Inhibitory Effect of Neem Seed Extract on Soil Nitrogen Mineralization under Chemical and Organic Amendments

Nitrogen (N) loss from cultivated land through the N transformation process causes many environmental pollutions, e.g., nitrate contaminations of surface- and groundwater and nitrous oxide emission (Benckiser et al., 2015). These pollutions consequently affect severe human health risks. The environmental contaminations of N are attributed both to chemical fertilizers and N-rich-organic amendments (Bijay and Craswell, 2021). Enhancing N use efficiency (NUE) by applying an N-release controller such as N mineralization inhibitors has been recommended to minimize environmental hazards due to N leaking (Singh et al., 2019). Economically, this strategy can save chemical fertilizer costs under its expensive crisis nowadays.

Many chemicals are employed as soil N mineralization inhibitors; nevertheless, they are costly and unavailable in traditional markets (Kumar et al., 2010). Neem extract has been reported as a natural N mineralization inhibitor and improves NUE of plant (Meena et al., 2021).

Several organic amendments were recently promoted for soil fertility improvement, substituting chemical fertilizers to address the negative environmental impact of industrial fertilizer production and ease the expensive cost crisis (Galic et al., 2020). Cricket-keeping farms have continuously grown up in many countries in Asia and Africa (Halloran et al., 2017). In Thailand alone, over 20,000 farms were observed, where released cricket feces at an average of 44 Mg farm^-1 year^-1 (Halloran et al., 2017). Recycling the feces into organic amendment can create economic value and simultaneously decrease environmental problems. However, a very high N content, 2.3-2.6% N, of cricket feces (Halloran et al., 2017) may be mineralized and lost rapidly. Application of inhibition inhibitor may be a solution to the aforementioned issues. Yet, to our knowledge, neem extract application as an N mineralization inhibitor in soil amended with N-rich organic material has not been reported.

The current paper hypothesized that combining cricket feces with neem seed extract would inhibit soil N mineralization and improve the NUE of the plant. Therefore, the objective of this paper aimed to estimate the effects of the combination uses of cricket feces with neem seed extract on soil N mineralization and plant’s NUE.
 

Soil, cricket feces and neem seed extract
 
The Roi-et soil series (isohyperthermic Aeric Kandiaquults) was collected from a research field in Sakon Nakhon Rajabhat University, Sakon Nakhon, Thailand, at a depth of 1-15 cm. Cricket (Acheta domesticus) feces was obtained from a farm located in Sakon Nakhon. The soil and cricket feces were air-dried and sieved through 2-mm mesh. The beginning characteristics of soil and cricket feces are shown in Table 1.



Neem (Azadirachta indica) seed extract was a commercially available agricultural product made from neem seed cake by a mill factory in Nakhon Ratchasima, Thailand. The extract contained 1375 mg N L^-1 and 12.6 mg azadirachtin mL^-1.
 
Pot trail
 
A pot trial was conducted under greenhouse from January to February 2020. A factorial treatment structure was employed with five soil amendments: unamended (Un); chemical fertilizer (CF); and three rates of cricket feces: 3.125 Mg ha^-1 (CrFlow), 6.25 Mg ha^-1 (CrFmedium) and 12.5 Mg ha^-1 (CrFhigh). These amendments were combined with two rates of neem seed extract: without (-Nm) and with (+Nm) extract. A total of ten treatment combinations were arranged in a completely randomized design (CRD) with three replications. Amaranth (Amaranthus tricolor) was used to evaluate NUE.

Two kilograms of air-dry soil were added to a pot (d=15 cm, h=14 cm and v=2085 cm3). Cricket feces rates were accordingly added, mixed thoroughly and incubated in the soil for 15 days before amaranth transplanting. Meanwhile, a recommendation rate of chemical fertilizer (312.5 kg N ha^-1, 100 kg P ha^-1 and 100 kg K ha^-1) regarding Chakhatrakan (2003) was applied equally two times to the CF treatments at 18 and 30 days after planting. A commercial amaranth variety was planted and nursed for 15 days and a single seedling was transplanted to each pot. Fifty milliliters of neem extract at a concentration of 0.1 mg azadirachtin ml^-1 was added to each +Nm pot once a week at 15, 22, 29 and 36 days after planting. The rate of neem extract was modified according to Sarawaneeyaruk et al. (2015). The soil moisture content of each pot was maintained to 65% of water holding capacity throughout the pot trail. At 39 days after planting, amaranth was cut to evaluate the NUE. Fresh soil was sampled on the same day for mineral N determination.
 
Laboratory analyses
 
Soil particle size distribution was determined by the pipette method (Kroetsch and Wang, 2008). The bulk density of soil and cricket feces was measured using the core method (Pansu and Gautheyrou, 2006). Electrical conductivity and pH were determined using the soil-to-water ratio of 1:5. Organic carbon determination was achieved by the Walkley and Black method (Nelson and Sommers, 1982). Determination of cation exchange capacity was performed following Pansu and Gautheyrou (2006).

Mineral N was extracted in 2 M KCl solution (Stevenson, 1982) and determined using the distillation method on a micro-Kjeldahl distillation (Pro-Nitro S 4002851, JP Selecta, Barcelona). Microbial activity was determined according to the fluorescein diacetate hydrolysis method described by Green et al. (2006).
 
Data calculations
 
The calculation of the net N mineralization rate (Net N min rate) was modified from Bi et al. (2017):
 

Where
[Mineral N]_t2 and [Mineral N]_t1 are soil mineral N concentrations at the harvest and the start of the experiment, respectively.

The N mineralization inhibition was computed using an equation modified from Aspelin and Ekholm (2017) as follows:
N mineralization inhibition (%) =
 

Where
(Net N min rate)_un = Net N mineralization rate of Un treatments. 
(Net N min rate)_am = Treatments of CF, CrF_low, CrF_medium and CrF_high.

Nitrogen recovery efficiency (NRC) and agronomic nitrogen use efficiency (AgrNUE) were calculated by modifying the method of Carranca (2012) through the following equations:
 

Where
(N uptake)_am = N uptake by amaranth under CF, CrF_low, CrF_medium and CrF_high treatments.
(N uptake)_un = N uptake under Un treatments; and N input is an amount of added N.
 

Where
(Shoot DW)_am = Shoot dry weight of amaranth under the CF, CrF_low, CrF_medium and CrF_high treatments.
(Shoot DW)_un = Shoot dry weight under Un treatments.
N input = An amount of added N.
 
Statistical analysis
 
A two-way analysis of variance based on a factorial arrangement in CRD was used to estimate the effects of combined uses of neem seed extract with soil amendments on soil N transformation and N use efficiencies of the amaranth. Treatment mean comparisons were carried out via the Tukey’s honest significant difference ‘‘(HSD)’’ test. The principal component analysis (PCA) using the PROC PRINCOMP model was performed to identify the relationships among parameters of soil N status and N use efficiency. Statistical analyses were done following SAS Institute Inc. (2004). Significant differences were at P≤0.05.

Inhibitory effect of neem seed extract on N mineralization occurring only in soils without cricket feces amendment
 
Soil mineral N concentrations and net N mineralization rates under Un and CF treatments significantly decreased in +Nm treatments relative to -Nm (Table 2). However, the opposite results were found in all cricket feces treatments. Soil mineral N concentrations and net N mineralization rates in -Nm and +Nm increased significantly with greater rates of cricket feces. Meanwhile, N mineralization inhibition occurred in Un and CF treatments under +Nm, but vice versa in the cricket feces treatments.

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The inhibitory effect on N mineralization under Un+Nm and CF+Nm was attributed to the neem active ingredients: azadirachtin, salanin, 14-epoxiazadiradione, meliantrol, melianone, gedunin, nimboline, nimbin, deacetilasalanin, azadiractol, azadirone, vilosinin and meliacarpine (Choudhury et al., 2016). These ingredients have been reported to inhibit the N mineralization sub-processes, i.e., urea hydrolysis (Mohanty et al., 2008) and nitrification (Alves et al., 2009). Among these ingredients, azadirachtin was reported to serve as an essential mineralization inhibitor (Sarawaneeyaruk, Krajangsang and Pringsulaka, 2015). The current study’s azadirachtin content of neem seed extract was 12.6 mg mL^-1.

Instead of inhibition, combinations of neem seed extract with cricket feces stimulated N mineralization, as elucidated in the negative values of N mineralization inhibition (Table 2). The denature of neem ingredients might be a consequence of malfunction of the inhibitory property. Sundaram et al. (1995) reported that azadirachtin was denatured via hydrolytic and microbial degradations. The higher degree of azadirachtin hydrolytic degradation occurred in the higher pH values in the following orders: pH 10 >>> pH 7 > pH 4. In the current study, pH values of soils under +Nm combined with CrF_low, CrF_medium and CrF_high were 5.90, 5.93 and 6.29, respectively (Table 2). Meanwhile, pHs of Un+Nm and CF+Nm were 5.90 and 5.82, respectively. Therefore, lower pH of the unamended and chemical fertilizer treatments than cricket feces amended soils (CrF_medium and CrF_high) implied that less hydrolysis degradation of azadirachtin existed in the former soils.

Another malfunction of inhibitory property of neem seed extract in the cricket feces amended soils might be a consequence of microbial degradation of azadirachtin. Microorganisms were reported to play a crucial role in decomposing azadirachtin (Stark and Walter, 1995). The current study showed significantly higher microbial activity, indicated by fluorescein released, in cricket feces amended treatments than the Un and CF treatments (Table 2). In addition, the higher rates of cricket feces brought about higher degrees of microbial activity, as shown by significantly higher fluorescein released in CrF_high than CrF_low and CrF_medium. Cricket feces was a vital source of energy and nutrients for soil microorganisms, as evidenced by the high contents of organic C and mineral N (Table 1). Our results agreed with Agyarko et al. (2006), who determined that higher degrees of azadirachtin degradation were rendered by increased amounts of poultry and cattle manures.

A significant increase in N mineralization inhibition under CrF_high relative to its lower cricket feces rates (Table 2) might be manifested by the decomposing products derived from the feces, such as humic substances and organic molecules. Humic acid was reported as a nitrification inhibiting compound (Benckiser, Schartel and Weiske, 2015). In addition, several organic molecules derived from the decomposition, e.g., quinines, catechols, ethylene, acetylene, gallic acid and tartaric acid, were reported to bring about microbial toxicity (Kaal et al., 2012). Even though organic molecule concentration was not determined in this paper, higher rates of cricket feces were assumed to increase the content of soil organic molecules. This assertion was in line with Nair et al. (2015) who stated that the greater input of farm yard manure brought about higher soil organic content.
 
Inhibition of N mineralization diminishing nitrogen use efficiencies of a vegetable amaranth
 
Significant decreases in N use efficiencies, involving NRE and AgrNUE, under CF+Nm compared to CF-Nm (Table 3) resulted from N mineralization inhibition. This effect was corroborated by the PCA results (Fig 1). The NRE and AgrNUE existed in the opposite PCA-quadrant to N mineralization inhibition but in the same quadrant with mineral N and net N mineralization rate (Fig 1A). In addition, CF+Nm (Fig 1B) was deposited on a similar quadrant with N mineralization inhibition (Fig 1B). This result was not the case for CF-Nm. The results indicated that CF+Nm decreased NRE and AgrNUE. Nitrification inhibition might stimulate N loss via gases such as NH3 volatilization. Soares et al. (2012) demonstrated increased NH3 volatilization by using dicyandiamide as a nitrification inhibitor in an acidic soil. Loss of N from the soil system in this study was verified, in part, by significantly lower mineral N in CF+Nm than CF-Nm (Table 3).


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This study has demonstrated that neem seed extract could inhibit the nitrogen mineralization only in unamended and chemically fertilized soils and vice versa for the cricket feces amended soils. Nitrogen use efficiencies of amaranth decreased only in the combined uses of neem seed extract with unamended and chemical fertilizer. Assessing the residual content of ingredients relating to the inhibitory effect of the neem extract on N mineralization is necessary to understand better the ineffectiveness of the extract in cricket feces amended soils.

This research was supported by the Research Fund for Researchers from Revenue FY 2020 [Project no. 7/2563] and the Research Career Development Fund FY 2022 [Project no. 7/2565] granted by Sakon Nakhon Rajabhat University, Sakon Nakhon, Thailand.

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