Biological nitrogen fixation by lucerne (Medicago sativa L.) in acid soils

Growth of lucerne( Medicago sativa L.) is poor in soils with values of pH-H2O below 6. This is often due to nitrogen deficiency, resulting from a hampered performance of the symbiosis with Rhizobium meliloti. This thesis deals with the factors affecting biological nitrogen fixation by lucerne in acid soils.In a field experiment, lucerne seeds were either inoculated with R.meliloti only,or inoculated and pelleted with lime, before sowing in a sandy soil of pH 5.2. Lime-pelleting significantly improved the establishment of the seedlings. This was caused by earlier nodulation, as evidenced by the formation of crown nodules, i.e.the nodules on the upper 10 mm of the seedling taproot. The number of seedlings carrying crown nodules tripled from 18% to 56%, 26 days after sowing as a result of lime-pelleting. The dry matter yield and nitrogen fixation of the plants at later stages showed a close correlation with crown nodulation.To minimize the problems inherent to field experiments,e.g.costs and labour, besides pots, rhizotrons (small root boxes made of plastic petri dishes) were developed to study the symbiosis under controlable conditions in the laboratory (Chapter 2). Comparative trials showed that crown nodulation in pots was practically similar to that in the field. In rhizotrons, the early root development and nodule formation could continuously be observed. moreover, lime- pelleting resulted in a greater increase of crown nodulation (5% to 90%) during a shorter incubation period (14 days).R.melilotibacteria are notorious for their acid sensitivity. As a consequence the increased crown nodulation by lime-pelleting might be the result of a better proliferation of these bacteria, thus resulting in a higher chance of root infection. To verify this hypothesis, the effect of lime-pelleting on the survival of inoculated R. meliloti around the seed was recorded during a period of 12 days following sowing. In the initial 12 hours a strong increase in rhizobial numbers coincided with the germination of the seed. Little differences in multiplication were measured in the presence or absence of a lime-pellet. To obtain optimal nodulation in this soil, however, lime-pelleting was a prerequisite and at least 10 5cells of R. meliloti per seed were required. In the absence of lime, only a few plants nodulated, even when the number of rhizobia was increased to 10 7per seed (Chapter 3). The nodulation effect of lime-pelleting could not be explained by a better rhizobial multiplication. The benefit of CaC0 3 can be due to either neutralization of soil acidity and/or to the input of calcium. To study the pH changes in the rhizosphere while leaving the spatial arrangement of the soil intact, the following method was deviced: The soil was covered with a thin layer of agar containing the pH indicator bromocresol purple. To prevent the contamination of agar with soil particles, a nylon gauze was used to keep the soil separated from the agar. Quantitative pH measurements were done by inserting a micro-electrode into the agar after 3 hours of contact with the soil. The validity of the data obtained by this method was investigated by comparing the results with those obtained using standard procedures for pH measurement in a soil-water suspension (Chapter 4). Using the agar-contact method, it was shown that the benefit of lime on the nodulation of lucerne was largely (80%) due to neutralization of acidity, and to a lesser extent (20%) to the supply of calcium. For nodulation, soil-pH measured around lime-pelleted seeds, should at least be 5.8. Untreated roots were also able to induce a pH-increase of 0.6 units at 6 days after sowing (d.a.s.) or later, but only very few plants nodulated. These results, obtained with soil-grown plants, confirm earlier findings with hydroponically grown plants, that nodulation is sensitive for acidity only during the early phase of the process (0 to 3 d.a.s.) (Chapter 5).The soil already contained 1.2 meq of calcium per 100 g. Hence, the contribution of calcium in the lime effect was rather small. When the native calcium was removed with a specific calcium chelator (EGTA). crown nodulation could further be reduced to 12%. However, an additional complication was the acidification that accompanied the chelation of soilcalcium, with soil-pH dropping from 6.1 to 5.2. This problem could be overcome by applying small agar blocks containing the chelator. This technique allowed local application and temporal treatment of the root with EGTA. It was shown that the nodulation could be inhibited by calcium depletion only during the first day (Chapter 6). Obviously, the early phase of the nodulation of lucerne in acid soil is transiently susceptible to low calcium availability, similar to the transient sensitivity to low pH.