Effect of Fertilization with N-Inhibitors on Root and Crop Development of Flaxseed Crop (Linum usitatissimum L.)

INTRODUCTION

Agricultural fertilization practice is one of the most important for crop production, especially nitrogen ferilizers (Scharf et al., 2005). That is why huge amounts of energy are being spent on the production of inorganic and organic fertilizers (Hoeppner, Entz, McConkey, Zentner, & Nagy, 2006). It is therefore judged to be as large as possible (fewer losses) and their sustainable utilization. The addition of N fertilizer increases oil crop seed yield and oil content in almost all olive oil crops (Xie et al., 2015). One of these crops is flax (Linum usitatissimum L.), which is grown for industrial use (oilseed flax/ linseed oil), for human consumption (flaxseed oil) and natural fiber production. The flax oil and seeds are rich in ω3 fatty acids and lignans (Dubey, Bhargava, Fuentes, Shukla, & Srivastava, 2020; Singh, Mridula, Rehal, & Barnwal, 2011;

Tonon, Grosso, & Hubinger, 2011).

The fiber and seed productions are increased with the application of N fertilizer (Easson & Long, 1992; Xie et al., 2015). The absorption of applied N depends directly on the root architecture. Regarding flax cultivation, it has been found that the addition of P increases root size (Thingstrup, Rubaek, Sibbesen, & Jakobsen, 1998). The root system of corn hybrids is not affected by N fertilizer (Mackay

& Barber, 1986) while the root system of winter wheat and rice was increased with applied N rise (Barraclough, Kuhlmann, & Weir, 1989; Etesami &

Alikhani, 2016).

Arbuscular mycorrhizal (AM) symbiosis is the parasitism of the root with the fungus. The presence of mycorrhiza in host plants improves the absorption of water and nutrients and therefore increases plant growth and biomass. However, the presence of AM fungi in poor soil is of great importance, where it uses the plant as many nutrients as possible for the sake of the plant from the poor soil. For example, ARTICLE INFO

Keywords:

Flaxseed Inhibitor

Nitrogen Indicator RootUrea

Article History:

Received: April 13, 2020 Accepted: August 18, 2020

*⁾ Corresponding author:

E-mail: i.kakabouki@gmail.com/

i.kakabouki@aua.gr

ABSTRACT

Flax is a crop whose products can be used in a variety of ways such as industrial use, human consumption, and fiber production. Nitrogen appears to have a positive effect on flaxseed growth and production.

In an experiment conducted in two consecutive years in Greece, it has been studied how three combinations of urea fertilization affect flaxseed cultivation (cv. ‘Everest’). More specifically, the experimental treatments with urea fertilizers were represented as follows: only urea; urea with urease inhibitor; and urea with urease inhibitor and nitrates inhibitors.

The value of root mass ranged from 0.780 to 1.182 mg/cm³ in the first year and from 0.872 to 1.267 mg/cm³ in the second year. The maximum value of plant height was 69.25 in urea with double inhibitors. Leaf Area Index (LAI) was double in fertilized plots compare to unfertilized. Oil content was significantly affected by year and noticed mostly 1% among treatments. Oil yield was affected by fertilizers and the maximum value was 513.96 kg/ha. A positive significant correlation coefficient was observed between oil yield and LAI (r=0.8699). A positive correlation was noticed between seed yield and NUE (r=0.6881). The most beneficial effects were mentioned under urea with UI and NI inhibitors.

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Cite this as: Kakabouki, I. P., Karydogianni, S., Zisi, C., & Folina, A. (2020). Effect of fertilization with N-inhibitors on root and crop development of flaxseed crop (Linum usitatissimum L.). AGRIVITA Journal of Agricultural Science, 42(3), 411–424. https://doi.org/10.17503/agrivita.v42i3.2650

Effect of Fertilization with N-Inhibitors on Root and Crop Development of Flaxseed Crop (Linum usitatissimum L.)

Ioanna P. Kakabouki^*), Stella Karydogianni, Charikleia Zisi and Antigolena Folina Department of Crop Science, Agricultural University of Athens, Greece

in chickpea cultivation, there was an increase in all nutrients with a simultaneous increase in biomass (Farzaneh, Vierheilig, Lössll, & Kaull, 2011). According to the literature, the element of P elevated with the presence of mycorrhiza (Smith

& Read, 2008). In flax cultivation, Arbuscular mycorrhizal fungi (AMF) colonization increased with decreasing P in soil (Thingstrup, Rubaek, Sibbesen,

& Jakobsen, 1998).

The absorption and utilization of N by the mycorrhiza depend on the rate of applied N and N in soil (Azcón, Rodríguez, Amora-Lazcano, &

Ambrosano, 2008). Most nitrate and nitrogen fertilizers are available with water and the ability to absorb nutrients is affected by the availability of water. AMF helps increase nutrient absorption even in adverse environmental conditions such as drought (Sánchez-Romera, Porcel, Ruiz-Lozano, &

Aroca, 2018). Some researchers are also studying the genetic modification of the fungus to increase the absorption of N from the root (Vázquez, Barea,

& Azcón, 2001) because the ability to absorb nutrients depends on the growth and maintenance of their hyphal networks. Maintaining this network is important because agricultural practices such as tillage negatively affect it (Bilalis & Karamanos, 2010) while cover crops increase the hyphal network (Boswell, Koide, Shumway, & Addy, 1998).

Decreased tillage creates the need to increase applied N so that there is no reduction in yields (Bilalis et al., 2012). Also, in flax cultivation, it was found that the colonization of mycorrhiza is not reduced with conventional tillage (Bilalis et al., 2010).

Uncontrolled use of fertilizers over the years has created pollution problems in surface and groundwater. Specifically, Adesemoye, Torbert,

& Kloepper (2008) report that nitrate leaching in groundwater is done by N fertilization. In fact, proof of this is the fact that in recent years some areas in Greece have joined denitrification programs to reduce water pollution. Some practices applied in the program are the cultivation of legumes to increase N₂ from biological fixation. Also, the reduction of N in the soil can be done application of inoculants, such as AMF and plant growth-promoting rhizobacteria (PGPR) (Adesemoye, Torbert, & Kloepper, 2008).

Fertilizers with inhibitors have been developed in the context of friendly environmental practices.

The timing of fertilizer application affects crop yields (Constable, Rochester, & Daniells, 1992).

However, recovery of applied N is not sufficient to reduce nitrate pollution (Freney et al., 1993).

Inhibitors used in cotton cultivation improved N absorption (Freney et al., 1993). The action of nitrite inhibitors is due to the retention of N in the form of ammonium which remains immobilized through soil microorganisms (Hauck, 1990). In canola, while high applied N values reduce seed quality, urea inhibitor fertilization increases yields and growth quality (Grant, Derksen, McLaren, & Irvine, 2011).

Oilseed rape bacterial activity did not decrease with the addition of nitrification inhibitor fertilizer (Li et al., 2008).

Since the application of N fertilizers is considered a given, it is important to check the proportion of this fertilizer was exploited-absorbed the plant and how much that remains in the soil that can cause environmental problems. One of the key pillars for the design of sustainable agricultural systems is the most efficient use of N efficiency with as few inputs and losses as possible (Spiertz, 2010).

That is why nitrogen indicators such as Nitrogen Use Efficiency (NUE) have been developed more recently. Also important is the transfer of N to the yield factors of each crop. In the cultivation of flax, that is, how much of the applied N was saved in the seeds compared to seed yield, or nitrogen total uptake on up-ground plant compared to total biomass.

Most studies on flaxseed cultivation have focused on P absorption. There is a gap in the effect of N fertilization on this crop and its effect on AM fungi. There is also a gap for the use of flax for human consumption, flaxseed, and not oilseed intended for industrial use. Our overall hypothesis was that inhibitors in fertilizers with inhibitors would boost the function of microbial inoculants AMF and increase plant growth and N absorption. In this experiment, we investigated the growth of the plant- agronomic traits, oil yield, N uptake, and indices of NUE, NHI, NAE, effect of uptake and effect of absorption concerning urea, and combination of urea with nitrification and urease inhibitors.

MATERIALS AND METHODS

In an experiment conducted in two consecutive years in Western Greece in Agrinio (38^o33’ N, 21^o24’ E), it has been studied how three combinations of urea fertilizations affect flaxseed cultivation (Linum usitatissimum L. cv. ‘Everest’).

They took place in 2018-2019. The soil was sand clay loam, with pH (1:1 H₂O) of 7.22 and organic matter containment was 2.25%. The experimental area was in total of 640 m². The field experiment was set up in Randomized Complete Block Design (RCBD), with four treatments (Control, Urea, Urea + Urease inhibitor (UI) and Urea + UI + nitrogen inhibitor (NI)) and therefore each plot was 40 m². The urease inhibitor was N-(n-butyl) thiophosphoric triamide (NBPT) and the nitrification inhibitor was dicyandiamide (DCD). The sown was took place for the first experiment on 20 March 2018 and for the

second one 2 April 2019. There were added 60 gr seeds. There were used 300 seeds per m² and they were sown by hand at a depth of 1 cm. The field area was irrigated three times with total amount of water 250 mm. A sprinkler irrigation system was set up on plots.

This practice is one of the basic weed control methods of Organic Agriculture (CEU, 2007). The meteorological data on the air temperature and the precipitation of the experimental space during the experimental periods are depicted in Fig. 1.

Fig. 1. Meteorological data (mean monthly temperature (°C) and precipitation (mm)) for the experimental site during the experimental periods (March-July, 2018-2019)

Root measurements were carried out at 60 days after sowing (DAS), at the flowering period.

Each root sample was taken at the middle point between successive plants within a row from a depth about 0–30 cm using a cylindrical auger 25 cm in length and 10 cm in diameter. Five plants were selected in two different places per plot. For each sample, the roots separated from the soil after the stay in water + (NaPO₃)₆ + Na₂CO₃ for 24 hours.

Roots were separated from the soil by wetting the samples during the night in 30 ml of a 0.5% solution of sodium hexametaphosphate. Then, the root samples were stirred for 5 minutes and washed over a 5 mm mesh-sieve. The roots thus were held on the sieves shed into a 0.1% trypan blue FAA staining solution (a mixture of 10% formalin, 50%

ethanol, and 5% acetic acid solutions). The stained root samples were placed on a high-resolution scanner (Hewlett Packard 4c, Palo Alto, CA, USA) and the images captured using Delta–T software (Delta–T Scan version 2.04; Delta–T Devices Ltd, Burwell, Cambridge, UK), to determinate the root length density (RLD). The other root samples were washed and stained with trypan blue in lactophenol (Phillips & Hayman, 1970). The percentage of root length colonized by AM fungi was determined microscopically with the gridline-intersection method at a magnification of × 30 to × 40 (Giovannetti &

Mosse, 1980). AMF was determined in five different sowing periods (20, 40, 60, 80, and 90).

Leaf Area Index (LAI) was measured using an automatic leaf area meter (Delta-T Devices Ltd). Plants height was measured at 80 DAS. The harvest was done by hand, for the first experiment on 10 July 2018 and for the second one on 15 July 2019. Dry matter, the weight of 1000 seeds, and seed yield were also observed.

The oil content was observed with a cold hydraulic press machine. These equipments were used to press the flaxseed samples in a single pass.

Samples were fed from the hopper to the press on demand by gravity (Gros, Lanoisellé, & Vorobiev, 2003). One kg seeds used one day after harvest.

The oil yield was estimated based on the equation 1 (eq. 1).

The measurement of nitrogen included percentage of N in stover and seeds, the total N in stover (eq. 2), and seeds (eq. 3), and the total

N uptake (eq. 4). The N percentage was measured by the Kjeldahl method (Bremner, 1960) using a Buchi 316 device. As well the samples were chosen randomly within each plot and all samples were ground to a fine powder and used for determination of nitrogen.

Moreover, there were calculated some nitrogen indicators to expose our results. Nitrogen Use Efficiency (eq. 5), was calculated base on equation 5.

The Nitrogen Harvest Index is an indicator which is defined as a ratio of the concentration of N in seeds to the total N in the plant (eq. 6). This indicator is positively related to seed yield.

Nitrogen Agronomic Efficiency shows the amount of seed produced per kg of N fertilizer (eq. 7).

Aside from the above parameters, the measurement was also carried out on the effects of nitrogen absorption and effects of uptake. The effects of nitrogen absorption the ratio of nitrogen total uptake to the quantity of N fertilizer (eq. 8) and effects of uptake is the ratio of yield to nitrogen total uptake (eq. 9).

Analysis of variance was performed using the Statistica (Stat Soft, 2011) logistic package as a Completely Randomized Design. The Analysis of

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Variance (ANOVA) used a mixed model, with years and replications as random effects of fertilization as fixed effects. Differences between means were separated using Tukey’s test. Correlation analyses were used to describe the relationships between growth parameters and yield components using Pearson’s correlation. All comparisons were made at the 5% level of significance (p ≤ 0.05).

RESULTS AND DISCUSSION

Table 1 presented the root characteristics of flaxseeds under various nitrogen treatments. The value of root mass ranged from 0.780 to 1.182 mg/

cm³ in the first year and increased from 0.872 to 1.267 mg/cm³ in the second year. The maximum value was 1.267 mg/cm³ in the treatment of urea with double inhibitors in the year B and the lowest was 0.780 mg/cm³ in control in the year A. The values of all treatments had statistically significant difference in both years. Concerning the root surface, the maximum value was 2.960 mm²/cm³ in urea + NU + NI in the second year and the lowest was 1.825 mm²/cm³ in control in the same year. The control had not statistically significant difference with the urea while, the urea with inhibitor urease had statistically significant difference with the urea + UI + NI, in the both years. In the second year, the values were higher than first year (Table 1). The values of root

diameter, ranged from 0.180 to 0.205 mm in the year A and from 0.185 to 0.210 mm in second year. There was negligible big difference in values between the two years. The root diameter under urea treatment showed insignificant difference with the urea + UI and similar phenomenon was observed on root diameter under urea + UI +NI and urea treatments in the both years. Moreover, in the root density the values ranged from 6.275 to 10.375 mm/cm³ in the first year and from 6.810 to 10.515 mm/cm³ in the second year, respectively. Also, the maximum value was 10.515 mm/cm³ in urea with double inhibitors and the lowest was 6.275 mm/cm³ in the second year. In both years, all treatments had statistically significant difference between them (Table 1).

Concerning the plant height, the values ranged from 56 to 68.75 cm, in the first year and increased from 57.75 to 69.25 cm, in the second year (Table 2). The values of plant height under control treatment showed insignificant difference with urea, neither did of the respected parameter under urea + UI + NI and urea in the both years. The maximum value was 69.25 in urea with double inhibitors in second year. On the side, the LAI’s values ranged from 1.823 to 2.293 in year A and from 1.95 to 2.355 in year B. The maximum value was 2.355 in urea + UI +NI in the second year and the lowest was 1.823 in control in first year. All treatments had statistically significant difference between them, in both years.

Table 1. The root characteristics as effected by fertilizer treatments

Fertilizer Root Mass (mg/

cm³) Root Surface

(mm²/cm³) Root diameter

(mm) Root Density

(mm/cm³) Year A

Control 0.780^a 1.825^a 0.180^a 6.275^a

Urea 0.895^b 1.952^a 0.193^bc 7.375^b

Urea UI 1.077^c 2.450^b 0.195^b 8.625^c

Urea UI+NI 1.182^d 2.875^c 0.205^c 10.375^d

Year B

Control 0.872^a 2.050^a 0.185^a 6.810^a

Urea 1.010^b 2.185^a 0.202^bc 7.638^b

Urea UI 1.145^c 2.643^b 0.204^b 9.140^c

Urea UI+NI 1.267^d 2.960^c 0.210^c 10.515^d

F_Fert 39.062^*** 27.014^*** 15.167^*** 50.910^***

F_Year 10.335^** 4.496^* 6.957^* 2.328^ns

FFert x Year 0.126^ns 0.154^ns 0.165^ns 0.166^ns

Remarks: Means within a column followed by the different letters are significantly different at P = 0.05; ‘ns’ = not statistically significant; * = statistically significant for a significance level of p < 0.05; ** = statistically significant for a significance level of p < 0.01; *** = statistically significant for a significance level of p < 0.001

Nitrogen is considered as the one of the most important nutrient in flax crop (Hocking, Randall, &

Pinkerton, 1987). Nitrogen plays an important role for the building protein structure (Frink, Waggoner,

& Ausubel, 1999). Urea with double inhibitors affected in the agronomic characteristics, like plant height which in the both years had the maximum value. Taller plants were observed in the second year in the average of 68.75 cm than that of the first year with the average of 69.25 cm. The rainfall was higher in the first year of experiment than in the second year as shown in Fig. 1. Less rainfall in the second year reduced nitrogen losses as nitrification thus, urease inhibitors were not affected. Contrary to our results, the study of Kariuki, Masinde, Onyango, Githiri, & Ogila (2014) report that there was no effect on plant height in any application of different amounts of nitrogen. The index has twice the fertilization values compared to the control. In other words, the fertilization had a significant effect on the leaf surface. Similar LAI values was reported by Bilalis et al. (2010) in the study of flax with different tillage systems, and different fertilizers. Giménez,

Sorlino, & Trápani (2007) also stated higher values LAI because of nitrogen application. In flax crop, improvement and agronomic efforts should be directed towards the long-term preservation of the leaf area, intense and long flowering period since the dry matter was significantly affected by urea during both years. The urea with double inhibitors and the urea + UI had the higher dry matters and the urea with double inhibitors had the highest among the treatments (Table 2).

Absorption of fertilizers with inhibitiors by plants causes some changes in metabolic pathways that reduce urease activity, resulting in increased nitrogen uptake. That is the reason why there is a difference among fertilization treatments in the components of flax growth (plant height, LAI, and dry weight) (Artola et al., 2011; Cruchaga et al., 2013).

In combination with the fact that flaxseed develops arbuscular mycorrhizae fungi, where these fungi help the plant absorb nutrients and water (Monreal et al., 2011). And in our study it is noted that the presence of the fungus has a significant positive correlation with the dry weight of the plant (Table 3).

Table 2. The agronomic characteristics as effected by fertilizer treatments Fertilizer Plant Height(cm) LAI DM

(kg/ha) 1000 Seed Weight

(g) Seed Yield

(kg/ha) Oil Content

(%) Oil Yield (kg/ha) Year A

Control 56^a 1.823^a 1,492.5^a 3.768^a 1,101.25^a 34.475^ns 379.67^a

Urea 59.5^ac 1.96^b 1,553.75^a 3.898^a 1,206.25^b 35.975^ns 433.937^b

Urea UI 64.75^b 2.095^c 1,650^b 3.985^b 1,307.5^c 36.3^ns 474.735^c

Urea UI+NI 68.75^c 2.293^d 1,745^c 4.077^b 1,403.75^d 36.575^ns 513.96^c

Year B

control 57.75^a 1.95^a 1,558.168^a 3.865^a 1,198.75^a 34^ns 407.325^a

Urea 60.75^ac 2.085^b 1,609.405^a 3.96^a 1,287.5^b 35^ns 450.587^b

Urea UI 66^b 2.173^c 1,693.564^b 4.295^b 1,388.5^c 35.5^ns 492.95^c

Urea UI+NI 69.25^c 2.355^d 1,766.089^c 4.167^b 1,420^d 35^ns 497.387^c

F_Fert 30.25^*** 51.66^*** 35.716^*** 10.155^*** 113.295^*** 2.864^ns 35.832^***

F_Year 1.465^ns 14.542^*** 7.531^* 8.131^** 40.787^*** 4.488^* 1.933^ns

FFert x Year 0.069^ns 0.413^ns 0.321^ns 1.356^ns 2.776^ns 0.261^ns 1.368^ns

Remarks: Means within a column followed by the different letters are significantly different at P = 0.05; ‘ns’ = not statistically significant; * = statistically significant for a significance level of p < 0.05; ** = statistically significant for a significance level of p < 0.01; *** = statistically significant for a significance level of p < 0.001

Table 3. Pearson’s correlation coefficient (r) of root characteristics, plant growth parameters and yields ( kg/ha)DM 1000 Seed Weight

(g) Seed Yield

(kg/ha) Oil Content

(%) Oil Yield (kg/ha) Root Mass (mg/cm³) .8891^*** .6298^*** .9197^*** .3292^*** .8611^***

Root Surface (mm²/cm³) .8246^*** .5811^*** .8560^*** .1905^ns .7602^***

Root Diameter (mm) .6778^*** .6057^*** .8078^*** .2676^ns .7463^ns Root Density (mm/cm³) .9491^*** .5699^*** .9026^*** .3338^ns .8510^***

AMF .8346^*** .6274^*** .8246^*** .3189^ns .7780^***

Plant height (cm) .7763^*** .5636^** .8207^*** .2592^ns .7540^***

LAI .9303^*** .7062^*** .9379^*** .3121^ns .8699^***

Remarks: ‘ns’ = not statistically significant; * = statistically significant for a significance level of p < 0.05; ** = statistically significant for a significance level of p < 0.01; *** = statistically significant for a significance level of p < 0.001

The differences between the percentage of 1000 seeds weight between control and urea with 2 inhibitors was 8.2%. The value under control treatment showed insignificant difference with urea and neither did urea + UI with urea + UI + NI in both years. The values of 1000 seed weight ranged from 3.768 to 4.077 g in first year and from 3.865 to 4.295 g in the second. The maximum value was 4.295 g in urea + UI, in the second year and the lowest was 3.768 g in control, in the first year. The 1000 seeds weight was not correlated with nitrogen uptake (N total stover) and nitrogen indices used for nitrogen evaluation (Table 4). During first year nitrogen with double inhibitors gave 27.46% higher seed yield than the control and 16.37% higher than urea. Respectively second year seed yield was 18.45% and 10.29% higher than the control and urea. Although the highest yield was in the second year, the percentage differences were higher.

Seed yield had a significant correlation with N total uptake (r = 8576). It can be concluded that the main factor that significantly affects the performance is the climate since Khajani, Irannezhad, Majidian, &

Oraki (2012) observed that almost double of seed yield (2384.28 kg/ha)was recorded on the climate of Iran. Goreeva, Korepanova, Fatykhov, & Islamova (2020) also mentioned that 91.5% of the seed yield in flax cultivation was affected by environment and abiotic factors and 3% from genetic factors.

Furthermore, in the seed yield, all treatments had statistically significant difference among them.

The values ranged from 1,101.25 to 1,403.75 kg/ha in the first year and from 1,198.75 to 1,420 kg/ha in the second year (Table 2). The maximum value of seed yield was 1,766.089 kg/hain urea with

double inhibitors, in the second year and the lowest was 1,492.5 kg/ha in control, in the first year. The maximum value was 1,420 kg/ha in the second year and the lowest 1,101.25 kg/ha in the first year. The urea with double inhibitors and the urea + UI had higher values than the other treatments. In the oil content, all treatments gave less variation in both years. The average values of oil content in the second year was lower than in the first year. The values ranged from 34.475 to 36.575% in the first year and from 34 to 35.5% in the second year. The highest value of oil content was recorded 513.96 kg/

haunder urea with double inhibitors and the lowest was under control treatment in the first year with the value of 379.67 kg/ha. The urea + UI had not statistically significant difference with the urea + UI + NI (Table 2).

Concerning the nitrogen indicators, percentages of N in stover in all treatments were less varied for N both years (Table 5). In the first year, the lowest value was observed 0.37% in control, while the highest was under urea with double inhibitors with the value of 0.447%. The percentage N ranged from 2.865 to 3.245 % in the first year and from 2.868 to 3.305% in the second year. The control had not statistically significant difference with the urea and with the urea + UI. Also, the urea with double inhibitors had statistically significant difference with the urea, in the both years. In the N total stover, the control had not statistically significant difference with the other treatments. The values ranged from 5.518 to 7.831 kg/hain the year A and from 6.747 to 7.864 kg/hain the year B. The lowest value was 5.518 kg/ha in the control, in the first year and the highest was 7.864 kg/hain urea + UI + NI, in the second year (Table 5).

Moreover in the total N seed, the control had not statistically significant difference with the urea and the urea + UI had not statistically significant difference with the urea + UI + NI, in the both years. The highest value was 46.952 kg/hain urea with double inhibitors, in the second year and the lowest was 31.555 kg/hain control, in the first year.

The urea + UI and the urea + UI+ NI had higher values than the other treatments in both years.

Also, in the total N uptake the highest value was 54.816 kg/hain urea + UI +NI, in the second year and the lowest was 37.073 kg/hain control in the

first year. The control had not statistically significant difference with the urea and the urea + UI had not statistically significant difference with the urea + UI + NI, in the both years. Concerning the NUE, the values ranged from 0.085 to 0.235 in the first year and from 0.028 to 0.189 in the second year. The urea had statistically significant difference with the other treatments in the both years. The highest value was 0.235 in urea with double inhibitors, in the first year and the lowest was 0.028 in control, in the second year. Furthermore, in the NHI none value were statistically significant, in the both years.

Table 4. Pearson’s correlation coefficient (r) of nitrogen indicators and yields N total

Stover N total

seed N total

uptake NUE NHI NAE Eff of

absorption (Nt/Ns)

Eff of uptake (Gw/Nt) 1000 seed weight (g) .2285^ns .4911^* .4747^* .3072^ns .2593^ns .3440^ns .4747^ns -.2270^ns Seed Yield (kg/ha) .6955^*** .8377^*** .8576^*** .6881^*** .1548^ns .7905^*** .8576^*** -.4462^* Oil content (%) .3676^ns .0982^ns .1454^ns .3642^ns -.2621^ns .5188^** .1454^ns -.0702^ns Oil Yield (kg/ha) .7494^*** .7331^*** .7726^*** .7426^*** -.0017^ns .8999^*** .7726^*** -.4006^ns N% in Stover .9219^*** .4003^ns .5022^* .5038^* -.5309^** .5126^* .5022^* -.2916^ns N% in seed .4177^* .8953^*** .8656^*** .8574^*** .6600^*** .4753^* .8656^*** -.9905^**

Remarks: ‘ns’ = not statistically significant; * = statistically significant for a significance level of p < 0.05; ** = statistically significant for a significance level of p < 0.01; *** = statistically significant for a significance level of p < 0.001

Table 5. The nitrogen indicators as effected by fertilizer treatments Fertilizer N% in

Stover N% in seed

N total stover (kg/ha)

N total (kg/ha)seed

N total uptake

(kg/ha) NUE NHI NAE Eff of

absorption (Nt/Ns)

Eff of uptake (Gw/Nt) Year A

Control 0.37^ns 2.865^a 5.518^a 31.555^a 37.073^a - 0.851^ns - - 29.712^ac Urea 0.405^ns 3.043^a 6.282^a 36.700^a 42.982^a 0.085^a 0.853^ns 1.75^a 0.716^a 28.073^a Urea UI 0.44^ns 3.142^ab 7.275^a 41.086^b 48.361^b 0.159^b 0.849^ns 3.437^b 0.806^b 27.058^ad Urea UI+NI 0.447^ns 3.245^b 7.831^a 45.603^b 53.435^b 0.235^b 0.854^ns 5.042^c 0.890^b 26.357^cd

Year B

Control 0.453^ns 2.968^a 7.069^a 35.580^a 42.649^a - 0.837^ns - - 28.208^ac Urea 0.42^ns 2.868^a 6.747^a 36.943^a 43.690^a 0.028^a 0.842^ns 1.480^a 0.728^a 30.042^a Urea UI 0.437^ns 3.212^ab 7.409^a 44.608^b 52.017^b 0.150^b 0.858^ns 3.163^b 0.866^b 26.704^ad Urea UI+NI 0.445^ns 3.305^b 7.864^a 46.952^b 54.816^b 0.189^b 0.857^ns 3.688^c 0.913^b 25.959^cd F_Fert 0.565^ns 5.987^** 3.138^* 28.285^*** 27.191^*** 26.083^*** 0.441^ns 65.422^*** 470.484^*** 4.087^* F_Year 0.938^ns 0.041^ns 1.786^ns 4.477^* 5.217^* 2.391^ns 0.252^ns 4.018^ns 1.552^ns 0.009^ns

FFert x Year 0.717^ns 0.812^ns 0.725^ns 0.686^ns 0.804^ns 0.631^ns 0.465^ns 1.603^ns 0.471^ns 1.012^ns

Remarks: Means within a column followed by the different letters are significantly different at P = 0.05; ‘ns’ = not statistically significant; * = statistically significant for a significance level of p < 0.05; ** = statistically significant for a significance level of p < 0.01; *** = statistically significant for a significance level of p < 0.001

The maximum value was 0.858 in urea + UI and the lowest was 0.837 in control in the second year. In the NAE, all treatments had statistically significant difference between them, in both years (Table 5).

The values ranged from 1.75 to 5.042 in the first year and from 1.480 to 3.668 in the second year.

The highest value was 5.042 in urea with double inhibitors, in the year A. Concerning the effect of absorption, the urea + UI had not statistically significant difference with the urea + UI + NI, in the both years. The maximum value was 0.913 Nt/Nsin urea with double inhibitors in the second year and the lowest was 0.716 in urea, in the first year. The second year the values were higher than the first year. Also, in the effect of uptake, the urea + UI + NI had statistically significant difference with urea, in the both years. The highest value was 30.042 Gw/

Nt in urea and the lowest was 25.959 Gw/Nt, in the second year, respectively (Table 5).

In our experiment the temperatures were higher in the second year and it is possible for this to be higher seed yield. According to the study of Homayouni, Souri, & Zarein (2013), nitrogen affects in the seed yield. Besides, high temperatures favor seed production. However, temperatures higher than 32^oC before and during flowering, when accompanied by drought, reduce the yield, size and oil content of the seed, as well as the quality of the oil (Bilalis et al., 2010). In our experiment, the temperatures were higher in the second year and it is possible for this to be higher seed yield. The oil content was not significantly affected by fertilization as opposed to oil yield. In the oil yield the urea with double inhibitors had not statistically significant difference with the urea + UI. Oil yield had a positive significant correlation with NAE index (r = 0.899).

This correlation is important because not only was there a higher yield in oil but also in nitrogen. El- Nagdy, Nassar, El-Kady, & El-Yamanee (2010) stated that the mineral fertilization had statistically significant difference in the oil yield.

Concerning the root characteristics, root mass, root surface, root diameter and root density, the fertilizers with inhibitors had highest values in both years. With regard to the agronomic characteristics mentioned above, the root characteristics had positive correlation with other parameters like root density with the dry matter (r = 0.9491, p = 0.000), the root mass with the oil yield (r = 0.8611, p = 0.000) (Table 3). The root developed more in the second year, with small to minimal differences with

the first year. The rainfall was lower in the second year as well as the temperature so there was less evaporation of ground water.

Fig. 2 below, shows the change in the percentage of arbuscular mycorrhizal fungi that were exposed on different fertilizer applications on different period of sowing in the two different years. In the first year (Fig. 2A), on the 20^th DAS the percentage arbuscular mycorrhizal fungi, had not statistically significant difference, between the treatments. Also, on the 40^th DAS the urea had not statistically significant difference with the control and the urea + UI had not statistically significant difference with the urea + UI+ NI. The highest value was 20.75 % in the urea with double inhibitors and the lowest was 8.5% in the control.

Then on the 60^th DAS, all treatments had statistically significant difference between them.

The urea + UI + NI had the highest value 29.5%, increased compared to the previous DAS and the lowest was 14.5% in the control. And the other treatments showed an increase in the percentage of arbuscular mycorrhizal fungi (Fig. 2A). As for the 80^th DAS, the urea + UI had not statistically significant difference with the urea + UI + NI. Values continued to rise and the highest was 29.75% in the urea with double inhibitors. Moreover, on the 100^th DAS, the urea + UI had not statistically significant difference with the urea + UI + NI and the urea had not statistically significant difference with the control. In all treatments the percentage had increased, the urea + UI had the same value with the 80^th DAS, highest rise had the control and the urea. The maximum value was 32.75% in the urea with double inhibitors and the lowest was 22.25 in the control (Fig. 2A).

In the second year (Fig. 2B), percentage of arbuscular mycorrhizal fungi was significantly higher was detected after 40 DAS than the first year.

The maximum value was 21.53% in the urea + UI + NI and the lowest was 12.8% in the control. Similar phenomena was also observed on 60 DAS in that the highest value was recorded 31.125% in the urea with double inhibitors and the lowest was 14.5% in the control (Fig. 2B). On the 100^th DAS the lowest value was 21.75% in the control and the maximum was 32.75% in the urea + UI +NI.

In the both years the highest value in all period of sowing, was in the urea with double inhibitors and the lowest was in the control. In the most sowing periods, the urea + UI + NI had not

statistically significant difference with the urea +UI.

Also urea had close values with the control in all period of sowing, except at 60 DAP.

According to Fig. 2, the percentage of arbuscular mycorrhizae fungi was higher in the urea with double inhibitors. It was observed that the urea + UI + NI had not statistically significant difference with the urea + UI in the different sowing period. It should be noted that the percentage of arbuscular mycorrhizae fungi was higher in fertilizers with inhibitors due to the fact that the action of urea kills fungi, while inhibitors work beneficially. The urea

with double inhibitors and the urea + UI increase the root growth (Qi et al., 2012). Nitrogen Use Efficiency, express how each application affects the nitrogen content of the seeds, compared to the control. In this study, it was shown that urea with double inhibitors had the best efficacy. This is because nitrogen was available for the plants for a longer period. Similar studies have suggested that double-acting urease may increase NUE due to loss reduction (Cantarella, Otto, Soares, & de Brito Silva, 2018).

Fig. 2. Changes in the percentage arbuscular mycorrhizal fungi as effected by fertilizer treatments, in different Days After Application (DAA), in 2018 and 2019. Different letters indicate significant differences according to LSD (p = 0.05)

Nitrogen Harvest Index is an indicator that shows how nitrogen is distributed in plants and in particular how much of it is available in the seeds.

NHI had higher value at the urea + NI + IU. This is due to the slow release of nitrogen, giving the crop the opportunity to use nitrogen for whatever reason is needed, depending on the stage of development.

On the other hand, the effects of uptake showed that although the intake nitrogen in application with double inhibitors had the greatest effect on the seed, the yield of the seeds was lower while the higher was in the control. This is explained by analyzing the factors that take place to calculate this indicator. From the Table 5, we can see that in the application with double inhibitors, the uptake nitrogen was significantly higher than the other applications. However, seed yield varies slightly among the treatments.

Nitrogen Agronomic Efficiency is the indicator informing each fertilizer effect on seed yield. The higher NAE was correlated with higher seed yield.

Table 4 showed that there were positive correlations between seed yield and NAE (r = 0.7905, p = 0.000).

In the present study, it was observed that urea with double inhibitors showed significant differences with the other treatments. This is because the difference in seed yield in this treatment from the seed yield in control was greater (Table 2), since the amount of fertilizer was the same for all treatments.

The Effects of Absorption express the amount of nitrogen absorbed from the amount applied.

Therefore, this index informs about the amount of nitrogen that was lost. In the present experiments, more losses occurred in the urea since it had the lowest value. According to Subbarao et al. (2006), the main reason for using NI is to reduce nitrogen leaching. Urease inhibitor also reduce volatility losses and double inhibitors application increased the value of effects of absorption.

CONCLUSION

Flax oil production is mainly affected by the influence of the environment and less by the genotype. Thus, in our study it was seen that flax was significantly affected by fertilization and climatic condition. The present study showed that urea with urease inhibitor and nitrification inhibitor and in combination with rainfall showed beneficial effects in almost all of the studied characteristics. Most of the growth characteristics of flaxseed, including

root parameters, showed higher values in urea with double inhibitors treatment. During 2 years observation, the less annual rainfall gave the crop to maximize the role of inhibitors. Also the higher temperatures gave higher seed yields. Finally, in terms of nitrogen indicators, apart from the effects of uptake, the rest had the higher value in the application with double inhibitors.

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