【作者】 张琴；

【导师】 张磊；

【作者基本信息】 西南大学 ， 微生物学， 2006， 硕士

【摘要】 紫花苜蓿是一种优质豆科牧草，有“牧草之王”的美称。近年来，随着对紫花苜蓿需求量的增加，紫花苜蓿产业化进程加快，苜蓿在酸性土壤上种植的呼声也愈高。在我国，紫花苜蓿主要种植在北方中性和碱性土壤上。随着西部大开发和退耕还林还草工作的进一步深入，产业结构的调整，紫花苜蓿的种植范围不断扩大，南方苜蓿种植已经成为苜蓿产业化的重要方面。然而，我国南方酸性土壤分布广、面积大，耐酸苜蓿根瘤菌资源缺乏。为此，筛选耐酸苜蓿根瘤菌十分必要。 国外在中度酸性土壤上种植紫花苜蓿的经验表明，酸性土壤上接种高效耐酸苜蓿根瘤菌是提高紫花苜蓿产量和品质的重要措施。借鉴国外经验，本研究从我国西南地区土壤分布类型和苜蓿种植情况入手，对紫花苜蓿耐酸根瘤菌进行了筛选，研究了耐酸苜蓿根瘤菌的生长特性、对紫花苜蓿生长和结瘤的影响，探讨了Ca²⁺作用下的耐酸机理。并通过田间试验，检测施Ca²⁺和接种耐酸苜蓿根瘤菌对酸性土上紫花苜蓿生长及产量和品质的影响，以期建立一套耐酸苜蓿根瘤菌筛选和鉴定体系，为在西南紫花苜蓿新种植区接种高效耐酸苜蓿根瘤菌奠定基础。研究的主要结果如下： 1．从在酸性土（pH5.2）上生长的紫花苜蓿根瘤中分离到若干苜蓿根瘤菌株，其中六株根瘤菌能够在pH4.8的固体培养基上稳定生长，分别编号为91511、91512、91521、91522、91531、91532，这六株根瘤菌均能使紫花苜蓿植株在9d左右结瘤，培养30d后，苜蓿植株平均每株结瘤数达10个以上，结瘤率也较高，约为66.67％-100％。 2．六株苜蓿根瘤菌在不同酸度（pH7.0、pH6.0、pH5.5、pH5.2、pH5.0、pH4.8）下生长曲线的测定结果表明，酸显著降低根瘤菌的生长速率，使其生长延滞期增长，对数生长期滞后，稳定期延迟。六株根瘤菌均能在pH5.0及以上的液体培养基中生长，菌株91532还能在pn4.8液体培养基中生长，说明，在低pH条件下，耐酸苜蓿根瘤菌在其耐受酸度范围内经过延长的延滞期后仍然能够较快地生长。平均代时的测定结果表明，酸性条件下各根瘤菌的平均代时均较中性条件下的长，环境pH越低，菌株平均代时越长。 3．采用正交试验法研究了两个苜蓿品种在不同环境pH（pH4.8、5.2、5.6）下接种六株苜蓿根瘤菌株的效果。结果表明，菌株、环境pH、植株品种对苜蓿整株鲜重的影响均极显著，影响程度依次为环境pH（F=71.8970）、苜蓿品种（F=27.0499）和菌株（F=11.8361）；环境pH、菌株对苜蓿有效根瘤数有极显著影响，植株品种的影响不显著，环境pH是影响苜蓿有效结瘤的最主要因素（F=8.0809）；苜蓿根瘤菌对苜蓿根瘤固氮酶活性的影响极显著（F=14.4269），环境pH的更多还原

【Abstract】 Alfalfa (Medicago sativa L.) is a fine forage legume, gaining the name of "king of pasture". In recent years, with the area of alfalfa plantation increasing, necessity to plant alfalfa on acid soils increases. In China, alfalfa mainly grows on northern neutral and alkali soils. As the program of developing western China and removing furrow to forest of grass progressing, industry structure regulating, and the growth area of alfalfa extending, planting on southern soils became important for the industrialization of alfalfa. However, there’s a lot of acid soil in South of China, and acid-tolerant rhizobia are in short. So, isolating acid-tolerant R. meliloti is necessary.The foreign experience of planting alfalfa on moderately acid soil showed that inoculating acid-tolerant rhizobium could improve the yield and quality of alfalfa. According to foreign experience, we started with the soil type and alfalfa growth on southwestern China, studied isolation of acid-tolerant R. meliloti, explored the growth characteristic of acid-tolerant rhizobium, and the effects on growth and nodulation of alfalfa, probed the acid-tolerance mechanism with the addition of calcium. In addition, field experiments were carried to examine influence on lucerne’s growth, yield and quality by applying calcium and inoculating acid-tolerant R. meliloti. All the measures taken above were in order to set up a system isolating and identifying acid-tolerant rhizobium, and to establish the foundation of effective inoculant for new planting areas in southwest Main results were listed as follows:1. Several R. meliloti were isolated from nodules of alfalfa grown on acid soils (pH5.2), six could grow on the solid media with pH4.8 steadily. The six rhizobia were numbered 91511, 91512, 91521, 91522, 91531, 91532, which could nodulate alfalfa in about 9d, make alfalfa gain high number of nodules (above 10 per plant) and high nodulation occupancy (about 66.67%-100%).2. Growth curve of the six R. meliloti were examined under different acidity (pH7.0, pH6.0, pH5.5, pH5.2, pH5.0, pH4.8). The results indicated that acidity slowed Rhizobia’s growth significantly, which made the rhizobia exhibit a longer lag phase, a lagged log phase and a delayed stationary phase. When incubated in liquid medium, the six strains showed the ability to grow at pH5.0 and above but could not grow at pH4.8 except strain 91532. So we can conclude that under low acidity, the acid-tolerant更多还原