Isolation and Characterization of Indole-3-acetic acid- and 1-aminocylopropane-1-carboxylyic Acid Deaminase-producing Bacteria Related to Environmental Stress

환경스트레스와 관련된 indole-3-acetic acid 및

1-aminocylopropane-1-carboxylyic acid deaminase 활성을 갖는 박테리아의 분리와 특성 연구

김희숙¹, 김지윤¹, 이송민¹, 박혜정¹, 이상현², 장정수¹*, 이문현¹

1

(

)

2신라대학교바이오산업학부제약공학전공

Received: April 1, 2019 / Revised: April 24, 2019 / Accepted: April 29, 2019

서 론

식물 생장 촉진 근권세균

(plant growth promoting rhizobacteria, PGPR)

^,

^·

^[1

^3].

^,

질소고정화를통해필요한영양분흡수를촉진하여식물생 장에도움을준다

^.

^,

^,

^,

^,

Arthrobacter

^, Bacillus

, Pseudomonas

, Streptomyces

되었다

^{[4, 5].}

태로가장많이존재하여생체이용률이현저히떨어지지만

^,

PGPR

Isolation and Characterization of Indole-3-acetic acid- and 1-aminocylopropane-1-carboxylyic Acid Deaminase-producing Bacteria Related to Environmental Stress

Hee Sook Kim

, Ji-Youn Kim

, Song Min Lee

, Hye-Jung Park

, Sang-Hyeon Lee

, Jeong Su Jang

*, and Mun Hyon Lee

1

Food Research Center, Angel Co., Ltd., Busan 46988, Republic of Korea

2

Major in Pharmaceutical Engineering, Division of Bioindustry, Silla University, Busan 46958, Republic of Korea

In this study, strains isolated from soil samples collected from Busan, Changwon, and Jeju Island were examined to verify their abilities of phosphate solubilization and nitrogen fixation, production of indole-3- acetic acid (IAA), siderophore, and 1-aminocylopropane-1-carboxylyic acid (ACC) deaminase in order to select strains that promote plant growth and play a role in biocontrol of pests or pathogens. According to the results of this study, most of the isolated strains were found to have ability of phosphate solubilization, nitrogen fixation, IAA production, siderophore production, and production of ACC deaminase. These iso- lated strains might help plant growth by directly improving absorption of nutrients essential for phosphate solubilization and nitrogen fixation. In addition, they can promote plant growth and control resistance to plant diseases through extracellular enzyme activity and antifungal activity. In addition, most of the selected strains were found to survive in various environmental conditions such as temperature, salinity, and pH. Therefore, Pseudomonas plecoglossicida ANG14, Pseudarthrobacter equi ANG28, Beijerinckia fluminensis ANG34, and Acinetobacter calcoaceticus ANG35 were finally selected through a comparative advantage analysis to suggest their potential as novel biological agents. Further studies are necessary in order to prove their efficacy as novel biological agents through formulation and optimization of effective microorganisms, their preservation period, and crop cultivation tests.

Keywords: 1-aminocyclopropane-1-carboxylic acid deaminase, indole acetic acid, phosphate solubilization, siderophores

*Corresponding author

Tel: +82-51-852-3636, Fax: +82-51-852-4477 E-mail: [email protected]

© 2019, The Korean Society for Microbiology and Biotechnology

산염형태로질소원을제공한다

^[6].

siderophore

^,

indole-3-acetic acid (IAA)

^.

siderophore

^·

^,

^Fe(OH)

^Fe

^,

^Fe

^.

^,

^,

알려져있다

^{[7, 8].}

에중요한역할을하며

^,

^0.05

^l/l

^,

^,

¹⁰⁰

^l/l

^.

.

^,

^,

^,

^,

^,

^.

^IAA

1-aminocyclopropane-1-carboxylic acid

(ACC) deaminase

다

^{. IAA}

IAA

^,

^IAA

^ACC

[9, 10].

^PGPR

ACC deaminase

^ACC

^ammonia

-ketobutyrate

^[11

^13].

따라서

^,

^,

^,

^,

^{, IAA}

, siderophore

ACC deaminase

^,

^.

재료 및 방법

토양 미생물의 분리

본연구에서사용한근권토양과뿌리는부산일대의신 라대학교인근야산

^,

^,

^,

^,

한

⁷

^.

²

^,

^{1 g}

^{9 ml}

. Serial dilution

6

Bennet agar (BA, 1% glucose, 0.2%

peptone, 0.1% beef extract, 0.1% yeast extract), glucose minimal salts agar (GMSA, 0.2% glucose, 1% yeast extract, 0.2% KH

PO

, 0.3% K

HPO

, 0.01% MgSO

·7H

O, 0.0002%

MnSO

), potato dextrose agar (PDA, Difco

), tryptic soy agar (TSA, Difco

) plate, actinomycetes isolation agar (AIA, 0.2% sodium caseinate, 0.01% L-asparagine, 0.4% C

H

O

Na, 0.05% K

HPO

, 0.01% MgSO

, 0.0001%

FeSO

, 1.5% agar), humic-vitamin agar (HV, 0.1%

humic acid, 0.05% KCl, 0.05% Na

HPO

, 0.05% MgSO

, 0.002% CaCO

, 0.002% FeSO

, 0.1% V

stock solution, 1.5% agar)

³⁰

²

^.

^{(Tween 80)}

(1.0% perchloric acid)

sonication

. Sonication

⁶

³⁰

²

^.

^colony

^.

건조

^,

^,

ACC deaminase

^Barnawal

^[11]

. Test tube

^TSB

³⁰

²

^.

^{DF salt}

^(0.4%

KH

PO

, 0.6% Na

HPO

, 0.02% MgSO

· 7H

O, 0.0001%

FeSO

, 0.000001% H

BO

, 0.00001% MnSO

· H

O, 0.000007% ZnSO

, 0.000005% CuSO

, 0.000001% MoO

, 0.2% glucose, 0.2% C

H

O

, 0.2% C

H

O

, pH 7.3)

^{3 mM ACC}

^{DF salt}

³⁰

²

^.

^,

600 nm

^ACC

증가를통해

ACC deaminase

^. Indole-3-acetic acid (IAA) 생성능 조사

식물성 호르몬인

^IAA

^0.1%

tryptophane

King’s B (2% proteose peptone, 0.25% K

HPO

, 0.6% MgSO

, 1.5% glycerol)

³⁰

²

^.

^(10,000 rpm, 15

⁾

, Salkowski’s reagent (35%

HClO

50 ml, 0.5 M FeCl

1 ml)

^{1:2 (v/v)}

혼합한다음

^,

³⁰

^.

(Spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland)

^{530 nm}

^.

^IAA

^,

^[14].

분리주의 다양한 환경조건에 대한 생육특성

분리주들에대한기후및토양환경에대한적용성과적 응능을알아보기위하여다양한온도

⁽¹⁰

⁵⁰

), pH (pH 4

12)

^{(NaCl 0}

^10%)

^.

^{600 nm}

^.

질소 고정화능 조사

질소 고정세균분리용

nitrogen free bromothymol blue (NFB, 0.5% (HO

CCH

CH(OH)CO

H, 0.05% K

HPO

, 0.001% MgSO

· 7H

O, 0.002% NaCl, 0.005% FeSO

· 7H

O, 0.0002% Na

MoO

, 0.001% MnSO

· 7H

O, 0.001%

CaCl

, 0.4% KOH, 0.2% bromothymol blue (in 0.5%

alcohol), 0.175% agar, pH 6.8)

^.

NFB

배지로서대기중의질소를고정할능력이있는분리주만이 생장할 수 있는 선택배지를 이용하였다

[15]. Test tube

NFB

^loop

³⁰

⁴

^.

인산 가용화능 조사

난용성인산염의분해능을조사하기위해

0.5% calcium

phosphate (10CaO·3P

O

·H

O)

Pikovskaya (PVK) agar

^[16].

^cork

borer

접종후

³⁰

⁴

clear zone

^. Siderophore 생성능 조사

식물병원균의길항기작중의하나인

siderophore

siderophore

chrome azurole S (CAS) blue agar plate assay

^{[17]. CAS}

³⁰

⁴

orange halo zone

^.

분리주들의식물병원성곰팡이에대한길항능을조사하기위

하여

Botrytis cinerea KACC 40574, Colletotrichum acutatum KACC 40801, Corynespora cassiicola KACC 44719, Fusarium sp. KACC 40241, Phytophthora capsici KACC 40180, Rhizoctonia solani AG-2-1 KACC 40124, Sclerotinia sclerotiorum KACC 40457

⁷

^.

^PDA

well diffusion

^{, PDA}

²⁵

^.

(inhibition rate)

^.

Inhibition rate (%) =

Where, R is the maximum radius of the fungal colony away from the bacterial colony; r is the radius of the fungal colony opposite the bacterial colony.

16S rRNA 유전자 염기서열 분석을 통한 분리주의 동정

분리주를동정하기위해서

^{16S rRNA}

시하였다

^.

^TSB

30

²

^.

^DNA

Wizard

Genomic DNA purification kit (Promega, USA)

. 16S rRNA

universal primer

27F (5’-AGA GTT TGA TCC TGG CTC AG-3’)

1492R (5’-GGT TAC CTT GTT ACG ACT T-3’)

^PCR

30 cycles (94.0

^-5

^, 55.0

^-30

^{, 72.0}

^-1

⁾

(TP600, Takara Bio Inc., Japan).

^PCR

PCR/Gel combo kit (Cat.

No. 3412, NucleoGen, Korea)

^.

^PCR

1% agarose gel

^.

Solgent Co. Ltd.

^,

^NCBI

^BLASTN

^.

^MEGA

(version 7.0, MEGA software)

^.

(SEM, scanning electron microscope)

^.

분리균주의 가수분해효소 생산

세포외 효소활성을 측정하기 위해서 각

^starch, carboxymethyl cellulose (CMC), skim milk, xylan

nutrient broth

³⁰

²

^{. Amylase}

Miller [18]

^,

^{1% starch} solution (50 mM sodium phosphate buffer)

^1:1

^.

color reagent solution

R r– ( ) --- 100R ×

합하여

¹⁰⁰

^{540 nm}

. Cellulase

xylanase

^Shin

^{Cho [19]}

^.

1% CMC solution (50 mM sodium phosphate buffer)

1% xylan solution (50 mM sodium phosphate buffer)

⁵⁰

10

^.

dinitrosalicylic acid

¹⁰⁰

¹⁰

^.

^{540 nm}

^{. Protease}

^Oh

^[20]

^,

0.65% casein (50 mM sodium phosphate buffer)

³⁷

¹⁰

^.

0.44 M trichloroacetic acid

³⁷

30

^.

^0.45

^m filter

³

^Folin- Ciocalteu’s phenol reagent

37

30

^.

^{660 nm}

^.

API ZYM kit (Biomérieux,

France)

진행하였다

^.

모든 분석은

³

^,

^±

(Mean

^SD)

^.

^SPSS (version 20.0 for Windows, SPSS Inc., Chicago, IL, USA)

(one-way ANOVA)

Duncan’s multiple range test

^.

p < 0.05

^.

결과 및 고찰

균주의 분리

유용미생물을선발하기위하여부산일대의신라대학교 인근야산

^,

^,

^,

^,

⁷

터순수분리하여

^colony

는

⁶⁶

^.

ACC deaminase

^{, IAA}

^,

^,

siderophore

^,

⁷

^.

ACC deaminase 및 IAA 생성능

에틸렌은식물의생장과발달에필수적인생장호르몬으로 서거의모든식물에서생성되지만

^,

^.

연구들에 의하면

^{, PGPR}

^ACC

deaminase

^ACC

^ammonia

^-

ketobutyrate

완화시켜주며

^,

^{[11, 12].}

^,

ACC deaminase

. ACC deaminase

^ACC

^,

ANG4, ANG14, ANG16, ANG25, ANG28, ANG34, ANG35

^ACC

^{DF salt}

(Table 1).

^Arshad

^[21]

ACC deaminase

Pseudomonas

ACC deaminase

의에틸렌의전구체인

^ACC

틸렌의합성을줄이며

^,

^,

^,

^[10].

^IAA

^,

Azospirllum, Pseudomonas, Rhizobium, Bacillus

^{[22], IAA}

ACC deaminase

^.

^IAA

^,

^IAA

^·

^IAA

농도는식물이생장함에있어중요한요인이될수있다

^[23].

본연구에서는환경적스트레스감소에영향을주는미생물

Table 1. Abilities of ACC deaminase production and survival concentration of NaCl and ranges of temperature and pH of isolated strains.

Strains

ACC deaminase production

Temperature

(

C) pH

NaCl concentration

(%)

ANG4 + 20-40 4-10 0-10

ANG14 + 20-50 5-10 0-10

ANG16 + 20-35 5-10 0-6

ANG25 + 20-35 5-9 0-3

ANG28 + 20-40 5-9 0-6

ANG34 + 20-40 5-9 0-4

ANG35 + 20-40 5-9 0-4

+: Positive reaction.

a

ACC deaminase production determined by growth on medium

with only ACC as nitrogen source.

결정인자인

^IAA

^{King’s B}

^{. ANG4}

^ANG25

^20.0

^g/ml

^IAA

^{, ANG16}

^ANG28

^40.8

^{g/ml, 42.8}

^g/ml

. ANG34, ANG35

^8.5

^g/

ml, 6.1

^g/ml

^IAA

^{. ANG14}

96.8

^g/ml

^IAA

^{. IAA}

^IAA

^ACC

^{(Fig. 1).}

다양한 환경조건하에 따른 생장조건 분석

식물은다양한생물적과비생물적스트레스요인에영향 을받으며

^,

^,

^,

^,

^,

[24].

ACC deaminase

^IAA

^.

^,

²⁰

^{, 40}

^ANG4, ANG14, ANG28, ANG34, ANG35

⁵

^{. 50}

^ANG14

^,

^ANG14

^.

^pH

^{pH 5.0}

^9.0

^{, ANG4}

^{pH 4.0}

10.0, ANG14

ANG16

^{pH 5.0}

^10.0

^.

^ANG25

⁰

^3%

육하며

^{, ANG34}

^ANG35

⁰

^4%

^.

^ANG16

^ANG28

⁰

^6%

^. ANG4

^ANG14

⁰

^10%

^{(Table 1).}

질소 고정능, 인산 가용화능

유용미생물의식물생장촉진활성을측정하기위해서질 소고정능과인산가용화능을확인하였다

^.

^,

^,

N

^,

^N

산염형태로질소원을제공한다

^[6].

^,

가지는분리주를분리하기위해서

^NFB

며

^{, NFB}

해양성판정을하였다

^.

^,

^NFB

^,

(Table 2).

0.05% (w/w)

^Ca

(PO

)

, Ca

(PO

)

(OH)

,

다

^[25].

비된유기산과무기산의작용으로가용화되며

^,

hydroxyl group

carboxyl group

Al, Fe, Ca

^pH

^[13].

^,

수분리한분리주를

^PVK

Table 2. Abilities of nitrogen fixation and phosphate solubili- zation.

Strains Nitrogen fixation

Phosphate solubilization

ANG4 ++ +

ANG14 ++ ++

ANG16 ++ ++

ANG25 + ++

ANG28 + +

ANG34 + ++

ANG35 + ++

++, +: Positive reaction.

a

Nitrogen fixation ability determined by color change of NFB broth.

b

Phosphate solubilization ability. ++ > 1.0 cm, + > 0.5 cm.

Fig. 1. Selection of IAA producing strain by 0.1% tryptophane King’s B test. Absorbance was measured with a spectropho- tometer at 530 nm. Data are mean ± SD of at least three replicates.

Means with the different letters (a-d) indicated significant

differences among samples at p < 0.05 by Duncan’s multiple

range test.

능을확인하였다

^.

clear zone

^{0.5 cm}

(Table 2).

^,

ANG14, ANG16, ANG25, ANG34, ANG35

^{1.0 cm}

^.

^,

^,

^(N

)

에도움을줄것으로기대된다

^[26].

Siderophore 생성능 조사

Siderophore

^,

^[27

^29].

^, Pseudomonas

siderophore

^PGPR

^Fe

^[30].

^,

siderophore

, siderophore

^Fig.

2

. ANG4, ANG14, ANG16, ANG25, ANG28, ANG34, ANG35

^{1 cm}

orange halo zone

^,

물제제에활용할수있을것으로판단된다

^.

분리주의식물병원성곰팡이에대한길항능은식물병원성 곰팡이

⁷

^,

^PDA

^.

Botrytis cinerea,

Colletotrichum acutatum,

Corynespora cassiicola,

Fusarium

^,

Phytophthora capsici,

Rhizoctonia solani,

Sclerotinia sclerotiorum

^.

(Table 3).

^{, ANG4}

^ANG14

Fusarium sp.

^{, ANG16}

^ANG25

^. ANG28

B. cinerea 16.7%, C. acutatum 19.0%, C. cassiicola 23.1%, R. solani 14.3%

^{. ANG34}

^ANG35

C. acutatum, C. cassiicola,

Fusarium sp.

는 것을 확인하였으며

^,

^ANG35

B. cinerea, P.

capsici, R. solani

^.

Fig. 2. Confirmation of orange halo zone on CAS medium by production of siderophore from selected strains. The strains were cultured on CAS agar medium at 30 ℃ for 4 days.

Table 3. Antifungal ability of isolated stains against phytopathogens.

Strains Inhibition rate (%)

ANG4 ANG14 ANG16 ANG25 ANG28 ANG34 ANG35

Botrytis cinerea - - - - 16.7 ± 3.4 - 33.3 ± 4.8

Colletotrichum acutatum - - - - 19.0 ± 4.8 33.3 ± 1.1 45.4 ± 3.9

Corynespora cassiicola - - - - 23.1 ± 0.6 34.8 ± 2.1 45.3 ± 2.4

Fusarium sp. 34.0 ± 1.2 38.1 ± 1.7 - - - 23.7 ± 0.7 28.8 ± 2.1

Phytophthora capsici - - - - - - 39.4 ± 3.8

Rhizoctonia solani - - - - 14.3 ± 0.6 - 28.6 ± 1.1

Sclerotinia sclerotiorum - - - - - - -

- : no activity. Data are mean ± SD of at least three replicates.

식물병원성곰팡이에대한길항능외에도

^ANG35

^Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes

^{(data not}

shown.).

^,

siderophore

가나타나는것으로사료된다

^.

선별된 다기능 분리주의 16S rRNA 동정

선별된분리주로부터

^{16S rRNA}

^{, NCBI}

^BLASTN

결과를

^{Table 4}

^.

^,

Pseudomonas plecoglossicida ANG14, Pseudarthrobacter equi ANG28, Beijerinckia fluminensis ANG34, Acinetobacter calcoaceticus ANG35

^,

여

^{16S rRNA}

(Fig. 3).

위해서

^SEM

^,

균형태를 나타내는

^ANG35

ANG14, ANG28,

Table 4. Identification of isolated strains by 16S rRNA gene analysis.

Strains Species Accession No. Similarity (%)

ANG4 Pantoea brenneri NR_116748 99%

ANG14 Pseudomonas plecoglossicida NR_114226 99%

ANG16 Enterobacter tabaci NR_146667 99%

ANG25 Chryseobacterium lactis NR_126256 98%

ANG28 Pseudarthrobacter equi NR_117032 99%

ANG34 Beijerinckia fluminensis NR_116306 99%

ANG35 Acinetobacter calcoaceticus NR_117619 99%

Fig. 3. Neighbor-joining phylogenetic tree of the isolated strains and other closely related based on the partial 16S rRNA gene

sequences. Bootstrap values (from 1,000 replicates) are indicated at the node. (A) Pseudomonas plecoglossicida ANG14, (B)

Pseudarthrobacter equi ANG28, (C) Beijerinckia fluminensis ANG34, (D) Acinetobacter calcoaceticus ANG35.