Responsive image
博碩士論文 etd-0101113-003332 詳細資訊
Title page for etd-0101113-003332
論文名稱
Title
香蕉園有機栽培與慣行農法農田土壤之細菌相研究
The study of soil bacterial communities between organic The study of soil bacterial communities between organic and conventional farming in a banana field conventional farming in a banana field
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
95
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-07-30
繳交日期
Date of Submission
2013-01-01
關鍵字
Keywords
微生物多樣性、有機栽培、聚合酶連鎖反應-變性梯度凝膠體電泳、香蕉園、土壤細菌相
Microbial diversity, Organic farming, PCR-DGGE, Banana field, The soil bacteria microbiota
統計
Statistics
本論文已被瀏覽 5725 次,被下載 1444
The thesis/dissertation has been browsed 5725 times, has been downloaded 1444 times.
中文摘要
中文摘要
基於維持土壤的永續健康與提升香蕉植株的抗病能力,台灣香蕉研究所於民國87年開始撥用部分區域試行有機栽培,並已證實有機栽培區黃葉病罹病率明顯低於慣行農法區。為了解有機栽培區與慣行農法區土壤中細菌菌相之差異,本研究分別在2010年8月、2010年12月及2010年5月採集試驗農場中蕉株的根圈土及非根圈土,進行理化特性檢測,並以分子生物學技術分析與鑑定土壤樣品中之細菌多樣性,使用之主要技術包括利用PCR-DGGE法分離各細菌之16S rDNA V6~V8區片段,並以pGEM-T Easy Vector 系統之重組DNA技術將各分離的DNA條帶加以定序和鑑定菌種。研究結果顯示,有機區的土壤屬於壤土,慣行區含砂量稍高些,屬於砂質壤土;有機13年區的土壤pH 值屬弱鹼性,慣行區則為中酸性至微酸性;所分析之土壤中各種養分,不見得有機13年區必然高於慣行區;各種有效性養分及有機碳在有機區含量的穩定性有高於慣行區之趨勢;施肥可能會影響土壤中有效性養分之含量;無法由施用之有機肥萃取到細菌DNA;土壤中的細菌相非常穩定,不因季節而變化;耕作制度之差異對土壤細菌相會有影響;香蕉品種對細菌相影響不大;根圈土與非根圈土菌相沒有差異;香蕉黃葉病生病植株附近之土壤菌相與健康植株附近之土壤菌相是否有差異不能十分肯定。Clone library 菌株中,有43個成功比對到對應的類別,其中有28個屬於真細菌類的Proteobacteria類,佔70% (其中又以Beta-proteobacteria類最多,Gamma-proteobacteria 次之);次多的是Acidobacteria(酸桿菌)類,尚Chlamydiae (披衣菌)、Actinobacteria(放線菌)、Nitrospirae(硝化螺旋菌)、Chloroflexi(綠彎菌);可能也有古細菌(Archea)。43個序列中,有34個為無法培養的(uncultured)菌種。各菌株之可能功能多是涉及元素循環的種類,包括參與N循環者、參與C循環者,也有參與S循環者(包含某些光合細菌)。將各樣品做系統分析樹,結果,有機13年區、有機3年、慣行區恰分成三個類群,有機13年區與慣行區之菌相差異最大。
Abstract
Abstract
Based on maintaining healthy soil for sustainable agriculture and enhancing banana disease resistance, Taiwan Banana Research Institute began to conduct organic cultivation on a trial basis in 1998. It had been proved that the morbidity of banana Fusarial wilt disease at organic cultivation plots was significantly lower than that of conventional farming. In order to study the differences of soil microbiota between the organic cultivation plots and the conventional farming areas, physical and chemical properties of the rhizosphere and non- rhizosphere soil samples were assayed during the period of Aug. 2010 to May 2011. The bacterial diversity was analyzed by molecular biology methods, including PCR-DGGE to separate the 16S rDNA V6 ~ V8 region of various bacteria and the recombinant DNA technology by using pGEM-T Easy Vector System to separate and sequence the DNA fragments. The results showed that organic plots was loam soil, but the conventional farming soil was sandy loam with higher sand content. The soil pH in 13 years organic area was mildly alkaline, but in conventional farming area was mildly acidic to slightly acidic. The content of various nutrients in organic 13-year area soil was not necessarily higher than the conventional farming area soil. The available nutrient contents in organic areas trend to be more stable than that in the conventional areas. Fertilization may affect the content of available nutrients in the soil. No bacterial DNA could be extracted from the organic fertilizer. The bacterial microbiota in soil was very stable, and was not related to the sampling seasons. The Banana strains had little effect on soil bacterial microbiota. There was no difference on the bacterial microbiota between the rhizosphere and non-rhizosphere soil samples. It is not sure whether there were any differences on the bacterial microbiota between the nearby soil of banana Fusarial wilt plants and the nearby soil of the healthy plants. By analyzing the DNA fragment clone library, 43 strains correspond to known category, of which 28 belonged to the Proteobacteria, and 34 were uncultured strains. The role of these microbial strains might involve in various element cycles, such as N cycles, C cycles, and S cycles (including some photosynthetic bacteria). The systematic cladogram showed that organic 13-year areas, organic 3-year areas and conventional farming areas represented three major categaries. The organic 13-year area and conventional area possessed the highest difference on the microbiota composition.
目次 Table of Contents
目次
目次 ⅰ
誌 謝 ⅲ
中文摘要 ⅳ
ABSTRACT ⅴ
圖目錄 ⅶ
表目錄 ⅷ
第一章 緒 論 1
1.1 土壤生態系 1
1.1.1 土壤生態系的非生物環境 1
1.1.2 土壤生態系的生物 4
1.1.3 微生物多樣性的重要 6
1.2 有機農業的緣起 10
1.3 香蕉簡介 11
1.4 香蕉黃葉病簡介 12
1.5 香蕉研究所 13
1.6 研就動機 14
第二章 材料與方法 15
2.1 採樣方法 15
2.1.1 採樣時間 15
2.1.2 採樣地點及方式 15
2.1.2.1 有機區的土樣: 15
2.1.2.2 慣行區的土樣: 15
2.1.2.3 根圈土(Rhizosphere soil): 15
2.1.2.4 非根圈土(Non-rhizosphere soil): 16
2.1.3 有機區施用的肥料樣品 16
2.1.4 樣品編號 16
2.1.5 樣品處理與保存 16
2.1.6 施肥施藥紀錄 16
2.2 土壤理化因子分析 16
2.2.1 土壤溫度 16
2.2.2 土壤pH值 16
2.2.3 土壤有機碳 17
2.2.4 土壤有效性氮 17
2.2.5 土壤有效性磷(Bray No.1) 18
2.2.6 土壤可交換性鉀、鈣、鎂 19
2.2.7 土壤質地分析 19
2.3 以分子生物技術分析土樣中之細菌菌相 20
2.3.1 土壤微生物群落Total Genomic DNA 之萃取 20
2.3.2 以PCR-DGGE法進行菌相比對 22
2.3.2.1 測試作PCR之適當Total Genomic DNA 稀釋濃度 22
2.3.2.2 以PCR反應取得並放大16S rDNA 片段 23
2.3.2.3 瓊脂膠體電泳檢視16S rDNA 片段 23
2.3.2.4 變性梯度膠體電泳(Denaturing Gradient Gel Electrophoresis,簡稱DGGE) 分析 24
2.3.3 細菌種類的分析與鑑定 25
2.3.3.1 以PCR反應取得並放大16S rDNA 片段 26
2.3.3.2 檢視16S rDNA 片段及回收PCR產物 26
2.3.3.3 Ligation 26
2.3.3.4 Transformation 26
2.3.3.5 確認Transformation 成功狀態 27
2.3.3.6 以DGGE 篩選剔除重複的clones 28
2.3.3.7 DNA定序及序列比對分析 28
2.3.3.8 特殊處理進行序列比對分析 29
2.4 以分子生物技術分析有機區施用肥料之細菌菌相 30
第三章 結果與討論 31
3.1 施肥施藥紀錄 31
3.2 土壤微生物群落TOTAL GENOMIC DNA 之萃取 31
3.3 土壤理化因子分析之結果 31
3.4 有機區使用之肥料的菌相分析 32
3.5 季節對土壤菌相的影響 33
3.6 耕作方式對土壤菌相的影響 34
3.7 香蕉品種對土壤菌相的影響 35
3.8 根圈土與非根圈土土壤菌相之差異 36
3.9 黃葉病病株附近土壤菌相與健康植株附近土壤菌相之差異 37
3.10 土樣中細菌種類的分析與鑑定結果 38
3.11 細菌多樣性分析及系統演化樹分析 40
第四章 結 論 41
參考文獻 42
圖附錄 49
表附錄 71
參考文獻 References
參考文獻
1. 王一雄。2000。土壤環境汙染與農藥。國立編譯館主編,明文書局印行。台北,台灣。pp.43,51~ 57。
2. 石濤。1998。環境微生物。鼎茂圖書出版。台北,台灣。pp.6-1~ 6-33。
3. 朱耀逢。1996。再見香蕉王國。旗山人文產業道導覽手冊。pp.93。
4. 李芳胤、陳士賢。2007。土壤分析實驗手冊。新文京開發出版。台北,台灣。pp.186~ 198。
5. 林良平。1987。土壤微生物學。 國立編譯館主編,南山堂出版社發行。台北,台灣。pp.39~ 117,193~ 268,291~ 297,383~ 405。
6. 孫守恭、黃振文。1996。台灣植物鐮胞菌病害。世維出版社。台北,台灣。pp.170。
7. 張春梅。2008。有機栽培對香蕉生育、後熟品質及土壤環境特性之影響。國立屏東科技大學環境工程與科學系碩士學位論文。
8. 莊再揚(A)。1988。香蕉黃葉病抑病土之研究(二)抑病土之特性。植物保護學會會刊 30: 125~ 134。
9. 莊再揚(B)。1988。香蕉黃葉病抑病土與導病土根圈微生物之研究。國科會研究報告。國立台灣大學植物病蟲害學系。
10. 莊再揚。1989。香蕉黃葉病抑病土與導病土根圈微生物之研究 (第二年)。國科會研究報告。國立台灣大學植物病蟲害學系。
11. 莊作權。2010。土壤肥料。三民書局。台北,台灣。pp.45~ 46,63~ 70,208~ 216。
12. 郭魁士。1979。土壤學。台北,台灣。pp.55~ 69,93,104~ 116,119~ 164,193~ 268。
13. 傅聲雷。2007。土壤微生物多樣性的研究概況與發展趨。 Biodiversity Science 15。pp.109~115。
14. 黃新川(A)。1988。香蕉-智者之果,營養泉源。果農合作486: 19~ 26。
15. 黃新川(B)。2000。香蕉黃葉病,植物疫情與策略。中華植物保護學會出版。台北,台灣。pp.45~ 58。
16. 黃新川(C)。2002。組培技術在香蕉黃葉病防治上之應用。植病會刊11: 57~ 61。
17. 黃新川(D)。2012。常綠果樹--香蕉。台灣香蕉研究所,http://www.banana.org.tw/Upload/20091129205759734.pdf
18. 葉茂生、羅紹麟。2000。農業概論。台北,台灣。 pp.13~ 19,51。
19. 農業統計年報(100年,農業生產-作物生產)。2012。行政院農委會,http://agrstat.coa.gov.tw/sdweb/public/book/Book.aspx.
20. 臺灣肥料公司, http://www.taifer.com.tw/02/index.htm.
21. 臺灣青果年報。1991。台灣省青果運銷合作社聯合社。pp.12。
22. 臺灣香蕉研究所(A) http://www.banana.org.tw/.
23. 臺灣香蕉研究所(B),http://www.banana.org.tw/Upload/2009121814856734.pdf.
24. 趙昌平、廖健勇。2001。農藥濫用影響國人健康及生態環境專案調查報告。pp.57。
25. Avrahami, S. and Conrad, R., 2003, Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures, Applied and Environmental Microbiology 69: 6152- 6164.
26. Azziz, G., Bajsa, N., Haghjou, T., Taule, C., Valverde, A., Jose Mariano Igual, J. M. and Arias, A., Abundance, diversity and prospecting of culturable phosphate solubilizing bacteria on soils under crop–pasture rotations in bacteria on soils under crop–pasture rotations in a no-tillage regime in Uruguay, Applied Soil Ecology, In Press, Corrected Proof, Available online 4 November 2011.
27. Bossio,D.A., Scow, K.M., Gunapala, N. and Graham, K.J., 1998, Determinants of soil microbial communities: effects of agricultural management, season, and soil type on phospholipid fatty acid profiles, Microb Ecol 36: 1-12.
28. Brady, N. C. and Weil, R. R., 2010, Elements of the nature and properties of soils, 3rd edition, International edition, Pearson, Prentice Hall, Upper Saddle River, NJ., 289.
29. Brock, 2009, Biology of microorganisms, 12th ed. 68-69, 456-463, 694-705.
30. Castro, H. F., Classen, A. T., Austin, E. E., Norby, R. J. and Schadt, C. W., 2010, Soil Microbial Community Responses to Multiple Experimental Climate Change Drivers, Applied and Environmental Microbiology 76: 999–1007.
31. Ceja-Navarro, J. A., Rivera-Orduña,F.N., Patiño-Zúñiga,L., Vila-Sanjurjo,A., Crossa,J., Govaerts, B. and Dendooven,L., 2010, Phylogenetic and multivariate analyses to determine the effects of different tillage and residue management practices on soil bacterial communities, Applied and Environmental Microbiology 76: 3685–3691.
32. Chang, Y. J., Stephen, J. R., Richter, A. P., Venosa, A. D., Bruggemann, J., Macnaughton, S. J., Kowalchuk, G. A., Haines, J. R., Kline, E. and White, D. C., 2000, Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation:effect of profiling method. Journal of Microbiological Methods 40: 19-31.
33. Corsaro D., Feroldi, V., Saucedo, G., Ribas, F., Loret,J.F. and Greub,G. , 2009, Novel Chlamydiales strains isolated from a water treatment plant. Environ Microbiol. 11: 188-200.
34. Fravel, D., Olivain, C., Alabouvette, C., 2003, Fusarium oxysporum and its biocontrol, New Phytologist 157: 493–502.
35. Gee,G.W.,and Bauder,J.W., 1986, Partical size analysis, In:Klut, A. (eds.), Methods of Soil Analysis. Part 1,2nd ed., ASA and SSSA, Madison, WI, USA., 383-411.
36. Getha, K. and Vikineswary, S., 2002, Antagonistic effects of Streptomyces violaceusniger strain G10 on Fusarium oxysporum f.sp. cubense race 4: Indirect evidence for the role of antibiosis in the antagonistic process, Journal of Industrial Microbiology & Biotechnology 28: 303-310.
37. Horn, M. and Wagner, M., 2001, Evidence for additional genus-level diversity of Chlamydiales in the environment, FEMS Microbiology Letters 204: 71-74.
38. Hu, S., van Bruggen, A. H. C., Wakeman, R. J. and Grünwald, N. J., 1997, Microbial suppression of in vitro growth of Pythium ultimum and disease incidence in relation to soil C and N availability, Plant and Soil 195: 43-52.
39. John, R. P., 1942, An ecological and taxonomic study of the algae of British soils: I. The distribution of the surface-growing algae_1, Annals of Botany 6: 323-349.
40. Katznelson, H. and Bose, B., 1959, Metabolic activity and phosphate-dissolving capability of bacterial isolates from wheat roots, rhizosphere, and non-rhizosphere soil,Canadian Journal of Microbiology 5: 79-85.
41. Keeney, D. R. and Nelson, D. W., 1982, Nitrogen-inorganic forms. In Page, A.L., Miller, R.H.and Keeney, D.R. (eds.) Methods of Soil Analysis. Part 2, 2nd ed., Agronomy Monograph, ASA and SSSA, Madison, WI, USA. 643-698.
42. Lloyd, A. B., 1969, Behaviour of Streptomycetes in soil, Journal of General Microbiology 56: 165-170.
43. Marschner, P., Crowley, D. and Yang, C. H., 2004, Development of specific rhizosphere bacterial communities in relation to plant species, nutrition and soil type. Plant Soil 261: 199–208.
44. Marschner, P., Kandeler, E. and Marschner, B., 2003, Structure and function of the soil microbial community in a long-term fertilizer experiment. Soil Biology & Biochemistry 35: 453–461.
45. McLean, E. O., 1982, Soil pH and lime requirement,In:Page, A.L.
Miller,R.H.,and KEEney,D.R.(eds.), Methods of Soil Analysis.Part 2,2nd ed., American Society of Agronomy and Soil Science society of America, Madison, WI, USA. , 199-224.
46. Miller, H. J., Henken, G. and Van Veen, J. A., 1989, Variation and composition of bacterial populations in the rhizospheres of maize, wheat, and grass cultivars, Canadian Journal of Microbiology 35: 656-660.
47. Muyzer,G., 1999, DGGE/TGGE a method for identifying genes from natural ecosystems, Current Opinion in Microbiology 2: 317–322.
48. Muyzer, G. and Smalla, K., 1998, Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology, Antonie van Leeuwenhoek 73: 127–141.
49. Muyzer, G., Waal, E. C. and Uitterlinden, A. G., 1993, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59: 695-700.
50. Neal, J. L., Larson, R. I. and Atkinson, T. G., 1973, Changes in rhizosphere populations of selected physiological groups of bacteria related to substitution of specific pairs of chromosomes in spring wheat, Plant and Soil 39: 209-212.
51. Nelson, D. W. and Sommers, L. E., 1982, Total carbon, OC, and organic matter, In:Page, A.L.Miller,R.H.,and KEEney,D.R.(eds.), Methods of Soil Analysis.Part 2,2nd ed., American Society of Agronomy and Soil Science Society of America, Madison, WI, USA., 539-577.
52. Nicol, G. W., Leininger, S., Schleper, C. and Prosser, J. I., 2008, The influence of soil ph on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria, Environmental Microbiology 10: 2966–2978.
53. Olsen, S. R. and Sommers, L. E., 1982, Phousphorus, In:Page, A.L.
Miller,R.H.,and KEEney,D.R.(eds.), Methods of Soil Analysis.Part 2,2nd ed., ASA and SSSA, Madison, WI, USA., 403-430.
54. Postma1, J., Schilder, M. and Speksnijder, A., 2007, Soil suppressiveness and functional diversity of the soil microflora in organic farming systems, 3rd QLIF Congress, Hohenheim, Germany, 20-23.
55. Reganold, J. P., 1995, Soil quality and profitability of biodynamic and conventional farming systems: A review, American Journal of Alternative Agriculture 10: 36-45.
56. Reganold, J. P., 1988, Comparison of soil properties as influenced by organic and conventional farming systems, American Journal of Alternative Agriculture 3: 144-155.
57. Reganold, J. P., Elliott, L. F. and Unger, Y.L., 1987, Long-term effects of organic and conventional farming on soil erosion, Nature 330: 370-373.
58. Reichenbach, H., Lang, E., Schumann, P. and Cathrin Spro¨er, C., 2006, Byssovorax cruenta gen. nov., sp. nov., nom. rev., a cellulose-degrading myxobacterium: rediscovery of‘Myxococcus cruentus’ Thaxter 1897, International Journal of Systematic and Evolutionary Microbiology 56: 2357–2363.
59. Rovira, A. D., 1953, Use of the warburg apparatus in soil metabolism studies, Nature 172: 29–30.
60. Seghers, D., Wittebolle, L., Top, E. M., Verstraete, W. and Siciliano, S. D., 2004, Impact of agricultural practices on the zea mays L. endophytic community, Applied and Environmental Microbiology 70: 1475–1482.
61. Sivamani, E. and Gnanamanickam, S. S., 1988, Biological control of Fusarium oxysporum f.sp.cubense in banana by inoculation with Pseudomonas fluorescens, Plant and Soil 107: 3-9.
62. Skinner, F. A., 1951, A method for distinguishing between viable spores and mycelial fragments of actinomycetes in soils, Journal of General Microbiology 5: 159-166.
63. Skyring, G. W. and Quadling, C., 1969, Soil bacteria: comparisons of rhizosphere and nonrhizosphere populations, Canadian Journal of Microbiology 15: 473-488.
64. Srivastava, S. C. and Lal, J. P., 1994, Effects of crop growth and soil treatment on microbial C, N, and P in dry tropical arable land, Biology and Fertility of Soils 17: 108-114.
65. Thomas, G. W., 1982, Exchangeable cations, In:Page, A.L.
Miller,R.H.,and KEEney,D.R.(eds.), Methods of Soil Analysis.Part 2,2nd ed., ASA and SSSA, Madison, WI, USA., 159-165.
66. Vanden Bygaart, A. J., Gregorich, E. G. and Angers, D. A., 2003, Influence of agricultural management on soil organic carbon:A compendium and assessment of Canadian studies. Canadian Journal of Soil Science 83: 363–380.
67. Watanabe, K. and Hayano, K., 1995, Seasonalvariation of soil protease activities and their relation to proteolytic bacteria and Bacillus spp in paddy field soil, Soil Biology and Biochemistry 27: 197–203.
68. Weller, D. M., Raaijmakers, J. M., McSpadden Gardener, B. B. and Thomashow, L. S., 2002, Microbial populations responsible for specific soil suppressiveness to plant pathogens1, Annual Review of Phytopathology 40: 309–348.
69. Widmer. F., Rasche, F., Hartmann, M. and Fliessbach, A., 2006, Community structures and substrate utilization of bacteria in soils from organic and conventional farming systems of the DOK long-term field experiment, Applied Soil Ecology 33: 294–307.
70. Wieland, G., Neumann, R., and Backhaus, H., 2001, Variation of microbial communities in soil, rhizosphere, and rhizoplane in response to cropspecies, soil type, and crop development, Applied and Environmental Microbiology 67: 5849–5854.
71. Wolsing, M. and Prieme, A., 2004, Observation of high seasonal variation in community structure of denitrifying bacteria in arable soil receiving artificial fertilizer and cattle manure by determining T-RFLP of nir gene fragments, FEMS Microbiology Ecology 48: 261–271.
72. Yang, Y. H., Yao, J., Hu, S. and Qi, Y., 2000, Effects of agricultural chemicals on DNA sequence diversity of soil microbial community: a study with RAPD marker, Microbial Ecology 39: 72–79.
73. Yeates, C., Gillings, M. R., Davison, A. D., Altavilla, N. and Veal, D. A., 1998, Methods for microbial DNA extraction from soil for PCR amplification., Biological Procedures Online 1: 40-47.
74. Zhou, J., Bruns M. A. and Tiedje, J. M., 1996, DNA recovery from soils of diverse composition, Applied and Environmental Microbiology 62: 316-322.
電子全文 Fulltext
本電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。
論文使用權限 Thesis access permission:校內校外完全公開 unrestricted
開放時間 Available:
校內 Campus: 已公開 available
校外 Off-campus: 已公開 available


紙本論文 Printed copies
紙本論文的公開資訊在102學年度以後相對較為完整。如果需要查詢101學年度以前的紙本論文公開資訊,請聯繫圖資處紙本論文服務櫃台。如有不便之處敬請見諒。
開放時間 available 已公開 available

QR Code