KR101127045B1 - A composition containing antagonistic rhizobacteria consortium for inhibiting phytophtor capsici and promoting red pepper growth - Google Patents

Abstract

The present invention is a composition for the control of pepper blight and pepper growth comprising Bacillus subtilis AH18 and Bacillus licheniformis K11 as an active ingredient, and after incubating Bacillus subtilis AH18 and Bacillus licheniformis K11 alone The present invention relates to a method for promoting pepper growth through the control of pepper blight, which is used in combination, and the method according to the present invention has much better control of pepper blight than the treatment of other pepper blight antagonism microorganisms after single culture or after co-culture. There is also an excellent effect in promoting growth of roots, stems, and leaves of red pepper.

Red pepper plague, red pepper growth, Bacillus subtilis AH18, Bacillus Richenformis K11

Description

A composition containing antagonistic rhizobacteria consortium for inhibiting phytophtor capsici and promoting red pepper growth}

The present invention relates to a composition for antagonizing microorganisms for controlling red pepper disease and promoting red pepper growth. More specifically, the composition for controlling red pepper disease and red pepper growth, comprising Bacillus subtilis AH18 and Bacillus licheniformis K11 as an active ingredient. And Bacillus subtilis AH18 and Bacillus licheniformis K11 after cultivating alone and pepper used to promote pepper growth by controlling pepper blight used in combination.

Antagonistic microorganisms, which directly or indirectly affect the growth and prevention of diseases, are called antagonistic microorganisms. Their antagonism is an antagonistic effect that inhibits the growth of phytopathogens by occupying habitat, inducing plant resistance and / or antibiotics, depending on the ability of microorganisms.

Many antagonist microorganisms have been selected and selected in the last 20 years, thanks to the movement of organic control and organic farming, which are trying to grow crops environmentally using root-strain microorganisms showing antagonistic ability against phytopathogens. However, only a few of the selected microorganisms have succeeded in commercializing biopesticides, but there is a problem in that the field reproducibility is inferior and the consistency of control effects is inconsistent. When developing the antagonist microorganisms, the control ability by one antagonism method was verified, and the effect was verified only in the laboratory environment. Therefore, the antagonist's reproducible control ability, which is required in various actual farmland environments, was not achieved. Because it is analyzed. In other words, because of its limited ability to control, it is thought that it was difficult to show various control effects only in one mechanism under various conditions. In addition, farmers want multi-functional drugs that are evenly controlled against a variety of diseases, even if a single crop, especially in the case of eco-friendly agricultural materials that can reduce the use of chemical pesticides, specific crops that can be controlled in one variety We crave exclusive farm materials.

Most microbial agents currently being researched or commercialized using one or two microbial strains, and even if the microbial strain is composed of three or more microbial strains, there is little research on the mutual relationship between the microbial strains used and the competition. As a matter of fact, it is virtually difficult to verify correct and lasting efficacy.

Therefore, the present inventors have intensively studied to develop agricultural materials for specific crops that can be variously controlled by one medicinal herb. As a result, the antagonists of eight antagonists whose various antagonisms have been confirmed as microbial control agents through conventional experiments are mutually By constituting the antagonistic microbial combinations by each mechanism, the combination was verified through the in vivo pot experiment to verify the pepper pest control disease and pepper growth promoting ability and led to the present invention,

Accordingly, it is an object of the present invention to provide an effective antagonistic microbial combination for controlling pepper blight and promoting pepper growth.

In addition, another object of the present invention is to provide an optimal combination of the antagonist microorganisms optimized for pepper pepper disease control and pepper growth promotion, and a method of using the same.

The purpose of the present invention as described above is to select individual strains having a high control ability against pepper blight and excellent plant growth promoting ability, the largest control ability against pepper blight and plant growth promoting ability, iron capture body production ability, through auxin production, Bacillus subtilus AH18 strain with antifungal β-glucanase production ability, phosphate solubilizing ability and plant growth promoting ability, phosphate solubilizing ability, iron capture body production capacity, antifungal β-glucanase production ability, antibiotics production ability Combination of 11 strains using two strains of Richenformis K11 as a key strain, and after culturing alone and co-culture, compared with the bacillus subtilis AH18 and in comparison with late blight control and growth promoting ability through in vivo pot experiments Combining Bacillus licheniformis K11 and combining it with red pepper after incubation is effective in controlling red pepper disease and promoting red pepper growth. Naemeurosseo found was achieved.

The present invention provides Bacillus subtilis AH18, Bacillus megaterium KL39, Bacillus licheniformis K11, Bacillus thuringiensis BK4, Pseudomonas fluoresense (Pseudo) fluorescens 2112, Pseudomonas maltophilia AM5, Serratia proteamaculans 3095, Chryseomonas luteola 5042, a combination of two selected from the group consisting of It provides a composition for controlling pepper blight.

The present invention particularly provides a composition for controlling red pepper disease comprising Bacillus subtilis AH18 and Bacillus licheniformis K11 as an active ingredient.

In another aspect, the present invention provides a method for promoting pepper growth through the control of pepper blight to treat the above-mentioned combination of pepper strains.

In particular, the method of the present invention is characterized in that the individual strains of the above strain combinations are cultured alone, and then mixed and treated with red pepper.

In particular, the method of the present invention is characterized in that the inoculation of the individual strains of the strain combination at the same time and co-cultivation to the pepper.

In particular, the method of the present invention is characterized by culturing Bacillus subtilis AH18 and Bacillus Richenformis K11 alone and using a mixture of peppers.

In the present invention, the present inventors have attempted to develop an environment-friendly agricultural antagonistic microbial combination using strains of antagonistic microorganisms researched and developed for biopesticides, antibiotic ability, iron trap and cellulose production capacity, auxin production capacity, As a result of constructing and combining a combination candidate group of complementary microbial strains for each mechanism such as phosphate availability, a combination of Bacillus subtilis AH18 and B. licheniformis K11 among the combination candidate groups. No. 11 showed the best pepper growth promoting activity and pepper pest control in the in vivo pot test. No. is the best combination 11 Individual cultures of B. subtilis AH18 and B. licheniformis K11 alone were used to investigate mutually uncompetitive mutual symbiosis between two antagonists in the combination. As a result of three treatments of inoculating two strains at the same time and mixing each incubation alone at the time of inoculation, the co-cultivation resulted in the control and growth promoting ability when compared to the culture of each alone. All showed no synergy. However, in the mixed treatment group after the single culture, synergistic effect was shown to be large, and the growth promoting effect of root, stem, and leaf was all higher than the post-culture treatment or co-culture treatment. .

According to the present invention, in the case of using pepper bacillus control and pepper growth promoting composition combining Bacillus subtilis AH18 and Bacillus licheniformis K11, when they are cultured alone and mixed with pepper, these or other pepper antagonist antagonists It is much better in controlling pepper disease than in post-culture or co-cultivation of microorganisms, and also has an excellent effect in promoting growth of roots, stems and leaves of red pepper. will be.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Complementary Complementary Combination of Individual Strain Mechanisms .

In this embodiment, using the strains of antagonism microorganisms (refer to Table 1) researched and developed for conventional biopesticides, each group of antibiotic ability, siderophore and cellulase production capacity, auxin production capacity, phosphate availability, etc. A combination candidate of farewell complementary microbial strains was constructed.

TABLE 1

Antagonistic microbial strain

Antagonistic microbial strain Antagonist activity References Bacillus subtilis AH18 Antibiotic action
PGPR Jung HK, Kim JR, Woo SM, and Kim SD (2006) An auxin producing plant growth promoting rhizobaterium Bacillus subtilis AH18 which has siderphore-producing biocontrol activity. Korean J Microbiol Biotechnol 34 , 235-244. Bacillus megaterium KL39 Antibiotic action Jung HK and Kim SD (2003) Purification and characteriztion of an antifungal antibiotic from Bacillus megaterium KL 39, a biocontrol agent of red-pepper Phytophtora blight disease. Kor J Microbiol Biotechnol 31 , 235-241 Bacillus licheniformis K11 Antibiotic action
PGPR Woo SM, Woo JW, and Kim SD (2007) Purification and characterization of the siderophore from Bacillus licheniformis Kll, a multi-functional plant growth promoting rhizobacterium. Kor J Microbiol Biotechnol 35 , 128-134. Bacillus thuringiensis BK4 Antibiotic action Jung HK and Kim SD (2003) Purification and characteriztion of an antifungal antibiotic from Bacillus megaterium KL 39, a biocontrol agent of red-pepper Phytophtora blight disease. Kor J Microbiol Biotechnol 31 , 235-241 Pseudomonas fluorescens 2112 Antibiotic action
PGPR Lee ET and Kim SD (2001) An antifungal substance, 2,4-diacetylphloroglucinol, produced from antagonistic bacterium Pseudomonas fluorescens 2112 against Phytophthora capsici . Kor J Microbiol Biotechnol 29 , 37-42. Pseudomonas maltophilia AM5 Antibiotic action Kim JR (2004) Isolation and characteristics research of an Auxin and hydrolytic enzyme by Plant Growth Promotion Rhizobacteria (PGPR). MS Thesis, Yeungnam University, Gyeongsan, Korea. Serratia proteamaculans 3095 Chitinase Lee ET and Kim SD (1999) Isolation and antifungal activity of the chitinase producing bacterium Serratia sp. 3095 as antagonistic bacterium against fusarium sp. J Korean Soc Agric Chem Biotechnol 42 , 181-187 Chryseomonas luteola 5042 Antibiotic action Yun GH, Lee ET, and Kim SD (2001) Identification and antifungal antagonism of Chryseomonas luteola 5042 against Phytophthora capsici. Kor J Appl Microbiol Biotechnol 29 , 186-193.

All of the above strains are disclosed by the above reference, and used those stored at −70 ° C. in the laboratory of Applied Microbiology of Yeungnam University. Bacillus subtilis AH18 (Accession No .: KACC 91205P), Bacillus licheniformis K11 (Accession No .: KACC 91206P), Pseudomonas fluorescens 2112 (Accession No .: KACC 8944P), and Serratia proteamaculans 3095 (Accession No .: KACC 8945P) and Bacillus thuringiensis BK4 (Accession No .: KACC 9116P) are each registered in Korean Patent No. 10-0747700. Nos. 10-0747701, 10-347721 and 10-312330, which were deposited with KACC under the corresponding accession number for the filing of the patent.

Bacillus megaterium KL39, Pseudomonas maltophilia AM5 and Chryseomonas luteola 5042 can also be obtained in the manner described in the above references.

The combination of individual strains for the composition of candidate candidates was preferentially selected for individual strains with high control ability against pepper blight and excellent plant growth promoting ability, and antagonism or growth promoting mechanisms could be supplemented in addition to the selected and secured individual antagonistic microorganisms. The method of combining the strains one by one was selected.

The composition of the combination was expected to be less effective in the plural combinations of three or more, and the study was conducted by limiting the number of antagonistic microbial strains of the combination to two strains. Two strains, Bacillus subtilis AH18, which have the greatest control against pepper blight and have plant growth promoting ability and phosphate solubilizing ability through auxin production, and Bacillus licheniformis K11, which have plant growth promoting ability and phosphate solubilizing ability, are the key strains. Thus, after performing various experiments on the combination group composed of other strains by permutation and combination as a candidate candidate group, the optimum combination component group was determined as the final and optimal combination.

These two strains were able to combine the seven individual antagonistic microorganisms with each other as shown in Table 2 below, and the combination candidate group that can be constructed is No. No. 1 in All 11 were 11 candidates. Both of these strains are Bacillus sp. Derived from the soil, and it is considered to be very advantageous in the commercial aspect since it is possible to formulate agricultural products for long-term preservation and distribution through sporulation.

TABLE 2

Antagonist combination

Strain Bacillus subtilus AH18 Bacillus Richenformis K11 Bacillus Megaterium KL39 No. One No. 6 Bacillus thuringiensis BK4 No. 2 No. 7 Pseudomonas Fluorescens 2112 No. 3 No. 8 Serratia proteinase probably cool lance 3095 No. 4 No. 9 Criseomonas Luteola 5042 No. 5 No. 10 Bacillus subtilus AH18 - No. 11 Bacillus Richenformis K11 No. 11 -

Example 2 Individual Antagonistic Microbial Strains and Combinations of Constructs in vivo  Comparison of late blight control and growth promotion by pot experiment

The combination of complementary functions between the strains against the previously obtained biological control bacteria of Table 1 was carried out experiments for pepper pest control and pepper growth promoting ability for the combination candidate group. The experiment was carried out in a pot (90.0 × 90.0 × 70.0 mm) in which pepper was transplanted in a constant temperature and humidity chamber at 28 ^° C. and 50% humidity. In the red pepper disease control and growth promotion experiments, 2 × 10 ⁵ CFU / ml of the red pepper phytophthora phytophthora capsici was inoculated in a pepper pot of the same size and length. 1 day wet (28 ^o C, 70% humidity) treatment, and two days later the combination strains were treated (10 ⁸ CFU / ml per port) to continue the growth while periodically checking the onset.

In addition, the pepper growth promoting experiment was carried out simultaneously with the control performance experiment, and was carried out continuously by selecting only the pepper pot having the control ability against pepper blight. After recovering only the cells of the combination strain group cultured again in the pot (combination treatment) for 3 days, suspended in distilled water and inoculated with irrigation, compared to the pepper pot treated with the same amount of distilled water (no bacillus treatment) as a control. . The cultivation of the combination strain group in principle was to cultivate each strain alone and mixed after treatment. The soil used for the pepper pot was sterilized soil ( Capsicumannum L., Buchon-Seminis Korea Co.) and all in vivo pot experiments were used for 30 weeks pepper per treatment, repeated three times. In addition, standard error was calculated | required at P <= 0.05 level.

As a result of the experiment, all eight individual strains showed more than 50% of the control of pepper blight, and also exhibited auxin production ability (see FIGS. 1 and 2). In addition, in pepper pepper disease control experiments of the 11 combination candidate group showed a symptom in the pepper seedlings not treated with the combination on the 6th day after the combination candidate group, all combination treatment showed more than 50% control ability (see Fig. 3).

TABLE 3

Bacillus subtilis AH18 and Bacillus licheniformis K11 combination No. 11 plant growth and antagonism

function Main substance Bacillus Richenformis K11 Bacillus subtilus AH18 Plant growth promoting ability IBA (MW 203 kDa) IAA (MW 175 kDa)
IBA (MW 203 kDa)
IPA (MW 189 kDa) Antifungal plant disease inhibitory activity Iron trap
(2,3-dihydroxybenzoate,
MW 200 kDa) Iron trap
(2,3-dihydroxybenzoyl glycine, MW 207 kDa) Fungal Cell Degeneration
β-glucanase
(MW 54 kDa) Fungal Cell Degeneration
β-glucanase
(MW 55 kDa) Antibiotic
(Iturin, MW 1.5 kDa) -

* IAA: indole-3-acetic acid, IBA: indole-3-butyric acid, IPA: indole-3-propionic acid

In addition, in all these treatments, the growth promoting ability between the combinations was confirmed after 15 days, and in particular, as shown in FIG. The combination of 11 Bacillus subtilis AH18 and Bacillus Richenformis K11 showed the best plant growth promoting ability. Although the growth was not as good as the control group treated with water without the pepper bacterium at all, despite the induction of late blight by the inoculation of pepper bacterium, the growth was similar to that of the pepper seedlings treated only with water. The results show that, as shown in Table 3, the optimal combination of mechanisms between the two strains is an excellent combination having the pepper growth promoting ability as well as the pepper pest control ability.

Example 3 Verification of Control Ability in General Arable Land Soils

In vivo pot test was conducted to verify pepper pest control ability through indoor pot test of general cultivated soil in the optimal combination group with strong control ability against pepper blight and excellent pepper growth promoting ability. The general soil used for the experiment was pepper arable land in Gyeongsan, Gyeongsangbuk-do, and was mixed with crushed soil and soil. Control force verification experiment was carried out in the same manner as the pepper pestilence control experiment.

In general, even if successful in the experiment on sterilized soil, there are many microbial agents that do not easily succeed when applied to actual packaging due to the lack of verification of control in general soil. Particularly, even in indoor conditions, soils of general soils are required to be tested on indoor soils because various phytopathogens, including red pepper bacterium, exist before spontaneous disease. Therefore, no. Indoor pot verification experiments were performed on 11 soils. As a result, 7 days after the inoculation of the pathogen on the port filled with unsterilized arable soil, the control ability against pepper blight was also excellent as shown in FIG. 5. These results demonstrate that a large number of pathogenic or non-pathogenic bacteria and fungi, which are present on a large number of common soils, are not only superior to other microorganisms, but also excellent in control, and also well combinatorial in combination strains. It is. In addition, it is excellent in controlling pepper disease and growth promoting ability in sterilized soil, and also excellent in controlling soil in general soil. Therefore, it is applied to agricultural field as an eco-friendly microbial agent that can overcome the weakness that is inactivated in soil. The probability of success is also high.

Example 4 Examination of Mutual Non-Competitive Mutual Symbiosis of Antagonist Microorganisms in Combination

In order to investigate mutually uncompetitive mutual symbiosis among the antagonistic microorganisms composed of combinations, co-cultures were inoculated and inoculated simultaneously with individual cultures in which individual strains were cultured alone and combined. . Two antagonistic microorganisms Bacillus subtilis AH18 and Bacillus licheniformis K11 were selected in combination and incubated for 4 hours at 37 ^o C, respectively, and divided into three treatment groups of single culture, co-culture and single culture. And growth promotion experiments were conducted.

Treatment of the combination strains resulted in Bacillus subtilis AH18 and Bacillus licheniformis K11 for single culture at 10 ⁸ CFU / ml per pot, respectively, for coculture, Bacillus subtilis AH18 and Bacillus liche for co-culture. Niformis K11 was treated so that the total volume per port was 10 ⁸ CFU / ml each at 10 ⁴ CFU / ml.

No. is the best combination 11 Individual strains of Bacillus subtilis AH18 and Bacillus licheniformis K11 were inoculated separately and inoculated at the same time to examine mutual noncompetitive mutual symbiosis between the two antagonists in the combination; And three cultures, each of which were cultured alone, were mixed. The co-culture did not show synergistic effects in both control and growth-promoting ability when compared with the culture of each alone. However, the mixed treatment after single culture showed a large synergy effect, and it showed more than 50% synergy effect after the single culture or co-cultivation in all of the root, stem, and leaf growth promoting effects with control ability. (See Figure 6 and Table 4).

TABLE 4

Growth Promotion Activity of Red Pepper by Single Culture and Simultaneous Culture

Live weight (mg) Stem Growth (cm) leaf amount size Untreated
14 ± 1.5 6.3 ± 0.6 10 ± 1 2.5 × 1.9 Water treatment
47 ± 1.1 11.3 ± 0.8 13 ± 1 4.5 × 1.9 Bacillus subtilus AH18 treatment 38 ± 1.2 7.4 ± 0.8 11 ± 1 3.1 × 2.1 Bacillus Richenformis K11 treatment 34 ± 1.5 7.5 ± 0.8 12 ± 1 3.2 × 2.0 Simultaneous treatment of two strains of individual culture 82 ± 1.2 10.9 ± 0.6 15 ± 1 4.2 × 3.1 Treatment of Two Strain Cocultures 37 ± 1.1 7.6 ± 0.8 11 ± 1 2.8 × 1.9

Therefore, the optimal combination No. 11 shows that synergistic use of Bacillus subtilis AH18 and Bacillus licheniformis K11 alone and in combination can be effective in controlling pepper disease and promoting pepper growth.

1 is a graph showing the antifungal activity of selected antagonist myopic microbes,

Antifungal activity (%) = [(total pepper seedlings)-(number of infected pepper seedlings) × 100.

Figure 2 is a graph showing the auxin production capacity of the selected antagonist rhizosphere microorganisms, IAA is the absorption capacity calculated at 540 nm by the Salkowski test, the concentration of IAA means the highest IAA value for 72 hours .

3 is a graph showing the inhibitory ability of phytophthora cappci by various antagonist microbial combinations.

Figure 4 is a photograph showing the antifungal activity and plant growth promoting ability of various combinations for peppers infected by phytophthora cappci, N group treated only with phytophthora cappci, C group treated only with water , Nos. 1 to 11 are groups treated with a combination of phytophthora cappci and antagonist microorganisms.

FIG. 5 shows combination No. 1 for phytophthora cappucci infected cropland soil. FIG. 11 shows the antifungal activity of N, the group treated only with phytophthora cappci, the group treated with water only, and port 1 and port 2 combined with phytophthora cappci. 11 treated group.

Figure 6 is a photograph showing the phytophthora blight inhibitory ability and pepper growth promoting ability of single culture and co-culture pepper, N is the group treated only with phytophthora cappci, C is the group treated only with water, 1 is phytope Group treated with Tora capsici and Bacillus subtilis AH18 incubated alone, 2 treated with phytophthora capcici and treated with Bacillus licheniformis K11 incubated alone, 3 with phytophthora capcici and Groups treated with Bacillus subtilis AH18 alone cultured and Bacillus licheniformis K11 cultured alone, 4 were phytophthora cappci and cocultured groups.

Claims (6)

delete delete delete delete delete Bacillus subtilis AH18 and Bacillus licheniformis K11 after culturing each alone and mixed with red pepper, characterized in that the pepper growth promoting method through the control of pepper disease.

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