Plant growth and biomass increment

At the beginning of the experiment seedlings transplanted into the different soil treatments showed no significant differences in plant height, diameter and biomass. During the two growing seasons, liming strongly affected plant development, whereas effects due to inoculation were less pronounced. The cultivation of seedlings in limed soils resulted in significantly higher plant growth and biomass increment after two years (Fig. 3.2, Tab. 3. 1).

The inoculated seedlings in the unlimed soil grew significantly slower than uninoculated seedlings in the first vegetation period, but due to higher growth (not significant) in the 30

second year, no differences between the two treatments were observed for the complete growing period. Also, in the soil with moderate CaCO3 application (treatment b) the inoculation with Rhizobium resulted in significantly higher seedling growth in the second growing season, but not in the first year or for the cumulative growth over two years. For soil treatment c (strongly limed soil), only the diameter increment of seedlings in inoculated pots was significantly higher in the second vegetation period. The height and diameter growth of inoculated seedlings in all soils was observed to be lower in the first year of cultivation than in soils without Rhizobium addition. The fixation of atmospheric nitrogen is a highly energy consuming process. The carbon costs for the fixation are between 4mg (Warembourg &

Roumet 1989) and 6mg C/mg N fixed (Werner 1987). Therefore growth of plants in an early stage is often lower, when soil nitrogen is limited and plants rely on fixation as the only N source (Johnsen & Bongarten 1992), which was not the case in this study. Balla et al. (1998) reported that a beneficial growth stimulation of black locust seedlings can occur later, depending on the strains of Rhizobia used for soil inoculation.

The highest nodule biomass increment and total nodule number, especially the number of small nodules (<2mm) of all treatments were found in the moderately limed soil with inoculation (b / Inoc.) (Table 3.1). Compared to the uninoculated treatment of this soil, the increment of the leaf biomass and the number of nodules with a diameter of 2-5mm (medium size) were significantly higher. Significant differences in the total number of nodules between unlimed and strongly limed soil were found for the uninoculated treatments.

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Absolute height growth in the different variants

Absolute diameter growth in the different variants

0

Figure 3.2: Absolute height and diameter growth of black locust seedlings cultivated in soils with different starting pH and Rhizobium application in 2002 (first year), 2003 (second year) and for the complete observation period from May 2002 to October 2003.

Data presented as medians (n=15) with quartiles (Q25, Q75). Bars denoted with different letters (a, b) are significantly different between inoculation treatments within the same lime treatment and measuring interval; bars denoted with * are significantly different in comparison to the unlimed soil variants within the same inoculation treatment and time interval.

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Liming of acid soils with moderate amounts of CaCO3 may promote the development of and infection with Rhizobium bacteria. According to Marschner (1995), N2 fixation of legumes can be affected indirectly or directly by mineral nutrients. Root infection and nodule initiation have a much higher calcium requirement than the root and shoot growth of the host plant.

Table 3.1: Mean (± standard error) absolute biomass increment of Robinia seedlings in soils with different starting pH and treatments (Inoc. = inoculated; Un-Inoc. = uninoculated) from May 2002 to October 2003 and mean number of nodules per plant in October 2003 (n=15).

Absolute biomass increment

Values denoted with ⁺ are significantly higher between different treatments within the same soil pH, values with different superscript letters (^{a, b}) are significantly different between soils of equal treatment (p<0.05)

May 2002-October 2003 Nodule number in October 2003 Initial Soil pH /

Treatment

Leaves Stems Roots Nodulestot Biomasstot Big Normal Small Total

Franco and Munns (1982 a, b) observed a decrease of soybean nodulation and a simultaneous decrease in root hair length by lowering the pH from 5.5 to 5.0. For white clover (Trifolium repens), changes in nodulation were more closely associated with changes in soil pH than soil Ca (Brauer 1998; Brauer et al. 2002). Furthermore, the availability of soil N (NO3-, NH4+) can enhance or depress nodulation and N2 fixation, depending on plant genotypes and the form and level of the N supply. For black locust, Roberts et al. (1983) observed that seedlings grown in nutrient solution with combined nitrogen (5mM NH4NO3) had significantly lower N2 fixation rates but higher relative growth rates than seedlings grown without additional nitrogen supply.

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To determine differences in the activity of nodules in all treatments, the CO2 production of fresh nodules was measured directly after harvesting (Table 3.2). Inoculation of soil had no effect on the measured CO2 concentrations (mg CO2 * g^-1 DW * h^-1) or on CO2 production per nodule biomass (mg CO2 * h^-1) within the same soil pH treatment.

Table 3.2: Mean CO2 production (± standard error) of different sized nodules per mg nodule dry weight (mg CO2* g^-1 DW * h^-1) and plant (mg CO2* h^-1) from soils with different starting pH and inoculation treatment, measured directly after harvesting the plants in October 2003 (n=15).

Mean CO2 production of nodules Initial Soil pH / Values with different superscript letters (^{a, b}) are significant different between soils of equal treatment (p<0.05)

Respiration of nodules from the strongly limed soil was higher than from the other two lime treatments, but these differences were not always significant. Liming of soil elevated the number and biomass of nodules (medium, small, and total) and therefore in some cases also the total CO2 production of the nodules per plant.

Measuring the CO2 production of fresh nodules as an indicator for nodule activity and efficiency of N2 fixation is just a rough technique to get an impression of possible differences due to variable growth conditions of the host plant. According to Hoffmann (1964), nodule number and biomass are not correlated to the nodule activity and efficiency of N2-fixation.

Statements about the efficiency of nodules are possible through the analysis of the N-content of the complete plant, only. In recent studies, nodule activity and amount of fixation are

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determined by the acetylene reduction assay (Roberts et al. 1983; Ibekwe et al. 1997) or the 15N isotope dilution method (Danso et al. 1995; Olesniewicz & Thomas 1999).