KR100974292B1 - Bacterial strains promoting plant growth isolated from larval gut of diamondback moth and method for promoting plant growth using the same - Google Patents

Abstract

The present invention is Chinese cabbage moth ( Plutella) xylostella ) bacterial strains that promote plant growth isolated from the larvae of the larvae, microorganisms and plant fertilizers for the growth of plants comprising the strain or its culture as an active ingredient, a method for producing the biofertilizer, the plant or the strain The present invention relates to a method of promoting plant growth by dipping or irrigation of seed of a plant.

Chinese cabbage moth, larval intestine, plant growth, microbial preparation, biofertilizer

Description

Bacterial strains promoting plant growth isolated from larval gut of diamondback moth and method for promoting plant growth using the same

The present invention relates to a bacterial strain that promotes plant growth isolated from the larvae of the Chinese cabbage moth and a method of promoting plant growth using the same. More specifically, the plant growth isolated from the larvae of the Plutella xylostella Promoting bacterial strains, microbial agent and plant fertilizer for promoting plant growth comprising the strain or its culture as an active ingredient, a method for producing the biofertilizer, the immersion or irrigation treatment of the strain in plants or plant seeds of the plant It is about how to promote growth.

Insects (insect rivers) are the main group of arthropods and the most diverse group of animals on earth. This enormous variety survives different ecological conditions and is due to its ability to metabolize several food sources. Insects also have numerous microorganisms in their intestines and are therefore used as a report on microbial diversity. In addition, the enormous adaptability of insects can be attributed to various roles by their microbial partners. For example, the presence of nitrogen-binding bacteria in the insect gut aids nitrogen uptake, and the production of indium 3-acetic acid (IAA) and iron siderophore by symbiotic insect gut bacteria has antagonistic activity against pathogenic bacteria and fungi. [Dillon RJ, Dillon VM (2004) Annu Rev Entomol 49: 71-92], the production of the extracellular hydrolase, chitanase, has shown the physical properties of the peritrophic membrane essential for nutrient diffusion. Helps to maintain.

In previous studies, different genera of bacteria related to insect intestines were closely related to bacteria from the rhizosphere, the phyllosphere, and the soil, which caused tritrophic interactions between herbivorous insects, crops and their microorganisms. Presence, where beneficial effects can include production of plant hormones, associated nitrogen fixation, enzyme activity, improved mineral uptake, and inhibition of pathogenic and harmful organisms. Therefore, the bacterium has been referred to as plant growth promoting bacteria (PGPB) (Cattelan et al., (1999) Soil Sci Soc Am J 63: 1670-1680).

Most reported PGPBs are rhizosphere, lobesphere, and soil isolation strains with the exception of some milk and cow dung. The microbial population (4 × 10 ⁵ to 2 × 10 ¹¹ / ml intestinal suspension) in the insect gut is larger than in the leaf zone (3 × 10 ⁵ / cm ² ), indicating that it is a suitable source for useful bacterial isolation. Many insect species are presumed to derive their microflora from the surrounding environment, for example from the plant surface of food crops. Perhaps the rate of nutrient cycling in the intestinal environment is much higher and the potential disturbances are even more pronounced than in soil and plants. Insects also have well-developed oral parts that are used to grind organic materials, making them more accessible to microbial colonization. Insect-intestinal bacterial strains are also more metabolically versatile than isolated strains from elsewhere. Therefore, the isolation and identification of microorganisms from these specialized dynamic environments represents a progress in the development of bioinoculants for increased crop production.

Recently, the inventors have reported on the isolation and characterization of bacterial strains from the Chinese cabbage moth (DBM), which is a Redodotera insect.

Korean Patent Registration No. 10-530885 discloses Pseudomonas florences B16 strain and a method for promoting growth and control of bacterial wilting disease, and Korean Patent Registration No. 10-755509 has a nitrogen fixing ability and crop growth promoting effect The new strain Azospirilum Brasilens CW301, a biological fertilizer using the same and a manufacturing method thereof are disclosed, Korean Patent Registration No. 10-686596 is a new strain KLP1 for promoting the growth of plants and a method for promoting plant growth using the same U.S. Patent No. 6,156,560 discloses the use of Pseudomonas corrugata as a biological control agent, but the plant growth of isolated from the larval intestine of Plutella xylostella of the present invention Different from bacterial strains.

The present invention has been made in accordance with the above requirements, and since the bacterial properties giving a beneficial effect on host insects may also have a good effect on crop growth, the present invention provides for plant growth of Chinese cabbage moth (DBM) intestinal strains. Promotional properties were intended to be tested. In addition, the ability of selected bacterial strains to promote plant growth under uninfected conditions was evaluated using canola and tomatoes as test plants.

In order to solve the above problems, the present invention is Chinese cabbage moth ( Plutella xylostella ) provides bacterial strains that promote plant growth isolated from the larval intestine.

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

The present invention also provides a bio-fertilizer for plant growth comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a method for producing a biofertilizer by culturing the strain.

In addition, the present invention provides a method for promoting plant growth by immersing or irrigating the strain into a plant or seed of the plant.

According to the present invention, the bacteria associated with the insect larvae gut of the present invention have plant growth promoting (PGP) properties and can positively affect plant growth, and thus insect gut bacteria as effective plant growth promoting bacteria. Can broaden the spectrum of beneficial bacteria useful for crop production. In addition, the novel strain of the present invention can be used to increase the yield and quality of crops by replacing the chemical fertilizer in eco-friendly organic farming where the use of fertilizer is limited.

In order to achieve the object of the present invention, the present invention provides a bacterial strain that promotes plant growth isolated from the larvae intestine of Plutella xylostella . The bacterial strains were Acinetobacter sp. PSGB03 strain (KACC 91364P), Acinetobacter sp. PSGB04 strain (KACC 91365P), Pseudomonas sp. PRGB06 strain (KACC 91362P), Serratia genus (Serratia sp.) PRGB11 strain (KACC 91363P), Acinetobacter genus (Acinetobacter sp.) PSGB05 strain, Acinetobacter genus (Acinetobacter sp.) PRGB15 strain, Acinetobacter genus (Acinetobacter sp.) PRGB16 strain , Serratia sp., Is a PSGB13 strain. These eight bacterial strains have been isolated from the larval intestine of Chinese cabbage moth and have the function of promoting plant growth. Preferably, Acinetobacter sp. PSGB03 strain (KACC 91364P), Acinetobacter sp. PSGB04 strain (KACC 91365P), Pseudomonas sp. PRGB06 strain (KACC 91362P), or Serratia sp.PRGB11 strain (KACC 91363P).

Eight bacterial strains isolated from the larvae of Plutella xylostella , the Chinese cabbage moth (DBM), were tested for their plant growth promoting (PGP) properties and their effect on early plant growth. All strains tested were positive for nitrogen fixation and indole 3-acetic acid (IAA) and salicylic acid (SA) production, but negative for hydrogen cyanide (HCN) and pectinase production. In addition, five strains showed significant levels of tricalcium phosphate (TCP) and zinc oxide (ZnO) solubilization, six strains could oxidize sulfur (S) in growth medium, and four strains were chitinase. And β-1,3 glucanase activity. Based on its IAA production, for six strains, including four 1-aminocyclopropane-1-carboxylate (ACC) deaminase-positive and two ACC deaminase-negative strains, uninfected PGP activity on early growth of its canola and tomato seeds under gnotobiotic conditions was tested.

Acinetobacter genus (Acinetobacter sp.) PSGB04, on the other hand was a significant increase in root length (41%), nurse vitality, and dried in vivo by weight (30%) of canola plants, Pseudomonas species (Pseudomonas sp.) PRGB06 are vitro Under conditions, Botrytis cinerea , Colletotrichum coccodes , C. gleospoiroides , Rhizoctonia solani , and Scleur The mycelial growth of Sclerotia sclerotiorum was inhibited. When the seeds were treated with PRGB06, a significant increase was observed in the growth parameters of the tomato plants compared to the control.

The present invention also provides a microbial agent for promoting plant growth and a biofertilizer comprising a bacterial strain or a culture medium thereof that promotes plant growth isolated from the larvae intestine of Plutella xylostella of the present invention.

The microbial preparations cabbage moth (Plutella xylostella) in the larvae of the bacterial strain to stimulate the plant growth isolated from intestinal Acinetobacter of (Acinetobacter sp.) PSGB03 strain (KACC 91364P), genus Acinetobacter (Acinetobacter sp.) PSGB04 strain (KACC 91365P), Pseudomonas sp. PRGB06 strain (KACC 91362P), or Serratia sp. PRGB11 strain (KACC 91363P) may be included 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 is a bacterial strain that promotes plant growth isolated from the larvae of Plutella xylostella ( Plutella xylostella ), Acinetobacter sp. PSGB03 strain (KACC 91364P), Acinetobacter sp. strain (KACC 91365P), Pseudomonas species (Pseudomonas sp.) PRGB06 strain (KACC 91362P), or Serratia genus (Serratia sp.) PRGB11 strain using (KACC 91363P) service How to use as a biological fertilizer to promote the growth of the crop do. Using the culture medium cultured the strain it can be used as it is irrigation in a liquid state, immersed or sprayed on the seed of the crop or coated on the seed.

The present invention also provides a method for producing a biofertilizer comprising culturing the four bacterial strains of the present invention. The method for culturing the bacterial 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.

The present invention also provides a method for promoting plant growth comprising immersing or irrigating each of the four bacterial strains of the present invention or a combination thereof into a plant or seed of the plant. The method of promoting the growth of the plant may be carried out by immersing or irrigating, i.e. spraying, a seed culture or a microorganism preparation using four strains of bacteria 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.

Plants capable of promoting growth by the method of the present invention may include most of the plants, particularly effective in canola, tomatoes and the like.

In a method according to one embodiment of the present invention, the strain of the present invention may promote plant growth through nitrogen fixation and the production of indole 3-acetic acid (IAA) and salicylic acid (SA).

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.

Bacterial and Fungal Strains

Hemolytic properties of bacterial strains isolated from the gut of DMB grown in cabbage under laboratory conditions were measured according to the Benson method [Benson, HJ (2002) Microbiological applications. Boston, Mass., McGraw Hill, pp 170-200. Five genus Acinetobacter genus (PSGB03, PSGB04, PSGB05, PRGB15, PRGB16), one Pseudomonas genus (PRGB06), and two genus Serratia (PRGB11, PSGB13) with nascent blood characteristics were selected for the present invention. All these bacterial strains were isolated from stage 4 larvae of DBM using nutrients, Luria Bertani (LB), and MacConkey agar medium (Difco Laboratories, Detroit, Michigan), and their physical, biochemical, and molecular characteristics Based on the identification, 16S rRNA sequences of each bacterium were registered in the National Center for Biotechnology Information (NCBI). Pure cultures of bacterial strains were maintained in 50% glycerol at −80 ° C. Phytopathogenic fungi listed in Table 2 were obtained from Korean Agricultural Culture Collection (KACC), Suwon, Korea, and maintained on a PDA (potato dextrose agar) plate.

Mineral solubilization

The mineral phosphate and zinc solubilizing ability of the bacteria was tested in Pikovskaya's medium supplemented with 0.5% TCP (Ca ₃ (PO ₄ ) ₂ (0.5 g per 100 mL medium containing 998.45 μg 'P' / mL) or 0.12% ZnO. Phosphate solubilization in liquid medium was quantified by Murphy and Riley's method [Murphy J, Riley JP (1962) Anal Chim Acta 27: 31-36], zinc solubilization efficiency (%) based on zone diameter / colony diameter × 100 [Nguyen et al., (1992) Plant Soil 143: 193-199], sulfur oxidation capacity was tested in mineral salt thiosulfate medium, sulfate concentration in the medium used was quantified by turbidity measurement at 340 nm. Massassoumi A, Cornfield AH (1963) Analyst 88: 321-322.

Nitrogen fixation, ACC  (One- Aminocyclopropane -One- Carboxylate ) Diaminase  Active and Plant Hormone Production ( IAA , SA ).

Acetylene reductive activity (ARA) of bacterial strains was performed using Hardy et al. [Hardy et al., (1968) Plant Physiol 43: 1185-1207], and the presence of ACC deaminase enzyme activity in bacteria Quantification by measuring the amount of α-ketobutyrate produced as a result of enzymatic cleavage, and quantification of indole-3-acetic acid (IAA) and salicylic acid (SA) were performed by Gordon and Weber [Gordon SA, Weber RP (1951) Plant Physiol, respectively. 26: 192-195 and Meyer and Abdallah [Meyer JM, Abdallah MA (1978) J Gen Microbiol 107: 319-328].

Iron siderophores, hydrolases, and antifungal metabolites (HCN, NH ₃ ), And in vitro antagonism.

Qualitative analysis of iron capture and chitinase production was performed in CAS (chrome azural S) agar and peptone agar medium supplemented with 1.5% colloidal chitin, respectively, as the carbon source, and β-1,3-glucanase activity was Nelson. Quantified according to Nelson, N (1944) J Biol Chem 153: 375-380, and pectinase production was tested in M9 medium. For a qualitative measurement of hydrocyanic acid (HCN) production, bacterial strains were streaked in King's B agar medium supplemented with 4.4 g glycine / L. Whatman no. 1 filter paper was cut into 8 cm long and 0.5 cm wide uniform strips, then saturated with alkaline picrate solution (0.5% picric acid + Na ₂ CO ₃ ; pH 13) and placed in the lid of a petri dish. The plate was then sealed with parafilm and incubated at 30 ° C. for 48 hours. Thereafter, the color change from yellow to reddish brown of sodium picrate present in the filter paper was considered as an indication of HCN production. The ammonia production capacity of the bacterial strains was tested in peptone broth, and the antifungal activity of the bacterial strains was tested for phytopathogenic fungi on PDA (potato dextrose agar) plates according to the dual culture technique. A suspension of 10 ⁶ conidia / mL was plated on a PDA with a 6 mm diameter hole in the center, and the hole was inoculated with 6 μl (10 ⁸ CFU / mL) of bacterial cells. The plates were then incubated at 30 ° C. until the fungal mycelium covered the agar surface of the control plate free of bacterial cells and examined for inhibition zones. If necessary, the protein content of the cells can be determined using standard Lowry methods [Lowry et al., (1951) J Biol Chem. 193: 265-275].

Gnotobiotic growth pouch analysis.

Four ACC deaminase-positive ( Acinetobacter sp. PSGB03, PSGB04, PSGB05 and PRGB15) and two ACC deaminase-negative ( Pseudomonas sp.PRGB06 and Serratia sp.PRGB11) strains using canola and tomato as test plants Were further selected to test plant growth promoting ability. Both plant species are sensitive to ethylene levels at an early stage of growth. Seed treatment and uninfected growth pouch analysis were performed according to Penrose and Glick [Penrose DM, Glick BR (2003) Physiol Plant 118: 10-15]. Primary root lengths of canola and tomato seeds were measured 5 and 10 days after sowing and naïve vitality and dry biomass (oven dried at 70 ° C. with constant weight) were recorded 15 days after sowing.

Statistical analysis.

Data from SAS institute Inc. Statistical analysis was performed by ANOVA using the general linear model developed by (Cary, NC, USA), version 9.1, and the mean was compared using the least significant difference (LSD) P ≥ 0.05.

Nucleotide sequence registration number.

NCBI GenBank accession numbers for the sequences of bacterial strains PSGB03, PSGB04, PSGB05, PRGB15, PRGB16, PRGB06, PRGB11, and PSGB13 are EF195353, EF195354, EF195355, EF195345, EF195346, EF195341, EF195344, and EF195352, respectively.

Example 1 Isolation of Bacterial Strains of the Invention

After measuring the hemolytic properties of bacterial strains isolated from the intestine of DMB grown in Chinese cabbage under laboratory conditions, five kinds of genus Acinetobacter (PSGB03, PSGB04, PSGB05, PRGB15, PRGB16) and one Pseudomonas genus ( PRGB06), and two Serratia genera (PRGB11, PSGB13) were selected for the present invention. All these bacterial strains were isolated from stage 4 larvae of DBM using nutrients, Luria Bertani (LB), and MacConkey agar medium (Difco Laboratories, Detroit, Michigan), and their physical, biochemical, and molecular characteristics Identified on the basis of identification.

Among them, two Acinetobacter genus (PSGB03, PSGB04), one Pseudomonas genus (PRGB06), and one Seratia genus (PRGB11), which are bacterial strains that have a remarkable effect on plant growth, were selected from the Korea Agricultural Microorganism Resource Center ( KACC) dated March 26, 2008 (accession number: KACC 91364P, KACC 91365P, KACC 91362P, KACC 91363P).

Example 2: Plant growth promoting properties of the bacterial strains of the present invention

The results for various plant growth promoting properties of the selected intestinal bacterial strains are shown in Table 1 below.

Table 1.Promoting Plant Growth of Bacterial Strains Isolated from Larva Intestine of Chinese Cabbage Moth (DBM)

All bacterial strains were shown to solubilize mineral phosphate (P) or zinc (Zn) in plate based analysis, as can be seen from the formation of clear halo around the colonies. In addition, in broth based assays, the maximum P solubilization was followed by the Acinetobacter spp. Strains (479-547 μg P / mL), followed by Pseudomonas (250 μg P / mL) and Serratia genus strains (120-215 μg P / mL). ) Was observed. Except for the two Acinetobacter spp strains (PRGB15 and PRGB16), all bacterial strains have been shown to accumulate sulfate in the culture medium.

The measurement of ARA in intestinal bacterial strains showed the ability to fix dinitrogen and was evidenced by the amount of ethylene (2-3 nmol / h / mg protein) generated as a result of acetylene reduction by the bacterial strain. All Acinetobacter spp. Strains (except PSGB13 and PRGB16) showed significant ACC deaminase activity as demonstrated by the production of α-ketobutyrate, but neither Pseudomonas or Seritia strains had ACC deaminase activity. Appeared.

All strains produced significant amounts of IAA and SA. All strains were also positive for iron trap and ammonia production, but negative for pectinase and HCN production. Acinetobacter spp strains PSGB03, PRGB15 and Serratia spp strains PRGB11, PSGB13 showed positive responses in the chitinase production test. Of the four strains positive for β-1,3-glucanase production, maximal activity was recorded for the chitinase negative Pseudomonas genus PRGB06 (8 μg glucose / min / mg protein).

Example 3: Evaluation of antagonistic activity against phytopathogenic fungi of bacterial strains of the present invention

Antagonistic activity of bacterial strains was evaluated in terms of zone of inhibition diameter as an indicator of the reduction in growth of various test phytopathogenic fungi (Table 2).

Table 2. Antagonistic activity of PRGB06 genus Pseudomonas in the Chinese cabbage moth gut bacteria against phytopathogenic fungi

Among the eight isolates, Pseudomonas genus PRGB06 was found to be the most effective biocontrol agent with inhibitory effect on five growth out of eight fungi tested. The maximum inhibition was C , the causative agent of anthrax and leaf blight in red pepper and rice, respectively. gloespoiroides (3.6 cm) and R. The growth of solani (3.0 cm) was observed. None of the isolated strains were C. acutatum , C. capsici , and F. Although it did not inhibit the growth of oxysporum , Seratia genus PRGB11 and all Acinetobacter spp. had inhibitory effects on Phytophthora capsici .

Example 4 Effect of Bacterial Strains of the Invention on Canola and Tomato Growth Under Uninfected Conditions

The effect of selected bacterial strains on the growth of canola and tomato under uninfected conditions is shown in Table 3 below.

Table 3. Effect of seed treatment of Chinese cabbage moth larvae intestinal bacteria strain on early growth of canola and tomato under uninfected condition

Acinetobacter spp. Strains with ACC deaminase activity significantly increased the canola root length to 35-41% compared to the control. Two isolated strains (PRGB06 and PRGB11) that do not produce ACC deaminase also increased tomato root length by 23%, similar to the increase in root length by PSGB05 of the genus Acinetobacter, which produces ACC deaminase. Do. However, the amount of ACC deaminase activity did not have a significant effect on canola and tomato root lengths. PSGB04 in each Acinetobacter And increased hair vitality and canola (30%) and tomato (26%) dry biomass due to seed inoculation of Pseudomonas genus PRGB06 reflected the effect of increased root length and stem length (data not shown).

Claims (9)

A microbial agent for promoting plant growth, comprising the Acinetobacter sp. PSGB03 strain (KACC 91364P) or its culture liquid isolated from the larvae of Plutella xylostella . A microbial agent for promoting plant growth comprising Acinetobacter sp. PSGB04 strain (KACC 91365P) or its culture solution isolated from the larvae of Plutella xylostella . A microbial agent for promoting plant growth, comprising Pseudomonas sp., PRGB06 strain (KACC 91362P) or its culture medium isolated from the larvae of Plutella xylostella . A microbial agent for promoting plant growth comprising Serratia sp., PRGB11 strain (KACC 91363P) isolated from the larvae of Plutella xylostella larvae as an active ingredient. delete Bio-fertilizer for plant growth comprising the strain according to any one of claims 1 to 4 or its culture solution as an active ingredient. delete A method for promoting the growth of a plant comprising the step of immersing or irrigating the strain according to any one of claims 1 to 4 in a plant or seed of the plant. The method of claim 8, wherein the strain promotes plant growth through nitrogen fixation and production of indole 3-acetic acid (IAA) and salicylic acid (SA).