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

A. Opoku 1 , 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 by neem seed oil, and 42% 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 N is a critical issue for crop production especially in sub-Saharan Africa

where soil fertility decline has been identified as a key constraint to Agricultural productivity

(Sanchez and Jama, 2002). Crop residues is vital resource which may release 15 (Mwato et al.,

1999) to 150 kg N ha−1 (De Neve and Hofman, 1998) upon mineralization. The NO3- so formed

is 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

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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 leached NO3- pollutes ground and surface water and creates potential health

risks to infants and livestock.

Strategies to reduce N2O emissions and NO3- leaching are 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 the activity 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-dimethylpyrazole

Phosphate 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 fertilizer or 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 effects on 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

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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 to evaluate 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 pH KCl 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 and N contents were determined by dry

combustion using a CNS 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 content of 111 g kg−1, a total N

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content of 48.7 g kg−1, C:N ratio of 8.28, lignin of 8.1% , Phenols of 1.9%,, NH4+ content of 2.11

g kg−1 and NO3- content of 1.38 g kg−1.

Laboratory incubation

Application rate for neem seed oil was 30 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. Fresh cauliflower leaves (36 t FM ha−1 ≈ 4 t DM ha−1) cut into

small pieces were treated with neem seed oil, or neem leaf extract or DCD and thoroughly mixed

with fresh soil equivalent to 283 g oven dry soil. The incubations were carried out in an

incubation cabinet at fluctuating temperatures reflecting actual field temperatures during fall and

winter 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.

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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 –1 NH4+ 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 –1 during 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--N by 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- formation for 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

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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 DAI and maintained a significantly higher

inhibitory effect (42%) until 45 DAI. 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 30 was

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

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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 et al., 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 avaialable was as

potent as DCD which is expensive to acquire. The observed superior inhibitory effect of neem

seed oil (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

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incubation. Although, the degree of inhibition by neem seed oil found in the present study were

higher than the 4 to 31% reported by Kumar 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 of these 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

extract from 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

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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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Figures

Error bars are LSD at 5%Figure 1: Effect of NIs on NH4

+ formation

Error bars are LSD at 5%Figure 2 Effect of NIs on NO3

- formation

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Error bars are LSD at 5%Figure 3 Effect of NIs on nitrification inhibitions

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0-14 0-30 0-700-45 0-90