Abstract
Introduction
Materials and methods
Results and discussion
Conclusions
Acknowledgements
References
Luyindula N. and Karabaranga L.
Departement de Microbiologie
CREN-K, B.P. 868, Kinshasa XI, Zaire
The symbiotic relationship between rhizobium and Leucaena leucocephala was studied using a strain of rhizobium isolated from the rhizospheric soil of a leucaena tree growing near the Regional Centre for Nuclear Studies at Kinshasa.
The isolate was purified on Yeast Extract Mannitol Agar and used to inoculate seedlings of a local cultivar of L. leucocephala grown in vermiculite moistened with N-free Fahraeus medium. Efficient nodules were observed 38 days after inoculation. The fresh weight of inoculated plants was 240% higher than that of the uninoculated control plants. The leaves of the uninoculated plants showed symptoms of N deficiency.
In a second experiment, three cultivars of L. leucocephala (local, Peru and Cunningham), two cultivars of Glycine max (local and Amsoy) and a local cultivar of Arachis hypogaea were inoculated with R. japonicum sp. CB 756, Rhizobium CB 81, the local isolate and a peat-based inoculum for leucaena. The broad-spectrum Rhizobium CB 756, used in Australia to inoculate most of the cowpea group of legumes, nodulated only Arachis hypogaea, while strain CB 81, the local isolate and the peat-based inoculum nodulated only leucaena.
In an experiment with two different soils, beneficial effects were observed when the local cultivar of L. leucocephala was inoculated with the local isolate. Fresh weight of inoculated plants was 140 % higher than that of the uninoculated control.
Biochemical analysis of leaves and seeds showed that the local cultivar of L. leucocephala was rich in proteins and other nutrients. Antitrypsic factors were also present but were inactivated by heating at 97 °C for 2 hours or autoclaving at 120 °C for 30 minutes.
Leucaena species belong in the subfamily Mimosoideae of the Leguminoseae. They are widely used in the tropics as a shade plant, for erosion control, for fuel and as a browse crop. As a forage crop, leucaena has yielded upto 20 t DM/ha per year. Alfafa (Medicago sativa) yields more green forage than leucaena but the feeding value of the two are about equal (USAID, 1981).
Leucaena is rich in protein and is an excellent forage for animals and is also eaten by humans. In Australia, cattle fed on a diet of 100% leucaena gain up to 1 kg per animal per day and can be fed at this level about 4 months. In Asia and Latin America, the slightly better leaves, flowers and young pods are eaten by humans (Brewbaker and Styles, 1982).
The use of leucaena to maintain soil fertility has been documented (Prussener, 1982: Parera, 1982; Kovitradhi and Yantasath, 1982: USAID, 1981). However, not all soils contain the correct strain nor the necessary amount of rhizobium for effective N fixation by leucaena (Halliday and Somasegaran, 1982).
This paper presents the findings of preliminary studies of the symbiotic relationship between rhizobium and a local variety of Leucaena leucocephala in Zaire, which were conducted in order to elucidate rhizobium strain affinities with local species of leucaena.
Isolation, purification and culture of bacteria
Indigenous rhizobia were isolated both from the rhizospheric soil and directly from nodules of leucaena seedlings, according to the method of Vincent (1970).
Data on the bacteria used in this investigation and their origins are given in Table 1. The isolates were maintained on Yeast Extract Mannitol (YEM) agar slants at 4°C. To provide inoculant, bacteria were grown in YEM liquid medium for 4 days for the fast-growing strains and for 7 days for the slow-growing ones.
Plant material
Three cultivars of L. leucocephala were used to determine rhizobium-legume compatibility: a local cultivar growing spontaneously in the surroundings of CREN-K and the varieties Peru and Cunningham, supplied by CSIRO, Australia. Two cultivars of Glycine max (local and Amsoy) and a local cultivar of Arachis hypogaea were also tested. The effectiveness of the nodules was tested only on the local cultivar of L. leucocephala.
Tube experiment
Plants were grown in glass tubes (200 mm x 35 mm) containing 15 g of vermiculite moistened with 60 ml of nitrogen-free Fahraeus medium (Vincent, 1970).
Seeds of Glycine max and Arachis hypogaea were surface-sterilised with HgCl2 0.2% according to the method of Vincent (1970). Seeds of L. leucocephala were soaked in hot water (80 °C) for 5 minutes before being surface-sterilised. The seeds were germinated and the seedlings transferred aseptically to the sterilised glass tubes when the radicles were about 1 cm long. The tubes were then inoculated with 5 ml of a rhizobium culture, providing about 5 x 10 rhizobia/tube. In the case of the solid inoculum, 1 g of peat was placed close to the young radicle. Uninoculated controls were treated with 5 ml of sterile YEM. When the seedlings reached the top of the culture medium, the vermiculite was covered with a layer of paraffined sand, prepared as described by Bonnier and Brackel (1969). Four tubes were used for each inoculation treatment and control. The experiment was arranged in a completely randomised design and the tubes were watered as required with sterile Fahraeus medium or sterile distilled water.
Table 1. Rhizobium strains used.
|
Strain |
Origin |
Characteristics |
|
Rhizobium japonicum CB1809 |
CSIRO, Australia |
Recommended for soya bean inoculation (Date, 1969) |
|
Rhizobium sp. CB756 |
CSIRO, Australia |
Recommended for inoculation of tropical legumes (Date, 1969) |
|
Rhizobium sp. CB81 |
Plant Patholon Konedon, Papua New Guinea |
Recommended for inoculation of L. leucocephala in acid soils (Diatloff, 1973). |
|
Rhizobium sp. CREN-K |
CREN-K collection of rhizobia |
Indigenous rhizobi from a nodule of a local L. leucocephala (this work). |
|
Peat-based inoculum |
CSIRO, Australia |
Inoculum for L. leucocephala |
Pot experiment
The nitrogen-fixing abilities of the different rhizobium strains were evaluated on the local cultivar of L. leucocephala in 15 cm diameter x 15-cm-deep pots containing 1200 g of soil taken from the surroundings of CREN-K or from a field at Ndjili (30 km from CREN-K). Pots were covered with aluminium foil and sterilised in a forced air oven at 180 °C for 4 hours. Six pregerminated seeds of leucaena were planted in each pot; seedlings were removed after emergence to leave four plants per pot. Treatments compared were inoculated plants and uninoculated controls, with or without N. Each treatment was replicated three times in a completely randomised design. Pots were inoculated with 100 mg of a vermiculite-based inoculum prepared in our laboratory, containing 2.4 x 1010 rhizobia/g of moist vermiculite.
After germination, all the pots were placed outside under natural conditions of light, temperature and humidity. The pots were watered as required with non-sterile tap water. Nitrogen was added to pots one week after planting as an aqueous solution of KNO3 at a rate of 100 kg N/ha.
Statistical analyses
Results of tube and pot experiments were subjected to analysis of variance and the means were compared using the new Duncan multiple range test (Dagnelie, 1975).
Chemical analyses
Chemical analyses were performed only on leaves and seeds of the local cultivar of L. leucocephala. Protein content was determined by the Kjeldahl method as modified by Villegas (1971). The ash content was determined according to Onyembe et al (1980b).
The Ca, Mg and P contents were determined according to the method of Didier de St. Amand et Cas (1967). Micronutrient contents (Mo, Cr. Zn, Fr, Co, Se, Na, Mn) were determined after neutron activation in the Triga Mark II reactor of the Regional Center for Nuclear Studies at Kinshasa. Lipids were extracted by the soxhelt method using ether as the solvent and the amount of lipids determined as the difference of weight before and after delipidation (Kabele et al, 1975; 1977).
Isolation, purification and characterization of isolates from leucaena nodules
The bacteria colonies on agar were whitish, convex, circular with regular contour, viscous and stuck easily to the inoculating loop. The colonies grew quickly and acidified the Wright agar medium containing bromothymos blue. The isolate did not absorb Red Congo dye. With a few exceptions, this property distinguishes between rhizobia and other bacteria (Barbara and Thomas, 1983).
Nodulation of L. leucocephala
Tube experiment: Thirty-eight days after inoculation, efficient root nodules (13 nodules per plant on average) were observed on inoculated plants, whereas uninoculated plants were without nodules. The nodules were elongated and measured 4 to 5 mm long and 1.5 to 2 mm wide. The inside of the nodules was dark red.
There were significant morphological differences between inoculated and uninoculated plants. The uninoculated plants were smaller than the inoculated plants and had yellowish leaves, whereas the leaves of the inoculated plants were dark green. Nodule numbers and plant fresh weights are shown in Table 2. i
Table 2. Effect of inoculating a local variety of Leucaena leucocephala with a local isolate of rhizobium on nodule numbers per plant and plant fresh weight 38 days after inoculation.
|
Treatment |
Number of nodules per plant |
Plant fresh weight(mg) |
|
Uninoculated
|
0 |
128 |
|
0 |
102 |
|
|
0 |
76 |
|
|
12 |
317 |
|
|
14 |
352 |
|
|
10 |
315 |
|
|
Inoculated |
9 |
310 |
|
16 |
388 |
|
|
15 |
388 |
|
|
12 |
345 |
Pot experiment: Uninoculated plants did not nodulate and gave smaller fresh-weight yields than inoculated plants even when loo kg N/ha was applied (Table 3). The fresh-weight yield of inoculated plants was more than double that of uninoculated plants in both the low-N soil (CREN-K, 0.02% N) and the N-rich soil (Ndjili, 0.23% N).
As in the tube experiment the internal colouration of the nodules was dark red, suggesting the presence of leghaemoglobin, which is related to nitrogen fixation efficiency (Bergersen, 1966; Bonnier and Brackel, 1969; Vincent, 1970).
Table 3. Effects of inoculation (local rhizobium isolate), N fertilizer (100 kg N/ha) and soil type on nodulation and fresh and dry weights per plant of a local variety of Leucaena leucocephala 55 days after sowing in a pot trial.
|
Soil |
Treatment |
Nodules per plant |
Fresh wt per plant (mg) |
Dry wt. per plant (mg) |
|
|
Uninoculated |
0 |
1425 |
275 |
|
(Control) |
0 |
975 |
225 |
|
|
CREN-K |
Uninoculated+ 100 kg N/ha |
0 |
2825 |
550 |
|
0 |
1750 |
325 |
||
|
0 |
1400 |
250 |
||
|
Inoculated |
10 |
3150 |
675 |
|
|
7 |
2550 |
550 |
||
|
|
|
|
||
|
Ndjili soil |
Uninoculated (Control) |
0 |
1075 |
550 |
|
0 |
1400 |
250 |
||
|
0 |
1475 |
275 |
||
|
Inoculated |
6 |
2975 |
675 |
|
|
10 |
3275 |
725 |
Study of local leucaena isolate specificity
Only the isolate obtained from the local variety of Leucaena leucocephala, strain CB 81 and the Australian peat inoculum for leucaena formed root nodules on the three leucaena cultivate used in the experiment. In each case, the fresh-weight yield of nodulated leucaena plants was more than double that of the non-nodulated plants. These results indicate that the local rhizobium isolate is effective in forming nodules and fixing N.
Biochemical studies
General analyses: The biochemical composition of the local variety of leucaena was similar to that found by other workers (see NAS, 1977; Riviere, 1978). The seeds contained more protein than the leaves (36.13 vs 25.56%), while leucaena leaves contained nearly twice as much protein as the leaves of Stylosanthes guianensis (Table 4).
Compared with other local legumes, leucaena seeds contained much less lipids than Glycine max and Psophocarpus tetragonolobus but more than Phaseolus vulgaris and Sphenotylis sternocarpa. Carbohydrate content was similar to that of P. vulgaris and S. sternocarpa seeds, and higher than that of P. tetragonolobus and Glycine max (Table 5).
Anti-trypsic activity: Leucaena contains antinutritive factors, such as trypsin inhibitors and mimosine, which reduce its feeding value. Antitrypsic activity was observed in leucaena seeds in this study, but it was found that this could be eliminated by heating the seeds at 97 °C for 2 hours or by autoclaving the seeds at 120 °C for 30 minutes, as previously reported for other legumes (Onyembe et al, 1980a; Gillespie et al, 1981).
Table 4. Biochemical composition of the leaves of a local variety of L. leucocephala and Stylosanthes guyanensis leaves.
|
|
L. leucocephala (local) leaves |
S. guianensis leaves |
|
($ DM) |
(% DM) |
|
|
Crude protein |
25.56 |
12.9 |
|
Fats |
1.81 |
2.8 |
|
Carbohydrate |
66.40 |
75.9 |
|
Ash |
6.23 |
8.4 |
|
Ca |
0.56 |
1.25 |
|
Mg |
0.20 |
0.21 |
|
P |
0.22 |
0.24 |
|
K |
1.31 |
1.44 |
|
Fibre |
20.3 |
32.2 |
Source: Riviere (1978).
Table 5. Biochemical composition of Leucaena leucocephala seeds compared with that of other legumes grown in Zaire.
|
Species |
Moisture |
Crude protein |
Fats |
Carbo- hydrates |
Fibre |
Ash |
|
$ DM |
||||||
|
L. leucocephala |
27.31 |
36.13 |
2.27 |
57.81 |
15.6 |
3.79 |
|
S. stenocarpa |
11.14 |
21.32 |
1.64 |
62.28 |
7.6 |
3.32 |
|
P. tetragonolobus |
8.70 |
29.8-40.0 |
16.80 |
39.00 |
4.1 |
4.40 |
|
Glycine max |
10.00 |
37.1-40.8 |
17.00 |
35.80 |
6.0 |
5.30 |
|
P. vulgaris |
10.00 |
20.3 |
1.2 |
69.9 |
5.0 |
4.79 |
Source: Onyembe et al (1982).
Leucaena was found to be nodulated only by strains of rhizobia that were isolated from Leucaena species. The broad-spectrum cowpea strain of rhizobium, CB 756, which nodulates most tropical legumes, failed to nodulate the three cultivars of leucaena used in this study. It was found that efficient nodulation more than doubled the fresh weight yield of leucaena compared with the non-nodulated control plants. It was also found that leucaena contains a large proportion of crude protein and other nutrients, and that the antitrypsic factor preset could be inactivated by heat treatments.
The authors are grateful to Dr Tshitenge K, Dr Ir Onyombe P M L, Dr Mbaya N and Dr Makoko M for useful discussions; to Mr Nsonsa N M and Mr Mama A for their technical assistance; to Mr Bashengezi K for reading and correcting the English manuscript, and to Mr Mpongo for typing the manuscript. This investigation was supported by IAEA Technical Assistance Project No. ZAI/5/006.
Barbara E K and Thomas A L. 1983. Congo red absorption by Rhizobium leguminosarum. Appl. Environ. Microbiol. 45(1): 340-342.
Bergersen J F. 1966. Nitrogen fixation in the legume root nodule. In: Proceedings of the 4th international congress on microbiology, Moscow. p. 97.
Bonnier C and Brackel J. 1969. Legumineuse-Rhizobium: lutte biologique contre la faim. Ed. Duculot, Gembloux.
Brewbaker J L and Styles B. 1982. Economically important nitrogen fixing tree species. NFTA Misc. Publ. 82-04.
Dagnelie P. 1975. Theorie et methodes statistiques. Applications agronomiques. Vol. 2. Les presses agronomiques de Gembloux.
Date R A. 1969. A decade of legume inoculant quality control in Australia. J. Aust. Inst. Agric. Sci. March 1969:27-37.
Diatloff A. 1973. Leucaena, needs Inoculation, Queens1. Agric. J. 99(12):642.
Didier de St Amand et Cas G. 1967. Dosage des elements mineraux majours chez les vegetaux. ORSTOM, Paris. 41 pp
Gillepsie Balangrone R J and Kortt A A. 1981. Characterisation of Winged bean seeds proteins. The Winged Bean Flyer 3(2).
Halliday J and Somaseganran P. 1982. Nodulation, nitrogen fixation, and rhizobium strain afinities in the genus Leucaena. Proced. 23-26 nov. 1982.
Kabele N. Vieux A and Poquet C. 1975. Une huile riche en acides lignocerique et cerotique. L'huile des graisses d'Adenanthera pavonina. Oleagineux 30:119-120.
Kabele N. Vieux A and Poquet C. 1977. Les plantes a huile du Zaire. III. Familles botaniques fournissant des huiles d'insaturation relativement elevee. Oleagineux 32(12):535-537.
Kovitradhi K and Yantasath P K. 1982. Research on Leucaena and other nitrogen-fixing Trees at the Thailand Institute of Scietific and Technological Research (TISTR). Proced. Workshop Singapore, 23-26 Nov.: pp. 133-136.
NAS (National Academy of Science). 1977 Leucaena: Promising forage tree crop for the future. NAS, Washington D.C.
Onyembe P M L, Kembola K and Paulus J J. 1982. Etude de la valeur nutritive des graines de Sphenostylis stenocarpa (Hochet et Rich) Harms. Rev. Zaire Sci. Nucl. 3/2:199209.
Onyembe P M L, Kembola K and Litua Gelonda. (1980). Influence du traitement thermique sur l'activite des inhibiteurs de trypsine de quelques varietes de soja cultivees a l'INEFRA-YANGAMBI. - Rev. Zaire Sci. Nucl. 1/1:105-112.
Onyembe P M L, Paulus J J. Mukienga K, Kembola K. 1980. Study of the food-value of Psophocarpus palustris Desvaux. Rev. Zaire Sci. Nucl. 1-2:51-64.
Parera V. 1982. Leucaena for erosion control and green manure in Sikka. Proceed. Workshop Singapour, 23-26 Nov.: pp. 169-172.
Prussener K A. 1982. A farmers' practical guide to growing leucaena. Proceeding of a workshop held in Singapour, 2326 Nov. 1982.
Riviere. 1978. Manuel d'alimentation des ruminants domestiques en milieu tropical.
USAID (United States Agency for International Development). 1981. Leucaena leucocephala a tree that "defies the wood cutter". USAID, Washington D.C.
Villegas. 1971. Methodes chimiques employees par CIMMYT dans la determination et l' evaluation de la qualite des proteines de mats. Bull.. d'Invest. n° 20. CIMMYT, Mexico.
Vincent J M. 1970. A manual for the practical study of root-nodule bacteria. IBP handbook No. 75. Blackwells, Oxford.
