Efficacy of chlorine dioxide fumigation on storage insects and fungi in stored rice

국문 초록 (Abstract)

곡물의 수확 후 저장 중 곤충과 곰팡이에 의한 곡식 피해를 막기 위하여 이산화염소 가스를 처리하였다. 수확 후 곤충과 곰팡이를 억제할 수 있는 최적의 이산화염소 가스 처리 조건을 찾기 위해서 다양한 농도와 처리 시간을 시험하였다. 또한 이산화염소 가스 처리 후, 곡물 씨앗의 활성과 이산화염소 잔류량도 확인해 보았다. 이산화염소 가스의 처리는 화랑곡나방의 모든 생장 단계에서 효과를 보였다. 알과 성충 단계의 화랑곡나방은 장시간의 저농도 처리나 단시간의 고농도 처리에도 높은 치사율을 보였다. 하지만, 유충과 번데기 단계의 화랑곡나방은 알과 성충 단계보다 감수성이 적었다. 유충과 번데기 단계의 경우에는 낮은 농도 처리에서는 낮은 치사율을 보였다, 하지만, 낮은 농도라 할지라도 장시간 처리될 경우에는 높은 치사율을 보였다. 모든 단계의 화랑곡나방에 이산화염소 가스를 처리한 후 장기간 관찰한 결과, 낮은 농도에 장시간 노출된 화랑곡나방은 높은 농도에 단시간 노출된 화랑곡나방과 비슷한 증상을 나타내었다. 그리고, 낮은 농도에서 처리된 유충 단계의 화랑곡나방은 유충 기간이 늘어나는 것을 보였으며, 이 유충은 번데기가 되거나 성충으로 우화하는 데 실패하였다. 이산화염소에 의해서 활성산소가 화랑곡나방 유충에 축적되게 된다. 그러나, iPLA2 가 발현되어 피해입은 지방산을 고치거나 유지시킨다. 그럼에도 불구하고, 지방산 산패 축적이 화랑곡나방에서의 살충 효과를 야기하게 된다. 이산화염소 가스를 통한 곰팡이 억제 실험을 위해서 Aspergillus ochraceus 가 사용되었다. 가스 처리가 된 곰팡이에서는 비정상적인 균사와 함께 낮은 균사 성장률을 볼 수 있었다. 또한, 이산화염소 가스가 처리된 곰팡이에서 얻은 분생포자는 낮은 발아율을 보이거나, 발아가 늦는 것을 확인할 수 있었다. 이산화염소 가스 처리는 곰팡이를 인위접종한 창고 실험에서 곰팡이를 완벽하게 억제시키는 것을 보여주었다. 가스 처리 이후, 곡물 종자 활성 또한 조사되었다. 종자 활성은 가스의 농도와 처리 시간에 영향을 받았다. 하지만 이산화염소 잔류량은 국제 기준보다 낮거나 검출되지 않았다. 또한 A. ochraceus의 균사 성장과 포자의 발아를 억제했다. 적절한 농도와 처리 시간은 곡물 종자 활성에 영향을 미치지 못했으며 종자에 잔유물을 남기지 않았다. 이산화염소 가스 처리는 수확 후 보관기간동안 곡물의 품질 관리에 기여할 수 있을 것이다.

다국어 초록 (Multilingual Abstract)

Gaseous chlorine dioxide (ClO2) treatment during the stored period against postharvest insects and fungi on grain was studied. Various gaseous ClO2 concentration and treatment time were tested to find optimum condition for the control of postharvest insect and fungi. Also, the viability of grain seeds and residue of ClO2 on grain after the gas treatment was investigated. Treatment of gaseous ClO2 controls all the life stages of Plodia interpunctella. Both egg and adult stage of P. interpunctella was showed high mortality rate on low concentration treatment for a long time or high concentration for short time treatment of ClO2 gas. However, larva and pupa stage P. interpunctella was less sensitive compared to egg or adult stage. Larva and pupa stage showed low mortality on low concentration treatment. However, low concentration long-time treatment showed high mortality. Long-term investigation after ClO2 gas treatment to all stages of P. interpunctella showed that low concentration for long time treatment showed the same effect to a high concentration for short time treatment. And low concentration gas treatment increased larva stage of P. interpunctella, and that larva failed to pupate or eclose. The reactive oxygen species (ROS) was induced by ClO2 treatment in P. interpunctella larva. However, expression of iPLA2 was increased to repairing damaged fatty acid. Nevertheless, the accumulation of lipid peroxidation showed to be resulted insecticidal to P. interpunctella. At gaseous ClO2 treatment to control fungi, ochratoxin producing fungi, Asperillus ochraceus was tested. The gas treated fungi showed abnormal mycelia and lower growth rate. Also, the conidia spores from ClO2 gas treated fungi showed a lower rate of germination or delay of germination. Gaseous ClO2 completely suppressed the A. ochraceus which was artificially inoculate in the field test. The viability of gas treated grain seed was also investigated. The viability was affected by the gas concentration and treatment time. However, The ClO2 residues on grain seed were lower than international standards or not detected. Furthermore, the treatment reduced the growth rate and germination rate of fungi, A. ochraceus. The treatment with proper concentration and duration did not lower the grain seed viability and did not leave chemical residue on the seed. The treatment of gaseous ClO2 contributed to keeping postharvest quality and quantity of grains during storing period.

목차 (Table of Contents)

• TABLE OF CONTENT
• ACKNOWLEDGEMENT i, 1 p
• ABSTRACT iii, 3 p
• KOREAN ABSTRACT v, 5 p
• TABLE CONTENTS vii, 7 p
• LIST OF FIGURES xii, 12 p
• LIST OF TABLES xiii, 13 p
• CHAPTER
• 1. Literature review 1
• 2. Antifungal activity of gaseous chlorine dioxide treatment against Aspergillus ochraceus from stored grain 18
• 2.1. Abstract 19
• 2.2. Introduction 20
• 2.3. Materials and Methods 22
• 2.3.1. Fungal materials and growth condition 22
• 2.3.2. Chlorine dioxide treatment 22
• 2.3.3. Evaluation of mycelial growth, germination rate, and sporulation 23
• 2.3.4. Statistical analysis 24
• 2.4. Results 25
• 2.4.1. Effect of gaseous ClO2 treatment to grown mycelia 25
• 2.4.2. Effect of gaseous ClO2 treated conidia spore germination rate 27
• 2.4.3. Effect of gaseous ClO2 treatment to young mycelia 29
• 2.4.4. Germination rate of conidia spore from gaseous ClO2 treated mycelia 31
• 2.4.5. Effect of gaseous ClO2 treatment to sporulation 33
• 2.5. Discussion 35
• 2.6. References 37
• 3. Sensitivity of different life stages of Indian meal moth Plodia interpunctella to gaseous chlorine dioxide 42
• 3.1. Abstract 43
• 3.2. Introduction 44
• 3.3. Materials and Methods 46
• 3.3.1. Rearing of test insects 46
• 3.3.2. Chlorine dioxide treatment 46
• 3.3.3. Preparing the Plodia interpunctella life stages for gas treatment 47
• 3.3.4. Mortality percentages of the different developmental stages 47
• 3.3.5. Statistical analysis 47
• 3.4. Results 49
• 3.4.1. Sensitivity of different developmental stages 49
• 3.4.2. Effect of gaseous ClO2 on the egg stage 51
• 3.4.3. Effect of gaseous ClO2 on the larval stage 53
• 3.4.4. Effect of gaseous ClO2 on the pupal stages 55
• 3.4.5. Effect of gaseous ClO2 on the adult stage 57
• 3.5. Discussion 59
• 3.6. References 61
• 4. Chlorine dioxide enhances lipid peroxidation through inhibiting calcium-independent cellular PLA2 in larvae of the Indian meal moth, Plodia interpunctella 66
• 4.1. Abstract 67
• 4.2. Introduction 68
• 4.3. Materials and Methods 70
• 4.3.1. Insect rearing and cell culture 70
• 4.3.2. Tissue preparation 70
• 4.3.3. ClO2 production and treatment 71
• 4.3.4. Bioinformatics analysis of gene structure 71
• 4.3.5. RNA extraction and RT-PCR 72
• 4.3.6. RT-qPCR 72
• 4.3.7. RNA interference of Pi-iPLA2 expression 73
• 4.3.8. Lipid peroxidation analysis 73
• 4.3.9. Treatment of Sf9 cells with chlorine dioxide or vitamin E 75
• 4.3.10. Treatment of P. Interpunctella with chlorine dioxide by injection 75
• 4.3.11. Treatment of P. Interpunctella with chlorine dioxide by fumigation 76
• 4.3.12. Hemocyte nodulation assay 76
• 4.3.13. Data analysis 76
• 4.4. Results 78
• 4.4.1. Identification of iPLA2 from P. Interpunctella 78
• 4.4.2. Expression profile of Pi-iPLA2 81
• 4.4.3. RNAi effect of Pi-iPLA2 on lipid peroxidation and cellular immunity 84
• 4.4.4. ClO2 increases lipid peroxidation via ROS 89
• 4.4.5. Fumigation with ClO2 inhibits Pi-iPLA2 expression and increases lipid peroxidation 92
• 4.5. Discussion 96
• 4.6 References 100
• 5. Effect of gaseous chlorine dioxide treatment on the quality of rice and wheat grain 105
• 5.1. Abstract 106
• 5.2. Introduction 107
• 5.3. Materials and Methods 109
• 5.3.1. Seed preparation 109
• 5.3.2. Chlorine dioxide treatment 109
• 5.3.3. Seed test 110
• 5.3.4. Residue analysis 110
• 5.3.5. Statistical analysis 111
• 5.4. Results 112
• 5.4.1. Effect of 50 ppm gaseous ClO2 on rice and wheat seed 112
• 5.4.2. Effect of 100 ppm gaseous ClO2 on rice and wheat seed 114
• 5.4.3. Effect of 200 ppm gaseous ClO2 on rice and wheat seed 116
• 5.4.4. ClO2 residues 118
• 5.5. Discussion 120
• 5.6. References 123
• 6. Conclusion 128
• APPENDIX 131