microorganisms have been actively attempted to control anthrax (Kang et al., 2015).

Recently, as interest in eco-friendly agriculture increases, the development of microbial preparations that control crop diseases using useful microorganisms is increasing. Studies on antagonistic microorganisms for controlling pepper anthrax include Pseudomonas koreansis, Bacillus sp., Paenibacillus polymyxa (Park et al., 2006; Kim et al., 2007; Kim et al., 2016), C. gloeosporioides for the control of C. gloeosporioides. There are Streptomyces padanus and Saccharomyces cerevisiae (Chi et al., 2012, Lopes et al., 2015) for the control of acutatum, but there are no studies on the biological control of C. scovillei that have occurred recently.

Korean Patent Registration No. 10-0815106

In order to solve the problems of the prior art as described above, the present invention is Burkholderia cepacia, which has an effect of inhibiting mycelial growth and spore formation against the microorganisms of the genus Coletotricum (Colletotrichum sp.) that cause pepper anthrax. An object of the present invention is to provide a KF1 strain and a composition for controlling pepper anthrax comprising the same.

Another object of the present invention is to provide a method for controlling pepper anthrax using the Burkholderia cepacia KF1 strain, which has an environmentally friendly control effect and can increase the marketability and productivity of pepper.

In order to achieve the object of the present invention as described above, the present invention, Burkholderia cepacia (Burkholderia cepacia) KF1 strain having antifungal activity against the microorganisms (Colletotrichum sp.) in the genus Colletotrichum causing pepper anthrax, and It provides a composition for controlling pepper anthrax comprising this.

In order to achieve another object of the present invention, the present invention is a method for controlling red pepper anthrax, characterized in that the Burkholderia cepacia KF1 strain or metabolites produced therefrom are treated in pepper plants and cultivated areas Provides.

Hereinafter, the present invention will be described in detail.

As an aspect of the present invention, the present invention relates to a strain of KF1 genus Burkholderia having antibacterial activity against pepper anthrax. Burkholderia cepacia KF1 strain of the present invention has antifungal activity against various strains that cause pepper anthrax. Although not limited thereto, in one embodiment of the present invention, the effect of inhibiting the mycelial growth and spores of the microorganisms of the genus Colletotrichum (Colletotrichum sp.) was confirmed. The KF1 strain of the genus Burkeholderia of the present invention was isolated using soil collected from red pepper cultivation areas not treated with a fungicide, and among the 12 colonies that were primarily isolated, two species of red pepper anthrax bacteria (C. nymphaeae, C. scovillei) was selected for microorganisms showing antimicrobial activity. As a result of selecting and identifying the strain with the best antibacterial activity, it was identified as B. cepacia. The relationship with other strains is shown in Fig. 1, named Burkholderia cepacia KF1, and deposited with the Korea Microbial Conservation Center on October 29, 2019. (Accession number: KFCC 11842P)

The KF1 microorganism of the genus Burkeholderia used in the present invention may be a live cell or a culture thereof. In addition, the microorganism may be a spore from the viewpoint of preservation as a product of a microbial pesticide composition. Therefore, in order to form spores, it is preferable to prepare culture conditions such as medium composition, medium pH, culture temperature, culture humidity, and oxygen concentration during culture to suit the spore formation conditions at the end of the culture. In addition, when using spores in the present invention, it is preferable to minimize the moisture content in the composition from the viewpoint of preservation of the biological pesticide formulation composition.

As another aspect of the present invention, the present invention provides a composition for controlling pepper anthrax, comprising the strain and an agrochemically acceptable carrier. As another term, in the present specification, the control composition is described as a biological agent or a microbial pesticide. “Microbial pesticide” means an agricultural control agent using live microorganisms such as fungi, bacteria, viruses and protozoa. The KF1 strain of the genus Burkholderia of the present invention is a microorganism having the ability to prevent the occurrence of anthrax by treating the plant with microorganisms, such as spraying it on the plant, or to cure the induced anthrax. It is a strain. In the composition of the present invention, the strain alone may be included, or a plurality of different microorganisms having the same effect may be mixed and used in the composition.

The amount of microorganisms contained in the biological preparation composition of the present invention is not particularly limited, but is preferably contained in an amount of 1 to 50% by weight based on the total amount of microorganisms and inorganic salts. In addition, the concentration of microorganisms contained in the biological composition of the present invention is a colony-forming unit, preferably 1×10 ³ to 1×10 ¹⁰ cfu/g, more preferably 1×10 ⁵ to 1×10 ⁸ cfu/ can be g When the strain is included at a concentration below the above range, the control effect by microorganisms is insignificant, and when it is included above the above concentration, the control effect may be lowered.

In addition, agrochemically acceptable carriers in the composition are, but are not limited to, surface activators, inert carriers, preservatives, wetting agents, supply accelerators, attractants, encapsulating agents, binders, emulsifiers, dyes, U.V. It can be obtained by adding other ingredients that are easy to apply depending on the target pathogen and crop, such as a protective agent and a buffer. The composition of the present invention may be in a form suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application, or in a concentrate or primary composition. In addition, the composition of the present invention according to a method of formulating a biological agent in a manner known in the art, for example, a culture solution of cultured cells, freeze-dried cells and starch, crude protein and rock powder, etc. It can be formulated as a capsule and used as it is.

The composition of the present invention may contain inorganic salts for the activity of microorganisms in the composition, and components are not particularly limited as long as the effect of the invention is not impaired, and for example, carbonates, sulfates, phosphates and nitrates may be included. . The content is not particularly limited, and may be included in an amount of 10 to 50% by weight based on the total amount of microorganisms and inorganic salts. The inorganic salt makes it possible to prepare a composition that is balanced in various aspects, such as the control effect on plant pathogens and the hydration properties of the biological agent composition.

As an auxiliary component, a surfactant or the like may be included in the composition to improve dispersibility and suspension properties when the composition is abraded. The surfactant is not limited within a range that does not affect the activity of the composition, and any type of surfactant may be included.

The biological preparation of the present invention can be prepared in the form of a variety of preparations including a culture solution or culture of the strain or strain of the genus Burkholderia of the present invention, and for example, it can be prepared as a solution, a wettable powder, a powder, a granule, an emulsion, etc. I can. The manufacturing method follows a general manufacturing method known in the art.

As another aspect of the present invention, the present invention provides a method for controlling pepper anthrax using the KF1 strain of the genus Burkholderia or a metabolite produced therefrom, the method comprising the KF1 strain of the present invention or the KF1 strain of the present invention By using the metabolite produced from the pepper as an environmentally friendly control agent when growing pepper and spraying it on the plant part or cultivation area, the damage to pepper caused by pepper anthrax can be reduced.

The Burkholderia sp. KF1 strain of the present invention has the effect of inhibiting mycelial growth and spore germination of pathogens against Colletotrichum species that cause pepper anthrax. The control of pepper anthrax using the above strains reduces harms such as ecosystem pollution caused by the use of chemical pesticides and effectively controls pests to reduce yield loss caused by anthrax, thereby reducing damage to farmers and further guaranteeing the stability of our food. And ecosystem conservation effects can be expected.

Figure 1 shows the relationship with other strains of the KF1 strain of the Burkholderia genus of the present invention.
Figure 2 shows the results of the enzyme activity analysis in vitro of the KF1 strain of the genus Burkholderia of the present invention. KF1 produced protease, cellulase and sideropore. The halo section of the cellulase assay plate was stained with 0.1% Congo Red. The protease and siderophore plates were incubated at 28° C. for 3 days in dark conditions. Cellulase plates and poorly soluble phosphoric acid solubility were incubated at 28° C. for 7 days in dark conditions.
Figure 3 shows the results of mycelium growth inhibition test for pepper anthrax bacteria of the KF1 strain of the genus Burkeholderia of the present invention. (A): Alternative culture test for inhibition of mycelial growth. Pathogens were incubated on PDA plates at 25° C. for 7 days. (B): The rate of inhibition of mycelial growth was measured 7 days after inoculation against C. scobili, C. nymph a, C. akutatum and F. vultisilliides. Error bars represent the standard deviation of three replicates.
Figure 4 shows the results of the germination inhibition analysis of the KF1 strain of Burkholderia genus of the present invention. The concentration of the spore suspension was 1 x 10 ⁵ spore count/mL. Controls were treated with sterilized distilled water. Photos were taken 16 hours after inoculation. Scale bar = 10 μm.
Figure 5 shows the results of measuring the germination rate of pepper anthrax bacteria according to the treatment concentration when the KF1 strain of the present invention is treated. (A): Germination rate of C. scobili. (B): Germination rate of C. nymphaeae. Error bars represent standard deviations, and the experiment was repeated 3 times 3 times.

Hereinafter, the present invention will be described in more detail by examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Experiment method>

1. Selection of red pepper anthrax and antagonistic microorganisms

The antagonistic microorganisms were isolated from the soil around Mt. Chunma, Hwado-eup, Namyangju-si, Gyeonggi-do, using soil not applied with a fungicide. For isolation of antagonistic microorganisms, the soil was placed in a 1.5 mL tube, ^{diluted with sterile distilled water, diluted to a concentration of 10 -3} to 10 ^-5 , spread on LB agar medium, and incubated for 1 to 2 days at 28°C. After incubation, a single colony was purely isolated. The isolated strains were suspended in 20% glycerol and stored in a deep freezer at -70°C. Pepper anthrax strain C. scovillei was used as a strain isolated in this laboratory, and C. nymphaeae was sold and used by the National Academy of Agricultural Sciences, Rural Development Administration.

2. DNA extraction, PCR and sequencing analysis of antagonistic microorganisms

Genomic DNA extraction of antagonistic microorganisms was performed in 3 mL of LB (Luria Bertani) liquid medium at 28°C and 185 rpm for 16 hours, and the culture solution was extracted according to the method of Wizard Genomic DNA Purification Kit (Promega, USA). Subsequently, the purified DNA was used as primers 27F (`5-AGAGTTTGATCMTGGCTCAG-3`) and 1492R (`5-TACGGYTACCTTGTTACGACTT-3`) for 16S rRNA sequencing, and recA forward (`5- AGGACGATTCATGGAAGAWAGC-3`) and recA reverse (`5-GACGCACTGGAYGMRTAGAACTT-3`) primers were used (Spilker et al., 2009). PCR was performed at 98° C. for 30 seconds after denaturation, 98° C. for 10 seconds, 58° C. for 30 seconds, and 72° C. for 1 minute, repeated 30 times, and then reacted at 72° C. for 7 minutes. The PCR product was electrophoresed for 15 to 20 minutes using 1X TAE (Tris-acetate-EDTA) buffer in 1% agarose gel, and then stained with Etbr and the band was identified using UV. The identified band was purified according to the method of DOKDO-prepTM Gel Extraction Kit (ELPIS BIOTECH, Korea), and then the purified DNA was subjected to sequencing service at Seoul National University (http://nicem.snu.ac. kr) was used. The analyzed nucleotide sequence was analyzed using the BLASTN program at NCBI (http://www.ncbi.nlm.nih.gov), and was performed according to the neighborjoining method with the bootstrap 1,000 times method using the Mega7.0 program.

3. Assay for antagonistic microbial enzyme activity

To assay the enzyme activity of the antagonist microorganism, the antagonist microorganism was cultured in 3 mL of LB liquid medium at 28° C. and 185 rpm for 16 hours to obtain a culture solution. In order to remove the components of the LB liquid medium, the obtained culture was dispensed into a 2 mL microtube, centrifuged at 5,000 rpm for 10 minutes, carefully poured out the supernatant, and then added 1 mL of 10 mM MgCl2 and vortexed to obtain an antagonistic microbial suspension. To confirm the protease activity, a hole was drilled in a medium made by adding 3% skim milk powder to LB agar medium using a cork borer with a diameter of 5 mm, and 10 μL of an antagonistic microorganism suspension at a concentration of ^{1×10 9 cfu/mL was inoculated.} After incubation at 28°C for 3 days, the formation of clear zones was observed to test protease activity (Sokol et al., 1979) CMC made by adding 1% Carboxymethyl Cellulose (CMC) to LB agar medium to confirm cellulase activity. After making an agar medium, inoculating antagonistic microorganisms in the same way as above, incubating for 7 days, staining the medium in 0.1% congo red solution for 15 minutes, immersing the medium in 1M NaCl solution for 15 minutes, bleaching, and observing the formation of clearzone to test cellulase activity. (Budi et al., 2000; Singh et al., 2013). Pikovskaya's agar medium supplemented with Ca3(PO4)2 was used to determine whether there was poorly soluble phosphate decomposition activity, and antagonistic microorganisms were inoculated as above, cultured for 10 days, and phosphoric acid soluble activity was assayed through clear zone formation. To examine the siderophore activity, a modified CAS (Chrome Azurol Sulfonate) medium was used (Han et al., 2015). After inoculating the antagonistic microorganism in the same manner as above and incubating for 3 days, the formation of a clear zone was observed to observe siderophore activity.

4. Antimicrobial activity range and mycelial growth inhibition test of antagonistic microorganisms

In order to determine whether the antagonistic microorganisms have an inhibitory effect on mycelial growth against three species of pepper anthrax bacteria C. scovillei, C. nymphaeae and C. acutatum and Fusarium verticillioides, the mycelial growth inhibition effect was tested by replacement culture. For the replacement culture, PDA medium was made in a 90 mm Petri dish, and three holes were made around the center with a 5 mm diameter cork borer. The antagonistic microorganisms were cultured in LB liquid medium at 28° C. and 185 rpm for 16 hours. The cultured culture was centrifuged at 5,000 rpm for 10 minutes to discard the supernatant, and then vortexed with _{1 mL of 10 mM MgCl 2} to obtain an antagonistic microbial suspension. The obtained antagonist suspension was ^{adjusted to a concentration of 1×10 9} cfu/mL, and then 20 μL was inoculated into three holes. In addition, two species of pepper anthrax were cultured in 55 mm PDA medium at 25°C for 4 days in dark conditions, and the end of the cultured pathogens were separated by a 5 mm diameter cork borer to separate the agar plug, and the PDA medium was made for replacement culture. It was inoculated in the middle. After inoculation, the cells were cultured at 25° C. for 7 days in dark conditions, and the length of the suppressed hyphae was measured. The rate of inhibition of mycelial growth was calculated as follows, and the experiment was repeated 3 times a total of 3 times.

5. Red pepper anthrax spore inhibition test

To obtain spores of pepper anthrax bacteria C. scovillei and C. nymphaeae, spore formation was induced by culturing in 55 mm OA (Oatmeal agar media) medium at 25° C. for 6 days under light conditions. 5 mL of sterilized distilled water was poured into the medium containing each pathogen, and the surface was carefully scraped with the lower part of the 1.5 mL tube, and then filtered through two layers of miracloth to remove the mycelial residues. The resulting suspension was centrifuged at 5,000 rpm for 10 minutes, the supernatant was slowly discarded, and a process of washing with 1 mL of sterilized distilled water was carried out twice. Thereafter, a spore suspension was obtained with a concentration of 1×10 ^{5 conidia/mL using a hemocytometer.} The antagonistic microbial suspension was ^{adjusted to 1×10 5} , 10 ⁶ , 10 ⁷ , and 10 ⁸ cfu/mL, respectively. In order to induce spore germination, 10 μL was inoculated on the hydrophobic glass surface, 10 μL of bacterial suspension at each concentration was inoculated thereon, and 10 μL of sterile distilled water was inoculated on 10 μL of the spore suspension and 10 μL of LB liquid medium as a control. The inoculated one was used. And 16 hours after inoculation, the rate of formation of adhesion groups for 100 spores was confirmed. The experiment was repeated 3 times 3 times.

<Experiment result>

Experimental Example 1. Selection and identification of antagonistic microorganisms

Microorganisms were separated using soil that did not contain a fungicide at Mt. Chunma, Hwado-eup, Namyang-si, Gyeonggi-do. First, 12 colonies of different types were selected, and microorganisms showing antibacterial activity were selected through replacement culture with two species of pepper anthrax (C. nymphaeae, C. scovillei). Among them, 16S rRNA, recA sequencing analysis of the antagonistic microorganism that showed antifungal activity against red pepper anthrax was identified as B. cepacia. The relationship with other strains is shown in FIG. 1. The strain isolated in this experiment was named Burkholderia cepacia KF1, and was deposited with the Korea Microbial Conservation Center on October 29, 2019 (accession number: KFCC 11842P), and used for the experiment.

Experimental Example 2. Assay for antagonistic microbial activity enzyme

Antagonistic microorganisms refer to microorganisms that have the ability to inhibit or block growth by using hydrolytic enzymes that degrade the outer membrane of plant pathogens. Therefore, it was confirmed through an in-flight experiment whether PGPR (Plant growth promoting rhizobacteria) can play a role in protease and cellulose activity and plant growth promotion among the hydrolytic enzymes secreted by the isolated antagonistic microorganisms. As a result, the enzyme activity of B. cepacia KF1 was confirmed by forming a clear zone in a medium capable of confirming protease, cellulase, and siderophore enzyme activities (FIG. 2). In addition, the enzymes secreted by Burkholderia spp. include amylase (Chatterjee et al., 2019), lipase (Xu et al., 2018), catalase (Khambalkar & Sridar, 2015), and chitinase (Suryadi et al., 2018). Is known. Siderophore acts as a PGPR that helps promote plant growth by specifically binding to iron ions and solubilizing iron that cannot be used by plants, while enhancing the ability to settle the root zone of crop plants. Can increase yield by promoting the growth of (Kloepper et al., 1980; Bagg & Neilands, 1987). Therefore, KF1 secretes cellulase and protease to show antibacterial activity against capsicum anthrax bacteria, and it is thought that it will help promote plant growth by producing siderophores.

Experimental Example 3. Antimicrobial activity range and mycelial growth inhibitory effect of antagonistic microorganisms

In order to find out the range of antimicrobial activity of antagonistic microorganisms, C. nymphaeae, C. acutatum, which are pepper anthrax bacteria closely related to C. scovillei, and F. verticillioides, which cause corn stump rot, were investigated through replacement culture with KF1 ( Fig. 3). As a result, it showed the highest antimicrobial activity with 66.0% inhibition of mycelial growth against C. scovillei, and 64.6% and 59.7% of inhibition against C. nymphaeae and C. acutatum, respectively, showing excellent activity. It also showed 44.7% inhibition of mycelial growth against F. verticillioides, which is thought to be applicable to the control of corn stump rot.

Experimental Example 4. Effect of antagonistic microorganisms to inhibit spore germination

The conidia of pepper anthrax bacteria recognize the surface of the plant to form a germination tube, and by forming an attachment group, the accumulation of sugars such as glycerol in the attachment period causes high tension, and the invading mycelium invades into the plant due to the increased tension and causes disease. . Therefore, inhibition of germination of pathogens is an important factor in disease control. In order to confirm the spore germination inhibitory effect of the antagonistic microorganism KF1, the antagonist suspension was treated at each concentration in the capsicum anthrax suspension, and the rate of adhesion formation was investigated after 16 hours (Fig. 4A). As a result ^{, spore germination rates according to concentrations of 1×10 5} , 10 ⁶ , 10 ⁷ , 10 ⁸ cfu/mL for C. scovillei were 7.8%, 4.3%, 1.2%, and 0%, respectively. The germination rate was 80.7%. C. nymphaeae was identified as 1.7%, 6.7%, 1.8%, and 0%, respectively, and the untreated group was identified as 78% (Fig. 4B). Therefore, KF1 is expected to be used as a microbial pesticide against red pepper anthrax by inhibiting mycelial growth, spore germination and adhesion formation of red pepper anthrax bacteria, preventing invasion into host plants.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Korea Microorganism Conservation Center KFCC11842P 20191029

Claims (5)

Buckholderia genus KF1 strain having antifungal activity against red pepper anthrax bacteria (accession number: KFCC11842P). The method of claim 1,
The strain is a strain characterized in that it has a control activity against the genus Colletotrichum (Colletotrichum). A composition for preventing or treating pepper anthrax infection comprising the strain of claim 1 and an agrochemically acceptable carrier. The method of claim 3,
The strain in the composition is a composition for preventing or treating pepper anthrax infection, characterized in that contained in a concentration ^{of 1 × 10 3} to 1 × 10 ^{10 cfu / g.} A method for preventing or treating an infection of pepper anthrax by treating the strain of claim 1 or a culture solution thereof to a plant or culture paper.

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