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Application of amino acids improves lettuce crop uniformity and inhibits nitrate accumulation induced by the supplemental inorganic nitrogen fertilization.

Byline: Pavlos Tsouvaltzis Athanasios Koukounaras and Anastasios S. Siomos

Abstract

Lettuce plants (cv. Simpson') were grown for 10 weeks during winter under greenhouse to investigate the possibility of replacing the supplemental nitrogen (N) fertilization by using Amino16(R). Amino16(R) is a hydrolyzed protein solution containing 11.3% L-amino acids (alanine arginine aspartic acid glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tyrosine and valine) 4% total N and 25% organic matter produced of plant origin raw materials. Three weeks after transplanting supplemental fertilization with ammonium nitrate or Amino16(R) was applied. Ammonium nitrate solution was applied to the soil while Amino16(R)either to the soil or foliage. Two and 4 weeks later the foliar application of Amino16(R) was repeated. Plants without any supplemental fertilization were used as control. The results indicated that crop uniformity was substantially increased when plants received supplemental fertilization with ammonium nitrate and even more when Amino16(R) was applied to soil. Nutritional quality of lettuce plants in terms of dry matter total soluble solid ascorbic acid and total phenol contents was not affected by supplemental fertilization either with ammonium nitrate or Amino16(R). On the other hand the application of Amino16(R) prevented the accumulation of nitrates observed in plants fertilized with ammonium nitrate. Moreover the foliar application of Amino16(R) resulted in significant increase of antioxidant capacity in comparison to the control. According to the above the use of Amino16(R) as alternative of inorganic supplemental fertilization is of high importance in order to improve lettuce crop uniformity minimize nitrate content in plants without negative effect on other nutritional components or yield. Copyright 2014 Friends Science Publishers

Keywords: Amino16(R); Antioxidant; Fertilization; Foliar; Nutritional quality

IntroductionNitrogen is the most essential mineral nutrient that promotes sufficient plant growth and consequently yield. It isabsorbed by roots either as ammonium (NH4 ) or nitrate(NO3 ) ion and incorporated in amino acids and eventuallyproteins (Blom-Zandstra 1989).Frequently farmers use excessive rates of nitrogen invegetables to avoid N-deficiency (Porto et al. 2008)ignoring environmental pollution increase in production cost as well produce quality deterioration problems (Blom-Zandstra 1989; Wang et al. 2008; Montemurro2010). For example Wang et al. (2008) reported that over-application of N decreased vitamin C soluble sugar Mgand Ca concentrations as well as soluble solids in tomato fruit while increasing titratable acidity and the acid:sugar ratio lead to decrease in commercial and nutritionalquality. Excessive nitrogen supplement may pollute ground water (Gonzalez et al. 2010). Additionally abundant NO - availability leads to excessive absorption by the roots inlarger quantities converted into NH4 via nitrate reductase (Blom-Zandstra 1989; Wang et al. 2008) resulting to NO3 accumulation in the vacuoles of the cells. This frequently takes place in lettuce plants to continue N uptake until harvest (Salomez and Hofman2009) and accumulate excessive concentrations of NO - intheir tissues. This is regarded as negative qualitativeaspect from a nutritional standpoint because NO3

intake by humans has been associated with various health hazards (Blom-Zandstra 1989). Accumulation of NO3 in field grown lettuce dependson nitrogen supply in terms of fertilizer form used anddoses applied (Porto et al. 2008). Manipulation of nitrogen fertilization in terms of rates of application and source type appears as the most applicable means toreduce NO3accumulation in plants (Wang et al. 2008).However caution should be taken in order to ensurethat a critical minimum level of mineral nitrogen for sustaining plant growth and crop yield is not surpassedand additionally mineral nitrogen manipulation doesn'tinduce nutritional quality deterioration (Salomez andHofman 2009; Stefanelli et al. 2012).Recent research has shown that the use of organicfertilizers in field grown lettuce can be considered asvalid and useful alternative source of nitrogen to themineral ones (Porto et al. 2008; Montemurro 2010) but information regarding the application of amino acids another source of organic nitrogen is scarce. El-Naggar et al. (2008) reported that glycine solution sprayed on lettuce plants and soil served as an effective fertilization strategy as inorganic N fertilizers but the individual effect of foliar or soil application is not elucidated neither any other study regarding the application of a mixed amino acids solution on soil of field grown lettuce exist. Although in hydroponic systems when amino acids were used as alternative for part of NO - supply plant growth and crop yield of lettuce (Gunes et al. 1994) were promoted similar to 100% mineral nitrate fertilization it is unknown whether application of amino acids solution is also effective when applied to the soil. Indeed according to Owen and Jones (2001) organic N applied in soil as amino acids solutions may not be as effective as inorganic fertilizers due to the slow movement of amino acids in soil the rapid turnover of amino acids by soil microorganisms and the poor competitive ability of plant roots to capture amino acids from the soil solution.Apart from the root supply amino acids can be successfully applied on plant foliage as foliar sprays. The use of foliar nutrient sprays in agriculture is increasinglywidespread since they are potentially more environmentfriendly and target-oriented as compared to root treatments (Fernandez and Eichert 2009; Gonzalez et al. 2010; Yasmeen et al. 2013). Foliar application of mixed amino acids solution increased fresh and dry weight of shoots in field grown radish plants while reducing nitrate content (Liu et al. 2008a b). Additionally foliar application of mixture of amino acids and seaweed extract at different growth stages had a positive effect on vegetative growth reproductive growth and berry quality of grapevines (Khan et al. 2012). However in order to be used in vegetables these should be tested carefully in various crops and cultivars.Amino16(R) is a hydrolyzed protein solution containing11.3% L-amino acids (alanine arginine aspartic acid glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tyrosine and valine) 4% total N and 25% organic matter (according to the manufacturer).Demand for environment friendly organic materials of plant origin suitably combined with inorganic fertilizers is increasing in order to preserve soil health crop yield and improved nutrient use efficiency (Rahman et al. 2013) the aim of this study is to investigate the possibility of replacing the supplemental mineral nitrogen fertilization on field grown lettuce by using an organic substitute like Amino16(R).

Materials and Methods

Plant Material and Fertilization Treatments

Lettuce plants (cv. Simpson') were grown in greenhouse condition at Aristotle University of Thessaloniki Greece from November 2010 to January 2011. Pre-plantincorporated application of synthetic fertilizer (12% N 12% P2O5 17% K2O) was made at 1000 kg/ha. Plants were transplanted at 2-3 true leaf stage at density of 12 plants m-2.During the growing period the plants were drip-irrigated and weeds were pulled by hands as required. Three weeks after transplanting supplemental fertilization with ammonium nitrate or Amino16(R) was applied. Amino16(R) is patented (EP2537823 A1) product of EVYP (Greek Industry of Hydrolyzed Protein L.L.P) produced of plant origin raw materials. Ammonium nitrate solution (1.2% w/v) was applied to the soil (100 mL plant-1) while Amino16(R) was applied either to the soil or the foliage (100 ml plant-1 of0.3% v/v or 6.25 mL plant-1 of 0.2% v/v respectively). Two and 4 weeks later the foliar application of Amino16(R) wasrepeated (12.5 mL plant-1 0.2% v/v). Plants without any supplemental fertilization were used as control.A randomised complete block design was usedcomprised from 4 blocks per treatment each block consisted of three rows with 14 plants in each row. The plants in the outside rows and the end plants of the remaining row were guards. The plants were hand-harvested at once 10 weeks after transplanting and fresh weight of each plant was taken. Plants were graded according to the following weighing scale: 50-99 100-149 150-199 200-249 250-299 300-349 and greater than 350 g. Two plants from each block were collected and stored in freezer at -20oC for the determination of the nutritional quality.

Nutritional Quality

Frozen samples were thawed and macerated in a Waring blender and the blended material was used for the determination of dry matter total soluble solid nitrate ascorbic acid and total soluble phenol content as well antioxidant capacity.The dry matter was determined after drying the pulp at70oC for 72 h. The total soluble solids were measured in the juice of the pulp using a portable digital refractometer (AtagoCo. Ltd. Tokyo model PR-1). The nitrates were determined according to Cataldo et al. (1975). For the extraction ofascorbic acid 20 g of the pulp was homogenized with 50 mL1% oxalic acid solution and filtered through Whatman No 1. The ascorbic acid was measured in the filtrate using RQflex reflectrometer (Merck Darmstadt Germany). Total soluble phenols were determined according to Scalbert et al. (1989) and expressed as mg gallic acid equivalents (GAE) kg-1 FW and antioxidant capacity according to Brand-Williams et al. (1995) and expressed as mg ascorbic acid equivalents (AEAC) kg-1 FW.

Statistical Analysis

Analysis of variance (ANOVA) was performed in MSTAT and mean separation was done with Duncan's multiple range test (Pless than 0.05). Percentages were statistically processed using arcsin square root transformation and actual percentage values are given in the figures.

Results

Plant Fresh Weight

The fresh weight of harvested plants was not affected by supplemental fertilization (Table 1). The plant fresh weight was not significantly different between the treatments indicating that pre-plant fertilization was sufficient to sustain plant growth.However the distribution of plant fresh weight in the various classes was affected by the treatments (Fig. 1). Particularly the percentage of 7 weight classes in controltreatment did not differ among each other (Fig. 1a) isindicative of low crop uniformity which is an undesirable trait for the market. On the other hand inorganic supplemental fertilization resulted in better crop uniformity compared to the control as long as 38% of plants were weighing 150-199 g although not significantly different than classes 200-249 g and 250-299 g with 20 and 18% of plants respectively (Fig. 1b). Ten percent of harvested plants were weighing 100-149 g and 15% were weighinggreater than 300 g. The application of Amino16(R) on the foliage was even more effective in improving crop uniformity as longas 44% and 28% of plants weighing 150-199 g and 200-249g respectively followed by 100-149 g (15%) significantly higher than classes 50-99 250-299 300-349 and greater than 350 g with 2% of plants each (Fig. 1c). However the best crop uniformity was obtained when Amino16(R) was applied to soil compared to all other treatments (Fig. 1d) as long as plant weight was grouped in 3 distinct weight classes. Particularly 55% of the harvested plants were weighed 200-249 g 38% 100-199 g and 5% 250-349 g without any harvested plant weighing less than 100 or greater than 350 g (Fig. 1d). In- between the 7 weight classes significant differences were only observed in 200-249 g class where the percentage of plants that received Amino16(R) to the soil was significantly higher (55%) than all other treatments (24-28%).Although plant fresh weight and consequently crop yield were not affected by supplemental fertilization (Table1) crop uniformity was substantially increased when plants received supplemental fertilization with ammonium nitrate and even more when Amino16(R) was applied and especially to the soil (Fig. 1).The application of Amino16(R) to the foliage also resulted in better crop uniformity compared either to the control or inorganically-fertilized plants but lower than itssoil application.

Nutritional Quality

Dry matter total soluble solid ascorbic acid and total phenol contents were not affected by supplemental fertilization (Table 1).The antioxidant capacity of control plants was 1.88 ascorbic acid equivalents (AEAC) 100 g-1 FW not significantly different than plants treated with inorganic fertilization or Amino16(R) applied to the soil (Fig. 2a). On theother hand the application of Amino16(R) to the foliage resulted in significant increase of antioxidant capacity (3.15 mg AEAC 100 g-1 FW) in comparison to the control.

The nitrate content of control plants was 202 mg kg-1FW not significantly different from plants receiving both Amino16(R) treatments (186-189 mg kg-1 FW) (Fig. 2b). Contrarily the inorganic fertilization resulted in significantly increased nitrate content reaching 261 mg kg-1 FW.

CorrelationsNo significant correlations between fresh weight andnutritional quality characteristics were observed (Table 2). Contrarily dry matter was correlated with soluble solids (r=0.54 P=0.031) and total soluble phenols (r=0.57 P=0.020) as well as soluble solids with nitrates (r=-0.71 P= 0.002).

Discussion

The application of amino acids did not affect lettuce fresh weight in our study (Table 1) in contrast to Liu et al. (2008a) the application of mixed amino acids solution on field grown radish resulted in increased fresh weight when plants were treated frequently. Similar to our results the replacement of 20% of nitrate in nutrient solutions with mixed amino acids in soilless culture had no effect on fresh weight of lettuce or onion (Gunes et al. 1994 1996). Likely fresh weight of pak-choi (Brassica chinensis L.) was either increased or reduced depending on the individual amino acids source (Wang et al. 2007).However the distribution of lettuce fresh weight in the various classes was affected by the treatments (Fig. 1) and indeed the application of amino acids improved lettuce crop uniformity especially when Amino16(R) was applied to soil. Lettuce crop uniformity is of high importance in the produce market and is therefore included as a critical marketing standard in the European Commission Regulation 543/2011 (2011).Although N concentration in ammonium nitrate or Amino16(R) solution was equal the above finding probably implies that amino acids were absorbed by the roots of plants more favourably than ammonium nitrate either due to higher affinity with the roots or due to higher adsorption of amino acids on soil particles. The form of N uptake is mainly determined by its abundance accessibility and sets of finely tuned transporters allow the plant to precisely adapt N uptake in response to plant demand and external N supply (Von WirACopyrightn et al. 1997). It is well documented that the nitrate adsorption on soil particles is low; leading tonitrate loss through leaching as well as assimilation of absorbed inorganic N into amino acids is an energy- requiring process (Von WirACopyrightn et al. 1997). The use ofmixed amino acids solutions as alternative part of nitratefertilization has successfully sustained lettuce plant growth and yield in soilless growing systems (Gunes et al. 1994) indicating the efficient absorption of amino acids by plants' roots. This is the first report though exhibiting the beneficial effect of amino acids application on field grown lettuce crop uniformity.The reduced effectiveness of foliar application of Amino16(R) in comparison to soil application is probably due to the lower amino acids concentration in the plant sap resulting either from a low penetration rate through the foliar cuticle (Fernandez and Eichert 2009) or from the low solution dispersion on the internal shielded leaves. Additionally the total N amount applied through the foliar sprays of amino acids solution was 5-times lower than soilapplications of ammonium nitrate or Amino16solutionswhich may explain the present study findings. Indeedaccording to Fernandez and Eichert (2009) given the low penetration rates of foliar nutrient solutions the concentration range applied should be higher as compared to root treatments. Foliar application of amino acids has been shown to increase fresh weight of field grown radish shoots which is a short-cycle crop only when application was done intensively i.e. 4 times in a period of 10 days (Liu et al. 2008a). Such practice though cannot be adopted in vegetable crops with a longer cycle such as lettuce due to the significant increase of production cost. Therefore the foliar application of amino acids can be properly timed and thus; limited to specific stages of plant growth when nutrient demand is likely to be high or when soil conditions are known to restrict nutrient uptake (Gonzalez et al. 2010).The use of mixed amino acids solutions as alternative of part of nitrate fertilization in NFT (nutrient film technique) grown lettuce plants did not affect dry matter content (Gunes et al. 1994) similar to field grown lettuce plants in our study. However dry weight of Pak-choi has been shown to be negatively affected when part of nitrate fertilization was replaced with amino acids in the nutrient solution in soilless culture but this effect was amino acid type-dependent (Wang et al. 2007). Furthermore our results corresponds with Stefanelli et al. (2012) who concluded that reduced nitrate N fertilization can be applied in soilless grown lettuce without affecting total phenolics and ascorbic acid content although N fertilization rates and form have been shown to significantly affect ascorbic acid content in vegetables (Wang et al. 2008).Interestingly antioxidant capacity was correlated with dry matter (r= 0.61 P= 0.013) and soluble solids (r=0.63 P= 0.009) but not with total soluble phenols contrary to other reports (Llorach et al. 2004; Oh et al. 2009; Boo et al. 2011) neither with ascorbic acid with the latter being in consistency with previous results in iceberg or Chinese lettuce (Szeto et al. 2002).The effect of amino acid application on NO - reduction found in this study has also been demonstrated in field grown Pak-choi (Wang et al. 2007) radish (Liu et al.2008a b) as well as lettuce (Gunes et al. 1994) and onion(Gunes et al. 1996) grown in NFT. The interpretation of this effect is contradictory among researchers with other stating that amino acids are preferably absorbed by plants as reduced N source (Gunes et al. 1994 1996) while others claim that the main role of amino acids on nitrate uptake and assimilation is the regulation of many processes and metabolic pathways of plant N metabolism such as nitrate and nitrite reductase and glutamine synthetase activities (Liu et al. 2008a b) or even the inhibition of the expression of HvNRT2 transcript in roots which induces the synthesis of the mRNA encoding nitrate transporter that is directly related to nitrate uptake (Vidmar 2000). Alternatively Aslam et al. (2001) suggested that induction of the NO - transporter in the presence of amino acids may be normal but the turnover of the mRNA encoding the transporter may be increased.Although the nitrate content of plants receiving inorganic fertilization was much lower than maximum permissible levels established by European Commission Regulation 466/2001 (2001) for lettuce produced in protected areas (4500 mg/kg FW) the use of amino acids as alternative of mineral supplemental fertilization is of high importance in order to minimize nitrate content in consumed vegetables. Indeed European Commission Regulation 1881/2006 (2006) implies that presence of contaminants must be reduced more thoroughly wherever possible by means of good agricultural practices in order to achieve a higher level of health protection especially for sensitive groups of the population.In conclusion crop uniformity was substantially increased when plants received supplemental fertilization with ammonium nitrate and even more when Amino16(R) was applied especially to the soil. Moreover the use of Amino16(R) as alternative of inorganic supplemental fertilization is of high importance in order to minimize nitrate content in lettuce without negative effect on other nutritional components or yield.

References

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Author:Tsouvaltzis, Pavlos; Koukounaras, Athanasios; Siomos, Anastasios S.
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Oct 31, 2014
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