PCR-DGGE Analysis of the Fungal Community of Red-pepper Fields Utilizing Eco-friendly Farming Methods

PCR-DGGE를 이용한 친환경 농법 적용 고추경작지 내 진균의 군집 다양성 분석

정병권, 김광섭, 송진하, 김상달*

영남대학교미생물생명공학과

Received : March 6, 2013 / Revised : June 18, 2013 / Accepted : June 19, 2013

서 론

토양은복잡하고역동적이며미생물들에의한생물학적활 성이지배적인환경이다

^.

^,

^{[15, 24].}

(mycorrhizae)

^,

기도한다

^{[16, 22].}

진균의군집에대한연구가필수적이다

^.

과거에는희석도말법과같이배양방법을통한

^colony

^.

(Basidiomycetes)

(Endomycorrhizae)

^,

^[3, 25].

때문에 최근에는

fingerprinting

denaturing gradient gel electrophoresis (DGGE)

는데

^{, DGGE}

해서 가장 먼저 사용되었으며

[1], Muyzer

^[18]

PCR-DGGE Analysis of the Fungal Community of Red-pepper Fields Utilizing Eco-friendly Farming Methods. Jung, Byung-Kwon, Gwang-Seop Kim, Jin-Ha Song, and Sang-Dal Kim*. Department of Applied Microbiology and Biotechnol- ogy, School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Korea

In this study, we analyzed the changes in fungal populations of red-pepper fields employing eco-friendly farming methods, such as microbial agents and crop rotation, by using polymerase chain reactions coupled with denaturing gradient gel electrophore- sis (PCR-DGGE). Primer specific for fungi were used to determine the contribution of domains to the microbial community.

Analysis of planted and non-planted soil samples applying PCR-DGGE technology offered evaluation of long-term patterns in fungal species richness. To evaluate the stability of DGGE patterns from different soils, comparison of planted and non-planted soil samples were compared using PCR-DGGE. The number of DNA fragments obtained from all planted soil samples by DGGE separation was far greater (14 to 15 bands) than that of the non-planted soil samples (3 to 4 bands). In addition, 14 bands were observed from crop continuation soil treated with agrochemicals and 18 bands from crop rotation soil treated with microbial agents. The PCR-DGGE analysis suggests that the use of crop rotation and microbial agents benefits the fungal com- munity more than crop continuation using agrochemicals. These results indicate that crop rotation with microbial agents was better able to support beneficial organisms, enable more effective biological control and maintain a healthier balance of nutri- ents, organic matter and microorganisms.

Keywords: Microbial agent, crop rotation, DGGE, fungal diversity

*Corresponding author

Tel: +82-53-810-3053, Fax: +82-53-810-4663 E-mail: [email protected]

© 2013, The Korean Society for Microbiology and Biotechnology

PCR-DGGE

^[18].

agarose gel

DGGE

polyacrylamide gel

urea, formamide

(denaturants)

polyacrylamide

^DNA

^guanine

^cytosine

정도가달라지며

^{, gel}

으로

^PCR

^DNA

[2, 6, 14].

^,

[17, 20, 21].

한편현대농업은경작면적에비해상대적으로많은노동 력과자본을이용하는경작방식인집약농업이대부분이루 어지고있으며

,

^,

^,

^{[5, 9].}

따라서본연구에서는고추의경작지를대상으로하여식물 생장촉진근권세균

(Plant growth promoting rhizobacteria, PGPR)

auxin, siderophore

bbb-glucanase

Bacillus licheniformis K11 [28], auxin, siderophore, celluase

Bacillus subtilis AH18 [8],

2,4-diacetylphloroglucinol (2,4-DAPG)

1-aminocyclopropane- 1-carboxylic acid deaminase (ACC deaminase)

Pseudomonas fluorescens 2112 [11-13]

^,

^DNA

물학적기법인

^PCR-DGGE

변화를비교하고자하였다

^.

재료 및 방법

시험포장지의 설치 및 컨소시엄 미생물제제의 처리 토양미생물의군집분석을위한시험포장지는경상북도경 산시대동영남대학교자연자원대학소재의실습포장지

⁽²³

33 m)

^,

(Capsicum annum L.)

⁶⁰⁰

²⁵

⁴

^. 2

(Phaseolus vulgaris var. humilis Alef.)

²⁵

⁴

^.

또한동일한양의컨소시엄미생물제제

(2 Bacillus sp., and 1 Pseudomonas sp., BBP),

(agrochemical, Ac)

(water, Wt)

^,

²

¹

^{100 ml}

^.

¹⁰

^CFU/ml

^,

^(A

^L

⁾

^.

(non-planting, NP)

⁶

⁶

^, 2 mm

^{(10 mesh)}

^.

고추 경작지 토양으로부터 total DNA의 추출

컨소시엄미생물제제와화학농약을처리한연작및윤작 지에존재하는진균의군집다양성을분석하기위해고추를 심기전

(N-P, non-planting)

⁶

^{total DNA}

. Total DNA

^,

^,

^DNA

^FastDNA

SPIN Kit for Soil (MP biomedicals, USA)

^.

^DNA

1.5% agarose gel

^loading

(100V, 30 min)

, UV-vis spectrophotometer (ND-1000, NanoDrop Technologies Thermo scientific, USA)

^.

^DNA

^PCR

²⁰

^C

^.

Fungal DNA 증폭을 위한 PCR 전략 및 조건

컨소시엄미생물제제와화학농약을처리한연작및윤작지 토양으로부터추출한

^{total DNA}

^DNA

primer set

^{test PCR}

. Test PCR

^Oomycetes

Phytophthora capsici

Sordariomycetes

Fusarium oxysporum

genomic DNA

^,

^Bacillus

subtilis AH18, Pseudomonas fluorescens 2112

^genomic

DNA

. Primer set (forward/reverse)

ITS1/ITS4 [23], FF390/FR1 [4], nu-SSU-0817/nu-SSU-

1196 [7]

^,

^DNA

primer set

^DGGE

melting temperature

^DNA

^reverse

primer

⁵

^{GC clamp}

(Table 1).

^DNA

^PCR

nested-PCR

^.

second-PCR

target gene

, nested-PCR

의과정은먼저선발된

primer set

¹

^PCR

^{GC clamp}

primer set

2

^PCR

^{. PCR}

pre-denaturation (94

C, 5 min)

denaturation (94

C, 1 min), annealing (50

C, 1 min 30 s), extension (72

C, 3 min)

35 cycles

final extention (72

C, 10 min)

⁴

^C

^.

^PCR

1.5% agarose gel

^loading

^{(100 V,} 30 min)

^.

PCR 증폭산물의 DGGE 분석

컨소시엄미생물제제와화학농약을처리한연작및윤작 지토양에 서식하는진균의 군집다양성을 분석하기위해

BioRad Dcode System (Bio-Rad Laboratories, Hercules, USA)

Denaturing gradient gel electrophoresis (DGGE)

^{. DGGE}

^gel

denaturing gel

9% polyacrylamide (37.5:1=

acrylamide:bisacrylamide)

(denaturants)

^for- mamide (Sigma, USA), urea (Sigma, USA)

30-50%

^{, PCR}

축을 위한

stacking gel

8% polyacrylamide gel (30%

acrylamide solution, 4x stacking gel buffer)

^.

^gel

1X TAE buffer (40 mM tris-acetate, 1 mM

EDTA) 8 L

⁶⁰

^C

^{, PCR}

⁵⁰

^l

6X loading dye (Solgent Co., Ltd. Korea) 10

^l

^gel

^loading

⁽⁶⁰

^{C, 60 V,}

18 h)

^,

밀도변화를분석하기위해식물병원성진균

⁶

^{(Table 2)}

^DNA

^marker

^.

^gel

EtBr

1X TAE buffer

²⁰

^{, 40}

^buffer

^{, UV}

^gel

band

^.

DGGE fragment elution

DGGE

^DNA

^gel

^band

^e-tube

^,

^gel

^DNA

^QIAEX

^II Gel Extraction Kit

^protocol

^.

^gel

^e-tube

^gel

2 volume diffusion buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA [pH8.0], 0.1% SDS)

⁴

^C

overnight

⁽⁴

C, 15,000 rpm, 10 min)

^{. DNA}

0.1 volume

3 M sodium acetate

^0.6 volume

iso-propanol

Table 1. PCR primers design for the amplication of fungal rDNA genes from soil total DNA.

Set No. Primer Sequence (5’-3’) mer Reference

No.1 ITS1

TCCGTAGGTGAACCTGCGG 19 Tao et al. [26]

ITS4

TCCTCCGCTTATTGATATGC 20 Tao et al. [26]

No.2 nu-SSU-0817

TTAGCATGGAATAATRRAATAGGA 24 Ian et al. [7]

nu-SSU-1196

TCTGGACCTGGTGAGTTTCC 20 Ian et al. [7]

No.3 FF390

CGATAACGAACGAGACCT 18 Eeva et al. [4]

FR1

AICCATTCAATCGGTAIT 18 Eeva et al. [4]

GC clamp CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGCACGGG 36 Tao et al. [26]

a

Forward primer

b

Reverse primer I, Inosine; R, A + G

Table 2. Phytopathogenic fungal strains used for marker of DGGE.

Strains KACC No. Plant disease

Fusarium oxysporum f. sp. Lycopersici 40032 Fusarium wilt

Rhizoctonia solani AG-1 (IA) 40101 Sheath blight

Rhizoctonia solani AG-1 (IB) 40111 Damping-off

Phytophthora capsici 40476 Blight

Colletotrichum acutatum 40042 Anthracnose

Corynespora cassicola 40964 Leaf blight, Brown spot

제거하였으며

, 70% ethanol

^TE buffer 20

^l

^DNA

^{. Elution}

^DNA

PCR

1.5% agarose gel

(100 V, 30 min)

^.

DNA fragment sequencing 및 처리구 간 상동성 분석

Elution

^DNA

Solgent Co., Ltd.

Korea

^,

^NCBI

^BLAST search

Genbank

^.

XLstat2008 (Addinsoft, USA)

^Dice

, unweighted pair group average

dendrogram

^.

결과 및 고찰

경작지 토양으로부터 total DNA 추출 및 확인

경작지토양으로부터진균의군집을분석하기위해

^fungal DNA

^{total DNA}

^DNA

FastDNA

SPIN Kit for Soil (MP biomedicals, USA)

, 1.5% agarose gel

^{(100 V,} 30 min)

^{3,000 bp}

^DNA

^{(Fig. 1).}

^{total DNA}

^PCR

^UV- vis spectrophotometer (ND-1000, NanoDrop Technologies Thermo scientific, USA)

였으며

^,

^PCR

⁽

^).

^PCR

^humic compounds

^.

진균의 DNA 증폭을 위한 PCR 전략 및 조건

DGGE

primer set

Oomycetes

Phytophthora capsici, Sordariomycetes

Fusarium oxysporum

genomic DNA

^Bacillus subtilis AH18, Pseudomonas fluorescens 2112

^genomic DNA

^{test PCR}

, ITS1/4 primer set

^PCR

^.

nu-SSU 0817/1196 primer set

, ITS1/4 primer set

^DNA

Fig. 1. Confirmation of total DNA extracted from crop contin- uation and rotation field soil.

(A), non-planting; (B), after 6 month; lane M, 100 bp DNA ladder;

lane 1-3, crop continuation field; lane 4-6, crop rotation field; lane1, 4: water treatment; lane 2, 5: consortium microbial agent treat- ment; lane 3, 6: agrochemical treatment.

Fig. 2. Comparison of aspect of PCR amplication using the various primer set.

A, primer set No. 1; B, primer set No. 2; C, primer set No. 3; lane

M, 100 bp DNA ladder; lane 1, Bacillus subtilis AH18;, lane 2,

Pseudomonas fluorescens 2112; lane 3, distilled water; lane 4,

Phytophthora capsici; lane 5, Fusarium oxysporum.

인되었으나증폭산물의농도가

ITS 1/4 primer

, FF390/FR1 primer set

^{(Fig. 2).}

^DGGE

^{total DNA}

^PCR

ITS 1/4 primer set

^.

DGGE 분석을 위한 PCR product 제작

Test PCR

^DNA

ITS 1/4 primer set

⁶

^,

^,

^,

^{total DNA}

nested-PCR

. Nested-PCR

pre-denaturation (94

C, 5 min)

denaturation (94

C, 1 min), annealing (50

C, 1 min 30 s), extension (72

C, 3 min)

35 cycles

final extention (72

C, 10 min)

^{, 1}

ITS1/4 primer set

^{total DNA}

ITS4 primer

GC-clamp

²

^PCR

^.

^,

⁶

total DNA

500-600 bp

^{(Fig. 3).}

진균의 군집조사를 위한 DGGE pattern 분석

고추의정식전과정식

⁶

total DNA

universal primer

^ITS

1/4 primer set

nested-PCR

500-600 bp

^DGGE

^{, Fig. 3}

산물의

^band

^.

고추경작지에서우점하고있는종으로판단되는

^band

^3-4

⁶

¹⁵

^,

^.

^band

¹⁸

장많은

^band

^,

구가

¹⁴

^band

^.

marker

⁶

^band

^,

R. solani AG-1 (IB)

^.

정적인영향을미칠수있다는것을확인할수있었다

^(Fig.

4).

DNA fragment

⁶

^,

Paraphaeosphaeria quadriseptata

, P. quadriseptata

monocillin

Fig. 3. Confirmation of the PCR product amplified by ITS 1/4 primer set in agarose gel.

(A), non-planting; (B), after 6 month; lane M, 100 bp DNA ladder;

lane 1-3, crop continuation field; lane 4-6, crop rotation field; lane1, 4: water treatment; lane 2, 5: consortium microbial agent treat- ment; lane 3, 6: agrochemical treatment.

Fig. 4. DGGE profiles of the fungal community fingerprints of the red-pepper field soil under different cropping system. The DGGE profiles of fungal communities were generated by sep- aration of 18S rRNA fragments amplified with primers ITS1 and ITS4-GC.

*, pattern of R. solani AG-1 (IB); lane lane M, from top and bottom:

R. solani AG-1 (IA), R. solani AG-1 (IB), C. cassicola, P. capsici, F. oxysporum f. sp. Lycopersici, C. acutatum; NP, non-planting;

AF6, after six month; Wt, water treatment; BBP, consortium micro-

bial agent treatment; Ac, agrochemical treatment; CC, crop con-

tinuation field; RC, crop rotation field.

등을생산하는것으로알려져있다

^[19].

Mortierella chlamydospora, Cucurbitaria berberidis

Chaetomium globosum

^,

M. chlamydospora

^[27].

^Kirk

^[10]

^C.

globosum

진균으로보고되었으며

^[10],

(Table 3, Fig. 4).

처리구 간 상동성 분석

DGGE

분석하기위해

^band

XLstat2008 (Addinsoft, USA)

unweighted pair group average

dendrogram

^.

^,

^.

^,

양상을띄는것으로나타났다

^{(Fig. 5).}

토양에서식하는진균의군집구성은연작및윤작과같은 경작체계보다제제의종류에의해영향을더크게받는것 으로확인되었다

^.

요 약

본연구에서는분자생물학적기법인

^PCR-DGGE

^.

^DNA

^DGGE

universal primer

ITS 1/4 primer set

nested-PCR

며

^,

^DGGE

군집을나타내는

^band

^3-4

¹⁵

^.

Table 3. Result of sequence analysis of DNA fragments from DGGE.

Band name Identification Taxon/Characteristics

Non-planting

NP1 Paraphaeosphaeria quadriseptata Ascomycetes/Monocillin produce

After 6 month

AF6C Mortierella chlamydospora Zygomycetes/leaf disintegration

AF6E Cucurbitaria berberidis Ascomycetes

AF6J Chaetomium globosum Ascomycetes

NP, non-planting; AF6, after six month

Fig. 5. Cluster analysis on fungal communities of crop con- tinuation and rotation in red-pepper field soil.

The dendrogram was based on the cluster analysis by the

unweighted pair group method using arithmetic averages. Wt,

water treatment; BBP, consortium microbial agent treatment; Ac,

agrochemical treatment; CC, crop continuation field; RC, crop

rotation field.

미생물제제를동시에적용한 고추경작지에서

^band

18

^.

^band

¹⁴

^.

^DNA

marker

^,

R. solani AG-1 (IB)

^.

^,

Paraphaeosphaeria quadriseptata,

Mortierella chlamydospora, Cucurbitaria berberidis

Chaetomium globosum

^.

^,

.

Acknowledgments

This research was supported by Basic Science Research Pro- gram through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012-0001795).

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