In Vitro and in Vivo Inhibitory Effect of Gaseous Chlorine Dioxide against Pathogenic Fungi Isolated from Stored Sweetpotato (Ipomoea batatas L.)
저장 고구마에서 분리한 병원균에 미치는 이산화염소 가스 처리의 억제효과
- 주제(키워드) Chlorine dioxide , Sweetpotato , Postharvest diseases , 이산화염소 , 고구마 , 저장병
- 발행기관 고려대학교 대학원
- 지도교수 김기덕
- 발행년도 2018
- 학위수여년월 2018. 2
- 학위구분 석사
- 학과 대학원 바이오시스템공학과
- 세부전공 식물병리학 및 원예과학전공
- 원문페이지 84 p
- 실제URI http://www.dcollection.net/handler/korea/000000080407
- 본문언어 영어
- 제출원본 000045932233
초록/요약
Sweetpotato (Ipomoea batatas Lam.) is one of the important food crops in Korea; however, postharvest fungi still cause a significant loss of quality during storage. In this study pathogenic fungi from sweetpotatoes were isolated and identified; Four fungal isolates, SP-d1, SP-f6, SP-p15, and SP-r1, were obtained from diseased sweetpotatoes from Muan, Korea. For identification of these isolates, internal transcribed spacer (ITS) region sequences were amplified using the primers ITS1 and ITS4. As a result, isolates SP-d1, SP-f6, SP-p15, and SP-r1 were identified as Diaporthe batatas (100% similarity), Fusarium oxysporum (99.10% similarity), Penicillium expansum (100% similarity), and Rhizopus oryzae (99.15% similarity), respectively. To test pathogenicity of the isolates, healthy sweetpotatoes were hole-inoculated with mycelial agar plugs. Isolates SP-d1, SP-f6, SP-p15, and SP-r1 caused symptoms on sweetpotato roots at 14, 10, 14 days, and 36 hours after inoculation, respectively. These isolates were re-isolated from the lesions to prove Koch’s postulates. In vitro and in vivo inhibitory effects of gaseous chlorine dioxide (ClO2) against isolates SP-d1 and SP-f6 were also evaluated. For in vitro tests, each fungus smeared on acidified potato dextrose agar (APDA) was treated with various ClO2 concentrations (1, 5, 10, and 20 ppm) for 0, 1, 10, 30, and 60 min. Fungal populations were decreased at the concentrations of ClO2 over the times. For isolate SP-d1, the decline of most spore development started at 30 min of 1 ppm ClO2; for isolate SP-f6, almost all spore development was inhibited at 5 ppm treatment of ClO2 for 5 min. The test fungal isolates were inhibited at the 20 ppm treatment of ClO2. For in vivo tests, spore suspension of each isolate was dropped on sweetpotato slices and treated with various ClO2 concentrations (5, 10, and 20 ppm) for 0, 10, 30, and 60 min. Lesion diameters of these isolates were not significantly different at the concentrations of ClO2 over the times. However, fungal populations were decreased at the ClO2 concentrations over times. These results indicate that D. batatas, F. oxysporum, P. expansum, and R. oryzae may be significant fungal pathogens on sweetpotatoes during storage in Korea and gaseous ClO2 can inhibit the fungal development and infection on sweetpotato. This also implies that ClO2 may be applied for controlling fungal diseases during sweetpotato storage.
more초록/요약
고구마는 한국에서 주요 식용 작물 중 하나이지만, 저장 기간 중 진균에 의해 발생하는 저장병은 고구마에 심각한 손실을 일으킨다. 본 연구에서는 고구마에서 분리한 병원균을 동정하고, 이산화염소 가스 처리가 분리된 병원균에 미치는 억제효과를 분석하였다. 무안에서 받은 병든 고구마 샘플에서 총 4개의 균주(SP-d1, SP-f6, SP-p15, SP-r1)를 분리하였다. 분리된 균주는 ITS1과 ITS4 primer를 이용하여 internal transcribed spacer (ITS) region을 증폭시켜 동정하였다. 동정 결과, 균주 SP-d1, SP-f6, SP-p15, SP-r1은 각각 Diaporthe batatas (100% similarity), Fusarium oxysporum (99.10% similarity), Penicillium expansum (100% similarity), Rhizopus oryzae (99.15% similarity)로 동정되었다. 분리된 균주의 병원성 테스트를 위하여 건강한 고구마 (‘주황미’)에 각각의 병원균의 균사를 상처접종한 결과, 균주 SP-d1, SP-f6, SP-p15, SP-r1은 각각 접종 10일, 14일, 10일, 36시간 후 고구마에 병징이 나타났다. 이 균주들은 병징으로부터 재분리됨으로써 코흐의 법칙을 충족시켰다. 다음으로, 균주 SP-d1과 SP-f6에 대한 이산화염소 가스 처리의 억제효과를 분석하였다. 먼저, in vitro 실험에서는 균주의 포자 현탁액을 각각 acidified potato dextrose agar (APDA)에 도말 후, 다양한 이산화염소 농도(1, 5, 10, 20 ppm)를 0, 1, 10, 30, 60분 동안 처리하였다. 그 결과, 가스처리의 농도와 시간이 커질수록 병원균의 밀도가 감소되었다. 균주 SP-d1에 대한 억제효과가 제일 컸고, 이 균은 1 ppm의 이산화염소 가스를 30분동안 처리하였을 때에도 포자 형성이 거의 대부분 억제되었다. 균주 SP-f6은 5 ppm의 이산화염소 가스를 5분 처리하였을 때 포자의 발달이 크게 저하되었다. 20 ppm의 이산화염소 가스를 처리하였을 때에는 두 균주 모두 생장이 억제되었다. In vivo 실험에서는 1 cm 간격으로 얇게 썬 고구마 슬라이스에 각 균주의 포자 현탁액을 10 µl씩 접종한 후, 5, 10, 20 ppm 농도의 이산화염소 가스를 0, 10, 30, 60분 처리하였다. 그 결과, 병징 길이를 평가한 값은 가스처리를 한 고구마와 하지 않은 고구마 간에 통계적으로 차이가 없었다. 하지만, 곰팡이의 밀도는 가스처리의 농도와 시간이 증가할수록 감소하였다. 이 연구의 결과를 종합해보면 D. batatas, F. oxysporum, P. expansum, R. oryzae는 수확 후 저장고구마에 중요한 병원균이라는 것을 보여주고, 이산화염소 가스 처리가 이 병원균에 의한 감염을 억제할 수 있다는 것을 알 수 있었다. 따라서, 이산화염소 가스 처리는 고구마의 저장기간 동안 저장병을 방제할 수 있는 하나의 방법이 될 수 있을 것이다.
more목차
ABSTRACT ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙i
KOREAN ABSTRACT ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙iii
CONTENTS ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙v
LIST OF TABLES ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ix
LIST OF FIGURES ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙xi
INTRODUCTION ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1
1. Sweetpotato (Ipomoea batatas L.) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1
2. Disease management postharvest diseases on sweetpotato ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙3
3. Chlorine dioxide (ClO2) treatment ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5
4. The objectives of this study ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙6
MATERIALS AND METHODS ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
1. Isolation and identification of pathogenic fungi from diseased sweetpotato (Ipomoea batatas L.) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
1.1. Isolation of fungi from diseased sweetpotato ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
1.1.1. Diseased sweetpotatoes ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
1.1.2. Isolation of fungi ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
1.2. Molecular-based identification of fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙8
1.2.1. The genomic DNA extraction ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙8
1.2.2. PCR amplification of gDNA ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10
1.2.3. Purification of PCR product ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙11
1.2.4. DNA sequence analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12
1.3. Pathogenicity test of the fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13
1.3.1. Fungal inoculum ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13
1.3.2. Pathogenicity tests ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13
1.3.3. Re-isolation of infected fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14
2. In vitro and in vivo inhibitory effect of gaseous chlorine dioxide (ClO2) against pathogenic fungi ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
2.1. In vitro inhibitory effects of gaseous chlorine dioxide ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
2.1.1. Preparation of spore suspensions ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
2.1.2 Gaseous chlorine dioxide treatment ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15
2.1.3. Statistical analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙16
2.2. In vivo inhibitory effects of gaseous chlorine dioxide ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙16
2.2.1. Preparation of sweetpotato slices ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙16
2.2.2. Preparation of spore suspensions ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙17
2.2.3 Gaseous chlorine dioxide treatment ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙17
2.2.4. Statistical analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18
RESULTS ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
1. Isolation and identification of pathogenic fungi from diseased sweetpotato (Ipomoea batatas L.) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
1.1. Isolation of fungi from diseased sweetpotato ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
1.2. Molecular-based identification of fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
1.3. Pathogenicity test of the fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙22
1.3.1. Pathogenicity tests ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙22
1.3.2. Re-isolation of infected fungal isolates ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27
2. In vitro and in vivo inhibitory effect of gaseous chlorine dioxide (ClO2) against pathogenic fungi ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙30
2.1. In vitro inhibitory effects of gaseous chlorine dioxide ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙30
2.1.1 Effect of gaseous ClO2 treatment on Diaporthe batatas SP-d1 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙30
2.1.2. Effect of gaseous ClO2 treatment on Fusarium oxysporum SP-f6 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙30
2.2. In vivo inhibitory effects of gaseous chlorine dioxide ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31
2.2.1 Effect of gaseous ClO2 treatment on Diaporthe batatas SP-d1 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31
2.2.2. Effect of gaseous ClO2 treatment on Fusarium oxysporum SP-f6 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙37
DISCUSSION∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44
1. Isolation and identification of pathogenic fungi from diseased sweetpotato (Ipomoea batatas L.) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙44
2. In vitro and in vivo inhibitory effect of gaseous chlorine dioxide (ClO2) against pathogenic fungi ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙49
LITERATURE CITED∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙52
