Studies on isolation of cowpea symbiotic nitrogen fixation bacteria, culture media and effect on cowpea (Vigna unguiculata) growth

• Main Authors: Chia-Chen Pan, 潘佳辰
• Other Authors: 黃裕銘
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/79936215889185260550

碩士 === 國立中興大學 === 土壤環境科學系所 === 101 === 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.