Soratto et al.82 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 NITROGEN FERTILIZATION OF FALL PANICUM CULTIVARS (Panicum dichotomiflorum Michx.): BIOCHEMICAL AND AGRONOMICAL ASPECTS Rogério Peres Soratto1*; Eduardo do Valle Lima1; Tiago Roque Benetoli da Silva1; Carmen Sílvia Fernandes Boaro2; Ana Catarina Cataneo3 1 UNESP/FCA - Depto. de Produção Vegetal, Fazenda Experimental Lageado, s/n, C.P. 237 - 18603-970 - Botucatu, SP - Brasil. 2 UNESP/IB - Depto. de Botânica, 18618-000 - Botucatu, SP - Brasil. 3 UNESP/IB – Depto. de Química e Bioquímica. *Corresponding author ABSTRACT: Information on improved fall panicum cultivars and agronomical pratices is scarce because of the relatively small importance of this crop in Brazil. The present study aimed to evaluate levels of nitrogen sidedressing on plant development and biochemical characteristics of two fall panicum cultivars. The experiment was set up under plastic tunnel conditions in Botucatu, São Paulo, Brazil, with plants growing inside asbestos-cement boxes. The experimental design was a randomized block setup and treatments consisted of six levels of nitrogen fertilization (0, 40, 80, 120, 160, and 200 mg L-1) applied at the onset of tillering on two fall panicum cultivars (AL Mogi and AL Tibagi). The application of increasing levels of nitrogen to fall panicum plants increased chlorophyll content, total amino acids, plant dry matter nitrogen content, plant height, tillering and grain yield, and decreased plant dry matter sugar content. The cultivars had similar performances in relation to the evaluated variables, as a response to nitrogen sidedressing. Key words: nitrogen, sidedressing fertilization, mineral nutrition ADUBAÇÃO NITROGENADA E ATRIBUTOS BIOQUÍMICOS E AGRONÔMICOS DE CULTIVARES DE PAINÇO (Panicum dichotomiflorum Michx.) RESUMO: São escassas as informações sobre cultivares melhoradas e técnicas culturais adequadas à cultura do painço. Este experimento objetivou avaliar o efeito da aplicação de níveis de nitrogênio (N) em cobertura no desenvolvimento e nos atributos bioquímicos de duas cultivares de painço. Para tanto, instalou-se um experimento em Botucatu (SP), em caixas de cimento amianto, mantidas em túnel plástico. O delineamento experimental foi em blocos casualizados, em esquema fatorial 2x6, com dois cultivares de painço (AL Mogi e AL Tibagi) e seis níveis de adubação nitrogenada (0, 40, 80, 120, 160 e 200 mg L-1), aplicada em cobertura, no início do perfilhamento. Os dois cultivares utilizados tiveram comportamento semelhante em relação às variáveis avaliadas, em resposta à aplicação de N em cobertura. A aplicação de doses de N na cultura do painço proporcionou incrementos nos teores relativos de clorofila, aminoácidos totais, nitrogênio total e altura das plantas, e redução dos teores de açúcares na matéria seca, até o intervalo entre 120 e 160 mg de N L-1. O perfilhamento e produção de grãos tiveram aumento linear com a aplicação de N. A adubação nitrogenada não afetou a produção de matéria seca. Palavras-chave: nitrogênio, adubação de cobertura, nutrição mineral INTRODUCTION Fall panicum (Panicum dichotomiflorum Michx.) is an annual grass, grown for grains, which are utilized as animal feed (Furuhashi, 1995), especially for birds in captivity. It is also used by the beer industry, mixed in small proportion to barley. Little known in Brazil and with a reduced economic expression, when compared to traditional crops, fall panicum is being tested as a soil cover crop species in the no-till system (Lima et al., 2000), and could become a crop option during the relay crop season and in the spring-summer period. However, for this plant to become a soil cover crop it is necessary to increase its straw and grain productivities, which can be achieved by the use of adequate cultural practices. Maximum productivity depends on environmen- tal factors, such as temperature, solar radiation and min- eral nutrition, as well as on genotype and cultural prac- tices (Maman et al., 1999). Therefore, nutrient supply is crucial to obtain high productivities (Clark, 1990), espe- cially in relation to N, in general the most required ele- ment by plants (Malavolta, 1979; Maman et al., 1999). Nitrogen fertilization of fall panicum 83 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 When plants show deficiency of this element, they be- come stunted and their leaves assume a coloration be- tween pale green and yellow. Nitrogen sidedressing, in addition to supplying all crop requirements, increases pro- ductivity (Oliveira et al., 1996). Crops such as millet, rice, maize and bean, which usually respond well to nitrogen sidedressing (Costa et al., 1983; Maman et al., 1999; Silva et al., 1999; 2000; Ferreira et al., 2001; Soratto et al., 2000; 2001). Even though no reports evaluating fall panicum were found in the literature, this could also be a species that is respon- sive to N. N is absorbed by the roots, especially in the form of NO 3 -, and is translocated to the leaves, where it is re- duced and incorporated into amino acids (Nelson & Cox, 2000). The process of reduction causes great energy ex- penditure to cells. Great part of this process takes place in leaves, which are responsible for the absorption and storage of light energy during photosynthesis. However, part of the reduction occurs in the root, at the expense of energy from carbohydrates produced in the leaves (Malavolta et al., 1997). In addition, plants utilize car- bon skeletons of sugars for amino acid synthesis (Turley & Ching, 1986). The biosynthetic pathways of amino ac- ids and nucleotides share nitrogen requirements; however, the soluble, biologically useful compounds of this element are scarce in natural environments (Nelson & Cox, 2000), hence the need for sidedressing supplementation. Since fall panicum is a little-known crop in Bra- zil, it is necessary to develop improved cultivars and ad- equate cropping techniques. Therefore, this work had the objective of evaluating the effect of application of dif- ferent levels of N sidedressing on the development and biochemical attributes of two fall panicum cultivars. MATERIAL AND METHODS The experiment was installed in March, 2002, in a plastic tunnel, in Botucatu, SP, Brazil, (22º53’09’’S, 48º 26’42’’W; altitude of 740 m), in asbestos cement boxes of 50 L capacity, with 30 cm effective depth and a hole on the bottom to drain the excess water, filled with a me- dium-textured Oxisol. The chemical characteristics of the soil are: 2 mg dm-3 phosphorus; 23 g dm-3 organic mat- ter; pH in CaCl 2 (0.01 mol L-1) = 4.7; 0.1; 6; 7; 34; 13 and 48 mmol c dm-3 of K, Ca; Mg, H+Al, BS, and CEC, respectively, and V% = 28, according to methodology de- scribed in Raij et al. (2001). The preparation of each box, before filling, con- sisted of preparing a mixture of soil and 62 g dolomitic lime (TNP = 90%) to raise base saturation to 70%, plus 5 mg L-1 N (urea), 150 mg L-1 P2O5 (single superphos- phate), 150 mg L-1 K 2 O, 1 mg L-1 B (boric acid), and 5 mg L-1 Zn as basic fertilization. The boxes were then filled, leveled, and moistened until approximately 80% of the container water capacity, and were then covered with a plastic tarp, remaining under wet incubation for 21 days. After this period, at seeding time, the soil pre- sented the following chemical characteristics: 88 mg dm-3 P; 20 g dm-3 O.M.; pH CaCl 2 (0.01 mol L-1) = 5.4; 2.2; 37; 16; 22; 55 and 77 mmol c dm-3 K, Ca, Mg, H+Al, BS, and CEC, respectively, and V%=71. The experimental was set up in a randomized blocks design, in a 2×6 factorial scheme, with two fall panicum cultivars, AL Mogi and AL Tibagi, and six lev- els of nitrogen fertilization: 0, 40, 80, 120, 160, and 200 mg L-1. Seeding was performed on April 9, 2002, sow- ing 12 seeds per box. Thinning was performed after seed- ling emergence, three days after seeding, leaving nine plants per box. The cycles of the utilized cultivars last from 60 to 80 days, so they can be sown during the regular crop- ping season, from September to December, or during the relay crop season, from January to March. As a major morphological difference, cultivar AL Mogi has open panicles, while those of AL Tibagi are closed. Sidedressing was performed 25 days after emergence (DAE), at the onset of tillering of the fall panicum plants. Irrigations were made by means of watering cans, with a watering interval of two days. Emerged weeds were controlled by hand and pest control was performed with one spray of deltamethrin insecticide at 25 DAE. When plants were at full bloom (44 DAE), the chlorophyll content and plant height were determined. Plants were then harvested for biochemical and physi- ological determinations: relative chlorophyll content, ure- ase activity, total amino acid content, total sugars and re- ducing sugars contents, total N content and dry mass in the above-ground part of the plant. The relative chloro- phyll content was evaluated by means of a portable Chlo- rophyll Meter (Minolta, 1989), model SPAD-502 (Soil and Plant Analysis Development), in ten samples per box, in the middle of the flag leaf. The flag leaf of one plant per pot at full bloom was collected for urease activity determination. Samples (200 mg) were cut into small fragments and transferred to a test tube containing a reaction mixture at 30ºC for 60 minutes (Hogan et al., 1983). Aliquots were extracted from each mixture to determine the amount of ammonia produced in the reaction by the indophenol method (Weatherburn, 1967). Urease activity was expressed as µg of NH 3 formed from urea, per gram of fresh leaf tissue per minute. Also at full bloom, two plants per pot were col- lected and packaged inside paper bags, properly identi- fied, and dried in a forced aeration oven at a mean tem- perature of 60-70ºC, until constant weight, and later, ground in a Willey-type mill to determine total amino ac- ids, nitrogen content, total soluble sugars, and reducing sugars. Total amino acids were extracted from dry mass samples with 80% ethanol at a temperature of 85ºC. Next, Soratto et al.84 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 0.2 mL of the extract and 0.4 mL of ninhydrin reagent were utilized, at a temperature of 85ºC, according to Kabat & Mayer (1967). The absorbance reading was made at 570 nm. The values obtained were compared to a glycine standard curve. The total amino acid content was expressed as percentage of the dry matter. The total soluble sugars and reducing sugars were extracted by water from dry leaves. The determination of soluble reducing sugars followed the technique described by Nelson (1944) using Somogy’s and Nelson’s reagents. Absorbance readings were performed at 545 nm and com- pared to a glucose standard curve. The method described by Dubois et al. (1956) was utilized for the determina- tion of total soluble sugars, by the reaction with phenol and sulfuric acid. Absorbance readings were performed at 545 nm. Results were compared to a glycine standard curve. The total soluble sugars and reducing sugars in the dry mass were expressed as percentages. The total N content in the dry matter of leaves was determined by sulfuric digestion and analysis accord- ing to specifications presented by Sarruge & Haag (1974). Results were expressed as grams of nitrogen per kg of dry mass. Dry mass of plants were expressed as grams per plant. Plant height corresponded to the distance between soil surface and plant apex. The total number of stalks divided by the number of plants per box was also evalu- ated. Plants from both cultivars had a 59-day cycle. Grain productivity was determined based on the panicles from all plants that remained in the boxes after being cut and left to dry in full sun. Grains were then manually removed from the panicles and weighed. Data were transformed into g per plant (13% wet basis). The results were submitted to analysis of vari- ance and the cultivar means compared by the Tukey test (P = 0.05). Means of different levels of N applied as sidedressing were evaluated by regression analysis. The criterion adopted to select a model was the magnitude of the regression coefficients at the 5% level. RESULTS AND DISCUSSION There were no influences of cultivar and of the cultivar × N level interaction on any of the biochemical variables (Table 1). The N levels affected all biochemi- cal variables, except urease activity (Table 1). The rela- tive chlorophyll content fitted a quadratic function (Fig- ure 1); as the level of sidedressing N increased, the rela- tive chlorophyll content also increased, as verified by di- rect reading on the equipment, up to the peak point, which occurred at the 157 mg L-1 N rate. This result corrobo- rates results obtained by Crusciol et al. (2001), who ob- served that the highest levels of N available in the soil and the greater uptake of this element increased the pig- ment content indicating, to a certain extent, that plants did not produce chlorophyll beyond their requirements. Urease activity was not influenced by application of N (Table 1). Similar results were verified by Cataneo (1989), who observed that nitrogen supplementation in the form of nitrate and urea to bean plants did not change urease activity. At the time of determination (full bloom), the enzyme activity was stabilized, since most of the ab- sorbed urea had already been hydrolyzed by urease, yield- ing ammonia, which is utilized for amino acid synthesis by the plants. noitairavfosesuaC llyhporolhC )DAPS( ytivitcaesaerU sdicaonimalatoT sraguSlatoT sragusgnicudeR µ HNg 3 hserfg/ etunim/ssam -----------------------MDni%----------------------- -------------------------------------------seulaVF------------------------------------------- )C(ravitluC sn73.0 sn21.0 sn61.0 sn22.1 sn20.0 )N(NgnisserdediS **03.13 sn94.1 **90.02 **24.6 **32.4 N*C sn44.1 sn82.0 sn80.2 sn44.1 sn94.0 NgnisserdediS .R.L **12.89 sn73.0 **21.44 **12.11 **90.9 .R.Q **10.54 sn13.0 **39.24 **54.71 *90.5 sravitluC igabiTLA 7.03 7100.0 16.6 61.4 65.2 igoMLA 1.13 7100.0 67.6 36.4 26.2 )%(VC 2.6 3.6 9.61 8.82 1.93 Table 1 - Relative chlorophyll content in leaves, urease activity, total amino acid content, total sugars and reducing sugars contents in the above-ground part of fall panicum plants, 44 days after emergence, for two cultivars and different sidedressing N levels. *, **, and ns are, respectively: significant at 5%, 1% and non-significant by the F test. L.R. = Linear regression. Q.R.= Quadratic regression. Nitrogen fertilization of fall panicum 85 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 The total amino acid content fitted a quadratic function (Table 1 and Figure 1), showing a point of maxi- mum at the 134 mg L-1 N rate. A similar result was ob- tained by Mengel & Kirby (1978), who mentioned that the amino acid content increased considerably in maize, as the amount of available N increased. Amino acids are derived from glycolysis intermediate compounds, from the citric acid cycle or from the pentoses-phosphate path- way, and N is incorporated into these pathways via glutamate and glutamine (Nelson & Cox, 2000). There- fore, the greater the amount of N available, the greater the amino acid synthesis, but only up to a certain point, above which inhibition occurs, as observed. Total sugar content and reducing sugar content fit- ted both quadratic equations (Table 1 and Figure 1). Sugar contents decreased as the chlorophyll and amino acids in- creased, up to a certain rate. The opposite occurred between rates of 120 and 160 mg L-1 N, i.e., there was a tendency of an increase in sugar contents as the chlorophyll and amino acid contents decreased. It becomes evident that plants uti- lize sugars both for supplying energy during N reduction, and for the carbon skeletons for amino acid synthesis, as re- ported by Turley & Ching (1986) and Cataneo (1989). Plant height presented differences between cul- tivars and between sidedressing N levels. The height data for sidedressing N levels fitted a quadratic func- tion (Figure 1), and maximum growth was attained at the 138 mg L-1 sidedressing nitrogen rate. Cultivar AL Mogi had higher growth as compared to AL Tibagi, which could cause lodging (Table 2). Figure 1 - Chlorophyll contents in leaves, total amino acids, total and reducing sugars in the above-ground part, plant height, number of stalks per plant, total N content in the above-ground part and grain yield of fall panicum plants, mean of two cultivars, as a function of sidedressing N levels (* and ** are, respectively, significant at 5% and 1%). y = 23.218 + 0.156x -0.0005x 2 R 2 = 0.91** 0 10 20 30 40 0 40 80 120 160 200 R el at iv e ch lo ro ph yl l c on te nt in th e le af ( S P A D ) y = 3.2011 + 0.0802x -0.0003x 2 R 2 = 0.87** 0 2 4 6 8 10 0 40 80 120 160 200 A m in o a ci d c o nt en t i n th e ab ov e- gr ou n d p ar t o f p la nt s (% in D M ) y = 4.0725 - 0.0314x + 0.0001x 2 R 2 = 0.67* 0 2 4 6 8 10 0 40 80 120 160 200 R ed uc in g su ga rs c o nt en t in t he a b ov e- gr o un d pa rt o f pl an ts ( % in D M ) y = 62.814 + 0.138x -0.0005x 2 R 2 = 0.57* 30 40 50 60 70 80 0 40 80 120 160 200 P la nt h ei gh t (c m ) y = 1.259 + 0.0038x R 2 = 0.75** 0 1 2 3 0 40 80 120 160 200 N um b er o f st al k s p er p la nt y = 18.371 + 0.3129x -0.0012x 2 R 2 = 0.85** 0 10 20 30 40 50 0 40 80 120 160 200 T ot al N in t he a b o ve -g ro un d p ar t o f p la nt s (g k g-1 ) Sidedressing N levels (mg L-1) y = 2.1757 + 0.0036x R2 = 0.35* 0 1 2 3 4 0 40 80 120 160 200 G ra in y ie ld (g p er p la nt ) y = 6.6021 - 0.0543x + 0.0002x 2 R 2 = 0.89** 0 2 4 6 8 10 0 40 80 120 160 200 T ot al s ug ar s co nt en t i n th e ab ov e- gr ou nd pa rt o f p la nt s (% in D M ) Soratto et al.86 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 The number of stalks per plant was influenced by the application of sidedressing N (Table 2). Data fitted a linear function, showing that the greater the application of sidedressing N, the greater the number of stalks per plant. This can be explained by the increase in vegeta- tive growth and by the promotion of tillering caused by the great amount of available N. These results agree with those by Purcino (1981), who verified vigorous growth and intense green color when this element was supplied, and when other factors were not limiting. However, dry mass of the cultivars did not vary under different nitro- gen levels or between cultivars, and no significant inter- action occurred between causes of variation, which can be justified by the high coefficients of variation observed (Table 2). The total N content in the above-ground part of the plants fitted a quadratic function, showing a point of maximum for total N content at the 130 mg L-1 N rate (Table 2 and Figure 1). Silva et al. (1999) also observed an increasing response in N content in the above-ground part of bean plants as consequence of sidedressing N ap- plication. Alejo & Crocomo (1981) observed an increase in total N content in the above-ground part of sugarcane as the application of urea increased. Maman et al. (1999) also observed an increase in dry matter N content of a millet hybrid with the application of mineral nitrogen. Ac- cording to those authors, the likely increase in the velocity of synthesis of nitrogen compounds occurred because of the greater NH 4 + availability, resulting from urea hydroly- sis. Since the appropriate total N contents in the above-ground part of fall panicum plants is unknown, the critical level for rice was utilized for comparison (Malavolta et al., 1980; Cantarella et al., 1997). With the exception of the treatment without N (control), which had a content below the critical level (27 g kg-1), plants of all treatments involving N applications presented adequate levels of this nutrient at blooming time, in the aerial part. This probably occurred because plant needs were supplied by the treatments containing N. Yield fitted a linear function as sidedressing N in- creased, grain yield also increased (Table 2). Several pa- pers emphasize the response of different crops, for ex- ample, millet, rice, wheat, maize, and bean, to sidedressing N application (Costa et al., 1983; Maman et al., 1999; Silva et al., 1999; Rodrigues et al., 2000; Silva et al., 2000; Soratto et al., 2000; Ferreira et al., 2001; Soratto et al. 2001). However, Furuhashi (1995), study- ing the effect of rates and seasons of nitrogen sidedressing for fall panicum, concluded that the sidedressing N ap- plication did not influence grain productivity. The absence of a response by the crop to the application of nitrogen can be explained by the fact that fall panicum was grown in succession to black velvet bean, a legume utilized as a green manure with high atmospheric N 2 fixation abil- ity, and according to Quaggio & Raij (1997), this area was characterized as having low response to N. Finally, the greatest differences observed for the evaluated variables were verified between the treatment without nitrogen application (control) and the smallest rate of this nutrient (40 mg L-1), while the differences veri- fied between the other applied rates were smaller (Fig- ure 1). CONCLUSIONS The fall panicum cultivars of this experiment had similar performance with regard to the evaluated vari- ables, as a response to sidedressing nitrogen application. The application of N rates to the fall panicum crop pro- noitairavfosesuaC thgiehtnalP tnalprepsklatS ssaMyrD NlatoT dleiyniarG mc tnalpg 1- gkg 1- tnalpg 1- -------------------------------------------seulaVF------------------------------------------- )C(ravitluC *66.6 sn200.0 sn70.0 sn100.0 sn100.0 )N(NgnisserdediS *04.3 **72.5 sn81.1 **24.74 *32.1 N*C sn94.2 sn93.2 sn16.0 sn42.2 sn42.0 NgnisserdediS .R.L sn54.2 **26.81 sn04.4 **62.101 *71.2 .R.Q *41.3 sn46.2 sn74.0 **25.99 sn90.3 sravitluC igabiTLA b7.66 66.1 90.4 9.13 35.2 igoMLA a9.27 56.1 22.4 9.13 45.2 )%(VC 2.01 3.12 9.53 1.9 1.43 *, **, and ns are, respectively: significant at 5%, 1% and non-significant by the F test. L.R. = Linear regression. Q.R.= Quadratic regression. Means followed by different letters, in the column, differ the 5% level by Tukey test. Table 2 - Plant height, number of stalks per plant, dry mass, N total content in the above-ground part and grain yield of fall panicum plants, for two cultivars and different sidedressing N levels. Nitrogen fertilization of fall panicum 87 Sci. Agric. (Piracicaba, Braz.), v.61, n.1, p.82-87, Jan./Fev. 2004 Received December 05, 2002 Accepted October 23, 2003 vided increases in the contents of relative chlorophyll, total amino acids, total nitrogen, and in plant height, and a reduction in dry matter sugars, up to the interval be- tween 120 and 160 mg N L-1. Tillering and grain yield experienced a linear increase with the application of N. Nitrogen fertilization did not affect dry matter yield. REFERENCES ALEJO, N.O.; CROCOMO, O.J. Biochemical and physiological aspects of sugarcane. II. Effect of NO 3 --N, NH 4 +-N and urea-N on carbohydrate level and growth of cv. NA56-79. Energia Nuclear na Agricultura, v.3, p.137-151, 1981. CANTARELLA, H.; RAIJ, B. van; CAMARGO, C.E.O. Cereais. In: RAIJ, B. van; CANTARELA, H.; QUAGGIO, J.A.; FURLANI, A.M.C. Recomendações de adubação e calagem para o Estado de São Paulo. 2.ed. Campinas: IAC, 1997. p.43-71, (Boletim Técnico, 100). CATANEO, A.C. Efeitos de fontes de nitrogênio (N-Uréia e N-Nítrico) na atividade de urease (E C 3.5.1.5), nos níveis de compostos nitrogenados e de carboidratos em feijoeiro (Phaseolus vulgaris L.) cultivar Carioca, cultivado em solução nutritiva. Botucatu: UNESP/IB, 1989. 96p. (Dissertação - Mestrado) CLARK, R.B. Physiology of cereals for mineral nutrient uptake, use and efficiency. In: BALIGAR, V.C.; DUNCAN, R.R. (Ed.) Crops as enhancers of nutrient use. San Diego: Academic Press, 1990. p.131- 209. COSTA, N.P.; GOMES, A.S.; PESKE, S.T.; POPINIGIS, F.; ZONTA, E.P. Influência da adubação nitrogenada sobre o vigor e conteúdo de proteínas de sementes de quatro cultivares de arroz irrigado. Revista Brasileira de Sementes, v.5, p.31-41, 1983. CRUSCIOL, C.A.C.; ANDREOTTI, M.; LIMA, E.V.; FURLANI JR., E.; NAKAGAWA, J. Teores de nutrientes, concentração de clorofila e produtividade de grãos do feijoeiro em função da adubação nitrogenada de semeadura ou em cobertura. Revista de Agricultura, v.76, p.101- 114, 2001. DUBOIS, M.; GILLES, K.A.; HAMILTON, J.K.; REBERS, P.A.; SMITH, F. Colorimetric method for determination of sugars and related substances. Analytical Chemistry, v.28, p.350-356, 1956. FERREIRA, A.C.B.; ARAÚJO, G.A.A; PEREIRA, P.R.G.; CARDOSO, A.A. Características agronômicas e nutricionais do milho adubado com nitrogênio, molibdênio e zinco. Scientia Agricola, v.58, p.131-138, 2001. FURUHASHI, S. Efeito de doses e de época de aplicação de nitrogênio na cultura do painço em sucessão a mucuna-preta. Jaboticabal: UNESP/ FCAV, 1995. 31p. (Monografia - Graduação) HOGAN, M.E.; SWIFT, I.E.; CONE, J. Urease assay and ammonia release from leaf tissues. Phytochemistry, v.22, p.663-667, 1983. KABAT, E.A.; MAYER, M.M. Experimental immunochemistry. Springfield: C.C. Thomas, 1967. p.560-563. LIMA, E.V.; CAVARIANI, C.; LIMA, P.L.; CRUSCIOL, C.A.C.; NAKAGAWA, J.; VILLAS BOAS, R.L. Qualidade fisiológica de sementes de painço (Panicum dichotomiflorum mix.) em função do tempo de mistura com o superfosfato triplo. Cultura Agronômica, v.9, p.177- 189, 2000. MALAVOLTA, E. ABC da Adubação. São Paulo: Ceres, 1979. p.26-30: Adubos nitrogenados MALAVOLTA, E.; DAMIÃO FILHO, C.F.; VOLPE, C.A.; MACHADO JR., G.R.; VELHO, L.M.S.; ROSA, P.R.F.; DE LAURENTIZ, S. Deficiências e excessos minerais no feijoeiro (Phaseolus vulgaris L., cv. Carioca). Anais da ESALQ, v.37, p.701-718, 1980. MALAVOLTA, E.; VITTI, G.C.; OLIVEIRA, S.A. Avaliação do estado nutricional das plantas: princípios e aplicações. 2.ed. Piracicaba: POTAFOS, 1997. 319p. MAMAN, N.; MASON, S.C.; GALUSHA, T.; CLEGG, M.D. Hybrid and nitrogen influence on pearl millet production in Nebraska: yield, growth, and nitrogen uptake, and nitrogen use efficiency. Agronomy Journal, v.91, p.737-743, 1999. MENGEL, K.; KIRBY, E.A. Principles of plant nutrition. Bern: International Potash Institute, 1978. 593p. MINOLTA CAMERA. Manual for chlorophill meter SPAD 502. Osaka: Minolta Radiometric Instruments Divisions, 1989. 22p. NELSON, N. A photometric adaptation of the Somogyi method for determination of glucose. Journal Biological Chemistry, v.153, p.375- 380, 1944. NELSON, D.L.; COX, M.M. Lehninger principles of biochemistry. 3.ed. New York: Worth Publishers, 2000. 1152p. OLIVEIRA, I.P.; ARAÚJO, R.S.; DUTRA, L.G. Nutrição mineral e fixação biológica de nitrogênio. In: ARAUJO, R.S.; RAVA, C.A.; STONE, L.F.; ZIMMERMANN, M.J.O. Cultura do feijoeiro comum no Brasil. Piracicaba: Potafós, 1996. p.169-221. PURCINO, A.A.C. Nitrogênio, correção e adubação do solo. Informe Agropecuário, v.7, p.16-20, 1981. QUAGGIO, J.A.; RAIJ, B. van. Produtividade esperada. In: RAIJ, B. van; CANTARELA, H.; QUAGGIO, J.A.; FURLANI, A.M.C. Recomendações de adubação e calagem para o Estado de São Paulo. 2.ed. Campinas: IAC, 1997. p.13-14, (Boletim Técnico, 100). RAIJ, B. van; ANDRADE, J.C.; CANTARELLA, H.; QUAGGIO, J.A. (Eds.). Análises Químicas para avaliação de fertilidade de solos tropicais. Campinas: IAC, 2001. 284p. RODRIGUES, O.; DIDONET, A.D.; GOUVEIA, J.A.; SOARES, R.C. Nitrogen translocation in wheat inoculated whit Azospirillum and fertilized wih nitrogen. Pesquisa Agropecuária Brasileira, v.35, p.1473- 1481, 2000. SARRUGE, J.R.; HAAG, H.P. Análises químicas em plantas. Piracicaba: ESALQ, 1974. 56p. SILVA, T.R.B.; SORATTO, R.P.; CHIDI, S.N.; ARF, O.; SÁ, M.E.; BUZETTI, S. Doses e épocas de aplicação de nitrogênio em cobertura na cultura do feijoeiro de inverno. Cultura Agronômica, v.9, p.1-17, 2000. SILVA, T.R.B.; SORATTO, R.P.; SILVA, L.C.; DOURADO, M.C.; ALVES, M.C. Diferentes doses e épocas de aplicação de N, e sua influência na matéria seca das plantas e N total em folhas de feijão. Cultura Agronômica, v.8, p.117-129, 1999. SORATTO, R.P.; SILVA, T.R.B.; ARF, O.; CARVALHO, M.A.C. Níveis e épocas de aplicação de nitrogênio em cobertura no feijoeiro irrigado em plantio direto. Cultura Agronômica, v.10, p.89-99, 2001. SORATTO, R.P.; SILVA, T.R.B.; CHIDI, S.N.; ARF, O.; SÁ, M.E.; BUZETTI, S. Feijoeiro irrigado e aplicação de nitrogênio em cobertura e molibdênio via foliar. Cultura Agronômica, v.9, p.115-132, 2000. TURLEY, R.H.; CHNG, T.M. Physiological responses of barley leaves to foliar applied urea-ammonium nitrate. Crop Science, v.26, p.987-993, 1986. WEATHERBURN, M.W. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry, v.39, p.971-974, 1967.