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Evaluation of N2 fixation and agroforestry potential in selected tree legumes for sustainable use in South Africa
PII:
Soil Biel. Biochem. Vol. 29, No. 516, pp. 993-998, 1997 0 1997 Elsevier Science Ltd. AI1 rights reserved Printed in Great Britain S0038-0717(%)002167 0038-0717/97 %17.00 + 0.00
EVALUATION OF N2 FIXATION AND AGROFORESTRY POTENTIAL IN SELECTED TREE LEGUMES FOR SUSTAINABLE USE IN SOUTH AFRICA T. H. MASUTHA, M. L. MUOFHE and F. D. DAKORA* Botany Department, University of Cape Town, P/B, Rondebosch, 7701, South Africa ( Accepted 4 July 1996) Summary-Seven
tree legumes with agroforestry potential were tested for their capacity to nodulate with indigenous rhizobia or bradyrhizobia in soils collected from different ecological zones in South Africa. Our findings show that al1 the tested species, except Acacia senegal, could effectively nodulate with root-nodule bacteria native to Venda and Klipspruit soils. Since Acacia seyal, Acacia gulpinii, Albiziu lebbe& and Acacia auriculiformis exhibited, in that order, a greater leve1 of growth and symbiotic performance, they manifest themselves as suitable species for agroforestry programs in Venda and Klipspruit soils. One indigenous species, Acacia araxuncunfha, was assessed to be a non-nodulating legume even when inoculated with a promiscuous strain of Rhizobium NGR234. 0 1997 Elsevier Science Ltd
INTRODUCTION
Globally, tree legumes are an important source of timber, fuel and fodder. Their ability to fix NZ 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 vita1 role in rehabilitation of degraded and marginal soils and restoration of nutrient fertility in fields exhausted by intensive cultivation. With the ever-increasing population in Africa, the need to exploit these tree legumes for sustained productivity of soil and increased crop yields has also increased (Dakora and Keya, 1996). The use of Nz-fixing trees in agroforestry systems in Africa has shown considerable promise in terms of benefit to crop and livestock production (Kang et al., 1990; Danso el al., 1992). An earlier study involving Faidherbiu albidu (formerly Acacia albidu) in Senegal showed a marked increase in nutrient fertility within the area covered by tree canopy, and an increased protein content in millet from that soil (Charreau and Vidal, 1965). N deficiency is therefore a major fertility constraint to agricultural productivity in Africa, a problem solvable through the development of agroforestry. The use of prunings from nodulated tree legumes has doubled cereal crop yields in Nigeria (Sanginga et al., 1986; Kang et al., 1990). In Malawi, Sesbanin *Author for correspondence: [email protected].
sesban is a fast-growing multipurpose legume that nodulates naturally with bradyrhizobia in soils, forms leaves that yield up to 80 kg N ha-’ and 6 kg P ha-’ after falling, and can produce 1.7 t ha-’ of fuelwood for rural communities (Sanchez, 1995). The nutrient-supplying capacity of Sesbaniu sesbun has therefore resulted in the development of a productive relay intercropping of maize with this species. Similarly, a doubling of maize yields was obtained in Zambia in Sesbaniu fallows compared with continuous unfertilized maize (Kwesiga and Coe, 1994). Thus, interest has increased in exploiting both local and exotic tree legumes for timber, firewood and other agroforestry products in African countries. This interest in agroforestry has increased rapidly in the new South Africa, where distribution of limited Government land to the many African farmers has resulted in extremely smal1 farm sizes, which preclude the possibility of fallow periods or livestock grazing. Consequently, the importante of Nz-fixing trees as multipurpose crops has been recognized. In South Africa, this has involved the planting of various shrub and tree legumes, namely Faidherbia albida, Acacia Senegal. Acacia seyal. Acacia galpinii. Acacia erioioba, Acacia auriculiformis, Albizia lebbeck, Cajanus cajan. Leucaena leucocephala, Virgilia divaritata and Dalbergia sissoo in al1 the former Black
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Homelands in order to provide low-tost energy and cheap timber for the poorer communities while maintaining adequate soil fertility. However, establishment of many of these species is poor in soils without a previous history of the legume’s presence (Sanginga et al., 1989). It is therefore not known whether the indigenous and exotic
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T. H. Masutha et al.
species currently used in the South African agroforestry program can nodulate effectively with native rhizobia and bradyrhizobia from the different localities. Furthermore, not al1 the African tree legumes form symbioses with root-nodule bacteria, a factor likely to affect growth rates. The woody legume, Acacia brevispica, which contains 17-19% crude protein in dry matter of leaves and pods (Bogden and Pratt, 1974) does not nodulate under Kenyan conditions (Odee and Sprent, 1992). Also, whether under South African conditions, nodulated species of selected tree legumes can grow fast enough to merit their inclusion in agroforestry programs has not been evaluated. Moreover, Dommergues (1987) has grouped woody legumes into low and high Nz.fixing species, with Faidherbia albida. Acacia Senegal and Acacia pellita belonging to the group of low fixers. However, the universality of that observation has not been tested, especially that the effectivity of a symbiosis can be enhanced or limited by factors unique to that particular environment. The purpose of this study was to assess native tree legumes and a few exotic species for growth and symbiotic performance with indigenous rhizobia and bradyrhizobia in soils collected from Venda and Klipspruit, South Africa, where two agroforestry plantations are sited. Additionally, the nodulation or non-nodulation status of Acacia ataxacantha was tested using Rhizobium NGR234, a promiscuous marker strain.
MATERIALS
AND METHODS
Six agroforestry plantations have been sited in South Africa by Forestek, CSIR, in different ecologica1 zones, which presently represent mainly the former Black Homelands. Soil samples collected from these sites (0-30 cm depth) were sieved and used as a source of inoculum in nodulation studies with selected tree legumes. The rationale was to determine the ability of native soil rhizobia or bradyrhizobia to effectively nodulate and fix Nz with these plant species. The data reported here cover only two of those six sites. Chemical analysis showed that the Venda soil had pH 4.3, organic matter 5.2%, nitrogen 0.20 mg g-’ soil, and cation exchange capacity 2.2 meq 100 g-’ soil. Klipspruit soil was similarly characterized as having pH 5.0, organic matter 5.0 mg g-‘, nitrogen 0.16 mg g-’ soil and cation exchange capacity 2.2 meq 100 g-’ soil. Prior to planting, seeds of selected tree legumes were scarified by soaking for 12 min in boiling water and left to imbibe ovemight. The seeds were then surface-sterilized in 70% ethanol for 2 min, and 50% sodium hypochlorite (bleach) for another 2 min, followed by rinsing 10 times with sterile deionized water. The seeds were either germinated in
sterile vermiculite prior to transplanting or sown directly into sterile rooting medium contained in 2-1 PVC tubes. Later, seedlings were thinned out to two per culture vessel. An inoculant was made from stirring 250 g soil in 1000 ml sterile de-ionized water, and 10 ml of the suspension used to inoculate each seedling growing in a sterile medium of sand and vermiculite (1:3) covered at the surface with sterile non-wettable cotton wool as an anti-contamination mulch. Al1 operations were done aseptically in a laminar flow hood. Plants were frequently irrigated to field capacity with N-free sterile Hoagland nutrient solution. In our studies, A. ataxacantha was identified as a woody legume unable to form root nodules in soils from the six locations in South Africa, including Venda and Klipspruit. Consequently, the nodulation status of this legume was further tested using Rhizobium strain NGR234 as a promiscuous marker. Rhizobium NGR234 is known to nodulate over 70 genera of legumes (Relic et al., 1993). The Rhizobium strain was cultured on agar slants using yeast mannitol agar medium (Vincent, 1970), and 5 ml culture suspension used to inoculate Acacia seedlings growing aseptically in ataxacantha Leonard jars with N-free nutrient solution (Vincent, 1970). Faidherbia albida, DaIbergia sissoo, Leucaena leucocephala and A. erioloba which have a track record of nodulation were included as controls. Plants were harvested at 6 months after planting. At harvest, plant growth was measured as plant height, stem diameter size and plant total biomass. These variables were recorded during harvest, and each plant was then separated into nodules, roots and shoots, and oven-dried at 65°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 differente between total N in inoculated (nodulated) plants and total N in uninoculated (non-nodulated) plants. Inoculated and uninoculated Acacia ataxacantha plants together with controls were also harvested and their root systems observed for nodule formation and nodule number per plant. REXJLTS AND DISXJSSION
Plant growth
Of the plants inoculated with Venda soil, height growth was highest in A. seyal, followed by A. Iebbeek, D. sissoo, A. galpinii and A. auriculiformis. Acacia erioloba exhibited the least growth in height (Table 1). Height in the fastest-growing A. seyal was about 4-fold that of the slowest growing A. erioloba. With Klipspruit soil, A. seyal and A. lebbeek again showed the greatest growth in height, closely followed by A. galpinii and A. Senegal and least A. erioloba. The response of height growth to
Nitrogen fixation in tree legumes
995
Table 1. Height growth (cm) and stem diameters (mm) of nodulated tree legumes following inoculation with soil suspensions from Venda and Klipspruit Legume species
Plant heights
Acacia auriculiformis Acacia erioloba Acacia galpinii Acacia senegal Acacia seyal Albìzia lebbeck Dalbergia sissoo
Stem diameters
Venda
K/spruit
19.9 f 1.6
Venda
K/spruit 2.2 f 0.2 2.5 fO.l 5.1 * 1.2 2.8 f 0.5 5.5*0.1 3.4 f 0.7 2.8 k 0.4
9.8 + 0. I 23.2 + 1.9
18.8 + 1.0
2.3 f 0.2
7.5 f 0.1 26.6 f 1.1
2.8 f 0.1 5.0 f 0.2
ND
21.7 f 4.7 35.3 f 1.0
36.6 + 1.2 28.2 f 1.1 23.3 f 0.9
ND 6.4 k 0.3 2.4 1: 0.4 2.5 f 0.5
31.2 f 3.0 19,6 f 1.4
ND = not determined. Values are Means +SE (n = 3-4).
inoculation with the two soil types was comparable in al1 species tested. Except for A. seyal and A. galpinii, measures of stem diameters in the remaining tree legumes were extremely close (Table 1). Like plant height, stem diameter growth in A. seyal and A. galpinii was about 2-fold that of any of the other species. At 6 months after planting and on the basis of plant height and diameter growth, A. seyal and A. galpinii appeared to be the fastest-growing species. Height increment and stem diameter enlargement are good traits for selecting fast-growing plants for agroforestry (Duguma and Tonye, 1994). Consequently, both these two indicators of plant growth correlated in this study. So as expected, the pattern obtained for diameter growth following inoculation with the two soils was similar to that for plant height. Estimates of total plant biomass following inoculation with soil suspensions also differed considerably among the species. Acacia seyal and A. galpinii produced significantly higher wood and leaf biomass compared with the other five species (Table 3), and showed relatively similar accumulation of dry matter from inoculation with the two soil types. In contrast, A. lebbeck produced about twice the amount of dry matter with Klipspruit soil compared to Venda, thus indicating greater growth-promoting effects of N2 fixation by bacteria in the former soil. With that exception, dry matter yields were similar between the two soils in al1 other species. Apparently, wood dry matter increment in tree
legumes is species-dependent (Lugo et al., 1988). Although age is also a factor, these plants were of the same age, and any differences in performance would relate to the plant’s genetic potential or differential growth-promoting effects from inoculation. Since these species grow in different ecological zones, they tend to evolve root adaptations to cape with specific environmental stresses. In Africa, drought is a major problem, and tree plants in particular tend to develop deep rooting systems or large root volumes for maximizing water and nutrient uptake. Of the species shown in Table 2, D. sissoo exhibits the greatest root-to-shoot ratio when inoculated with either soil type, with a disproportionately higher investment in root mass. Albizia lebbeek and A. erioloba form the next group with high root-to-shoot ratios. Our data, suggest that these species with a high root-to-shoot ratio are probably better adapted to deep nutrient and water Capture, especially that they are typically found in arid and semi-arid environments in Africa. Thus, high rootto-shoot ratio may be an important adaptive trait for agroforestry trees growing in drier areas. Nodulation and N2 jxation
With the exception of Acacia Senegal which failed to nodulate with Venda soil, al1 the other species were effectively nodulated with native rhizobia or bradyrhizobia from the two soils (Table 2), resulting in high nodule dry mass. Acacia erioloba was how-
Table 2. Dry matter yield (g plant-‘) and root-to-shoot ratio and nodule dry weights (mg plant-‘) of tree legumes inoeulated with soil suspension from Venda and Klipspruit locations in South Afriea Total DM
Legume species
Acacia
Root-to-shoot
Venda
K/spruit
Venda
1.4 f 0.31
1.6 f 0.23
0.6 5 0.09
ratio K/spruit
Nodule DM Venda
Klipspruit
0.5 * 0.07
10.0 f 2.0
10.0 f 1.8
1.4*04 lO.Of 1.0 ND 8.8 * 0.1 10.0 f 1.5 4.0 f 0.3
1.3 f 9.0 * 15.6 f 6.5 f 17.0 f 28.0 *
auriculiformis
Acacia erioloba
1.0 + 0.04
1.3 f 0.03
1.8 f 0.04
1.9 f 0.06
Acacia galpinii Acacia Senegal Acacia seyal Albi:ia lebbeek Dalberaia sissoo
3.8 f 0.03
3.7 f 0.76
0.7 f 0.05
0.8 f 0.09
ND
0.8 f 0.22
ND
4.0 f 0.31
3.6 f 0.25
0.7 f 0.03
1.7* 0.66
3.1 f 1.14
0.7 f 0.03
0.8 * 0.01
1.1 f 0.26 9.3 + 0.45
0.7 f 0.51 1.1 *0.31 1.5 i: 0.08 10.7 i: 0.61
ND = not determined. Values are Means #.E.
SBB 29/5-&-H
(n = 3-4)
0.6 0.3 0.3 0.5 2.7 0.1
T. H. Masutha et al.
996 Table
3. Nodulation response Rhizobium NGR234.
of five tree legumes to inoculation with promiscuous Plants were harvested at 43 days after planting
Tree legume
Nodule
Nodulation”
Faidherbia albida Dolbergia sissoo Acacia erioloba Leucaena leucocephalu Acacia utaxacantha “Three replicate plants had 5 replicales
-Rhizohium
+ Rhizobrum
_ _ _
+ + + + _
_ were used m al1 nodulation
ever poorly nodulated in both soils, and accumulated the least dry matter in nodules. In some experiments, a few replicates failed to nodulate; and such unnodulated plants could be used to estimate N fixed as opposed to seed N. Although nodule effectiveness is often associated with higher N2 fixation, the nodulation data obtained in this study (Table 2) did not necessarily correlate with the amounts of NZ fixation. The woody legumes tested in this study differed significantly in their amounts of N2 fixation. In Venda soil, Acacia seyal and A. galpinii outperformed the other species, followed by A. auriculiformis and A. lebbeck. Acacia erioloba showed the lowest amount of N2 fixation (Fig. 1) due to poor nodulation (Table 2). With Klipspruit soil however, A. galpinii and A. lebbeck were the highest in N2 fixation, closely followed by A. seyal and A. auriculiformis (Fig. 2). Although nodule dry matter was highest in D. sissoo when nodulated with Klipspruit soil, the actual amount of N fixed was extremely low, suggesting ineffectiveness in nodulation, or a larger investment in cortical tissue at the expense of bacteria-infected tissue.
60
no./plant
10.3 8.0 2.0 2.0 0.0
assays. except A. utasacantha which
The pattern of nodulation and N2 fixation 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). Thus, the poor nodulation of A. erioloba in both soils or nodulation failure of A. Senegal in Venda soil might relate to paucity of rhizobial or bradyrhizobial numbers. Acacia Senegal nodulates only with strains of the genus Rhizobium while A. seyal which was among the best in nodulation and N2 fixation when inoculated with the two soils, nodulates with (Dommergues, both Rhizobium and Bradyrhizobium 1987). In that regard, A. seyal is a relatively more promiscuous legume compared with A. Senegal which is genus-specific. Of the seven woody legumes tested, A. seyal and A. galpinii have proved to be the fastest-growing species in terms of plant height (Table l), stem diameter growth (Table l), plant biomass (Table 2) and N fixed (Figs 1 and 2). 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 this study al1 variables of 60r
T
50 z m 40 a òñ .$ 30 -z z 10 z
200 i
Aa
Ds
As
Ag
Ae
Al
Legume tree spécies Fig. 1. Amounts of N fixed by selected tree legumes from inoculation with Venda soil. NI fixation was measured as the differente between total N in nodulated plants and uninoculated non-nodulated plants. Aa = Acacia auriculiformis; Ag = Acacia galpinii; Ds = Dalbergia sissoo; Ae = Acacia erioloba; As = Acacia seyal; Al = Albizia lebbeck.
0
Aa
Ds
As
Legume
Ag
Ae
Al
Asn
tree species
Fig. 2. Amounts of N fixed by selected tree legumes from inoculation with Klipspruit soil. N2 fixation was measured as the differente between total N in nodulated plants and uninoculated non-nodulated plants. Aa = Acacia auriculiformis; Ag = Acacia galpinii; Ds = Dalbergia sissoo; Ae = Acacia erioloba; As = Acacia seyal; Al = Albizia lebbeck.
Nitrogen fixation in tree legumes
plant growth have correlated with Nz fixation. On that basis, A. seyal and A. galpinii therefore appear to be the most promising elite material for use in agroforestry in both Venda and Klipspruit areas of South Africa. If however establishment of effective symbiosis with indigenous soil strains is the only consideration for species selection, then al1 seven tree legumes, except A. Senegal in Venda soil, are suitable for use in the agroforestry program. There is however no doubt that when dealing with agroforestry systems, criteria such as competitivity of the root systems are also important in addition to an active Nz fixation by the legume. It would be interesting to know how these glasshouse results relate to actual field performance. Studies are in progress, involving the use of 15N natura1 abundance technique, to measure the dependence on symbiotic fixation of these tree legumes growing in agroforestry plantations in Venda and Klipspruit. Such data, together with measurements of growth (e.g. plant heights and diameters), are vita1 for comparing field performance with glasshouse evaluation of these species. Additionally, we are interested in determining the populations and biodiversity of the root-nodule bacteria nodulating these tree legumes. Acacia ataxacantha, South Africa
a non-nodulating
legume
997
ate in legumes such as A. ataxacantha which is commonly found growing adjacent to nodulating Acacia species. Consequently, we suggest that non-nodulation in A. ataxacantha might be due to alterations in the biochemical pathways leading to synthesis of transcriptional regulators which induce nodulation (nod) genes in the homologous microsymbiont. Thus, identifying the nod gene-inducing molecules in nodulating Acacia species could provide a basis for determining the missing nod gene inducers in A. ataxacantha. Alternatively, inhibitors of nod gene induction from plant tissues could be responsible for non-nodulation in A. ataxacantha and other non-nodulating acacias. Experiments are in progress to test these hypotheses. Hopefully, the molecular basis of non-nodulation in Acacia ataxacantha and similar species can be established.
Acknowledgements-This work was supported by grants received from Forestek (CSIR), the URC of University of Cape Town, and from the Foundation for Research Development. The authors are grateful to the Protein Research Trust for a generous contribution which financed one of US (FDD) to present this paper at a Symposium organized in honour of Dr Johanna Dobereiner in Brazil. The Chairman’s Fund of Anglo-American is also thanked for contributing to the tost of travel.
in
In the course of this study, A. ataxacantha was found not to nodulate with indigenous bacteria present in soils from six different ecological zones within South Africa (Table 3). This lack of nodulation in the species was earlier noted by Grobbelaar and Clarke (1975) in their survey for field nodulation in indigenous legumes. The fact that soils from different ecological zones in South Africa have successfully nodulated most indigenous tree legumes in our current wider study, is a further indication of the non-nodulation of A. ataxacantha. Inoculating A. ataxacantha with Rhizobium NGR234 as a promiscuous nodulating marker, stil1 failed to induce nodule formation in this woody legume. The inability of A. ataxacantha to be incited into nodulation by Rhizobium NGR234, which is known to form nodules with over 75 genera of legumes (Relic et al., 1993), confirms in our view the non-nodulation status of this tree legume. Non-nodulation in Acacia has recently been reported in another African species, namely Acacia brevispica (Odee and Sprent, 1992). This is in addition to the well-documented case of non-nodulation in Acacia greggii in North America (Eskew and Ting, 1978). Interestingly, some legumes can lose their nodulation trait (Sprent and Raven, 1992) while stil1 retaining the ability to form other symbioses such as VA-mycorrhizal infection (Odee and Sprent, 1992). Clearly, the evolutionary advantage in not having a nodulation trait is difficult to evalu-
REFERENCES
Bogdan A. V. and Pratt D. J. (1974) Common Acacias of Kenya. Ministry of Agriculture, Animal Husbandry and Water Resources Grassland Reasearch Stations, Kitale and Marigat, Kenya. Charreau C. and Vidal P. (1965) Influence de /‘Acacia albida Del sur le sol, nutrition minerale et rendements des mils Pennisetum au Senegal. Agronomie Tropicale 20, 600-626.
Dakora F. D. and Keya S. 0. (1996) Contribution of legume nitrogen fixation to sustainable agriculture in Sub-Saharan Africa. Soil Biology & Biochemistry 29, 809-817.
Danso S. K. A., Bowen G. D. and Sanginga N. (1992) Biological nitrogen fixation in trees in agroforestry systems. Plant and Soill41, 177-196. Dommergues Y. R. (1987) The role of biological nitrogen fixation in agroforeestry. In Agroforestry, a Decade of Development (H. A. Steppler and P. K. R. Nair, Eds), pp. 245-271. ICRAF, Nairobi. Duguma B. and Tonye J. (1994) Growth of ten multipurpose tree species on acid soils in Sangmehna, Cameroon. Agroforestry Systems 27, 107-119.
Eskew D. L. and Ting 1. P. (1978) Nitrogen fixation by legumes and blue green algal-lichen trusts in Colorado desert environment. American Journal of Botany 65, 850-856.
Grobbelaar N. and Clarke B. (1975) A qualitative study of the nodulating ability of legume species: list 3. Journal of South African Botany 41, 29-36.
Kang B. T., Reynolds L. and Atta-Krah A. N. (1990) Afiey farming. Ädvances in Agronomy 43, 3 15-359. Kwesiga F. and Coe R. (1994) The effect of short rotation Sesbania sesban planted fallows on maize yields. Forest Ecology and Management 64, 199-208.
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Lugo A. E., Brown S. and Chapman J. (1988) An analytical review of production rate and stem wood biomass of tropical forest plantations. Forest Ecology anti Management 23, 179-200. Odee D. W. and Sprent J. 1. (1992) Acacia brevispica: a non-nodulating mimosoid legume? Soil Biology & Biochemistry 24, 717-719. Relic B., Fellay R., Lewin A., Perrent X., Price N. P. J., Rochepeau P. and Broughton W. J. (1993) Nod genes and Nod factors of Rhizobium species NGR234. In Neu Horizons in Nitrogen Fixation (R. Palacios, J. Mora and W. Newton, Eds), pp. 1833189. Kluwers Academie Publications, Dordrecht. Sanchez P. A. (1995) Science in agroforestry. Agroforestry Systems 30, 5-55. Sanginga N., Mulongoy K. and Ayanaba A. (1985) Effects of inoculation and mineral nutrients on nodulation and growth of Leucaena leucocephala. In Biological Nitrogen
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Fixation in Africa (H. Ssali and S. 0. Keya, Eds), pp. 419427. MIRCEN, Nairobi. Sanginga N., Mulongoy K. and Ayanaba A. (1986) Inoculation of Leucaena leucocephala (Lam) de Wit with Rhizobium and its nitrogen contribution to subsequent maize crop. Biological Agriculture and Horticulture 3, 341-347. Sanginga N., Mulongoy K. and Ayanaba A. (1989) Effectivity of indigenous rhizobia for nodulation and early nitrogen fixation with Leucaena leucocephala grown in Nigerian soils. Soil Biology & Biochemistry 21, 231-235. Sprent J. 1. and Raven J. A. (1992) Evolution of nitrogenfixing symbiosis. In Biological Nitrogen Fixation (G. Stacey, R. H. Burris and H. Evans, Eds), pp. 461496. Chapman and Hall, New York. Vincent J. M. (1970) A Manual for the Practica1 Study of Root-Nodule Bacteria. Blackwell Publications, Oxford.
Soil Biel. Biochem. Vol. 29, No. 516, pp. 993-998, 1997 0 1997 Elsevier Science Ltd. AI1 rights reserved Printed in Great Britain S0038-0717(%)002167 0038-0717/97 %17.00 + 0.00
EVALUATION OF N2 FIXATION AND AGROFORESTRY POTENTIAL IN SELECTED TREE LEGUMES FOR SUSTAINABLE USE IN SOUTH AFRICA T. H. MASUTHA, M. L. MUOFHE and F. D. DAKORA* Botany Department, University of Cape Town, P/B, Rondebosch, 7701, South Africa ( Accepted 4 July 1996) Summary-Seven
tree legumes with agroforestry potential were tested for their capacity to nodulate with indigenous rhizobia or bradyrhizobia in soils collected from different ecological zones in South Africa. Our findings show that al1 the tested species, except Acacia senegal, could effectively nodulate with root-nodule bacteria native to Venda and Klipspruit soils. Since Acacia seyal, Acacia gulpinii, Albiziu lebbe& and Acacia auriculiformis exhibited, in that order, a greater leve1 of growth and symbiotic performance, they manifest themselves as suitable species for agroforestry programs in Venda and Klipspruit soils. One indigenous species, Acacia araxuncunfha, was assessed to be a non-nodulating legume even when inoculated with a promiscuous strain of Rhizobium NGR234. 0 1997 Elsevier Science Ltd
INTRODUCTION
Globally, tree legumes are an important source of timber, fuel and fodder. Their ability to fix NZ 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 vita1 role in rehabilitation of degraded and marginal soils and restoration of nutrient fertility in fields exhausted by intensive cultivation. With the ever-increasing population in Africa, the need to exploit these tree legumes for sustained productivity of soil and increased crop yields has also increased (Dakora and Keya, 1996). The use of Nz-fixing trees in agroforestry systems in Africa has shown considerable promise in terms of benefit to crop and livestock production (Kang et al., 1990; Danso el al., 1992). An earlier study involving Faidherbiu albidu (formerly Acacia albidu) in Senegal showed a marked increase in nutrient fertility within the area covered by tree canopy, and an increased protein content in millet from that soil (Charreau and Vidal, 1965). N deficiency is therefore a major fertility constraint to agricultural productivity in Africa, a problem solvable through the development of agroforestry. The use of prunings from nodulated tree legumes has doubled cereal crop yields in Nigeria (Sanginga et al., 1986; Kang et al., 1990). In Malawi, Sesbanin *Author for correspondence: [email protected].
sesban is a fast-growing multipurpose legume that nodulates naturally with bradyrhizobia in soils, forms leaves that yield up to 80 kg N ha-’ and 6 kg P ha-’ after falling, and can produce 1.7 t ha-’ of fuelwood for rural communities (Sanchez, 1995). The nutrient-supplying capacity of Sesbaniu sesbun has therefore resulted in the development of a productive relay intercropping of maize with this species. Similarly, a doubling of maize yields was obtained in Zambia in Sesbaniu fallows compared with continuous unfertilized maize (Kwesiga and Coe, 1994). Thus, interest has increased in exploiting both local and exotic tree legumes for timber, firewood and other agroforestry products in African countries. This interest in agroforestry has increased rapidly in the new South Africa, where distribution of limited Government land to the many African farmers has resulted in extremely smal1 farm sizes, which preclude the possibility of fallow periods or livestock grazing. Consequently, the importante of Nz-fixing trees as multipurpose crops has been recognized. In South Africa, this has involved the planting of various shrub and tree legumes, namely Faidherbia albida, Acacia Senegal. Acacia seyal. Acacia galpinii. Acacia erioioba, Acacia auriculiformis, Albizia lebbeck, Cajanus cajan. Leucaena leucocephala, Virgilia divaritata and Dalbergia sissoo in al1 the former Black
Fax: 27.2 650 4041; e-mail: 993
Homelands in order to provide low-tost energy and cheap timber for the poorer communities while maintaining adequate soil fertility. However, establishment of many of these species is poor in soils without a previous history of the legume’s presence (Sanginga et al., 1989). It is therefore not known whether the indigenous and exotic
994
T. H. Masutha et al.
species currently used in the South African agroforestry program can nodulate effectively with native rhizobia and bradyrhizobia from the different localities. Furthermore, not al1 the African tree legumes form symbioses with root-nodule bacteria, a factor likely to affect growth rates. The woody legume, Acacia brevispica, which contains 17-19% crude protein in dry matter of leaves and pods (Bogden and Pratt, 1974) does not nodulate under Kenyan conditions (Odee and Sprent, 1992). Also, whether under South African conditions, nodulated species of selected tree legumes can grow fast enough to merit their inclusion in agroforestry programs has not been evaluated. Moreover, Dommergues (1987) has grouped woody legumes into low and high Nz.fixing species, with Faidherbia albida. Acacia Senegal and Acacia pellita belonging to the group of low fixers. However, the universality of that observation has not been tested, especially that the effectivity of a symbiosis can be enhanced or limited by factors unique to that particular environment. The purpose of this study was to assess native tree legumes and a few exotic species for growth and symbiotic performance with indigenous rhizobia and bradyrhizobia in soils collected from Venda and Klipspruit, South Africa, where two agroforestry plantations are sited. Additionally, the nodulation or non-nodulation status of Acacia ataxacantha was tested using Rhizobium NGR234, a promiscuous marker strain.
MATERIALS
AND METHODS
Six agroforestry plantations have been sited in South Africa by Forestek, CSIR, in different ecologica1 zones, which presently represent mainly the former Black Homelands. Soil samples collected from these sites (0-30 cm depth) were sieved and used as a source of inoculum in nodulation studies with selected tree legumes. The rationale was to determine the ability of native soil rhizobia or bradyrhizobia to effectively nodulate and fix Nz with these plant species. The data reported here cover only two of those six sites. Chemical analysis showed that the Venda soil had pH 4.3, organic matter 5.2%, nitrogen 0.20 mg g-’ soil, and cation exchange capacity 2.2 meq 100 g-’ soil. Klipspruit soil was similarly characterized as having pH 5.0, organic matter 5.0 mg g-‘, nitrogen 0.16 mg g-’ soil and cation exchange capacity 2.2 meq 100 g-’ soil. Prior to planting, seeds of selected tree legumes were scarified by soaking for 12 min in boiling water and left to imbibe ovemight. The seeds were then surface-sterilized in 70% ethanol for 2 min, and 50% sodium hypochlorite (bleach) for another 2 min, followed by rinsing 10 times with sterile deionized water. The seeds were either germinated in
sterile vermiculite prior to transplanting or sown directly into sterile rooting medium contained in 2-1 PVC tubes. Later, seedlings were thinned out to two per culture vessel. An inoculant was made from stirring 250 g soil in 1000 ml sterile de-ionized water, and 10 ml of the suspension used to inoculate each seedling growing in a sterile medium of sand and vermiculite (1:3) covered at the surface with sterile non-wettable cotton wool as an anti-contamination mulch. Al1 operations were done aseptically in a laminar flow hood. Plants were frequently irrigated to field capacity with N-free sterile Hoagland nutrient solution. In our studies, A. ataxacantha was identified as a woody legume unable to form root nodules in soils from the six locations in South Africa, including Venda and Klipspruit. Consequently, the nodulation status of this legume was further tested using Rhizobium strain NGR234 as a promiscuous marker. Rhizobium NGR234 is known to nodulate over 70 genera of legumes (Relic et al., 1993). The Rhizobium strain was cultured on agar slants using yeast mannitol agar medium (Vincent, 1970), and 5 ml culture suspension used to inoculate Acacia seedlings growing aseptically in ataxacantha Leonard jars with N-free nutrient solution (Vincent, 1970). Faidherbia albida, DaIbergia sissoo, Leucaena leucocephala and A. erioloba which have a track record of nodulation were included as controls. Plants were harvested at 6 months after planting. At harvest, plant growth was measured as plant height, stem diameter size and plant total biomass. These variables were recorded during harvest, and each plant was then separated into nodules, roots and shoots, and oven-dried at 65°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 differente between total N in inoculated (nodulated) plants and total N in uninoculated (non-nodulated) plants. Inoculated and uninoculated Acacia ataxacantha plants together with controls were also harvested and their root systems observed for nodule formation and nodule number per plant. REXJLTS AND DISXJSSION
Plant growth
Of the plants inoculated with Venda soil, height growth was highest in A. seyal, followed by A. Iebbeek, D. sissoo, A. galpinii and A. auriculiformis. Acacia erioloba exhibited the least growth in height (Table 1). Height in the fastest-growing A. seyal was about 4-fold that of the slowest growing A. erioloba. With Klipspruit soil, A. seyal and A. lebbeek again showed the greatest growth in height, closely followed by A. galpinii and A. Senegal and least A. erioloba. The response of height growth to
Nitrogen fixation in tree legumes
995
Table 1. Height growth (cm) and stem diameters (mm) of nodulated tree legumes following inoculation with soil suspensions from Venda and Klipspruit Legume species
Plant heights
Acacia auriculiformis Acacia erioloba Acacia galpinii Acacia senegal Acacia seyal Albìzia lebbeck Dalbergia sissoo
Stem diameters
Venda
K/spruit
19.9 f 1.6
Venda
K/spruit 2.2 f 0.2 2.5 fO.l 5.1 * 1.2 2.8 f 0.5 5.5*0.1 3.4 f 0.7 2.8 k 0.4
9.8 + 0. I 23.2 + 1.9
18.8 + 1.0
2.3 f 0.2
7.5 f 0.1 26.6 f 1.1
2.8 f 0.1 5.0 f 0.2
ND
21.7 f 4.7 35.3 f 1.0
36.6 + 1.2 28.2 f 1.1 23.3 f 0.9
ND 6.4 k 0.3 2.4 1: 0.4 2.5 f 0.5
31.2 f 3.0 19,6 f 1.4
ND = not determined. Values are Means +SE (n = 3-4).
inoculation with the two soil types was comparable in al1 species tested. Except for A. seyal and A. galpinii, measures of stem diameters in the remaining tree legumes were extremely close (Table 1). Like plant height, stem diameter growth in A. seyal and A. galpinii was about 2-fold that of any of the other species. At 6 months after planting and on the basis of plant height and diameter growth, A. seyal and A. galpinii appeared to be the fastest-growing species. Height increment and stem diameter enlargement are good traits for selecting fast-growing plants for agroforestry (Duguma and Tonye, 1994). Consequently, both these two indicators of plant growth correlated in this study. So as expected, the pattern obtained for diameter growth following inoculation with the two soils was similar to that for plant height. Estimates of total plant biomass following inoculation with soil suspensions also differed considerably among the species. Acacia seyal and A. galpinii produced significantly higher wood and leaf biomass compared with the other five species (Table 3), and showed relatively similar accumulation of dry matter from inoculation with the two soil types. In contrast, A. lebbeck produced about twice the amount of dry matter with Klipspruit soil compared to Venda, thus indicating greater growth-promoting effects of N2 fixation by bacteria in the former soil. With that exception, dry matter yields were similar between the two soils in al1 other species. Apparently, wood dry matter increment in tree
legumes is species-dependent (Lugo et al., 1988). Although age is also a factor, these plants were of the same age, and any differences in performance would relate to the plant’s genetic potential or differential growth-promoting effects from inoculation. Since these species grow in different ecological zones, they tend to evolve root adaptations to cape with specific environmental stresses. In Africa, drought is a major problem, and tree plants in particular tend to develop deep rooting systems or large root volumes for maximizing water and nutrient uptake. Of the species shown in Table 2, D. sissoo exhibits the greatest root-to-shoot ratio when inoculated with either soil type, with a disproportionately higher investment in root mass. Albizia lebbeek and A. erioloba form the next group with high root-to-shoot ratios. Our data, suggest that these species with a high root-to-shoot ratio are probably better adapted to deep nutrient and water Capture, especially that they are typically found in arid and semi-arid environments in Africa. Thus, high rootto-shoot ratio may be an important adaptive trait for agroforestry trees growing in drier areas. Nodulation and N2 jxation
With the exception of Acacia Senegal which failed to nodulate with Venda soil, al1 the other species were effectively nodulated with native rhizobia or bradyrhizobia from the two soils (Table 2), resulting in high nodule dry mass. Acacia erioloba was how-
Table 2. Dry matter yield (g plant-‘) and root-to-shoot ratio and nodule dry weights (mg plant-‘) of tree legumes inoeulated with soil suspension from Venda and Klipspruit locations in South Afriea Total DM
Legume species
Acacia
Root-to-shoot
Venda
K/spruit
Venda
1.4 f 0.31
1.6 f 0.23
0.6 5 0.09
ratio K/spruit
Nodule DM Venda
Klipspruit
0.5 * 0.07
10.0 f 2.0
10.0 f 1.8
1.4*04 lO.Of 1.0 ND 8.8 * 0.1 10.0 f 1.5 4.0 f 0.3
1.3 f 9.0 * 15.6 f 6.5 f 17.0 f 28.0 *
auriculiformis
Acacia erioloba
1.0 + 0.04
1.3 f 0.03
1.8 f 0.04
1.9 f 0.06
Acacia galpinii Acacia Senegal Acacia seyal Albi:ia lebbeek Dalberaia sissoo
3.8 f 0.03
3.7 f 0.76
0.7 f 0.05
0.8 f 0.09
ND
0.8 f 0.22
ND
4.0 f 0.31
3.6 f 0.25
0.7 f 0.03
1.7* 0.66
3.1 f 1.14
0.7 f 0.03
0.8 * 0.01
1.1 f 0.26 9.3 + 0.45
0.7 f 0.51 1.1 *0.31 1.5 i: 0.08 10.7 i: 0.61
ND = not determined. Values are Means #.E.
SBB 29/5-&-H
(n = 3-4)
0.6 0.3 0.3 0.5 2.7 0.1
T. H. Masutha et al.
996 Table
3. Nodulation response Rhizobium NGR234.
of five tree legumes to inoculation with promiscuous Plants were harvested at 43 days after planting
Tree legume
Nodule
Nodulation”
Faidherbia albida Dolbergia sissoo Acacia erioloba Leucaena leucocephalu Acacia utaxacantha “Three replicate plants had 5 replicales
-Rhizohium
+ Rhizobrum
_ _ _
+ + + + _
_ were used m al1 nodulation
ever poorly nodulated in both soils, and accumulated the least dry matter in nodules. In some experiments, a few replicates failed to nodulate; and such unnodulated plants could be used to estimate N fixed as opposed to seed N. Although nodule effectiveness is often associated with higher N2 fixation, the nodulation data obtained in this study (Table 2) did not necessarily correlate with the amounts of NZ fixation. The woody legumes tested in this study differed significantly in their amounts of N2 fixation. In Venda soil, Acacia seyal and A. galpinii outperformed the other species, followed by A. auriculiformis and A. lebbeck. Acacia erioloba showed the lowest amount of N2 fixation (Fig. 1) due to poor nodulation (Table 2). With Klipspruit soil however, A. galpinii and A. lebbeck were the highest in N2 fixation, closely followed by A. seyal and A. auriculiformis (Fig. 2). Although nodule dry matter was highest in D. sissoo when nodulated with Klipspruit soil, the actual amount of N fixed was extremely low, suggesting ineffectiveness in nodulation, or a larger investment in cortical tissue at the expense of bacteria-infected tissue.
60
no./plant
10.3 8.0 2.0 2.0 0.0
assays. except A. utasacantha which
The pattern of nodulation and N2 fixation 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). Thus, the poor nodulation of A. erioloba in both soils or nodulation failure of A. Senegal in Venda soil might relate to paucity of rhizobial or bradyrhizobial numbers. Acacia Senegal nodulates only with strains of the genus Rhizobium while A. seyal which was among the best in nodulation and N2 fixation when inoculated with the two soils, nodulates with (Dommergues, both Rhizobium and Bradyrhizobium 1987). In that regard, A. seyal is a relatively more promiscuous legume compared with A. Senegal which is genus-specific. Of the seven woody legumes tested, A. seyal and A. galpinii have proved to be the fastest-growing species in terms of plant height (Table l), stem diameter growth (Table l), plant biomass (Table 2) and N fixed (Figs 1 and 2). 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 this study al1 variables of 60r
T
50 z m 40 a òñ .$ 30 -z z 10 z
200 i
Aa
Ds
As
Ag
Ae
Al
Legume tree spécies Fig. 1. Amounts of N fixed by selected tree legumes from inoculation with Venda soil. NI fixation was measured as the differente between total N in nodulated plants and uninoculated non-nodulated plants. Aa = Acacia auriculiformis; Ag = Acacia galpinii; Ds = Dalbergia sissoo; Ae = Acacia erioloba; As = Acacia seyal; Al = Albizia lebbeck.
0
Aa
Ds
As
Legume
Ag
Ae
Al
Asn
tree species
Fig. 2. Amounts of N fixed by selected tree legumes from inoculation with Klipspruit soil. N2 fixation was measured as the differente between total N in nodulated plants and uninoculated non-nodulated plants. Aa = Acacia auriculiformis; Ag = Acacia galpinii; Ds = Dalbergia sissoo; Ae = Acacia erioloba; As = Acacia seyal; Al = Albizia lebbeck.
Nitrogen fixation in tree legumes
plant growth have correlated with Nz fixation. On that basis, A. seyal and A. galpinii therefore appear to be the most promising elite material for use in agroforestry in both Venda and Klipspruit areas of South Africa. If however establishment of effective symbiosis with indigenous soil strains is the only consideration for species selection, then al1 seven tree legumes, except A. Senegal in Venda soil, are suitable for use in the agroforestry program. There is however no doubt that when dealing with agroforestry systems, criteria such as competitivity of the root systems are also important in addition to an active Nz fixation by the legume. It would be interesting to know how these glasshouse results relate to actual field performance. Studies are in progress, involving the use of 15N natura1 abundance technique, to measure the dependence on symbiotic fixation of these tree legumes growing in agroforestry plantations in Venda and Klipspruit. Such data, together with measurements of growth (e.g. plant heights and diameters), are vita1 for comparing field performance with glasshouse evaluation of these species. Additionally, we are interested in determining the populations and biodiversity of the root-nodule bacteria nodulating these tree legumes. Acacia ataxacantha, South Africa
a non-nodulating
legume
997
ate in legumes such as A. ataxacantha which is commonly found growing adjacent to nodulating Acacia species. Consequently, we suggest that non-nodulation in A. ataxacantha might be due to alterations in the biochemical pathways leading to synthesis of transcriptional regulators which induce nodulation (nod) genes in the homologous microsymbiont. Thus, identifying the nod gene-inducing molecules in nodulating Acacia species could provide a basis for determining the missing nod gene inducers in A. ataxacantha. Alternatively, inhibitors of nod gene induction from plant tissues could be responsible for non-nodulation in A. ataxacantha and other non-nodulating acacias. Experiments are in progress to test these hypotheses. Hopefully, the molecular basis of non-nodulation in Acacia ataxacantha and similar species can be established.
Acknowledgements-This work was supported by grants received from Forestek (CSIR), the URC of University of Cape Town, and from the Foundation for Research Development. The authors are grateful to the Protein Research Trust for a generous contribution which financed one of US (FDD) to present this paper at a Symposium organized in honour of Dr Johanna Dobereiner in Brazil. The Chairman’s Fund of Anglo-American is also thanked for contributing to the tost of travel.
in
In the course of this study, A. ataxacantha was found not to nodulate with indigenous bacteria present in soils from six different ecological zones within South Africa (Table 3). This lack of nodulation in the species was earlier noted by Grobbelaar and Clarke (1975) in their survey for field nodulation in indigenous legumes. The fact that soils from different ecological zones in South Africa have successfully nodulated most indigenous tree legumes in our current wider study, is a further indication of the non-nodulation of A. ataxacantha. Inoculating A. ataxacantha with Rhizobium NGR234 as a promiscuous nodulating marker, stil1 failed to induce nodule formation in this woody legume. The inability of A. ataxacantha to be incited into nodulation by Rhizobium NGR234, which is known to form nodules with over 75 genera of legumes (Relic et al., 1993), confirms in our view the non-nodulation status of this tree legume. Non-nodulation in Acacia has recently been reported in another African species, namely Acacia brevispica (Odee and Sprent, 1992). This is in addition to the well-documented case of non-nodulation in Acacia greggii in North America (Eskew and Ting, 1978). Interestingly, some legumes can lose their nodulation trait (Sprent and Raven, 1992) while stil1 retaining the ability to form other symbioses such as VA-mycorrhizal infection (Odee and Sprent, 1992). Clearly, the evolutionary advantage in not having a nodulation trait is difficult to evalu-
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