臺灣博碩士論文加值系統

木黴菌是生物防治真菌之一，木黴菌的二次代謝產物在拮抗作用中扮演了一個重要能夠去抑制植物病原真菌的角色。本實驗是利用抗真菌活性來進行篩選分析，並利用稻草培養後的T. koningii RIS 3-8二次代謝產物，來有效的抑制植物病原菌。由本實驗結果可知，在體外的拮抗測試中，從23株木黴菌中顯示出T. koningii RIS 3-8可以最有效地抑制Fusarium solani, F. oxysporum, Rhizoctonia solani and Botrytis cinerea。利用加熱處理以及蛋白酶來移除蛋白質後，發現T. koningii RIS 3-8能有效抑制F. solani，主要是由於二次代謝產物，而蛋白質在抗真菌測試中完全沒有活性。本實驗結果顯示T. koningii RIS 3-8在固態培養第九天之代謝物，以及利用分配萃取後之氯仿層部分有最好的抗真菌活性。更進一步地利用活性導向和色譜分析法來分離和純化T. koningii RIS 3-8的二次代謝產物，分別為1-hydroxy-3-methylanthracene-9,10-dione，methyl 4-hydroxybenzoate，methyl 4-hydroxycinnamate，4-hydroxybenzaldehyde和4-hydroxyacetophenone共五個化合物，其中methyl 4-hydroxybenzoate，methyl 4-hydroxycinnamate，4-hydroxybenzaldehyde和4-hydroxyacetophenone是第一次從Trichoderma中被分離出來的。

此外，我們的研究從ethoxy ether functionalized imidazolium salts合成的化合物中的imidazolium上陽離子不同的10、12、14、16和18碳鏈長度的衍生物，去篩選能對抗植物病原菌立枯絲核菌的活性物質並且探討其機制。本實驗結果顯示imidazolium salts在14碳鏈長度的im-009 1-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-3-tetradecyl-1H-imidazol-3-ium chloride最具有抑制立枯絲核菌的生長能力。探討立枯絲核菌在處理im-009後，利用倒立式螢光顯微鏡去觀察立枯絲核菌的細胞壁和細胞核在形態學上的作用機制。

Secondary metabolites play a pivotal role in the antagonistic activities of some biocontrol species of Trichoderma resulting in the suppression of plant pathogens. T. koningii RIS 3-8, among other 22 strains, was the best Trichoderma spp. tested against phytopathogens, Fusarium solani, F. oxysporum, Rhizoctonia solani and Botrytis cinerea in vitro. Removing proteins by heat-treatment and protease-treatment showed proteins had no role in the antagonistic tests that lead to the secondary metabolites be the primary candidates. To discern the determinants of the T. koningii RIS 3-8 secondary metabolites, antifungal oriented assays were performed. The metabolites from the 9th day chloroform extract processed best antifungal activity against pathogens. Five bioactive compounds of T. koningii RIS 3-8 were isolated chromatographically: 1-hydroxy-3-methylanthracene-9,10-dione, methyl 4-hydroxybenzoate, methyl 4-hydroxycinnamate, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone that all but 1-hydroxy-3-methylanthracene-9,10-dione were firstly isolated from T. koningii.

Meanwhile, [Cn-im-3OEG][Cl], amphiphilic ionic liquids and ionic liquid crystals were tested their fungicial potency. [C14-im-3OEG][Cl] possesses the best antifungal activity against R. solani with an IC50 of 130 μM. Rupture of the bioenvelop appears to be operative in the process of antifungal activity.

Table of contents
Acknowledgement ................................................................................... I
Abstract ................................................................................................... II
中文摘要 ................................................................................................ III
Table of contents .................................................................................... V
Figure legends ........................................................................................ X
Table contents .................................................................................... XIV
Part I Fungicides screening from the Trichoderma secondary metabolites .............................................................................................. 1
1. Introduction ..................................................................................... 3
2. Experimental materials and methods ........................................... 7
2.1 Fungal strains ............................................................................... 7
2.2 Trichoderma cultivation ............................................................... 8
2.3 Metabolites preparation .............................................................. 9
2.4 Plate confrontation test .............................................................. 10 2.5 Exclude proteins from the Trichoderma culture ...................... 10
2.6 Bioactivity of the metabolite of Trichoderma ............................. 11
2.7 Purification the antifungal molecules from Trichoderma ....... 12
2.8 Structure determination by NMR ............................................ 19
3. Results ............................................................................................ 21
3.1 Antagonistic potency of Trichoderma strains ........................... 21
3.2 T. koningii RIS 3-8 cultivation on PDA .................................... 23
3.3 Secondary metabolites contribute major potency of T. koningii
RIS 3-8 against phytopathogens ............................................... 25
3.4 T. koningii RIS 3-8 cultivation on straw residue medium ........ 27
3.5 Isolation and characterization of bioactive secondary
metabolites .................................................................................. 28
3.5.1 The variation of metabolite between dry weight and
cultivation on days .............................................................. 28
3.5.2 The antifungal activities of Extraction ................................ 30
3.5.3 The antifungal activity of partitioned EG ........................... 35
3.6 Structure determination of isolated compound ...................... 35
3.7 The antifungal assay of compounds ......................................... 38
3.8 Effect of the compounds on the growth of Trichoderma ......... 40

4. Discussion ....................................................................................... 43
5. Conclusion ...................................................................................... 45
6. References ...................................................................................... 47


Part II Antifungal potency of the organic ethoxy ether functionalized imidazolium salts .................................................................................. 49
1. Introduction ................................................................................... 51
2. Experimental Materials and Methods ......................................... 55
2.1 Preparation of ethoxy ether functionalized imidazolium salts 55
2.2 Rhizoctonia cultivation ............................................................... 56
2.3 Antifungal assays ........................................................................ 56
2.4 IC50 determination ...................................................................... 56
2.5 Morphological observation by fluorescent stereomicroscope 57
3. Results ............................................................................................ 59
3.1 Antifungal activity ...................................................................... 59
3.2 Morphological observation ........................................................ 60

4. Discussion ..................................................................................... 65
5. Conclusion ...................................................................................... 67
6. References ...................................................................................... 69
7. Appendix ........................................................................................ 71
7.1 Fungicides screening from the Trichoderma secondary
metabolites .................................................................................. 71
7.1.1 Characteristics of Common Organic Solvents ................... 71
7.1.2 The secondary metabolites of the most important fungal
Trichoderma, include classes and structures ........................ 73
7.1.3 Purification the antifungal molecules by TLC and HPLC 97
7.1.4 1H-NMR spectra of Compound A ...................................... 104
7.1.5 1H-NMR spectra of Compound B ...................................... 105
7.1.6 1H-NMR spectra of Compound C ...................................... 106
7.1.7 1H-NMR spectra of Compound D ...................................... 107
7.1.8 1H-NMR spectra of Compound E ...................................... 108
7.1.9 1H-NMR spectra of Compound F ...................................... 109

7.2 Antifungal potency of the organic ethoxy ether functionalized
imidazolium salts ...................................................................... 110
7.2.1 The R. soloni morphology cell wall observation ............... 110
7.2.2 The R. soloni morphology nucleus observation ................ 112
7.2.3 Calculate the hyphal length and the hyphae amount ...... 113


Figure legends
Part I
Fig. 1 The strategy of isolation compounds A, B, C, D, E, and F. .... 18
Fig. 2 Morphology development of cultivated T. koningii RIS 3-8 on
the PDA plates at 28 oC. ........................................................... 24
Fig. 3 The antifungal activity assays of culture media without
treatment (■), heat (■), and protease K (■). .......................... 26
Fig. 4 Cultivation of Trichoderma on pulverized rice straw residue
medium soaked with liquid mineral medium. Trichoderma
mycelia side view (a) and (b); mycelia side view and green
conidia (c); bottom view mycelia and green conidia (d). ...... 27
Fig. 5 The dry weight of chloroform/methanol extracted fractions
from T. koningii RIS 3-8 in pulverized rice straw medium
supplement with Extraction C (◆), Extraction G (■) and
Extraction F (▲). ...................................................................... 29
Fig. 6 The antifungal activities of metabolite extraction of 3rd, 6th, 9th,
12th, 15th, 18th and 21st days from EC (a), EG (b), and EF (c).
Fungicide (╳), sample concentrations 15 μg plug-1 (◆), 150 μg
plug-1 (▲), 1500 μg plug-1 (■). Values are means of three
replications ± SE. ...................................................................... 32
Fig. 7 The antifungal activities of metabolite of 3rd, 6th, 9th, 12th, 15th,
18th and 21st days from EC1500 (◆), EG1500 (■), and EF1500 (▲).
Values are means of three replications ± SE. ......................... 33
Fig. 8 The antifungal activities of metabolite of 9th days from EC1500,
EG1500, and EF1500. Values are means of three replications ± SE
(P < 0.05). ................................................................................... 34
Fig. 9 Scheme of each purified compound.
1-hydroxy-3-methylanthracene-9,10-dione (a), methyl
4-hydroxybenzoate (b), methyl 4-hydroxycinnamate (c),
4-hydroxybenzaldehyde (d), 4-hydroxyacetophenone (e). ... 37
Fig. 10 Mycelia growth and sporulation promotion of Trichoderma by
the 4-hydroxybenzaldehyde (HBA) and
4-hydroxyacetophenone (Piceol) in 500μg mL-1 . .................. 42
Fig. 11 The fractions A1-1-1, and observed by 254 nm (a). The
wavelengths of UV light were 254 nm (green background). 97
Fig. 12 The pure compound A14-7-2 was separated from A14-7. The
fractions A14-7, and observed by 254 nm (a); The HPLC
chromatogram (b). ................................................................. 98
Fig. 13 The pure compound A14-7-3 was separated from A14-7. The
fractions A14-7, and observed by 254 nm (a); The HPLC chromatogram (b). ................................................................. 99
Fig. 14 The distribution of components from fractions A15-1 to
A15-6. Lane 1: A15-1, lane 2: A15-2, lane 3: A15-3, lane 4:
A15-4, lane 5: A15-5, lane 6: A15-6, and observed by 254 nm
(a) and burning TLC (b). The wavelengths of UV light were
254 nm (green background) and burning TLC (gray
background). ......................................................................... 100
Fig. 15 The pure compound A15-4-1 was separated from A15-4. The
HPLC chromatogram (a); The wavelengths of UV light were
254 nm (b). ............................................................................ 101
Fig. 16 The distribution of components from fractions A16-1 to
A16-6. Lane 1: A16-1, lane 2: A16-2, lane 3: A16-3, lane 4:
A16-4, lane 5: A16-5, lane 6: A16-6, and observed by 254 nm
(a), 365 nm (b) and burning TLC (c). The wavelengths of UV
light were 254 nm (green background), 365 nm (blue
background) and burning TLC (gray background). ......... 102
Fig. 17 The pure compound A16-5-1 was separated from A16-5. The
HPLC chromatogram (a); The wavelengths of UV light were
254 nm (b); burning TLC
(c). .............................................. 103


Part II
Fig. 1 The inhibition capacity of various alkyl length of the
[Cn-im-3OEG][Cl] compounds to R. soloni. .......................... 59
Fig. 2 The R. soloni morphology observation in the present of 130
μM , [C14-im-3OEG][Cl] under the 600 x Fluorescent
stereomicroscope. Scale bars = 0.5 mm. The more septa and
hyphae swelled along with longer period of treatment. Many
tiny blue dots and blue smear area were debri of lysed hyphae.
Arrow indicated the position of the septum . (A), (C), (E)
Control groups: (B), (D), (F) [C14-im-3OEG][Cl] treatment
groups. (A), (B) 24hpi; (C), (D) 72hpi; and (E), (F) 144hpi.
Treatment the present of 2000 μM, 144hpi (G). .................... 62
Fig. 3 The R. soloni nuclei morphology observation in the present of
130 μM [C14-im-3OEG][Cl] 72hpi under the 600 x
Fluorescent stereomicroscope. Scale bars = 0.5 mm. More
bright spots, represented nuclei, showed in the
[C14-im-3OEG][Cl] treated hyphae 72 hpi (D) then control
group (B). Also, the septa shorter and hyphae swelled can be
observed. .................................................................................... 63


Table contents
Part I
Table. 1 Antagonistic potency of Trichoderma strains ...................... 22
Table. 2 Information of isolated compounds ..................................... 36
Table. 3 Inhibition bioactivity of purified compounds from
Trichoderma against F. solani, F. oxysporum, R. solani
and B. cinerea. Values are inhibition percentage in means of
three replicates in percentage ± SD. ..................................... 39
Table. 4 Among the various pathogens growth length of the
Trichoderma of compounds 1 to 5. ........................................ 41

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