Abstract

Recently, with numerous environmental problems being caused by chemical fertilizer overuse, agricultural practices are shifting toward the development of environmentally friendly N fertilizers. In this study, pot and field experiments were simultaneously conducted to investigate the effect of poly(y-glutamic acid) (γ-PGA) on the yield, N use efficiency, and soil microenvironment of wheat. Our study demonstrates a statistically significant increase in winter wheat, number of tillers, seed number per spike, yield, soil microbial biomass N (SMBN), and soil enzymes after γ-PGA application. The highest grain yield of 7435.69 ± 55.91 kg ha^-1 was obtained after γ-PGA application in the field experiment, which was 7.17% higher than the urea control. The N recovery efficiency increased by 11.81%-14.00% and 11.30%-11.38% after the application of γ-PGA in pot and field experiments, respectively. More mineral nitrogen in soil was immobilized by the microbes after γ-PGA application at the early growth stage of wheat. The immobilized nitrogen was gradually released at the late growth stage. The results demonstrate that γ-PGA can be used as a fertilizer synergist.

Keywords: Poly (γ-glutamic acid), fertilizer synergist, N use efficiency, soil microbial biomass nitrogen, soil enzyme

1. Introduction

Modern agriculture now feeds 6 billion people in the global world. To meet the recent growing demand for food, investments on agriculture are increasing. Consequently, synthetic fertilizer application is also increasing (Thakur et al, 2013). Globally, fertilizer nitrogen (N) application has increased rapidly over the past several decades, from 32 million tons in 1970 to around about 100 million tons in 2010; it is expected to increase to 130-150 million tons/year by 2050 (Matson et al, 1998). However, the overuse of chemical fertilizers in farmlands is a major contributor to environmental problems such as ground water eutrophication, nitrate pollution, phosphate pollution, and others (Datta et al., 2010; Mahmoud and Abd EL-Kader, 2012). Due to increasing human health concerns and pressure in meeting stringent consumer standards, agricultural practices are shifting toward the development of nitrogen (N) fertilizers that are environmentally friendly, possess high N utilization efficiency, and can sustain crop production. Several strategies have been proposed to reduce N losses, such as the use of urease and nitrification inhibitors or coated fertilizers (Bremner, 1995; Han et al, 2008; Khalil et al., 2009). However, all these fertilizer technologies have disadvantages. For example, coated fertilizer is too expensive for wide agricultural use. Several nitrification inhibitors are also not easily available and likely to be toxic to animals and other microorganisms (Iizumi et al., 1998). Therefore, an environmentally friendly fertilizer synergist is required.

Poly (γ-glutamic acid) (γ-PGA) is a water-soluble, biodegradable biopolymer produced by microbial fermentation that consists of D- and L-glutamic acid monomers connected by amide linkages between á-amino and γ-carboxyl groups. γ-PGA can be used in a broad range of industrial fields such as food, medicine, cosmetics, water treatment, and agriculture (Shih and Van, 2001; Ashiuchi, 2010). Given the non-toxicity, high biodegradability, and environment friendliness of γ -PGA, it is attracting interest for potential application as an environment-friendly fertilizer synergist like thermal polyaspartate (TPA) and other polyamino acids (Koskan et al., 1998; Chen et al, 2005; King et al., 2011). Winter wheat (I aestivum L.) is one of the most important food and feed grain crops in the world. However, wheat production requires high levels of chemical fertilizer, which often leads to nutritional imbalance in soil and declined crop productivity. Soil microbes are the living part of soil organic matter that play critical roles in soil N cycling as well as overall soil quality and fertility (Doran, 1987; Tu et al, 2006; Goulding, 2007; Ulrich et al, 2010). They serve as both source and sink of plant nutrients (Dalal, 1998; Yang et al., 2013). Previous studies have shown that polyaspartate can enhance plant productivity and the efficient utilization of plant growth nutrients. Wang et al. (Wang et al., 2008) found that γ-PGA can significantly increase the dry weight of both roots and shoots and the roots to shoots ratio (R/S) of the cucumber seedlings. To date, there is no report about the effects of γ-PGA on soil microbes and soil enzymes. Therefore, the relationship between N transformation and crop productivity after fertilizer synergist addition must be determined. In the present study, the effects of γ -PGA on wheat productivity and soil microbes were evaluated.

2. Materials and Methods

2.1. Pot experiment

A pot experiment was conducted in a greenhouse in the Institute of Agricultural Resource and Environmental Sciences at Jiangsu Academy of Agricultural Sciences (119°13'21" E, 31° 44'03" N), Nanjing, China. The experiment lasted from November 24, 2010 to May 15, 2011. The greenhouse utilized natural light. The soil was air-dried, ground and sieved through a 2 mm sieve. The characteristics of the experimental soil were as follows: pH 6.27 (1:5 soil-water ratio), organic matter of 13.70 g kg^-1 soil, alkaline hydrolysis N of 104.72 mg kg^-1 soil, Olsen-P of22.30 mg kg^-1 soil, and available K of 97.47 mg kg^-1 soil.

2.2. Experimental design of pot experiment

The pot experiment consisted of six different treatments involving γ-PGA. The fertilizer treatments were as follows: no urea control (control, na), urea (nb), PGAU1 (nc), PGAU2 (nd), PGAU1 with a reduction of 15% nitrogen content, and PGAU2 with a reduction of 15% nitrogen content (nf). PGAU1 was urea mixed with purified γ-PGA. PGAU2 was urea coated with purified γ-PGA. The addition level of γ-PGA in PGAU1 and PGAU2 were both 0.10%. Turn on the blower power of the coating machine and then adjust the heating power to 400 W. The coating agent concentration of γ-PGA is 20 g/L and the rotate speed of coating machine is 30 r min^-1. 2 kg of urea was coated with 100 ml of γ-PGA by BY-400 coating machine. After coating the coated urea was stored in a desiccator for use. Seed were sown in s of winter wheat (Triticum aestivum L. cv. Ningmai 13) were sown in plastic pots (21.00 cm diameter ÷ 23.00 cm height) filled with 2.50 kg of sieved air-dried topsoil. Eleven seeds per pot were planted and later thinned to five plants. The experiment was arranged in a completely randomized design with four replicates. All pots were randomized weekly during the experiment. Urea, as the N fertilizer, was added to each pot at the rate of 150.00 mg N per kilogram soil before sowing stage. Superphosphate and potassium sulfate were applied in each treatment at the rates of 100.00 mg P₂O₅ and 150.00 mg kg^-1 K₂O soil, respectively, before sowing. All chemical regents used were analytical grade. Deionized water was used throughout the experiment.

2.3. Soil, plant sampling and analysis in pot experiment

At the tillering, jointing, booting, and maturity stages, the plants in each pot were harvested at ground level. The total N content of each sample was determined using the Kjeldahl method on a whole wheat flour sample prepared using a disc mill (0.20 mm setting). Total protein concentration was estimated as N ÷ 5.70. Apparent N recovery efficiency was calculated using 'difference method' following Motavalli et al. (Motavalli et al, 1989) as below:

Apparent N recovery (%) = [(N accumulation in treatment - N accumulation in control)/total N applied] x 100%.

Each soil sample was separated into two parts. One part was air dried and sieved through a 2.00 mm sieve for physical and chemical analyses, and the other part was passed through a 2.00 mm sieve and frozen until biochemical analysis. To determine their ammonium N (NH₄+-N) and nitrate N (NO₃^--N) contents, 10.00 g of fresh soil was extracted with 50.00 ml of 2.00 M KCl by shaking for 1 h. Filtered soil extracts were analyzed using a Lachart Flow Injection Analyzer (Henriksen and Selmer-Olsen, 1970; Krom, 1980). Soil microbial biomass N (SMBN) was determined by the chloroform-fumigation-extraction method (Ross, 1992).

2.4. Site description of field experiment

This research was conducted at the Luhe Agricultural Station (118°38״342׳ E, 32°29״118׳ N) of the Jiangsu Academy of Agricultural Sciences, Jiangsu Province, China. The soil type was alluvial. The chemical characteristics of the soil were as follows: organic matter of 16.10 g kg^-1 soil, alkaline hydrolysis N of 151.19 mg kg^-1 soil, Olsen-P of 10.56 mg kg^-1 soil, available K of 126.00 mg kg^-1 soil, and pH 6.68.

2.5. Experimental design of field experiment

Four fertilizer treatments were arranged in a randomized complete block design with four replications. Each plot was 5.00 m ÷ 4.00 m with a buffer zone of 0.50 m between each plot. There were four treatments as follows: no urea control (control, NA), urea (NB), PGAU1 (NC), and reduced 15.00%PGAU1 application (ND). Urea was the N source and broadcasted at the rate of 225.00 kg ha^-1 N. Superphosphate was the P source and broadcasted at the rate of 90.00 kg ha^-1 P₂O₅. Potassium sulfate was the K source and applied a t the rate of 90.00 kg ha^-1 K₂O. PGAU1 was urea mixed with purified γ-PGA. The addition level of γ-PGA in PGAU1 was 0.10%.

2.6. Plant sampling and analysis of field experiment

At physiological maturity, plants within 1 m² of each plot were manually cut at ground level to measure dry matter and total N. Plant samples were digested by the Kjeldahl procedure and analyzed for total N by routine methods (Chapman and Pratt, 1961).At maturity, the grain yield and yield components, including the number of effective tiller, plant height, seeds per spike, and 1000-seed weight, were determined.

2.7. Statistical analysis

ANOVA was used to test the difference in each parameter among different treatments. Statistical analysis was performed by one-way ANOVA followed by Duncan's test (p < 0.05). Statistical analyses of all data were conducted using SPSS17.0.

3. Results and Discussion

3.1. Spike number, spike dry weight, 1000-seed weight, and grain yield

In the pot experiment, γ-PGA application at maturity in all treatments resulted in increased spike number per pot, 1000-seed weight, seed number per spike, and total seed weight per pot (Table 1). The seed number per spike and total seed weight per pot significantly increased with the application of γ-PGA. The highest crop yield of PGAU2-treated plants significantly increased by 11.15%, i.e., from 8.79 ± 0.61 g pot^-1 in the urea treatment to 9.77 ± 0.41 g pot^-1 in the PGAU2 treatment. With 15.00% decreased nitrogen fertilizer application, the grain yield increased by 1.82%-2.05% after γ-PGA application compared with treatment with urea only.

Table 1. Evaluations of wheat growth under the various treatments in the pot experiment

NA, no urea control; NB, urea; NC, PGAU1; ND, reduced 15.00% PGAU1 application. Values are means ± SD (n=4). The statistical analysis was performed by Duncan's test (at level p<0.05). Means in the same column and with different letters are statistically different.

In the field experiment, similar results were observed after γ-PGA application (Table 2). Compared with treatment NB, plants treated ־with γ -PGA in all treatments showed a significant increase in the spike number, seed number per spike, and yield of winter wheat. PGAU1 significantly increased the spike number, seed number per spike, and yields of wheat by 2.60%, 4.20%, and 7.17%, respectively, compared with those in treatment NB. With 15.00% decreased PGAU1 application, the crop yield increased by 0.51% compared with that in treatment NB.

Table 2. Evaluations of wheat growth under the various treatments in the field experiment

NA, no urea control; NB, urea; NC, PGAU1; ND, reduced 15.00% PGAU1 application. Values are means ± SD (n=4). The statistical analysis was performed by Duncan’s test (at level p<0.05). Means in the same column and with different letters are statistically different.

Tables 1 and 2 showed that seed yields of wheat were improved significantly after application of γ-PGA when nitrogen contents were at a normal level. After the nitrogen contents were decreased by 15%, seed yields were no significantly differences compared with

3.2. Total N accumulation and N recovery efficiency

The pot experiment results presented in Table 3 indicate that dry matter production, grain concentration of N, and total N accumulation were significantly affected by γ-PGA in all treatments. The straw production, grain production, and grain N concentration significantly from increased 9.38%-9.58%, 11.07%-11.53%, and 5.35%-6.79%, respectively, after γ-PGA application under the same level of N treatments. The application of γ-PGA in all treatments increased the N recovery efficiency by 11.81%-14.00% compared with that treatment nb. The N recovery efficiency was also obviously higher with 127.50 mg poH N than 150.00 mg pot^-1 N after γ-PGA application.

Table 3. Effects of γ-PGA on nitrogen use efficiency of wheat in the pot experiment

na, no urea control; nb, urea; nc, PGAU1; nd, PGAU2; ne, reduced 15.00% PGAU1 application; nf, reduced 15.00% PGAU2 application. Values are means ± SD (n=4). The statistical analysis was performed by Duncan's test (at level p<0.05). Means in the same column and with different letters are statistically different. TNA: total N accumulation; NRE: N recovery efficiency.

In the field experiment, similar trends were observed after γ-PGA application (Table 4). Compared with treatment NB, plants treated with γ-PGA showed a significantly increase in dry matter production, concentration of N, and total N accumulation. PGAU1 significantly increased the straw production, grain production, and grain N concentration by 2.50%, 7.17%, and 7.14%, respectively, compared with treatment NB. The application of γ-PGA in all treatments increased the N recovery efficiency by 11.30%-11.38% compared with that in treatment NB. In general, γ-PGA application leads to significantly increased dry matter production and N concentration of wheat crop. Tables 3 and 4 show that the N use efficiency can be significantly increased by γ-PGA application. Similarly, N use efficiency was obviously higher in low N condition than in high N condition after γ-PGA application in both pot and field experiments. Therefore, γ-PGA can improve the growth of wheat and increase nutrient utilization, especially

3.3. Total protein and starch

γ-PGA application in all treatments resulted in obviously increased total protein concentration and starch. The total protein of wheat grain significantly increased from 3.88% to 6.75% with the application of γ-PGA compared with treatment nb in the pot experiment (Figure 1(a)). The total protein in plants treated with γ-PGA in the field experiment increased from 4.99%-7.14% compared with treatment NB (Figure 1(b)). The starch content of wheat grain increased from 0.11%-1.95% after γ-PGA application compared with treatment nb in the pot experiment (Figure 1(c)). Starch treated with PGAU1 in the field experiment increased by 0.28% compared with treatment NB (Figure 1(d)).