KR20040033225A - Psychrotolerant bacillus species isolated from soil - Google Patents

Abstract

PURPOSE: Psychrotolerant Bacillus species isolated from soil is provided. A microorganism-cultured medium has low freezing point and inhibits formation of ice core by an ice core-producing microorganism to protect plants from cold-weather damage. CONSTITUTION: Psychrotolerant Bacillus species(KCCM 10409) isolated from soil is provided, wherein the psychrotolerant Bacillus species(KCCM 10409) can grow at 0 to 40 deg. C and pH 5 to 12, and is Gram positive, positive catalase activity, negative oxidase activity, and lecithinase activity. It is isolated by the steps of: collecting a soil sample; diluting the soil sample with 0.85% sodium chloride solution; inoculating the soil sample on a nutrition agar medium; culturing the sample at 4 deg. C for 3 to 4 days; isolating and inoculating colonies grown on the agar plate; and culturing one colony in a nutrition medium at 4 deg. C. A composition for protecting plants from cold-weather damage contains the psychrotolerant Bacillus species(KCCM 10409) as an effective component.

Description

Psychrotolerant Bacillus species isolated from soil

The present invention relates to a novel Bacillus strain, and more particularly to a new cold-resistant Bacillus strain isolated from the soil.

Psychotololerant microorganisms are microorganisms that can grow at temperatures close to 0 ° C., and usually exhibit optimal growth at temperatures above 20 ° C. These cold-resistant microorganisms have generally been characterized after isolation in permanently low temperature environments below 5 ° C. (MK Mannisto, JA Puhakka, FEMS Microbiol. Ecol. , 2002 , 1, 1364-1371). Bacteria that produce heat-resistant endospores are classified into various genus of the Bacillaceae family, except for bacteria that form anaerobic and endogenous spores. The genus Bacillus is the largest and best known (FG Priest, M. Goodfellow and C. Todd., J. General Micorbiol. , 1988 , 134, 1847-1882). Cold-resistant microorganisms, particularly species of the genus Bacillus, have been studied in various fields, including the characteristics of the species and the structure, function and properties of the products produced from the species (Davail, S. et al. , J. Biol. Chem. , 1994 , 269). (26), 17448-17453; Narinx E. et al. , Protein Engineering , 1997 , 10, 1271-1279; Yoko Okubo et al ., Biochem.Biophysical Res. Communications , 1999 , 256, 333-340; Kumio Yokoigawa et al., J. Catalysis B: Enzymatic , 2001 , 12, 27-35), several refractory Bacillus species isolated from soil are currently reported. Among these are Bacillus insolitus (Larkin JM and Stokes, JL, J. Bacteriol , 1967 , 94, 889-895) and Bacillus macquariensis (Marshall BJ, and isolated from Antarctic soils ). Ohye DF, J. General Microbiol. , 1966 , 44, 41-46) and Bacillus marinus (Ruger HJ, Fritze D., and Sproer C., Int. J. Syst. Evol. Microbiol. , 2000 , 50, 1305-1313).

The inventors of the present invention were investigating the strains of cold-resistant Bacillus species isolated from soils in Ganghwa-do in early March, late winter, by separating novel microorganisms belonging to the novel Bacillus genotype whose genotyping and phenotypic data are different from the known strains. Was completed.

An object of the present invention is to provide a cold-resistant Bacillus strain isolated from the soil and an anti-frost agent of a crop containing the strain as an active ingredient.

1 is a micrograph showing the morphology of the novel cold resistant Bacillus strain of the present invention,

Figure 2 A is a microscopic photograph of the new cold-resistant Bacillus strain of the present invention showing a cold resistance 100 times, the frozen ice distilled control and observed the surface of the ice crystals (a), after frozen control distilled water Observing the growth end of the ice crystals (b), after freezing the culture filtrate of Bacillus B4 of the present invention observed the surface of the ice crystals (c),

FIG . 2B is a graph illustrating the freezing point of the culture filtrate of the control group and Bacillus B4 through a differential irradiation physical calorimetry analysis,

Figure 3 is a graph of the ice nucleus activity of the control and treated with the culture filtrate of Bacillus B4 ice nucleus capacity was measured by the number of ice nucleus floating graph.

In order to achieve the above object, the present invention provides a cold-resistant Bacillus strain isolated from the soil.

In addition, the present invention provides an anti-frost agent of a crop containing the cold-resistant Bacillus strain as an active ingredient.

The novel cold-resistant strain isolated from the soil of the present invention has a rod shape with a width of 0.9-1.0 μm and a length of 2.5-4.0 μm, and has egg-shaped spores at the terminal part (see FIG. 1 ). ).

The strain isolated in the present invention is Gram-positive, can grow at a temperature of 0 to 40 ℃, and does not grow in the temperature and 45 ℃ or more anaerobic environment. Growth pH was pH 5 to pH 12, catalase activity was positive, oxidase activity was negative, and activity of lecithinase was detected. Arginine dihydrolase and indole production are negative. Acids such as citrate and propionate can be used, and acids can be produced using sugars such as D-glucose, D-mannitol and D-fructose. However, it does not produce acid from L-arabinose or D-xylose. In addition, the microorganism of the present invention can hydrolyze gelatin, casein and Tween 80, but has no hydrolytic ability to starch. It also does not reduce nitrate to nitrite (see Table 1 ).

Major fatty acids constituting the strain isolated in the present invention include C _{15: 0} anteiso and C _{15: 0} iso , C _{11: 0} , C _{14: 0} iso , C _{14: 0} , C _{15: 0} , Fatty acids such as C _{16: 1} w7c alcohol, C _{16: 0} iso , C _{16: 1} w11c, C _{16: 0} , iso C _{17: 1} w10c, C _{17: 0} iso , C _{17: 0} anteiso exist. In addition, the most abundant major fatty acid is C _{15: 0} anteiso , which is 34.49% (see Table 2 ).

After analyzing the 16s rDNA gene sequence of the novel strain of the present invention, a sequence similar to the gene sequence was searched by using a sequence database of existing strains registered in GENBANK. It was confirmed that the nucleotide sequence is very closely related to the nucleotide sequence of the gene present in the species of the genus Bacillus. Among them, the sequence similarity with the gene of the Bacillus simplex was the highest (about 99%) and low correlation with other species (see Table 3 ). In addition, it was confirmed that the G + C content of the gene DNA is 40 mol%.

As a result of analyzing the entire sequencing of 16s rDNA, it showed 99% similarity to Bacillus simplex, and the similarity between species was examined by DNA-DNA hybridization experiment. 35% similarity with Rex. Thus, considering 70% threshold value in species identification according to Wayne et al . (Wayne LG et al ., Int. J. Syst. Bacteriol. , 1987 , 37, 463-464), It can be seen that the isolated strain of the present invention is not associated with Bacillus simplex in the range of species. From the above results, it was confirmed that the strain of the present invention isolated from the soil is a novel species belonging to the genus Bacillus, and designated as B4 as the type 'Bacillus B4' and the strain was named Korea microorganisms on July 25, 2002. Deposited to a conservation center (Accession No. KCCM 10409).

The Bacillus strain of the present invention is a cold resistant strain having a low freezing point. In a specific embodiment of the present invention, the culture of Bacillus B4, that is, the product has a function of inhibiting the growth of ice crystals (see A of FIG. 2 ), and the control water begins to freeze at -15.96 ° C. while the product of Bacillus B4 The treated sample was found to start freezing at -18.37 ° C (see B and Table 4 in Figure 2 ).

Therefore, the Bacillus strain of the present invention is a cold resistant strain with a freezing point of -18.37 ℃.

The present invention provides an anti-chilling agent for crops containing the cold-resistant Bacillus strain as an active ingredient.

The culture filtrate of the cold-tolerant Bacillus strain of the present invention has a low freezing point and can inhibit the ice-nucleus activity of the ice-nucleus-activated bacterium, which has a freezing activity and has a major influence on the freezing of crops. Therefore, the culture filtrate of the cold-resistant Bacillus strain of the present invention can be effectively used to prevent the freezing of the crop by treating the crop.

Thus, it is possible to produce a crop anti-corrosive agent containing the cold-resistant Bacillus strain of the present invention as an active ingredient.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Cold-Resistant Strains

We suspended the soil samples collected at Ganghwa Island in 0.85% sodium chloride solution and then inoculated them in nutrient agar plates. After the soil samples were incubated at 4 ° C. for 3 to 4 days, colonies formed from bacteria were plated on agar plates and separated into strains. After repeating the above process, single colonies were isolated and cultured, and then grown in a nutrient medium at 4 ° C. The isolated strains were then bred to enhance vitality through subculture, and then inoculated in a general bacterial culture solution medium and cultured at 4 ° C. While confirming the proliferative properties of the cold-resistant strains. To maintain the isolated microorganisms for a long time, cell suspensions were stored at −40 ° C. on broth culture containing 20% (v / v) glycerol.

Example 2 Characterization of Isolated Strains

<2-1> Morphology analysis and Gram staining

The present inventors incubated the strain in a nutrient agar plate medium for 30 hours, 24 hours to analyze the form of the isolated strains and then observed the form and colony form of the bacteria through a microscope, Sigma Gram staining experiments were performed using a Gram stain kit (Sigma Diagnostics, kit HT 90-A) prepared by.

As a result, this strain was a Gram-positive strain, and the cell shape was rod-shaped with a width of 0.9-1.0 μm and a length of 2.5-4.0 μm, and had sporulation ability ( FIG. 1 ).

The colony was round, opaque white, colony viscous, convex, elevated, and colony rounded.

<2-2> Physiological Properties of Strains

The present inventors observed the growth of the bacteria by culturing the bacteria in a nutrient medium under a temperature condition of 0 to 40 ℃ in order to find out whether the growth by temperature of the isolated strain.

100 mM citrate / 100 mM sodium citrate buffer at pH 3 to 5, 100 mM sodium phosphate buffer at pH 6.0 to 8.0 (Na ₂ HPO ₄ / NaH) ₂ PO ₄ buffer), 100 mM sodium carbonate buffer (NaHCO ₃ / Na ₂ CO ₃ buffer) at pH 9.0 to 10.0, 50 mM sodium phosphate (Na ₂ HPO ₄ ) / 100 mM sodium hydroxide (NaOH) buffer at pH 11 and At pH 12-13, growth of bacteria was observed in nutrient medium containing 100 mM potassium chloride / sodium hydroxide buffer (KCl / NaOH buffer).

In order to determine the growth of bacteria under anaerobic conditions, inoculation was carried out in the test tube of anaerobic agar to the bottom of the test tube and inoculated, and the growth of the bacteria was observed. Growth was observed in nutrient medium treated with sodium chloride.

To determine the catalase activity was determined by observing the foam production from 3% (v / v) hydrogen peroxide, the oxidase activity was 1% (w / v) tetramethyl-p-phenylenediamine Resitinase activity was determined by incubating the bacteria in yolk agar medium (0.5% glucose, 1% sodium chloride, 1.5% agar, 5% egg yolk emulsion, nutrient medium) and decomposing the lecithin of egg yolk It was judged by observing the degree of turbidity around.

Arginie dihydrolase activity was tested by culturing in Bacto Decarboxalase Base Moeller (Difco) containing 1% arginine. Indole production ability was inoculated with SIM medium (sulfide, Indole, motility) (Difco Inc.) and incubated after incubation was added to observe the indole production capacity (Kovac's reagent).

Nitrate reduction was used as the nitrate (Nitrate) medium, the nitrate or nitrite in the absence of the nitrate or nitrite was observed by reducing the gas was observed to determine whether or not the nitrate reduction ability. The availability of sugar was observed by adding sugar to the nutrient medium in 10 mM concentration by disinfection (0.2 μm microfilter). The availability of citrate and propionate was determined by preparing the citrate and propionate using a slanted medium, inoculating the bacteria, and incubating for 14 days to observe the production of pigments.

The Vogues-Proskauer test was conducted on VP broth (Dipotassium hydrogen phosphate (0.5%, D (+)-glucose 0.5%, peptone 0.7%, sodium chloride 0.7%, Sigma). After inoculation, the culture was determined by mixing 40% sodium hydroxide and adding 0.5 to 1 mg of creatine to observe the formation of pigment at room temperature.

Starch resolution was prepared by adding 0.5% soluble starch to nutrient agar medium to prepare starch agar plate medium, inoculating bacteria, incubating at 30 ° C for 3 days, and then pouring 95% ethanol into plate medium and flowing it through 15 to After 30 minutes, the state of starch was observed to determine whether a clear zone was formed around the growth of the bacteria. The degradation of casein was prepared by adding 10% skim milk to nutrient agar to prepare skim milk agar medium, and then inoculating the bacteria and incubating for 7 days, and then observed the state of casein around the growth of the bacteria. Degradation of gelatin was observed after inoculating the bacteria in a nutrient gelatin (Difco) medium and incubated at 28 ℃. Tween 80 resolution was determined using 3.5 ml of Tween 80 supplemented with saturated blue Nile blue sulfate solution, tryptic soy agar, TSB agar, Difco; pancreatic digest of casein 1.7%, enzymatic digest of soybean meal 0.3%, glucose 0.25%, sodium chloride 0.5%, dipotassium phosphate 0.25%, agar 1.5%) and mixed with 70 ml to prepare a medium, inoculated with the bacteria and incubated the bacteria at 30 ℃ after the growth of the blue light around (blue halo) was observed to determine whether it was positive.

As a result, the growth possible temperature range is 0 to 40 ℃, growth pH range is pH 5 to 12, growth under aerobic conditions, catalase activity is positive, oxidase activity is negative, Lecithinase (Lecithinase) Activity is positive, arginie dihydrolase activity and indole generating ability are negative, salt resistance was able to grow in medium containing 2% to 8% sodium chloride, glucose, mannitol and fructose in sugar use , Arabinose and xylitol were not used, and propionic acid and citric acid were used in using the acid. In addition, casein, gelatin and tween 80 could be hydrolyzed, but starch did not hydrolyze, had no nitrate reducing ability, and showed Boes-Proscaurer test negative ( Table 1 ).

Physiological Properties of Bacillus Strains Stick shape + Sporulation + Growth temperature 4 ℃ 10 ℃ 30 ℃ 40 ℃ 45 ℃ ++++- pH range pH 5pH 6pH 7pH 8pH 9pH 10 ++++++ Growth in NaCl 2% 5% 8% 10% +++- Oxidase - Catalase + Anaerobic growth - Nitrate reducing ability - VP reaction - PH in VP broth 6.8 Preparation of Acid D-Glucose L-Arabinose D-Xylose D-Mannitol D-Fructose +-++ Gas production from glucose - Recitase + Arginine Dihydrolase Activity - Indole resolution - Hydrolyzed Starch Gelatin Casein Twin 80 -+++ Use of citric acid Use of propionic acid ++

<2-3> Fatty Acid Pattern Analysis of Strains

In order to analyze the fatty acid pattern of the strain, the present inventors used a pancreatic digest of casein 1.7%, enzymatic digest of soybean meal 0.3%, glucose 0.25%, sodium chloride 0.5%, dipotassium phosphate 0.25 for 24 hours at 37 ° C. %, Agar 1.5%: strains were cultured on Difco. After dissolving the bacteria in the cell membrane, extracting the fatty acid and methylation, the fatty acid composition was analyzed by GC.

As a result, the major fatty acids constituting the strain of the present invention contained C _{15: 0} anteiso and C _{15: 0} iso, and C _{11: 0} , C _{14: 0} iso , C _{14: 0} , C _{15: 0} , Fatty acids such as C _{16: 1} w7c alcohol, C _{16: 0} iso , C _{16: 1} w11c, C _{16: 0} , iso C _{17: 1} w10c, C _{17: 0} iso , C _{17: 0} anteiso Present ( Table 2 ).

Profile of intracellular FAMA (fatty acid methyl ester) fatty acid content(%) Saturated fatty acid C _{14: 0} 1.92 C _{15: 0} 1.76 C _{16: 0} 4.75 C _{17: 0} 1.5 C _{18: 0} 0.46 Unsaturated fatty acid C _{14: 1} w5c 0.18 C _{16: 1} w7c alcohol 4.39 C _{16: 1} w11c 1.58 Fatty acid C _{13: 0} iso 0.13 C _{13: 0} anteiso 0.21 C _{14: 0} iso 12.9 C _{15: 0} iso 25.41 C _{15: 0} anteiso 34.49 C _{15: 1} iso 0.39 C _{16: 0} iso 7.07 C _{17: 0} iso 0.97 C _{17: 0} anteiso 0.84 iso C _{17: 1} w10c 0.66 C _{18: 1} w9c 1.51

<2-4> Biochemical Analysis of Strains

The present inventors extracted and purified genomic DNA with a DNA extraction kit (QIAamp DNA mini kit, QIAGEN) to analyze the gene sequence of the strain isolated in the present invention, and then the primers described in SEQ ID NO: 1 and SEQ ID NO: 2 Was used to amplify the 16S rDNA gene. The amplified PCR product was directly ligation using pGEM-T Easy Vector System II (Promega) and transformed with DH5α Escherichia coli. Transformants purified plasmid DNA was screened by any of the conversion clones, plasmid DNA purification kit ^(QIAprep? Spin spin mini kit, QIAGEN Inc.), and the sequence analyzed by the DNA sequence analyzer.

As a result, it was confirmed that the strain of the present invention had a nearly complete gene sequence of 1,500 or more nucleotides as set forth in SEQ ID NO: 3 , using the sequence database for the existing strains registered in the Geneva Bank (GENBANK). As a result of searching for a nucleotide sequence similar to the gene sequencing, it was confirmed that the nucleotide sequence is closely related to the nucleotide sequence of the gene present in the species of the genus Bacillus. Among them, the sequence similarity with the gene of Bacillus simplex was about 99%, and showed the low correlation with other species ( Table 3 ).

Sequence similarity with known Bacillus strains based on 16s DNA sequence Type of strain Similarity (%) Bacillus simplex 1515/1523 (99%) Bacillus macroids 1462/1473 (99%) Bacillus psychrosaccharolyticus 1418/1450 (97%) Bacillus maroccanus 1393/1430 (97%) Bacillus niacini 1468/1528 (96%) Bacillus flexus 1462/1529 (95%) Bacillus benzoevovans 1455/1526 (95%) Bacillus fumarioli 1437/1505 (95%) Bacillus megaterium 1426/1498 (95%)

In addition, after the DNA isolated above was digested with nucleosides, the G + C content of the gene was analyzed using Reverse Phase HPLC, and the G + C content of the DNA was 40 mol%.

<2-5> Species Similarity Survey

The inventors of the present invention analyzed the entire sequencing of the 16s rDNA. As a result, it was confirmed that the Bacillus simplex showed 99% similarity, and thus the similarity between species was examined by DNA-DNA hybridization experiment. Specifically, DNA was isolated by chromatography using hydroxyapatite according to the method of Cation et al . (Cashion P. et al ., Anal. Biochem. , 1977 , 81, 461-466). Huss et al. (Huss VAR et al. , J. Syst. Appl. Microbiol. ) According to the method of De Ley J. et al ., Eur, J. Biochem. , 1970 , 12, 133-142 . , 1983 , 4, 184-192) and modified methods such as Escara et al . (Escara JF et al ., Biopolymers , 1980 , 19, 1315-1327) applying model 2527-R thermoprogrammers and plotters DNA-DNA hybridization was performed using a spectrophotometer (Model 2600, Gilford Instrument Laboratories Inc., Oberlin, Ohio, USA) equipped with. The denatured rate was calculated by the TRANSFER.BAS program by Jahnke.

As a result, DNA / DNA hybridization showed 35% similarity to Bacillus simplex, unlike the sequencing results. Thus, considering 70% threshold value in species identification according to Wayne et al . (Wayne LG et al ., Int. J. Syst. Bacteriol. , 1987 , 37, 463-464), It can be seen that the isolated strains of the present invention are not associated with Bacillus simplex in the range of species. In addition, since the strains of the present invention have different physiological properties from the remaining strains except for the Bacillus simplex of Table 3 ( Table 1 ), the strains of the present invention were classified as novel species belonging to the genus Bacillus, Designated as B4, the strain was named Bacillus B4 and the strain was deposited with the Korea Microorganism Conservation Center on July 25, 2002 (Accession Number: KCCM 10409).

Example 3 Antifreeze of Bacillus B4

<3-1> Freezing Point Survey of Bacillus B4

The present inventors investigated the freezing point of Bacillus B4 to determine whether the Bacillus B4 has the effect of inhibiting the ability to form ice nuclei, that is, floating activity. Specifically, Bacillus B4 was incubated at 4 ° C. for 3 days, and the culture solution was centrifuged at 6000 rpm for 20 minutes, and the culture supernatant was ultrafiltered using Centriprep YM 10 membrane (Millipore), and then frozen at -20 ° C. for 18 hours. The ice crystals were observed under a microscope, and the freezing point was measured using differential scanning calorimeter (Perkin Elmer).

As a result, the culture filtrate of Bacillus B4 possessed the function of inhibiting the growth of ice crystals ( FIG . 2A ), while the control water began to freeze at -15.96 ° C., while the sample treated with the culture filtrate of Bacillus B4 Started to freeze at −18.37 ° C. ( B and Table 4 in FIG. 2 ).

Freezing Point Investigation of Bacillus B4 Production by Differential Investigation Temperature Points in the Chemical Fever Curve Control (water) Bacillus B4 Tf1 (start point) -15.96 -18.37 Tf2 (highest point) -17.22 -19.19 Tf3 (Extinction Point) -21.75 -21.84

<3-2> Antifreeze Activity of Bacillus B4

The present inventors found that Bacillus B4 is a genus of Pseudomonas that causes thePsudomonas),Erwinia), Genus XanthomonasXanthomonasThe purpose of this study was to determine whether it has the function of inhibiting ice nucleus activity, which is characteristic of ice nucleus active bacteria present in the leaves of plants and causing freezing at relatively high temperatures. Specifically, Escherichia coli DH5α strains produced by ice nucleus active bacteria and possessing ice nucleus active protein freezing at high temperature were precultured at 30 ° C. for 18 hours and main culture at 23 ° C. for 6 hours. . Centrifuging the culture solution at 15000 g, only the precipitated cells were taken, washed with tertiary distilled water, and centrifuged again three times.⁷To 10⁸It was adjusted to dogs and suspended in tertiary distilled water. Bacillus B4 strain with antifreeze in the suspension was incubated at 4 ° C. for 3 days, and the culture supernatant was centrifuged at 6000 rpm for 20 minutes. The degree of ice nucleation activity by bacteria was analyzed by the drop freezing method. Samples diluted in steps were dropped into 20 blocks of 5 μl of drops, and then the number of drops in each subcooled state was lowered from 0 ° C. to -15 ° C. at 0.2 ° C. per minute. Was calculated.

As a result, it was confirmed that the sample treated with the culture filtrate of Bacillus B4 inhibited the ice nuclei activity by the ice nucleus active bacteria ( FIG. 3 ).

As described above, the culture filtrate of the novel cold-resistant Bacillus strain isolated from the soil of the present invention has a low freezing point, and the strain of the present invention shows the effect of inhibiting the ability of ice nucleus active bacteria to form ice cores. It can be usefully used as an antifreeze agent.

Claims (2)

Cold-resistant Bacillus strains isolated from soil (Accession No .: KCCM 10409). An anti-icing agent containing the strain of claim 1 as an active ingredient.