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Phosphorus requirement and nodulation of herbaceous and shrub legumes in low P soils of a Guinean savanna in Nigeria
Applied Soil Ecology ELSEVIER
Applied Soil Ecology 3 (1996) 247-255
Phosphorus requirement and nodulation of herbaceous and shrub legumes in low P soils of a Guinean savanna in Nigeria N. Sanginga *, J.A. Okogun, I.O. Akobundu, B.T. Kang International Institute of Tropical Agrieulture (IITA), Oyo Road, PMB. 5320 lbadan, Nigeria
Accepted 29 September 1995
Abstract
There is increasing interest to improve the N2 fixation and P use efficiency of herbaceous and shrub legumes currently being introduced in cereal-based cropping systems in the moist savanna zones of West Africa. Knowledge about N and P nutrition of these legumes can assist in adapting them to new areas where fertilizer use is not feasible by the smallholder farmers. Pot and field experiments were carried out to examine variation among potential herbaceous and shrub legumes for their ability to nodulate and to use P efficiently. These legumes were grown at two P levels (0 and 7 kg P ha - I ) in soils collected from two fields with different cropping histories (compound and degraded fields) at Yamrat in the northern Guinea savanna of Nigeria. In the compound field, animal manure and household residues are applied annually whereas in the degraded field, cereals are grown continuously with minimal organic inputs. Significant differences in growth, P content and mycorrhizal infection rate occurred among the different legumes as affected by P and cropping history. Legumes grown in soil from the degraded field responded more to P application than those grown in soil from the compound field. Phosphorus responsive legume species e.g. Mucuna pruriens var. utilis (black seed) and Crotolaria ochroleuca, had a low mycorrhizal infection rate compared to lower P responsive species such as Lablab purpureus and Cajanus cajan. A significant relationship was found between mycorrhizal infection rate and plant growth but not with nodulation of these legumes. All legumes nodulated naturally and the proportion of N derived from atmospheric N2 ranged from 38% for Centrosema brasilianum to 90% for L. purpureus. Application of P increased the weight of nodules but did not improve the proportion of N 2 fixation. This study showed that species such as L. purpureus has a high N 2 fixing capability and can also adapt to low P soils conditions in the northern Guinea savanna of West Africa. Keywords: Compoundfield; Degraded field; P content; Nodulation;N2 fixation; Mycorrhiza
1. I n t r o d u c t i o n Low levels of soil available N and P and large crop responses to N and P applications are common
* Corresponding author: c/o Lamboum and Co., 26 Dingwall Road, Croydon CR9 3EE, UK. Elsevier Science B.V. SSDI 0929- 1393(95)00083-6
in the northern Guinea savanna of West Africa (Bationo et al., 1986; Mughogho et al., 1986). The establishment, nodulation and growth of legumes being introduced in cereal-based cropping systems will also benefit from P fertilizer application. Preliminary observations in the northern Guinea savanna of Nigeria have shown that legumes require about 30 kg P h a - l for optimal growth and N 2
248
N. &mgingaet al./Applied Soil Ecology 3 (1996)247-255
fixation (Weber et al., personal communication). Small-scale farmers, however, only use limited amounts of fertilizer for their main food crops. Under these circumstances, it would be beneficial to select legume species or cultivars with low P requirements for growth, nodule development and N 2 fixation. Recent studies have shown large variability in growth, P content and use efficiency and N 2 fixation in legumious trees used in alley cropping system (Sanginga et al., 1994) and annual legumes (Gunawardena et al., 1993). Kang et al. (in press) also showed large differences in early growth of woody species in response to P application. Level of P response was correlated to seed P content. However, only limited information exists on P responses of herbaceous and shrub legumes currently being tested into the moist savanna zone of West Africa. Relatively few data exist on the external P and N requirements of these legumes in P deficient soils. A better understanding of leguminous species or cultivar differences in P and N nutrition may help in adapting these plants to new areas where fertilizers are not readily available. The objectives of this study were: (i) to determine specific differences in P uptake and use between various herbaceous and shrub legumes (ii) to assess the response of these legumes to P application and (iii) to select those which have high nodulation and N 2 fixation potential at low levels of P in the soil.
2. Materials and methods This investigation was conducted both in the field at Yamrat in the northern Guinea Savanna of Nigeria (9°81'N 10°13'E) and in pot experiments using Yamrat soil. The pot experiment was undertaken at the International Institute of Tropical Agriculture (IITA) at Ibadan, Nigeria. Field trials were installed in two farmers' fields with different cropping histories. Experimental field one was a compound field, where animal manure and other household residues are applied annually and the field is most often used for cereals (maize and sorghum) production. The second experiment was on a degraded field, where cereals were continuously grown for 10 or more years with minimal organic material inputs. Selected soil
Table 1 Selected physico-chemicalcharacteristics of degraded and compound fields at Yanarat Compound field Degradedfield pH (H20) Organic C (%) Total N (%) N03-N (/.tg g.l soil) NH4-N (/zg g~ soil) Extr. P ( p,g g-i soil) Mycorrbizal spores (number per 100 grams soil) Sand (%) Silt (%) Clay (%)
8.29 1.76 0.13 32.40 8.25 85.44 56
5.72 0.50 0.04 9.49 5.08 1.10 54
80 13 7
81 12 7
physico-chemical characteristics (0-15 cm depth) of these two fields measured according to IITA analytical procedures (IITA, 1989) are given in Table 1. 2.1. Pot experiment 2.1.1. Experimental design A randomized complete block design with three replications was used . Treatments included; (i) the ten legume species and maize, (ii) two P levels (with and without P) and (iii) the two soils (degraded and compound fields). Soils for the pot experiment were collected from 0 - 1 5 cm depth from each of the two fields. The soil was air dried and sieved ( < 2 mm screen) and 5 kg soil per pot was used for the trial. A basal application of 60 mg K k g - 1 soil as muriate of potash and 1 ml of a combination of micronutrients (Vincent, 1970) per kilogram soil was applied to each pot before planting. Phosphorus was applied as KHePO 4 a rate of 7 kg P h a - I 2.1.2. Legumes species and maize seeds Ten legume species (Table 2) and maize cultivar Oba super (as a reference plant) were used in this trial. Psophocarpus palustris seeds were scarified with H 2 S O 4 for 5 min, while the other seeds were only surface sterilized with ethanol (1 min) and H202 (5 min), and then rinsed with sterile water. The sterilized seeds were soaked for 24 h in sterile water and then pregerminated in Petri dishes containing sterile cotton wool. Eight seeds were sown in
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
each pot and thinned to two plants per pot at 1 week after emergence.
2.1.3. Harvesting All plants in the pot were harvested 8 weeks after planting (WAP) and assessed for dry matter production (shoots and roots), nodulation, and total N and P content. Mycorrhizal infection was rated on fresh roots using the method by Giovanetti and Mosse (1980). The proportion and amount of N derived from atmospheric N 2 were estimated by the total N difference method using maize as a reference plant. 2.2. Field experiment A split-plot design with three replications was used. Main-plots were two P rates, 0 and 7 kg P ha -~ (as single superphoshate) and the sub-plots were eight legume species and maize as control treatment. Subplot size was 10 m X 5 m, including 2 m X 5 m area reserved for destructive sampling (at one end of each subplot). Legume seeds were surface sterilized as described for the pot experiment. The seeds of small seeded legumes were drilled within each plot at 50 cm inter-row spacing at a seeding rate of 10 kg seed ha-~. For large size seeds e.g. maize, M. pruriens, L. purpureus, and C. cajan, 50 cm inter-row and 25 cm intra-row spacings were
249
used. Two seeds were planted per hill. Five plants were harvested at 6 and 14 WAP. The same plant parameters were assessed as in the pot experiment.
2.3. Chemical analysis Analysis of P in the plant shoots was done using the vanadomolybdate yellow method and total N was determined by the Automatic N analyzer following wet acid digestion (IITA, 1989).
2.4. Statistical analyses Analysis of variance (ANOVA) was done using the SAS program (Statistical Analysis Systems Institute Inc., 1986) to determine treatment and interaction effects. Least significant differences (LSD) were calculated at the 5% level to assess treatment differences.
3. Results 3.1. Pot experiment 3.1.1. Nodulation and N 2 fixation All plants nodulated in the two soils, but the number and nodule mass at 8 WAP varied signifi-
Table 2 Total N and percentage N derived from the atmosphere of the legumes grown in the greenhouse in soils collected from degraded and compound fields Legume species
L. purpureus a C. cajan b C. verrucosa ~ C. pascuroum a P. argentea a M. pruriens (black) a C. brasilianum a C. rotundifi~lia a A. histrix a P. palustris a LSD 5% a (1) (2)
N derived from the atmosphere (%)
Total N (mg per plant) Degraded field
Compound field
Degraded field
Compound field
91 50 52 78 23 107 16 43 31 47
157 42 81 94 62 81 34 34 78 44
90 52 81 87 59 91 43 78 70 80
87 50 73 77 65 73 38 38 72 51
5 4
13 12
Mean values averaged over P treatments; P effect was not significant. a Herbaceous species. b Shrub species.(1) For comparing plant species within sites; (2) For comparing plant species between sites.
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247 255
250
cantly between legume species. Nodule number per plant ranged from 1 for Aeschynomene histrix to 32 for Centrosema pascuorum. Mucuna pruriens in spite of its low number of nodules had the highest nodule mass followed by L. purpureus. Aeschynomene histrix recorded the lowest nodule mass (Fig. 1). Phosphorus application significantly increased the number and mass of nodules of most species except P. argentea, C. rotundifolia and A. histrix (Fig. 1). Herbaceous and shrub legumes derived 69% (average) of their plant total N from atmospheric N 2.
1 0 LSD 5% 4 [] ~
2 ILSD 5%
3
%
2
~ - = . ~
~ ~
40
I I] LSD 5%
2
~
G
~
~2 G6
n 3( 1. 2.
¢~ 2C Z
LSD 5% for comparing plant species at different P levels LSD 5% for comparing plant species at same P levels
Fig. 2. Effect, at 8 WAP in the greenhouse, of P level on shoot dry weight of legume species grown in soil collected from degraded and compound fields at Yamrat.
Z 1(
d
4
~J
G
~j
& ,,.
~j
I~ LSD5% rl
&
2
2 II I.l LSD 5%
[ o { .[ .i ~J LSD 5% 1. 2.
{J
For comparing plant species at different P levels For comparing plant species at same P levels
Fig. 1. Effect, at 8 WAP in the greenhouse, of P on the number and mass of nodule weight of legume species grown in soils collected from degraded and compound fields at Yamrat.
The proportion of N z fixed depended largely on the field history and legume species (Table 2). Percentage N 2 fixed varied between 38% and 90% for C. brasilianum and L. purpureus, respectively and it was higher in soil from the degraded field (76%) than in soil from the compound field (62%). There was a significant correlation ( r = 0.91, P = 0.05) between the proportion and amount of N 2 fixed per plant. Unlike the amount of N 2 fixed, the proportion of N 2 fixed was not significantly affected by P application. 3.1.2. Shoot dry weight and total N accumulation
Shoot dry weight differed significantly between legume species; ranging from a mean of 1.21 g per plant for C. brasilianum to 4.15 g per plant for M. pruriens (Fig. 2). Shoot dry weight response to P application varied between legumes species. The highest P response was observed with C. verrucosa (197%) > A. histrix (105%), > C. rotundifolia (78%), > M. pruriens (28%) C. pascuorum (26%). Cajanus cajan and C. brasilianum showed small responses while P. argentea and L. purpureus shoot yields were reduced with P application. Shoot dry matter yield was correlated to total N accumulation in the legumes ( r = 0.99; P = 0.05).
P
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
251
49
42
35
.o 28
.fi 21
8 14
o ~
~
g.
.~
~
,~
~
t5
t5
.
_~
~t
,5 Fig. 3. Mycorrhizal infection (%) of legume species, at 8 WAP in the green house. Mean values across sites and P levels.
3.1.3. Total P accumulation and mycorrhizal infection
0.98; P = 0.05). It ranged from 0.98 mg P per plant for P. argentea to 4.99 mg P per plant for M. pruriens in degraded field and from 1.76 mg P per plant for A. histrix to 8.01 mg P per plant for L.
Total P accumulation varied between species and was significantly correlated to shoot dry matter (r =
Table 3 Nodule dry weight of legume species grown with or without P addition in degraded and compound fields in Yamrat at 14 weeks after planting (WAP) Legume species
Nodule dry weight (mg per plant) Degraded field
Lablab purpureus Crotolaria ochroleuca Centrosema pascuorum Cajanus cajan Mucuna pruriens (black) Mucuna pruriens (white) Centrosema brasilianum Chamaechrista rotundifolia LSD 5% ( 1) (2)
Compound field
-P
+p
-p
+p
2.01 0.01 0.17 1.90 1.16 2.01 2.04 0.00
2.16 0.06 0.16 0.55 5.51 0.03 0.16 0.84
16.62 12.66 3.06 20.95 25.58 12.18 1.28 2.78
22.49 24.54 11.98 18.00 29.02 I 1.67 1.08 4.40
5.00 4.00
8.00 9.00
(1) For comparing legumes at different P levels; (2) For comparing legumes at same P levels.
252
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
purpureus in the compound field. The relationships between P content and P application also varied between species and field history and was also related to shoot dry matter. Mycorrhizal infection rate of plants grown in pots ranged between 10% (average) for C. verrucosa, A. histrix and Psophocarpus palustris and 27% (average) for C. rotundifolia, L. purpureus, and P. argentea (Fig. 3). Maize infection rate was the highest (40%). There were significant correlations between mycorrhizal infection rate and shoot dry weight (r = 0.64; P = 0.05: n = 80) but not with nodulation parameters. 3.2. Field experiment 3.2.1. Nodulation and N 2 fixation Nodulation was affected by legume species and sampling time. At 6 W A P , L. purpureus nodulated profusely and had more nodules than the other plants (data not shown). At 14 WAP, when most of the legumes were flowering, L. purpureus again produced the highest number of nodules especially in the degraded field (Table 3). The application of P increased the nodule weight of a few species (e.g. M. pruriens in the degraded soil and L. purpureus, C. Ochroleuca and C. pascuorum in the compound field) but did not improve the number of nodules. Legume species grown in the compound field had on
average lower nodule numbers, higher mass and weight than those in the degraded field (Table 3). The proportion of N 2 fixed by the legume species was estimated by the total N difference method using maize as a control (data not shown). Lablab purpureus had again the highest percentage N 2 fixed (72% average). Estimations of N 2 fixed in the other legumes were not determined because their early growth rate and total N accumulation was lower than that of maize control.
3.2.2. Shoot dry weight total N accumulation Shoot dry weight differed significantly between legume species and P application levels (Table 4), M. pruriens (black) having more shoot biomass than that of the other species. It was followed by C. ochroleuca while C. brasilianum and C. rotundifolia had the lowest shoot biomass. There was a significant correlation between shoot dry and total N (r = 0.93; P = 0.05). Phosphorus application increased shoot dry weight and total N of legumes grown in degraded and compound farmer's field by an average of 91% and 38%, respectively. Crotolaria ochroleuca and the two M. pruriens grown in degraded farmer's field had their shoot dry weight increased by about 4 to 6 fold, respectively by P application. Such large responses to P application did not occur in the compound farmer's field.
Table 4 Shoot dry weight and total N (mg per plant) in shoots of legume species grown with or without P application in degraded and compound fields in Yamrat at 14 WAP Legume species
Lablab purpureus Crotolaria ochroleuca Centrosema pascuorum Cajanus cajan Mucuna pruriens (black) Mucuna pruriens (white) Centrosema brasilianum Charnaechrista rotundifolia LSD 5% (1) (2)
Shoot dry weight (g per plant)
Total N (mg per plant)
Degraded field
Compound field
Degraded field
Compound field
-p
+p
-P
+p
-p
+p
-p
+p
44 34 60 51 21 35 60 30
69 130 65 59 55 205 58 25
22 48 38 34 108 60 8 14
47 76 61 34 123 83 2 34
1201 I0 l 3 1480 1735 521 637 794 707
1747 3323 t 013 1832 3884 2399 604 602
601 1231 614 993 3466 1214 102 219
595 2432 1833 1021 3745 1228 61 701
13 24
19 38
421 1421
(1) For comparing legumes at different levels; (2) For comparing legumes at same levels.
596 1193
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
253
Table 5 Mycorrhizal infection rate (%) and total P (mg per plant) content in plant shoots of legume species grown with or without P application in degraded and compound fields in Yamrat at 14 WAP Legume species
Lablab purpureus Crotolaria ochroleuca Centroserna pascuorum Cajanus cajan Mucuna pruriens(black) Mucuna pruriens (white) Centrosema brasilianum Chamaechrista rotundifi~lia LSD 5% (l) (2)
Total P (mg per plant)
Mycorrhizal (%) Degraded field
Compound field
Degraded field
Compound field
-p
+P
-p
+P
-p
+p
-p
+p
8 13 4 4 3 7 4 7
15 3 11 3 0 15 8 10
26 10 4 20 9 19 6 9
33 27 36 26 4 19 17 3
52 44 52 50 18 27 44 17
76 115 51 81 188 153 34 14
21 65 23 40 138 52 8 12
32 I19 24 58 201 61 5 35
7 18
8 19
21 48
29 62
(1) For comparing legumes at different P levels; (2) For comparing legumes at same P levels.
3.2.3. Total P accumulation Phosphorus application increased total P of the legumes by 73% and 84% (average) when grown in the degraded or compound farmer's fields, respectively, at 6 WAP (data not shown). At 14 WAP, increases of 147% and 49% due to P application were obtained in the two fields, respectively. Total P content at low P varied between 8 and 138 mg P per plant while that at high P ranged from 5 to 201 mg P per plant at high P (Table 5). The two M. pruriens (black and white seeds) and C. ochroleuca required P addition to grow well as shown by their significant increase in total P content due to P application in both fields. Cajanus cajan and L. purpureus had similar total P content at both zero and P application while C. brasilianum and C. rotundifolia grew poorly at both zero and P application. 3.2.4. Mycorrhizal infection Mycorrhizal infection rate was affected by plant species and the field history but not by P application. Legume species grown in the compound field were more infected than those grown in the degraded field (Table 5). Mucuna pruriens (black seed), C. brasilianum and C. rotundifolia showed the lowest rate of mycorrhizal infection while, L. purpureus and M. prurieus (white seed) were highly infected by mycorrhizal fungi. As in the pot experiment, significant
correlation was found only between mycorrhizal infection rate and shoot dry weight.
4. Discussion
Three types of growth response to P application were evident for herbaceous and shrub legumes. For example, C. cajan and L. purpureus achieved maximum yield at inherent P levels in both degraded and compound fields. With M. pruriens and C. ochroleuca, maximum yield was obtained only when P was added in the degraded field. Pseudovigna argentea had its shoot dry weight reduced by P application in the greenhouse. This suggests that the P requirements for herbaceous and shrub legumes to achieve maximum growth vary. It can be argued that if the ability to grow in low P soils is of overriding importance, species such as M. pruriens and C. ochroleuca that require P application for maximum biomass production would be at a disadvantage when grown in low P such as in the degraded field (Tables 4 and 5). On the other hand, species such as L. Purpureus and C. cajan did not respond significantly to P in both fields indicating that the inherent soil P concentration in these fields was adequate for their growth.
254
N. Sanginga et al./Applied Soil Ecoh~gy 3 (1996) 247-255
Species are known to differ greatly in their ability to assimilate P especially at low P levels; opinion differs, however, concerning the mechanisms of these differences in ability to grow at low P levels in soils. Studies by F~Shse et al. (1988) indicate that species differ in their ability to extract soil P depending upon the potential of roots to absorb P, their active lifetime and the amount of root per unit of shoot. On the other hand, results of Sanginga et al. (1991) and Chisholm and Blair (1988) indicate that species able to use absorbed P more efficiently in the production of biomass should be better adapted to low P soils. Our investigation shows that differences between several species in their ability to be infected by mycorrhizae and the difference in P uptake and growth response to P could largely account for the difference in adaptation of these species to low P soils. For example, at the lowest P level, species such as L. Purpureus and C. cajan had the highest mycorrhizal infection rate (average 20%) compared to 4 - 1 0 % for the other legumes, with M. prurieus and C. Ochroleuca having the lowest infection rate. Phosphorus responders species had on average lower mycorrhizal infection rate than the P-non responders. The influence of mycorrhizal fungi on P uptake is well documented. Habte and Manjunath (1987) demonstrated that plants with stronger mycorrhizal association have a lower external P requirement than plants of the same type without an effective mycorrhizal association. Data presented also indicate that leguminous species tested also differ in their ability to fix N 2 under low P conditions. Percentage of N 2 fixed by these legumes was generally high (on average 80%) and is comparable to values reported for herbaceous legumes used as cover crop or pasture elsewhere (Date, 1991; Sylvester-Bradley, 1984). The best species in terms of percentage and total N fixed under low P soil level was L. purpureus. Such species should be better suited N and P deficient soils. Although other species such as M. pruriens presently being adopted by farmers in some areas of the moist savanna showed comparable percentage N 2 fixation, their optimum amount of N fixation and shoot dry matter production strongly depend on the availability of P application. When considering a number of species to be screened for their adaptability to low P, differences
in response to P application in pot experiments may not always correspond with differences in field behaviour (Sanginga et al., 1994). However, similar responses to P are obtained by some species in both pot and field experiments. A comparison of the results of L. purpureus and M. pruriens shows that the behaviour of these species was similar both in the pot and in the field experiments. The similarity of results suggests, between pot experiments can be used in pre-screening work of legume species.
5. Conclusion Large species differences were observed in almost all the major parameters (growth, N accumulation, nodulation and N 2 fixation) examined. Legume species also differed widely in their P requirements for growth and N 2 fixation. Lablab purpureus with high N 2 fixation ability and high P use efficiency under low P-conditions should be ideal for low P soils as observed in the savanna region. However, as L. purpureus also removed the largest amount of P from the soil, further testing is needed to examine the effect on P status of soil and subsequent crops. Species such as M. pruriens and C. ochroleuca which require P application for maximum yield will be more suitable for soils with high P level. These results suggest the need to take the P requirements of these legume species into account in plant introduction and plant selection for the moist savanna zone soils.
References Chisholm, R.H. and Blair, G.J., 1988. Phosphorus efficiency in pasture species. I. Measures based on total dry weight and P content. Aust. J. Agric. Res., 39: 807-816. Bationo, S., Mughogho, S.K. and Mokwunye, A., 1986. Management of nitrogen fertilizers for tropical African soils. In: A. Mokwunye and P.L.G. Vlek (Editors), Management of Nitrogen and Phosphorus Fertilizers in Sub-Saharan Africa, Martinus Nijhoff, Dordrecht, pp. 283-318 Date, R.A., 1991. Nodulation success and persistence of recommended inoculum strains for subtropical and tropical forage legumes in northern Australia. Soil Biol. Biochem., 23: 533541. F~hse, D., Classen, N. and Jungk, A., 1988. Phosphorus efficiency of plants. Plant Soil, 110: 101-109.
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255 Giovanetti, M. and Mosse, B., 1980. An evaluation of methods for measuring vesicular-arbuscular infection in roots. New Phytol., 84: 489-500. Gunawardena, S.F.B.N., Danso, S.K.A. and Zapata, F., 1993. Phosphorus requirements and N2 accumulation by three mungbean (Vigna radiata (L) Wewlzek cultivar). Plant Soil, 147: 267-274. Habte, M. and Manjunath, 1987. Soil solution P status and mycorrhizal dependency in Leucaena leucocephala. Appl. Environ. Microbiol., 53 (4): 797-801. Kang, B.T., Ladipo, D.O. and Ofeimu, O., in press. Phosphorus and liming effects on early growth of selected plant species grown on an Ultisol. J. Comm. Soil Sci. Plant Anal., in press. Mughogho, S.K., Bationo, A., Christianson, B. and Vlek, P.L.G., 1986. Management of nitrogen fertilizers for tropical African soils. In: A. Mokwunye and P.L.G. Vlek (Editors), Management of Nitrogen and Phosphorus Fertilizers in Sub-Saharan Africa, Martinus Nijhoff, Dordrecht, pp. 117-172.
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Sanginga, N., Bowen., G.D. and Danso, S.K.A., 1991. lntraspecific variation in growth and N 2 fixation of Leucaena leucocephala and Gliricidia sepium at low levels of soil P. Plant Soil, 133: 201-208. Sanginga, N., Danso, S.K.A., Zapata, F. and Bowen, G.D., 1994. Field validation of intraspecific variation in phosphorus use efficiency and nitrogen fixation by provenances of Gliricidia sepium grown in low P soils. Appl. Soil Ecol., 1: 127-132. Statistical Analysis Systems Institute Inc., 1986. SAS User's Guide: Statistics, SAS Institute Inc., Cary, NC. Sylvester-Bradley, R., 1984. Rhizobium inoculation trails designed to support a tropical forage selection programme. Plant Soil, 82: 377-386. Vincent, J.M., 1970. A Manual for the Practical Study of RootNodule Bacteria, IBP Handbook No. 15, Blackwell, Oxford.
Applied Soil Ecology 3 (1996) 247-255
Phosphorus requirement and nodulation of herbaceous and shrub legumes in low P soils of a Guinean savanna in Nigeria N. Sanginga *, J.A. Okogun, I.O. Akobundu, B.T. Kang International Institute of Tropical Agrieulture (IITA), Oyo Road, PMB. 5320 lbadan, Nigeria
Accepted 29 September 1995
Abstract
There is increasing interest to improve the N2 fixation and P use efficiency of herbaceous and shrub legumes currently being introduced in cereal-based cropping systems in the moist savanna zones of West Africa. Knowledge about N and P nutrition of these legumes can assist in adapting them to new areas where fertilizer use is not feasible by the smallholder farmers. Pot and field experiments were carried out to examine variation among potential herbaceous and shrub legumes for their ability to nodulate and to use P efficiently. These legumes were grown at two P levels (0 and 7 kg P ha - I ) in soils collected from two fields with different cropping histories (compound and degraded fields) at Yamrat in the northern Guinea savanna of Nigeria. In the compound field, animal manure and household residues are applied annually whereas in the degraded field, cereals are grown continuously with minimal organic inputs. Significant differences in growth, P content and mycorrhizal infection rate occurred among the different legumes as affected by P and cropping history. Legumes grown in soil from the degraded field responded more to P application than those grown in soil from the compound field. Phosphorus responsive legume species e.g. Mucuna pruriens var. utilis (black seed) and Crotolaria ochroleuca, had a low mycorrhizal infection rate compared to lower P responsive species such as Lablab purpureus and Cajanus cajan. A significant relationship was found between mycorrhizal infection rate and plant growth but not with nodulation of these legumes. All legumes nodulated naturally and the proportion of N derived from atmospheric N2 ranged from 38% for Centrosema brasilianum to 90% for L. purpureus. Application of P increased the weight of nodules but did not improve the proportion of N 2 fixation. This study showed that species such as L. purpureus has a high N 2 fixing capability and can also adapt to low P soils conditions in the northern Guinea savanna of West Africa. Keywords: Compoundfield; Degraded field; P content; Nodulation;N2 fixation; Mycorrhiza
1. I n t r o d u c t i o n Low levels of soil available N and P and large crop responses to N and P applications are common
* Corresponding author: c/o Lamboum and Co., 26 Dingwall Road, Croydon CR9 3EE, UK. Elsevier Science B.V. SSDI 0929- 1393(95)00083-6
in the northern Guinea savanna of West Africa (Bationo et al., 1986; Mughogho et al., 1986). The establishment, nodulation and growth of legumes being introduced in cereal-based cropping systems will also benefit from P fertilizer application. Preliminary observations in the northern Guinea savanna of Nigeria have shown that legumes require about 30 kg P h a - l for optimal growth and N 2
248
N. &mgingaet al./Applied Soil Ecology 3 (1996)247-255
fixation (Weber et al., personal communication). Small-scale farmers, however, only use limited amounts of fertilizer for their main food crops. Under these circumstances, it would be beneficial to select legume species or cultivars with low P requirements for growth, nodule development and N 2 fixation. Recent studies have shown large variability in growth, P content and use efficiency and N 2 fixation in legumious trees used in alley cropping system (Sanginga et al., 1994) and annual legumes (Gunawardena et al., 1993). Kang et al. (in press) also showed large differences in early growth of woody species in response to P application. Level of P response was correlated to seed P content. However, only limited information exists on P responses of herbaceous and shrub legumes currently being tested into the moist savanna zone of West Africa. Relatively few data exist on the external P and N requirements of these legumes in P deficient soils. A better understanding of leguminous species or cultivar differences in P and N nutrition may help in adapting these plants to new areas where fertilizers are not readily available. The objectives of this study were: (i) to determine specific differences in P uptake and use between various herbaceous and shrub legumes (ii) to assess the response of these legumes to P application and (iii) to select those which have high nodulation and N 2 fixation potential at low levels of P in the soil.
2. Materials and methods This investigation was conducted both in the field at Yamrat in the northern Guinea Savanna of Nigeria (9°81'N 10°13'E) and in pot experiments using Yamrat soil. The pot experiment was undertaken at the International Institute of Tropical Agriculture (IITA) at Ibadan, Nigeria. Field trials were installed in two farmers' fields with different cropping histories. Experimental field one was a compound field, where animal manure and other household residues are applied annually and the field is most often used for cereals (maize and sorghum) production. The second experiment was on a degraded field, where cereals were continuously grown for 10 or more years with minimal organic material inputs. Selected soil
Table 1 Selected physico-chemicalcharacteristics of degraded and compound fields at Yanarat Compound field Degradedfield pH (H20) Organic C (%) Total N (%) N03-N (/.tg g.l soil) NH4-N (/zg g~ soil) Extr. P ( p,g g-i soil) Mycorrbizal spores (number per 100 grams soil) Sand (%) Silt (%) Clay (%)
8.29 1.76 0.13 32.40 8.25 85.44 56
5.72 0.50 0.04 9.49 5.08 1.10 54
80 13 7
81 12 7
physico-chemical characteristics (0-15 cm depth) of these two fields measured according to IITA analytical procedures (IITA, 1989) are given in Table 1. 2.1. Pot experiment 2.1.1. Experimental design A randomized complete block design with three replications was used . Treatments included; (i) the ten legume species and maize, (ii) two P levels (with and without P) and (iii) the two soils (degraded and compound fields). Soils for the pot experiment were collected from 0 - 1 5 cm depth from each of the two fields. The soil was air dried and sieved ( < 2 mm screen) and 5 kg soil per pot was used for the trial. A basal application of 60 mg K k g - 1 soil as muriate of potash and 1 ml of a combination of micronutrients (Vincent, 1970) per kilogram soil was applied to each pot before planting. Phosphorus was applied as KHePO 4 a rate of 7 kg P h a - I 2.1.2. Legumes species and maize seeds Ten legume species (Table 2) and maize cultivar Oba super (as a reference plant) were used in this trial. Psophocarpus palustris seeds were scarified with H 2 S O 4 for 5 min, while the other seeds were only surface sterilized with ethanol (1 min) and H202 (5 min), and then rinsed with sterile water. The sterilized seeds were soaked for 24 h in sterile water and then pregerminated in Petri dishes containing sterile cotton wool. Eight seeds were sown in
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
each pot and thinned to two plants per pot at 1 week after emergence.
2.1.3. Harvesting All plants in the pot were harvested 8 weeks after planting (WAP) and assessed for dry matter production (shoots and roots), nodulation, and total N and P content. Mycorrhizal infection was rated on fresh roots using the method by Giovanetti and Mosse (1980). The proportion and amount of N derived from atmospheric N 2 were estimated by the total N difference method using maize as a reference plant. 2.2. Field experiment A split-plot design with three replications was used. Main-plots were two P rates, 0 and 7 kg P ha -~ (as single superphoshate) and the sub-plots were eight legume species and maize as control treatment. Subplot size was 10 m X 5 m, including 2 m X 5 m area reserved for destructive sampling (at one end of each subplot). Legume seeds were surface sterilized as described for the pot experiment. The seeds of small seeded legumes were drilled within each plot at 50 cm inter-row spacing at a seeding rate of 10 kg seed ha-~. For large size seeds e.g. maize, M. pruriens, L. purpureus, and C. cajan, 50 cm inter-row and 25 cm intra-row spacings were
249
used. Two seeds were planted per hill. Five plants were harvested at 6 and 14 WAP. The same plant parameters were assessed as in the pot experiment.
2.3. Chemical analysis Analysis of P in the plant shoots was done using the vanadomolybdate yellow method and total N was determined by the Automatic N analyzer following wet acid digestion (IITA, 1989).
2.4. Statistical analyses Analysis of variance (ANOVA) was done using the SAS program (Statistical Analysis Systems Institute Inc., 1986) to determine treatment and interaction effects. Least significant differences (LSD) were calculated at the 5% level to assess treatment differences.
3. Results 3.1. Pot experiment 3.1.1. Nodulation and N 2 fixation All plants nodulated in the two soils, but the number and nodule mass at 8 WAP varied signifi-
Table 2 Total N and percentage N derived from the atmosphere of the legumes grown in the greenhouse in soils collected from degraded and compound fields Legume species
L. purpureus a C. cajan b C. verrucosa ~ C. pascuroum a P. argentea a M. pruriens (black) a C. brasilianum a C. rotundifi~lia a A. histrix a P. palustris a LSD 5% a (1) (2)
N derived from the atmosphere (%)
Total N (mg per plant) Degraded field
Compound field
Degraded field
Compound field
91 50 52 78 23 107 16 43 31 47
157 42 81 94 62 81 34 34 78 44
90 52 81 87 59 91 43 78 70 80
87 50 73 77 65 73 38 38 72 51
5 4
13 12
Mean values averaged over P treatments; P effect was not significant. a Herbaceous species. b Shrub species.(1) For comparing plant species within sites; (2) For comparing plant species between sites.
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247 255
250
cantly between legume species. Nodule number per plant ranged from 1 for Aeschynomene histrix to 32 for Centrosema pascuorum. Mucuna pruriens in spite of its low number of nodules had the highest nodule mass followed by L. purpureus. Aeschynomene histrix recorded the lowest nodule mass (Fig. 1). Phosphorus application significantly increased the number and mass of nodules of most species except P. argentea, C. rotundifolia and A. histrix (Fig. 1). Herbaceous and shrub legumes derived 69% (average) of their plant total N from atmospheric N 2.
1 0 LSD 5% 4 [] ~
2 ILSD 5%
3
%
2
~ - = . ~
~ ~
40
I I] LSD 5%
2
~
G
~
~2 G6
n 3( 1. 2.
¢~ 2C Z
LSD 5% for comparing plant species at different P levels LSD 5% for comparing plant species at same P levels
Fig. 2. Effect, at 8 WAP in the greenhouse, of P level on shoot dry weight of legume species grown in soil collected from degraded and compound fields at Yamrat.
Z 1(
d
4
~J
G
~j
& ,,.
~j
I~ LSD5% rl
&
2
2 II I.l LSD 5%
[ o { .[ .i ~J LSD 5% 1. 2.
{J
For comparing plant species at different P levels For comparing plant species at same P levels
Fig. 1. Effect, at 8 WAP in the greenhouse, of P on the number and mass of nodule weight of legume species grown in soils collected from degraded and compound fields at Yamrat.
The proportion of N z fixed depended largely on the field history and legume species (Table 2). Percentage N 2 fixed varied between 38% and 90% for C. brasilianum and L. purpureus, respectively and it was higher in soil from the degraded field (76%) than in soil from the compound field (62%). There was a significant correlation ( r = 0.91, P = 0.05) between the proportion and amount of N 2 fixed per plant. Unlike the amount of N 2 fixed, the proportion of N 2 fixed was not significantly affected by P application. 3.1.2. Shoot dry weight and total N accumulation
Shoot dry weight differed significantly between legume species; ranging from a mean of 1.21 g per plant for C. brasilianum to 4.15 g per plant for M. pruriens (Fig. 2). Shoot dry weight response to P application varied between legumes species. The highest P response was observed with C. verrucosa (197%) > A. histrix (105%), > C. rotundifolia (78%), > M. pruriens (28%) C. pascuorum (26%). Cajanus cajan and C. brasilianum showed small responses while P. argentea and L. purpureus shoot yields were reduced with P application. Shoot dry matter yield was correlated to total N accumulation in the legumes ( r = 0.99; P = 0.05).
P
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
251
49
42
35
.o 28
.fi 21
8 14
o ~
~
g.
.~
~
,~
~
t5
t5
.
_~
~t
,5 Fig. 3. Mycorrhizal infection (%) of legume species, at 8 WAP in the green house. Mean values across sites and P levels.
3.1.3. Total P accumulation and mycorrhizal infection
0.98; P = 0.05). It ranged from 0.98 mg P per plant for P. argentea to 4.99 mg P per plant for M. pruriens in degraded field and from 1.76 mg P per plant for A. histrix to 8.01 mg P per plant for L.
Total P accumulation varied between species and was significantly correlated to shoot dry matter (r =
Table 3 Nodule dry weight of legume species grown with or without P addition in degraded and compound fields in Yamrat at 14 weeks after planting (WAP) Legume species
Nodule dry weight (mg per plant) Degraded field
Lablab purpureus Crotolaria ochroleuca Centrosema pascuorum Cajanus cajan Mucuna pruriens (black) Mucuna pruriens (white) Centrosema brasilianum Chamaechrista rotundifolia LSD 5% ( 1) (2)
Compound field
-P
+p
-p
+p
2.01 0.01 0.17 1.90 1.16 2.01 2.04 0.00
2.16 0.06 0.16 0.55 5.51 0.03 0.16 0.84
16.62 12.66 3.06 20.95 25.58 12.18 1.28 2.78
22.49 24.54 11.98 18.00 29.02 I 1.67 1.08 4.40
5.00 4.00
8.00 9.00
(1) For comparing legumes at different P levels; (2) For comparing legumes at same P levels.
252
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
purpureus in the compound field. The relationships between P content and P application also varied between species and field history and was also related to shoot dry matter. Mycorrhizal infection rate of plants grown in pots ranged between 10% (average) for C. verrucosa, A. histrix and Psophocarpus palustris and 27% (average) for C. rotundifolia, L. purpureus, and P. argentea (Fig. 3). Maize infection rate was the highest (40%). There were significant correlations between mycorrhizal infection rate and shoot dry weight (r = 0.64; P = 0.05: n = 80) but not with nodulation parameters. 3.2. Field experiment 3.2.1. Nodulation and N 2 fixation Nodulation was affected by legume species and sampling time. At 6 W A P , L. purpureus nodulated profusely and had more nodules than the other plants (data not shown). At 14 WAP, when most of the legumes were flowering, L. purpureus again produced the highest number of nodules especially in the degraded field (Table 3). The application of P increased the nodule weight of a few species (e.g. M. pruriens in the degraded soil and L. purpureus, C. Ochroleuca and C. pascuorum in the compound field) but did not improve the number of nodules. Legume species grown in the compound field had on
average lower nodule numbers, higher mass and weight than those in the degraded field (Table 3). The proportion of N 2 fixed by the legume species was estimated by the total N difference method using maize as a control (data not shown). Lablab purpureus had again the highest percentage N 2 fixed (72% average). Estimations of N 2 fixed in the other legumes were not determined because their early growth rate and total N accumulation was lower than that of maize control.
3.2.2. Shoot dry weight total N accumulation Shoot dry weight differed significantly between legume species and P application levels (Table 4), M. pruriens (black) having more shoot biomass than that of the other species. It was followed by C. ochroleuca while C. brasilianum and C. rotundifolia had the lowest shoot biomass. There was a significant correlation between shoot dry and total N (r = 0.93; P = 0.05). Phosphorus application increased shoot dry weight and total N of legumes grown in degraded and compound farmer's field by an average of 91% and 38%, respectively. Crotolaria ochroleuca and the two M. pruriens grown in degraded farmer's field had their shoot dry weight increased by about 4 to 6 fold, respectively by P application. Such large responses to P application did not occur in the compound farmer's field.
Table 4 Shoot dry weight and total N (mg per plant) in shoots of legume species grown with or without P application in degraded and compound fields in Yamrat at 14 WAP Legume species
Lablab purpureus Crotolaria ochroleuca Centrosema pascuorum Cajanus cajan Mucuna pruriens (black) Mucuna pruriens (white) Centrosema brasilianum Charnaechrista rotundifolia LSD 5% (1) (2)
Shoot dry weight (g per plant)
Total N (mg per plant)
Degraded field
Compound field
Degraded field
Compound field
-p
+p
-P
+p
-p
+p
-p
+p
44 34 60 51 21 35 60 30
69 130 65 59 55 205 58 25
22 48 38 34 108 60 8 14
47 76 61 34 123 83 2 34
1201 I0 l 3 1480 1735 521 637 794 707
1747 3323 t 013 1832 3884 2399 604 602
601 1231 614 993 3466 1214 102 219
595 2432 1833 1021 3745 1228 61 701
13 24
19 38
421 1421
(1) For comparing legumes at different levels; (2) For comparing legumes at same levels.
596 1193
N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255
253
Table 5 Mycorrhizal infection rate (%) and total P (mg per plant) content in plant shoots of legume species grown with or without P application in degraded and compound fields in Yamrat at 14 WAP Legume species
Lablab purpureus Crotolaria ochroleuca Centroserna pascuorum Cajanus cajan Mucuna pruriens(black) Mucuna pruriens (white) Centrosema brasilianum Chamaechrista rotundifi~lia LSD 5% (l) (2)
Total P (mg per plant)
Mycorrhizal (%) Degraded field
Compound field
Degraded field
Compound field
-p
+P
-p
+P
-p
+p
-p
+p
8 13 4 4 3 7 4 7
15 3 11 3 0 15 8 10
26 10 4 20 9 19 6 9
33 27 36 26 4 19 17 3
52 44 52 50 18 27 44 17
76 115 51 81 188 153 34 14
21 65 23 40 138 52 8 12
32 I19 24 58 201 61 5 35
7 18
8 19
21 48
29 62
(1) For comparing legumes at different P levels; (2) For comparing legumes at same P levels.
3.2.3. Total P accumulation Phosphorus application increased total P of the legumes by 73% and 84% (average) when grown in the degraded or compound farmer's fields, respectively, at 6 WAP (data not shown). At 14 WAP, increases of 147% and 49% due to P application were obtained in the two fields, respectively. Total P content at low P varied between 8 and 138 mg P per plant while that at high P ranged from 5 to 201 mg P per plant at high P (Table 5). The two M. pruriens (black and white seeds) and C. ochroleuca required P addition to grow well as shown by their significant increase in total P content due to P application in both fields. Cajanus cajan and L. purpureus had similar total P content at both zero and P application while C. brasilianum and C. rotundifolia grew poorly at both zero and P application. 3.2.4. Mycorrhizal infection Mycorrhizal infection rate was affected by plant species and the field history but not by P application. Legume species grown in the compound field were more infected than those grown in the degraded field (Table 5). Mucuna pruriens (black seed), C. brasilianum and C. rotundifolia showed the lowest rate of mycorrhizal infection while, L. purpureus and M. prurieus (white seed) were highly infected by mycorrhizal fungi. As in the pot experiment, significant
correlation was found only between mycorrhizal infection rate and shoot dry weight.
4. Discussion
Three types of growth response to P application were evident for herbaceous and shrub legumes. For example, C. cajan and L. purpureus achieved maximum yield at inherent P levels in both degraded and compound fields. With M. pruriens and C. ochroleuca, maximum yield was obtained only when P was added in the degraded field. Pseudovigna argentea had its shoot dry weight reduced by P application in the greenhouse. This suggests that the P requirements for herbaceous and shrub legumes to achieve maximum growth vary. It can be argued that if the ability to grow in low P soils is of overriding importance, species such as M. pruriens and C. ochroleuca that require P application for maximum biomass production would be at a disadvantage when grown in low P such as in the degraded field (Tables 4 and 5). On the other hand, species such as L. Purpureus and C. cajan did not respond significantly to P in both fields indicating that the inherent soil P concentration in these fields was adequate for their growth.
254
N. Sanginga et al./Applied Soil Ecoh~gy 3 (1996) 247-255
Species are known to differ greatly in their ability to assimilate P especially at low P levels; opinion differs, however, concerning the mechanisms of these differences in ability to grow at low P levels in soils. Studies by F~Shse et al. (1988) indicate that species differ in their ability to extract soil P depending upon the potential of roots to absorb P, their active lifetime and the amount of root per unit of shoot. On the other hand, results of Sanginga et al. (1991) and Chisholm and Blair (1988) indicate that species able to use absorbed P more efficiently in the production of biomass should be better adapted to low P soils. Our investigation shows that differences between several species in their ability to be infected by mycorrhizae and the difference in P uptake and growth response to P could largely account for the difference in adaptation of these species to low P soils. For example, at the lowest P level, species such as L. Purpureus and C. cajan had the highest mycorrhizal infection rate (average 20%) compared to 4 - 1 0 % for the other legumes, with M. prurieus and C. Ochroleuca having the lowest infection rate. Phosphorus responders species had on average lower mycorrhizal infection rate than the P-non responders. The influence of mycorrhizal fungi on P uptake is well documented. Habte and Manjunath (1987) demonstrated that plants with stronger mycorrhizal association have a lower external P requirement than plants of the same type without an effective mycorrhizal association. Data presented also indicate that leguminous species tested also differ in their ability to fix N 2 under low P conditions. Percentage of N 2 fixed by these legumes was generally high (on average 80%) and is comparable to values reported for herbaceous legumes used as cover crop or pasture elsewhere (Date, 1991; Sylvester-Bradley, 1984). The best species in terms of percentage and total N fixed under low P soil level was L. purpureus. Such species should be better suited N and P deficient soils. Although other species such as M. pruriens presently being adopted by farmers in some areas of the moist savanna showed comparable percentage N 2 fixation, their optimum amount of N fixation and shoot dry matter production strongly depend on the availability of P application. When considering a number of species to be screened for their adaptability to low P, differences
in response to P application in pot experiments may not always correspond with differences in field behaviour (Sanginga et al., 1994). However, similar responses to P are obtained by some species in both pot and field experiments. A comparison of the results of L. purpureus and M. pruriens shows that the behaviour of these species was similar both in the pot and in the field experiments. The similarity of results suggests, between pot experiments can be used in pre-screening work of legume species.
5. Conclusion Large species differences were observed in almost all the major parameters (growth, N accumulation, nodulation and N 2 fixation) examined. Legume species also differed widely in their P requirements for growth and N 2 fixation. Lablab purpureus with high N 2 fixation ability and high P use efficiency under low P-conditions should be ideal for low P soils as observed in the savanna region. However, as L. purpureus also removed the largest amount of P from the soil, further testing is needed to examine the effect on P status of soil and subsequent crops. Species such as M. pruriens and C. ochroleuca which require P application for maximum yield will be more suitable for soils with high P level. These results suggest the need to take the P requirements of these legume species into account in plant introduction and plant selection for the moist savanna zone soils.
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N. Sanginga et al./Applied Soil Ecology 3 (1996) 247-255 Giovanetti, M. and Mosse, B., 1980. An evaluation of methods for measuring vesicular-arbuscular infection in roots. New Phytol., 84: 489-500. Gunawardena, S.F.B.N., Danso, S.K.A. and Zapata, F., 1993. Phosphorus requirements and N2 accumulation by three mungbean (Vigna radiata (L) Wewlzek cultivar). Plant Soil, 147: 267-274. Habte, M. and Manjunath, 1987. Soil solution P status and mycorrhizal dependency in Leucaena leucocephala. Appl. Environ. Microbiol., 53 (4): 797-801. Kang, B.T., Ladipo, D.O. and Ofeimu, O., in press. Phosphorus and liming effects on early growth of selected plant species grown on an Ultisol. J. Comm. Soil Sci. Plant Anal., in press. Mughogho, S.K., Bationo, A., Christianson, B. and Vlek, P.L.G., 1986. Management of nitrogen fertilizers for tropical African soils. In: A. Mokwunye and P.L.G. Vlek (Editors), Management of Nitrogen and Phosphorus Fertilizers in Sub-Saharan Africa, Martinus Nijhoff, Dordrecht, pp. 117-172.
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Sanginga, N., Bowen., G.D. and Danso, S.K.A., 1991. lntraspecific variation in growth and N 2 fixation of Leucaena leucocephala and Gliricidia sepium at low levels of soil P. Plant Soil, 133: 201-208. Sanginga, N., Danso, S.K.A., Zapata, F. and Bowen, G.D., 1994. Field validation of intraspecific variation in phosphorus use efficiency and nitrogen fixation by provenances of Gliricidia sepium grown in low P soils. Appl. Soil Ecol., 1: 127-132. Statistical Analysis Systems Institute Inc., 1986. SAS User's Guide: Statistics, SAS Institute Inc., Cary, NC. Sylvester-Bradley, R., 1984. Rhizobium inoculation trails designed to support a tropical forage selection programme. Plant Soil, 82: 377-386. Vincent, J.M., 1970. A Manual for the Practical Study of RootNodule Bacteria, IBP Handbook No. 15, Blackwell, Oxford.