African Journal of Biotechnology Vol. 1 (1), pp. 17-22, November 2002

ISSN 1684-5315  © 2002 Academic Journals  

Biological nitrogen fixation in Crotalaria species estimated using the ¹⁵N isotope dilution method

R.T. Samba¹, S.N. Sylla², M. Neyra¹, M. Gueye³, B. Dreyfus⁴ and I. Ndoye^2*

¹Laboratoire de Microbiologie des Sols IRD/ISRA/UCAD, B.P. 1386, Dakar-Sénégal

²Département de Biologie Végétale, Université Cheikh Anta Diop, B.P. 5005 Dakar, Sénégal / Laboratoire de Microbiologie des Sols IRD/ISRA/UCAD, B.P. 1386, Dakar-Sénégal

³MIRCEN-Centre IRD/ISRA/UCAD Laboratoire de Microbiologie des Sols, B.P. 1386, Dakar-Sénégal

*Corresponding author; e-mail: Ibrahima.Ndoye@ird.sn

Accepted 16 October 2002

A greenhouse experiment was conducted to measure nitrogen fixation in three Crotalaria species : C. ochroleuca, C. perrottetii and C. retusa growing in Senegal by using ¹⁵N direct isotope dilution technique. Two non-fixing plants, Senna obtusifolia and Senna occidentalis served as reference plants. The amount of nitrogen fixed two months after planting was obtained using the average of the two reference plants. The atom % ¹⁵N excess in the Crotalaria species was significantly lower than that of the reference plants, indicating that significant nitrogen fixation occurred in the three plants. Significant differences were observed between the Crotalaria species; C. ochroleuca yielded more dry matter weight and total nitrogen than did C. perrottetti and C. retusa. The % nitrogen derived from atmosphere (%Ndfa) in leaves and stems was also higher in C. ochroleuca. There was no significant difference in %Ndfa in the whole plant between the three Crotalaria species (47% to 53%). In contrast, interspecific variability was observed based on the %Ndfa. C. ochroleuca significantly exhibited the higher amount of total nitrogen fixed, equivalent to 83 kg of nitrogen fixed per hectare. Based on these data, it was concluded that C. ochroleuca could be used in multiple cropping systems in Senegal for making more nitrogen available to other plants.

Key words: Crotalaria spp, isotope dilution, ¹⁵N, nitrogen fixation, reference plant.

In the tropics, soil erosion and depletion are becoming a problem of global proportions and few farming systems are totally immune to it. Legumes, nitrogen fixing trees with high fixing potential, can be used in agricultural systems for replenishing nitrogen, the most limiting growth factor in the soil. Based on the current knowledge (Giller and Wilson, 1991), the challenge is to choose the appropriate legume in a given niche. In tropical areas, spontaneous legumes play an important role in the maintenance and improvement of soil fertility, but they remain largely unexploited. Among them, the legumes belonging to the genus Crotalaria are active in fixing nitrogen through the nodules they form on their roots in association with rhizobia. Crotalaria is widespread in tropical regions and includes about 550 species in Africa and Madagascar (Polhill, 1982), of which about 33 species are found in Senegal. Crotalaria plants have a high dry matter production potential and are able to grow on poor soil with low nitrogen content (Daimon et al., 1995). They have also been reported as good intensive fallowing cover crops to regenerate the soil (Müller-Sämann and Kotschi, 1994). These annual or perennial plants can be used as green manure or in intercropping farming systems. Although several Crotalaria nodulated species have been found (Allen and Allen, 1981), no information is available on how much nitrogen can be fixed by these plants. The present experiment was designed to estimate the nitrogen fixing potential of three Crotalaria species native to Senegal using the ¹⁵N isotope dilution method in view to use an elite one in Senegal cropping systems.

A greenhouse experiment was carried out at the IRD Bel-Air experimental station at Dakar, using a local sterilized sandy soil (Psamment; vernacular name: Dior), with 93% of sand. This soil contains approximately 10² native Bradyrhizobium per gram counted by infection test method (Brockwell, 1982; Vincent, 1970) using Crotalaria seedlings. The soil was pH 7.0 with 1.9% total carbon and 0.025% total nitrogen (Bremner, 1965). The soil was sieved (<1 mm), homogenized and 10 kg was placed into pots of 30 cm diameter.

The seeds of the three fixing species of Crotalaria and that of the non-nodulating Senna occidentalis and Senna obtusifolia (reference plants) were used in these experiments. They were surface-sterilized with sulfuric acid and germinated in petri dishes containing soft agar (0.8%). Seedlings were transplanted one per container after two days, followed by inoculation (5 ml/pot) with a liquid rhizobial inoculum strain containing 10⁹ cells/ml. All Crotalaria seedlings were then inoculated with appropriate liquid inoculum: C. perrottetii was inoculated with ORS 1924 rhizobial strain isolated from C. perrottetii, while C. ochroleuca and C. retusa were inoculated with ORS 1929 rhizobial strain isolated from C. comosa (Samba et al., 1999). A solution of (¹⁵NH₄)₂SO₄ containing 10.9 ¹⁵N atom % excess was applied to all pots to supply 0.2 g nitrogen/pot. A basal fertilizer was then applied to each pot equivalent to 0.14 g of K₂HPO₄. The pots were arranged randomly and watered such that soil moisture was kept close to field capacity (14%, v/w). Five replicates of these experiments were made.

The plants were harvested after two months after transplanting into pots. Dry weights of the different plant parts (leaves, stems, roots and nodules) were recorded. Nitrogen content (%N) and atom %¹⁵N excess (%¹⁵NAE) were determined for each plant part by the Central service of analysis of CNRS at the University of Lyon.

Nitrogen fixation (%Ndfa) was estimated using the isotope dilution equation (Fried and Middelboe, 1977):

                         %¹⁵NAE in fixing crop

  %Ndfa = 1 –                                                 

                        %¹⁵NAE in non fixing crop

A weighted %¹⁵N atom excess (WAE) for the whole plant was estimated as follows:

Where AE(sh), AE(st), AE(r) and AE(p) refer to atom excess in shoots, stems, roots, and pods, respectively. While TN(sh), TN(st), TN(r) and TN(p) designate total nitrogen in shoots, stems, roots and pods, respectively.

Then the %Ndfa for the whole plant was:

                          WAE in fixing crop

    %Ndfa = 1–                                                

                          WAE in non-fixing crop

 

Abstract

Introduction

Materials and Methods

Results

Discussion

References

Dry matter weight and total nitrogen accumulation

There were significant variations in dry matter weight and total nitrogen uptake between the Crotalaria species (Table 1). C. ochroleuca accumulated five and two times more dry matter than C. perrottetii and C. retusa, respectively. C. ochroleuca also accumulated more nitrogen than C. perrottetii and C. retusa when either the whole or part of the plant is considered.

Table 1. Dry matter weight, % ¹⁵N AE, proportions and amounts of total nitrogen in plant parts of three species of Crotalaria  cultivated in 10 kg pots using Senna as reference plants

Values in the same column and for each plant part, values followed by the same letter do not differ significantly at

P= 0.05. CV = coefficient of variation

Nodulation and nitrogen fixation

Nodules were found in all three Crotalaria plants studied. C. ochroleuca exhibited significantly higher nodule dry weight (1.54 g/plant) than the two others plants (0.63 and 0.21 g/plant for C. retusa and C. perrottetii respectively) (Table 1). The % ¹⁵N atom excess in each plant part and in the whole plant was higher in the two reference Senna plants than in Crotalaria species indicating the occurrence of nitrogen fixation in Crotalaria. No statistically significant differences were observed in the % ¹⁵N atom excess between the Crotalaria species for each plant part and the whole plant, although the lowest atom % ¹⁵N excess was recorded in C. ochroleuca. 

Table 2. Proportions of nitrogen from fixation, fertilizer and soil of three species of Crotalaria cultivated in 10 kg pots of soil using two species of Senna (S. occidentalis et S. obtusifolia) as reference plants