Biological Nitrogen Fixation by Mucuna Bracteata Under Oil Palm Field Environments

Oil Palm Bulletin 60 (May 2010) p. 22 - 27 Biological Nitrogen Fixation by Mucuna bracteata under Oil Palm Field Environments

Cheah, S S*; Zaharah, A R** and Aminuddin, H**

INTRODUCTION ABSTRACT Nitrogen (N) is known to be the most limiting In recent years, there has been a tremendous renewal nutrient in oil palm crop production. It has been of interest in incorporating the N-fixing creeping estimated that to produce 30 t fresh fruit bunches legume, Mucuna bracteata, in the oil palm (FFB) ha-1 yr-1, oil palms of 9-12 years planted on production system. Although a considerable amount Bungor Series soil would require 129.4 kg N ha-1 of work has been done on M. bracteata, the N-fixing yr-1 (Tarmizi and Mohd Tayeb, 2006). Much of capacity of this legume has not been fully studied. this N requirement is fulfilled by the application 15 Hence, an experiment using the N isotopic dilution of inorganic N fertilizer with the remaining technique was laid out to quantify the N fixed by supplied by soil-N. Given the large dependence M. bracteata in a 3-year-old oil palm plantation of FFB production on N, it is understandable where P. conjucatum and mixed broad-leaf weeds why inorganic N fertilizer always tops the list as were used as non-N-fixing reference plants. The one of the most expensive variable costs in FFB results revealed that M. bracteata planted on production. As a consequence of its critical role, Jawa Series soil derived 67%-84% of its N from N resource management has become an extremely N-fixation. The finding confirmed thatM. bracteata important aspect in FFB production of oil palm. has the potential to contribute to the N pool of the oil palm cropping system, and has potential to be used In the face of increasing fertilizer prices, low as a green manure or even as an alternative N source crude palm oil price and growing concerns for to inorganic N. The high percentage of N fixed by environmental quality, the oil palm industry has M. bracteata also indicates that competition for been under tremendous pressure to seek cheaper applied N and soil N between M. bracteata and oil and more environmental-friendly alternatives palm, if any, would be insignificant. to N fertilizer. This has inadvertently resulted in a renewal of interest in incorporating N-fixing ABSTRAK creeping legumes in the production system of oil palm (Lee et al., 2005; Goh et al., 2006; Mathews Penggunaan kekacang penutup bumi, M. bracteata and Saw, 2006; Shaharudin and Jamaluddin, 2006), dalam sistem pengeluaran sawit semakin mendapat especially since a new species of creeping legume, perhatian kebelakangan ini. Walaupun banyak M. bracteata, was introduced to the oil palm penyelidikan telah dijalankan ke atas M. bracteata, industry (Mathews, 1998). keupayaan untuk mengikat N daripada atmosfera belum dikaji dengan sepenuhnya. Sehubungan Credited to its many desirable positive ini, satu kajian telah dijalankan untuk mengkaji characteristics, such as the potential capability keupayaan pengikatan N oleh M. bracteata di of accumulating and returning large amounts of ladang sawit yang berumur tiga tahun. Dalam N (Mathews and Leong, 2000; Chiu et al., 2001; 15 kajian ini, kaedah N isotopi digunakan untuk Ng et al., 2005; Goh et al., 2006; Shaharudin and mengukur jumlah N yang diikat oleh M. bracteata Jamaluddin, 2006), many oil palm plantations yang ditanam di celah-celah barisan sawit, di mana have started planting M. bracteata as a cover crop P. conjucatum dan campuran rumpai berdaun during the replanting of oil palm. Although a lebar digunakan sebagai tumbuhan rujukan. Hasil considerable amount of work has been done on M. kajian menunjukkan M. bracteata yang ditanam di bracteata, there are still many questions remaining. atas tanah siri Jawa berupaya mengikat 67%-84% Moreover, the competition between M. bracteata keperluan N daripada atmosfera. and oil palm for soil-N and fertilizer-N is a concern as well among the planters and the agronomists. Keywords: M. bracteata, N- fixation, oil palm. Hence, quantifying the amount of N fixed by M. bracteata is required to ensure that M. bracteata

* Sime Darby Research Sdn Bhd, Lot 2664 Jalan Pulau Carey, is a net contributor of atmospheric N to the system P. O. Box 207, 42700 Banting, Selangor, Malaysia. and is not just mining N from soil and applied ** Universiti Putra Malaysia, Department of Land Management, fertilizers, thus competing with oil palm. In order 43400 UPM Serdang, Selangor, Malaysia. to address the various concerns and the void in our

22 Biological Nitrogen Fixation by Mucuna bracteata under Oil Palm Field Environments knowledge on M. bracteata, this study was guided all above-ground green vegetation. The mixed by the following objectives: broad-leaf weeds, MBW (comprising Asystasia intrusa > Borreria latifolia > Cleome rutidosperma), • to determine the biological N-fixation of that had grown voluntarily within the microplots M. bracteata planted on Jawa Series soil in an of M. bracteata were harvested and treated as an oil palm field environment; and additional non-N-fixing control. All harvested • to evaluate the influence of two different plant materials of M. bracteata, P. conjucatum and reference plants on the estimates of N-fixation MBW were then weighed, cleaned with distilled by M. bracteata planted on Jawa Series soil in an water and oven-dried at 70˚C to constant weight. oil palm field environment. Dry weights of the respective plant species and components were recorded, and P. conjucatum was MATERIAL AND METHODS sub-sampled before all samples were milled to pass through a 1-mm sieve. All milled plant materials of The experiment was carried out from April to June M. bracteata, P. conjucatum and MBW were then kept 2007 within an experimental plot of M. bracteata. in sealed containers and stored in a dessicator with The 15N isotope dilution method was used to silica gel before being analysed for total N content quantify biological N-fixation of M. bracteata by the semi-micro Kjeldahl digestion method under oil palm field conditions. Thirty-month-old (IAEA, 2001). The 15N analysis was conducted to M. bracteata and Paspalum conjucatum were used in measure the proportion of the applied 15N taken this study. P. conjucatum and weeds regenerating up by M. bracteata, P. conjucatum and MBW. The 15N within the microplots of M. bracteata were used as analysis was carried out by first digesting the plant the non-N-fixing reference plants. materials using the semi-micro Kjeldahl method. This was followed by distillation and titration Preparation of Microplots (Horwitz, 1980) and then the measurements of the 15N/14N ratio using an optical emission Six microplots (1 m2), representing replicates, spectrometer (IAEA, 2001) were carried out at the of M. bracteata and P. conjucatum were established, Malaysian Nuclear Agency, Bangi. respectively. All green leaves of M. bracteata were pruned off and removed from the microplots Calculation of Percent N Derived from together with leaf litter. Only the stems of the Atmosphere (%Ndfa) M. bracteata plants were kept intact in the microplots for regeneration of new leaves. All leaves including The percentage of N derived from atmospheric the stems of P. conjucatum were pruned off to N (%Ndfa) was computed using the following approximately 1.0 cm above the soil surface. All formula (Fried and Middleboe, 1977; Fried and green leaves, stems and litter of P. conjucatum were Broeshart, 1981): removed from the microplots as well. atom% 15N excess F 15 %Ndfa = (1 - ) x 100 Application of N Labelled Ammonium Sulphate atom% 15N excess NF Solution where, The 15N-labelled ammonium sulphate

[(NH4)2SO4] fertilizer was used for soil enrichment. F denotes the N2-fixing plant; and 15 The N-labelled fertilizer was applied at the NF denotes the non-N2-fixing reference plant. rate of 20 kg N ha-1 enriched with 10% 15N atom excess equivalent to 2 g N m-2 of 10% 15N atom Data Analysis excess (IAEA, 2001). Thus, 9.5 g of 15N-labelled ammonium sulphate were dissolved in 1 litre of All data were subjected to analyses of variance distilled water. The resultant 15N-labelled solution (ANOVA) using the software package SAS version was then uniformly applied to each microplot by 9.1 for Windows (SAS, 2005). Mean separation was spraying onto the soil surface. Every microplot of done by the Least Significant Difference (LSD) test M. bracteata and P. conjucatum received 1 litre of (Steel and Torrie, 1980). All comparisons between 15N-labelled solution each. means were done at the 0.05% significance level. The %Ndfa was calculated accordingly based Harvesting and Preparation of Plant Samples on %15N atom excess (%15Na.e) of each reference plant and the mean %15Na.e of two reference All microplots of M. bracteata and P. conjucatum plants (P. conjucatum and MBW) following the were harvested at 60 days after application of the recommendations by Boddey et al. (1990). 15N-labelled ammonium sulphate by removing

23 Oil Palm Bulletin 60

RESULTS AND DISCUSSION accumulated significantly higher amounts of total N compared to M. bracteata and MBW (Table 1). Its Dry Matter Yield and Nitrogen Accumulation total N content (5.41 g m-2) was much higher than the highest total N content reported by Zaharah et There were significant differences in dry matter al. (1986), which was 1.52 g m-2 over a three-month yield and total nitrogen accumulation between period. By contrast, M. bracteata and MBW recorded M. bracteata, P. conjucatum and mixed broadleaf lower total N content due to their correspondingly weeds (MBW) (Table 1). Nonetheless, large variations low dry matter yields. in the measurements of dry matter produced were obvious as indicated by the high coefficients of Percent 15N Atom Excess, Percent Nitrogen variation. In this study, P. conjucatum accumulated Derived from Fertilizer and Soil 16 times and 25 times more dry matter than MBW and M. bracteata, respectively over a 60-day period. As expected, the %15Na.e in M. bracteata was On the other hand, MBW was found to accumulate found to be significantly lower than in both the only 1.5 times more dry matter than M. bracteata, reference plants (Table 2). However, the %15Na.e but the difference was not statistically significant. recovered by both reference plants varied The significantly lower dry matter yield of significantly. A similar trend was observed for M. bracteata was attributed to the progressive %Ndff where both reference plants and M. bracteata dieback of its stems in five replicates. This was accumulated significantly different amounts of one of the reasons leading to a higher coefficient of labelled fertilizer-N. On the other hand, %Ndfs variation for dry matter yield. The reason for this in the reference plants were found to be similar progressive dieback was not clear. The lower dry while accumulation of soil-N by M. bracteata was matter produced by MBW may be linked to the significantly smaller. quantity of seeds that were in the soil to begin with and their germination rate. The lower %15Na.e in M. bracteata was expected because the N obtained by M. bracteata from the As expected, being a legume, the N concentration soil and the atmosphere reduced the 14N/15N in M. bracteata was significantly higher than in ratio in the tissues (Hardason et al., 1983; Zaharah P. conjucatum, but was not significantly different et al., 1986; Viera-Vargas et al., 1995). However, from that of MBW (Table 1). The high N differences in the pattern of N accumulation and concentration in M. bracteata was in accordance with the rate of growth between the reference plants the results from other studies (Kothandaraman et and M. bracteata can also influence the %15Na.e in al., 1989; Shaharudin and Yow, 2000), while the N their plant tissues (Witty, 1983; Chalk, 1985; Boddey concentration in P. conjucatum was consistent with et al., 1995). This effect was demonstrated in this data reported by Zaharah et al. (1986). Similarly, experiment where plants with a lower dry matter MBW which was dominated by Asystasia intrusa yield also accumulated a lower %15Na.e. A similar also recorded a similar N concentration as that observation has been reported by Ståhl et al. (2005). reported by Rajaratnam et al. (1977). Attributable The %15Na.e enrichment in the soil tends to decrease to its much higher dry matter yield, P. conjucatum over time as the soil-N pool is continuously

Table 1. Dry matter yield, % N and total N in M. bracteata, P. conjucatum and MBW1 on Jawa Series soil

Specie Dry matter yield % N Total N (g m-2) (g m-2) P. conjucatum 398.47a 1.38a 5.41a MBW 24.46b 3.94b 0.97b M. bracteata 16.16b 3.77b 0.35b CV (%) 48.7 13.6 55.3

Note: means in a column followed by the same letter are not significantly different from one another according to the Least Significant Difference test at P=0.05. CV = coefficient of variation. 1MBW = mixed broad-leaf weeds, comprising Asystasia intrusa, Borreria latifolia and Cleome rutidosperma.

24 Biological Nitrogen Fixation by Mucuna bracteata under Oil Palm Field Environments

TABLE 2. %15N a.e, percent of N from fertilizer (%Ndff) and soil (%Ndfs) IN M. bracteata, P. conjucatum and MBW1 on Jawa Series soil

Specie %15Na.e %Ndff %Ndfs P. conjucatum 0.564a 5.64a 94.36a MBW 0.302b 3.02b 96.99a M. bracteata 0.085c 0.40c 15.43b CV (%) 35.5 38.6 8.56

replenished by unlabelled N from mineralization native Bradyrhizobia of M. bracteata in the soil. This of soil organic matter (Fried et al., 1983; Witty, 1983; confirmed the findings of Mathews et al. (2006). Smith et al., 1992; Boddey et al., 1995). Under these conditions, if the reference plants as well as the Accuracy of Measurements of N-fixation legume have different N-uptake patterns, they will inevitably obtain different %15Na.e enrichments. It is usually impossible to obtain a well-matched This was supported by the different %Ndff in reference plant for accurate quantification of M. bracteata and in both reference plants (Table 2). N-fixation due to spatial and temporal variations in isotope distribution as well as differences in Percent Nitrogen Fixed by M. bracteata N uptake patterns of the reference plant and the N-fixing plant (Chalk et al., 1983, Rennie et al., 1988; The %Ndfa estimated using the %15Na.e of Smith et al., 1992; Boddey et al., 1990; 1995; 2000). each reference plant and the mean %15Na.e of two Recognizing the inherent errors associated with reference plants was very high but not significantly the reference plant, the strategy of using several different (Table 3). The estimated %Ndfa ranged reference plants was advocated to minimize these from 67.36%-83.98% being equivalent to 0.26-0.31 g errors (Rennie et al., 1988; Boddey et al., 1990; 1995; N fixed per square meter over a 60-day period. Ståhl et al., 2005).

On average, the estimated %Ndfa in M. bracteata In this experiment, although there were was slightly higher than the estimate reported differences in the amounts of N accumulated and by Goh et al. (2006), who found that M. bracteata %Ndff amongst M. bracteata and the reference planted on Bungor Series soil derived about 70% of plants, a high percentage of N was fixed by its N requirement from the atmosphere. The high M. bracteata. Thus, the data on %Ndfa implies that estimates of %Ndfa in M. bracteata planted on the this N symbiosis is relatively insensitive to the acidic Jawa Series soil also suggested the presence errors associated with the reference plant as well of effective, infective and low soil pH-tolerant as isotopic non-uniformity. This confirmed the

Table 3. Percent of N derived from the atmosphere (%Ndfa) IN M. bracteata planted on Jawa Series soil using P. conjucatum and MBW1 as reference plants

Reference plant %Ndfa P. conjucatum 83.98a MBW 67.36a P. conjucatum + MBW2 79.36a CV (%) 21.4

Note: means followed by the same letter are not significantly different from one another according to the Least Significant Difference test at P=0.05. CV = coefficient of variation 1MBW = mixed broad-leaf weeds, comprising Asystasia intrusa, Borreria latifolia and Cleome rutidosperma. 2The mean %15Na.e of two reference plants (P. conjucatum and MBW) was used to calculate the %Ndfa.

25 Oil Palm Bulletin 60 findings of Reichardt et al. (1987) and Hardason et al. grain legumes: a strategy for the practical (1988), who found that any potential errors over the application of the 15N isotope dilution technique. lack of concurrence in N uptake patterns amongst Soil Biology and Biochemistry, 22: 649-655. the reference plants and the N-fixing plants will be minimal when the %Ndfa is consistently high. BUSSE, M D (2000). Suitability and use of the Busse (2000) even concluded that the requirement 15N-isotope dilution method to estimate nitrogen for a well-matched reference plant is unnecessary fixation by actinorhizal shrubs. Forest Ecology for highly effective symbiosis. Hence, the %Ndfa in Management, 136: 85-95. M. bracteata planted on Jawa Series soil is potentially accurate despite the considerably high coefficients CHALK, P M (1985). Review: estimation of of variation observed in this experiment. N2-fixation by isotope dilution: an appraisal of techniques involving 15N enrichment and their CONCLUSION application. Soil Biology and Biochemistry, 17: 389- 410. In this study, M. bracteata planted on Jawa Series soil has a high N-fixing ability. Depending on the CHALK, P M; DOUGLAS, L A and BUCHANAN, S reference plant used, 67%-84% of its N was found A (1983). Use of 15N enrichment of soil mineralizable to originate from N-fixation. The results suggest N as a reference for isotope dilution measurements that M. bracteata has the potential to contribute to of biologically fixed nitrogen. Canadian Journal of the N pool of the oil palm cropping system, and Microbiology, 29: 1046-1052. has the potential to be used as a green manure or even as an alternative N source to inorganic N. CHIU, S B; SIOW, A and FARIDA, E (2001). Mucuna The high percentage of N fixed by M. bracteata bracteata – a super legume cover crop revisited. The also indicates that competition for applied N and Planter, 77 (907): 575-583. soil-N between M. bracteata and oil palm, if any, would be insignificant. The study indicates that it is FRIED, M and BROESHART, H (1981). A further better to use several reference plants for estimating extension of the method for independently N-fixation rather than selecting or recommending measuring the amount of nitrogen fixed by a a single ‘suitable’ reference plant for a specific legume crop. Plant and Soil, 62: 331-336. legume, in this case M. bracteata. This is because the use of several reference plants rather than one FRIED, M; DANSO, S K and ZAPATA, F (1983). specific plant may help elucidate and validate The methodology of measurement of N2 fixation conclusively the results obtained. by non-legumes as inferred from field experiments with legumes. Canadian Journal of Microbiology, 29: ACKNOWLEDGEMENT 1053-1062.

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