臺灣博碩士論文加值系統

在農業生產中，氮是重要且大量存在的元素。然而大量施用的化學氮肥被植物吸收利用率只有50%左右，流失的氮肥不僅浪費且對生態環境影響巨大。一條根學名為闊葉大豆(Glycine tomentella)，現今金門農試所積極推廣當地農民種植做為經濟作物，施用之化學肥料一旦流失將直接影響海洋生態。因此可利用根瘤菌與豆科植物形成共生固氮根瘤，固定氮氣轉換成植物可直接利用態氮(NH3)。本研究由不同的土壤中篩選出能與一條根共生之根瘤菌，分析菌株種類及特性，找出適合接種且固氮能力最佳之菌株。篩選菌株前，必須先克服種子發芽率不佳的問題，將種子浸泡於濃硫酸中10分鐘，發芽率可以提升至80%左右。而種子經過脫水乾燥保存於7℃環境下，於90天後仍可維持相同的發芽率。以三種土壤分別種植一條根，篩選並純化自然結瘤之根瘤菌株。三種土壤中共純化出30株菌，其中有三株生長快速。經過初步回接測驗後，共有12株菌可以成功以人工接種方式結瘤。將成功結瘤之菌株進行16S rDNA鑑定分析，分別為Bradyrhizobium elkanii、Bradyrhizobium iriomotense、Bradyrhizobium iaoningense及Rhizobium multihospitium，共2屬4種根瘤菌株。以Murashige and Skoog培養基種植一條根並測試此4種菌在不同pH下之族群數及共生結瘤能力，發現在pH 6.8時有最大族群數及結瘤數。為了找出在自然土壤環境中也能有良好共生且固氮活性高之菌株，分別以4株菌接種一條根種植於3種土壤上。其中Br. iriomotense有良好的結瘤數且固氮活性為4株菌中最高。但若種植於無機態氮含量高(327 mg/kg)之土壤時，則所有菌株的結瘤能力及固氮活性都會被抑制。

Nitrogen is abundant and an important element in agricultural production. However, profusely applying chemical nitrogen fertilizer may not enhance the absorbability efficiency of plants (only about 50 % efficiency), but leads to waste and tremendous impact on the environment. Broadleaf soybean (Glycine tomentella) is called “I-Tiao-Gung” in local name in Chinese. Now Kinmen County Agricultural Research Institute actively promotes the local farmers to plant “I-Tiao-Gung” as commercial crop, nevertheless, once the applied fertilizer washed away, it would directly affect the marine ecology. Symbiosis of legumes and Rhizobium would form nodule to convert atmospheric nitrogen into ammonia (NH3), which plants could use directly. This study selected the most suitable Rhizobium from different soils that was able to be symbiotic with “I-Tiao-Gung” and analyze the type and characteristics of isolated Rhizobium. For overcoming the problem regarding germination rate, soaking the seeds of “I-Tiao-Gung” into condensed sulfuric acid for 10 minutes could increase the germination rate to 80 %. “I-Tiao-Gung” was planted in three soils, the one that could develop nodule with Rhizobium were screened and purified. In three soils, there were 30 strains were isolated and three of them grew faster among all 30 strains. After preliminary inoculation test, a total of 12 strains had successful nodule. The results of 16S rDNA identification indicated they were Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium iaoningense, and Rhizobium multihospitium, respectively, which gave a total of 4 species and 2 genera. Four strains identified were tested to compare the population number and the ability of symbiotic nodulation under different pH. Results showed that at pH 6.8 would give the best population and nodule number. To examine the ability of symbiotic nodulation in soil environment, “I-Tiao-Gung” were inoculated with four strains in three soils. Among four inoculated strains, Br. iriomotense had the best nodule quantity and the highest nitrogenase activity. However, high available nitrogen would inhibited ability of nodulation and nitrogen fixation activity.

摘要......................................................I
Abstract................................................III
目錄......................................................V
表次.....................................................IX
圖次......................................................X
一、 前言.................................................1
二、 前人研究.............................................3
(一) 一條根...............................................3
1. 金門一條根之品種來源...................................3
2. 植物分類學之特徵.......................................3
3. 生長特性...............................................4
(二) 根瘤菌...............................................4
1. 生物固氮作用之研究背景.................................4
2. 根瘤菌之分類...........................................6
3. 豆科植物與根瘤菌之共生感染、發育及功能.................7
(三) 一條根共生根瘤菌.....................................9
三、 材料與方法..........................................11
(一) 種子發芽率..........................................11
1. 不同處理方法對種子發芽之影響..........................11
2. 處理時間對種子發芽之影響..............................11
3. 種子保存時間對種子發芽之影響..........................12
(二) 土壤基本性質分析....................................12
1. 酸鹼值................................................12
2. 有機質................................................12
3. 無機態氮..............................................13
4. 有效性磷 (Mehlich-III P)..............................13
5. 質地..................................................13
(三) 根瘤菌分離與鑑定....................................16
1. 根瘤菌之純化與培養....................................16
2. 菌株之 16S rDNA 萃取與純化............................18
3. 16S rDNA 序列比對與演化樹.............................23
4. BOX-PCR 建立指紋圖譜..................................23
(四) 根瘤菌回接一條根之結瘤測試..........................25
1. 在不同pH環境下根瘤菌族群生長數目......................25
2. 根瘤菌在不同pH環境下結瘤能力..........................25
3. 接種根瘤菌對一條根生長影響............................27
(五) 根瘤固氮能力測定....................................27
四、 結果................................................29
(一) 一條根種子處理與保存對發芽率之影響..................29
(二) 根瘤菌分離與鑑定....................................33
1. 根瘤菌之純化與培養....................................33
2. 16S rDNA 序列分析與演化樹.............................33
3. BOX-PCR 建立指紋圖譜..................................38
(三) 根瘤菌在不同環境下之結瘤能力........................40
1. 在不同 pH 下根瘤菌族群數目............................40
2. 根瘤菌在不同 pH 環境下接種能力........................42
3. 接種根瘤菌對一條根生長影響............................45
五、 討論................................................48
(一) 一條根種子處理與保存對發芽率之影響..................48
(二) 根瘤菌分離與鑑定....................................50
(三) 根瘤菌在不同環境下之結瘤能力........................54
六、 結論................................................58
七、 參考文獻............................................60

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