臺灣博碩士論文加值系統

胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)在世界上分布範圍廣泛，寄主繁多，目前已知1200種以上，跨越100多科之植物皆為其寄主，加之CMV在田間可被75種以上之蚜蟲傳播，若缺乏適當之防治，易導致產量減少造成經濟上損失，為經濟上極為重要之植物病毒，然而目前對CMV所引起之病害仍然缺乏長期、有效之防治方法。本研究擬利用交叉保護以及土壤微生物分泌之代謝物誘發植物產生抗性，來對CMV所引起之病害進行防治。在交叉保護上，利用接種已截切掉CMV 2b蛋白之弱系CMV於圓葉菸草上，已證實此弱系之CMV可以交叉保護多種強系之CMV。在利用土壤微生物分泌之代謝物誘發植物產生抗性上，發現自土壤中分離之菌株NTU1F8培養後之過濾液，於圓葉菸草上可以有效抑制胡瓜嵌紋病毒之感染以及病程之發展。此外，於圓葉菸草及芥菜上，培養濾液亦可抑制蕪菁嵌紋病毒之感染。

Cucumber mosaic virus (CMV) is an economically important plant viruses with a worldwide distribution and a very wide host range. We aim to use two strategies to manage disease caused by CMV on crops. One is to develop cross protection by utilization of mild strain virus and the other is to induce plant resistance in plant by treatment of culture filtrate derived from fungi (NTU1F8) isolated from soil. We first conducted our strategies on Nicotiana benthamiana. The result showed that 2b gene-partial deleted CMV induced only mild symptom on N. benthamiana and protect plant from infection by different severe strains of CMV. In addition, the treatment of culture filtrate of NTU1F8, can obviously decrease infection rate, delay symptom development and decrease virus accumulation. We also found pretreatment of N. benthamiana and Brassica juncea with culture filtrate of NTU1F8 following inoculation of Turnip mosaic virus could also decrease the infection rate and disease severity.

Acknowledgement i
中文摘要 ii
Abstract iii
Table of content iv
Chapter Ι. Introduction 1
Chapter ΙΙ. Materials and methods 7
2.1 Plant materials 7
2.2 Incubation of soil microorganism 7
2.3 Preparation of culture filtrate derived from soil microorganisms 7
2.4 In vitro transcription 8
2.5 Preparation of virus inoculum 8
2.6 Inoculation methods 9
2.6-1 Mechanical inoculation 9
2.6-2 Agro-inoculation 9
2.7 Treatment of culture filtrate on plant sample 11
2.8 Disease index 11
2.9 RNA extraction 12
2.10 Detection of CMV and TuMV by indirect ELISA 12
2.11 Detection of CMV and TuMV by RT-PCR 13
2.12 Phylogenetic trees and similarity analyses 14
Chapter ΙΙΙ. Results 16
3.1 Symptom and inoculation of mild strain Cucumber mosaic virus (CMV) 16
3.2 Cross protection effect of mild strain Cucumber mosaic virus (CMV) in Nicotiana benthamiana 16
3.2-1 Cross protection of CMV 20Δ2b against CMV 20 16
3.2-2 Cross protection against distal related-CMV strains 17
3.3 Effect of application of culture filtrate on virus on Nicotiana benthamiana 17
3.3-1 Effect of culture filtrate on CMV 17
3.3-2 Effect of culture filtrate of NTU1F8 on TuMV 18
3.4 Effect of application of culture filtrate on virus on Brassica juncea 19
Chapter ΙV. Discussion 21
Reference 24
Tables and Figures 37
Table 1. The ratio of plants showing symptoms on plants with different pre-inoculation then inoculate with Cucumber mosaic virus 20 (CMV 20). 37
Table 2. Nucleotide sequence identities between CMV 20 and other CMV strains. 38
Table 3. The ratio of plants showing symptoms on plants with different pre-inoculation then inoculated with CMV CA. 39
Table 4. The ratio of plants showing symptoms on plants with different pretreatments and then inoculated CMV. 40
Table 5. The ratio of plants showing symptoms on plants with different pretreatments and then inoculated Turnip mosaic virus (TuMV). 41
Figure 1. Schematic representation of genome of CMV 20 and CMV 20 Δ2b. 42
Figure 2. Symptoms of Nicotiana benthamiana inoculated with CMV. 43
Figure 3. The agarose gel separate the products of RT-PCR designed to detection of CMV 20Δ2b in N. benthamiana. 44
Figure 4. Symptoms on CMV inoculated plants with different pre-inoculation. 45
Figure 5. The agarose gel separate the products of RT-PCR designed to detection of CMV 20Δ2b in N. benthamiana and the symptoms of N. benthamiana with different pre-inoculation then inoculated with CMV 20. 46
Figure 6. Percentages of CMV 20Δ2b- inoculated plants showed severe symptoms after inoculated with CMV 20. 48
Figure 7. Phylogenetic tree of CMV CA, CMV Fny, CMV Y, CMV LS, CMV LY, CMV Q and CMV 20. 49
Figure 8. Symptoms of N. benthamiana with pre-inoculation then inoculated with CMV CA. 51
Figure 9. Percentages of CMV 20Δ2b- inoculated plant showed severe symptoms after inoculated with CMV CA. 52
Figure 10. Disease development of N. benthamiana after inoculation with CMV. 53
Figure 11. Symptoms of N. benthamiana with different pretreatments and then inoculated with CMV 20. 54
Figure 12. The agarose gel separate the products of RT-PCR designed to detection of CMV 20 in N. benthamiana with different pretreatments. 55
Figure 13. Symptoms development of N. benthamiana with different pretreatments and then inoculated with CMV. 56
Figure 14. Disease development of N. benthamiana after inoculation with TuMV. 57
Figure 15. Symptoms of N. benthamiana with different pretreatments and then inoculated with TuMV. 58
Figure 16. The agarose gel separate the products of RT-PCR designed to detection of TuMV in N. benthamiana with different pretreatments. 59
Figure 17. Symptoms development of N. benthamiana with different pretreatments and then inoculated with TuMV. 60
Figure 18. Disease development of Brassica juncea after inoculation with TuMV. 61
Figure 19. Symptoms development on Brassica juncea with different pretreatments and then inoculated with TuMV. 62
Figure 20. Detection of TuMV in Brassica juncea by ELISA in different treatments. 63
Appendix 64
Table A-1. Primer of PCR and RT-PCR used in this study. 64

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