Patent Application
20240016158
The present disclosure has been made with the support of the Rural Development Administration under Project ID. No. PJ014978012020, which was conducted by the Industry Foundation of Chonnam National University in the research program named “Development of New-Concept Eco-Friendly Disinfectant Using Microbe-Derived Material” as a branch of the research project titled “Technology for Improving Agricultural Environment Using Microorganisms (R&D)” under the research management of the Rural Development Administration, from Jan. 1, 2020 to Dec. 31, 2020.
This application claims priority to and the benefit of Korean Application Number 10-2020-0153115, filed in the Korean Intellectual Property Office on Nov. 16, 2020, the entire content of which is incorporated herein by reference.
This application claims priority to and the benefit of Korean Application Number 10-2021-0153374, filed in the Korean Intellectual Property Office on Nov. 9, 2021, the entire content of which is incorporated herein by reference.
The present disclosure relates to a composition including a culture of Streptomyces sp. JCK-6141 strain or an extract from the culture for controlling plant diseases, a method for producing same, and a method for controlling plant diseases and, more particularly, to a disease control efficacy identified in a culture of Streptomyces sp. JCK-6141 having an antifungal activity or an extract from the culture against phytopathogens.
Phytopathogenic fungi settle inside or on the surface of plants to siphon nutrients from the plants and propagate, causing serious damage to various crops worldwide. Plant diseases caused by fungi are more diverse and more damaging than other pathogens, to the extent that 17 out of the 33 cases of severe damage by plant diseases in the past were caused by fungi.
In addition, ten types of phytopathogenic fungi, including Magnaporthe oryzae, Botrytis cinerea, Puccinia spp., Fusarium graminearum, Fusarium oxysporum, Blumeria graminis, Mycospaerella graminicola, Colletotrichum spp., Ustilago maydis, and Melampsora lini, have been reported to be scientifically and economically important worldwide.
Plant diseases caused by phytopathogenic fungi are very difficult to control for the following reasons. First, many phytopathogenic fungi efficiently and sustainably produce an excessively large number of spores to infect other healthy plants. Second, it takes a short time for spores to be produced in the host after infection and the mobility of spores is also good, so their infectivity is high. Third, phytopathogenic fungi have good ability to absorb nutrients from the host and sometimes secrete toxic substances or enzymes that are harmful to the host. Due to these characteristics, phytopathogenic fungi parasitize the host plant in spite of continuous control efforts, causing enormous economic damage.
For the past several decades, chemical fungicides have been used to control plant fungal diseases, but their repeated use and abuse thereof have given rise to serious problems, such as contamination of the environment including soil and surrounding water, reduction of beneficial microorganisms, residual toxicity, and occurrence of resistant strains. Accordingly, in order to solve these problems, eco-friendly biocontrol agents using microorganisms and microorganism-derived natural substances that can replace chemical pesticides have been proposed as an alternative, and related studies have been actively ongoing to date.
However, since the efficacy and type of the previously developed biocontrol agents are insufficient compared to chemical pesticides, there is an urgent need to develop better biocontrol agents.
Streptomyces is the largest genus of Actinomycetota. Like other Actinomycetota, Streptomyces is gram-positive and has genomes with a high GC content. Most Streptomyces found primarily in soil and decaying vegetation produce spores and are characterized by complex secondary metabolism. They are also known to produce clinically useful antibiotics of natural origin, including neomycin, cephamycin, grisemycin, bottromycin, and chloramphenicol. As various physiologically active substances as well as antibiotics are isolated from the Streptomyces spp., the strains are being treated as industrially and medically very important microorganisms. Research on these antibiotics advances from finding substances effective against bacterial or fungal infections to searching for substances effective against viral diseases, but most studies have focused on medicine.
Previous in vitro studies using the genus Streptomyces have reported the inhibitory effects of Actinomycetes against the growth of various phytopathogenic microorganisms and stated that chitinases secreted from Streptomyces spp. could act on the cell walls of fungi to repress the growth of fungi. Given these characteristics, the genus Streptomyces has excellent potential as an antimicrobial biocontrol agent.
Therefore, there is an urgent need to develop an antifungal biocontrol agent using the genus Streptomyces.
Research conducted by the present inventors results in the finding that when applied to pathogens causative of plant disease, a culture of Streptomyces sp. JCK-6141 or an extract from the culture exhibited significantly high antifungal activity.
Accordingly, an aspect of the present disclosure is to provide a Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP having antifungal activity.
Another aspect of the present disclosure is to provide a composition for controlling plant diseases, the composition including a culture of Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP or an extract from the culture.
Another aspect of the present disclosure is to provide a method for preparing a composition for controlling plant diseases, which includes a culture of Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP or an extract from the culture.
Another aspect of the present disclosure is to provide a method for preventing plant diseases, which includes steps of treating a plant with a composition containing a culture of Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP or an extract from the culture to control plant diseases.
The present disclosure relates to a composition containing a culture of Streptomyces sp. JCK-6141 strain or an extract from the culture of the strain for controlling plant diseases, a preparation method therefor, and a method for controlling plant diseases. The composition for controlling plant diseases exhibits excellent antifungal activity when applied to pathogens of plant diseases.
The present inventors found that a culture of Streptomyces sp. JCK-6141 strain or an extract from the culture exhibited excellent antifungal activity when applied to pathogens of plant diseases.
Hereinafter, a detailed description will be given of the present disclosure.
An aspect of the present disclosure is a Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP and having antifungal activity.
Another aspect of the present disclosure is a composition containing a Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP or an extract from the culture to control plant diseases.
As used herein, the term “culture” refers to a culture medium containing microorganisms after culturing microorganisms. The term “culture supernatant” means an upper liquid obtained by removing most of microorganisms from the culture solution through centrifugation, and also referred to as “supernatant”. The term “culture filtrate” is meant a liquid that is obtained by centrifugation and filtration from the culture and is free completely of microorganisms.
As used herein, the term “extract” refers to a matter extracted from a culture of the strain and is intended to encompass fractions.
In the present disclosure, plant diseases may be caused by one or more strains selected from the group consisting of fungi and oomycetes, but with no limitations thereto.
Herein, the fungus may be at least one selected from the group consisting of Athelida rolfsii, Botryosphaeria dothidea, Botrytis cinerea, Colletotrichum caudatum, Colletotrichum cocodes, Colletotrichum horii, Curvularia lunata, Endothia parasitica, Fusarium fujikuroi, Fusarium graminearum, Fusarium oxysporum f. sp. raphani, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum F. sp. cucumerinum, Gaeumannomyces graminis, Magnaporthiopsis poae, Magnapothe oryzae, Pseudocercospora circumcissa, Raffaelea quercus-mongolicae, Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Valsa cerasperma, but is not limited thereto.
In the present disclosure, the fungus may be Puccinia recondita, but is not limited thereto.
In the present disclosure, Puccinia recondita is an obligate biotrophic fungus and parasitizes only in living plants.
In the present disclosure, the oomycete may be at least one selected from the group consisting of Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Pythium graminicola, and Pythium ultimum, but is not limited thereto.
In the present disclosure, the extract from the culture of the strain may include at least one selected from the group consisting of reveromycin E, reveromycin E 1-methyl ester, and reveromycin E derivatives, but is not limited thereto.
Another aspect of the present disclosure is a method for preparing a composition for controlling plant diseases, which comprises a culturing step of preparing a culture by culturing the Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP.
In the present disclosure, plant diseases may be caused by one or more strains selected from the group consisting of fungi and oomycetes, but with no limitations thereto.
In the present disclosure, the fungus may be at least one selected from the group consisting of Athelida rolfsii, Botryosphaeria dothidea, Botrytis cinerea, Colletotrichum caudatum, Colletotrichum cocodes, Colletotrichum horii, Curvularia lunata, Endothia parasitica, Fusarium fujikuroi, Fusarium graminearum, Fusarium oxysporum f. sp. raphani, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum F. sp. cucumerinum, Gaeumannomyces graminis, Magnaporthiopsis poae, Magnapothe oryzae, Pseudocercospora circumcissa, Raffaelea quercus-mongolicae, Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Valsa cerasperma, but is not limited thereto.
In the present disclosure, the fungus may be Puccinia recondita, but is not limited thereto.
In the present disclosure, Puccinia recondita is an obligate biotrophic fungus and parasitizes only in living plants.
In the present disclosure, the oomycete may be at least one selected from the group consisting of Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Pythium graminicola, and Pythium ultimum, but is not limited thereto.
In the present disclosure, the culturing step may include the following fractionation steps:
In the present disclosure, the first fractionation step may be conducted using at least one solvent selected from the group consisting of ethyl acetate, ethyl ether, ethanol, and butanol, for example, ethyl acetate, but with no limitations thereto.
In the present disclosure, the second fractionation step may include the following processes:
In the present disclosure, the concentrate preparation step may include a process of concentrating with a reduced pressure concentrator, but with no limitations thereto.
In the eluate preparation step of the present disclosure, a silica gel column may serve as a stationary phase, but with no limitations thereto.
In the eluate preparation step of the present disclosure, at least one selected from the group consisting of chloroform, methanol, water, and acetic acid may be used as a mobile phase, but with no limitations thereto.
In the present disclosure, the fraction preparation step may be carried out by silica gel thin layer chromatography (TLC) while the eluate is loaded, but with no limitations thereto.
In the fraction preparation step of the present disclosure, at least one selected from the group consisting of chloroform, methanol, water, and acetic acid may be used as a mobile phase, but with no limitations thereto.
In the present disclosure, the fractions eluted in the second fractionation step may be used alone or in combination, but with no limitations thereto.
In the third fractionation step of the present disclosure, elution may be eluted using distilled water containing 0.1% trifluoroacetic acid:acetonitrile at a ratio of 20:80 to 50:50 or 30:70 to 50:50, but with no limitations thereto.
In the pure substance identification step of the present invention, an Xbridge column may be used as a stationary phase but with no limitations thereto.
In the pure substance identification step of the present invention, a 50 to 100% acetonitrile solution may be used as a mobile phase, but with no limitations thereto.
In the present disclosure, the pure substance may be at least one selected from the group consisting of reveromycin E, reveromycin E 1-methyl ester, and reveromycin E derivatives, but is not limited thereto.
In the present disclosure, reveromycin E has a molecular weight of 688 and a molecular formula of C38H56O11.
In the present disclosure, reveromycin E has the following Chemical Formula (I):
In the present disclosure, reveromycin E 1-methyl ester has a molecular weight of 702 and a molecular formula of C39H58O11.
In the present disclosure, reveromycin E 1-methyl ester has the following Chemical Formula (II):
In the present disclosure, reveromycin E derivative has a molecular weight of 716 and a molecular formula of C40H60O11.
Another aspect of the present disclosure is a method for controlling plant diseases, which comprises a step of treating a plant with a composition including a culture of Streptomyces sp. JCK-6141 strain deposited with accession number KCTC 14333BP or an extract from the culture to control plant diseases.
In the present disclosure, the plant disease may be caused by at least one selected from the group consisting of fungi and oomycetes, but with no limitations thereto.
In the present disclosure, the fungus may be at least one selected from the group consisting of Athelida rolfsii, Botryosphaeria dothidea, Botrytis cinerea, Colletotrichum caudatum, Colletotrichum cocodes, Colletotrichum horii, Curvularia lunata, Endothia parasitica, Fusarium fujikuroi, Fusarium graminearum, Fusarium oxysporum f. sp. raphani, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. cucumerinum, Gaeumannomyces graminis, Magnaporthiopsis poae, Magnapothe oryzae, Pseudocercospora circumcissa, Raffaelea quercus-mongolicae, Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Valsa cerasperma, but is not limited thereto.
In the present disclosure, the fungus may be Puccinia recondita, but is not limited thereto.
In the present disclosure, Puccinia recondita is an obligate biotroph fungus and parasitizes only in living plants.
In the present disclosure, the oomycete is at least one selected from the group consisting of Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Pythium graminicola, and Pythium ultimum, but is not limited thereto.
In the present disclosure, the extract from the culture of the strain may include at least one selected from the group consisting of reveromycin E, reveromycin E 1-methyl ester, and reveromycin E derivatives, but is not limited thereto.
In the present disclosure, the composition-treating step may be carried out in a manner selected from the group consisting of soil drenching, soil irrigation, foliar spray, trunk injection, foliage treatment, rhizosphere treatment, and seed treatment, but with no limitations thereto.
As used herein, the term “drenching” refers to a method of injecting or spraying a drug by injecting a chemical solution into a hole dug into soil or trees.
As used herein, the term “soil drenching” is a method of injecting or spraying a chemical solution into crop cultivation soil.
The overlapping common contents between the preparation methods for a composition for controlling plant diseases and the plant disease-controlling method composition and the composition for controlling a plant disease are omitted to avoid undue complexity of the specification.
The present disclosure relates to a composition containing a culture of Streptomyces sp. JCK-6141 strain or an extract from the culture for controlling a plant disease, a preparing method therefor, and a method for controlling a plant disease. Since they exhibit excellent antifungal activity upon application to pathogens of plant diseases, the culture of the strain and the extract from the culture can effectively control plant diseases.
The present disclosure relates to Streptomyces sp. JCK-6141 strain, deposited under accession number KCTC 14333BP, having antifungal activity.
Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for illustrating the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these examples according to the gist of the present disclosure.
Unless otherwise stated, “%” used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (volume/volume) % for liquid/liquid throughout the specification.
Strains with plant disease control activity were isolated from the weed rhizosphere (soil environment in which growing roots absorb and store substances and thus make various changes to the proportion of constituent organisms) in Gokseong, Jeollanam-do, South Korea. After being sampled, 1 g of soil was diluted in phosphate buffered saline (pH 7.0), spread on a tryptic soy agar (TSA) plate, and incubated for 3 to 7 days at 30° C. to isolate strains.
Molecular biological identification was made of the strain isolated from the soil by sequencing 16S rRNA. The strain was inoculated into ISP2 medium and then cultured with shaking at 180 rpm at 28° C. for 7 days. Then, genomic DNA (gDNA) of the strain was extracted using the 1-genomic BYF DNA Extraction Mini kit (iNtRON Biotechnology, Korea) according to the protocol. Specifically, the extracted gDNA of the strain was mixed with PCR-premix (polymerase chain reaction-premix, iNtRON Biotechnology, Korea) and the primer set 9F/1512R capable of amplifying the 16S rRNA of the strain, listed Table 1 below, before PCR amplification.
PCR started at 95° C. for 5 minutes and proceeded with 30 rounds of 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30 seconds, followed by extension at 72° C. for 7 minutes and termination at 12° C.
The PCR product of 16S rRNA gene was based sequenced by Genotech (Daejeon, Korea).
The 16S rRNA encoding nucleotide sequence of the isolated JCK-6141 strain was determined to have a total nucleotide sequence of 1338 bp, and the results are shown in Table 2.
Based on the nucleotide sequence, the homology test of the nucleotide sequence was performed using the Blast program (http://www.ncbi.nlm.nhi.gov) for registered information (GenBank database). As a result, as shown in Table 3, the strain was identified as Streptomyces sp., named Streptomyces sp. JCK-6141, and was deposited with accession name KCTC 14333BP.
Streptomyces sp. HPF1204 (LC515800)
Streptomyces sp. Z2 (AF068055)
Streptomyces sp. MR602 (LC381941)
In order to examine the effect of the isolated and selected Streptomyces sp. JCK-6141 strain on the growth of plant pathogens, measurement was made of inhibitory activity against the growth of the three plant pathogens Fusarium oxysporum f. sp. cucumerinum, Pythium ultimum, and Rhizoctonia solani. Mycelial fragments of the plant pathogens obtained from cultures growing at high growth rates were inoculated into one part of PDA (Potato Dextrose Agar) medium which was separately inoculated in a straight line with Streptomyces sp. JCK-6141 at a site 2.5 cm away from the inoculated part, then incubated at 28° C. When the plant pathogens grew to the edge under monitoring, degrees of inhibition against mycelial growth of the plant pathogens by the Streptomyces sp. JCK-6141 strain were measured, and the results are shown in
As shown in
The separated and selected Streptomyces sp. JCK-6141 strain was measured for minimum inhibitory concentrations against the mycelial growth of plant pathogens. Streptomyces sp. JCK-6141 strain was inoculated into a Glucose-Soybean meal-Sodium chloride (GSS) broth and then cultured with shaking at 180 rpm at 28° C. for 7 days. After centrifuging the culture at 4500 rpm for 20 minutes, the supernatant thus obtained was filtered through a 0.2 μm sterile filter to obtain the culture filtrate (hereinafter referred to as the sample). The sample was applied at concentrations of 0.16, 0.32, 0.63, 1.25, 2.5, 5, and 10% to plant pathogens mycelia. Mycelia of the plant pathogens Fusarium oxysporum f. sp. lycopersici, Pythium ultimum, Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum f. sp. cucumerinum were cultured stationarily at 25° C. for 7 days in a PDB medium. After the concentration of the mycelia was adjusted into 500 μg/ml (w/v), the mycelia were ground using a blender (DBIHAN, HG-15D).
The ground mycelia were added in an amount of 180 μL to each well of 96-well plates to which 20 μL of the sample was dispensed to form a concentration of 10%. Then, 100 μL was taken from each well treated at a concentration of 10% and mixed with 100 μL of the ground mycelia in a neighbor well to form a concentration of 5%. The dilution was repeated in such a serial dilution manner to prepare concentrations of 0.16, 0.32, 1.25, 2.5, 5, and 10%. After incubation for 24 hours at 25° C., degrees of mycelial growth were measured, and for each group, the experiment was performed in triplicate.
Fusarium
Fusarium
oxysporum
oxysporum
Pythium
Rhizoctonia
Botrytis
lycopersici
ultimum
solani
cinerea
cucumerinum
As shown in Table 4, the culture filtrate of Streptomyces sp. JCK-6141 strain exhibited potent inhibitory activity against the mycelial growth of various plant pathogens such as Fusarium oxysporum f. sp. lycopaceae, Pytium Ultium, Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum f. sp. cucumerinum. Although slightly differing in the concentration of 100% growth inhibition from one pathogen to another, the strain showed MIC values (%) of 0.31 to 0.63.
5-1. Isolation of Antimicrobial Active Substance
In order to isolate the antimicrobial active substance produced by Streptomyces sp. JCK-6141 strain, the strain was inoculated in a Glucose-Soybean meal-Sodium chloride (GSS) broth and then cultured at 28° C. for 7 days with shaking at 180 rpm. Thereafter, a supernatant was obtained by briefly spinning the culture medium (total 3 L) at 400 rpm for 10 minutes, as shown in
The prepared ethyl acetate fraction (first active fraction) was concentrated using a vacuum concentrator. For further purifying antimicrobial active substances, silica gel column chromatography was performed. In this regard, the fraction was loaded into a silica gel column (500 g, 200-400 mesh), followed by elution with chloroform:methanol (8:2, 1 L), chloroform:methanol (7:3, 1 L), and chloroform:methanol:water:acetic acid (30:9:1:0.25, 2 L). Drops of the eluted fractions were loaded to the silica gel plate for thin layer chromatography (0.25 mm film thickness) and then developed with chloroform:methanol:water:acetic acid (30:9:1:0.25). Absorbance was read at 254 nm. After para-anisaldehyde reagent was sprayed thereto, fractions with similar color development were collected and concentrated under reduced pressure.
Among the fractions concentrated under reduced pressure, one fraction showing antifungal activity against Rhizoctonia solani (second active fraction) was selected. Substances were separated from the fraction using Atlantis® T3f prep OBDTM (5 μm, 19×250 mm) column-equipped preparative high-performance liquid chromatography (Waters, Japan). For this, 0.1% trifluoroacetic acid (TFA)-added distilled water and acetonitrile were used as an eluent. Distilled water containing 0.1% trifluoroacetic acid was added to acetonitrile to prepare 50% to 70% acetonitrile solutions. Elution was performed with 50% to 70% acetonitrile solutions at a flow rate of 15 ml/min for 50 minutes. As a result of elution, a total of 14 fractions (F1 to F14, the third active fraction) were obtained.
Among them, the three fractions F6, F7, and F8 that exhibited antifungal activity were fractioned by preparative thin layer chromatography (silica gel thin layer chromatography, 0.5 mm film thickness). Chloroform:methanol:water:acetic acid (30:9:1:0.25) was used as the developing solvent. As a result, active F61 (35.2 mg), F71 (15.3 mg), and F81 (9.2 mg) (fourth activity) fraction) were obtained.
Active pure substances were identified in F61, F71, and F81 thus obtained. In this regard, high-performance liquid chromatography was performed while the amount of organic solvent was started from 50% acetonitrile solution on Xbridge C18 (4.6×250 mm, 5 um) column, increasing to 100% acetonitrile solution in 35 minutes. Active substances were detected by a PDA (photodiode array) detector at a detection wavelength of 240 nm. The results are shown in
5-2. Molecular Weight Identification of Active Antimicrobial Substances
The three substances F61, F71, and F81 isolated from Streptomyces sp. JCK-6141 strain were lyophilized and measured for molecular weights through liquid chromatography electrospray mass method (LC-ESI-MS, API2000, ABSCIEX, USA) in negative and positive ion modes, and the results are depicted in
As shown in
5-3. Chemical Structures of Active Antifungal Substances
In order to identify the chemical structures of F61 and F71, various modes of NMR analysis were performed. Among them, NMR data for F61 are summarized in Table 5 below.
1′
2′
3′
4′
As shown from the data of Table 5 and
NMR data of F71 are summarized in Table 6 below.
1′
2′
3′
4′
As shown in Table 6 and
6-1. Pour-Plating Method
A pour-plating method was used to observe the inhibitory effect of Streptomyces sp. JCK-6141 strain culture containing the active antifungal substance reveromycin E against the mycelial growth of 21 species of plant pathogenic fungi (16 species) and oocytes (5 species). Briefly, PDA (potato dextrose agar) plates were treated with predetermined concentrations (0 μg/ml, 1 μg/ml, and 5 μg/ml) of the ethyl acetate fraction of a Streptomyces sp. JCK-6141 strain culture. The centers of the plates were inoculated with the pathogenic fungi and oomycetes listed in Table 7, including Athelida rolfsii, Botryoshpaeria dothidea, Colletotrichum caudatum, Colletotrichum cocodes, Colletotrichum horii, Curvularia lunata, Endothia parasitica, Fusarium fugikuroi, Fusarium graminearum, Gaeumannomyces graminis, Pseudocerocospora circumcissa, Raffaelea quercus-mongolicae, Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, Valsa ceratosperma, Phytophthora capsici, Phytophthora cambivora, Phytophthora cinnamomi, Phythium graminicola, and Phythium ultimum, which were then incubated at 25° C. under the monitoring of mycelial growth.
Athelida rolfsii
Botryoshpaeria dothidea
Colletotrichum caudatum
Colletotrichum cocodes
Colletotrichum horii
Curvularia lunata
Endothia parasitica
Fusarium fugikuroi
Fusarium graminearum
Gaeumannomyces graminis
Pseudocerocospora circumcissa
Raffaelea quercus-mongolicae
Rhizoctonia solani
Rhizoctonia cerealis
Sclerotinia homoeocarpa
Valsa ceratosperma
Phytophthora capsici
Phytophthora cambivora
Phytophthora cinnamomi
Phythium graminicola
Phythium ultimum
As shown in
6-2. 96-Well Microplate Method
The minimum inhibitory concentration of active antifungal substance reveromycin E (F61) (C38H56O11), which was isolated and identified in Example 5, was measured against the mycelial growth of phytopathogenic fungi. First, reveromycin E (F61) was applied at a concentration of 0.0244 to 3.125 μg/ml to mycelia of the phytopathogens listed in Table 8, including Botryoshpaeria dothidea, Botryritis cinerea, Colletotrichum horii, Curvularia lunata, Endothia parasitica, Fusarium graminearum, Fusarium oxysporum f. sp. raphanin, Fusarium oxysporum f. sp. cucumerinum, Gaeumannomyces graminis, Magnaporthiopsis poae, Magnapothe oryzae, Phythium ultimum, Rhizoctonia solani, Sclerotinia homoeocarpa, Valsa ceratosperma, Phytophthora cactorum, Phytophthora cambivora, and Phytophthora cinnamomi.
Botryoshpaeria dothidea
Botryritis cinerea
Colletotrichum horii
Curvularia lunata
Endothia parasitica
Fusarium graminearum
Fusarium oxysporum f. sp. raphanin
Fusarium oxysporum f. sp. cucumerinum
Gaeumannomyces graminis
Magnaporthiopsis poae
Magnapothe oryzae
Phythium ultimum
Rhizoctonia solani
Sclerotinia homoeocarpa
Valsa ceratosperma
Phytophthora cactorum
Phytophthora cambivora
Phytophthora cinnamomi
The strains listed in Table 8 were stationarily cultured at 25° C. for 7 days in PDA (Potato Dextrose Agar) plates. The mycelia thus obtained were adjusted into a concentration of 500 μg/ml (w/v) and then ground using a blender (DBIHAN, HG-15D).
The ground mycelia were added 180 μl to each well of 96-well plates, and then 20 μl of the sample was dispensed to form a concentration of 3.125 μg/ml. Subsequently, 100 μL taken from the well where a concentration of 3.125 μg/ml had been prepared was mixed with 100 μl of the ground mycelia in a neighbor well to form a concentration of 1.56 μg/ml. The dilution was repeated in such a serial manner to prepare concentrations of 0.78 to 0.0224 μg/ml. After incubation for 24 hours at 25° C., degrees of mycelial growth were measured, and for each group, the experiment was performed in triplicate. The results are shown in Table 9 below.
Botryoshpaeria dothidea
Botryritis cinerea
Colletotrichum horii
Curvularia lunata
Endothia parasitica
Fusarium graminearum
Fusarium oxysporum f. sp. raphani
Fusarium oxysporum f. sp.
cucumerinum
Gaeumannomyces graminis
Magnaporthiopsis poae
Magnapothe oryzae
Phythium ultimum
Rhizoctonia solani
Sclerotinia homoeocarpa
Valsa ceratosperma
Phytophthora cactorum
Phytophthora cambivora
Phytophthora cinnamomi
As shown in Table 9, reveromycin E (F61) exhibited potent antifungal activity to inhibit the mycelial growth of various plant pathogens. Although slightly differing in the concentration of 100% growth inhibition from one pathogen to another, reveromycin E exhibited excellent antifungal activity against the pathogens, ranging in MIC value from 0.0224 to 3.125 μg/ml. In particular, the substance exhibits excellent antifungal activity against the oomycetes Phytophthora cactorum, Phytophthora cambivora and Phytophthora cinnamomi for which MIC values were measured to range from 0.195 to 0.39 μg/ml.
Usually, Fusarium strains are more resistant to chemicals than other species. However, reveromycine E (F61) exhibited very high antifungal activity against fungi such as Fusarium graminearum and Fusarium graminearum, with MIC values therefor ranging from 1.56 and 0.195 μg/ml, respectively.
In order to assay the lytic enzyme activity of Streptomyces sp. JCK-6141 strain culture, the activity of the lytic enzymes protease, chitinase, and cellulase were measured. Strains having lytic enzyme activity are known to be high in chitinase activity. This characteristic confirms inhibitory activity against fungi.
For use in an assay for protease activity, agar was added to 1% skim milk to make a plate onto which Streptomyces sp. JCK-6141 strain was then inoculated. After inoculation, the plate was stationarily incubated at 30° C. Two days after incubation, the clear zone by protease activity was observed. For chitinase activity, Streptomyces sp. JCK-6141 strain was inoculated onto PDA (Potato Dextrose Agar) plates containing 1.5% colloidal chitin and then stationarily incubated at 28° C. for one week to create a clear zone thereon. Cellulase enzyme activity was measured by culturing Streptomyces sp. JCK-6141 strain in Mendel-Leeds agar plates using carboxymethyl cellulose (CMC) as the sole carbon source. Plate containing 0.4 g/L KH2PO4, 0.02 g/L CaCl2), 0.02 g/L NaCl, 0.02 g/L FeSO47H2O, 2.5 g/L CMC, and 15.0 g/L agar was adjusted with 1 M NaOH to have a pH of 7.2. Streptomyces sp. JCK-6141 strain was inoculated onto the plate and stationarily incubated 28° C. for one week while cellulase release was monitored. The activity of each protease, chitinase, and cellulase was measured, and the results are summarized in Table 10 below.
As shown in Table 10, it was confirmed that Streptomyces sp. JCK-6141 strain not only had chitinase activity, but, in particular, possessed excellent cellulase activity. Oomycete cell walls contain cellulose, and other fungi contain chitin as a cell wall component. Therefore, through the measurement of cellulase and chitinase activities of Streptomyces sp. JCK-6141 strain, it can be revealed whether Streptomyces sp. JCK-6141 strain has higher antimicrobial activity against oomycetes or fungi. Previously, the results of Example 6-2 demonstrated that treatment with reveromycin E (F61) had higher inhibitory activity against mycelial growth of phytopathogenic oomycetes than phytopathogenic fungi. As such, the culture of Streptomyces sp. JCK-6141 strain possesses chitinase and cellulase activity as well as the low-molecular weight active antimicrobial material reveromycin E (F61), thereby exhibiting excellent antimicrobial activity against various phytopathogenic fungi and oocytes.
8-1. Plant Preparation
“Nebakja” cucumber seeds (Syngenta, Korea), a sensitive cultivar of cucumber, were germinated for two days at 25° C. on filter paper (Petri dish with 9 cm diameter) soaked in sterile water and then grown for 5 days to prepare plants (5-day-old cucumber seedlings), performing a pot experiment for an in vivo biocontrol of damping-off in cucumbers by JCK-6141 strain.
8-2. Sample Application and Inoculation
A total of four samples to be applied to the plants by soil drenching were prepared by diluting Streptomyces sp. JCK-6141 strain culture 100-fold and 10-fold with distilled water and fractionating the dilutions with ethyl acetate to final concentrations of 50 μg/ml and 200 μg/ml.
Rhizoctonia solani (hereinafter referred to as “Rs”), which causes damping-off in cucumbers, was prepared as an inoculum to be inoculated into the plant. Agar covered with active-grown Rs mycelia was cut and inoculated into a 250 ml Erlenmeyer flask containing 100 mL of PDB (potato dextrose broth). After incubation of the flask at 28° C. for 4 days with shaking at 150 rpm, the cultured mycelia were filtered through 4-layer gauze and washed with sterile water. The soil for Rs mycelial inoculation was prepared by mixing 0.75 g of Rs mycelia per 1 kg of the gardening soil mix.
Five days after the germination, the soil in which cucumber seedlings were planted was drenched with 10 ml of each of the four samples. After one day of soil drenching, the soil for Rs mycelium inoculation was aliquoted into 150 g per pot and inoculated with cucumber seedlings to prepare 1 DBI. 1 DBI could reveal the preventive effect of Streptomyces sp. JCK-6141 on damping-off in cucumbers because the sample was pre-treated before inoculation.
The soil for Rs mycelium inoculation was aliquoted into 150 g per pot. After 5 days of germination, cucumber seedlings were transplanted, and after 1 hour, 1 HAI was prepared by drenching the seedlings with 10 ml of each of the four samples. 1 HAI can confirm the therapeutic effect because the sample was drenched after transplanting the seedlings into the soil mixed with Rs mycelium.
For a non-treated group, a solution added with Tween 20 (concentration 250 μg/ml) was used, and as a control drug, a dispersive solution of Janrokend (active ingredient Hymexazole 30%, Penthiopyrad 5%, Korea Ginseng Corporation, Korea) was diluted 1000-fold and applied. All treatments were carried out in a soil drenching manner with 10 ml per pot.
8-3. In Vivo Bioassay
Cucumber seedlings transplanted into Rs mycelium-inoculated pots were grown at 25° C. for 7 days and then harvested to count diseased plants. Each treatment was repeated with triple replicates, and a replicate consisted of 10 pots. The experiment was conducted with two replicates to verify the efficacy. The disease control value was calculated using Equation 1 below, and the results of 1 DBI and 1 HAI are summarized in Tables 11 and 12, below.
As shown in Tables 11 and 12, and
9-1. Plant Preparation
The control efficacy of Streptomyces sp. JCK-6141 strain against Fusarium wilt was evaluated in vivo. Horticultural gardening soil No. 5 (Punong Sangtosa, Korea) was put into 8×8 pots (15 ml/pot, Bumnong Co. Ltd., Korea), and one seed of the cucumber species “Jungboksamcheok” (Syngenta, Korea) was sown per pot and cultivated in a greenhouse for 7 days to prepare plants to be used for the control effect evaluation.
9-2. Sample Treatment
Fusarium oxysporum f. sp. cucumerinum (Foc) was inoculated into PDA plates and cultured at 25° C. for 7 days. From the cultured plate, mycelial fragments of Foc were removed and then were inoculated into V8 broth (V-8 juice 200 ml, CaCO3 3 g, distilled water 1 L) and cultured at 25° C. in a dark condition for 7 days with shaking at 150 rpm. After shaking the culture, the pathogen culture was harvested and filtered through quadruple gauze to remove mycelia, followed by centrifugation (5000 rpm, 10 minutes, 4° C.). The spore pellet obtained by removing the supernatant was added with sterile water and suspended. For use as an inoculum, the Foc spore suspension was diluted with sterile water under an optical microscope to give a spore (conidium) concentration of 1.0×107 conidia/ml as countered by a hemacytometer.
9-3. In Vivo Bioassay
After being transplanted at a density of one seed per pot, pregerminated cucumber seeds were grown at 25° C. into cucumber seedlings which were then treated with the sample in three ways 5 days later. The culture of Streptomyces sp. JCK-6141 strain was diluted 100-fold and 10-fold with distilled water, and fractionated with ethyl acetate to final concentrations of 50 μg/ml and 200 μg/ml to prepare a total of four samples.
The each samples prepared from Streptomyces sp. JCK-6141 strain was applied in 10 ml to 5 days-old cucumber seedlings through soil drenching. One day after the soil drenching treatment of Streptomyces sp. JCK-6141 strain sample, the treated group was inoculated with 10 ml of the spore suspension of the Foc strain to prepare 1 DBI. In addition, 5 days-old cucumber seedlings were inoculated with 10 ml of the spore suspension of Foc strain by soil drench. One hour after inoculation, 10 ml of the Streptomyces sp. JCK-6141 strain sample was applied to seedlings by soil drenching to prepare 1 HAI.
For a non-treated group, a solution added with Tween 20 (concentration 250 μg/ml) was applied, and as a control drug, a dispersive solution of Janrokend (active ingredient Hymexazole 30%, Penthiopyrad 5%, Korea Ginseng Corporation, Korea) was diluted 1000-fold and applied. All treatments were carried out in soil drenching manner with 10 ml per pot.
The inoculated cucumber seedlings were cultured in a humid room at 25° C. for 24 hours, then transferred to a constant temperature room of 25° C., irradiated with light for 12 hours a day, and grown for 3 weeks. Each treatment was repeated with triple replicates, and a replicate consisted of 10 pots. The experiment was conducted with two replicates to verify the efficacy of the treatment. The disease control value was calculated using Equation 1 and the results are summarized in Tables 13 and 14, below.
As shown in Tables 13 and 14, and
The control efficacy of Streptomyces sp. JCK-6141 strain against rice sheath blight (RSB) was examined in vivo. As an experimental plant, the rice cultivar Nakdong (Oryza sativa L.) was used.
One day before pathogen inoculation, the Nakdong rice plants were treated by soil drenching with 10 ml of each of the 3-fold and 9-fold dilutions of the Streptomyces sp. JCK-6141 strain culture in distilled water. After putting an appropriate amount of Nakdong rice bran treated with the dilutions was put into a 1-L culture bottle and sterilized, followed by evenly inoculating finely ground mycelial mass of cultured Rhizoctonia solani into the pots where the rice plants had been grown. After inoculation, incubation was carried out for 3 days in a humid chamber (25° C.) and then for 4 days in an incubator with a relative humidity of 80%. Examination was made of the onset of the disease. As a control, the synthetic chemical fungicide Flutolanil was used at 20 μg/ml or 50 μg/ml concentration. For incidence rate, infected areas in the rice sheath were examined, and the results are summarized in Table 15, below.
As shown in Table 15 and
The control efficacy of Streptomyces sp. JCK-6141 strain on wheat leaf rust was examined in vivo. As an experimental plant, the wheat cultivar Eunpa (Triticum aestivum L.) was used.
One day before pathogen inoculation, the Eunpa wheat plants were treated by soil drenching with 10 ml of each of the 3-fold and 9-fold dilutions of the Streptomyces sp. JCK-6141 strain culture in distilled water. Since the pathogen Puccinia recondite is an obligate biotroph, uredospores formed on wheat seedlings as a result of passages of the plant in the laboratory were used as an inoculum. A uredospore suspension (spore 0.67 g/L) was prepared and sprayed over the wheat seedlings, which were then incubated at 20° C. for one day in a humid room and subsequently in an incubator with a relative moisture of 70% to induce the onset of the disease. As a control, the synthetic chemical fungicide Flutolanil was used at 20 μg/ml or 50 μg/ml concentration. For incidence rate, infected areas in the rice sheath were examined 7 days after inoculation, and the results are summarized in Table 16, below.
As shown in Table 16 and
The control efficacy of Streptomyces sp. JCK-6141 strain on Fusarium head blight was examined in vivo. As an experimental plant, the wheat cultivar Eunpa (Triticum aestivum L.) was used.
First, Streptomyces sp. JCK-6141 strain was inoculated into a sterile TSB broth and then cultured at 30° C. for 3 days with shaking at 150 rpm, to prepare a culture. Each of the 5- and 20-fold dilutions of the Streptomyces sp. JCK-6141 strain culture in distilled water was sprayed over ears of wheat, then left for 2 hours. The plants were inoculated with a spore suspension of Fusarium graminearum, which is a pathogen causing Fusarium head blight, and the entire pot was covered with a vinyl sheet to maintain humidity for 3 days. The sample was applied to the plants when the ears flowered. As a positive control, a synthetic pesticide Almuri (2000-fold dilution, Syngenta, Switzerland) was applied while Tween-20 (500 μg/ml) was used as a negative control. Ten ears of wheat were treated three times as a basic unit. The disease severity and incidence rate were examined 2 weeks after inoculation, and the results are summarized in Table 17 below.
As shown in Table 17 and
The control efficacy of Streptomyces sp. JCK-6141 strain against dollar spot disease was evaluated in vivo.
13-1. Plant Preparation
As an experimental plant, seeds of creeping bentgrass (Agrostis palustris), which is a type of cool-season grass, were used. In a pot with a diameter of 7 cm and a height of 6 cm, sterilized sand and horticultural gardening soil were mixed at a ratio of 3:1. The seeds were sown and grown for 30 days before use in experiments.
13-2. Sample Treatment
After diluting Streptomyces sp. JCK-6141 strain culture 3- and 9-fold with distilled water, Tween-20 (250 μg/ml) was added thereto, and then 10 ml per pot was applied by soil drenching 1 day before inoculation with the pathogen. An untreated group was treated with Tween-20 (250 μg/ml) solution while as a control agent, a Horikuo (active ingredient tebuconazole, 25% by weight) emulsion, commercially available for prevention of grass fungus, was diluted 2000-fold and applied by soil drenching to the pots in an amount of 10 ml per pot one day before inoculation. The pathogen Sclerotinia homoeocarpa was cultured on PDA (potato dextrose agar) plates for 5 days. The colonies thus formed were cut into pieces, each 1 cm wide and 1 cm long. For use as an inoculum, five pieces were added into each wheat bran-rice husk medium (36 g of wheat bran, 6 g of rice husk, and 40 ml of distilled water in 1 L Erlenmeyer flask) and then cultured for 7 days in a 25° C. incubator. After incubation, 400 ml of sterile water (including 200 μg/ml of Streptomycin sulfate) was added. The mixture was ground using a blender and then applied by soil drenching with 4 ml per pot. The treated turfgrass was placed under 25° C. and 95% relative humidity to develop the disease for 7 days. The disease severity was examined, and the results are summarized in Table 18, below.
As shown in Table 18 and
The present disclosure relates to a composition including a culture of Streptomyces sp. JCK-6141 or an extract from the culture for controlling plant diseases, a method for producing the same, and a method for controlling plant diseases and, more particularly, to a controlling efficacy identified in a culture of Streptomyces sp. JCK-6141 having an antimicrobial activity or an extract from the culture against phytopathogens.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0153115 | Nov 2020 | KR | national |
| 10-2021-0153374 | Nov 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/016450 | 11/11/2021 | WO |
