chlorophyll leaf concentration and its relationships

Published:

with N use efficiency in linseed

Abstract

Plant-based diagnostic techniques are used to determine the level of crop N nutrition but there is limited comparative research of the different methods that can be used on the different plant species and especially on linseed. In addition, the effect of N level on the relationship among chlorophyll meter (CM) readings, N nutrition index (NNI), and N use efficiency (NUE) has not been studied in linseed. The objectives of this study were to determine whether there is a relationship between chlorophyll meter (CM) readings, N nutrition index (NNI) of linseed, and N use efficiency, and to compare CM readings and NNI as diagnostic tools for predicting seed yield response to N fertilization. A two-year field study was therefore conducted with the objective to determine the effect of N fertilization (0, 40, and 80 kg ha-1) on CM readings, NNI and NUE and its components of three linseed cultivars (Livia, Lirina, Creola). N fertilization affected N concentration of the different plant parts and there was no difference between the applied N rates. CM readings and RCM readings were affected by the N treatment and were higher at both N levels compared with the control. N nutrition index varied from 0.58 to 1.25 across years, growth stage, and cultivar and was affected by the fertilization level. Also, NUE, N uptake efficiency, and N utilisation efficiency were affected by the cultivar, N fertilization, growing season, and their interaction. N utilization efficiency accounted for more of the variation of N use efficiency than the N uptake efficiency and was higher at the 0 kg N ha-1 than at the 80 kg N ha-1 in both years. CM readings at anthesis was correlated with NUE, NNI at anthesis, at green capsule stage, and at harvest, seed yield, and relative seed yield. This study provides new information about the effect of N application on chlorophyll meter readings, relative chlorophyll meter readings, NNI, and NUE of linseed. Therefore, in order to obtain high yields of linseed, the crop must receive adequate amounts of N which can be diagnosed using CM, RCM readings, and NNI.

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Keywords: linseed, chlorophyll meter readings, nitrogen efficiency, uptake, utilization.

1. Introduction

Flax (Linum usitatissimum L.) and linseed for seed production have emerged as an alternative crop species to increase diversification of European cropping systems. Linseed is an important source of essential fatty acids for human diets (Millis, 2002) and has several human health benefits (Millis, 2002). Thus, there is growing interest in linseed for food, feed, and industrial products and more attention is now being given to meeting the growing demand for this crop. However, the great variability of seed yields has been one of the major limiting factors to the increase of linseed area (Weiss, 2000). Therefore, it is important for scientists to develop a better understanding of the factors that can affect seed yield and especially nitrogen (N) fertilization which is one of the most important crop management technique which affects seed yield (Lafond et al., 2008; Dordas, 2010).

One of the most important diagnostic tools that have developed are the plant-based diagnostic methods of N deficiency which are used to improve N management and decrease the risk of N loss to ground and surface waters (Lemaire et al., 2008; Naud et al., 2009; Scharf, 2001; Schröder et al., 2000; Zebarth et al., 2002). Chlorophyll meter (CM) readings is one of the most popular approaches and have proven to be effective as a rapid diagnostic method to determine the N status of many crops, including spring wheat (Triticum aestivum L.) (Follett et al., 1992; Vidal et al., 1999; Arregui et al., 2006), rice (Oryza sativa L.) (Turner and Jund, 1991; Peng et al., 1993; Ladha et al., 1998), safflower (Carthamus tinctorius L.) (Dordas and Sioulas, 2008), and corn (Zea mays L.) (Ziadi et al., 2008b; Piekielek and Fox, 1992; Schepers et al., 1992; Dordas et al., 2008). However, CM readings have several disadvantages as it depends on cultivar, developmental stage, management techniques, site characteristics, disease or insect damage, plant density, and other nutrient deficiency (Lemaire et al., 2008; Blackmer and Schepers, 1995; Piekielek et al., 1995; Waskom et al., 1996). Therefore the relative CM readings (RCM) have been recommended to account for the influence of these factors (Ziadi et al., 2008b; Prost and Jeuffrey, 2007). The RCM readings are calculated by dividing the readings from the test area by the reading from a saturated plot that has received a high N rate.

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Another approach to assess crop N nutrition has been proposed using the N nutrition index (NNI). NNI is calculated by dividing the actual N concentration by the critical N concentration (Nc). The critical N concentration is defined as the minimum N concentration in the shoot biomass required for maximum growth rate, has been established for linseed (Flenet et al., 2006): Nc = 4.69W-0.53 where W represents the shoot biomass in Mg ha-1 DM. This model is derived from the critical N dilution curve which is different in linseed compared with other C3 plants (Flenet et al., 2006), and the derived NNI is considered a reference tool for assessing crop N nutrition. However, a major limitation in using the NNI at the farm level is the need to determine the actual crop biomass and its N concentration. Therefore, to simplify the evaluation of crop N status, a quicker method of estimating NNI is needed. That's why chlorophyll measurements have been proposed as an alternative tool for estimating the level of N nutrition. There are studies which showed that although CM and RCM readings were related to NNI, but they did not provide a valid and robust estimation of the plant N status because the relationships of CM and RCM readings with NNI varied with sites and years (Ziadi et al., 2008b). In addition, CM, RCM, and NNI were not used in linseed to determine the N status of the crop and it is not know how those criteria for N diagnosis relate with each other and the seed yield.

Nitrogen is one of the main inputs in high-input agricultural systems. It is also responsible for an important part of agriculture related pollution through leaching or denitrification (Fageria and Baligar, 2005). During at least the past 40 years, the nitrate content of water has increased in the intensive cropping area of many countries due to N fertilization (Fageria and Baligar, 2005). To address both economic and ecological issues, plant breeders would have to release cultivars that minimise pollution risks and maximise farmers' revenue. Limited pollution risks could be achieved either with low fertiliser rates or cultivars that better absorb and utilize N (Marino et al., 2004). Concerning N, high revenue should be obtained with a maximum yield and quality per unit of N applied. Plant breeding programmes must produce varieties that absorb N more efficiently and use it more efficiently to produce grain (Moll et al., 1982; Dhugga and Waines, 1989). Field experiments have shown that genetic variability for N uptake exists in small grains (Loffler et al., 1985; Van Sanford and MacKown, 1986; Moll et al., 1982; Dhugga and Waines, 1989). The factors that affect N utilization of linseed is important for the successful introduction of the crop to a given cropping system. This information can be used by the growers for adopting the appropriate cultural practices and also by the breeders for choosing the most efficient selection criteria in order to improve N exploitation. However, such information is limited for linseed.

The main objectives of this study were: (i) to establish the relationship between CM, RCM readings, and NNI for linseed, (ii) to compare both methods as diagnostic tools for predicting yield response to N fertilization, and (iii) to determine NUE and its components N utilization efficiency and N uptake efficiency and its relationship with CM, RCM readings and NNI for linseed.

2. Materials and methods

2.1 Experiment set-up

The experiment was carried out at the experimental farm of the Aristotle University of Thessaloniki, Greece during the 2005-2006 (2006) and 2006-2007 (2007) growing seasons. The farm is located in Northern Greece (22o59'6.17" N, 40o32'9.32" E). Three different cultivars were used Livia, Lirina, and Creola, with different characteristics and were obtained from different German seed companies (Deutsche Saatveredelung Lippstadt-Bremen GmbH Zu Lippstadt and De-Vau-Ge Gesundkostwerk GmbH). Creola was an early flowering cultivar, while Livia and Lirina were later flowering cultivars. Creola also had larger seeds of lighter color.

The soil type where the experiment took place was a calcareous sandy loam (Typic Xerorthent) with wheat (Triticum turgidum subsp. durum L.) as the preceding crop. Wheat straw was baled and removed after harvest. Seedbed preparation included moldboard ploughing, disk harrowing, and use of cultivator. Soil samples (0 to 30 cm depth) were taken prior to the application of fertilizers and analyzed. Briefly, the soil contained an average of 28 % clay, 15% silt, and 57% sand, with a pH of 7.96 (1:2 water), organic matter content 0.72%, N-NO3 15.56 ppm, P (Olsen) 6.69 ppm, and K 51.00 ppm. Weather data (rainfall, maximum and minimum temperatures) were recorded daily and are reported as mean monthly data for the two years that the study was conducted (Table 1). The weather conditions during the growing season in the years 2006 and 2007 were different. The 2006 growing season was mild during the spring and there was higher rainfall. In contrast, in 2007 the spring was hotter with significantly lower rainfall (Table 1). Therefore, there were differences in the response of the three cultivars to N fertilization during the two years of the study, which was probably because of the different weather conditions.

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2.2 Crop management and experimental design

The experimental design was split plot with the N levels the main plots and the subplots the different cultivars (Livia, Lirina, Creola) and five replications. The treatments were the following: 0, 40, and 80 kg N ha-1 applied preplanting in the form of (NH4)2SO4 (N-P-K 20.5-0-0). The (NH4)2SO4 form was selected as it is the major N fertilizer used in the area and also is less likely than the NO3 form to leach from the soil. The fertilizer was incorporated into the soil with a disk harrow. Also P and K were applied pre-planting at a rate of 60 kg P2O5 ha-1 and 100 kg K2O ha-1 respectively, in the form of superphosphate (0-46-0). Seeds were hand-planted on 7 of November 2005 and on 10 of November 2006 at the rate of 700 seeds m-2. The crop was kept free of weeds by hand hoeing when necessary. Plants were grown without supplemental irrigation in both growing seasons.

2.3 Crop sampling for measurements

The following parameters were determined: chlorophyll content at anthesis and green capsule stage, total above ground biomass at anthesis, at green capsule stage, and at maturity. Anthesis was defined when 50% of the plants of the plot bearing flowers or capsules (Smith and Froment, 1998). Also the green capsule stage was defined when 50% of the plants of the plot bearing capsules and 50% of the capsules have reached full size (Smith and Froment, 1998). At each harvest 2m of the central row was randomly selected and separated into leaves and stems (at anthesis), leaves, stems and capsules at green capsule stage, and leaves+ capsule vegetative components, stems, and seeds (at harvest); samples were dried at 80 oC and then weighed. The dry vegetative samples were first ground in a hammer mill and then reground finely using a 1 mm screen. N content was determined by the Kjeldhal method (Dordas and Sioulas, 2009).

One thousand seed weight (TSW) was determined by measuring the weight of 100 seeds from each plot and multiplying by 10 in order to express one thousand seed weight. Seed protein content was determined by multiplying by 6.25 the seed N concentration and protein yield was determined by multiplying the seed yield by the seed protein content. Seed yield was determined by harvesting the six central rows with a research plot combine (Wintersteiger AG, Austria) in the last week of June in both years. Relative seed yields at each plot were computed as the ratio of seed yield at a given N rate by the highest seed yield among all N treatments.

2.4 Chlorophyll measurements

Chlorophyll meter (CM) readings were taken with a hand-held dual-wavelength meter (SPAD 502, Chlorophyll meter, Minolta Camera Co., Ltd., Japan). For each plot the 20 youngest fully expanded leaves per plot were used when the plants were at anthesis and green capsule stage (Smith and Froment, 1998). The instrument stored and automatically averaged these readings to generate one reading per plot. Relative chlorophyll meter (RCM) readings were calculated by dividing any SPAD reading by the maximal value from the 80 kg N ha-1. This index, ranging from 0.5 to 1, is also called the sufficiency index (Varvel et al., 1997).

2.5 Nitrogen Nutrition Index

The Nitrogen Nutrition Index (NNI) of the crop at each sampling date was determined by dividing the N concentration of the shoot biomass by the critical N concentration (Nc) (Ziadi et al., 2008a, b). Critical N concentration, the minimum N concentration required to achieve maximum shoot growth, was defined as a function of shoot biomass as proposed for linseed by Flénet et al., (2006; Nc = 4.69 Ã- W−0.53 where W is the total shoot biomass expressed in Mg DM ha−1).

2.6 Nitrogen efficiency and its components

Nitrogen use efficiency (NUE) was defined as seed production per unit of N available in the soil. NUE is Sw/Ns in which Sw is the seed weight and Ns is N supply expressed in the same units (e.g. kg ha-1) (Moll et al., 1982). There are two primary components of NUE (1) N uptake efficiency (Nt/Ns) and (2) N utilization efficiency which describes how the N that is absorbed is utilized to produce seeds (Sw/Nt), where Nt is the total N in the plant at maturity. Therefore, the NUE can follow the equation:

Sw/Ns=(Nt/Ns)(Sw/Nt)

The expression can be expanded to include additional factors. For example, N uptake during seed filling and translocation of N to seed.

Sw/Ns=(Nt/Ns)(Sw/Ng)(Na/Nt)(Ng/Na)

where Sw/Ng=seed produced per unit of seed N

Ng/Nt=fraction of total N that is translocated to seed

Na/Nt=fraction of total N that is accumulated after anthesis

Ng/Na=ratio of N translocated to seed to N accumulated after anthesis.

where Ng is the N uptake by seeds and Na is the N uptake after anthesis.

2.7 Component analysis

Various expressions were constructed and analyzed according to the method suggested by Moll et al. (1982) and Dhugga and Waines (1989). The analysis involves linearizing the multiplicative relationships by taking logs and then determining the contribution of each component trait to the sum of squares of the resultant trait. The sum of cross products of each component trait by the resultant trait (Sxiyi) divided by the sum of squares of the resultant trait (Syi2) gives the relative contribution of each component variable to resultant variable. This analysis describes the net contribution of each component variable both directly and indirectly through the other variable (Moll et al., 1982). The following expressions were analyzed:

log(N use efficiency (Seedw/Ns))= log(Uptake efficiency (Nt/Ns)) + log(Utilization efficiency (Seedw/Nt))

2.8 Statistics

The data were analyzed by the ANOVA method according to a 2Ã-3Ã-3 factorial design (Growing season Ã- N levels Ã- Cultivars) with 5 replications per treatment combination. More specifically, the experiment was set up as a Randomized Complete Block Design for the N levels (main plots), with cultivars as split plots. A combined analysis over growing season was carried out according to the aforementioned design (Steel et al., 1997). Tukey's post hoc procedure was used for testing the differences between treatment means. The significance level of all hypotheses testing was preset at P<0.05. Pearson correlation analyses across years were done to find the relationship between the different characteristics. All statistical analyses were performed using the SPSS ver. 17 software package (SPSS Inc., USA, IL: Chicago).

3. Results

Cultivars affected seed N concentration, seed protein content and yield, thousand seed weight (TSW), seed yield and relative seed yield, chlorophyll meter (CM) and relative chlorophyll meter (RCM) readings, N use efficiency, N utilization efficiency, and seeds produced per unit of seed N (Table 2). Also N treatments affected all the characteristics except the TSW, fraction of total N that is translocated to seed, fraction of total N that is accumulated after anthesis, and ratio of N translocated to seed N and is accumulated after anthesis. Growing season affected all the characteristics except the N and protein concentration of seeds and TSW. The interaction between cultivars and treatments affected seed yield and relative seed yield, protein yield, CM and RCM readings, N use efficiency and its components, and seed produced per unit of seed N. The interaction between growing season and treatment affected most of the characteristics that were studied except from the N concentration of leaves + flowers and stems at anthesis, TSW, N utilization efficiency, fraction of total N that is translocated to the seed, ratio of N translocated to seed to N accumulated after anthesis. Also the interaction between the cultivar and growing season affected seed yield and relative seed yield, seed protein content, CM and RCM readings, N utilization efficiency and its components, seed produced per unit of seed N and fraction of total N that is translocated to seed. Finally the interaction among cultivars, growing seasons, and treatments affected the seed yield, relative seed yield, CM and RCM readings, and also N use efficiency. Therefore, these characteristics are presented in greater detail, whereas the rest of the characteristics where there was no interaction among the treatments, growing seasons, and cultivars only the main effects are presented.

3.1 Nitrogen concentration

Nitrogen concentration of the different plant parts was not affected by the cultivar with the only exception of the seed N concentration was higher in the Creola cultivar. N fertilization affected N concentration of the leaves+flowers and stems at anthesis and also the leaves + capsule vegetative component, stem, and seeds N at harvest (Table 3). There was an increase in N concentration of the leaves+flowers and stems at anthesis with N application by an average of 20 and 26% respectively in the three cultivars and in both years compared with the control. Also the leaves + capsule vegetative components, stem, and seeds N concentration at harvest were increased by 40, 65, and 15% respectively compared with the control. In most cases, N concentration in linseed plants was not different between the applied N rates. Also N concentration of the different plant organs was higher during the 2007 compared with the 2006 growing season and with the only difference that the N seed concentration was not affected by the growing season. Similar trend was found at the green capsule stage (data not shown).

3.2 Seed yield, relative seed yield, seed protein content

Seed yield was higher at Creola cultivar and was increased with N fertilization in the three cultivars by an average of 35% (Figure 1a and 1b). Relative seed yield was lower at Livia cultivar and higher at Creola cultivar and ranged between 0.74 to 1.0. Relative seed yield was increased by 34% with N fertilization compared with the control. Seed protein content was higher at Creola cultivar compared with the Livia and Lirina (Table 4). Similarly, seed protein yield was higher at Creola compared with the other two cultivars. Seed protein content was increased with N application by an average of 14 % in all cultivars over the two years and seed protein yield was increased by an average of 59% compared with the control (Table 4). Also, the protein yield was higher during the 2007 compared with the 2006. In addition, TSW was higher at Creola than at Livia and Lirina. However, the TSW was not affected by N fertilization.

3.3 Chlorophyll meter and relative chlorophyll meter readings

Chlorophyll meter (CM) readings were affected by the N treatments and were higher at both N levels compared with the control (Figure 2a and 2b). At anthesis, CM readings were higher by an average of 15 % in the fertilized treatments compared with the control. At green capsule stage, there were significant differences between the control and the N treatments especially during the first year, where the chlorophyll level was by 22% higher compared with the control. RCM readings were higher at the fertilization treatments compared with the control and it was in the range of 0.75 to 1.03 (Figure 3a and 3b). At anthesis, relative chlorophyll meter (RCM) readings were higher by an average of 15 % in the fertilized treatments compared with the control. At green capsule stage, there were significant differences between the control and the N treatments especially during the first year, where the RCM level was by 22% higher compared with the control (Figure 3a and 3b).

3.4 Nitrogen nutrition index

Nitrogen nutrition indices varied from 0.65 to 1.25 across years, growth stages, and cultivars and were affected by the fertilization level (Table 5). Values of NNI ≥ 1.0 indicate that N supply to the crop is nonlimiting or in excess, while values of NNI < 1.0 indicate N deficiency. Nitrogen nutrition indices were generally significantly affected by N fertilization (Table 5). At anthesis NNI were higher by an average of 58% at the fertilization treatments compared with the control and also it was higher during the 2007 than the 2006. At green capsule stage it was higher at the fertilization treatments by an average of 42% and the values ranged from 0.79 to 1.16.

3.5 Nitrogen efficiency, its components, and N translocation indices

Nitrogen use efficiency (NUE) and its components N uptake efficiency and N utilisation efficiency were affected by N fertilization. NUE, N uptake efficiency, and N utilization efficiency was higher at the control compared with the two fertilization treatments (Table 6). Livia and Lirina had higher N uptake efficiency at both N levels, while Creola had the lowest during the first year. Also, Creola had high N utilization efficiency at the control treatment. N utilisation efficiency ranged from 8.48 to 14.33 kg kg-1 N. Livia was the only genotype that showed no significant difference between the three N levels. The fraction of total N that is translocated to seed (Ng/Nt) was affected only by the growing season and was higher during 2006 (Table 7). The fraction of total N that was accumulated after anthesis was higher at the Livia cultivar compared with the Creola cultivar. Also, it was higher at the control and 40 kg N ha-1 compared with the 80 kg N ha-1 and was higher at the 2006 than in 2007. The ratio of N translocated to seed to N accumulated after anthesis was higher at the Creola and Livia and lower at the Lirina cultivar and was higher during the 2007 growing season. Seed produced per unit of seed N was higher at the Creola compared with the other two cultivars. In addition, it was higher at the control compared with the other two treatments and also during the 2007 compared with the 2006.

3.6 Component analysis of the different traits.

The relative contributions of NUE components are presented in Table 8. N utilization efficiency accounted for more of the variation of NUE than the N uptake efficiency and was higher at the 0 kg N ha-1 than the 80 kg N ha-1 in both years. The variation attributed to the N uptake efficiency was higher at the 80 kg ha-1 compared with the other two levels which indicates that there was significant interaction in NUE among cultivars, treatments, and years. The highest variation at 80 kg N ha-1 was found in N utilization efficiency during 2006. However, in 2007 the highest variation was found at the N uptake efficiency (Table 8).

3.7 Correlations

The most important characteristics were tested to determine their relationship using correlation analysis (Table 9). CM readings at anthesis were correlated with chlorophyll at green capsule stage, RCM at anthesis, RCM at green capsule stage, NNI at anthesis, NNI at green capsule stage, NNI at harvest, seed yield, and relative seed yield. Also CM readings at green capsule stage were correlated with all of the above characteristics except from NNI at harvest. RCM at anthesis and RCM at green capsule stage were correlated with all the characteristics. Seed yield was negatively correlated with NUE indicating that NUE can not be used for predicting high seed yield because of the negative correlation. Also the other characteristics such as CM and RCM readings or NNI showed a negative correlation with NUE and could not be used for higher NUE.

4. Discussion

4.1 Nitrogen concentration

Nitrogen fertilization affected N concentration of the different plant organs at anthesis and at harvest. In most cases, N concentration in linseed plants was not different between the applied N rates. This indicates that the N level in the soil was adequate when N was applied even at the lowest N rate (40 kg N ha-1) and at the higher N rate (80 kg N ha-1) did not increase N concentration. When the N level is marginal, as in the present study, there was an increase in N concentration with N fertilization (Dordas et al., 2008). However, even if the soil N concentration is marginal there are studies which showed no increase in N concentration and also in seed yield (Grant et al., 1999; Nuttall and Malhi, 1991). This can be because of the response of linseed to N fertilization is affected by the soil type, linseed cultivar, climate, growing season moisture conditions, N fertilizer form and placement, and seeding rate and seeding date (Lafond et al., 2008; Grant et al., 1999; Lafond, 1993; Nuttall and Malhi, 1991).

4.2 Seed yield, relative seed yield, protein content, and protein yield

Seed yield and relative seed yield were increased by an average of 35 and 34 % respectively. Despite the fact that linseed does not have very high requirements for N and other nutrients like canola, wheat, and barley (Nuttall and Mahli, 1991), it was shown that it responds to N fertilization (Dordas, 2010; Lafond et al., 2008; Diepenbrock and Porksen, 1992). However, the response of seed yield to N fertilization can also vary with site and year as Grant et al. (1999) reported and as the soil N content increased from 46 to 110 kg ha-1, there was an increase in seed yield. Low rainfall and high temperatures during anthesis and seed filling stage can have a significant effect on seed yield (Hocking et al., 1997; Casa et al., 1999) as it occurred during 2006 growing season when the seed yield was lower compared with the 2007. In addition, Lafond (1993) observed limited response of linseed to N application where soil NO3 levels were above 25 mg kg-1 whereas in this study the NO3 concentration was 15.56 mg kg-1 which is much lower than the sufficient level. Grant et al., (1999) found an increase in seed yields up to applied 80 kg N ha-1. However, in some locations there was a significant decrease in seed yield at high N levels because of lodging (Grant et al., 1999). The increase in seed yield can be due to the increase in the main yield components such as seed weight per plant, number of capsules per plant and per m2, and the effect of N on growth rate and N uptake rate of the crop (Dordas, 2010).

Seed protein content was increased with N application by an average of 14 % in all cultivars over the two years and seed protein yield was increased by an average of 59% compared with the control. The highest increase in seed protein yield compared with the seed protein content was because N fertilization affected more the seed yield than the seed protein content. Seed protein content was found to increase with N fertilization (Hocking et al., 1997; Hocking and Pinkerton, 1993) when the weather conditions are favorable. But when there was low rainfall and with high temperatures there was no response to N fertilization (Hocking et al., 1997). The TSW was not affected by N fertilization. Similar responses was found by Hocking and Pinkerton (1993) who reported that N fertilization did not affect TSW. However, Lafond et al., (2008) found that N fertilization increased the TSW.

4.3 Chlorophyll meter and relative chlorophyll meter readings

Chlorophyll meter readings were affected by the N treatment and were higher at anthesis and at green capsule stage at the N treatments compared with the control and were higher at anthesis than at green capsules stage. The lower amount of N available to the control was perhaps N was remobilized for seed growth, causing the leaves to senesce quicker and lowering the amount of chlorophyll (HYPERLINK "http://agron.scijournals.org/cgi/content/full/92/6/1228?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexacttitle=and&titleabstract=chlorophyll+and+maize&andorexacttitleabs=and&andorexactfulltext=and&searchid=1140084831934_283&FIRSTINDEX=0&sortspec=relevance&journalcode=agrojnl&journalcode=cropsci#BIB14BIB14"Dordas et al., 2008; Dordas and Sioulas, 2008; Ziadi et al., 2008a, 2008b;HYPERLINK "http://agron.scijournals.org/cgi/content/full/92/6/1228?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexacttitle=and&titleabstract=chlorophyll+and+maize&andorexacttitleabs=and&andorexactfulltext=and&searchid=1140084831934_283&FIRSTINDEX=0&sortspec=relevance&journalcode=agrojnl&journalcode=cropsci#BIB14BIB14" HYPERLINK "http://agron.scijournals.org/cgi/content/full/92/6/1228?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&andorexacttitle=and&titleabstract=chlorophyll+and+maize&andorexacttitleabs=and&andorexactfulltext=and&searchid=1140084831934_283&FIRSTINDEX=0&sortspec=relevance&journalcode=agrojnl&journalcode=cropsci#BIB14BIB14"Schepers et al., 1992). This clearly indicates that when there is an adequate N supply in the soil leaf senescence is slower and the plant supplies the seed with N and photoassimilates for longer time which results in higher yields (Dordas et al., 2008; Ziadi et al., 2008b).

Chlorophyll meter readings varied from 36 to 52 which is quite typical since there is variability in chlorophyll content (Dordas et al., 2008; Dordas and Sioulas, 2008; Ziadi et al., 2008b; Masoni et al., 1996). Chlorophyll meter readings generally increases during the growing season up to a maximum and then gradually decreases (Ziadi et al., 2008a, 2008b; Dordas et al., 2008). This variation over the growing period is important as there is a need to specify the developmental stage at which CM readings are taken. At the earlier growing stages the CM readings are not significantly affected by N treatments which can be because of the residual N in the soil and the low requirements of N for the plant (Ziadi et al., 2008b; Dordas et al., 2008). At anthesis is generally a time where the differences in the response to N fertilization are more pronounced. As the plants reach maturity there is loss of chlorophyll due to senescence and CM readings decrease (Ziadi et al., 2008b). The CM readings can be affected by cultivar, site characteristics, developmental stage, disease or insect damage, plant density and other nutrient deficiencies (Masoni et al., 1996). RCM readings have been recommended to account for the influence of the above mentioned factors on CM readings and the RCM variation found in this study was smaller and can be used instead of the CM readings for N status diagnosis (Blackmer and Schepers, 1995; Piekielek et al., 1995; Waskom et al., 1996).

4.4 Nitrogen nutrition index

The main objective of this study was to determine whether there is a relationship between NNI and N concentration and also CM and RCM readings. Nitrogen nutrition indices varied from 0.58 to 1.25 and were affected by the growing season, N treatments, and developmental stage. Similar ranges have been reported for a number of different crops such corn (Ziadi et al., 2008a; approximately 0.29-1.3; Plénet and Cruz 1997; approximately 0.55-1.45; and Justes et al. 1997; approximately 0.45-1.30), annual ryegrass (Marino et al., 2004; 0.4-1.6), spring wheat (Ziadi et al., 2010; 0.34-1.43) and durum wheat (Debaeke et al., 2006; 0.25-1.5). Values of NNI ≥ 1.0 indicate that N supply to the crop is nonlimiting or in excess, while values of NNI < 1.0 indicate N deficiency. Nitrogen nutrition indices were generally significantly affected by N fertilization. This increase in NNI values with increasing N fertilization has been reported in corn and other crops but has not reported in linseed (Plénet and Cruz, 1997; Justes et al., 1997). NNI is recognized as a reference method for detecting N deficiency in wheat and corn (Ziadi et al., 2008a; Justes et al., 1997). NNI can be used as a priori diagnosis of plant status during crop growth to determine the necessity of applying additional fertilization. However, a major difficulty in using the NNI as a diagnostic tool is the need to determine the actual crop biomass and its N concentration. Therefore, it was suggested for many crops and it seems that the same exists for linseed that NNI can be used as a reference for simpler procedures (eg chlorophyll measurements, leaf transmittance or nitrate concentration in stem base extract) to determine crop N status (Justes et al., 1997; Prost and Jeuffroy, 2007; Naud et al., 2009). The NNI was also used in crop models to account for the effect of N on growth and yield of winter wheat (Devienne-Barter et al., 2000).

4.5 Nitrogen efficiency and its components

Nitrogen use efficiency and its components were generally higher in 2006 than in 2007. NUE was decreased with increasing N fertilization rate (Marino et al., 2004). Genotype differences were observed in NUEs with Creola to have the highest values in both years compared with the other two cultivars (Livia and Lirina). NUE was negatively correlated with seed yield and it was also negatively correlated with the relative seed yield, indicating that high yield was not associated with more efficient exploitation of N. Additional correlation analysis indicated that there was a negative correlation between NUE and CM and RCM readings. These relationships suggest that high yield is the result of better exploitation of N or high seed N concentration, high CM and RCM readings, and consequently, may be accompanied by low NUEs. Therefore, linseed breeders should select for both high seed yield and NUEs in order to ensure an improvement in both traits.

Nitrogen uptake efficiency was higher for 2007 than for 2006. Under field conditions, soil N availability shows high spatial and temporal heterogeneity that affects plants' N uptake. During the winter and early spring of 2006 lower rainfall during seed filling period (especially in May) could have determined short periods of soil water deficiency and, consequently, a decreased N uptake of the applied fertilizer (Bloom et al., 1985). In contrast, the higher water availability during the 2007 growing season in relation to the same period of 2006 might have favored a greater N uptake from the fertilizer applied. The uptake rate of a given element depends on its external concentration and on the plants absorption capacity (Lee, 1993). It was found that the maximal nutrient absorption capacity of plants is higher than that required to obtain the maximum yield (Jarvis and Macduff, 1989). Justes et al. (1994) observed that, for a certain amounts of aerial biomass, the N concentration could be up to 160% of the N concentration considered critical. Nutrients such as N can be accumulated (stored) in plants during periods of external abundance and consumed in subsequent growth when they are externally limited (Bloom et al., 1985).

Higher values of N utilization efficiency were found in 2007 than in 2006. This means that a higher amount of biomass and seed yield per unit of N uptake was produced in 2007. The amount of N uptake required to produce the maximum seed yield was estimated as 11.93 and 9.87 kg N ha-1 for 2007 and 2006, respectively. According to the previous discussion, the lower N utilization efficiency during 2006 in relation to 2007 reflects a luxury consumption in 2007. In other words, in 2007, plants acquired N in excess for its current growth.

4.6 Component analysis of the different traits

Analysis of the log NUE and its components N utilization efficiency and N uptake efficiency revealed differences in the magnitude of the contribution of each component to the variation in NUE among genotypes, growing season, and also among the N treatments. Nitrogen utilization efficiency was the most important component of NUE and accounted for more of the variation of N use efficiency than the N uptake efficiency and was higher at the 0 kg N ha-1 than the 80 kg N ha-1 in both years. Like Moll et al. (1982), Dhugga and Waines (1989), Ortiz-Monasterio et al. (1997), and the present study showed that the contribution of N uptake efficiency and seed N utilisation efficiency were dependent on N level. Ortiz-Monasterio et al. (1997) found that N uptake efficiency accounted more for the variation in N use efficiency at the control than at the N fertilization treatments which disagrees with the present study where the N utilization efficiency accounted more for the variation of the N use efficiency. When N is rare, the ability to absorb N is certainly of paramount importance and would then be related to root characteristics. It may be hypothesised that differences for the ability to explore the soil or to absorb N existed in the material that were tested. When N is not the limiting factor, N utilisation efficiency have to be more determinant as N will be available for each genotype independent of the efficiency of their root system. Ortiz-Monasterio et al. (1997) proposed selecting in medium-high fertility environments to improve for both low and high fertility conditions. NUEs was negatively correlated with (leaf + stem) N concentration at maturity, CM and RCM readings suggesting that low straw N concentration and CM and RCM readings may be indication of higher NUEs.

4.7 Correlations

Linseed is a species that has not been studied extensively and many characteristics that are used to describe responses to different treatments are not known, e.g. N deficiency or use of N to increase the productivity and the selection of new cultivars. Therefore, it is important to know whether the selected characteristics that were chosen in this study can be used to describe the response of other cultivars to N deficiency, or whether we can use them to improve selection of new cultivars and hence increase productivity. Also the characteristics that were studied can be used for the diagnosis of N requirements in linseed and also the relationships between these characteristics can help us for better N management and for increasing N use efficiency and use N more sustainable. It was found a linear relationship between CM readings at anthesis and at green capsule stage with NNI, seed yield, and relative seed yield. NUE was negatively correlated with seed yield indicating that there is a need for more research to try to understand better those characteristics and try to increase both in new cultivars. This is the first report were the effect of N supply on certain physiological characteristics was determined for linseed and also their relationship with seed yield. It is obvious that CM can be used as a tool for selection of new cultivars with high yield. However, more research is needed to explore these tools for linseed breeding and also for better linseed management especially under rainfed conditions.

5. Conclusion

Nitrogen is one of the most important nutrients needed for plant growth and development and is important to use diagnostic methods for determining N deficiency and ensuring high seed yield. CM and RCM readings were affected by the N treatment and were higher at both N levels compared with the control and can be used for determining N status of the plant. Nitrogen nutrition indices varied from 0.58 to 1.25 across years, growth stage and cultivar and were affected by the fertilization level. In addition, NUE and its components N uptake efficiency and N utilisation efficiency were affected by N fertilization and were negatively correlated with most of the characteristics. Moreover, CM and RCM readings at anthesis were correlated with NNI, seed yield, and relative seed yield indicating that CM and RCM can be used as diagnostic tools for N requirements. In conclusion, the interrelations found among the various NUE-related traits suggest that using simple selection criteria to improve NUE of linseed might have negative implications on seed yield and quality. Therefore, evaluation and selection of different genotypes for NUE should be based on multiple criteria rather than just one criterion and also should be accompanied by evaluation for seed yield.

Acknowledgements

The author is grateful to Deutsche Saatveredelung Lippstadt-Bremen GmbH Zu Lippstadt and De-Vau-Ge Gesundkostwerk GmbH for providing seeds for the experiment.