Abstract: Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield in response of Leucaena leucocephala and Sesbania sesban to inoculation with auxotrophic mutants of fast growing Rhizobium strains was explored in short-term field trials. All the strains formed nodules and fixed nitrogen in both hosts with some relative differences. The diversity of rhizobia that form symbioses with the roots of both hosts, an economically important leguminous tree species, was examined by inoculating seedling root zones with samples of auxotrophic mutants of rhizobia derived from both mutagens, acridine and ascorbic acid. Nitrogen fixation, total nitrogen accumulation, and plant growth varied significantly among both hosts seedlings inoculated with the representative isolates. All auxotrophic mutants derived from the parental strain FFAMU-8, stimulated chlorophyll (a) formation in Sesbania sesban, relative to the negative control. Although, three of auxotrophic mutants, asc2-FFAMU-8, asc3-FFAMU-8 and asc4-FFAMU-8 affected to significantly increase chlorophyll (b) formation than their negative and positive control plants. Both auxotrophic mutants, asc3-FFAMU-8 and asc3-ARCG-10 affected to significantly greater below ground biomass components in Leucaena leucocephala than their in the positive control plants. In addition, some of auxotrophic mutants derived from the strain HRT-27 affect to significantly increase woody, aerial and root biomass over the positive control of Sesbania sesban. Some of auxotrophic mutants appeared reliable ranking for nodule development/plant biomass among both hosts. Many of significant correlations were obtained among both tree legumes between nodulation, nitrogen fixation parameters with plant growth criterion. The results support the use of efficient rhizobial strains to inoculate woody legumes for improving plant survival and biomass development.
Introduction
Nitrogen fixing trees (NFTs), like their herbaceous counterparts, may enhance soil fertility through the return of N-rich litter if nodulated with effective rhizobia. The integration of NFTs in crop production systems is seen as a viable alternative to N fertilizer application, especially for the resource poor farmers in the tropics (Nyamai, 1992). In addition, they can also provide protein (seed and leaves) for man and livestock (Topps, 1992). The formation of nitrogen-fixing root nodules on legumes by rhizobia is a complex developmental process that involves constant communication between the partners. Products of the phenylpropanoid biosynthetic pathways, flavonoids (Rolfe, 1988), have been widely identified in leguminous plants as being involved in the initial signalling steps between plant and rhizobia, including chemotaxis towards a potential host (Kape et al., 1991) and induction of the bacterial nodulation (nod) genes which are necessary to elicit a corresponding signal to the plant from the bacterium (Dahiya, 1991).
Globally, tree legumes are an important source of timber, fuel and fodder. Their ability to fix N2 in association with Rhizobium, Bradyrhizobium and Azorhizobium bacteria means they can meet their N requirements directly from symbiosis. Additionally, they can improve the N fertility of soils in which they grow through release of symbiotic N from decomposing organic residues. Consequently, tree legumes play a vital role in rehabilitation of degraded and marginal soils and restoration of nutrient fertility in fields exhausted by intensive cultivation. The objective of the present study, therefore, were II) to evaluate the ability of the auxotrophic mutants of Rhizobium spp. derived from acridine and ascorbic acid to renodulate Leucaena leucocephala and Sesbania sesban, and (ii) to determine whether inoculation actually increases biomass of tree legumes.
Materials and Methods
Bacterial strains and media: Rhizobiam strains and their auxotrophic mutants induced which used in the present study are listed in Table 1.
Yeast extract mannitol medium (YEM) was used for culture maintenance according to Vincent (1970). Tryptone yeast extract medium (TY) was used as a complete medium to ensure the independence of mutations according to Beringer (1974). Evaluation of the symbiotic performance of auxotrophic mutants in pots experiment: Pots were filled with sand and then washed with hydrochloric acid for 24 hours, after that with tap water for 48 hours. The gars were sterilized with chloroform 0.25%, then washed three times with tap water until all chloroform disappear from all pots. Five sterilized Sesbania sesban and Leucaena leucocephala seeds were cultivated in each of three replicate systems and thinned to three seedlings after 15 days of growth (Vincent, 1970). Then they were inoculated with three ml of rhizobial broth culture per plant. The plants were irrigated using free nitrogen nutrient solution of Bond's modified Arone's stock mixture (Allen, 1959). Plants were harvested after three months of planting and growth was measured as plant height, stem diameter and plant total biomass. Nodules, roots and shoots were oven dried at 70°C prior to dry matter determination. The plant samples were finely ground and weighed out. Total N in each sample was analyzed following kjeldahl digestion, and N2 fixation measured as the difference between total N in inoculated (nodulated) plant and total N in uninoculated (non-nodulated) plants. Inoculated and uninoculated Sesbania sesban and Leucaena leucacepala plants together with controls were also harvested and their root stems were observed for nodule formation and nodule number per plant.
Nodule numbers and plant dry weight: The plants of three replicates each containing two plants from tube method experiment, and three plants from pot system experiment were removed and washed by tap water. Then the number of nodules per plant was calculated for both Sesbania sesban and Leucaena leucocephola. The plants were dried at room temperature for two days, then oven dried at 70°C for two days and put in the desiccator before measurement of dry weight. The dry weight per plant was calculated for each inoculation treatment.
Photosynthetic activity: Chlorophyll a, b and total chlorophyll were determined after 13 weeks of planting using a spectrophotometric method, for measuring optical density (OD) according to Mackinney (1941).
Determination of nitrogen content in plant: Nitrogen content in dried plant materials was determined by the wet digestion of dried and finely pulverized plant material using the macrokjeldahl method (Jackson, 1958).
Nitrogenase activity: N2(C2H2)-fixation potential of root nodules was estimated by the acetylene reduction assay according to Hardy et al. (1968) and Dart et al. (1972).
Statistical analysis: Data were subjected to statistical analysis of variance using the Statistical Analysis System (SAS). When analysis of variance showed significant effect between treatments, the least significant difference (LSD) test was applied to make comparisons among the means at 0.05 and 0.01 levels of significance (Steel and Torrie, 1980).
Results and Discussion
Effect of inoculation with Rhizobium on chlorophyll content:The effect of several inocula of Rhizobium auxotrophic mutants on the chlorophyll content in Sesbania sesban and Leucaena leucocephala are shown in Table 2 and 3, respectively. As shown from Table 2, relative to the control, all auxotrophic mutants, derived from parental isolate FFAMU8, stimulated chlorophyll (a) content. In addition, three auxotrophic mutants, asc2 FFAMU8, asc3-FFAMU8 and asc4-FFAMU8; were significantly increase chlorophyll (b) content than their negative and positive control plants. Non of auxotrophic mutants showed significant increase in total chlorophyll content, if comparison with the positive control; but all of them showed significant increase in total chlorophyll content, if comparison with the negative control.
In respect of relative increase in total chlorophyll content (a+b), all of auxotrophic mutants and their parental wild type isolate showed significant relative increase than uninoculated plants. On the other hand, there is no significant increase in total chlorophyll content was obtained by any of auxotrophic mutants induced from ARCGio if compared with their positive and negative control. The results obtained here concerning the effect of Rhizobium inoculation on the significant increase of chlorophyll formation indicated that nitrogen fixation by Rhizobium wild type and auxotrophic mutants has a stimulatory effect on chlorophyll formation. This are in agreement with Gupta et al. (1992), who reported that NO2 has a stimulatory effect on photosynthesis (PN).
The results obtained in Table 3 did not show any significant differences between treatments with different Rhizobium inoculation for chlorophyll content in Leucaena leucocephala. This indicated that there were no clear differences between treatments in the mechanisms of chlorophyll formation. Sims et al. (1998) reported that nitrogen supply primarily affected photosynthetic capacity per unit volume of tissue, whereas photosynthetic capacity per unit nitrogen was significantly affected only by nitrogen supply. Treshow and Anderson (1989) reported that high concentration of NO2 (above 0.5 μmol mol–1) are known to inhibit plant growth. Low levels (up to 0.2 μmol mol–1) of NO2, on the other hand, increase the rate of photosynthesis, N and chlorophyll both in soybean [Glycine max (L.) Merr.] (Sabaratnam and Gupta, 1988) and in black turtle bean (Phaseolus vulgaris L.) (Sandhu and Gupta, 1989).
Biomass production: Table 4 shows that belowground biomass components in Sesbania sesban was significantly greater after the plants were inoculated with both auxotrophic mutants asc-3 FFAMU-8 and asc-3 ARCG-10, if compared with the positive control. The plants inoculated with different auxotrophic mutants showed insignificant differences for the aboveground biomass components. The present results are in agreement with Lal and Khanna (1996a), who found that biomass yield of all inoculated plants of L. leucocephala at the end of five years showed a significant increase over the uninoculated plants, among the three strains tested, one strain gave the maximum response (45% more dry matter) in biomass production and w as followed by another strain, which showed 27% increase over the negative control (uninoculated plants). The results summarized in Table 5 demonstrated that some of the auxotrophic mutants derived from the isolate HRI-27 revealed a significant increase over the positive control plants for woody, aerial and root biomass. This indicated that these auxotrophic mutants may efficient nitrogen fixers resulting in higher biomass production of Leucaena leucocephala, whereas the others proved to be a poor nitrogen fixers (Lal and Khanna, 1996a). These observations are in agreement with previous reports on inoculation of woody legumes with selected rhizobial strains, which showed shoot biomass yield was the major criterion in the selection of rhizobial inoculants. Short-term observation on the effect of Rhizobium on biomass yield of tree legumes do not give a true picture, a long-term study is essential to assess the potential of Rhizobium in increasing the biomass yield of tree legumes. The obtained results are in accordance with Lal and Khanna (1996b), who found that in nursery and short-term field trials of Acacia nilotica and Leucaena leucocephala the inoculation of these tree legumes with specific and effective strains of Rhizobium spp. had a positive effect on tree biomass (expressed as total dry matter). Shoot biomass yield was the major criterion in the selection of rhizobial inoculants.
Nodulation of woody legumes: Results in Table 6 show that auxotrophic mutants of rhizobia capable of eliciting nodules more than 40 per plant by both isolates derived from strains FFAMU-8 and ARCG-10.
| Table1: | Source of Rhizobium strains used in the present study |
| Table 2: | Effect of inoculation with Rhizobium biochemical mutants on chlorophyll content (mg/g fresh weight) in Sesbania sesban |
| Table 3: | Effect of inoculation with Rhizobium biochemical mutants on chlorophyll content (mg/g fresh weight) in Leucaena leucocephala |
| Table 4: | Cumulative biomass production (g plant–1) after three months in Sesbania sesban tree legume inoculated with Rhizobium wild type and biochemical mutants |
| Table 5: | Cumulative biomass production (g plant–1) after three months in Leucaena leucocephala tree legume inoculated with Rhizobium wild type and biochemical mutants |
| Table 6: | Nodulation and relative symbiotic effectiveness of Rhizobium sp. (Sesbania sesban) wild type and auxotrophic mutants |
| Table 7: | Nodulation and relative symbiotic effectiveness of Rhizobium sp. (Leucaena leucocephala) wild type and auxotrophic mutants |
Auxotrophic mutant acr-1 FFAMIJ-8 revealed significant increase in number of nodules per plant. In addition, most of auxotrophic mutants derived from ARCG-10 shows significant increase in number of nodules per plant. Sanginga et al. (1991) reported a wide range of nodulation among provenances of inoculated Gliricidia sepium in Austrian soils. The present study clearly demonstrates the ability of Rhizobiumauxotrophic mutants induced to survive and renodulate under field conditions and their effect on biomass yield of tree legumes. Shoot biomass yield was the major criterion in the selection of rhizobial inoculants. Short-term observation on the effect of Rhizobium on biomass yield of tree legumes do not give a true picture; a long-term study is essential to assess the potential of Rhizobium in increasing the biomass yield of tree legumes. In Sesbania sesban, plants inoculated with auxotrophic mutant asc-4 ARCG-10 gained maximum acetylene reduction, possibly because of high nitrogenase activity.
| Table 8: | Nitrogen fixation abilities of Rhizobium wild type and auxotrophic mutants at 13 week-old Sesbania sesban plants and corresponding estimates of annual N2-fixation per hectare |
| Table 9: | Nitrogen fixation abilities of Rhizobium wild type and auxotrophic mutants at 13 week-old Leucaena leucocephala plants and corresponding estimates of annual N2-fixation per hectare |
These observation are in agreement with previous reports on inoculation of woody legumes with selected rhizobial strains, which showed increased survival percentage in seedlings and greater biomass production in all inoculated trees (Herrera et al., 1993; Galiana et al., rhizobial inoculants. Short-term observation on the effect of Rhizobium on biomass yield of tree legumes do not give a true picture, a long-term study is essential to assess the potential of Rhizobium in increasing the biomass yield of tree legumes. In Sesbania sesban, plants inoculated with auxotrophic mutant asc-4 ARCG-10 gained maximum acetylene reduction, possibly because of high nitrogenase activity. These observation are in agreement with previous reports on inoculation of woody legumes with selected rhizobial strains, which showed increased survival percentage in seedlings and greater biomass production in all inoculated trees (Herrera et al., 1993; Galiana et al., 1994).
As shown from the results presented here the auxotrophic mutants inoculated woody legume, Sesbania sesban, differed significantly in their effect on plant heights and stem-diameter. Most of auxotrophic mutants derived from FFAMU-8 showed significant increase in plant heights due to effective nodulation. In addition, some of auxotrophic mutants derived from ARCG-10 showed significant increase in stem-diameter, suggesting effectiveness in nodulation. The pattern of plant height and stem diameter obtained in this study are consistent with published data. Where there is no history of the presence of a tree legume in a particular locality, native bacteria capable of nodulating that species are likely to be few or completely absent (Sanginga et al., 1985). Total numbers of nodules and nodule occupancy data, after inoculation L. leucocephala with strains of Rhizobium sp. (Leucaena) and their auxotrophic mutants, are shown in Table 7. The results revealed that many of auxotrophic mutants derived from both isolates show significant increase in nodule number, nodule dry weight and nodulation index. The woody legume tested in this study revealed that auxotrophic mutants tested in this investigation did not differed significantly in both average weight / nodule and acetylene reduction. The present results are in agreement with Masutha et al. (1997), who reported that nodule effectiveness is often associated with higher N2 fixation, the nodulation data obtained in their study did not necessarily correlate with the amounts of N2 fixation. The pattern of nodulation and N2 fixation obtained in this study are consistent with published data.
Of the woody legume tested here the obtained results revealed insignificant differences in plant heights and stem-diameter among both isolates NRC-19 and HRI-27. The results have proved to be the fastest-growing Leucaena leucocephala and Sesbania sesban in terms of plant height, stem diameter growth. So, although it has been suggested that fast-growth in woody legumes is not necessarily an index of N2 fixation in the species (Danso et al., 1992), in the study of Masutha et al. (1997) all variables of plant growth have correlated with N2 fixation.
| Table 10: | Correlation coefficient (r) between plant estimates of N2 fixation for Sesbania sesban grown in sterilized sandy soil |
If however establishment of effective symbiosis with auxotrophic mutants is the only consideration for species selection, then both tree legumes, are suitable for use in the agroforestry program. There is however no doubt that when dealing with agroforestry systems, criteria such as competitively of the root systems are also important in addition to an active N2 fixation by the legume.
Nitrogen fixation: The present study clearly demonstrates (Table 8, 9) the ability of introduced Rhizobium isolates of Sesbania sesban and L. leucocephala to significantly increase nitrogen fixation by some of auxotrophic mutants used in plants inoculation. The results showed that the inoculation of these tree legumes with specific and effective strains of Rhizobium spp. had a positive effect on nitrogen fixation. This are in accordance with Herrera et al. (1993), who reported that inoculation of woody legumes with selected rhizobia and arbuscular mycorrhizal fungi improves outplanting performance, plant survival and biomass development.
| Table 11: | Correlation coefficient (r) between plant estimates of N2 fixation for Leucaena leucocephala grown in sterilized sandy soil |
The present study with Sesbania sesban and L. leucocephala, over 13 weeks, clearly indicated that these' tree legumes responded significantly to inoculation with some of Rhizobiumauxotrophic mutants, they showed more nodulation, nitrogenase activity, N2-fixation than those inoculated with wild type isolate (positive control) and uninoculated trees (negative control). The woody legumes tested in this study differed significantly in their relative increase in N2-fixation. Although, nodule effectiveness is often associated with higher N2-fixation also and the nodulation data obtained in this study (Table 10, 11) did not necessarily correlate with the amounts of N2-fixation (Masutha et al., 1997). The pattern of N2-fixation obtained in this study are consistent with published data. Of the two woody legumes tested, Sesbania sesban and L. leucocephala proved to be the fastest-growing species in terms of plant height, stem diameter growth (Table 6, 7), plant biomass (Table 4, 5) and nitrogen fixed (Table 8, 9). So, although it has been suggested that fast-growth in woody legumes is not necessarily an index of N2-fixation in these species (Danso et al., 1992). On that basis, both tree legumes therefore appear to be the most promising elite Material for use in agroforestry. If however establishment of effective symbiosis with Rhizobium-auxotrophic mutants and/or indigenous soil strains is the only consideration for species selection, then both tree legumes, are suitable for use in the agroforestry program. There is however no doubt that when dealing with agroforestry systems, criteria such as competitively of the root systems are also important in addition to an active N2-fixation by the legume. Kwesiga and Coe (1994) obtained a doubling of maize yields in Zambia in Sesbania follows compared with continuous unfertilized maize. Moreover, Dommergues (1987) has grouped woody legumes into low and high N2 fixing species. However, the universality of that observation has not been tested, especially that the effective of a symbiosis can be enhanced or limited by factors unique to that particular environment.
Correlation: In respect of plants inoculated with rhizobial isolates derived from FFAMU8, it is evident from the results presented in Table 10 that chlorophyll (a) was significantly correlated with; total chlorophyll (Chl) (r = 0.901, p<0.01), nodule dry weight (r = 0.569, p<0.01) and stem diameter (r = 0.71, p<0.01). Whereas, leaves dry weight (DW) was significantly correlated with woody DW (r = 0.813, p<0.01), aerial DW (r = 0.93, p<0.01), N2 fixation (mg/plant) (r = 0.759, p<0.01) and aerial protein % (r = 0.662, p<0.05). However, woody DW was significantly correlated with aerial DW (r = 0.47, p<0.01) and acetylene reduction (r = 0.64, p<0.05), as well as aerial DW was significantly correlated with total DW (r = 0.739, p<0.01), acetylene reduction (r = 0.652, p<0.01) and N2 fixation (mg/plant) (r = 0.637, p<0.01). In addition, root DW was significantly correlated with total DW (r = 0.669, p<0.01), nodulation index (r = 0.658, p<0.01), average weight per nodule (r = 0.666, p<0.01) and N2 fixation (mg/plant) (r = 0.666, p<0.01). The present results are in agreement with Dobert and Blevins (1993), who found strong correlation between; shoot and nodule dry weights, plant height with shoot DW, nodule mass and mass per nodule, nodule number with both shoot DW and nodule mass. Although, total DW was significantly correlated with average weight per nodule (r = 0.68, p<0.01), N2 fixation (r = 0.668, p<0.01) and aerial protein % (r = 0.667, p<0.01). However, nodule number was significantly correlated with stem diameter (r = 0.632, p<0.05), as well as, nodule OW with both nodulation index, average weight per nodule and nodule efficiency. In addition, nodulation index was significantly correlated with average weight per nodule, whereas, acetylene reduction was significantly correlated with nodule efficiency, nodule efficiency with stem diameter and root length with plant height.
The results obtained in this study are in accordance with those reported. Who found that fixed N2 was highly correlated with maturity (r = 0.96, p<0.01), since late-maturing lines fixed more N2 than earlier-maturing lines. The parameters related to nitrogen fixation in Sesbania sesban plants inoculated with rhizobial isolates derived from ARCG 10 showed a significant correlations between; chlorophyll b with root length (r = 0.632 , p<0.01), leaves DW with both woody DW (r = 0.70, p<0.01), aerial DW (r = 0.934, p<0.01), total DW (r = 0.813, p<0.01) and plant height (r = 0.817, p<0.01). Although, woody DW was significantly correlated with both aerial DW (r = 0.909, p<0.01), plant height (r = 0.945, p<0.01) and aerial protein % (r = 0.602, p<0.05). However, aerial DW showed the same trend of positive correlation with both root DW (r = 0.605, p<0.05), total DW (r = 0.71, p<0.05), plant heights (r = 0.949, p<0.01) and N2 fixation (mg/plant)(r = 0.619, p<0.05).
In addition, root DW showed the same trend with both plant heights (r = 0.684, p<0.01) and N2 fixation (mg/plant) (r = 0.649, p<0.01), as well as, total OW with both plant heights (r = 0.979, p<0.01), N2 fixation (mg/plant) (r = 0.693, p<0.01) and N2 fixation % (r = 0.581, p<0.01). On the other hand, nodule DW showed similar nature with both nodulation index (r = 0.939, p<0.01) and average weight per nodule (r = 0.666, p<0.01), as well as, nodulation index with average weight per nodule (r = 0.669, p<0.01), acetylene reduction with stem diameter (r = 0.849, p<0.01), nodule efficiency with both plant heights (r = 0.657, p<0.05) and N2 fixation (mg/plant) (r = 0.672, p<0.01). In addition, plant heights was significantly correlated with N2 fixation (mg/plant) (r = 0.715, p<0.01).
The results obtained herein are in agreement with those reported by Dobert and Blevins (1993), who found strongest relationship (r = 0.85) between nodule mass and shoot DW, although nodule number was correlated closely with shoot DW. The same authors also reported that nodule mass and often number were closely related to shoot biomass.
As shown from the results tabulated in Table 11, in the respect of plants inoculated with the isolates derived from NRC19, that the strongest relationship was existed between chlorophyll (a) and nodulation index (r = 0.924, p<0.01), chlorophyll (b) with both total chlorophyll (r = 0.972, p<0.01) and woody DW (r = 0.671, p<0.05), as well as, total chlorophyll with woody DW (r = 0.664, p<0.01). In addition, the same trend of significant correlation was existed between reaves DW with both total DW (r = 0.884, p<0.01) and N2 fixation (mg/plant) (r = 0.711, p<0.01). However, woody OW showed the same trend with both aerial OW (r = 0.77, p<0.01), root DW (r = 0.702, p<0.05), total DW (r = 0.791, p<0.01), acetylene reduction (r = 0.654, p<0.01), root length (r = 0.789, p<0.01) and N2 fixation (mg/plant) (r = 0.721, p<0.01).
Similar nature of significant correlations was obtained between aerial DW with both root DW (r = 0.633, p<0.05), total OW (r = 0.965, p<0.01), root to shoot ratio (r = 0.682, p<0.01), root length (r = 0.876, p<0.01) and N2 fixation (mg/plant) (r = 0.881, p<0.01). Although, the same trend was existed between root OW with both total DW (r = 0.807, p<0.01), nodule number (r = 0.705, p<0.01), nodule efficiency (r = 0.666, p<0.01) and N2 fixation (mg/plant) (r = 0.702, p<0.01). Total DW also showed the same trend with both root length (r = 0.898, p<0.01), as well as, root to shoot ratio showed the same trend with root length (r = 0.743, p<0.01).
On the other hand, nodule number was significantly correlated with both nodule DW (r = 0.953, p<0.01), nodulation index (r = 0.67, p<0.01) and average weight per nodule (r = 0.638, p<0.05), as well as, nodule DW with both nodulation index (r = 0.683, p<0.01), average weight per nodule (r = 0.706, p<0.01) and stem diameter (r = 0.705, p<0.01). However, similar nature was existed between average weight per nodule with both stem diameter (r = 0.643, p<0.01), nitrogen fixation % (r = 0.708, p<0.01) and aerial protein % (r = 0.714, p<0.01), as well as, nodule efficiency with nitrogen fixation % (r = 0.715, p<0.01) and aerial protein % (r = 0.641, p<0.01). Although, the similar nature was existed between root length with N2 fixation (mg/plant) (r = 0.731, p<0.01).
The results obtained here are in agreement with those reported by Buttery and Dirks (1987), who found that nodule fresh weight was correlated with the rate of acetylene reduction per plant of soybean, as well as, plant weight per unit of nodule fresh weight was positively correlated with acetylene reduction rate per unit nodule fresh weight for both strains of B. japonicum and cultivars. The same authors also reported that the increase in plant dry weight is probably the best indicator of nitrogen fixing capacity (Materon and Vincent, 1980). In addition, acetylene reduction rate and nodule mass are two widely used indicators of nitrogen fixation capacity (Rys and Mytton, 1985). The most practical method for estimating nitrogen fixing ability would seem to be some combination of nodule mass and of nodule efficiency (such as acetylene reduction rate) (Buttery and Dirks, 1987). Rhizobial isolates derived from HRI-19, also revealed significant correlations between some parameters related to nitrogen fixation. The present results are in accordance with Mytton and Rys (1985), who reported that the numbers of nodules formed on white clover in the presence of nitrate was a heritable character well correlated with nitrogenase activity. Rys and Mytton (1985) suggested that both nodule formation and nitrogenase activity should be used as selection criteria in any breeding program aimed at improving fixation in the presence of combined N.
In conclusion, the selection of highly efficient strains remains one of the main tasks for inoculant procedures. In developing countries, the demand for fuel wood, timber and fodder are increasing. It is unlikely that such a demand can be met by expanding tree planting on fertile land as this is required for food crop production. Thus, extensive areas of marginal lands on which trees and shrubs can be planted are available in developing countries, to became fertile lands enough for food crop production.