Plant and Soil 184: 111-116, 1996. 111 (~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Long term field study shows increased biomass production in tree legumes inoculated with Rhizobium

Banwari Lal and Sunil K h a n n a 1 Microbiology and Molecular Genetics Unit, Tata Energy Research Institute, Habitat Place, Lodhi Road, New Delhi-110 003, India. t Corresponding author*

Received 5 March 1996. Accepted in revised form 10 August 1996

Key words: Acacia nilotica, biomass, Leucaena leucocephala, nitrogenase activiy, nodulation, soil nitrogen

Abstract

Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield response of Leucaena leucocephala and Acacia nilotica to inoculation with 6 selected fast growing Rhizobium strains was explored in long-term (5 year) field trials. All the strains formed nodules and fixed nitrogen in L. leucocephala and A. nilotica. Seasonal effects on nitrogenase activity was observed and in winter (ambient temperature about 20 °C), nitrogenase activity could not be detected. However, with the onset of spring and a rise in temperature, fresh nodulation (renodulation) by all the inoculant rhizobial strains was observed in both the tree legumes. In L. leucocephala, maximum renodulation was exhibited by strain A1 while in A. nilotica, strain AB3 formed the maximum renodulation 24 months after transplantation. Dry matter yield of all the inoculated plants demonstrated a significant increase over that of the uninoculated plants at the end of five years after transplanting. In L. leucocephala, strain NGR8 gave the maximum response (45% more dry matter yield) in dry matter production while in A. nilotica, strain USDA 3325 showed a 25% increase in total dry matter yield five years after transplantation.

Introduction

The overexploitation of forests, leading to scarcity of fuelwood, fodder and timber for human beings and animals, coupled with increasing wasteland formation is a major concern of developing countries. Legumi- nous trees and shrubs are a renewable source of fuel and wood and many are known to have symbiotic association with rhizobia. Of the tree legumes, Leu- caena leucocephala is found in dry tropical regions whereas Acacia nilotica is usually found in arid and semi-arid regions. Both are renowned for their fast growth even on marginal lands and are popular plants for use in land rehabilitation. Though A. nilotica and L. leucocephala are nodulated by a wide range of slow- and fast-growing strains of rhizobia, most of the nod- ules formed were found to be ineffective (Dreyfus and Dommergues, 198 la,b). To achieve maximum growth of tree legumes in a nitrogen-deficient soil, it is recom- mended that tree legumes be inoculated with specific

* FAX No: +91 11 4621770. E-mail: sunilk@.teri.ernet.in

and effective strains of rhizobia which may not occur naturally at all sites. Nursery and short-term field trials of A. nilotica and L. leucocephala (Lal and Khanna, 1993; Khanna et al., 1992) showed that the inocula- tion of these tree legumes with specific and effective strains of Rhizobium spp. had a positive effect on tree biomass (expressed as total dry matter). Inoculation of woody legumes with selected rhizobia and arbuscular mycorrhizal fungi improves outplanting performance, plant survival and biomass development (Herrera et al., 1993). Also, the height of all inoculated plants of Aca- cia mangium showed a significant increase of 9 to 26% compared to that of uninoculated trees (Galiana et al. 1994). However, most of these results were obtained either under laboratory conditions or from short-term field trials. There are no reports on the long-term sur- vivability and renodulation potential of the introduced rhizobia strains of tree legumes. The objectives of the present study, therefore, were (1) to evaluate the ability of the introduced strains of Rhizobium spp. to renodu- late L. leucocephala and A. nilotica over a period of

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2 years; (2) to determine whether inoculation actually increases biomass of tree legumes (3) to study if there is any change in the nitrogen content of the soil.

Materials and methods

Source of Rhizobium and culture conditions

Fast growing strains of Rhizobium spp. used in this study were USDA 3325, AB3, AD4, A1, A3 and NGR 8. These strains were selected for field trials after various screening processes in the laboratory. Strain USDA 3325 for A. nilotica was obtained from the USDA culture collection while NGR 8 for L. leuco- cephala was from NIFTAL, Hawaii. Strains AB3 and AD4 were isolated from the nodules of A. nilotica growing at Barkha and Dhanwas in northern India. A1 and A3 were obtained from root nodules of L. leuco- cephala growing at Gual Pahari (TERI's field station). Strains USDA 3325 was resistant to tetracycline (25 #g mL- l ) , NGR 8 was resistant to neomycin (125 #g m L - 1), isolate AB3 was resistant to nalidixic acid (300 #g mL -1), AD4 was resistant to penicillin (400 #g m L - l), A 1 was resistant to sodium azid (400/zg m L - l) and A3 was resistant to streptomycin (300 #g mL -1). The resistance was stable in these isolates over at least one year during storage. Antibodies were developed against these strains in white rabbits. Cross-reactivity among these strains was observed by microimmun- odiffusion (Ball, 1990). The inoculum strains USDA 3325, AB3, AD4, NGR 8, A1 and A3 belonged to different serotypes and did not cross-react with each other, as observed by microimmunodiffusion. Further, the antiserum of none of the isolates cross-reacted with the native population of slow-growing rhizobia. The inoculum of Rhizobium spp. was grown on yeast extract mannitol (YEM) broth (Vincent, 1970) on a rotary shaker at 30 °C for 24 h. The cultures were harvested by centrifugation at 7500 g, washed twice with YEM broth and resuspended in sterile YEM broth devoid of carbon source. The seeds ofA. nilotica and L. leucocephala were obtained from the Forestry group of the Tata Energy Research Institute (collected from just one 'plus' tree each of A. nilotica and L. leucocepha- la). Seeds that were uniform in shape, size and weight were selected (to reduce variation) for field trials. The seeds were pelleted with the respective Rhizobium spp. isolates (10 7 cfu seed-t) , were raised in a nursery and, after 8 weeks, were transplanted into the field.

Field plots

The 5 year field trials of L. leucocephala and A. nilot- ica were conducted at Gual Pahari in northern India. The field trial of L. leucocephala was conducted from July 1989 to June 1994 and that of A. nilotica from June 1990 to May 1995. The experimental design was a completely randomized block design with 100 repli- cates in each treatment, planted in plots of 5 m × 5 m with a 0.5 m gap between replications and 2 m gap between treatments. During the first year 50 replicates (alternate trees) from each treatment were removed by destructive sampling, increasing the dis- tance between each replicate to 1 m. Root systems of one year old plants did not spread more than 0.5 m, therefore, cross contamination through direct contact of roots, did not occur. The soil at the experimental site was sandy loam. The nitrogen content was 0.020- 0.024%, available phosphorus was 2-5 mg kg -1 soil, and organic carbon was 0.2%. The pH was 8.2-8.9. The indigenous rhizobial population at both the sites was negligible (one cell per gram dry soil estimated by the MPN method); the land had not been cultivated for the last 10 years and no tree legumes were found at the site. Apart from a few species of grass, which grew in the rainy season, it was a barren land.

Sampling

Nitrogenase activity and nodule occupancy by introduced rhizobial strains The plants were harvested 4, 8, 12, 18 and 24 months after transplantation and nitrogenase activity in root nodules plus feeder roots were estimated in the field by the acetylene reduction assay (ARA). At the same time nodule number, nodule fresh weight and dry weight were also recorded. The nodule occupancy by intro- duced rhizobial strains were quantified at zero time (at the time of transportation) and from 12 and 24 months old plants. The methods of harvesting of tree legumes and collection of nodules for the acetylene reduction assay and for nodule occupancy has been published elsewhere (Lal and Khanna, 1993).

Biomass Shoot biomass (expressed as dry matter yield) of L. leucocephala and A. nilotica was recorded at 1, 2, 3, 4 and 5 years after transplantation. One-and two-year- old plants were cut at ground level, chopped into small pieces (5-10 cm), put in muslin cloth bags and dried

to a constant weight at 50 °C. Dry weight of 3-4-and 5-year-old plants was obtained by using a regression equation between oven-dry weight and the height and collar diameter at 50 cm and 137 cm.

Soil nitrogen Soil samples were taken of 15 cm and 45 cm horizons with a sampler. The soil sampling was done at zero month (at the time of transplanting), 1, 2, 3, 4 and 5 years after transplantation. Soil was dried in an oven at 40 °C and powdered by a soil pulverizer. Total soil nitrogen was determined by the method of Bremmer and Mulvaney (1982).

Results

Nitrogenase activity

Leucaena leucocephala L. leucocephala was transplanted in July 1989. Nitro- genase activity was assayed at 4, 8, 12, 18 and 24 months after transplantation. All three strains exhib- ited nitrogenase activity in root nodules in 4-12-and 24-month old plants but no activity in 8 and 18-month- old plants, which coincided with the onset of winter and senescence of the nodules (Figure 1A). Enhanced nitrogenase activity was recorded in root nodules of 12-and 24-month old plants as compared to 4-month- old plants, due to the increased number of nodules (Table 1) leading to increased nitrogen fixation.

Acacia nilotica A. nilotica was transplanted in June 1990. Nitrogenase activity, nodule number, nodule fresh weight and dry weight in 4-8-12-18 and 24-month-old plants indicated that all three Rhizobium isolates fixed dinitrogen in root nodules ofA. nilotica but values were much lower than that in L. leucocephala (Figure 1). Senescence of nodules as observed in L. leucocephala was also recorded in A. nilotica in 8- and 18 months plants leading to a greatly reduced nitrogenase activity nodule number and nodule dry weight during these periods (Figure 1B, Table 1).

Nodule occupancy by introduced strains

Leucaena leucocephala Immunological probes as well as the characteristic resistance to specific antibiotics of the introduced

113

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Figure 1. Nitrogenase activity in root nodules of Leucaena leuco- cephala and Acacia nilotica harvested at 4, 8, 12, 18 and 24 months after transplanting in the field. A. nitrogenase activity in root nod- ules ofL. leucocephala O, Control; 0, AI; V, A3; V,NGR8. B. Nitrogenase activity in root nodules of A. nilotica. O, Control; 0, AB 3; V, AD4; T, USDA 3325. Values are means of four replicates. Bars represent standard errors.

strains of L. leucocephala and A. nilotica were used to determine the extent of survival of the introduced strains over a period of 24 months. As shown in Figure 2A, among the three strains, the indigenous strain A1 showed maximum survival and renodulation (forma- tion of fresh nodules after senescence of old nodules) up to 24 months after transplantation in that ecosystem. The renodulation capability of strain A3 was also high (76% nodule occupancy) 24 months after transplanta- tion. NGR8 was the least competitive and renodulated poorly (71% nodule occupancy).

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Table 1. Nodule number, fresh weight and dry weight (mg plant- ] ) of Leucaena leucocephala and Acacia nilotica

Species Nodule Fresh wt. Dry wt. Nodule Fresh wt. Dry wt. Nodule Fresh wt. Dry wt.

No. No. no.

4 Months 8 months 12 months L. leucocephala 21.6 4- 3.7 123.3 ± 7.8 38.2 4- 2.0

N. nilotica 18.3 4- 1.4 128.3 4- 0.8 41.2 4- 0.7

18 months

L. leucocephala 18.6 -4- 3.3 72.3 4- 18.6 15.8 q- 4.5

N. nilotica 15.3 4- 3.1 41.6 4- 1,6 11.2 4- 0.6

13.3 4- 2.4 77 4- 10.9 18.2 4- 4.9

14.6 4- 0.8 44.3 4- 4.7 10.6 -4- 0.8

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41.6 4- 2.0 202.6 -4- 12.1 68.9 4- 4.1

43.3-4-4.3 351.64- 10.5 112.24-9.5

20 4- 5.2 191.3 4- 8.5 66.9 4- 3.2

Values are mean of 12 replicates. ± represents standard errors.

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Figure 2. Nodule occupancy (%) by introduced Rhizobium strains of Leucaena leucocephala (A) and Acacia nilotica (B) at 0 month (at the time of transplanting from the nursery), 12 months and 24 months after transplantation. Values are mean of 100 replicates. Bars represent standard errors.

Acacia nilotica Nodulation and renodulation (reinfection) data with USDA 3325, AB3 and AD4 of A. nilotica showed that among these strains, AB3 was the most com- petitive (72% nodule occupancy), followed by AD4 (66% nodule occupancy) and USDA 3325 (60% nod- ule occupancy) even after 24 months of transplantation (Figure 2B). In the nursery (zero month), the survival percentage of all three strains was very high and more than 85% of the nodules were formed by the introduced rhizobial strains. The movement of Rhizobia strains in the soil was minimal, and the maximum renodulation occured near the old nodules (data not shown).

Biomass

Leucaena leucocephala Biomass yield of all inoculated plants at the end of 5 years showed a significant increase over the uninocu- lated plants. Among the three strains, NGR 8 gave the maximum response (45% more dry matter) in biomass production and was followed by strain A3, which showed 27% increase over uninoculated control (Fig- ure 3A). The indigenous strain A1 did not prove as effective on L. leucocephala, despite the fact that it was recorded the highest survival percentage among the three inoculants. However, NGR 8 and A3 were efficient nitrogen fixers resulting in higher biomass production of L. leucocephala, whereas A1 proved to be a poor nitrogen fixer.

Acacia nilotica In A. nilotica among the three strains USDA 3325 showed 25% more yield in total dry matter yield over the uninoculated control 5 years after transplantation. Rhizobium strain AD4 also showed a 23% increase in dry matter yield but strain AB3 showed no significant effect (Figure 3B).

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Figure 4. Soil nitrogen content (%) of experiment plots of Leucaena Leucocephala (A) and Acacia nilotica (B) at 0 month (at the time of transplanting from the nursery) and 60 months after transplantation to the field. Values are means of 10 replicates. Bars represent standard e r r o r s .

Soil nitrogen content At zero month (at the time of transplanting), the soil nitrogen content of all the experimental plots was 0.020-0.022%. After 5 years the soil nitrogen con- tent of the 15 cm horizon increased to 0.065-0.075%, i.e. more than a three-fold increase. There was no sig- nificant increase in soil nitrogen content of the 45 cm horizon in both the experiments. The inoculated plots of A. nilotica, also showed a significant increase in soil nitrogen of 15 cm horizon over that of the uninoc- ulated plots (Figure 4B), whereas inoculated plots of L. leucocephala did not exhibit a significant increase in soil nitrogen content over the uninoculated control plots during this period (Figure 4A).

D i s c u s s i o n

Our study clearly demonstrates the ability of intro- duced Rhizobium strains of L. leucocephala and A. nilotica to survive and renodulate (reinfect) under field conditions. This is the first report on L. leucocepha- la to show the renodulation potential of introduced strains and their effect on biomass yield of tree legumes beyond 5 years after transplantation on barren land. Renodulation in tree legumes over a long period is important because with tree legumes repeated inoc- ulation year after year is neither feasible nor desir- able. Our previous field study with A. nilotica (Lal and Khanna, 1993) demonstrated very high renodulation

116

capability in the introduced strains under field condi- tions but this study was limited to 12 months. Among crop plants, McLoughlin et al. (1984) with Rhizobium trifolii and Kucey and Hynes (1989) with Rhizobium leguminosarum showed that inoculated isolates persist for several years after harvest of the legume crops. On the contrary, McLoughlin et al. (1986) with Rhizobium fredii and McLoughlin et al. (1990) with Bradyrhizo- bium japonicum showed loss of the inoculated popu- lation of rhizobia in subsequent years.

Shoot biomass yield was the major criterion in the selection of rhizobial inoculants. Short-term observa- tions on the effect of Rhizobium on biomass yield of tree legumes do not give a true picture; a long-term study is essential to assess the potential of Rhizobium in increasing the biomass yield of tree legumes. Our stud- ies with L. leucocephala and A. nilotica, over 5 years, clearly indicated that A. nilotica responded poorly to inoculation in contrast to L. leucocephala. Rhizobium strains of L. leucocephala showed more nodulation and nitrogenase activity than those of A. nilotica. L. leu- cocephala is a shallow-rooted tree with more feeder roots, resulting in high nodulation (Table 1) leading to high nitrogenase activity. However, A. nilotica, being a deep-rooted tree has fewer feeder roots and thus fewer nodules, resulting in less nitrogen fixation. In L. leu- cocephala, plants inoculated with NGR8 gained maxi- mum dry matter and showed high apparent nitrogenase activity while those inoculated with A1 strain showed lower gains, possibly because of low nitrogenase activ- ity. These observations are in agreement with previous reports on inoculation of woody legumes with selected rhizobial strains, which showed increased survival per- centage in seedlings and greater biomass production in all inoculated trees (Galiana et al., 1994; Herrera et al., 1993). Nitrogenase activity could not be detected in root nodules of 8 and 18 month old plants. These two observations were taken in winter when senescence of nodules owing to the comparatively low temperature led to the release of bacteroids and or undifferentiated bacteria to the soil. With the onset of spring and a rise in temperature, these bacteria formed fresh nodules (renodulation) near the old ones. Total soil nitrogen content of the 15 cm horizon increased significantly over a period of 5 years. This could be due to the leaf fall that occurred during winter and also due to the addition of nodule and root litter to the soil, thereby enriching the soil with nitrogen.

Acknowledgements

We thank Dr R K Pachauri, Director, Tata Energy Research Institute, New Delhi, for providing the infra- structure to carry out this work, Mr Yateen Joshi for critically reading and correcting the manuscript and Neena Mata for typing the manuscript. We are also thankful to European Economic Community for the financial assistance extended during this work.

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