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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 土壤環境科學系 > 依資料類型分類 > 碩博士論文 >  豇豆根瘤菌篩選、固氮能力評估培養基比較及 對豇豆生長影響之研究

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/151644

標題: 豇豆根瘤菌篩選、固氮能力評估培養基比較及 對豇豆生長影響之研究
Studies on isolation of cowpea symbiotic nitrogen fixation bacteria, culture media and effect on cowpea (Vigna unguiculata) growth
作者: 潘佳辰
Pan, Chia-Chen
Contributors: 黃裕銘
土壤環境科學系所
關鍵字: 豇豆;固氮細菌;栽培介質
Cowpea;nitrogen fixation bacteria;cultivation media
日期: 2013
Issue Date: 2013-11-07 11:39:22 (UTC+8)
Publisher: 土壤環境科學系所
摘要: 製作氮肥消耗之能量占總農業操作50%以上。豇豆接種優良豇豆固氮菌株,可以提供豇豆生長但源所需。豇豆共生性根瘤菌在台灣的研究較少。豇豆回接試驗發現用蛭石介質時,植株生長受到抑制。本研究目的可分為一、篩選豇豆共生性固氮菌。二、篩選及評估適合豇豆生長之培養介質。三、評估豇豆共生性固氮菌施用於實驗室、盆栽及田間試驗中對於豇豆生長促進效率。四、於田間試驗中進行種子拌種並比較豇豆生長情形。
由豇豆根瘤篩選固氮菌結果中,由49種土壤以豇豆 (栽培種:101) 培養28天,經初步回接試驗篩選出7株豇豆共生性固氮根瘤菌,經過16s rDNA鑑定得知六株為Bradyrhizobium sp.,一株為Ensifer sp.。結瘤能力及乙炔還原能力測試顯示TY5具有最高的根瘤數目為25顆,根瘤新鮮重以6243最高為0.369 g,而MY2-23 (2) 在乙炔還原法有最高的表現分別為890 nmole C2H4 plant-1 hr-1及8715 nmole C2H4 g-1 hr-1。菌株耐酸鹼能力試驗將菌株接種至pH 4.8、5.8、6.8、7.8、8.8YEM培養液,MY2-23 (2) 於pH 7.8 O.D.600測值最高;DL4 (2) 於pH 6.8最佳;TY5於pH 6.8最佳;PT2於pH 4.8最佳;6242於pH 6.8最佳;6243於pH 6.8最佳;632於pH 7.8最佳;菌株耐電導度能力試驗將菌株接種至含NaCl 0.02、1、3、5、7% YEM培養液,MY2-23 (2)、PT2能生長至1% NaCl YEM培養液中;DL4(2)、TY5、6242、6243、632能生長至0.02% NaCl YEM培養液中。介質試驗中以泥炭土、真珠石、洋菜培養基、酸化蛭石為栽培介質。豇豆在酸化蛭石中生長受到抑制,而在泥炭土、真珠石、洋菜培養基處理則生長情況較好。泥炭土、真珠需將根系拔出才可觀察根瘤生長;洋菜培養基則易於觀察,但根瘤生長空間僅洋菜膠與空氣的交接處,易低估微生物效率。由介質試驗中,泥炭土處理由植株鮮、乾重中顯示適度的氮肥、充足的養分可以增加植株生長,且在根瘤數上並無明顯減少。由於回接試驗中事先去除養液配方之硝酸鉀故需額外添加4.38 g L-1氯化鉀,結果顯示添加氯化鉀後可顯著增加豇豆鮮、乾重,卻會降低乙炔還原能力及根瘤數。為使植體內養分間平衡及提高乙炔還原能力的準確度,額外添加鉀源是必要的,然鉀濃度需要進行修正以降低對鈣、鎂的擷抗作用。豇豆栽培種與菌株試驗中,選用豇豆栽培種為101、131、白皮 (White);菌株為TY5、MY2-23 (2)。101為菌株篩選宿主。接種MY菌株中101有最高鮮重及乾重;接種TY菌株以131鮮重最高、White乾重最高。131與MY2-23(2)及TY5有較高的乙炔還原能力表現,而非101品系,顯示根瘤菌與豇豆間存在不同的親和性。
盆栽試驗及田間試驗探討氮肥、接種根瘤菌及施用殺菌劑對豇豆 (品種 101) 接踵固氮菌之生長差異。在盆栽試驗中添加氮肥與否對於豇豆鮮重、乾重沒有明顯差異。田間試驗結果發現,添加豇豆固氮菌可以提升根瘤數目,但無提升產量。可能原因為本試驗進行時間為冬季,根瘤消耗較多碳水化合物以維持固氮效率;或是當地土生豇豆固氮菌較接踵之菌株提供較多氮源。土壤添加氮肥及殺菌劑會降低根瘤數目;接踵豇豆固氮細菌會增加根瘤數目。添加豇豆固氮細菌及殺菌劑皆會降低白粉病發病率。
The production of nitrogen fertilizer is the most energy consuming activity in agricultural sector. A good symbiotic nitrogen fixation bacteria can contribute main part of nitrogen requirement for legume crops. In Taiwan, there was few reports about cowpea symbiotic bacteria. In preliminary experiment, the growth of cowpea was seriously inhibited in non pH adjusted vermiculite medium. The purposes in this thesis were: First to isolate and identify the cowpea symbiotic nitrogen fixation bacteria for cowpea at first. Secondly, to develop a suitable culture medium formula for easier handle in bacteria re-inoculation test. Thirdly, to evaluate the plant promoting ability of the bacteria selected on the growth of cowpea in indoor controlled system, greenhouse, and field experiments. Last, to evaluate the effect of the fungicide and nitrogen fertilizer on the efficiency of the selected cowpea symbiotic nitrogen fixation bacteria.
There were 7 effective cowpea symbiotic nitrogen fixation strains selected from 49 soil samples by using cowpea cultivar 101. MY2-23(2) strain is Ensifer sp. and six strains are Bradyrhizobium sp.. Nodule growth showed strain TY5 had largest number of nodules per plant, strain 6243 had the highest fresh nodule weight per plant. MY2-23(2) showed the highest ethylene reduction ability in 890 nmole C2H4 plant-1 hr-1 and 8715 nmole C2H4 g-1 hr-1. The 7 selected strains were cultured in pH of Yeast Extract Medium (YEM) at 4.8, 5.8, 6.8, 7.8, 8.8 and their growth were detected under O.D. 600. The results showed MY2-23(2) and 632 strains were grown better in alkaline range, DL4(2), TY5, 6242, 6243 strains were grown better in neutral range, PT2 strain was grown better in acid range, growth well in pH 4.8. In salinity tolerance test, the selected strains were cultured in YEM prepared in 0.02, 1, 3, 5, and 7% NaCl strengths. The results showed MY2-23(2)、PT2 can growth in 1% NaCl YEM solution. DL4(2)、 TY5、 6242、6243、632 can growth only in 0.02% NaCl YEM solution. In culture medium experiment, peat, perlite, agar, and acidified vermiculite were used in growing cowpea inoculated with the selected strain. The growth of cowpea in vermiculite medium was inhibited even whose pH was already pre-acidified, the growth of cowpea other three media were acceptable. However, it seems more convenient as cowpea growing in agar medium for easier observing the development of nodule for its partial transparent character. Additionally, peat used in this study containing more available nitrogen than other media but the number of nodules formed didn’t reduce in significant level. An extra potassium source added with 4.38 g L-1 chloride potassium into nutrient solution which can rise the fresh and dry weights significantly but reduce the ability of ethylene reduction and number of nodules. Three cowpea cultivars (101, 131, and white) and two nitrogen fixation bacteria (TY5, MY2-23(2)) were used to examine the cross infection between the cowpea and strains although both strains were selected through cultivar 101. In the result of inoculation, 101 had more fresh and dry weights inoculated by MY2-23(2), and cultivar 131 had more fresh weights and cultivar White had more dry weights as inoculated by TY5. The ethylene reduction ability of MY2-23(2) and TY5 strains in cultivar 131 were better than in cultivar 101.
The combination effects of nitrogen fertilizer, nitrogen fixation bacteria, and fungicide on cowpea growth were examined in factorial combination of these three factors in pot and field experiments. In pot experiment, results showed that a higher fresh and dry weights in inoculating treatments no matter with nitrogen application or or not. In field experiment, although the formed nodules in inoculating treatments is higher, but lower cowpea pod productions was found. This may be due to the cowpea is not suitable for the winter season and more nodules may consume more carbohydrate from the host or may be the native nitrogen fixation bacteria is more efficient in nitrogen fixation than the introduced bacterium. The use of fungicide showed a decrease of the nodule formation. The disease severity of powdery mild were decreased by applying fungicide and cowpea nitrogen fixation bacteria. This will be a good practice in plant disease control and worth to pay attention to.


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