KR100995308B1 - Paenibacillus lentimorbus strains having antifungal, nematicidal activity and plant growth-promoting effect and method for controlling plant disease complex and promoting plant growth using the same - Google Patents

Abstract

The present invention has an antimicrobial activity, nematode impact and plant growth promoting effect ( Penibacillus lentibus) ( Paenibacillus lentimorbus ) strain, the antimicrobial, nematode, or microbial agent for the control of complex diseases of the plant comprising the strain or its culture as an active ingredient, the microbial agent for promoting plant growth comprising the strain or its culture as an active ingredient, the strain It relates to a method for controlling a complex disease of plants by treating the plant, and a method for promoting the growth of the plant by treating the strain to the plant.

Fusarium oxysporum f. sp. lycopersici, Meloidogyne incognita, complex disease, plant growth-promoting myobacterial bacteria, control effect, antibacterial activity, nematode impact

Description

Penibacillus lentimobus strains having antimicrobial, nematicidal and plant growth promoting effects and methods for controlling plant growth and plant growth using the same {Paenibacillus lentimorbus strains having antifungal, nematicidal activity and plant growth-promoting effect and method for controlling plant disease complex and promoting plant growth using the same}

The present invention relates to a penicillus lentismobus strain having an antibacterial activity, nematicidal force and plant growth promoting effect, and a method for promoting plant growth control and complex growth of plants using the same, more specifically, antibacterial activity, nematicidal force and plant growth promotion Penibacillus lentimorbus strain having an effect, the microorganism agent for the control of antimicrobial, nematicidal, or complex diseases of plants comprising the strain or its culture medium as an active ingredient, the strain or its culture medium as an active ingredient The present invention relates to a microbial agent for promoting plant growth, a method of treating a plant by controlling the strain, and a method of promoting plant growth by treating the plant with the strain.

The interaction of plant parasitic nematodes with fungi is reported to produce greater losses than caused by pathogens alone (Francl LJ, Wheeler TA. (1993) In: Khan MW (eds), Nematode interactions , Chapman and Hall , India pp 79-103). Infection of roots by root-knot nematodes results in the development of root rot and wilting disease.

Complex diseases in soil caused by soil infectious fungal pathogens, such as the root-knot nematodes, Meloidogyne spp. And Fusarium spp., Often damage plants more seriously. Make disease control more difficult than disease.

In the past decades, plant disease control has been based primarily on the use of chemicals. However, most chemical controls are banned due to adverse effects on human health, phytotoxicity and groundwater contamination. Given the above limitations, there is a recent need for the use of bioefficiency, economical, biodegradable and environmentally safe eco-friendly methods.

Some studies have shown that the interaction between plants and some endophytic bacteria is associated with beneficial effects such as promoting plant growth and biological control against plant pathogens (Hallmann et al. (1995) Phytopathology 85 : 1136).

Paenibacillus The genus is Gram-positive, aerobic, rod-shaped, spore-forming bacteria. The main property of this group is the secretion of extracellular enzymes in neutral and alkaline growing conditions. Some species can also produce polysaccharides, amino acids and secondary metabolites such as antibiotics, pigments, toxins, enzyme inhibitors, pheromones and plant and animal growth promoters (Dasman et al. (2003) Int J Syst Evol Microbiol 52: 1669-1674). The genus Penibacillus is currently the subject of much research and its antibiotic activity has been identified for a wide spectrum of microorganisms (Rosado AS, Seldin L. 1993. World J Microbiol Biotechnol 9 : 521-528). Some strains of Penibacillus have been used for biocontrol of plant diseases (Kim YK. (1995) Biological control of phytophtora blight of red pepper by antagonistic Bacillus polymyxa 'AC-1'. Seoul National University. Ph D Thesis. 78 pp), Its ability to degrade chitin, the major compound present in fungal cell walls, has made it an important antimicrobial biocontrol agent.

The present invention has been made in accordance with the above requirements, screening and evaluating the antimicrobial and nematicidal activity of penibacilli strains isolated from rotten ginseng roots, Meloidogyne incognita and Fusarium in tomato plants Oxyforum F. Esp. To evaluate the biocontrol capacity of selected strains for complex diseases caused by Fusarium oxysporum f. Sp. Lycopersici interaction.

In order to solve the above problems, the present invention provides a strain of Penibacillus lentimorbus having an antibacterial activity, nematicidal force and plant growth promoting effect.

In addition, the present invention provides an antimicrobial microbial agent comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a nematicidal microorganism preparation comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a microbial preparation for controlling a complex bottle of plants comprising the strain or its culture as an active ingredient.

The present invention also provides a microbial agent for promoting plant growth comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a method for controlling a complex disease of plants by treating the strain to plants.

The present invention also provides a method for promoting the growth of a plant by treating the strain to the plant.

According to the present invention, the GBR462, GBR508 and GBR158 strains selected in the present invention have excellent antibacterial and nematicidal properties, inhibited egg hatching of nematodes, and have a high disease control rate in tomato and promote plant growth. In addition, the GBR158 strain is considered to be efficient for commercial use due to its high control rate even when treated with tomato seeds. If these strains are used in agriculture, they can be effectively used to control nematodes and fungi in soils that are difficult to promote and control plant growth.

In order to achieve the object of the present invention, the present invention provides a Penibacillus lentimorbus GBR158 strain (Accession No. KACC 91374P) having an antimicrobial activity, nematicidal force and plant growth promoting effect.

The pathogen Fusarium oxyphorum f causing wilt on tomatoes. Esp. Lycopersicis ( Fusarium oxysporum f. Sp. Lycopersici ) acts on plants with the root-knot nematode Meloidogyne incognita , which forms a nodules on plant roots, causing a complex disease. At this time, the synergistic effect shows a much larger hospital potential than the sum of the two pathogens, resulting in severe disease on the plant. Accordingly, the present invention selects strains with strong antibacterial and nematicidal effects among the bacteria belonging to the plant growth-promoting rhizobacteria (PGPR) group for the control of complex diseases to check the control effect and effectively The purpose is to use.

For the experiments, the root-knot nematodes Meloidogyne incognita and tomato wilt Fusarium oxysporum f.sp. The lycopersici test for the incidence of complex disease in tomato was performed, indicating that the severity index was 0.4 in the pathogen-only infection and 3.4 in the case of the complex infection. Decided. Selection of useful microbial strains was carried out in F. oxysporum f.sp. in vitro in 32 strains belonging to the PGPR group isolated from the root rot of ginseng during storage. Strains with strong antimicrobial activity against lycopersici were selected according to the size of the low support on the medium, and strains with M. incognita 2 larvae were treated with bacterial strain culture medium. As a result, it was confirmed that the strains of Paenibacillus polymyxa GBR462, GBR508 and P. lentimorbus GBR158 were both strong antibacterial and nematicidal. Three strains selected in the laboratory experiments were tested in F. oxysporum f. sp. The control effect of lycopersici and M. incognita- combined tomato irrigated with culture broth was 90-98%.

Therefore, the present inventors deposited the P. lentimorbus GBR158 strain to the Korea Agricultural Microbial Resources Center (KACC) on April 14, 2008 (Accession Number: KACC 91374P).

The present invention also provides an antimicrobial microbial agent comprising the strain of the present invention or a culture thereof as an active ingredient. Molds that can be controlled by the microbial agent of the present invention are F. oxysporum f. sp. lycopersici may be exemplified, but is not limited thereto.

The present invention also provides a nematicidal microorganism preparation comprising the strain of the present invention or a culture thereof as an active ingredient. Nematodes that can be controlled by the microbial agent of the present invention may include, but are not limited to, M. incognita , a root-knot nematode.

The present invention also provides a microbial agent for the control of complex diseases of plants caused by fungi and nematodes comprising the strain of the present invention or a culture thereof as an active ingredient. In the microbial preparation according to an embodiment of the present invention, the fungus is Fusarium oxyforum F. Esp. May be beaded between lycopene (Fusarium oxysporum f. Sp. Lycopersici), nematodes may be an Melo Ido Guinea encoding other cognitive (Meloidogyne incognita), but is not limited thereto. In addition, the combination bottle may be tomato wilting disease, but is not limited thereto.

In the microbial preparation according to an embodiment of the present invention, the microbial preparation may be used for plant seed coating. In an experiment for selecting a strain that can be used as a seed treatment agent among the three strains of the present invention, the penibacillus lentibus GBR158 had the highest fixation rate in the roots of tomato seeds and germinated seedlings, which was confirmed by scanning electron microscopy. Was treated with tomato seeds to investigate the combined control effect of 91.2%. The control effect of root nodule formation was also high as 92.0%. In addition, as in the irrigation treatment, the seed treatment also showed some effects on the growth of above-ground and below-ground tomatoes.

The present invention also provides a microbial agent for promoting plant growth and a biofertilizer comprising the strain of the present invention or a culture thereof as an active ingredient. The plant to which the microbial agent and the biofertilizer of the present invention can be applied is not particularly limited, but is preferably a tomato.

The microbial agent may include a penisbacillus lentimorbus GBR158 strain (Accession No. KACC 91374P) having an antibacterial activity, nematicidal force and plant growth promoting effect as an active ingredient. The microbial preparation according to the present invention may be prepared in the form of a liquid fertilizer and may be used in the form of powdered powder by adding an extender thereto or granulated by formulating it. However, the formulation is not particularly limited. In other words, it can be formulated as a biofertilizer to overcome this in eco-friendly organic farming where the chemical fertilizer supply is limited.

The present invention also provides a method for producing a biofertilizer comprising the step of culturing the strain of the present invention. The method of culturing the penivacillus strain and the method for producing the biofertilizer may use any method known in the art, and is not particularly limited to a specific method.

Treatment of the microbial strain of the present invention, as a result of the growth of the ground and underground parts of the plant more vigorously than the control group, as well as the pathogen-free inoculation control group, it was shown to have a growth promoting effect.

The present invention also provides a method for controlling a complex disease caused by fungi and nematodes comprising the step of immersing or irrigating a strain of the present invention into a plant or seed of a plant. In the method according to an embodiment of the present invention, the complex bottle may be tomato wilted disease, but is not limited thereto.

In the method according to an embodiment of the present invention, the strain controls the complex disease by disrupting the mycelia of the fungus or inhibiting growth, inhibiting hatching of nematode eggs, or inhibiting the formation of giant cells. can do.

In order to know what mechanisms of the present invention the combined disease control effect by the mechanism, the strain treated F. oxysporum f. sp. Mycelia of lycopersici , eggs of nematodes, inoculated plants, etc. were observed by light microscopy and scanning electron microscopy. The pathogenic fungal mycelium treated with microorganisms was swollen with normal tip growth, but the middle of the mycelium was broken and destroyed. Root-knot nematodes hatched 98% in water after 3 days, whereas they did not hatch at all in 1% of bacterial cultures, and the egg shells were damaged and the larvae inside the eggs died. In the roots of plants infected with root-knot nematodes, giant cells are formed. By the treatment of useful bacteria, the size and number of giant cells become smaller and smaller, indicating that their formation was suppressed. This suggests that useful bacteria have antimicrobial and nematicidal properties as well as penetrating effects on plants.

In the method according to one embodiment of the present invention, inhibition of hatching of the nematode eggs may be carried out through the production of proteinase of the strain of the present invention. Pheniba silus lentimobus GBR158 strain was investigated for its ability to produce proteinase as one of the mechanisms of inhibiting hatching of root nodules eggs. In the plate in which the Penivacillus lentibus GBR158 bacterial culture solution was dropped on skim milk-agar, a large halo was formed around the bacteria, indicating that proteinase was produced.

The present invention also provides a method for promoting plant growth, comprising the step of immersing or irrigating the strain of the present invention into a plant or seed of the plant. As a method for promoting the growth of the plant can be carried out by immersing or irrigating the seed or plant in the culture medium culturing the strain of the present invention and the microbial preparation using the strain. In the case of the dipping method, the culture and the preparation may be poured into the soil around the plant or the seeds may be immersed in the culture and the preparation.

The plant which can be applied to the method of the present invention is not particularly limited, but is preferably a tomato.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Nematode inoculum

Root-knot nematode Meloidogine incognita race-1 was obtained from pure cultures maintained on tomato plant roots ( Lycopersicon esculentum ). Whenever you need an inoculum. The roots of the plants were pulled, soaking the entire root in water to remove the attached soil. M. incognita egg masses were collected using tweezers. Baermann funnel method (Southey JF. (1986) Laboratory Methods for work with Plant and Soil Nematodes.Ministry of Agriculture Fisheries and Food, modified to use the eggs in experiments or to obtain a two-stage juvenile (J2) Incubation for 3-5 days using HMSO, London, UK). The nematode inoculum was used for in vitro and pot experiments. The colony density of nematodes was measured from 5 replicates of 2-ml aliquots of inoculum suspension and adjusted to the desired density.

Fungal Pathogen Inoculation

Rice bran medium (rice bran 1: sand 4: distilled water 1.5) was added to Fusarium oxysporum f. sp. It was used to prepare lycopersici . The medium was autoclaved at 121 ° C. for 20 minutes. F. oxysporum f. Grown in potato dextrose agar (PDA; 26.5 g Difco potato dextrose broth, 18 g agar, 1 L sterile water) for 7 days at 25 ° C. sp. Mycelial plug of lycopersici was inoculated into rice bran medium. The inoculated medium was ground in a blender and used as a fungal pathogen inoculum.

Collection and Cultivation of Penivacillus Strains

Penibacillus strains tested for biocontrol were isolated from four years old roots of ginseng with rot symptoms collected at market and storage facilities. The isolated Penivacillus strain was stored at −70 ° C. in sterile distilled water containing 20% glycerol until use. The bacteria were grown in brain heart infusion (BHI) broth at 28 ° C. for 2 days with shaking at 200 rpm. The resulting culture was considered a standard and an additional dilution was prepared by the addition of sterile distilled water to test its nematicidal activity against egg hatching and larval lethality of root nodules under in vitro conditions.

In vitro experiment

Antimicrobial activity

Penny Bacillus 40 strains of Paenibacillus polymyxa and Paenibacillus lentimorbus were grown on PDA at 25 ° C. in F. oxysporum f. sp. Screened for antimicrobial activity against lycopersici . Seven days after incubation, F. oxysporum f. sp. Mycelial discs of lycopersici (1 cm in diameter) were placed in the center of the PDA plate, the paper discs (8 mm in diameter) were soaked with each culture of the Penivacillus strain, dried overnight and placed on the plate 3 cm away from the fungal mycelium discs. . A disc soaked in BHI medium without bacteria was used as a control. After incubation for 7 days, the presence of the inhibitory region was established and the inhibitory activity was measured as the size of growth hypotermination between the bacterial colony and the fungal pathogen (mm). Each treatment was repeated five times and the experiment repeated.

Nematode activity

To test nematode activity, LC ₅₀ values (culture concentrations required to kill 50% of the nematode population 2 days after exposure) were determined by F. oxysporum f. sp. Eleven strains of Penibacillus spp. showing antimicrobial activity against lycopersici were measured (FIG. 3). Various concentrations of 300 μl bacterial cultures were transferred to 96-well Microtest ^™ Tissue Culture Plates (Becton Dickinson, NJ, USA) where 50 freshly hatched M. incognita J2 were located. B2 liquid medium without bacteria or J2 located in sterile distilled water was used as a control. Two days after exposure, immobilized J2 was counted under low magnification stereomicroscopy. The nematode looks straight and stiff under the microscope and is considered dead if it does not move when stabbed with a thin needle (Cayrol et al. (1989) Rev Nematol 12: 331-336). Each treatment was repeated five times and the experiment repeated.

Inhibitory effect on egg hatching

To determine the inhibitory effect of bacterial cultures on egg hatching of M. incognita , Penivacillus Polymix GBR-462; GBR-508 and 500 μl cultures of varying concentrations (1, 10, 50, and 100%) of Penivacillus lentimobus GBR-158 were prepared using 24-well Microtest ^TM Tissue Culture Plates (Becton Dickinson, NJ, USA). Eggs placed in bacteria-free BHI broth or sterile distilled water were used as controls. Five days after exposure, hatched larvae were counted under low magnification stereomicroscopy. Each treatment was repeated five times and the experiment repeated.

Scanning electron microscope

To observe the direct interaction of Penibacillus against the pathogen, fungal mycelium and nematode eggs were treated with a culture of Penibacillus polymyx GBR-508 and incubated for 24 hours. After incubation, F. oxysporum f. sp. Mycelial plugs and nematode eggs of lycopersici were fixed at 4 ° C. with Karnovsky's fixative in 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M cacodylate buffer at pH 7.2. After 12 hours, the fixed samples were washed three times with 0.05 M cacodylate buffer for 15 minutes each. The sample was post-fixed in 2% OsO ₄ in 0.01 M cacodylate buffer (pH 7.2) at 4 ° C. for 4 hours and then washed briefly with distilled water three times. The samples were then dewatered with 30, 50, 70, 80 and 90% ethanol series for 10 minutes each and finally dehydrated three times for 10 minutes in 100% ethanol. The sample was then passed through 100% isoamyl acetate twice every 10 minutes at room temperature. The sample was critically dried and coated with gold using a Sputter Coater (JFC-1110E, JEOL, Tokyo, Japan). The sample was observed with a scanning electron microscope (JSM-5410LV, JEOL, Tokyo, Japan).

Port experiment

Synergy

M. incognita and F. oxysporum f. sp. The interaction of lycopersici was determined under greenhouse conditions. A plastic pot 6 cm in diameter was filled with 500 g sterile sand and pot mixture. 3 week tomato seedlings cv. Seon-Myeong (root-sensitive nematode) was planted in the pot. Each plant was F. oxysporum f. sp. Inoculated with stage 2 larvae of lycopersici and / or M. incognita . The fungus was grown in rice bran medium for 15 days and 20 g of the medium was mixed with 5 cm of soil top. Two stage larval (J2) suspensions were dispensed by pipette at a rate of 1000 J 2 / port. In combination treatment, the fungal culture and J2 suspension were prepared in three different orders, namely F. oxysporum f. sp. Two days before lycopersici treatment, concurrent with and two days after treatment were inoculated into pot soil inoculated with M. incognita . Plants grown in untreated soil were used as controls. Each treatment was repeated five times and the experiment repeated. The pots were arranged in a random block design on a bench in a greenhouse at 25 ± 2 ° C. and watered daily with field doses.

Seven weeks after fungal and / or nematode inoculation, the plants were carefully uprooted from the pot and the soil attached to the roots was removed by gentle stirring in water. The extent of root galling in M. incognita- infected tomato plants was evaluated based on a 0-5 rating according to the percentage of galled tissue (0 = 0-10% galled root; 1 = 11-20%; 2 = 21- 50%; 3 = 51-80%; 4 = 81-90%; and 5 = 91-100% .The wilting degree was also graded using a 0-5 scale (0 = no visible symptoms; 1 = 1 Upper leaf growth (leaf sagging) and yellowing / wilting of tea leaves; 2 = yellowing / wilting of secondary and tertiary leaves, primary leaves may be lost; 3 = withering, second and third leaves of tertiary leaves Tertiary leaves may be lost; 4 = sulphation / wilting of the entire plant, and 5 = complete plant death (Marley PS, Hillocks RJ. (1996) Field Crops Res 46: 15-20).

Effects of Penivacillus Strains on Combination Diseases

In in vitro assays, among the tested strains, Penivacillus polymixes GBR-508, GBR-462 and Penivacillus lentibus GBR-158 were identified as F. oxysporum f. sp. It was found that it has the highest inhibitory activity against lycopersici and M. incognita . The strains were prepared from F. oxysporum f. sp. It was used for biocontrol of complex diseases caused by lycopersici and M. incognita .

A plastic pot 6 cm in diameter was filled with 500 g sterile sand and pot mixture. 3 week tomato seedlings cv. Seon-Myeong was planted in the port. Each plant was treated in the same manner as described above in the Synergy section of F. oxysporum f. sp. It was inoculated simultaneously with J2 of lycopersici and M. incognita . Penny Bacillus polymix grown in BHI broth for 2 days Cultures of GBR-508, GBR-462 and Penivacillus lentibus GBR-158 were added separately to the pot soil around the plant roots at a rate of 5 ml / port to measure their biocontrol efficacy. Plants grown in untreated soil were used as controls. Each treatment was repeated five times and the experiment repeated. The pots were arranged in a random block design on a bench in a greenhouse at 25 ± 2 ° C. and watered daily with field doses. Seven weeks after treatment, the plants were carefully rooted from the pot. Root nodules and wilting incidence were evaluated as described above.

Effect of Penivacillus Polymyx GBR-508 on the Formation of Giant Cells

M. incognita and F. oxysporum f. sp. Root nodules caused by lycopersici interaction Taken from plants treated with GBR-508 and untreated control plants and fixed in Karnovsky's fixative solution in 0.01 M cacodylate buffer (pH 7.0) for 24 hours. Root lump samples were post-fixed with 0.01 M OsO ₄ in the same buffer solution for 2 hours and then washed three times with the buffer. The samples were dehydrated in ethanol series and embedded in Spurr's epoxy resin. Nematodes were clearly visible through embedding material. Embedded samples were sectioned with a glass knife (MT 800 nm) on an MT-X ultra-cutter (RMC, Inc., Tucson, AZ, USA). The sections were stained with 1% toluidine blue O in 30% ethanol and observed under a compound optical microscope (Axiophot, Zeiss, Germany).

Statistical analysis

All experiments were performed twice. The analysis did not show any significant interaction between the two trials for any treatment. Therefore, the test results were combined for final analysis. Analysis of variance was performed using Statistix 7.0 (NH Analysis software, Roseville, MN). Duncan's multirange test was performed to test the significant difference between treatments at P ≦ 0.05.

Effects on Seed Treatment of Phenivacillus Polymyxa GBR462 and Phoebacillus Lentimobus GBR158 Strains

(1) bacterial culture

Phenibacillus Polymixsa Resistant to Rifampicin Mutants of GBR462 and Penivacillus lentibus GBR158 (represented as GBR-462-Rif + and GBR-158-Rif +, respectively) were obtained by culturing them on medium supplemented with 100 ppm rifampicin for mutation. The mutants were cultured and stored at −70 ° C. in sterile distilled water containing 20% glycerol until use. Each bacterium was grown in Brain Heart Infusion (BHI) (CONDA, Madrid, Spain) broth at 28 ° C. for 2 days with shaking at 200 rpm. Bacterial biomass was collected by centrifugation at 12,000 rpm for 20 minutes, and the pellet was again suspended in distilled water and adjusted to 10 ⁸ CFU / ml (OD ₆₀₀ = 0.8).

(2) seed treatment

Tomato seeds were immersed in a mixture of bacterial suspension and 0.05% CMC (carboxymethyl cellulose sodium) for 1 hour and air dried on filter paper for 12 hours at room temperature. Five seeds treated with GBR158-Rif + and GBR158-Rif + separately were placed on the filter paper (Whatman No. 2) at the bottom of the petri plate (d = 11 cm), poured with 5 ml sterile distilled water, sealed with a polypropylene wrap and placed in a petri plate. Constant moisture was maintained. After 7 days incubation at 28 ° C., the effect of seed treatment on seed germination and phytotoxicity was investigated. In addition, the population densities of GBR462-Rif + and GBR158-Rif + on germinated seeds were investigated to determine their establishment capacity for the plant root system. To this end, 1 cm of hypocotyl segments from the top were cut with sterile razor blades, called and diluted in sterile distilled water. Colony forming units (CFU) of bacteria in each of the embryonic sections were measured by smearing dilutions on BHI agar supplemented with 100 ppm rifampicin and incubating at 28 ° C. for 2 days.

Test of Proteinase Production Ability of Penivacillus Lentimobus GBR158

Phenibacillus polymixes GBR508, GBR462 and Penivacillus Lentimobus GBR158 bacterial cultures were incubated in BHI broth for 48 hours, then the bacterial density was adjusted to 10 ⁸ CFU / ml (OD ₆₀₀ = 0.8) and each bacterial culture was adjusted to 10 After dropping in skim milk-agar plate (Difco ^™ Skim Milk, 100 g agar, 1 L sterile water) medium, the solution was dried for 10 minutes. And each plate was observed after incubating for 24 hours at 28 ℃.

Example 1: Antimicrobial Activity

Of the 40 strains of the genus Penibacillus tested, 11 strains were F. oxysporum f. sp. It showed antimicrobial activity against lycopersici (Table 1).

Table 1. Fusarium oxysporum f. sp. Antimicrobial Activity of Penibacillus Strains against lycopersici

Penivacillus strain Restraining area (mm) ^x Penny Bacillus Polymix GBR-11 3.0 ± 0.0 Penivacillus Polymix GBR-27 2.0 ± 0.2 Penivacillus Lentimobus GBR-158 7.2 ± 0.2 Penny Bacillus Polymix GBR-192 1.7 ± 0.6 Penny Bacillus Polymix GBR-447 4.3 ± 0.5 Penny Bacillus Polymix GBR-462 7.4 ± 0.5 Penny Bacillus Polymix GBR-477 3.7 ± 0.6 Penny Bacillus Polymix GBR-485 1.3 ± 0.6 Penivacillus Polymix GBR-508 7.5 ± 0.4 Penny Bacillus Polymix GBR-602 2.7 ± 0.6 Penny Bacillus Polymix GBR-603 1.3 ± 0.6

^x Antimicrobial activity was tested using the paper disk method.

The antimicrobial strains could be classified into three groups based on their inhibitory strengths: strong antibacterial with an inhibitory area of at least 7.0 mm, moderate antibacterial at 3.0-7.0 mm and weak antimicrobial at 3.0 mm or less. Penivacillus Polymix GBR-462, GBR-508 and Penivacillus Lentimobus GBR-158 belongs to the group with strong inhibitory activity against fungal pathogens (FIG. 1). Phenibacillus polymix yarn GBR-508 and F. oxysporum f. sp . Electron microscopy observations of the direct interaction between lycopersici showed alteration and distortion of mycelial cell walls (FIG. 2).

Example 2: Nematode Effect

Culture concentrations required for LC ₅₀ were different for different strains (FIG. 3). No J2 was killed in sterile distilled water and bacteria free BHI medium control (data not shown). Cultures of Penivacillus lentibus GBR-158 and Penibacillus polymixes GBR-447, GBR-462, GBR-477 and GBR-508 had lower LC ₅₀ values (LC ₅₀ <0.1%), among which , GBR-158, GBR-462 and GBR-508 are described in F. oxysporum f. sp. It has already shown strong antibacterial activity against lycopersici . Therefore, using these three strains for further studies, F. oxysporum f. For tomato plants in pot soil under greenhouse conditions. sp. Its biological control against multiple diseases caused by lycopersici and M. incognita was evaluated.

Example 3: Suppression of Egg Incubation

Various concentrations (1-100%) of the Penivacillus polymixes GBR-508, GBR-462 and Penivacillus lentibus GBR-158 cultures completely inhibited egg incubation. Nearly all eggs hatched in sterile distilled water for 3 days, while no bacterial enrichment occurred in all bacterial treatments even at low concentrations (1%) 3 days after treatment (data not shown). Electron microscopy observations of the direct interaction between the eggs of GBR-508 and M. incognita showed that the egg shells rotted and destroyed and numerous bacterial cells attached to their surface (FIG. 4). Nematode larvae inside the egg seemed dead.

Example 4: Synergistic Interaction

The synergistic effect of M. incognita was compared by F. oxysporum f. sp. It was confirmed in Fusarium wilt of tomatoes caused by lycopersici (FIG. 5). Seven weeks after inoculation, F. oxysporum f. sp. The wilting index for tomato plants caused by lycopersici alone was estimated to be 0.4, and no wilting symptoms occurred with M. incognita alone (Table 2). However, it was markedly increased by the combined inoculation of the two pathogens.

Table 2. Fusarium oxysporum f. sp. Meloidogine incogita against wilting caused by lycopersici (FO) (MI) effect

process Withering degree ^x MI inoculated 2 days before FO 2.2 (± 0.4) Simultaneous inoculation of MI and FO 3.4 (± 0.8) MI inoculated 2 days after FO 3.0 (± 1.2) Only inoculates FO 0.4 (± 0.5) Only inoculated with MI 0 Untreated control 0

^The wilting degree was graded using a 0-5 scale (0 = no visible symptom; 1 = upper leaf growth of the primary leaf (leaf drop) and yellowing / wilting; 2 = secondary and tertiary leaf yellowing) Wither, primary leaves may be lost; 3 = withered or higher leaves of tertiary leaves, secondary and tertiary leaves may be lost; 4 = yellowing / wiping of the entire plant, and 5 = plant death.

The wilting level was F. oxysporum f. sp. When M. incognita was inoculated 2 days prior to lycopersici inoculation, and 2 days after inoculation, it was estimated to be 2.2, 3.4 and 3.0 in the fussium withering of tomato plants, respectively.

Example  5: In a compound bottle  About Penibacilli  effect

In the pot experiment, the wilting degree of the Fusarium wilted-root-hox nematode complex was drastically reduced by treatment with Peniva Sealus Polymix GBR-508, GBR-462, and Penivacillus Lentimobus GBR158, and their control effects were 97.6, respectively. It was evaluated as%, 92.7% and 90.2% (Table 3). The treatment also reduced nodules by 64-88% compared to untreated controls (Table 3).

Table 3. Fusarium oxysporum f. sp. lycopersici Control Effects of Penibacillus Strains on Combination Diseases in Tomatoes Caused by (FO) and Meloidogine Incognita (MI)

process Withering Lump formation Stem kidney
(mm) Stem weight
(g) Root weight
(g) Index ^x CE (%) Index ^y CE (%) FO + MI 4.1a ^z - 5.0a - 194.2c 2.58c 1.30b FO + MI + GBR-508 0.1b 97.6 0.6c 88.0 392.5a 14.85a 2.47a FO + MI + GBR-462 0.3b 92.7 1.1b 78.0 423.9a 14.12a 2.33a FO + MI + GBR-158 0.4b 90.2 1.8b 64.0 445.8a 13.54a 2.30a Untreated control 0.0b - 0.0b - 287.5b 6.52b 1.95a

^The degree of ^x withering was graded using the 0-5 scale as described in Table 2.

^{The y-} hox index was evaluated based on a 0-5 rating (0 = 0-10%; 1 = 11-20%; 2 = 21-50%; 3 = 51-80%; 4 = 81-90%; and 5 = 91-100% root nodule formation)

^{In the z} column, the mean of the same letter is not significant at P = 0.01 by Duncan's multirange test.

CE = control effect

Stem and root growth of tomato plants was markedly reduced by infection of two pathogens, but treatment of these bacterial strains improved plant growth, particularly showing significantly more stem growth than uninoculated healthy plants (Table 3 and FIG. 6). Tomato plants infected by the two pathogens showed vascular bundle rot and tremendous galling of the stem (FIG. 7), but the treated plants did not show the vascular bundle rot and root nodule formation.

Example 6: Inhibitory Effect of Penivacillus Polymyxa GBR-508 on Giant Cell Formation

Through embedded material, untreated and penivacillus polymix M. incognita and F. oxysporum f. sp . Roots caused by lycopersici or 7 weeks after inoculation were observed. In the untreated control group, M. incognita and F. oxysporum f. sp . It consisted of 6-14 giant cells in the root or central line induced by lycopersici , mostly occupying the woody part (FIG. 8A). In contrast, few or few (1-3) small giant cells were formed in plants treated with GBR-508 (FIG. 8B), indicating inhibition of giant cell formation. Female nematodes were typically located near the central column area. M. incognita and F. oxysporum f. sp. The site of infection of root (root nodules) using lycopersici had giant cells characterized by hypertrophy and dense cytoplasm. At the center of the roots, the cell wall of the giant cells was thick and stained differently in the toluidine blue staining of that of the duct or phloem fibers, indicating that the cell wall was not woody. The cell wall of giant cells is stained red or purple like the primary cell wall of milk tissue cells, while the water and phloem fibers are light blue.

Example 7 Seed Treatment Effects of Penivacillus Polymixsa GBR462 and Penivacillus Lentimobus GBR158 Strains

Seed germination, phytotoxicity, and bacterial population densities recovered from germinated seeds (implantation) were investigated to determine the relevance of seed treatment with bacterial strains GBR158-Rif + and GBR462-Rif + for biocontrol. The germination rate of the tomato seeds treated with the two bacterial strains was as high as the untreated control, with no symptoms showing phytotoxic effects (Table 4). However, the bacterial population density recovered from GBR158-treated seeds (implantation) at 7 days after treatment was significantly higher than that treated with GBR462 (1.3x10 ³ cfu / cm embryonated) (5.9x0 ⁴ cfu / cm embryonated) (Table 4 ).

Table 4. Effect of seed treatment with Penivacillus lentibus GBR158-Rif + and Penivacillus polymix GBR462-Rif + on seed germination, phytotoxicity, and antagonistic bacteria density settled at 1 cm hypocotyl.

GBR158-Rif GBR462-Rif 0.05% CMC No treatment Germination rate 98% 97% 96% 98% Phytotoxic effect none none none none Number of bacteria recovered 5.9 x 10 ⁴ 1.3 x 10 ³ 0.0 0.0

SEM observation of seeds and hypocotyl showed that GBR158 bacterial cells proliferated abundantly, covering their surface (FIGS. 9 and 10).

In pot experiments under greenhouse conditions, seed treatment with Penivacillus lentibus GBR158 was associated with the degree of wilting caused by Fusarium withering and root-knot nematodes and M. incognita compared to the untreated control. It reduced root nodule formation and increased tomato plant growth, in particular not only the control group but also showed significantly more stem growth than the inoculated healthy plants (Table 5 and FIG. 11).

Table 5. Fusarium oxysporum f. sp. lycopersici ( F ) and effect of seed treatment with Penivacillus lentibus GBR158 on tomato seedlings infected by Meloidogyne incognita (MI).

process Withering Lump index Stem kidney
(mm) Stem weight
(g) Root length
(mm) Root weight
(g) Index ^x CE (%) Index ^y CE (%) FO + MI 3.6a ^z - 2.5b - 454.5c 13.6b 128.9b 3.1b MI 0.0b - 3.3a - 632.5a 20.1a 166.3a 4.9a FO + MI +
GBR-158 0.3b 91.7 0.2c 92.0 717.7a 24.1a 186.0a 5.3a No treatment 0.0b - 0.0c - 645.0a 23.4a 175.4a 4.3a

^The wilting degree was graded using a 0-5 scale (0 = no visible symptom; 1 = epidermal growth (leaf sagging) and sulphation / wilting of the primary leaf; 2 = sulphation of secondary and tertiary leaves) Wither, primary leaves may be lost; 3 = withered or higher leaves of the tertiary leaves, secondary and tertiary leaves may be lost; 4 = withered whole plants, and 5 = plant death.

^The root nodules for ^y M. incognita were assessed based on a grade of 0-5 according to the percentage of galled tissue (0 = 0-10%; 1 = 11-20%; 2 = 21-50%; 3 = 51 -80%; 4 = 81-90%; and 5 = 91-100% root nodules).

^{In the z} column, the mean of the same letter is not significant at P = 0.01 by Duncan's multirange test.

The withering index was 3.6 and the root nodules index was 2.5 in tomato seedlings caused by the combined infection of two pathogens in the untreated control group. However, when treated with GBR158, the incidence was reduced to 0.3 and 0.2, and its control effects were 91.7% and 92.0%, respectively. Untreated tomato plants infected by two pathogens showed vascular rot of the stem, but treated plants did not show vascular rot.

Example 8 Confirmation of Proteinase Generating Ability of Penivacillus Lentimobus GBR158

Previous studies have shown that the strains of Penibacillus polymixes GBR508, GBR462 and Penivacillus lentibus GBR158 inhibit the hatching of root nodules eggs. One of the inhibitory mechanisms was the ability to produce proteinases. Nematode shells account for about 59% of protein, so if proteinases were produced, it would have a significant effect.

Plates in which Penibacillus lentibus GBR158 bacterial culture was dropped on skim milk-agar produced large halo around the bacteria, indicating that proteinases were produced. Symptoms did not appear, it can be seen that no proteinase was produced (Fig. 12).

1 is Fusarium oxysporum f. sp. lycopersici For The antibacterial test of the Penivacillus strains (A: GBR-158, B: GBR-462, C: GBR-508) is shown. Asterisks are paper discs soaked in BHI medium without bacteria (control).

2 is penivacillus Polymix Fusarium oxysporum f. Showing broken (arrow) and warped (arrowhead) mycelia (A) and intact mycelia (B) in untreated controls affected by GBR-508 f. sp. Scanning electron micrograph of lycopersici . BC: bacteria.

FIG. 3 shows the culture concentrations of different strains of Penivacillus required for LC ₅₀ of Meloidogine Incognita 48 hours post inoculation.

4 is the penivacillus Scanning electron micrographs of nematode eggs treated with Polymix GBR-508. Numerous bacteria (BCs) are attached to nematode eggs. AB: show dead nematode larvae (NJ) inside the warped egg; C, D: Twisted eggshell.

FIG. 5 shows the melodogine incogita and Fusarium oxysporum f. sp. It shows synergistic effect of lycopersici . A: unvaccinated wholesome plants; B: F. oxysporum f. sp . inoculation of lycopersici alone; C: inoculation with M. incognita alone; D: M. incognita and F. oxysporum f. sp . Co-inoculation with lycopersici .

6 is Fusarium oxysporum f. sp. It shows the control effect of the penivacillus strain on the combined disease caused by lycopersici and M. incognita . FO: F. oxysporum f. sp. lycopersici ; MI: M. incognita ; Control: Unvaccinated tender plants.

7 is Meloidogine incogita and Fusarium oxysporum f. sp. lycopersic i Phenibacillus for Combination Disease Show the effect of Polymix GBR-508. A: untreated control showing vascular bundle rot; B: Penivacillus polymix yarn Treated with GBR-508 to show healthy growth.

8 is Meloidogine Incogita (N) and Fusarium oxysporum f. sp. It shows the giant cells (GC) induced in the wood (X) of the tomato root by lycopersici . A: Untreated control (X 200) showing severe giant cell formation; B: Penivacillus without giant cells Polymix GBR-508 treatment group (X 200).

Degree 9 is a scanning electron micrograph of tomato seeds. A: untreated control; B: Treatment with Penivacillus lentibus GBR158-Rif + showing seed surface with bacteria attached.

Degree 10 is a scanning electron micrograph of the hypocotyls of tomato seeds. A: untreated control; B: Treatment with Penivacillus lentibus GBR158-Rif + showing the surface of the embryonic axis to which bacteria were attached.

11 shows F. oxysporum f. sp. lycopersici (FO) and M. incognita Effect of Seed Treatment with Penivacillus Lentimobus GBR158 on Tomato Seedlings Infected by (MI). FO + MI: inoculated with two pathogens; FO + MI + GBR158: inoculated with two pathogens and treated with Penivacillus lentibus GBR158; Controls: Unvaccinated and untreated control.

FIG. 12 shows the results of treatment of skim milk-agar plates with Penivacillus lentibus GBR158 and Penivacillus polymix GBR508, GBR462 bacterial cultures.

Claims (13)

delete delete delete Fusarium oxyforum F, comprising a penisbacillus lentimorbus GBR158 strain (Accession Number KACC 91374P) or a culture thereof as an active ingredient. Esp. Between lycopene Persie (Fusarium oxysporum f. Sp. Lycopersici ) fungi, and other cognitive Melo Ido Guinea Inco (Meloidogyne incognita) tomato wilt disease control complex microbial agent for infections caused by a complex of nematodes. delete delete 5. The microbial preparation according to claim 4, wherein the microbial preparation is used for coating tomato plant seeds. delete Fusarium oxyforum f comprising the step of immersing or irrigation of the seed of the Paenibacillus lentimorbus GBR158 strain (Accession No. KACC 91374P) in tomato plants or tomato plants. Esp. Between lycopene Persie (Fusarium oxysporum f. Sp. Lycopersici ) fungi, and other cognitive Melo Ido Guinea Inco (Meloidogyne incognita) how to control tomato wilt disease complex caused by a complex of nematode infection. delete delete delete delete

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