臺灣博碩士論文加值系統

現今的耕種面積下所生產的農作物已無法滿足全球人口爆增後所需的食物。多年來，人們為了生產更多的糧食而盲目的過量使用化學肥料以應對糧食不足的危機。不料，許多研究數據顯示過量的使用化學肥料，會帶來了嚴重的環境污染和土壤劣化的問題，最終反而導致農業生產量大幅度的降低。因此，本研究透過接種溶磷菌於玉米台農一號並配合化學肥料減量之處理，探討其對植物生長及養分吸收的影響。從楊秋忠院士菌種庫中挑選非病原性之微生物進行磷酸溶解能力測試篩選後，取得12株有效菌株，其中包含6株新種微生物。此12株有效菌株皆未曾被發表具有溶磷特性，其中菌屬Chitinophaga，Cohnella和Ochrobactrum更是首次被發現擁有此特性。針對這些菌株進行各形態之固定性磷的溶解能力，吲哚-3-乙酸 (indole-3-acetic acid, IAA) 的生產能力，酸鹼度的改變能力，及種子發芽生物分析試驗的分析，篩選出高潛力的菌株Burkholderia phytofirmans CC-S-L25及Rhizobium lusitanum CC-S-L19延續溫室盆栽試驗。試驗數據顯示，菌株B. phytofirmans CC-S-L25具有磷酸鈣，磷酸鋁和磷酸鐵的溶解能力、色氨酸 (tryptophan, Trp) 依賴性IAA生物合成能力、降低酸鹼度的能力及促進胚芽和胚根的生長能力。菌株R. lusitanum CC-S-L19具有磷酸鈣和磷酸鐵的溶解能力、色氨酸依賴性和非依賴性IAA生物合成能力、降低酸鹼度的能力及促進胚芽和胚根的生長能力。在溫室盆栽試驗中，將此兩株溶磷菌接種至玉米台農一號28天後發現，無論接種B. phytofirmans CC-S-L25或R. lusitanum CC-S-L19配合半量施肥之處理，皆可超越單獨施用全量化學肥料之處理的植體株高，葉片數量，鮮重及乾重。在植體分析的結果上 ，氮、磷、鈣、鎂、錳、鋅、銅之含量在接種B. phytofirmans CC-S-L25配合半量施肥之處理，顯著的比全量化學肥料之處理高，且鉀、鐵、硼之含量也不比全量化學肥料之處理差；然而，在R. lusitanum CC-S-L19配合半量施肥之處理下，磷、鈣、鎂之含量顯著的比全量化學肥料之處理高，且氮、錳、鋅、硼之含量與全量化學肥料之處理相等。綜合以上所有試驗之結果，溶磷菌B. phytofirmans CC-S-L25及R. lusitanum CC-S-L19具有植物生長促進之效果，且可以大量減少玉米化學肥量施用量。因此，接種溶磷菌B. phytofirmans CC-S-L25及R. lusitanum CC-S-L19不但可以加強農業管理免於化學肥料之濫用，還可以促進永續農業之發展。

Nowadays, the agricultural productions are no longer sufficient to feed the world's population. Over the years, people excessive application of chemical fertilizer in order to increase agricultural production to cope with the food crisis. Unfortunately, several research showed that excessive use of chemical fertilizer will cause the deterioration of environmental pollution and soil quality, and lead to significant reduction in agricultural production eventually. This study was therefore undertaken to investigate the efficiency and characteristic of PSBs and evaluate their plant growth promoting activities to maize (Zea mays L. cv. Tainung No. 1) cultivation under greenhouse conditions, while achieving the purpose of minimization on chemical fertilizer application rate at the same time. First, sceening phosphate solubilizing bacteria (PSBs) from Prof. Young’s microbial library, a total 12 potential non-pathogenic microorganisms were selected, which including 6 novel species. None of them has yet been reported as PSBs in any scientific journals, and we also found that Chitinophaga, Cohnella, and Ochrobactrum are the novel genus of PSBs from this research study. The abilities of phosphates solubilization, indole-3-acetic acid (IAA) production, alteration of pH, and seed germination bioassays were analyzed, and Burkholderia phytofirmans CC-S-L25 and Rhizobium lusitanum CC-S-L19 showing extraordinary performance, which were chosen as bioinoculant for greenhouse experiment. Data showed that B. phytofirmans CC-S-L25 has abilities to solubilize calcium-phosphate, aluminum-phosphate, and iron-phosphate; tryptophan-dependent IAA biosynthesis capability; pH acidification; hypocotyl and root growth promoting. While, R. lusitanum CC-S-L19 has calcium-phosphate and iron-phosphate solubilizing abilities; tryptophan-dependent and tryptophan-independent IAA biosynthesis capability; pH acidification; hypocotyl and root growth promoting. Plant height, leaf numbers, fresh weight, and dry weight of maize were significantly increased compare to 100% chemical fertilizer (CF) treatment no matter in case of 50% CF + B. phytofirmans CC-S-L25 or 50% CF + R. lusitanum CC-S-L19 treatments under greenhouse experiments. Regarding plant analysis, 50% CF + B. phytofirmans CC-S-L25 treatment was able to increase the uptake of N, P, Ca, Mg, Mn, Zn, and Cu compared to 100% CF treatment, and the performance of K-, Fe-, and B-uptake as good as 100% CF treatment. On the other hand, 50% CF + R. lusitanum CC-S-L19 treatment was able to increase the uptake of P, Ca, and Mg compared to 100% CF treatment, and the performance of N-, Mn-, Zn-, and B-uptake as good as 100% CF treatment. Based on the results, B. phytofirmans CC-S-L25 and R. lusitanum CC-S-L19 obviously has the plant growth promotion of maize, simultaneously, could significantly minimize the application of chemical fertilizer. Therefore, the inoculation of B. phytofirmans CC-S-L25 and R. lusitanum CC-S-L19 not only give a hand to reduce environmental pollution, but also promoting the development of sustainable agriculture.

誌謝…………………………………………………………………………………i
摘要………………………………………………………………………………...ii
ABSTRACT…………………………………………………………………….....iv
CONTENT………………………………………………………………………...vi
LIST OF ABBREVIATIONS…………………………...……………………….viii
LIST OF TABLES…………………………………………………………………x
LIST OF FIGURES..…………………………………………………………….xii
CHAPTER 1. INTRODUCTION…………………………………………………1
CHAPTER 2. REVIEW AND LITERATURE……………………………...……3
2.1 Phosphorus…………………………………………….……………………..3
2.2 Phosphate solubilizing bacteria…………………………………................7
2.2.1 Mechanisms of phosphorus solubilization………………………………9
2.2.2 Plant growth promoting attributes of PSBs…………………………….11
CHAPTER 3. MATERIALS AND METHODS………………………………...15
3.1 Research framework………………………………………………………..15
3.2 Laboratory condition: characteristics of PSBs…………………………...15
3.2.1 Screening phosphate solubilizing bacteria…………………………….15
3.2.2 Different forms of phosphate solubilization…………………………….18
3.2.3 Indole-3-acetic acid producing capability……………...…….. ………..18
3.2.4 Phosphate solubilizing activity, EC, and pH…………………. ………..22
3.2.4.1 Preparation of HNO3-Vanadate-Molybdate solution……................22
3.2.4.2 Preparation of standard curve of soluble phosphate……………….22
3.2.4.3 Mo-blue spectrophotometry method……………………...................24
3.2.5 Seed germination bioassay.……………………………….…………….24
3.2.5.1 Preparation of PSBs bioinoculant…………………………………….24
3.2.5.2 Equations and formulas…………………………...………………..…25
3.3 Greenhouse experiments: plant growth promoting test.………………..25
3.4 Plant analysis……………………………………………...………………..27
3.4.1 Kjeldahl digestion method for total nitrogen analysis…...……………27
3.4.1.1 Digestion of samples…………………………..……...……………….27
3.4.1.2 Distillation of filtration solutions………….…………...……………….28
3.4.1.2.1 Preparation of boric acid indicator solution………………………..28
3.4.1.3 Titration of final solutions…………………...…………………………28
3.4.1.3.1 Preparation of standard ammonium solution……………………...29
3.4.1.3.2 The recovery rate of distillation apparatus…...……………………29
3.4.1.3.3 The calculation of nitrogen concentration………………………….30
3.4.2 Nutrients analysis for P, K, Ca, Mg, Fe, Mn, Zn, Cu, and B………….30
3.5 Statistical analysis…………………………………………………………..31
CHAPTER 4. RESULTS AND DISCUSSIONS………………………………32
4.1 Laboratory condition: characteristics of PSBs……………………..........32
4.1.1 Screening phosphate solubilizing bacteria…………………………….32
4.1.2 Different forms of phosphate solubilization…………………………….36
4.1.3 Indole-3-acetic acid producing capability ……………………………...42
4.1.4 Phosphate solubilizing activity, EC, and pH…………………………...43
4.1.5 Seed germination bioassays…………………………...………………..45
4.2 Greenhouse experiments: plant growth promoting test..……………….50
4.2.1 Effect on plant height…………………………….……………………….51
4.2.2 Effect on leaf numbers………………………....…………….................51
4.2.3 Effect on fresh and dry weight…………………………………………..51
4.3 Plant analysis…………………………………………...…………………..56
4.3.1 Major elements: N, P, K, Ca, and Mg…………………………………..56
4.3.2 Trace elements: Fe, Mn, Zn, Cu, and B………………………………..60
4.3.3 Efficiency of nutrients uptake of PSBs inoculation…………...............64
CHAPTER 5. CONCLUSIONS………………………………………………...67
CHAPTER 6. REFERENCES………………………………………………….68

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