臺灣博碩士論文加值系統

由病原真菌Phellinus noxius所引起的褐根病，會感染許多種植物，尤其以木本植物最為嚴重。染病的樹木通常初期難以察覺，一旦感染嚴重時就很少會有存活者，因此褐根病又被稱為「樹癌」。本病原菌主要透過植物根系與根系的接觸進行傳播，會由喬木傳染喬木，還會傳染灌木甚至草本植物。2017年，中興大學校園中有三棵細葉榕(Ficus microcarpa) 嚴重感染褐根病；且病原菌透過榕樹根系接觸傳染到附近一種名為錫蘭葉下珠(Phyllanthus myrtifolius)的灌木，造成植株枯死的現象。往昔錫蘭葉下珠沒有被褐根病菌感染的記錄，因此假設其為新的寄主，並透過完成柯霍氏法則加以證實。被接種過的錫蘭葉下珠苗木出現葉子小化、生長停滯、枝枯、植株弱化等症狀。雖可觀察到明顯症狀，但弱化的植株仍可存活很長的時間。接種後一年才有少量植株枯死的情況。相較另一種接種的植物馬拉巴栗，僅接種二週就100%死亡，錫蘭葉下珠顯得非常地耐病。在臺中市共採集了15株褐根病菌株，將部分菌株的ITS及28S序列與前人發表序列作親緣分析,結果發現臺灣和東南亞地區的褐根病菌株序列與Zhou等在2018年發表的Pyrrhoderma noxium並非在同一分類群。在生物防治方面，經過拮抗測試篩選後得到2株拮抗菌，Streptomyces padanus PMS702與Bacillus velezensis BB118。以S. padanus PMS702 的拮抗能力最佳，它的拮抗物質Fungichromin會造成褐根病菌細胞受損及細胞質滲漏甚至死亡的情況，其濃度在6至8 ppm 就可抗制半數菌絲生長。為進一步測試在植物上的防治效果，將2株拮抗菌分別培養發酵液，稀釋10倍和50倍，直接施用於扦插在含有褐根病木屑菌種之介質的錫蘭葉下珠枝條上。結果顯示，2株拮抗菌都可降低錫蘭葉下珠扦插苗的罹病度由0.51降至0.37(PMS702 50倍稀釋液)和0.14 (BB118 50倍稀釋液);此外，2株拮抗菌都有促進植物生長的功效，尤其可促進根系的發育。顯然S. padanus PMS702及B. velezensis BB118都具有潛力進一步研發用於防治褐根病或其他土媒病害，或開發成為培育樹苗的生物肥料。

Brown root rot is caused by Phellinus noxius (Corner) G. H. Cunningham that infects over two hundred species of plant, especially wooden trees. Another name Tree Cancer was given to the disease because of hardly discovered and scarcely surviving of infested trees. Pathogen disperses through root to root connection, not only from arbor to arbors, but also from arbor to shrubs or even to herbs. In 2017, three Chinese banyans (Ficus microcarpa) in the campus of National Chung Hsing University were killed by P. noxius. A shrub plant named Ceylon myrtle (Phyllanthus myrtifolius) which is near by the tree Chinese banyans was killed by the same disease too. We demonstrated that Ceylon myrtle is a new host of P. noxius by completing Koch’s postulates. Several major symptoms on Ceylon myrtle were observed, including leaf size reduction, dieback of branches, growth suspended of young leaf. Mortality of inoculated seedlings can be observed after 1 year, but most of seedlings are still surviving. Ceylon myrtle is significantly tolerance to P. noxius compared to another plant Malabar-chestnut (Pachira microcarpa), which 100% mortality rate was observed within 2 weeks after wound inoculation. Total 15 strains of P. noxius were collected in Taichung city, some of them were applied to phylogenetic analysis with the ITS and 28S sequences, the data demonstrate that the strains of P. noxius from Taiwan were not classified in the same clade of Pyrrhoderma noxium named by Zhou et al. in 2018. In bio-control assays, three agents were tested for bio-control efficacy against P. noxius. In vitro antagonistic assay shows that all strains were able to inhibit P. noxius’s growth. Among the tested strains Streptomyces padanus PMS702 expressed the strongest inhibition. A major antibiotic fungichromin of S. padanus caused cytoplasmic leakage and cell death of P. noxius. The concentration in the range of 6 to 8 ppm inhibits 50% mycelium growth of P. noxius. To evaluate the effect of the antifungal activity in planta, culture broth of S. padanus PMS702 and Bacillus velezensis BB118 was applied on cuttings of Ceylon myrtle planted on the substrate inoculated with P. noxius. Both bio-control agents reduced disease severity from 0.51 to 0.37 (PMS702 50-fold dilution) and 0.14 (BB118 50-fold dilution). Besides, the two biocontrol agents exhibited a well growth-promoting effect on cutting seedlings, especially on root development. With double advantages, S. padanus PMS702 and B. velezensis BB118 have great potential for further study to control P. noxius and other soil pathogens in field application or develop as biological fertilizers.

中文摘要 i
Abstract iii
Table of contents v
List of Tables and Figures vii
1. Introduction 1
2. Backgrounds and literature review 2
3. The Motivation and objectives 6
4. Materials and Methods 7
4.1. Isolation of the pathogen 7
4.2. DNA extraction 7
4.3. Amplification of partial sequences of 28s and ITS region 8
4.4. Phylogenic analyzing of Phellinus noxius 8
4.5. Cultivation of P. noxius under different light wavelengths 9
4.6. Sample preparation for SEM (Scanning Electron Microscopy) 10
4.7. Soil pH evaluation 10
4.8. Preparation of P. noxius inoculum with wheat grain and sawdust 10
4.9. Preparation of cutting seedlings of Ceyon myrtle 11
4.10. Wounding inoculation method 12
4.11. Koch’s postulates performance 12
4.12. Determination of disease severity 12
4.13. The mortality of inoculated seedlings 13
4.14. Antagonistic assay 13
4.15. Preparation of cultural broth SSM and SMGC-2 13
4.16. Properties assays of culture broths 14
4.17. Evaluating biocontrol agents for controlling BRR on cuttings of Ceyon myrtle 15
4.18. Investigating the effect of fungichromin on P. noxius 16
4.19. Data analysis 18
5. Results 19
5.1. Disease symptoms of BRR of Chinese banyan and Ceylon myrtle in NCHU campus 19
5.2. Morphological and molecular identification of P. noxius 19
5.3. Alignment and phylogenetic relationships of Phellinus noxius 20
5.4. Acidification of soil from rhizosphere of infected plants 21
5.5. The effect of various light wavelengths on the growth of P. noxius 21
5.6. Symptoms on the inoculated seedlings 21
5.7. Fulfill Koch’s postulates and prove Ceylon myrtle as a new host 22
5.8. Disease severity of inoculated seedlings of Ceylon myrtle and Malabar chestnut 22
5.9. Biocontrol agents selection 23
5.10. Properties of cultural broth of biocontrol agents 23
5.11. Evaluating biocontrol agents for controlling BRR on cuttings of Ceylon myrtle 24
5.12. The mode of action of Streptomyces padanus PMS-702 inhibits P. noxius 24
6. Discussion 26
7. Conclusion 31
8. References 32
Tables and Figures 38

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