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1 This article was downloaded by:[usda Natl Agricultul Lib] On: 26 March 2008 Access Details: [subscription number ] Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Plant Nutrition Publication details, including instructions for authors and subscription information: Nitrogen Use Efficiency in Cacao Genotypes M. A. Q. Ribeiro a ; J. O. da Silva b ; W. M. Aitken c ; R. C. R. Machado c ; V. C. Baligar d a Department of Physiology Cepec/Ceplac, Itabuna, Bahia, Brazil b Center of Science and Technology and Agriculture, UENF, RJ, Brazil c Almirante Centre of Study of Cacao, Itajuipe, Bahia, Brazil d USDA-ARS-Sustainable Perennial Crop Laboratory, Beltsville, Maryland, USA Online Publication Date: 01 February 2008 To cite this Article: Ribeiro, M. A. Q., da Silva, J. O., Aitken, W. M., Machado, R. C. R. and Baligar, V. C. (2008) 'Nitrogen Use Efficiency in Cacao Genotypes', Journal of Plant Nutrition, 31:2, To link to this article: DOI: / URL: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Journal of Plant Nutrition, 31: , 2008 Copyright Taylor & Francis Group, LLC ISSN: print / online DOI: / Nitrogen Use Efficiency in Cacao Genotypes M. A. Q. Ribeiro, 1 J. O. da Silva, 2 W. M. Aitken, 3 R. C. R. Machado, 3 and V. C. Baligar 4 1 Department of Physiology,Cepec/Ceplac,Itabuna, Bahia, Brazil 2 Center of Science and Technology and Agriculture, UENF, RJ, Brazil 3 Almirante Centre of Study of Cacao, Itajuipe, Bahia, Brazil 4 USDA-ARS- Sustainable Perennial Crop Laboratory, Beltsville, Maryland, USA ABSTRACT Cacao (Theobroma cacao L) is mostly grown on soils with low natural fertility. On such soils nitrogen (N) is one of the most yield limiting nutrients for cacao. Information is lacking on N use efficiency in cacao. A greenhouse experiment was conducted to evaluate growth response and N use efficiency by two cacao genotypes. The genotypes used were TSH-565 and and N rates adapted were 0, 120, 240, 360, and 480 mg N /pot. In both genotypes, increasing levels of applied N improved growth (stem girth, dry weight of shoot and roots and shoot/root ratio), and concentration and uptake of N. Genotypes differed significantly for stem girth and ICS -9 produced greater stem girth compared with TSH-565. Nitrogen uptake had a linear relationship with root dry weight of the two genotypes. In both genotypes, increasing levels of applied N overall increased N-uptake efficiency (NEFF = N concentration in shoot x shoot/root), but decreased N- use efficiency by shoot and roots (NUE = g dry matter of shoot or root/mg N) and N- use efficiency of carbohydrate (NUEC = mg of total carbohydrates in shoot/mg of N in shoot). Both genotypes responded differently to applied N, despite the existence of close genetic relatedness between them. The method used here appears to be suitable method for identification of cacao genotypes that are efficient in uptake and utilization of N. Keywords: Theobroma cacao L, N uptake efficiency, N-use efficiency of carbohydrate Stem girth INTRODUCTION Even though cacao is grown on a wide range of soils, the most of these soils are nutrient poor (Wood and Lass, 2001). In Brazil, cacao is grown mainly on Received 27 November 2006; accepted 15 April Address correspondence to V. C. Baligar, USDA-ARS- Sustainable Perennial Crop Laboratory, Beltsville, MD , USA. vbaligar@asrr.arsusda.gov 239

3 240 M. A. Q. Ribeiro et al. Alfisols, Oxisols and Ultisols often strongly leached sandy silt to sandy clay soils with, moderate percent base saturation (38 70%), acidic ph ( ), and low available phosphorus (P) (<15 g/g), low N (0.4 to 1.8 mg/g), low organic carbon (C) (1 to 18 mg g 1 ) and other essential nutrients. (Wood and Lass, 2001). With exception of potassium (K), there is a decline in all-essential nutrients in soils under long term cacao cultivation (Wood and Lass, 2001; Wilson, 1999). In Bahia, Brazil, early planting of cacao was done on Oxisols that were of medium to high natural fertility, but recent expansion of cacao cultivation was done predominately on soils with low natural fertility. Such soils require fertilizer application for a reasonable cacao production which results in increased fertilizer input cost. Low cacao prices coupled with the cacao disease, called Witches broom, have led farmers to reduce applications of high cost fertilizers and this has resulted in a further decline of cacao productivity. Long term cultivation, lack of fertilizer inputs, leaching losses and low soil organic matter content have contributed for reduced soil fertility, especially low levels of soil N. Cabala-Rosand et al. (1989) states that under field condition the most common deficiencies noted in cacao are N, K, zinc (Zn), iron (Fe), and boron (B). Nitrogen is the most limiting nutrient for crop production in many of the tropical soils, and its efficient use is important for the economic sustainability of cropping systems (Fageria and Baligar, 2005). It is well known for other plant species that, factors such as ph, water deficit, and/water logging in soils limit nutrient availability resulting in reduced crop production. However, inherent plant traits may also be limiting factors of productivity, even under conditions of adequate essential nutrient availability in soil (Swietlik, 1989). Due to low-uptake, translocation, and/or inefficient nutrient utilization, several crops show nutrient deficiency symptoms and low productivity even when essential nutrients availability in the soil is adequate (Hacisalihoglu et al., 2001). Nutrient use efficiency is defined as the ability of a plant to maintain a good growth and yield on a nutrient poor soil (Graham, 1984). Usually, fertilizer application rates to cacao are based on results obtained from chemical analysis of the soil for essential nutrients and field trials with and without fertilizer (Cabala-Rosand, 1999). But such determinations do not allow for an adequate evaluation of efficiency and/or sufficiency of nutrient uptake and utilization or to ensure optimal growth and productivity. Cacao trees appear to respond more promptly to applied P and N than K, mainly in early developmental stages of their growth (Cabala-Rosand et al., 1982). However, response to applied K by cacao in Amazon region has been reported (Acquaye et al., 1965; Ojeniyi and Egbe, 1985). Cabala-Rosand and Mariano (1985) observed clear differences in vegetative growth among four catongo cacao hybrids and varieties in response to P application. Differences in N uptake and utilization efficiency among and within field crop species has been extensively investigated (Fageria and Baligar, 2005). Information on genotypic differences for N uptake and use efficiency among plantation crop species such as cacao is still lacking.

4 N Use Efficiency in Cacao 241 Cacao genotypes that are efficient N utilizers are most desirable because they can produce well at low soil N levels and respond well to applied N. Many of the agricultural soils in the world are deficient in one or more of the essential nutrients to support healthy and productive plant growth. Therefore, there is a compelling need to increase nutrient use efficiency in crops especially with long duration crops such as cacao (Baligar et al., 2001). The only alternative solution that seems desirable for successful crop production on infertile tropical soils is to adapt plants to the soil that we have rather than altering the soil to fit the plant needs (Chude, 1988). High cost soil amendments are needed to improve the soil, and such practices are unworkable solutions for resource poor farmers of tropical regions. Suitable cacao cultivar that grow and produce well at lower levels of soil nutrients may be an important component of a cacao improvement program aiming to increase its productivity. In spite of experimental findings and economic importance of fertilizer for sustainable cacao production, information is still lacking on genotypic difference in nutrient uptake, translocation and utilization in cacao (Dominguez, 1985). So, understanding morphological traits, physiological mechanisms, and biochemical processes that control nutrient use efficiency in cacao may be a key step in the selection of suitable genotypes for the improvement of cacao yields in infertile tropical soils. The objective of this greenhouse experiment was to evaluate the responses of two cacao genotypes to varying N levels and assess genotypic difference for uptake and use efficiency of N. MATERIALS AND METHOD Genotypes Two cacao genotypes TSH-565 and were used in this study. The genotype TSH-565 (Trinidad Selected Hybrids), was the product of a crossing program developed by the Department of Agriculture of Trinidad and Tobago. TSH-565 is a clone derived from a cross between SCA-6 and ISC-1 and has intermediate yield potential (>600 kg ha 1 ). Genotype (Imperial College Selections) is derived from tree selection from cross between Trinitarios and is late maturing and low yielding clone (<300 kg ha 1 ). Seedlings of both genotypes were obtained from seeds of previously protected pods, but from natural pollination. Seeds were planted in washed sand in small polyethylene cups and after fifteen days were transferred to plastic pots containing 6 kg of washed sand. The application of nutrient solution started 10 days after the loss of cotyledon leaves at this stage plants were 40 days old. Growth Conditions Experiments were carried out under green house condition for seven months from the initial transplanting by planting in a six-kg capacity plastic pots. Each

5 242 M. A. Q. Ribeiro et al. pot received one cacao seedling. During the growth, green house temperature varied from C and relative humidity varied from 70 90%. Plants grew, under artificial shade, and received nine hours of 60% full sunlight. There were no serious occurrences of pests and diseases during the experiment, therefore any fungicides or insecticides were applied. Plants were harvested 7th month after transplanting into the larger pots. Roots and shoot were separated, washed in distilled water and rinsed with deionized water. Shoot and root from individual seedling were separately oven dried at 65 C for 72 hours and dry matter was recorded. Nitrogen was analyzed by the Kjeldahl method as described by Kiehl (1985). Total carbohydrate contents were determined by the method suggested by Clegg (1956). Nitrogen Levels Five levels of nitrogen were used at rate of 0, 120, 240, 360 and 480 mg N/ pot. Nitrogen was supplied as N-nitrate (NO 3 )+ N-ammonium (NH+ 4 ) and levels of these in a Hoagland nutrient solution were (mmol L 1 ) ; ; ; ; and , respectively. Desired levels of N were achieved by adding nutrient solution at the rate of 60 ml per pot every 10th day with a total of 17 applications. Hogland nutrient solution (Monte et al., 1985) also contained (mmol L 1 ) 0.26 P, 1.51 K, 1.25 calcium (Ca), 0.5 magnesium (Mg), 0.5 sulfur (S), and (µmol L 1 )of5.5 B, 0.08 copper (Cu), Fe, 1.46 manganese (Mn), 0.05 molybdenum (Mo), and 0.46 Zn. The seedlings were watered every other day with deionized water. After each watering, leachate from the pot was recovered in a plate placed under each pot and put back into the respective pot. A complete randomized design was adopted with a factorial arrangement consisting of two genotypes, five N levels and four replications (2 5 4). Data analysis was undertaken using SAS (SAS, Inc.,Version 8.1, Cary, North Carolina). Nitrogen Use Efficiency Parameters Nutrient uptake efficiency [(NEFF), N-concentration in shoot, (mg/g) shoot/root ratio] was defined by Soon (1992) for estimating Phosphorus transport to shoot in other plant species. It was adopted here for nitrogen as NEFF (mg g 1 ). Nitrogen-use efficiency (NUE) = units of shoot or root dry matter produced per unit of N present in the shoot or in root (g mg 1 ). Nitrogen-use efficiency for carbohydrate (NUEC) = units of total carbohydrates in shoot per unit of N present in shoot (mg mg 1 ). Nitrogen concentration = N content per unit of plant tissue dry matter (mg g 1 ). Nitrogen-uptake = N concentration plant tissue dry matter (mg plant 1 )

6 N Use Efficiency in Cacao 243 RESULTS AND DISCUSSION Plant Growth Parameters In both of the genotypes, increasing N levels improved stem girth, shoot and root dry weight and shoot/root ratio (Table 1). In both the genotypes, stem girth and shoot (stem and leaves) dry weights were significantly affected (P = 0.05) by N levels up to 240 mg N/pot (Table 2). Although had shown higher values of shoot dry weight than TSH-565, no significant differences were observed between the two at higher N levels. Increasing N - levels from120 mg to 480 mg N/pot, stem girth of increased over TSH-565. It has been reported that under field conditions, mature trees of showed larger trunk cross-section area (94.6 cm) when compared to TSH-565 (75.6 cm) (Daymond et. al., 2001). For, a significant increase in root dry weight was observed only between the N levels 0 to120 mg N/pot, where as for TSH-565 significant difference in root weight was observed from 0- to 240 mg N/pot. Genotype TSH- 565 appears to require more N for the root growth. Over all, at all N levels, gave higher root weight than TSH-565 although the differences were only significant at 360 and 480 mg N/pot (Table 2). Shoot/root ratio showed a contrasting pattern from that of root wt, i.e., significantly higher values were observed for TSH-565. The lower shoot/root ratio for are related to higher root dry matter shown by this clone, since among both genotypes and all levels of N there was no significant difference observed for shoot dry weight (Table 2). Table 1 Growth parameters of two cacao genotypes under different nitrogen levels Nitrogen levels Stem girth Shoot dry matter Root dry matter Shoot/root (mg pot 1 ) Genotypes (cm) ( g plant 1 ) (g plant 1 ) ratio 0 TSH c 7.68 c 4.07 b 1.87 b 3.04 b 7.92 d 4.17 b 1.90 ab 120 TSH bc b 5.57 ab 2.83 a 3.58 ab c 7.87 a 1.82 b 240 TSH ab b 7.40 a 2.53 ab 3.97 a b 8.67 a 2.34 ab 360 TSH a a 8.00 a 2.95 a 4.07 a a a 2.43 ab 480 TSH a a 8.03 a 3.20 a 4.19 a a a 2.57 a Data are means from four replications. For any given N levels, means for the genotypes within a column not followed by the same letter differ at the 0.05 level of probability by Tukey s test.

7 Table 2 Statistical significance for growth parameters and concentration, uptake, NUE, NEFF and NUEC Nitrogen levels Stem girth Shoot DW. Root DW. Shoot/root N-conc. root N-Conc. shoot (mg pot 1 ) Genotypes (cm) (g plant 1 ) (g plant 1 ) ratio (mg g 1 ) (mg g 1 ) 0 TSH-565 NS NS NS NS NS NS 120 TSH-565 NS NS NS * NS NS 240 TSH-565 * NS NS NS NS NS 360 TSH-565 NS NS * * * NS 480 TSH-565 NS NS * * NS * Nitrogen levels N-Uptake Root N-Uptake Shoot NUE Root NUE Shoot NEEF NUEC (mg pot 1 ) Genotypes (mg plant 1 ) (mg plant 1 ) DW (g mg 1 ) DW (gmg 1 ) (mg g 1 ) (mg mg 1 ) 0 TSH-565 NS NS NS NS NS NS 120 TSH-565 NS NS NS NS NS NS 240 TSH-565 NS NS NS NS NS NS 360 TSH-565 * NS * NS NS NS 480 TSH-565 * NS NS * NS * For any given N levels statistical significance (F test) between two genotypes is given in each column. NS = no significant difference between genotypes within N level; * significant at

8 N Use Efficiency in Cacao 245 Nitrogen Concentration and Uptake With the exception of 120 mg N/pot, concentration of N in roots of both genotypes was not significantly affected by the levels of N applied (Table 3). Invariably at all levels of applied N, had higher N concentration in roots than TSH-565; however, significant differences between the two genotypes were only observed at N levels of 360 mg N/pot (Table 2). Nitrogen-concentration in shoots followed a similar trend as that of roots, but a significant increase for TSH-565 was observed only for the 480 mg N/pot treatment (Table 2). Significant differences in shoot N concentration for were observed between Nlevels of zero and 360 and 480 mg N/pot (Table 3). Differences between genotypes for shoot N concentration were significant only at 480 mg N/pot (Table 2). Results of N-uptake in roots and shoots followed similar pattern as that observed for concentrations of N in roots and shoots (Table 3). Both genotypes responded to the increased level of N application. But at all levels of applied N, had higher N uptake in roots than TSH-565, but the differences were significant only at 360 and 480 mg N/ pot (Table 2). Such differences were probably due to higher root dry weight of (Table 1). Cabala-Rosand and Mariano (1985) found a close relation between P- content in cacao seedlings and root size, e.g., cultivars with larger root length and surface area absorbed much higher P from the growth medium. The same inference may be applied Table 3 N-concentration and N-uptake in the roots and shoot of two cacao genotypes under different N-levels N- Concentration (mg g -1 ) N-Uptake (mg plant 1 ) N- levels (mg pot 1 ) Genotypes Root Shoot Root Shoot 0 TSH a b b d 7.35 ab b c c 120 TSH a b ab c 7.00 b ab bc b 240 TSH a b ab c 8.20 ab ab b b 360 TSH a 16.90b a b 9.90 a a a a 480 TSH a a a a 9.30 a 18.27a a a Data are means from four replications. For any given N levels, means for the genotypes within a column not followed by the same letter differ at the 0.05 level of probability by Tukey s test.

9 246 M. A. Q. Ribeiro et al. for N-uptake in the roots of. Baligar and Duncan (1990) state that length, thickness, surface area and volume of roots have a profound effect on a plant s ability to acquire and absorb nutrients from the soil. In case of large trees and perennial plant species, like cacao, nutrient concentration in the roots may not be an appropriate parameter for screening efficient cacao genotypes, at least in young growth stages since nutrient concentration is also influenced by growth conditions and plant size. In both genotypes regression analysis showed a significantly close relationship between N-uptake (defined here as the amount of N present in the shoot) and root dry matter. Significant correlation (r > 0.80) between these parameters was observed for both genotypes (Figure 1), even when higher values of N-uptake in roots for existed (Table 3). Therefore root size may be a promising growth parameter for identification of efficient nutrient absorbing cacao genotypes. At any given root weight, TSH-565 accumulated more N in shoots than (Figure 1). This indicates that TSH-565 may be more efficient as an N-transporter than (Figure 1). However, may be more efficient in N-accumulation in roots than TSH-565. If this is the case, root size may be a promising parameter for detecting genotypes that can take up higher nutrients which could be used to select rootstock with higher nutrient uptake capacity. From the obtained results, it appears that this method could be used to select genotypes that have high capacity to transport nutrients such as TSH-565. In perennial crops, like coffee and cacao, it is highly advantageous to select or breed root stock of genotypes that have efficient root system for higher nutrient uptake capacity (Dechen et al.,1999). Nitrogen Use Efficiency Nitrogen-uptake efficiency (NEFF) is defined and used by Soon (1992) for P transport to shoot. Nitrogen-uptake efficiency was adapted as the formula to estimate N-transport to shoots. In both genotypes, increasing N generally increased NEFF. With some exceptions, at any given N levels TSH-565 recorded higher NEFF than. (Table 4). Since NEFF indicates efficiency of nutrient transport (Soon, 1992), and according to the obtained results, TSH-565 may be more a efficient N transporters than, although no significant differences for NEFF were observed at the tested levels of N (Table 2). The NUE-shoot or NUE-root significantly decreased in both genotypes with increasing levels of applied N (Table 4). Similar to NEFF, with the exception of NUE at 480 mg N/pot there was no significant differences observed between genotypes for NUE. (Table 2). Both genotypes, generally showed decreasing NUE- for root and shoot with increasing N-levels. In relation to N- use for total carbohydrate production (NUEC), a significant difference between genotypes was observed at 480 mg N/pot (Table 2). Overall, both the genotypes NUEC reduced with increasing levels of applied N (Table 4).

10 N Use Efficiency in Cacao 247 Figure 1. Relationships between root dry matter and nitrogen uptake in shoot of two cacao genotypes. On whole plant basis both cacao genotypes incorporated around 72% of the applied N at 120 mg N/pot. But at 360 mg N/pot, used around 20% more N than TSH-565. Overall, absorbed a much higher percentage of N in its roots than TSH-565 (18.5 against 9.4%). The NUE in roots did not change significantly in TSH-565 with increasing N levels (Table 4). The similarity between these two genotypes (TSH-565, ) in relation to several of the response parameters to applied- N, such as shoot dry matter and N- concentration, may be explained by the existence of close genetic relatedness

11 248 M. A. Q. Ribeiro et al. Table 4 Influence of N-levels on N-use efficiency of two cacao genotypes NUEC shoot Nitrogen levels NEFF NUE root NUE shoot DW(CHO)n (mg pot 1 ) Genotypes (mg g 1 ) DW (gmg 1 ) DW(gmg 1 ) (mg mg 1 ) 0 TSH b a a 2.98 a a ab a 3.51 a 120 TSH b a b 1.55 b a a ab 1.53 b 240 TSH b a ab 1.47 bc a ab ab 1.24 b 360 TSH b a bc 1.00 bc a b b 0.98 b 480 TSH a a c 0.66 c a ab b 0.77 b Data are means from four replications. For any given N levels, means for the genotypes within a column not followed by the same letter differ at the 0.05 level of probability by Tukey s test. between them, since TSH-565 is a descendent of a cross between ICS-1 with SCA-6. REFERENCES Acquaye, D. K., R. W. C. Smyth, and R. G. Lockard Potassium deficiency in unshaded Amazon cocoa (Theobroma cacao L.) in Ghana. Journal Horticultural Science 40: Baligar, V. C. and R. R. Duncan. Eds Crops as enhancers of nutrient use. San Diego, CA: Academic Press. Baligar, V. C., N. K. Fageria, and Z. L. He Nutrient use efficiency in plants. Communications in Soil Science and Plant Analysis 32: Cabala-Rosand, P Requirements and fertilisation of cocoa trees in different production systems. In: Proceedings of 12th International Cocoa Research Conference Salvador, Bahia, Brazil. Cabala-Rosand, P. and A. H. Mariano Differential P-absorption in cocoa cultivar. In: Proceedings of 9th International Cocoa Research Conference Lomé, Togo. Cabala-Rosand, P. C., C. L. de Santana, and E. R. Miranda Responses of cocoa trees to increasing doses of applied fertilizers in South of Bahia, Brazil. Revisit Theobroma. (Brazil), 12:

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