Agroforestry Systems 40: 297307, 1998. 1998 Kluwer Academic Publishers. Printed in the Netherlands.

Improvement of nitrogen fixation in Acacia mangium through inoculation with rhizobium

A. GALIANA 1, *, G. M. GNAHOUA2, J. CHAUMONT1, D. LESUEUR1, Y. PRIN1 and B. MALLET1
1

CIRAD-Fort, Campus international de Baillarguet, Montferrier-Sur-Lez, BP 5035, 34032 Montpellier, Cedex 1, France; 2 Institut des Forts-Dpartement de la Foresterie (IDEFOR/DFO), 08BP33 Abidjan 08, Cte dIvoire (*Author for Correspondence: E-mail: galiana@cirad.fr) Key words: fallow, humid tropics, nodules, soil fertility, symbiotic association

Abstract. Acacia mangium, a N2-fixing tree legume, has become a major plantation tree species in the tropical humid and sub-humid zones. In addition to being a major pulp-wood producer, the tree has a good potential to restore soil fertility as a fallow species in agroforestry systems, and as a fuel species. In this paper, we report an overview of the results from several rhizobium inoculation field trials in different edaphic and ecological conditions, conducted by the CIRAD-Fort (The Forest Program of Centre de Coopration Internationale en Recherche Agronomique pour le Dveloppement) in partnership with national research organizations of various countries of the humid and sub-humid lowlands of West Africa and Cook Islands. Rhizobium inoculation had a positive effect on tree growth up to 39 months after tree planting. Immunological identification of the Bradyrhizobium strains present in the nodules confirmed the persistence of the more efficient introduced strains up to 42 months after transfer of the inoculated trees to the field. In Cte dIvoire, nitrogen derived from atmospheric N2 fixed symbiotically by A. mangium was 50% in the whole trial and up to 90% in plots with less fertile soils when the trees were inoculated with an efficient strain.

Introduction The increasing introduction of Acacia mangium in industrial plantations during the last decade is largely due to its good silvicultural potential and its ability to grow on degraded soils, particularly nitrogen-depleted ones. Large-scale plantations of A. mangium have been successfully established in tropical humid zones, especially in eastern Malaysia (Sabah) and in Indonesia where most of them were set up on poor soils and grasslands infested by a noxious weed, Imperata cylindrica. In 1985, 55,000 hectares were already planted in Sabah (Udarbe and Hepburn, 1987). To a lesser extent, expansion of Acacia plantation in the Philippines, Papua-New Guinea, China and in tropical humid regions of Africa is also documented (National Academy of Science, 1983). The introduction of the species into agroforestry systems has been reported to be promising in the humid and sub-humid tropics of Asia and Africa (De Taffin et al., 1991; Dupuy and NGuessan Kanga, 1991). Due to the increasing importance of the species, CIRAD-Fort initiated a research program on the A. mangium-rhizobium symbiosis in 1986. The program, BSFT (Biotechnology

298 Laboratory on Tropical Forest Symbioses, common laboratory between CIRAD-Fort and ORSTOM, Nogent-sur-Marne, France) in association with several countries mainly of the West Africa humid lowlands, aims at studying the Acacia-rhizobium association in order to increase the productivity of the species through nitrogen fixation. Our experience in this domain over the last decade is summarized below.

Symbiotic characteristics of A. mangium At the inception of the study in 1986, only one A. mangium strain of rhizobium was available. The initial aim of the study was thus to isolate a large number of strains from A. mangium root nodules collected from a range of tropical countries. A. mangium was found to nodulate spontaneously in its native area (North Queensland, Australia) and in countries to which it had been introduced. About 50 strains were isolated by the BSFT from the nodules collected in the native area of the species (Souvannavong and Crmire, 1986) and range of other countries (West Africa, French Guyana, China and Malaysia). All these strains were identified as slow-growing Bradyrhizobium strains. The host spectrum of these strains was established under controlled conditions by testing their infectivity (ability to form nodules) and effectivity (ability to fix nitrogen) on A. mangium and A.auriculiformis. Prior results showed that A. mangium was a specific host since the effectiveness of the different strains varied considerably according to their origin compared to the non-specific hosts like Acacia auriculiformis which nodulate and fix nitrogen regardless of the strain (Galiana et al., 1990). Moreover, when inoculated to A. mangium, the strains originating from Australia were more efficient than those from other origins or the collection of strains isolated from other host species.

Effect of inoculation on Acacia mangium growth The efficiency of the different rhizobium strains was tested in field experiments conducted in three countries Cte dIvoire, Cook Islands and Benin. The trial locations differ in both climatic annual rainfall ranging from 1500 mm to 2500 mm and edaphic soil types ranging from sandy to clay as well as acid to neutral conditions. The results showed that among the five strains tested, the Bradyrhizobium strains originating from Australia were the most efficient. Inoculating seedlings in the nursery with Aust 13c and Aust 11c had a positive and significant effect on plant height and basal area development, up to 39 months after transplanting in the field. This was particularly the case in the trials set up at Port-Bout in Cte dIvoire, at Seme in Benin and at Turoa and Hospital Hill in Cook Islands. The data obtained from greenhouse experiments and in vitro conditions in laboratory were in

299 agreement with that obtained under field conditions. This observation was well illustrated in the trial conducted at Seme in Benin. The ranking of the seven Bradyrhizobium strains according to their efficiency on plant shoot height, shoot dry weight, number and dry weight of root nodules obtained at three months after sowing the seeds in the nursery, with or without a preliminary disinfection of the nursery soil was the same as that obtained in controlled in vitro and greenhouse conditions with plants cultured on nitrogenfree medium (Souvannavong and Galiana, 1991). In the trial set up at Anguededou in Cte dIvoire, the positive and significant effect of inoculation on tree height observed four months after transplanting in the field did not persist at 21 month after transplanting. However, the effect on basal area development was still significant (Table 1). Another experiment was conducted at Anguededou in Cte dIvoire to test the effect of interaction between different rhizobium strains and four different A. mangium provenances on tree growth. The result showed a significant interaction effect. The Oriomo provenance (originating from Papua New Guinea) had a mean height (13 m) that was 25% greater than that of the other provenances (San Pedro-Cte dIvoire, Rex Range-Australia and Piru CeramIndonesia) after 19 months of growth in the field (results per provenance not detailed in Table 1). However, some strain by provenance combinations such as AG3 San Pedro, RMBY Rex Range and RMBY Piru Ceram had a significant (P < 0.05) effect on tree height, with performances similar to the combinations involving the Oriomo provenance. By contrast, other combinations such as AG3 Oriomo had a negative effect on tree growth. Identification and survival of Bradyrhizobium strains introduced in field experiments In order to confirm the validity of the in situ inoculation experiments and the positive effect of certain strains on tree growth, we collected root nodules at a soil depth of 015 cm in the field trials established in Cte dIvoire (Anguededou and Port-Bout sites). The sampling was done at 23 and 42 months after transplanting at the Anguededou site and at 19 months for the Port-Bout site. The purpose was to study the survival of the introduced strains and their competitive ability against native rhizobium strains. The results of identification of the introduced strains with serological typing (indirect immunofluorescence method described by Somasegaran and Hoben, 1985) showed that the most efficient strain, i.e. Aust 13c, persisted in 100% of the collected nodules up to two years after transplanting of the inoculated trees (Galiana et al., 1994). At Anguededou station, the majority of the collected nodules contained the Aust 13c strain at 23 and 42 months after tree transplanting. This was the case in all blocks for all A. mangium provenances analyzed (Table 2). All the nodules collected from trees inoculated with Aust 13c strain exclusively

300
Table 1. Effect of rhizobium inoculation on Acacia mangium growth: results obtained from several inoculation field trials set up by the CIRAD-Fort in humid and sub-humid tropics of West Africa and Cook islands.a Location of trials Area of trial Date of Plant age at planting observation (month/year) (months) Rhizobium strains tested Disinfection of nursery soilb Height c (% Increment/ control) Basal aread (% Inc./control)

Cte DIvoire Anguededou (study of the interaction provenance strain)

1.07 ha

3/88

04

Aust 13c TAL 72 RMBY AG3 Control Control Aust 13c TAL 72 RMBY AG3 Control Control PBG3 Aust 13c TAL 582 Control Aust 13c CB 756 Control

+ + + + + + + + + + + + + + + + +

0+99% +124% +106% 0+82% 0+28% 000 00+4% 00+3% 00+8% 005% 001% 0 (10.90 m) 00+7% 00+3% 00+3% 000 0+15% 0000.1% 00 (5.37 m)

000 000 000 000 000 000 0+24% 0+23% 0+12% 0+15% 0+13% 000 000 000 000 000 0+20% 002% 000

21

Abidjan (study of the interaction strain soil type) Port-Bout

(nursery)

4/90

42

0.78 ha

4/90

20

BENIN Seme

(nursery)

3/89

03

Aust 13c Aust 11c RMBY AG 3 PBG 3 TAL 72 CB 756 Control Aust 11c Aust 11c Control Aust 13c Control CB 756

+/ +/ +/ +/ +/ +/ +/ +/ + + + +

0+41% 0+30% 0+14% 00+1% 00+9% 006% 006% 000 0+28% 009% 00 (2.58 m) 00+5% 00 (2.38 m) 00+9%

000 000 000 000 000 000 000 000 000 000 000 000 000 000

Seme

1.56 ha

6/89

20

Cook Islands Turoa

590 m2

7/88

39

Aust 11c AG 3 Control Aust 11c AG 3 Control

00+7% 0+11% 00 (7.87 m) 0+37% 0+16% 00 (4.90 m)

0+30% 0+18% 000 +128% 0+40% 000

Hospital Hill

590 m2

7/88

39

Sources: Ehrhart (1991); Galiana (1990); Galiana et al. (1994); Lesueur et al. (1994); Mallet and Gnahoua (1989); Messant (1991). + = soil disinfected, = soil not disinfected, +/- = disinfected and not disinfected soil treatments combined, ( ) = Height of uninoculated control trees. c Percentages shown in bold characters refer to values significantly higher than those of the control treatments. d Control = Uninoculated control trees.
b

301

302

Table 2. Nodule occupancy of inoculated and uninoculated trees in field inoculation trial at Anguededou, Cte dIvoire. Months after tree transplanting Block Tree provenance Inoculated strain Number of nodules reacting positively with antiseruma Aust 13c 23 I San-Pedro Aust 13c RMBY TAL 72 AG3 Uninoculated Db Uninoculated NDc Aust 13c RMBY TAL 72 AG3 Uninoculated D Uninoculated ND Aust 13c RMBY TAL 72 AG3 Uninoculated D Uninoculated ND 10 06 09 10 09 09 10 10 10 10 nd nd 10 10 07 10 09 07 RMBY 00 04 00 00 00 00 ndd 02 01 00 nd nd 01 00 00 00 00 00 TAL 72 00 05 00 00 00 00 nd 02 00 01 nd nd 00 00 00 00 00 00 AG3 00 00 00 00 00 00 nd 00 02 00 nd nd 00 00 00 00 00 00

42

Rex range

42

II

Rex range

Nodules were analyzed by the indirect immunofluorescence technique (Somasegaran and Hoben, 1985); 10 nodules per tree were tested and each nodule was analyzed with each of the four antisera. b Uninoculated D; uninoculated control trees initially grown at the nursery on soil disinfected with metam sodium. c Uninoculated ND; uninoculated control trees initially grown at the nursery on non disinfected soil; trees inoculated with the four Bradyrhizobium strains were grown on soil initially disinfected with metam sodium. d nd = not determined.

303 contained Aust 13c and no native strain was detected. Among the other three introduced strains, only RMBY strain was recovered at a significant rate, with 40% of the nodules exclusively containing the RMBY inoculant strain at 23 months after field transplanting. All the other plots that were inoculated with TAL 72 and AG3 strains and the plots with uninoculated control trees were broadly contaminated by Aust 13c strain in all the blocks for all A. mangium provenances that were analyzed 70 to 100% of the nodules contained Aust 13c from each randomly sampled tree. The overall contamination and nodulation of trees by Aust 13c in this experiment could explain the gradual reduction in differences between treatments with time as the positive effect of inoculation on height growth observed at 4 months was no longer visible 19 months after tree transplantation. In the Port-Bout field experiment, the immunological identification of strains in the nodules also showed the persistence of Aust 13c at 19 months after transplanting of the inoculated trees in all the blocks for all the trees analyzed (Table 3). All the nodules collected from the trees inoculated with Aust 13c reacted positively with the homologous antiserum. All of the observed bacteroids under UV microscope were identified as Aust 13c strain.
Table 3. Nodule occupancy of inoculated and uninoculated trees in field inoculation trial at Port-Bout, Cte dIvoire, after 19 months of field growth. Inoculated strain Block Tree no. Number of nodules reacting positively with antiseruma Aust 13c Aust 13c I II III CB 756 I II III Uninoculated I II III 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 10 10 10 10 10 ndb 00 00 00 02 02 09 00 01 01 00 06 02 CB 756 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 2 0 0 PBG 3 00 00 00 00 00 00 01 00 00 00 00 00 00 00 00 00 00 nd

Nodules were analyzed by the indirect immunofluorescence technique: 10 nodules per tree were tested and each nodule was analyzed with each of the three antisera. b nd = not determined.

304 The two plots of block III containing trees inoculated with the CB756 strain and uninoculated control, respectively, were largely contaminated by Aust 13c. By contrast, CB756 was not identified in the nodules collected from trees inoculated with the corresponding strain. However, CB 756 was found in few nodules from two uninoculated control trees located in block II. All other nodules collected from trees inoculated with CB 756 or those not inoculated contained a majority of indigenous strains. At Port-Bout, contrary to the trial at Anguededou, the plots inoculated with CB 756 and the uninoculated controls were still slightly contaminated by Aust 13c that occurred in the adjacent plots after 19 months. This is probably due to the fact that, the adjacent plots at Port-Bout were separated from each other by three to five rows of border Eucalyptus urophylla whereas a single row of A. mangium separated the different strain treatments in the Anguededou field trial. Consequently, the positive effect of inoculation with Aust 13c on tree growth could still persist after 19 months at Port-Bout. This suggests that indigenous rhizobium strains are less efficient than the introduced Aust 13c strain.

Estimation of nitrogen fixed by Acacia mangium in plantation The percentage of nitrogen derived from atmospheric N2 (Ndfa %) and fixed by A. mangium was evaluated in the rhizobium inoculation field trial set up at Port-Bout that is reported above (see Tables 1 and 2). The treatments were trees inoculated with Aust 13c, CB 756 and control (uninoculated trees). Each treatment was replicated three times in 3 blocks in a Randomized Complete Block Design. The Ndfa% was calculated following the 15N natural abundance method described by Amarger et al. (1977) and Bardin et al. (1977). Eucalyptus (Eucalyptus urophylla) was included in the trial both as a border row to avoid cross contamination of Bradyrhizobium species strains between plots and as a non-fixing reference plant for the determination of Ndfa%. Table 4 shows a positive and highly significant effect (P < 0.05) of inoculation with Aust13c on tree growth at 19 months after transplanting in the field. The height of trees inoculated with Aust 13c was about 10% greater than that of the trees inoculated with the CB 756 strain and the uninoculated ones. Similarly, the diameter at ground level of trees inoculated with Aust 13c was about 15% greater than that measured in the two other strain treatment plots. Furthermore, the growth of the trees inoculated with Aust 13c was the greatest compared to that of the trees in the other treatment plots in each of the three blocks. The block effect on tree growth was also highly significant (P < 0.01 for height and P < 0.05 for diameter, respectively). Tree height was greater in block I than in blocks II and III by 6.7% and 15.8%, respectively whereas tree diameter was greater in block II than in blocks I and III by 10% and 18.3%, respectively. The percentage of nitrogen derived from atmospheric N2 (Ndfa %) contained in A. mangium leaves at 19 months after field planting varied from 19.4

305
Table 4. Effects of Bradyrhizobium strain and block factors on tree height, diameter and percentage of nitrogen derived from nitrogen fixation in Acacia mangium, 19 months after tree transplantion at Port-Bout, Cte dIvoire. Height (m. tree1) Strain effecta Aust 13c CB 756 Uninoculated Block effectb Block I Block II Block III Diameter at ground level (cm. tree1) Nitrogen fixed (%)

5.92 a 5.34 b 5.37 b 5.93 a 5.56 b 5.12 c

11.1 a 09.6 b 09.7 b 10.0 b 11.0 a 09.3 c

61.7 54.9 41.5 27.1 64.4 66.5

In each column, the values (means obtained from 90 trees for height and diameter and from 24 trees for nitrogen fixed) followed by a same letter are, according to Newman and Keuls test (Dagnlie, 1969) not significantly different at P = 0.01. a All block treatments combined. b All strain treatments combined.

to 90.6% depending on the different blocks and the Bradyrhizobium strain treatments. The trees inoculated with Aust 13c strain had Ndfa % slightly higher (61.7%) than that of the trees inoculated with CB 756 strain (54.9%) and the uninoculated trees (41.5%). However, the difference was not statistically significant at P = 0.05. Nitrogen fixation was markedly enhanced on the N-deficient blocks (Blocks II and III). The Ndfa % of the A. mangium in blocks II (64.4%) and III (66.5%) was 2.4 times higher than that of the trees in Block I (27.1%). The non-significant differences were due to the large variation in Ndfa% values within each plot as shown by the relatively high coefficients of variation (Galiana et al., 1996). This could be due to the heterogeneity of the soils and/or high genetic variability within the A. mangium provenances as in the E. urophylla that was used as reference tree to calculate the Ndfa%. Although the A. mangium inoculated with Aust 13c fixed more nitrogen and had a better growth than the A. mangium inoculated with CB 756 and the uninoculated ones, in each of the three blocks of the trial, there was no significant correlation between Ndfa % and the growth parameters height and diameter measured for A. mangium. By contrast, we observed highly positive and significant correlation coefficients between A. mangium height and the concentration of different soil chemical properties including total N (r = 0.85 at P 0.005), nitrate-N (r = 0.88 at P < 0.005), available P (Olsen method) (r = 0.86 at P 0.005), and other soil properties such as organic C or the percentage of organic matter. Although nitrogen fixation by leguminous plants is known to be inhibited by high concentration of combined nitrogen, especially nitrate-N (Vessey and Waterer, 1982) in the soil, correlation between the soil nutrients and Ndfa % was not statistically significant.

306 Ndfa % was negatively correlated (r = 0.72 at P 0.05) to height and diameter of the intercalary E. urophylla close to the A. mangium plots. The eucalyptus, which does not fix N2 but only assimilates mineral N is known to be a reliable indicator of soil fertility. This implies that nitrogen fixation by A. mangium is inversely proportional to the soil fertility status as in a given plot the A. mangium and the adjacent eucalyptus were planted on soils of the same soil fertility conditions. The absence of correlation between Ndfa % and growth parameters measured in A. mangium thus suggests the possibility of simultaneous action and the interdependency of the N2-fixing and N-assimilating processes complementary and synergistic during the 19 months of growth. The result showed that A. mangium had a high nitrogen-fixing ability as Ndfa % reached more than 50% over the whole trial in a soil which is not particularly poor in nitrogen. This N2-fixing potential was especially high in the less fertile plots of the trial, located in block III, and when the trees were inoculated with the most efficient strain Aust 13c as Ndfa % reached 90% when these two conditions were fulfilled.

Conclusion All the field trials conducted in different countries and on different soil types showed a positive effect of inoculation on tree growth with A. mangium rhizobium strains isolated from the natural distribution area of the host species (Australia). Moreover, this positive effect of inoculation was maintained after two to three years of planting the trees in the field. Immunological identification of the rhizobium strains that were present in the nodules confirmed the persistence and the high competitivity of the most efficient introduced strains (originating from Australia) two to three years and half after inoculation. The studies showed in particular the high N2-fixing potential of A. mangium. The percentage of nitrogen derived from atmospheric N2 fixed by A. mangium and then from a symbiotic origin was 50% over the whole trial and reached 90% on the less fertile plots and when trees were inoculated with an efficient strain. Further research is necessary to quantify the N fluxes and N input to the soil with particular reference to N derived from symbiotic N2 fixation after litter decomposition. References
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