The aim of the current study was to determine the chlorophyll relative index (CRI) in different leaves and phenological stages of the crop in order to diagnose the status of nitrogen (N) in corn (Zea mays L.) hybrids as a function of N rates applied in bands correlating them with N content in the leaves and crop productivity. The field experiment consisted of two corn hybrids (P30R50 and AG8025) and 6 N rates applied in bands (0, 75, 150, 225, 300 and 375 kg N ha-1), under a factorial 2 × 6 experimental design, arranged in a randomized block with 4 replications. The dose of 295 kg N ha-1 allowed estimating crop yields corresponding to 13.033 kg ha-1. Hybrids and N rates influenced concomitantly CRI in several leaves and phenological stages. The chlorophyllometer is shown to be quite sensitive to nutritional status in corn hybrids as a response to N rates applied in bands since the early stages of the crop growing season for early diagnosis. At the end of the vegetative phase, as well as the reproductive phenological stage the chlorophyllometer performed well as an indicator of efficiency of nitrogen fertilizer application.
The field experiment was conducted using a no tillage system at the region of Entre Rios, in the municipality of Guarapuava, PR, Brazil [latitude 25º32’S, longitude 51º28’W and altitude of 1,126 m] throughout the period between October 1st and March 20th of 2010. The climate of the site in the study according to the Köppen’s classification is of the humid subtropical type without a dry season and with frequent severe frosts (Peel et al., 2007). Mean annual rainfall in the region is of 1.942 mm. Monthly mean air temperature is of 16.8°C with maximum and minimum values corresponding to 23.1 and 12.4oC, respectively (Simepar, 2011).
Throughout of the crop growing season the overall amount of rainfall was above the 35 year historical average along with variations of 5.1 mm for the month of November and of 114.7 mm for the month of January. Crop rotations over the last year at the experimental area were for the winter/summer year season wheat/soybean (2006), oat/maize (2007), barley/soybean (2008), and in 2009 oat preceded maize crop. The type of soil prevailing in the experiment area is latossol with a depth of roughly 2 m, good physical conditions, and with a high potential for agricultural use. The chemical characteristics of the soil might be seen in the Table 1.
Treatments resulting from the combination of two simple hybrids classified as a precocious cycle for maize - Pioneer 30R50 and Agroceres 8025 (P30r50 and AG8025) - and 6 doses of N to be applied in cover (0, 75, 150, 225, 300 and 375 kg of N ha-1 in the form of urea), comprising a factorial design 2×6 designed in randomized blocks with four replications. Plots consisted of 8 lines 5 m long spaced 0.75 m occupying a total area of 30 m2. Sowing was performed manually on October 1st of 2009 shortly after the incorporation of the 8-30-20 fertilizer formula plus 0.4% of Zn in order to reach a plant population of 69.722 plants ha-1. Cultural practices were implemented according to its occurrence and recommendation for the crop in the field.
Nitrogen doses were applied manually in the total area in just one application on September 9th of 2009 with the plants at V5 stage. Under the V3 stage out of the two central lines 2 plants plot-1were selected and identified for determination of CRI by means of the chlorophyllometer-chlorophy LOG.
Fifteen readings of CRI were made in compliance with the following sequence: 1st reading: Leaf 3 at stage V3 (L3V3); 2nd reading: L5V5; 3rd reading: L3V5; 4th reading: L5V7; 5th reading: L7V7; 6th reading: L7V9; 7th reading: L9V9; 8th reading: L5V9; 9th reading: L9 grain filling stage (L9GFS); 10th reading: corn tassel emission L11CTE; 11th reading: L11R1; 12th reading: L13R2; 13th reading: L15R2; 14th reading: L13R3; 5th reading: L15R3. For all analyzed leaves readings were taken in two different stages, except for the leaf 5, which were made in V5, V7 and V9.
For each evaluation of CRI one single leaf plant-1 was used from four localized points in the central part of the leaf, between the edge and central nerve, obtaining the average from 8 readings per plot (2 plants). For the first assessment of CRI leaves were identified for the second reading, leaf 3 was the one taken into consideration for the identification of the other leaves.
Ten leaves per plot at the R1 stage were used for the determination of leaf N content, always taking into account the below and opposite leaf oriented to the primary ear according to Malavolta et al. (1997). Leaves were stored in plastic bags, dried in a greenhouse with air circulation at 65°C up until the constant DPS was reached, then ground in a knife mill and analyzed by the semi-micro Kjeldhal method. Final productivity was assessed by means of the manual harvest of the ears from a useful area of 13.5 m2, mechanical threshing, determination of DPS and extrapolation of the values for kg ha-1, correcting it to a water content of 13%.
Experimental data obtained from each variable was subjected to analysis of variance through the SAS statistical program (SAS, 2008). Whenever the interaction between the hybrids and N doses was significant, and also when the effect of N doses was observed, a study of regression was carried out by means of illustrations provided by graphs made by an Excel program. The degree of correlation and agreement between the variables measured herein was expressed by the coefficient of the Pearson correlation.
Interactions between the maize hybrids and doses of N to be applied in bands for the variable CRI occurred throughout the phenological stages: L05V7, L07V7, L07V9, L09V9, L11VT, L11R1, L13R2, L15R2 e L15R3. Subedi and Ma (2005) obtained interactions only for the CRIs whenever N was applied in bands at different phenological stages of maize hybrids at V8 four weeks after flowering.
A significant influence of the hybrids was observed over the CRIL03V4, CRIL05V9, CRIL11R1, CRIL13R2, CRIL13R3 and CRIL15R2. However, this occurred with a higher frequency for the determinations performed throughout the reproductive phase. The only assessment of the CRI performed shortly before the application of N in bands was CRIL03V4. Nevertheless, the maize hybrid P30R50 showed a CRIL03V4 higher in 2.24 in relation to the AG8025 (Table 2). Average values obtained for the hybrids P30R50 and AG8025 was higher and similar to a CRI of 45.4% as cited by Argenta et al. (2003) at the stage V3 to V4, indicating that the content of N in the plant is quite adequate. The reading CRIL03V4 did not correlate with the N content in the leaf, as well as with yield (Table 3), corroborating with Argenta et al. (2002) and Hurtado et al. (2010).
The CRIL03V5 was remarkably affected by the doses of N applied in bands to the leaves, observing, therefore, an increasing linear effect (Figure 1a), occurring 4 days after its application (DAA). According to Godoy et al. (2007), a CRI of 46.6 between V4 and V5 triggered a more pronounced accumulation of DPS in maize. Once the mean value obtained for CRIL03V5 was of 46.87, such a value may be considered as perfect for the phenological stage in question.
The CRIL03V5 responded to the doses of N; however, during the assessment of the CRIL05V5 such a response did not show any statistical significance. This might be due to the fact that F05 is a very young phenological stage, suggesting then that the N added to the tissues will be utilized first to increase the number of cells of new leaves in conjunction with the cellular differentiation process. This final process will enhance the production of chloroplasts - the site of synthesis of chlorophyll - being confirmed during the evaluation of CRIL05V7 and CRIL05V9. Since the F03 was to be in a more advanced growth stage along with the fact that its final size was not reached at all, its cells were at a more advanced differentiation stage, possessing more chloroplasts per cell and as a result of that having the synthesis of chlorophyll intensified by the supply of N. First of all N is used to assure cellular division plus cellular differentiation processes in the new leaves of the plants. Throughout the differentiation process cells get their perimeter enlarged, as well as the number organelles such as chloroplasts and photosynthesizing parenchyma. As a result of that, the content of chlorophyll rises concomitantly with cellular differentiation from tissue to organ. In this particular case, the organ in consideration is the leaf, being therefore incorporated into the membranes of the thylakoids as reaction centers called components of photo systems II and I (Taiz and Zeiger, 2013).
Mean values of CRIL03V5 (46.87) and CRIL05V5 (55.69) were to be very close to that one obtained by Hurtado et al. (2010) at V4 to V5 phenological stages, causing the maize crop to reach yields of 11.300 kg ha-1 found by such scientists, as well as by the current research (roughly corresponding to 11.684 kg ha-1). However, either CRIL05V5 or mean yields were similar to the outcomes obtained by these authors. This might be explained by the fact that the amount of 32 kg ha-1 of N applied to the crop during the sowing date was sufficient to meet the initial physiological needs of the plants up to the stage of V5, as well as owing to the fact that soil type in study possessed a content of organic matter capable of making available a higher amount of N to the soil solution. CRIL03V5 correlated positively with leaf N content and yield, showing coefficients equivalent to 0.42** and 0.36*, respectively, as opposed to the CRIL05V5 (Table 3). Similarly, Hurtado et al. (2010) observed a positive correlation between CRIV4-V5 with yield.
Nitrogen doses applied in bands resulted in a linear increase of CRIL05V7 (Figure 1b). Mean CRIL05V7 was of 63.7, regardless of the hybrid and N dose in consideration, revealing a superiority of 22.26% in relation to the CRI of 52.1 obtained by Argenta et al. (2003) for maize at V6 to V7, which corresponded to a rise of 6.7% in productivity. With a rise in availability of N an increment of CRIL05V7 occurred in conjunction with a rise in yield, a fact that might be firmed up by the positive correlation established between CRIL05V7 readings and leaf N content, as well as productivity (Table 3).
The P30R50 hybrid showed a CRIL05V9 3.8% higher in relation to the one yoked to the AG8025 hybrid, corresponding to a CRI difference of 2.33 (Table 2). Genetic variability between maize hybrids and discrepancies in chlorophyll synthesis was also observed by Sunderman et al. (1997) and Subedi and Ma (2005). In so far as N in bands increased up to an estimated dose of 325 kg of N ha-1 CRIL05V9 increased as well, resulting in a fraction of 66.73 of CRIL05V9 (Figure 1c). CRIL05V9 was associated to a higher coefficient of correlation concerning leaf N content (0.72**) rather than with yield (0.57**) (Table 3).
Nitrogen doses unfolding for CRIL07V7 allowed to identify the differential responsiveness of hybrids to the N doses applied in bands, since the P30R50 hybrid responded well up to an estimated dose of 205 kg of N ha-1, which would be corresponding to a CRI of 65.33, and the AG8025 hybrid responded linearly to the increasing N doses applied (Figure 1d). The estimated value for the hybrid P30R50 was superior to the mean value of 59.7, a fact that was evidenced also by Hurtado et al. (2010) between V7 and V8.The correlation of CRIL07V7 with leaf N content and yield was of 0.39** and 0.33**, respectively, being therefore quite below the one for CRIL05V9 (Table 3).
Faced with the unfolding of N doses applied in bands within maize hybrids for the variable CRIL07V9, P30R50 did not respond to the N doses, leading to a mean CRI of 65.3, whereas for the AG8025 hybrid there was a consistent rise in the CRIL07V9 up to an estimated dose of 245 kg of N ha-1 when CRI was expected to be of 69.29 (Figure 1e). The coefficient of correlation between CRIL07V9 and leaf N content and yield was the same (0.58**) (Table 3).
The differentiated responsiveness of CRIL07 for the hybrids at all N doses observed between V7 and V9stages might be ascribed to the fact that AG8025 hybrid was more productive than P30R50 (Table 2), once for such a genotype CRIL07 between V7 and V9 was roughly the same. Such a similarity was established throughout the time and for the AG8025 hybrid an increment of 3.9 was achieved in such a fashion as to enhance the positive correlation between the CRI and leaf N content and yield at the stage V9 in comparison to V7 (Table 3). The difference between the CRI values estimated at F07 between the Stages V7 and V9 for the AG8025 hybrid demonstrated that there was an increase in the chlorophyll synthesis due to N incorporation into the tissues.
CRIL09V9 readings obtained for the P30R50 hybrid were not remarkably influenced by the N doses, showing an average of 58.43, whereas for the AG8025 hybrid CRIL09V9 expressed an increasing linear response to the N doses (Figure 1f). CRIL09V9 was strongly correlated to leaf N content in 0.41** (Table 3). The lack of correlation between CRIL09V9 and yield might be attributed to the fact that CRI readings for the P03R50 hybrid were not influenced by the N doses, in spite of the linear responsiveness of the AG8025 hybrid. Nevertheless, low amplitude of CRIL09V9 among the maize hybrids was observed herein. Such a fluctuation translates that while for the P30R50 hybrid the mean value was of 58.43, the estimated CRIL09V9 in 61.7 by means of the fitted equation with a dose of 375 kg of N ha-1 would have been obtained for the AG8025 hybrid.
The mean value of CRIL09V9 was of 57.85 irrespective of the genotype and N dose to be applied, being higher in 9.15% to that one observed by Godoy et al. (2007) between V8 to V9, as well as lower in 8.03% of that obtained by Hurtado et al. (2010) between V9 and V10. The comparisons revealed that the indicator leaf of the phenological stage, no matter how young it is, usually does not respond to N applied in bands as to the CRI likely due to its utilization firstly for cellular division and differentiation. The CRIL09GFS regardless of the hybrid responded linearly to the increment of N applied in bands (Figure 1g). CRIL09GFS readings were positively correlated to both leaf N content and yield, showing coefficients of 0.73** and 0.67**, respectively (Table 3).
The AG8025 hybrid responded in an increasing manner up to the estimated dose of 250 kg of N ha-1 for the CRIL11CTE, corresponding to a CRI of 72.66. The CRIL11CTE obtained for the P30R50 hybrid increased linearly with the increment of N applied in bands (Figure 1h). Since N takes part in the chlorophyll molecule with an overall of four atoms, it is well known that a rise in the requirements of N to the plants up to a certain point provides an increase in the tonality of the green color of the leaves, which will increase the chlorophyll content or CRI, as well as leaf N content and yield. Such a fact might be confirmed by means of the correlation analysis, once the coefficients between CRIL11CTE and leaf N content and yield were of 0.63** and 0.60**, respectively (Table 3).
Argenta et al. (2003) obtained a CRI of 58 in the leaves below and opposite to the stalks at the filling physiological stage of maize crop. Therefore, the mean CRIL11CTE of 67.84 was 16.96% higher. Chlorophyll synthesis is triggered by the light, being iron the co-factor of some enzymes, depending upon N, Mg and carbonic skeletons from the photosynthesis itself, in conjunction with the extrinsic factors (availability of N, Mg, Fe, water, solar radiation, photoperiod, management practices, etc.) and intrinsic factors (genetic variability). Water availability was an average of 8 mm day-1 throughout the month of December during the CTE stage. According to Matzenauer et al. (1998) such a water supply to the maize plants coincides with the crop needs and contributes to the plants in order to synthesize and maintain a high content of chlorophyll, expressed by the CRI.
The AG8025 hybrid is the one that would reach a technical efficiency maximum dose (TEMD) in 375 kg of N ha-1 under an estimated CRIL11R1 of 68.70 (Figures 1h), and for the P30R50 hybrid there was an increment of the CRIL11R1 with N doses applied in bands (Figure2a). The Pearson correlation coefficients between CRIL11R1 and leaf N content and yield were of 0.67** and 0.82**, respectively (Table 3).
The mean value found for CRIL11R1 was of 60.13, an index higher than the mean CRI of 57.9 and lower than the maximum CRI of 68.9, as evidenced by Sunderman et al. (1997) for hybrids of maize at the same phenological stage under controlled water supply conditions. Such environmental conditions led to a mean yield of 14.455 kg ha-1, showing a significant superiority to the maximum estimated yield observed in the current trial as 13.033 kg ha-1 (Figure 3).
For the variable CRIL13R2the unfolding of the interaction revealed a squared response for the P30R50 hybrid, having estimated a technical efficiency maximum dose in 235 kg of N ha-1 and a CRI in 66.37 (Figure 2b). The Pearson correlation coefficients between CRIL13R2 and leaf N content and yield were of 0.65** and 0.80**, respectively. The CRIL13R3 obtained for the AG8025 hybrid was higher in 4.37 for the P30R50 hybrid, corresponding to a value of 6.77% (Table 2). Regression analysis on the CRIL13R3 allowed to estimate the dose that would be quite conducive to the highest CRIL13R3 (75.4) as the one equivalent in 335 kg of N ha-1 (Figure 2c). In the current study, there was a positive correlation of the CRIL13R3 with the leaf N content given by a Pearson correlation coefficient of 0.69**, along with a higher correlation coefficient of 0.86** for yield. Hurtado et al. (2010) obtained in R3 a CRI of 65.6, having been similar to the mean CRI of 66.76, as well as to the values obtained for each one of the hybrids in study (Table 2), but lower than the estimated value as a function of N doses (Figure 2c).
The highest CRIL13R3 obtained for the AG8025 hybrid evidences a more accentuate chlorophyll synthesis (Table 2). The fact that the plants show a higher content of chlorophyll, mainly of chlorophyll a, results in the highest photosynthetic rates. Consequently, yields will be the highest as shown in Table 3, for the AG8025 hybrid was the most productive. In order to reinforce such evidence in terms of physiological responsiveness, it may be observe in Table 3 that the highest Pearson correlation coefficient obtained for yield was just corresponding to the CRIL13R3, indicating that F13 is an important resource of photoassimilates for the grains.
Those doses estimated by means of equations that would be conducive to the maximum CRIL15R2 were of 260 and 275 of N ha-1 for the P30R50 and AG8025 hybrids, respectively, corresponding to a CRI of 71.69 and 77.77 (Figure 2d). The effect of N doses applied in bands for each hybrid on the CRIL15R3, shortly after the unfolding of the interaction, allowed to identify the doses of 375 and 245 kg of N ha-1 as being those that caused the P30R50 and AG8025 hybrids to present the highest values of CRIL15R3 (83.45 and 79.44, respectively) (Figure 2e).
CRI increased significantly throughout the maize crop growth season, corroborating the outcomes obtained by Argenta et al. (2010). Moreover, the results found in the current research, irrespective of the maize hybrid, leaf or phenological stage of the crop, showed higher CRI values than those obtained by Argenta et al. (2010), Godoy et al. (2007), Hurtado et al. (2009) and Hurtado et al. (2010). Fluctuations in the values of CRI reported by the literature might be ascribed to the equipment used, age of the leaf, position of the leaf, position reading on the leaf, phenological stage, cultural practices, and soil and climate conditions.
The mean leaf N content of 26.56 g kg-1 or 2.66% of the DPS determined in the reference leaf R1 was found to be roughly above the adequate threshold (27.5 to 32.5 g kg-1) in agreement with Malavolta et al. (1997). Ferreira et al. (2009) in a site under no-tillage system for 18 years observed for the AG9020, AG6018 and AG8021 hybrids mean leaf N contents of 23.17, 24.13 and 25.53 g kg-1, respectively. Such indices were also above the range prescribed by Malavolta et al. (1997).
The leaf N content determined at R1 was influenced by the N doses applied in bands (Figure 2f) without therefore expressing interactions with maize hybrids. Increments in N supplementation up to the estimated dose as TEMD was of 330 kg of N ha-1, in which the reference leaf would reach a leaf N content of 31.26 g kg-1. Hurtado et al. (2009) obtained a leaf N content of 30.25 g kg-1 by the time TEMD for yield was replaced with 242 kg of N ha-1, corresponding therefore to a yield of 9.210 kg ha-1. In the current work the estimated dose for maximum yields was of 295 kg of N ha-1, culminating to a yield of 13.033 kg ha-1. However, when such a dose was replaced with the one obtained by the fitted equation for the leaf N content (Figure 2f) in compliance with the proposition of Hurtado et al. (2009), the leaf N content to be obtained would be of 31.09 g kg-1, reaching an index quite similar to that found by the aforementioned authors, but with a difference of 3.823 kg ha-1 in the grain yield.
By assessing four distinct field experiments, Argenta et al. (2010) obtained the following Pearson correlation coefficients between the readings of CRI performed in the reference leaf corresponding to the filling stage and maize yield: 0.69*, 0.80*, 0.87* and 0.93*. Table 3 reveals that readings of CRIL11CTE, CRIL11R1, CRIL13R2 and CRIL13R3 were associated to Pearson correlation coefficients of 0.60**, 0.82**, 0.80** and 0.86**, respectively. The correlation coefficient of 0.69** obtained between leaf N content and yield was lower than those coefficients reported by Argenta et al. (2002) in their four field trials (0.73*, 0.76*, 0.83* and 0.91*).
Argenta et al. (2010) obtained better correlations between CRI and leaf N content at more advanced phenological stages of the crop. Nevertheless, it can be noticed that such correlations get stronger with the physiological age of the leaves. This was observed during three evaluations at F05 for there was an increase of the correlation at the stage V5 in relation to the V7, as well from such stage to V9 when the leaves 7, 9, 11 and 13 expressed the same behavior. This physiological responsiveness allows obtaining high correlations throughout the initial crop growth phases, such as vegetative phenological stage. Thus the observation on the age of the leaves for the assessment of CRI readings infers the efficiency of evaluation of N status in corn plants by means of CRI, regardless of the phenological stages related to vegetative and reproductive phases (Table 3).
The AG8025 hybrid productivity was higher than that of P30R50 in 1.510 kg ha-1, corresponding to a difference of 13.8% (Table 2). Argenta et al. (2010) obtained a difference of 2.900 kg ha-1 between the P32R21 and Premium hybrids with final yields of 12.400 and 9.500 kg ha-1, respectively. Discrepancies in productivity among maize hybrids were also found by Ferreira et al. (2009), whose outcomes report that hybrids such as AG9020 and AG8021 were more productive than AG6018.
The identification of the dose 295 kg of N ha-1 that would express maximum yields, equivalent to 13.033 kg ha-1 (Figure 3), corroborates the assumptions of Fontoura and Bayer (2009), since both scientists report that for yields above 12.000 kg ha-1 it is necessary an application within the range from 130 to 300 kg of N ha-1 in bands. The maximum yield estimated in 9.210 kg ha-1 obtained by Hurtado et al. (2009), which corresponded to 242 kg of N ha-1 in bands, resulted in a saving of 53 kg of N ha-1 causing therefore a reduction of 3.823 kg ha-1 in the productivity of grains. Different results reported by the literature might be attributed to differences in climate and soil conditions, management and cultural practices among studied sites in conjunction with genetic variability among hybrids. An inferior responsiveness was also found by Silva et al. (2005) with a TEMD estimated in 166 kg of N ha-1, leading to a final yield of 6.709 kg ha-1.
Over three years of evaluation, Holand and Schepers (2010) evidenced that the dose of 200 kg of N ha-1 brought about yields of 11.530, 12.110 and 13.660 kg of N ha-1 for maize crops. In the work published by Sangoi et al. (2009) the application of two doses of 100 kg of N ha-1 in bands at the phenological stages V4 and V10 resulted in a yield of 12.634 kg of N ha-1 for the P30F53 simple hybrid. Such outcome was quite similar to that obtained for the AG8025 hybrid (Table 2) and for the maximum estimated yield with the dose of 295 kg of N ha-1 in bands (Figure 1) but with only one application at V5. This appears to be an outstanding observation to surmise because P30F53 simple hybrid reveals an important participation in cultivated areas of the region of Campos Gerais of Paraná in order to assure the sustainability of agriculture in the southern regions of Brazil.
