Beneficial microorganism exhibiting antifungal activity against flower-infecting plant pathogen causing fruit decay, and use thereof

Abstract

The present invention relates to: a composition and a method for biologically controlling plant or crop diseases, the composition comprising three antagonistic microorganisms such as Bacillus velezensis CT-1, Bacillus amyloliquefaciens PT-1, and Paenibacillus polymyxa BW-1. The germination ability of pathogenic conidia that causes infection by adhering to and inhabiting flowers (floral organs) is suppressed by treating with a culture medium or extract of the novel strain, whereby the proliferation of pathogens is suppressed or the infiltration of bacteria into tissues is blocked, and thus the present invention can be effectively used for the stable production and environmentally friendly quality maintenance of fruits.

Description

Beneficial microorganisms with antifungal properties against flower-infecting plant pathogens causing fruit rot and their uses

[0001] The present invention relates to an effective control technology for dealing with disease infection in plants or crops through flower (floral) infection based on inhibiting the germination of conidia of pathogens that cause disease in plants or crops using three novel antagonistic microorganisms, including Bacillus velezensisCT-1, Bacillus amyloliquefaciensPT-1, and Paenibacillus polymyxaBW-1.

[0002] In the case of floral infection, pollen becomes infected with pathogens, and the pathogens that invade through the infected pollen gradually grow inside the fruit during the growth and ripening process of the crop or plant fruit. Eventually, the flesh rots, or in severe cases, the pathogens invade and attach to the inside and outside of the seeds, transmitting the pathogens to the next generation. Representative plant diseases that cause floral infection include barley smut caused by stigma infection due to the structure of the flower, pear and apple fire blight (bacterial) caused by anther infection, and wheat ergot and fire blight caused by nectar infection.

[0003] Seeds serve as the most important source for the perpetual survival of pathogens. They can survive for a long time within the seeds, and if contaminated seeds are sown in a new area, widespread dispersal occurs easily. Furthermore, they cause various external symptoms in the seeds, such as shriveling, discoloration, and fading, leading to commercial losses. Internally, they cause biochemical degradation and changes in the quality of nutrients, and some species, such as Asperillus flavus, produce toxins, causing significant harm to livestock fed with contaminated feed or to humans who consume the food.

[0004] Task 1 is to provide an appropriate strain and method for responding to flower infection by pathogens so that, when plant or crop pathogens infect flowers during the flowering period of crops, antagonistic microbial agents inhibit the germination of pathogen spores or regulate the growth of germ tubes or early hyphae prior to the colonization of pathogens in flowers or invasion into tissues, thereby enabling stable production of fruits or berries without damage from pathogens.

[0005] Task 2 is to provide biomass of antagonistic bacteria against pathogens such as Trichothecium roseum and Penicillium oxalicum, which cause fruit rot by infecting the flowers (floral organs) of crops or plants, or seed-transmitting pathogens that cause seed transmission.

[0006] Solution 1 comprises one or more of the following: the Bacillus velezensis CT-1 strain deposited at KCTC19188P, the Bacillus amyloliquefaciens PT-1 strain deposited at KCTC19189P, and the Paenibacillus polymyxa BW-1 strain deposited at KCTC19190P.

[0007] It is an antagonistic microbial agent against pathogens that cause fruit rot and seed-borne pathogens that cause seed transmission.

[0008] Solution 2 is, in solution 1,

[0009] The above-mentioned antagonistic microbial preparation is characterized in that the pathogen causing the fruit rot disease is Trichothecium roseum or Penicillium oxalicum.

[0010] Solution 3 is,

[0011] A biomass or culture of at least one of the following strains as an active ingredient: Bacillus velezensis CT-1 strain deposited at KCTC19188P, Bacillus amyloliquefaciens PT-1 strain deposited at KCTC19189P, and Paenibacillus polymyxa BW-1 strain deposited at KCTC19190P.

[0012] This is a composition for preventing or controlling plant diseases caused by pathogens that cause fruit rot and seed-borne pathogens that cause seed transmission.

[0013] Effect 1 is that novel strains of Bacillus velezensis CT-1, Bacillus amyloliquefactionis PT-1, and Pennybacillus polymyxa BW-1 were isolated and identified, and deposited with the Korea Research Institute of Biotechnology and Bioengineering (deposit numbers KCTC 19188P, 19189P, 19190P), which can help prevent and control rot disease of crops or fruit, which can be caused by contamination, decay, and disease of flowers through infection by plant pathogens during the flowering period.

[0014] Figure 1 shows the phylogenetic tree of closely related species analyzed using BLAST (Basic Local Alignment Search Tool) and 16S rDNA gene amplification of the P. polymyxaBW-1 strain.

[0015] Figure 2 shows the DNA gyrase subunit B (gyrB) gene amplification of the P. polymyxaBW-1 strain and the phylogenetic tree of closely related species analyzed using BLAST (Basic Local Alignment Search Tool).

[0016] Figure 3 shows the phylogenetic tree of closely related species analyzed using DNA gyrase subunit B (gyrB) gene amplification of the B. velezensisCT-1 strain and BLAST.

[0017] Figure 4 shows the phylogenetic tree of closely related species analyzed using DNA gyrase subunit B (gyrB) gene amplification and BLAST of the B. amyloliquefaciens PT-1 strain.

[0018] Figure 5 shows the disease symptoms appearing on young fruits after pollination following the artificial spray inoculation of pathogen conidia into melon flowers during the flowering period. 1) is the Trichothecium roseum inoculation group, and 2) is the Penicillium oxalicum inoculation group.

[0019] Figure 6 shows the disease symptoms that appeared in the flesh of a melon during the process of fruit development and maturation following an incubation period of pathogens that had infected the flower through artificial inoculation during the melon flowering period. 1) shows the fungal bodies developed on the surface of the flesh and the internal decay state at the end of growth, about 40 to 45 days after infection by Trichothecium roseum at the time of pollination, and 2) shows the disease symptoms that appeared severely from the beginning of flesh ripening, 10 to 13 days after fertilization, in a fruit infected with Penicillium oxalicum, resulting in the fruit disappearing from the pod.

[0020] Figure 7 shows the melon flowering time. 1) is a control group in which conidia of the pathogen Trichothecium roseum were diluted in distilled water and 1×10 6The disease control effect was investigated by first spraying cells at a CFU / ml level onto flowers, and 2) spraying conidia of the above pathogen, and then, when the moisture dried, spraying a 200-fold diluted YPD broth culture solution in which the antagonist microorganism B. amyloliquefaciens PT-1 was cultured with shaking at 35°C, three times at one-day intervals, and then culturing until harvest.

[0021] Figure 8 shows the physiological and chemical characteristics of antagonistic microorganisms through the utilization of organic and inorganic substances required for growth and fermentation.

[0022] Figure 9 shows the results of investigating the growth of mycelia after inoculating a colony of mycelia of 1) Trichothecium roseum, 2) Penicillium oxalicum, which are melon fruit rot pathogens, and 3) Sclerotinia minor, which are lettuce sclerotinia pathogens, onto a YPDA medium in which antagonistic microorganisms were cultured for 3 days. It shows that the growth of the mycelia is strongly inhibited and cannot adhere to the surface of the growth medium due to various physiologically active metabolites, such as antimicrobial substances produced by the antagonistic microorganisms in the medium.

[0023] Figure 10 shows the results of investigating the germination status of conidia of the melon rot pathogen at different temperatures (6 hours of culture). The arrows indicate the form in which conidia germinate and form germ tubes. Although there are differences in growth depending on the species of each pathogen, it can be determined that when conidia are exposed to normal temperature conditions (20–30°C), they elongate germ tubes in a short period of time (4 hours) and can easily invade the tissue while growing.

[0024] Figure 11 shows the results of confirming the germination status of pathogen conidia after streaking them onto a medium containing physiologically active metabolites, such as antibiotics, produced by antagonistic microorganisms cultured and grown on YPDA medium for 3 days, and leaving them for 144 hours. It can be observed that in these media, conidia failed to germinate or exhibited atypical germination states where they did not grow normally. 1) is a Trichothecium roseum conidia, 2) is a Penicillium oxalicum conidia, and from left are strains P. polymyxaBW-1, B. velezensisCT-1, and B. amyloliquefaciensPT-1.

[0025] Solution Principle: Provision of antagonistic bacteria against the pathogens of fruit rot disease

[0026] To complete the present invention, Bacillus velezensisCT-1 strain (Accession No.: KCTC19188P), Bacillus amyloliquefaciensPT-1 strain (Accession No.: KCTC19189P), and Paenibacillus polymyxaBW-1 strain (Accession No.: KCTC19190P) are provided to establish a biological control technology using the biomass of the following antagonistic bacteria against pathogens such as Trichocecium roseum and Penicillium oxalicum, which infect flowers (flowers) of crops or plants and cause fruit rot disease, or seed-transmitting pathogens that cause seed transmission.

[0027] The above antagonistic bacterial strain forms rough colonies on YPDA (yeast peptone dextrose agar) medium, and optimal growth occurs at a temperature of 35°C. The microbial agent containing biomass, produced as a culture during shaking culture in YPD broth for 48 to 60 hours, can be prepared in liquid, powder, or granular form. However, there are no specific restrictions on other types of formulations.

[0028] The culture of the antagonistic microorganism contains at least one or more effective components, such as enzymes, antibiotics, and hormones, which are physiologically active substances produced in the bacterial cells and culture filtrate, thereby exhibiting a control effect against plant diseases.

[0029] In addition to the plant pathogens described in the examples, the strain also exhibits antibacterial activity against various fungi and bacteria, such as anthracnose pathogens (Colletotricumspp.), wilt pathogens (Fusariumspp.), gray mold pathogens (Botrytisspp.), late blight pathogens (Phytophthoraspp.), damping-off pathogens (Rhizoctoniaspp.), and leaf blight pathogens (Stemphylliumspp.), as well as fruit rot pathogens (Acidovorax avenaesubsp. citrulli, soft rot pathogens (Pectobacterium carotovorasubsp. carotovora)).

[0030] Regarding control methods for plant pathogen infections and the composition of plants or crops, watermelons, melons, pumpkins, cucumbers, tomatoes, etc. are included, but are not limited to these.

[0031] The cell concentration of antagonistic microorganisms in the composition is 10 9 It is produced to be at least CFU / ml, and there are no restrictions even if it contains a higher concentration. When applying a diluted solution of an eco-friendly microbial agent as a control method, the concentration is 10 7 It is characterized by CFU / ml or more.

[0032] In addition to the technology for preventing and controlling flower infections by treating flowers during the flowering period of crops or plants, for soil-borne diseases, a microbial preparation prepared using biomass containing cells or endospores in which the above strain has been cultured can be applied to the surface of crops or plants, or to the soil of cultivation sites or farmlands.

[0033]

[0034] Example 1. Isolation and Identification of Three Antagonistic Microorganisms

[0035] Bacillus velezensisCT-1 was isolated from the rhizosphere of cherry trees, Bacillus amyloliquefaciensPT-1 from the rhizosphere of fir trees, and Paenibacillus polymyxaBW-1 from the roots of morning glory plants. The 16S rDNA and DNA gyrase subunit B (gyrB) gene sequences of these strains were performed, and their molecular genetic phylogenetic trees were compared. These antagonistic microbial strains were identified based on genetic characteristics of their DNA sequences and physiological and biochemical functional characteristics, such as urea production (see Figures 1–4, Tables 4–7).

[0036]

[0037] Example 2. Isolation of pathogens and pathogenicity testing

[0038] Trichothecium roseum and Penicillium oxalicum were isolated from diseased melon tissue. The strains were phylogenetically classified into the aforementioned species based on morphological characterization and molecular genetic elongation factor 1-alpha (EF1α) sequencing results. To confirm the pathogenicity and infection pathways of the pathogens, they were cultured on potato agar (PDA) medium for 7 days, and then 10 4-5 As a result of inoculating spores at CFU / ml levels once each—first on flowers during the flowering period and second on the surface at the time when the net was forming—pathogenicity and infection pathways were confirmed, with flesh decay occurring in the treatment group inoculated on flowers (Figs. 5–6). However, since the tissue at the time when the net was forming was firm, consequently, no invasion by fungi from the outside and disease development occurred.

[0039]

[0040] Example 3. Investigation of the physiological and chemical characteristics of three antagonistic microorganisms

[0041] The utilization of organic and inorganic compounds by antagonistic microorganisms was investigated. In Figure 8, 1) is the reduction reaction of nitrate to nitrite, with all three types showing a positive reaction; 2) is the lactic acid degradation ability, with Phenibacillus polymyxa BW1 showing a positive reaction and Bacillus velezensis CT-1 and Bacillus amyloliquefactionis PT-1 showing a negative reaction; and 3) is the sucrose utilization, with all three types showing a positive reaction.

[0042] All methyl red tests, which produce acid by fermenting glucose, were negative, and all three strains were also negative in the voges-prosakuer test (VP reaction) to check for acetoin production (Table 1).

[0043] Characteristics P. polymyxa BW-1B. velezensis CT-1B. amyloliquefaciens PT-1 Sucrose utilization+++Lactic acid fermentation+--Nitrate reduction+++Indole formation---Urea utilization---VP reaction---MR test---

[0044] +; Positive (Available), -; Negative (Unavailable)

[0045]

[0046] / *37 Example 4. Effect of antagonistic microorganisms on the growth of pathogen hyphae

[0047] To establish a biological control method, antagonistic strains of Bacillus velezensisCT-1, B. amyloliquefaciensPT-1, and Paenibacillus polymyxaBW-1 were placed on cellophane paper on YPDA medium, inoculated in the center, and cultured at 35°C for 3 days. Subsequently, colonies of the two pathogenic strains were placed in the center of the medium and cultured for 7 days. As shown in Figure 9, antimicrobial activity was confirmed, with the growth of pathogenic hyphae being strongly inhibited in the medium cultured with antagonistic strains compared to the control group. 1) represents the pathogenic strain Trichothecium roseum, 2) represents Penicillium oxalicum, A) represents the antagonistic strain Bacillus velezensisCT-1, B) represents B. amyloliquefaciensPT-1, and C) represents Paenibacillus polymyxaBW-1.

[0048]

[0049] Example 5. Effect of temperature on the germination of pathogen conidia

[0050] Conidial germination is a critical factor for invading host plant tissues. In particular, temperature, as well as moisture, has a significant influence on conidial germination. Since the time between pollination and flowering is short, the pathogen's conidia must be able to invade immediately. To determine the optimal germination conditions, the effect of temperature was examined using slide culture. As shown in Table 2 and Figure 10, the optimal germination temperature for Trichothecium roseum was in the range of 25–30°C, and germ tubes developed within 4 hours of inoculation. For Penicillium oxalicum, germ tube elongation began to appear within 4 hours of inoculation at 25–35°C. At 15°C, germ tubes appeared after 6 hours for Trichothecium roseum and 12 hours for Penicillium oxalicum. However, at 35℃, the spores of Trichothecium roseum did not germinate at all for up to 48 hours. Penicillium oxalicum showed excellent germination ability at slightly higher temperatures. Overall, in a favorable environment, conidia of the pathogens germinated within 4 hours, confirming the possibility of invasion.

[0051] Temperature(℃)Trichothecium roseumPenicillium oxalicum2 *) 46812(24)48246812(24)485- **) ----+----±10-----(+)-----(+)15--+----+20-+--±25-+-±30-+-+35-------+

[0052] *) Incubation time, **) +; Spore germination, ±; Weak spore germination, -; No spore germination

[0053]

[0054] Example 6. Evaluation of the effect of an antagonistic culture medium on the inhibition of pathogen conidial germination

[0055] To examine the applicability of biological control, spores of the pathogen were inoculated into a medium containing antibiotics produced by antagonistic bacteria, placed at 25°C and 30°C, which are the optimal germination temperatures for conidia, and the germination status of the spores was monitored at 24-hour intervals up to 144 hours. The pathogen Penicillium oxalicum did not germinate at all up to 144 hours after inoculation in the medium cultured with antagonistic bacteria P. polymyxaBW-1, B. velezensisCT-1, and B. amyloliquefaciensPT-1 (Table 3, Fig. 11).

[0056] In the medium in which the pathogen Trichothecium roseum was cultured with P. polymyxaBW-1 and B. amyloliquefaciensPT-1, the conidial cells did not swell significantly or produce germ tubes at all. In the medium in which the antagonist B. velezensisCT-1 was cultured, the formation of initial germ tubes was confirmed after 24 hours, but normal growth did not proceed even after 144 hours, and the cells remained in the initial germ tube state, confirming that hyphal elongation was strongly inhibited.

[0057] antagonistic bacteria * T. roseum P. oxalicum24 ** 4896120144244896120144Pp- *** ---------Bv±---------Ba----------

[0058] *) Pp=P. polymyxaBW-1, Bv=B. velezensisCT-1, Ba=B. amyloliquefaciensPT-1, **) Incubation time, ***) ±; Weak spore germination, -; No spore germination

[0059]

[0060] Example 7. Gene sequences of three antagonistic microorganisms

[0061] Table 4 shows the 16S rDNA gene sequence of the P. polymyxaBW-1 strain. Table 5 shows the DNA gyrase subunit B (gyrB) gene sequence of the P. polymyxaBW-1 strain. Table 6 shows the DNA gyrase subunit B (gyrB) gene sequence of the B. velezensisCT-1 strain. Table 7 shows the DNA gyrase subunit B (gyrB) gene sequence of the B. amyloliquefaciensPT-1 strain.

[0062]

[0063]

[0064]

[0065]

[0066] Example 8. Novel strains: Bacillus velezensisCT-1 strain (Accession No.: KCTC19188P), Bacillus amyloliquefaciensPT-1 strain (Accession No.: KCTC19189P), Paenibacillus polymyxaBW-1 strain (Accession No.: KCTC19190P)

[0067] As revealed in the above solution principles and experimental examples, this is a novel strain as an antagonistic strain against pathogens that cause fruit rot, such as Trichothecium roseum and Penicillium oxalicum, or seed-transmitting pathogens that cause seed transmission.

[0068]

[0069] Example 9. Microbial preparation and composition capable of preventing or controlling fruit rot and seed-borne pathogens

[0070] The above is a microbial preparation and composition for preventing or controlling fruit rot and seed-transmitting pathogens, comprising the strain biomass of Example 8 or a culture thereof as an active ingredient. The biomass includes bacterial cells, endospores, and a culture medium in which these antagonistic bacteria are cultured.

[0071] The microbial formulation or composition of the present invention may comprise an agriculturally acceptable carrier and may comprise fillers, solvents, excipients, surfactants, suspending agents, spreaders, adhesives, defoaming agents, dispersants, wetting agents, drift reducing agents, suxiliaries, adjuvants, or mixtures thereof.

[0072] The microbial preparation or composition of the present invention may be formulated into types such as concentrates, solutions, sprays, aerosols, immersion baths, dips, emulsions, suspension concentrates, gels, granules, etc. It may also be granules, powders, or pellets.

[0073] The microbial preparation or composition of the present invention may be used alone or in combination with other agricultural preparations, pesticides, insecticides, acaracides, fungicides (fungicides harmless to the fungi of the present invention), bactericidal agents, herbicides, antibiotics, antimicrobial agents, nematocides, rodenticides, entomopathogens, pheromones, attractants, plant growth regulators, plant hormones, insect growth regulators, chemosterilants, microbial pest control agents, repellents, viruses, phagostimulants, plant nutrients, plant fertilizers, and biological control agents, or used sequentially.

[0074]

[0075] Example 10. Prevention or control method using the strain of Example 8

[0076] This is a method of preventing or controlling plant fungal diseases by preparing the strain biomass of Example 8 in the form of granules, powder, pellets, etc. and spraying it.

[0077]

[0078] Depository: Korea Research Institute of Bioscience and Biotechnology

[0079] Trustee Number: KCTC19188P

[0080] Date of Trust: 20240613

[0081]

[0082] Depository: Korea Research Institute of Bioscience and Biotechnology

[0083] Trustee Number: KCTC19189P

[0084] Date of Trust: 20240613

[0085]

[0086] Depository: Korea Research Institute of Bioscience and Biotechnology

[0087] Trustee Number: KCTC19190P

[0088] Date of Trust: 20240613

[0089]

Claims

1. Including one or more of the following strains: Bacillus velezensis CT-1 deposited at KCTC19188P, Bacillus amyloliquefaciens PT-1 deposited at KCTC19189P, and Paenibacillus polymyxa BW-1 deposited at KCTC19190P Antagonistic microbial preparation against pathogens causing fruit rot and seed-transmitting pathogens causing seed transmission.

2. In Claim 1, An antagonistic microbial preparation characterized in that the pathogen causing the above fruit rot disease is Trichothecium roseum or Penicillium oxalicum.

3. A biomass or culture of at least one of the following as an active ingredient: Bacillus velezensis CT-1 strain deposited at KCTC19188P, Bacillus amyloliquefaciens PT-1 strain deposited at KCTC19189P, and Paenibacillus polymyxa BW-1 strain deposited at KCTC19190P. A composition for preventing or controlling plant diseases caused by pathogens that cause fruit rot and seed-transmitting pathogens that cause seed transmission.

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