臺灣博碩士論文加值系統

傳統上之慣行農法為使用化學肥料與化學農藥克服病蟲害及地力衰竭等問題。而永續農耕旨在建立一個不過度依賴農用化學合成製劑之栽培管理方式，以達到兼顧作物產量品質與保護環境之雙贏目的。本研究旨在探討長期有機與慣行耕作下對蕉園土壤微生物多樣性之影響，於台灣香蕉研究所已建立之長期有機耕作與慣行耕作試驗田區進行樣品之採集，針對土壤基本性質、微生物群落代謝多樣性、不同類別可培養微生物數量、土壤可培養之細菌族群進行分析，此外亦利用未經培養之核酸分析法得知試驗樣品中之優勢微生物。於2011年至2014年間共五次採樣之土壤性質中，有機耕作因採用草生栽培管理，土壤水分含量較長期慣行耕作高。有機耕作17年、7年與長期慣行耕作土壤pH值分別為7.37 ~ 8.01、6.43 ~ 6.99與4.99 ~ 6.58，代表長期施用有機資材會使土壤pH值提高，而長期慣行施肥將導致土壤酸化。有機耕作長期施用有機質肥料與採用宿根栽培，可累積土壤中的有機質。微生物代謝多樣性之研究中，經由EcoPlateTM碳源利用測試結果顯示生長速度較快之微生物以及全部微生物，在四次採樣時間點下的代謝潛勢及代謝多樣性在有機耕作7年土壤最高，在GN2 MircoPlateTM和GN2 MircoPlateTM分析結果中亦有同樣趨勢。在不同採樣時間點下，土壤中生長速度較快之微生物群落利用碳源之能力經PCA統計分析後，可將長期慣行耕作與長期有機耕作區隔開來，四次採樣結果中一致與有機耕作處理呈正相關之碳源為α-Malic Acid、β-Methyl-D-Glucoside、D-Cellobiose、L-Serine與N-Acetyl-D-Glucosamine共有五種；長期慣行耕作則是L-Asparagine和Pyruvic Acid Methyl Ester共兩種碳源。微生物族群多樣性研究中，2011~2013年間蕉園土壤之可培養微生物菌數均達106以上，耕作管理方式與採樣時間點的不同對可培養微生物菌數所造成之差異並不明顯。本研究分離之可培養微生物歸類於Actinobacteria、Bacteroidetes、Firmicutes和Proteobacteria菌門，其中不論長期有機耕作或長期慣行耕作的土壤中，均以Firmicutes為優勢菌門，其中又以Bacillius spp.為優勢菌屬，綜合2013年4月TSA和SMA分離出Firmicutes，不論是數量或種類都相當多樣化，又以Bacillius aerophilus菌種被發現最多，Bacillius luciferensis菌種被發現最多樣化有6個不同的分離株。利用16S rDNA選殖株基因庫之建構與定序分析，得知長期有機耕作的土壤中，含有至今尚未被歸類微生物的比例較高，而Proteobacteria菌門微生物是此試驗田區中主要存在的微生物菌門，並在三種耕作管理方式下均能維持相當程度的優勢。其他尚包含Acidobacteria、Actinobacteria、Bacteroidetes、Firmicutes與Gemmatimonadetes菌門。長期慣行耕作之Acidobacteria比例最高。綜合結果顯示長期有機耕作之蕉園土壤之微生物多樣性確實比長期慣行耕作高，在生長速度較快之微生物群落代謝多樣性以有機耕作7年者最高，經培養法與未經培養之核酸分析法觀察之族群多樣性以有機耕作17年為優勢，說明有機耕作時間增加可提升生物多樣性及生態穩定性，產生該環境下之優勢族群而使其代謝多樣性趨於一穩定值。

Chemical fertilizers and chemical pesticides are commonly used to increase soil fertility and reduce plant pathogen in conventional farming. To achieve agricultural production in a more sustainable way, integrated managements instead of overdose use of agricultural chemicals are adopted to obtain high quality and yield of crops meanwhile maintain soil quality. The distribution of microbes which participate in nutrient transformation may be changed spatially or temporally in a dynamic soil ecosystems. In the present studies soil microbial diversity as influenced by long-term organic and conventional farming were evaluated in banana plantation soils. Soil samples were collected from the established fields in Taiwan Banana Research Institute during 2011-2014. Basic properties of soil such as water content, pH, EC and organic matter content were determined. Approaches to study metabolic and population diversity in banana plantation soils were conducted. These included the culture-dependent and culture-independent methods to obtain viable number of different bacterial groups, community level of physiological profiles and microbial community structure under different long-term farming systems. The results demonstrated that water content, pH and organic matter content were the highest in 17-year organic farming soils and lowest in long-term conventional farming soils. Metabolic potentials of fast-growing microbes in 7-year organic farming soils were generally higher than that in 17-year organic farming soils or conventional farming soils, as revealed by richness, average well color development and diversity index. The SAWCD value demonstrated that carbohydrates were well utilized by fast-growing microbes in soils under organic farming than that under conventional farming. α-Malic acid, β-Methyl-D-Glucoside, D-Cellobiose, L-Serine and N-Acetyl-D-Glucosamine were positively correlated with organic farming, while L-Asparagine and Pyruvic Acid Methyl Ester were positively correlated with conventional farming, which were verified by principle component analysis. Soils under organic farming harbored slight higher number of heterotrophs, free-living nitrogen fixers and tricalcium phosphate solubilizers than that in conventional farming soils. Members belonging to the phylum Firmicutes were dominated in all the three farming soils, and diverse Bacillus lineages at species or strain level were resolved by cultivated-based method. Microbial community structure of soils under three farming systems was compared through 16S rDNA clone library construction and sequence analysis. The 17-year organic farming soils have the highest percentage of unclassified bacteria (at the phylum level) while members belonging to Proteobacteria were dominated in 7-year organic farming soils and conventional farming soils. A higher percentage of clones was occupied by the phylum Acidobacteria in conventional farming soils, which may be related to soil deterioration.

摘要 i
Abstract ii
目次 iii
圖次 v
表次 viii
前言 1
文獻回顧 3
一、 香蕉簡介 3
(一) 香蕉起源 3
(二) 台灣香蕉產業發展現況 4
(三) 香蕉營養價值 5
(四) 台灣香蕉栽培品種 6
(五) 香蕉研究所介紹 7
二、 有機耕作與慣行耕作方式對土壤之影響 7
三、 土壤微生物多樣性之研究方法 11
(一) 代謝多樣性 12
(二) 族群多樣性 12
材料與方法 16
一、 試驗地區背景資料與土壤性質分析 19
(一) 樣區設計與田間管理資訊 19
(二) 土壤採樣與前處理 25
(三) 土壤性質分析 25
二、 微生物代謝多樣性研究 31
(一) 接種液製備 31
(二) 接種與培養 31
(三) 結果判讀 35
(四) 統計分析 35
三、 微生物族群多樣性研究 42
(一) 不同類別可培養微生物之菌數分析 42
(二) 微生物族群種類分析：培養法 44
(三) 微生物族群種類分析：未經培養之基因選殖法 48
結果與討論 62
一、 土壤基本物化性質 62
二、 微生物代謝多樣性之研究 67
(一) 利用EcoPlateTM分析微生物代謝多樣性 67
(二) 利用GN2 MircoPlateTM分析微生物代謝多樣性 82
(三) 利用GP2 MircoPlateTM分析微生物代謝多樣性 90
三、 微生物族群多樣性研究 95
(一) 培養法 95
(二) 未經培養之基因選殖法 133
結論 150
參考文獻 151

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