【作者】 韩华君；

【导师】 谢德体； 张磊；

【作者基本信息】 西南大学 ， 土壤学， 2007， 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，我国实施了“北草南引”的计划。“北草南引”是推进紫花苜蓿在我国南方种植的重大举措。然而，我国南方酸性土壤分布广、面积大，紫花苜蓿是对酸最敏感的豆科牧草，酸性土壤的存在严重阻碍了紫花苜蓿的“北草南引”及其产业化进程。因此在南方酸性土壤上接种耐酸根瘤菌建立高效共生抗逆系统显得尤为重要。苜蓿根瘤菌与苜蓿的共生是长期以来研究苜蓿的重要方面，高效耐酸苜蓿根瘤菌接种苜蓿将有助于在我国南方建立抗逆的苜蓿高效固氮系统。随着豆科植物在酸性土壤中的应用日益推广，接种耐酸根瘤菌已经作为提高酸性土上豆科牧草产量和品质的重要途径。而所接种根瘤菌在土壤中生存定殖能力的强弱直接影响着接种的效果。同时，根瘤菌施入土壤以后，对根际其他微生物和微生物生物量均有一定的影响。因此，加强根瘤菌根部定殖的研究对于揭示有益细菌在根圈的微生态学特征、提高其根际适应性和增产效果的稳定性具有十分重要的意义。同时，Ca²⁺作为一种信号传递因子，是一种能促进苜蓿根瘤菌耐酸的重要因子，其促进苜蓿根瘤菌及其共生系统耐酸的作用已得到了一定的研究。因此，本研究在缙云山酸性土壤上接种耐酸苜蓿根瘤菌，并施用不同浓度的Ca²⁺，探讨其在酸性土壤上的定殖及其对苜蓿根际土壤微生态环境的影响，进而分析了施Ca²⁺和接种耐酸苜蓿根瘤菌对紫花苜蓿共生效应的影响。可以为在南方紫花苜蓿新种植区接种高效耐酸苜蓿根瘤菌提供一定的理论和技术依据。研究的主要结果如下：1．采用裂区设计缺三区处理，在缙云山酸性土壤中（pH4.6）接种三株由本实验室分离的耐酸苜蓿根瘤菌91522、91512、91532，并施用不同浓度的Ca²⁺（0mmol/L、5mmol/L、10mmol/L），结果表明：三种耐酸苜蓿根瘤菌均能在酸性土中定殖，且定殖动态基本一致，三种供试菌株与对照相比，其定殖数量没有显著性差异，但三种供试菌株的定殖数量均高于对照，以接种菌株91512和91532的效果最好。施Ca²⁺处理对根瘤菌定殖影响不明显，但仍以施5mmol/LCa²⁺时，定殖效果最佳。说明接种耐酸根瘤菌并施用适当浓度的Ca²⁺，对根瘤菌在酸性土壤中的定殖有一定的促进作用；2．施Ca²⁺和接种耐酸苜蓿根瘤菌对苜蓿根际土壤微生物数量及其生物量的影响明显，其差异达到显著和极显著水平。其中，接着耐酸苜蓿根瘤菌后，细菌、真菌和微生物生物量均显著增加，而放线菌数量稍增加，未达到显著性差异。而三种菌株（91522、91512、91532）之间差异不显著。施Ca²⁺仅对细菌数量有显著性增加，而对其它微生物未产生显著性影响。可见在酸性土壤上，接种耐酸苜蓿根瘤菌和施Ca²⁺，改变了根际营养成分，有利于微生物的生长繁殖，从而使土壤的微生态环境得到改善，提高了微生物数量和微生物生物量；3．接种耐酸苜蓿根瘤菌后土壤微生物数量及其微生物生物量与对照相比，在大部分月份均明显增加，达到显著和极显著水平。四个处理的三大类微生物数量及其生物量均在6月份达到最大值。可见，随着根瘤菌的定殖，植株根系的生长发育，根系分泌物随之增加，为根际微生物提供了大量的能源，再加上土壤适宜的温湿条件，有利于各种微生物的生长繁殖。因而，使微生物的数量和微生物生物量达到了最大值；4．检测了施Ca²⁺和接种耐酸苜蓿根瘤菌对紫花苜蓿共生效应的影响。结果表明，接种耐酸苜蓿根瘤菌和施Ca²⁺对苜蓿植株瘤重、根鲜重、株高和植株上部鲜重的影响显著。其中接种菌株91512对苜蓿瘤重、株高和地上部鲜重的影响最大，根鲜重则以接种菌株91532的效果最好；而施Ca²⁺处理则以施5mmol/LCa²⁺时对共生效应的影响最大。说明接种耐酸苜蓿根瘤菌、施用适当浓度的Ca²⁺以及共生系统良好的结瘤性能能够提高酸性土上紫花苜蓿的产量和品质；5．施Ca²⁺和接种耐酸苜蓿根瘤菌对酸性土壤中土壤的养分状况有所改善，接种耐酸苜蓿根瘤菌和施Ca²⁺5mmol/L对土壤氮有明显的影响。各处理中，土壤交换性钙的含量均减少，而施5mmol/L的Ca²⁺处理，土壤中交换性钙减少最少。总体上看，除土壤中交换性钙含量减少，土壤全磷保持不变外，其余土壤养分含量均有所增加，但影响不显著。说明施Ca²⁺和接种耐酸苜蓿根瘤菌，改善了苜蓿植株的营养条件，促进了苜蓿根系的生长和代谢，进而促进根际微生物的生长和繁殖，使土壤的微生态环境得到改善，从而加速土壤养分的积累，提高了土壤的肥力。更多还原

【Abstract】 Alfafa is a high quality leguminous pasture, which is known as "kingdom of pasture". Recent years, the industrialization of alfafa is developing quickly with the increasing requirement, thus the demand is more that planting alfafa on the acid soil. In our country, alfafa mainly grow on the neutral and alkalescent soil in the north. With western exploition and the development of returning farmland to forestry and grassland, our county implied the plan of "bringing north pasture to south". This implement is an important action to boost the planting of alfafa. However, acid soils have a wide distribution and vast area in the south of China, in addition, alfafa is the most sensitive leguminous pasture, the existence of acid soils badly hampered the implement of "bringing north pasture to south" and the industrialization of alfafa. So it is important to establish a accrete system of highly effective and adversity resistance.The symbiosis of rhizobium and alfafa is an important aspect in the field of alfafa research, inoculating highly effective acid-tolerant rhizobium of alfalfa will help to establish an alfafa accrete system of highly effective and adversity resistance in the south of our country. With the increasingly spread of leguminous plant on acid soil, inoculating acid-tolerant rhizobium has already been an important approach to improve the yield and quality of leguminous pasture on the acid soil. However, the degree of viability and rhizobium colonization directly affects the inoculating effect. At the same time, rhizobium will affects other microorganism or microbial biomass to some distance after inoculating in the soil. So, it has a very important significance to strengthen the research of rhizobium colonization on the root, and it will help to discover the microecology characters of plant growth-promoting rhizobacteria, improve the adaptability in . rhizosphere and the stability of increase production.Meanwhile, as an ingredient that can transfer signal, Ca²⁺ is a vital factor which can promote the acid-tolerant ability of rhizobacteria. Thus, we studied that acid-tolerant rhizobium was inoculated on the acid soil in Jinyun mountain, and Ca²⁺ solution of three concentrations were applied on the soil, and discussed the rhizobium colonization on the acid soil and the effect that applying Ca²⁺ affected the soil microecological environment, then analyzed the affection of applying Ca²⁺ and inoculating acid-tolerant rhizobium to the symbiosis effect of rhizobium and alfafa. These can supply some theory and technology gist on the inoculating highly effective acid-tolerant rhizobium on the new south alfafa plantation. The main results followed:1.Bifida zone design which was lack of three zones was used, three strains of acid-tolerant rhizobium of alfalfa （91522、91512、91532） our lab had got were inoculated on the alfalfa, while Ca²⁺ solution of three concentrations （0mmol/L、5mmol/L、10mmol/L） were applied on the soil（pH4.6）. The results showed that all the three strains of acid-tolerant rhizobium of alfalfa could colonize on the acid soil, and the trends of colonization were basically consistent. Compared with the control, the colonization amount of all the three strains had no significant difference, but the three strains in the experiment all had higher colonization amount than that of control, the best effects appeared in inoculating the strains named 91512 and 91532. The treatments which were applied Ca²⁺ didn’t affect the rhizobium colonization significantly, but applying 5mmol/LCa²⁺ showed a best colonization effect. These indicated that inoculating acid-tolerant rhizobium and applying Ca²⁺ of suitable concentration will promote the colonization of rhizobium on the acid soil.2.Applying Ca²⁺ and inoculating acid-tolerant rhizobium of alfalfa could evidently affected the number of microorganism and microbial biomass in the .rhizosphere. The difference reaches a significant and very significant level. After inoculating acid-tolerant rhizobium of alfalfa, the number of bacteria、fungi and biomass increased significantly, actinomycetes increased a little but didn’t reaches the significant level. The difference among the three strains wasn’t significant. Applying Ca²⁺ only increased the number of bacteria significant, but not affected other microorganism significantly. It is obvious that inoculating acid-tolerant rhizobium of alfalfa and applying Ca²⁺ changed the nutrition ingredient of rhizosphere on the acid soil, it helped the microorganism to grow and propagation, so the microecological environment in the soil was meliorated, the number of microorganism and microbial biomass has also been increased.3.Compared to the control, after inoculating acid-tolerant rhizobium of alfalfa, the number of microorganism and microbial biomass had significant and very significant difference in most of the evaluating months. The number of microorganism and microbial biomass of three big types in the four treatments reached the maximum in June. It was obvious that with the colonization of rhizobium and the growth of plant roots, the increasing rhizosphere secretion provide large energy for rhizosphere microorganism, addition with the warm and wet condition of soil, all kinds of microorganism could grow and propagate quickly. Thus the number of microorganism and microbial biomass reached the maximum.4.Test the symbiosis of rhizobium and alfafa by inoculating acid-tolerant rhizobium and applying Ca²⁺. The results showed that inoculating acid-tolerant rhizobium and applying Ca²⁺ affected significantly on root fresh weight, plant height, fresh weight upground and nodule weight. Strain 91512 affected greatly on plant height, fresh weight upground and nodule weight, root fresh weight was affected greatly by strain 91532; by applying calcium 5mmol/L, the best symbiosis effect on alfalfa were gained, which showed that moderately calcium and acid-tolerant rhizobium of alfalfa were able to improve yield and quality of alfalfa.5.Inoculating acid-tolerant rhizobium and applying Ca²⁺can ameliorated the nutrition condition in the acid soil, acid-tolerant rhizobium and Ca²⁺ 5mmol/L greatly affected the total nitrogen of soil. In every treatment, exchangeable calcium of acid soil decreased, the least of decreased exchangeable calcium of acid soil was discovered in the Ca²⁺ 5mmol/L treatment. All soil nutrition increased not significantly, except that exchangeable calcium decreased and total P keep the number. It indicated that acid-tolerant rhizobium and Ca²⁺ ameliorated the nutrition condition that alfafa needed during their growing, promoted the growth and metabolism of alfafa roots, thus accelerated the upgrowth and propagation of rhizosphere microorganism, which can ameliorated the microecologica! environment and speed up the accumulation of soil nutrition, accompanied the improvement of soil fertility.更多还原