1Department of Crop and Soil Sciences, KNUST, Kumasi, Ghana

NEEM SEED OIL: A POTENT NITRIFICATION INHIBITOR TO CONTROL NITRATE LEACHING AFTER INCORPORATION OF CROP RESIDUES.

A. Opoku1, B. Chaves2 and S. De Neve 2.

1Department of Crop and Soil Sciences, KNUST, Kumasi, Ghana

2Department of Soil Management and Soil Care, Ghent University, Coupure Links 653, Ghent, Belgium

Abstract

The effect of neem seed oil and neem leaf extract as organic nitrification inhibitors on the accumulation of NH4+, NO3-, and nitrification inhibition after incorporation of crop residue was investigated in an incubation experiment. Dicyandiamide (DCD) applied at 15 and 30 kg active ingredient ha-1 were used as low and high doses of a standard synthetic nitrification inhibitor. Soil samples were amended with 21 g kg-1 cauliflower leaves and treated with nitrification inhibitors at a rate of 30 kg ha-1 of neem seed oil, 60 kg ha-1 of neem leaf extract, 15 kg ha-1 of DCD, and 30 kg ha-1 DCD. Samples were incubated at temperatures corresponding to field temperatures during fall and winter in Flanders, Belgium.

Neem seed oil increased NH4+ accumulation by 8.9 mg kg-1 and decreased NO3- by 13.5 mg kg-1 within a month. High and low doses of DCD increased NH4+ accumulation by 48.2 mg kg-1 and 1.6 mg kg-1 respectively. Nitrification was inhibited by 29% for 30 days by low dose of DCD, 58% in 30 days byneem seed oil, and42% in 45 days by high dose of DCD.Nitrification was not inhibited by the use of neem leaf extract.

Key words: Neem seed oil, neem leaf extract, nitrification inhibitors, crop residue, DCD

Introduction

The management of soil Nis a critical issue for crop production especially in sub-SaharanAfrica where soil fertility decline has been identified as a key constraint to Agricultural productivity (Sanchezand Jama, 2002). Crop residues is vital resource which mayrelease15(Mwato et al., 1999)to 150 kg N ha−1(De Neve and Hofman, 1998) upon mineralization.The NO3- so formedis vulnerable to leaching and denitrification losses, resulting in low N use efficiency (Abbasi et al., 2011). Globally, only 33% of the total N applied in cereal production is actually recovered in the grain. The unaccounted 67% representing $15.9 billion is loss annually (Raun and Johnson 1999). Besides the reduction in N use efficiency, increase in atmospheric N2O concentrations contributes substantially to global warming and stratospheric ozone depletion (IPCC, 2007). Furthermore, the leachedNO3-pollutes ground and surface water and creates potential health risks to infants and livestock.

Strategies to reduce N2O emissions and NO3- leachingare priority areas of research to increase N use efficiency.Fortification of organic or mineral fertilizers with nitrification inhibitors is a feasible strategy for reducing these loses. Nitrification inhibitors delay the bacterial oxidation of NH4+ to NO2- for a certain period by suppressing theactivity of Nitrosomonas spp. In this way, mineral N is retained as NH4+ form which is not affected by denitrification and seldom leaches (Chaves et al., 2006). During last decade different synthetic NIs such as dicyandiamide DCD (Cichota et al., 2010, Hoogendoorn et al.,2008 and Menneer et al.,2008) 3,4-dimethylpyrazolePhosphate DMPP (Zerulla et al., 2001, Diez-Lopez et al., 2008 and Hua et al., 2008) have been used to increase N use efficiency of mineral fertilizeror crop residues (Chaves et al., 2006). However,the use of many of these synthetic NIs has been restricted to experimental purposes as a result of their high cost and limited availability (Patra and Sukhmal,2009). Moreover, synthetic nitrification inhibitors may not be ecologically friendly as adverse effectson beneficial soil microorganisms and enzyme activities have been reported in previous studies (Patra et al., 2006, Hua et al., 2008).

Nitrification inhibitory properties of plant materials such as Karanj (Pongamia glabra), neem (Azadirachta indica) and tea (Camellia sinensis) waste have been identified (Majumdar, 2002; Kiran and Patra, 2003). Extracts from neem leaves, seeds, and bark possess a wide spectrum of antibacterial action against Gram-negative and Gram-positive microorganisms, and also act as an active nitrification inhibitor (Biswas et al., 2002; Kumar et al., 2007). Several studies have been conducted to evaluate the efficacy of aromatic plant materials (Kiran and Patra, 2003), neem seed cake and oil (Mohanty et al., 2008; Abbasi et al., 2011) and karanja seed powder(Majumdar, 2002) to suppress nitrification of urea. To our knowledge no study has been conducted to determine the effect of natural nitrification inhibitors on N transformation after the incorporation of crop residues.

The objective of the study was toevaluate effect of neem seed oil, neem leaf extract and DCD on NH4+ oxidation after incorporation of crop residues into the soil. We hypothesized that crop residues treated with neem seed oil, neem leaf extract or DCD would reduce the accumulation of NO3- in the soil and moderate leaching losses.

Materials and methods

Soil, crop residues and nitrification inhibitors

The soil used for the incubations was a silty loam soil (Aquic Hapludalf, USDA Classification) obtained from a vegetable farm in Poeke, Belgium. It consisted of 34.6% sand, 51.8% silt and 13.6% clay. The soil had a total C content of 15 g kg−1, N content of 1.7 g kg−1 and a pHKCl of 6.The soil collected was neither air- dried and nor sieved, stones and visible plant debris in order to minimize disturbance of microbial activity.

Cauliflower leaves were chosen as crop residues. Total C andN contents were determined by dry combustion using aCNS elemental analyser (Variomax CNS, Elementar, Germany). Lignin was determined by Stevenson fractionation method modified by Hofman and De Neve (1996). The polyphenol content of the plant residues was determined by the method of Folin–Denis (King and Heath, 1967). The cauliflower residues had a dry matter contentof 111 g kg−1, a total N content of 48.7 g kg−1, C:N ratio of 8.28, lignin of 8.1% , Phenols of 1.9%,, NH4+content of2.11 g kg−1 andNO3- content of 1.38 g kg−1.

Laboratory incubation

Application rate forneem seed oil was30 kg ha−1 (12% of total N in crop residue) as recommended by Slangen and Kerkhoff (1984) and 60 kg ha−1(24% of total N in crop residue) for neem leaf extract prepared from dry leaves. Also according to the recommendation of Solansky(1982), DCD was applied at 15 and 30 kg active ingredient ha-1 as low and high doses respectively of a standard synthetic NI. In addition to the NI treatments,crop residue only and unamended soil treatments were imposed.The experimental design was complete randomised design with three replications.Freshcauliflower leaves(36 t FM ha−1≈ 4 t DM ha−1) cut into small pieceswere treated with neem seed oil, or neem leaf extract or DCD and thoroughly mixed with fresh soilequivalent to 283 g oven dry soil. The incubations werecarried out in an incubation cabinet at fluctuating temperaturesreflecting actual field temperatures during fall andwinter in Flanders. Soil samples were collected on 7, 14, 30, 45, 70 and 95 days after incubation. Fresh soil samples were extracted with 1 M KCl (extraction ratio 1:2) for NH4+ and NO3- determinations. Percentage nitrification inhibition was calculated with the formula of Crawford and Chalk (1992).

Statistical analysis

Data on NH4+ NO3-, and nitrification inhibition % were analysed with GenStat discovery edition 3 (Payne et al., 2009) using the one–way analysis of variance without blocks procedure. Mean separations were performed using LSD. Treatment comparisons were deemed significant at P< 0.05.

Results

Effect of NIs on NH4+concentrations

The pattern of NH4+ evolution from the crop residue amended soil with or without NI is shown by Fig. (1).Ammonium released from the crop residue only treatment attained a peak of 9.6 mg kg –1NH4+ within the first week of incubation and diminished sharply to zero until the end of the incubation. The application of neem oil significantly (p < 0.05) increased the accumulation of NH4+by 12 -22 mg kg –1during 30 DAI. Neem leaf extract on the hand had no effect on NH4+ concentration throughout the study. The amounts of NH4+ accumulated by the application of DCD were higher than neem seed oil.The use of DCD 15 significantly (p < 0.05) increased the NH4+ accumulation by 14 -27 mg kg –1 during 30 DAI while a surge of 18 -48 mg kg –1 in NH4+ accumulation were found in DCD 30 amended soils. The pattern of ammonium –N build up in the DCD 15 and DCD 30 amended soils were closely related, except that the activity of DCD 15 lagged behind that of DCD 30.

Insert Fig. (1)

Effect of NIs on NO3-concentrations

The pattern of NO3- evolution from the crop residue amended soil with or without NI is shown by Fig (2). Nitrate was rapidly released from the crop residue following its incorporation. Without the use of any NI about 35 % of the applied N (37.4 mg NO3- -N kg-1) was transformed into NO3--Nby day 14. The evolution of NO3--N depressed slightly thereafter and leveled out to 49% of the total N applied (51.58 mg kg-1) by the end of incubation. The use of neem seed oil significantly suppressed NO3- formationfor a duration of 30 days while neem leaf extract had no effect on the NO3- formation. Significant (p < 0.05) inhibitory effect on NO3- accumulation was found for duration of 30 days in DCD 15 treated soils and 55days in DCD 30 treated soils. A significant (p < 0.05) jump in NO3- accumulation was observed in DCD 15 treated soils relative to the residue only soil between 30 DAI and 45 DAI whereas a related jump occurred in DCD 30 treated soils from 45 DAI to 70 DAI suggesting a loss of inhibition effect by the doses of DCD at different times.

Insert Fig. (2)

Influence of NIs on nitrification

Figure(3) shows the Nitrification inhibition percentage of DCD and neem based inhibitors. Nitrification inhibition percentage decreased with the duration of incubation. The use of DCD 30 exhibited highest nitrification inhibition of 82% at 14 DAIand maintained a significantly higher inhibitory effect (42%) until 45DAI. The nitrification inhibition by neem seed oil (58-66%) were significantly higher than DCD 15 (29-51%) on 14 and 30 DAI, The nitrification inhibition effect of DCD 15 and neem seed oil disappeared before day 45 while effect of the DCD 30was loss prior to day 70. Neem leaf extract had no inhibitory effect and showed negative inhibition percentages throughout the study.

Insert fig. (3)

Discussion

The observation that in the absence of any NI the highest concentration of NH4+ was found at day 7 followed by rapid release of NO3- indicates the incubation conditions were favorable for the ammonification and nitrification of the organic N in the crop residue. It was evident in the results that lower doses of DCD had moderate inhibitory effect (29%) during the first 30 days of incubation given rise to a relatively higher concentration of NH4+ and a corresponding lower concentration of NO3-. Whereas Hoogendoorn et al. (2008) and Cichota et al. (2010)observed a similar effect of DCD on NH4+ oxidation from ploughed in pastures, Rodgers et al. (1985) and Webb et al. (1991) observed no such inhibitory effect of DCD either in short–term grass leys or long-term grasslands. The discrepancy in these results can however be attributed to the fact that in the studies where DCD had no effect on NH4+ oxidation, the NI was applied after the pasture had been ploughed and extensive N mineralisation had occurred masking any subsequent effect of DCD.

Doubling the dose of DCD doubles inhibitory effect (64%) during the first 30 and further prolonged the duration of the effect until 45 days after incubation. The behavior of DCD can be explained by the nature and mode of action of DCD. The basic constituent of DCD (HN−C(NH2)–NH–CN) is a ligand, which inhibits the activity of the enzyme ammonium monooxygenase (AMO) by interfering with the electron transport in the cytochromes of AMO (Hyman etal., 1995). With increasing concentration of DCD, the degree of interference increases, resulting in a larger reduction in nitrification. The concentration of DCD in the soil decreases with time as the enzyme amidase hydrolyses DCD into urea (Tabatabai 1994).

The results indicated that neem seed oil which is less expensive and locally avaialablewas as potent as DCD which is expensive to acquire. The observed superior inhibitory effect of neem seedoil (58%) at day 30 relative to the lower dose of DCD (29%) concurs with the findings of Kiran, and Patra (2003) that treating urea with Artemisia oil led to a higher recovery of the added N (63%) than the use of DCD (46%). Abbasi et al. (2011)also found a satisfactory nitrification inhibitory effect (54%) of neem seed cake on urea-N transformation after 20 to 30 days of incubation.Although, the degree of inhibition by neem seed oil found in the present study were higher than the 4 to 31% reported byKumar et al. (2007). The differences in the efficacy of the neem seed oil may be due heterogeneity in their azadirachtin contents (Olfat and El-Shiekh , 2012). Even though neem seed oil is a potent nitrification inhibitior, its mode of action and the active compound for the inhibition remains unclear. Several studies have attributed the nitrification inhibition effect to azadirachtin (tetranortriterpenoids) partly because of its strong insecticidal effect (Neem Foundation,1997; Mohanty et al. 2008; Abbasi et al. 2011 Vyas et al.,1993). Conversely, studies by Baldwin et al.(1983) identified tannins and polyphenols as chemicals responsible for the inhibition of nitrification by neem product. Perhaps the observed nitrification inhibition was derived from the combined effect ofthese chemicals. The lack of nitrification inhibition by neem leaf extract could be attributed to the low concentration of azadirachtin in the leaves as the secretory cells for the synthesis of azadirachtin are more abundant in the seeds than in the leaves (Neem Foundation, 1997).In addition,azadirachtin content decreases with drying (Olfat and El-Shiekh, 2012), hence the preparation of the leaf extractfrom dry leaves also contributed to the poor inhibition by the extract.

Conclusion

The pre-treatment of crop residue with DCD at a rate of 30 kg ha-1 inhibited nitrification by 42% over a period 45 days and prevented 31% of the NO3- mineralised from the crop residues from leaching during the period. The pre-treatment of crop residue with DCD at a rate of 15 kg ha-1 on the other hand inhibited nitrification by 29% over a period 30 days and prevented 16% of the NO3- mineralised from the crop residues from leaching during the period. Furthermore, the addition of neem seed oil at a rate of 30 kg ha-1 to crop residues before incorporation inhibited nitrification by 58% over a period 30 days and prevented 33% of the NO3- mineralised from the crop residues from leaching during the period. Lastly neem leaf extract at a rate of 60 kg ha-1 had no effect on nitrification inhibition. It is concluded that, the inhibitory effect of neem seed oil was better than lower dose of DCD and therefore can be used as nitrification inhibitor to control NO3- leaching losses. Neem leaf extract on the other hand had no inhibitory effect and cannot be used to moderate NO3- leaching losses.

Acknowledgements

The authors are grateful to the Research and Development Division of Belgian Ministry of Small Enterprises and Traders and Agriculture, for funding this research (project S-6059). We also thank M. Remue, V. Van De Vyvere, L. Bauwens, T. Coddens and S. Schepens for their skilful technical assistance.

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