Semina: Ciências Agrárias ISSN: 1676-546X ISSN: 1679-0359 Universidade Estadual de Londrina

Gazola, Diego; Zucareli, Claudemir; Ringenberg, Rudiney; Graça, José Perez da; Hoffmann-Campo, Clara Beatriz Nitrogen fertilization in the contents of secondary compounds in cultivars Semina: Ciências Agrárias, vol. 39, no. 3, 2018, May-June, pp. 1015-1027 Universidade Estadual de Londrina

DOI: https://doi.org/10.5433/1679-0359.2018v39n3p1015

Available in: https://www.redalyc.org/articulo.oa?id=445760189009

How to cite Complete issue Scientific Information System Redalyc More information about this article Network of Scientific Journals from Latin America and the Caribbean, Spain and Journal's webpage in redalyc.org Portugal Project academic non-profit, developed under the open access initiative DOI: 10.5433/1679-0359.2018v39n3p1015 Nitrogen fertilization in the contents of secondary compounds in cassava cultivars

Adubação nitrogenada nos teores de compostos secundários em cultivares de mandioca industrial

Diego Gazola1*; Claudemir Zucareli2; Rudiney Ringenberg3; José Perez da Graça4; Clara Beatriz Hoffmann-Campo3

Abstract

The production of chemical substances is a strategy of plants to defend against the attack of pest . The synthesis of secondary compounds in plants is influenced by genotype and cultural management, such as the use of nitrogen fertilizers. Thus, the objective of this work is to evaluate the effect of doses of nitrogen fertilizer over time on the production of secondary compounds in two industrial cassava cultivars. The experiment was carried out in a greenhouse (25 ± 5 °C) of Embrapa Soybean in Londrina, Paraná, with the cultivation of potted plants (4l) in a 2 × 4 × 3 factorial scheme, with five replications. Two genotypes of industrial cassava (‘Baianinha’ and ‘Caiuá’) and four different nitrogen doses (0, 30, 60, and 90 kg ha-1) were used, with sampling at 15, 30, and 45 d after fertilization. Nitrogen fertilization was performed 50 d after sprouting. In the apical leaves, the levels of rutin, caffeic, p-coumaric and ferulic acids were evaluated in a high performance liquid chromatograph (HPLC). Exploratory analyses were performed on the data variables to verify the assumptions for analysis of variance (ANOVA), such as the independence and normality of errors, homogeneity of treatment variances and non-additivity of the model (they were submitted to analysis of homogeneity and normality, respectively. After these tests, ANOVA and Tukey’s test (p ≤ 0.05) were performed. For the quantitative data concerning the N rates, regression study analyzes were performed. The Caiuá cultivar was found to have a higher ferulic acid content. The caffeic acid content decreased over time after the application of N in both cultivars. The rutin content in both cultivars did not increase with an increase in nitrogen dose. At a dose of 90 kg ha-1, both cultivars presented a lower p-coumaric acid content after 30 d. Key words: Caffeic acid. P-coumaric acid. Ferulic acid. Defensive compounds. Manihot esculenta. Rutin.

Resumo

A produção de substâncias químicas é uma estratégia de defesa das plantas ao ataque de insetos praga. A síntese de compostos secundários em plantas é influenciada pelo genótipo e pelo manejo cultural, como o uso de fertilizantes nitrogenados. Deste modo, o trabalho tem como objetivo avaliar o efeito de doses de nitrogênio sobre a produção de compostos secundários em duas cultivares industriais de mandioca ao longo do tempo. O experimento foi realizado em casa de vegetação semiclimatizada (25°±5°C) da 1 Dr. em Agronomia, Universidade Estadual de Londrina, UEL, Londrina, PR, Brasil. E-mail: [email protected] 2 Prof. Dr., Adjunto, Centro de Ciências Agrárias, UEL, Londrina, PR, Brasil. E-mail: [email protected] 3 Pesquisadores, Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA Soja, Londrina, PR, Brasil. E-mail: rudiney. [email protected]; [email protected] 4 Pós-doutorando, Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA Soja, Londrina, PR, Brasil. E-mail: perezparr@ gmail.com * Author for correspondence Received: Oct. 18, 2017 - Approved: Mar. 13, 2018 1015 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Gazola, D. et al.

Embrapa Soja em Londrina/PR, com cultivo das plantas em vasos (4 litros), em esquema fatorial 2x4x3, com cinco repetições. Foram utilizados dois cultivares de mandioca industrial (Baianinha e Caiuá) e quatro doses de nitrogênio (0, 30, 60 e 90 kg ha-1), com avaliações aos 15, 30 e 45 dias após a adubação. A adubação nitrogenada foi realizada com ureia, 50 dias após a brotação das manivas. Nas folhas apicais foram avaliados os teores de rutina e de ácidos caféico, p-cumárico e ferúlico em cromatógrafo líquido de alto desempenho (HPLC). Foram realizadas análises exploratórias com os dados das variáveis e verificado os pressupostos para a Análise de Variância (ANOVA) como a independência e normalidade dos erros, homogeneidade de variâncias dos tratamentos e não-aditividade do modelo (SHAPIRO; WILK, 1965; PARENTE, 1984; BURR; FOSTER, 1972; TUKEY, 1949), respectivamente. Após estes testes foram realizadas as Anovas e os testes de comparações múltiplas de médias por Tukey (0,05), (STEEL; TORRIE, 1960). Para os dados quantitativos referentes as doses de N foi realizada análises de regressão. A cultivar Caiuá acumulou maior teor de ácido ferúlico. O teor de ácido caféico diminui ao longo do tempo após a aplicação de N em ambas cultivares. O teor de rutina nas cultivares de mandioca Baianinha e Caiuá não foi influenciada com acréscimo das doses de nitrogênio. Na dose de 90 kg ha-1 as cultivares apresentam menor teor de ácido p-cumárico após os 30 dias. Palavras-chave: Ácido caféico. Ácido p-cumárico. Ácido ferúlico. Compostos de defesas. Manihot esculenta. Rutina.

Introduction lotaustralin (SOLOMONSON; BARBER, 1990; VETTER, 2000). Cassava production in Brazil plays an important socioeconomic role, with crops concentrated In a study conducted by Oliveira et al. (2012) in areas belonging to small producers, that are it was concluded that the hydrocyanic acid (HCN) characterized by the use of few inputs and low content in the ‘Aciolina’ cultivar had a positive levels of technology. This is primarily a result of response to increased doses of topdress N (30, the plant’s ability to develop and produce in soils 60, 150, and 330 kg ha-1). The synthesis of plant with low fertility (CARVALHO; FUKUDA, 2006). secondary compounds is more influenced by The crop absorbs large amounts of nutrients, and agricultural practices (fertilizers, application of practically all absorbed nutrients are translocated to manure, and burning of crop residues) than by the tuberous roots (MATTOS; BEZERRA, 2003). seasonal factors. The synthesis of alkaloids and cyanogenic glycosides increases with an increased In general, N is the most required nutrient availability of nitrogen fertilizers, whereas the by crops, and has numerous functions, such as synthesis of phenolic compounds increases when promoting the formation and development of the plant is cultivated in a soil poor in nitrate flower and fruit buds and increasing the size of the fertilizer (GERSHENZON, 1984; WATERMAN; vegetative parts and protein content. In addition, MOLE, 1989). it is a structural component of amino acids and proteins, nitrogenous bases and nucleic acids, Phenolic compounds are a class of plant enzymes, co-enzymes and vitamins, pigments, and secondary metabolites with recognized biological some secondary metabolites (MALAVOLTA et al., activity. They provide plants protection against 1997). attacks by pest insects and pathogens (LORENZI et al., 2003; SCHALLER, 2008). These compounds In addition to the effects of N on the growth, may also serve as a warning to generalist pests development, and yield performance of plants, it is that the plant is unpalatable (ZAGROBELNY et involved in the synthesis of secondary compounds al., 2004). Santos et al. (2008) observed that the and amino acids in plants. In cassava, a greater higher the HCN content in the roots of cassava the availability of N in the soil also favors the synthesis lower the incidence of nymphs and adults of Vatiga of cyanogenic glycosides such as linamarin and 1016 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Nitrogen fertilization in the contents of secondary compounds in cassava cultivars illudens Drake (: Tingidae). Calatayud population below the level of economic damage, and Múnera (2002) concluded that the addition of in addition to reducing yield losses, and thus may the flavonol rutin to an artificial diet reduced the be included as an integrated pest management tool growth and development of the (BELLOTTI et al., 1999; PIETROWSKI et al., Phenacoccus manihoti Matile-Ferrero (Hemiptera: 2010). Pseudococcidae). In this context, the objective of the present study In cassava, the mealybug insect that feeds on was to assess the effect of nitrogen fertilization over the aerial part of the plant, P. manihoti, is one of time on the production of secondary metabolism the most severe pests attacking this crop worldwide compounds in two cultivars of cassava. (PARSA, et al., 2012), and because there are no effective control methods, nor a registered chemical product for use on cassava, implementing an Material and Methods appropriate and efficient management method is a The experiment was conducted in the semi- priority to achieve high crop yields. controlled climate greenhouse of Embrapa Soja The Caiuá (Olho junto) cassava cultivar, which is in Londrina Paraná (23°18′36′′ S and longitude widely cultivated on sandy soils in the northwest of 51°09′46′′ W) during 2016, at a temperature of 25 Paraná, is highly susceptible to the mealybug insect ± 5 °C. The experiment was implemented using P. Manihoti when grown in the field, exhibiting cassava stems from a commercial agricultural area explosions of populations especially during the in the west and northwest regions of Paraná; these second crop cycle. However, under laboratory were measured and cuttings of approximately 10 conditions, this cultivar has been shown to be cm long with five to seven buds were used. Vertical moderately resistant to this insect. The Baianinha planting was performed using the cassava stems cultivar, which is grown in the western region of in pots with a capacity of 5 L of soil. Water was Paraná and is better adapted to more fertile and provided to plants using drip irrigation thrice a day -1 -1 clayey soils, was susceptible to P. manihoti under (volume of 400 mL d pot ). The chemical analysis laboratory conditions, but high insect infestations of the soil used as a substrate led to the following 3 were not observed in the field (TAKAHASHI; results: P = 16.89 cmol/dm ; pH (CaCl2) = 5.5; Al = GONÇALO, 2005; RHEINHEIMER, 2013). 0 cmolc/dm³; H + Al = 4.61 cmolc/dm³; Ca = 6.99 cmolc/dm³; Mg = 1.35 cmolc/dm³; K = 0.85 cmolc/ These contradictory data may be associated with dm³; V% = 66.59; CEC = 13.8; MO = 3.15 g/dm³. the nutrient conditions of the soils in which these cultivars are traditionally grown. Depending on The experimental design was completely the time of application, nitrogen fertilization may randomized, with a 2 × 4 × 3 factorial arrangement, increase or decrease the production of secondary using two cultivars of cassava (Baianinha and compounds, thus making the plant more tolerant Caiuá) and four doses of topdressed N (0, 30, 60, -1 or susceptible to pests, with responses varying and 90 kg ha ). Leaf collections for analysis were according to the genotype. performed at three different times (15, 30, and 45 d after nitrogen fertilization) with five repetitions In addition to inducing plants to produce defense each, making up a total of 120 pots. Nitrogen compounds, nitrogen fertilization may decrease fertilization was applied as urea to moist soil in the natural physical barriers of plants. Therefore, all pots, according to the determined dose, 50 d inducing the production of secondary compounds after the sprouting of cassava stem cuttings. P may be a strategy for crop management because it has and K fertilization was not performed because the an inhibitory effect on pest insects, and keeps their soil analysis showed that these elements were not 1017 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Gazola, D. et al.

required. Two fully developed apical leaves from out in the UV region at a selected wavelength was each repetition were used in the assessments. The 260 nm. The identification and quantification of leaves were cut, labeled, wrapped in aluminum the compounds were performed by comparing the paper, and immediately frozen in liquid nitrogen. HPLC column retention times and the peaks in the They were subsequently sent to the Laboratory of spectra obtained in the experiment with those of Chemical Ecology of Embrapa Soja, Londrina, standard phenolic acids and flavonoids. The areas Paraná, and stored in an ultra-freezer at -86 °C. of each peak in the spectra were recorded, and the results were corrected using the initial weight of the For sample preparation in the laboratory, the plant tissue of each sample converted into µg/g. leaves were removed from the ultra-freezer, ground in a mortar with liquid nitrogen, transferred to a 15- The data regarding the variables were submitted mL Falcon tube, and weighed. The samples were to tests to analyze the homogeneity of variance added to 5 mL of methanol (MeOH; 90%) and the (BURR; FOSTER, 1972), normality of the residuals extracts were vortex-mixed for 10 seconds, placed using the Shapiro–Wilk test (SHAPIRO; WILK, in an ultrasonic bath for 20 min, and centrifuged 1965), and non-additivity of the model (TUKEY, at 5,650 rpm for 10 min at 4 °C. The supernatants 1949). The transformation was performed for were collected with a Pasteur pipette, transferred data regarding ferulic acid and p-coumaric acid. to glass tubes, and vacuum-dried. The samples Subsequently, the results were submitted to an were resolubilized in methanol (80%; 1.5 mL) and analysis of variance (ANOVA) test, and the means centrifuged for 10 min at 10,000 rpm at 4 °C. The were compared using Tukey’s test ( < 0.05). The samples were then filtered through Acrodisc® syringe data regarding the doses of N were submitted to filters with a 0.45 μm membrane (Millipore®) and regression analysis. The statistical software systems transferred to the automatic injector vials of the used were Statistical Analysis System (SAS, 2009) high-performance liquid chromatography (HPLC) version 9.2, and SISVAR, version 5.0. system (Shimadzu Prominence). The metabolic extracts from the samples were analyzed using a C18 reversed-phase column (250 mm length and 4.6 mm Results and Discussion internal diameter, 5 μm particles). Aliquots of 20 µL The effect of the triple interaction between the were automatically injected in the system equipped cultivars, doses of N, and time after application of with a CBM-20A system controller, a SPD-20A N on the concentrations of rutin and p-coumaric detector, a DGU 20A5 degasser, a LC-20AT pump, acid was significant. Ferulic acid content was a SIL-20A automatic sampler, and a CTO 20A only significantly affected by cultivars and by the column oven. The mobile phase was composed of interaction between fertilization and time, whereas two solvents: (A) 2% acetic acid (HOAc) and (B) a caffeic acid content was significant affected by the mixture of MeOH, acetic acid, and Milli-Q® water interactions between cultivars and time and between (MeOH:HOAc:H2O; 18:1:1). The initial condition doses of N and time (Table 1). of the linear gradient program used in the analysis The Caiuá cultivar exhibited higher was 75% of A and 25% of B, which was then changed concentrations of ferulic acid than the Baianinha to the reverse after 40 min, i.e., 25% of A and 75% cultivar (Table 2). This suggests that the content of B, which was maintained for 5 min. The program of this secondary compound is related to genetic returned to the initial condition for 5 min before the factors of the cassava plants, as indicated by Ramos next injection to rinse the column. The flow rate of et al. (2011). This acid is present in plant tissues, and the solvent was 1 ml/min and detection was carried

1018 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Nitrogen fertilization in the contents of secondary compounds in cassava cultivars has inhibitory effects on the growth of fungi and the et al., 1998; SOUZA et al., 2010). Thus, cultivars production of mycotoxins. Moreover, studies have with higher contents of this compound may be used reported its importance as a mechanism of self- in genetic improvement programs, with the aim preservation, because it strengthens the resistance of selecting materials that are resistant/tolerant to of the cell wall and protects it from damage caused agricultural pests or abiotic factors. by microbes and solar radiation (EKMEKCIOGLU

Table 1. Mean square values of the variables rutin, caffeic acid, p-coumaric acid, and ferulic acid in cassava cultivars according to the doses of N, at distinct collection times after nitrogen fertilization. Londrina, 2016.

G.L Ferulic acid Caffeic acid p-Coumaric acid Rutin Cultivar 1 0.00687* 0.000042* 0.01021* 2.29728* Fertilization 3 0.00036n/s 0.000043* 0.00178* 1.88372* C × F 3 0.00041n/s 0.000007n/s 0.00248* 1.00349* Time 2 0.00022n/s 0.000271* 0.00132* 0.26197n/s C × T 2 0.00007n/s 0.000024* 0.00045* 1.93567* F × T 6 0.00045* 0.000043* 0.00119* 0.62982* C × F × T 6 0.00040n/s 0.000007n/s 0.00157* 1.19713* Mean 0.0160 0.0087 0.1754 1.3215 CV (%) 43.46 32.50 37,57. 29.30 *, significant at 5% probability by Tukey’s test; n/s, non-significant at 5% probability by Tukey’s test.

Table 2. Ferulic acid contents in apical leaves of the Baianinha and Caiuá cassava cultivars. Londrina, 2016.

Cultivar Ferulic acid Baianinha 0.0086 b Caiuá 0.0239 a Means followed by the same lowercase letters in the column were not significantly different by Tukey’s test (p ≤ 0.05) .

In the interaction between the fertilization and the hot aqueous extract content and a 16% increase in time of leaf collection, a quadratic fit was only the ethanolic extract content between data obtained obtained at 30 d after nitrogen fertilization (Figure with and without poultry litter. According to these 1), with the minimum response of the compound studies, organic fertilization increases the soil’s occurring at a dose of 70 kg ha-1. Data obtained by cation exchange capacity (CTC), thereby increasing Ramos et al. (2011) showed that the contents of the pH and decreasing the aluminum content. antioxidant flavonoids, including ferulic acid, in Therefore, there is an increase in the availability the ethanolic extract and in the hot aqueous extract of nutrients introduced through mineral fertilizers, of rosella leaves were significantly affected by the which contributes to plant health and diversifies the addition of poultry litter (substrate rich in N) to the production of active substances by bacteria, such as plant crop. On average, there was a 18% increase in phenols and antibiotics (KIEHL, 2008).

1019 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 through mineralthrough fertilizers, mineral which fertilizers, contributes which to contributesplant health to andplant diversifies health and the diversifies production the of productionactive of active substances by bacteria,substances such by asbacteria, phenols such and as antibiotics phenols and (KIEHL, antibiotics 2008). (KIEHL, 2008). Regarding caffeicRegarding acid, a significantcaffeic acid, difference a significant between difference the two between cultivars the was two only cultivars observed was onlyat 15 observed d at 15 d Gazola, D. et al. after nitrogen fertilization,after nitrogen with fertilization, the highest with values the highestbeing detected values beingin the detected leaves of in Baianinha the leaves (Table of Baianinha 3). In this (Table 3). In this cultivar,Regarding there cultivar,was caffeic a decrease there acid, was in a caffeica significant decrease acid incontentdifference caffeic over acid attime content15 ,and and 30over the d than lowesttime at, and45 value d the after occurred lowest the application value at 45 occurred d afterof N. at 45 d after the applicationbetween thethe of two N.application Incultivars the Caiuá of was N. culti onlyIn thevar, observed Caiuá the contents culti at 15var, of the Thecaffeic contents effect acid of ofwere applying caffeic significantly acid N was were observed higher significantly at in 15 the and highershort- 30 at 15 and 30 d after nitrogen fertilization, with the highest values term, and did not persist in the long-term. This may d thanbeing at 45 detected d afterd than inthe atthe application 45 leaves d after of the ofBaianinha N.application The effect (Table of of N. 3). applying The be effect explained N wasof applying observed by the N way inwas the it observed wasshort applied-term in ,the and to short the did pots, -notterm , and did not persistIn thisin the cultivar, longpersist-term. there in the Thiswas long amay decrease-term. be explained This in caffeic may by be acid the explained waybecause it wasby the althoughappli wayed itto the was the irrigation applipots, edbecause toperformed the althoughpots, afterbecause the the although the irrigationcontent performed overirrigation time, after and performed thethe additionlowest after value of the N occurredfavoredaddition theatof Navailabilityaddition favored ofthe of N availabilitythefavored nutrient the ofavailabilityto thethe nutrientplants of during theto thenutrient thisplants during this 45 d after the application of N. In the Caiuá cultivar, to the plants during this period, daily irrigation may period,the dailycontents irrigationperiod, of caffeic daily may acid irrigationhave were favored significantly may leaching have favored higherover time.leaching have favored over time. leaching over time.

Figure 1. FerulicFigure acid 1 .contents Ferulic acidin leaves contents of thein Caiuáleaves cassava of the Caiuá cultivar cassava submitted cultivar to increasing submitted doses to increasing of doses of nitrogenFigure at 1different. Ferulicnitrogen timesacid at contents different of the assessment.in times leaves of of the theLondrina, assessment. Caiuá cassava 2016. Londrina, cultivar submitted2016. to increasing doses of nitrogen at different times of the assessment. Londrina, 2016.

0,033 0,033

ŷ = 0,0306 - 0,0007xŷ = 0,0306 + 0,000005x² - 0,0007x + 0,000005x² ) ) 1 1 - - 0,028 0,028 R² = 0,9308 R² = 0,9308 µg g µg g 0,023 0,023

0,018 0,018

0,013 0,013 erulic acid ( erulicacid erulic acid ( erulicacid F F 0,008 0,008 0 0 30 30 60 60 90 90 Doses of N (kgDoses ha-1 )of N (kg ha-1) 15 (d) 1545 (d) 4530 (d) 30

TableTable 3. 3.Contents ContentsTable of of3. caffeicContents acid acid ofin inapical caffeic apical leaves acid leaves of in the apicalof cassava the leaves cassava cultivars of cultivars at the distinct cassava at times distinct cultivars after timesthe at application distinct after the timesof after the applicationnitrogen ofertilization.f nitrogenapplication fertilization. Londrina, of nitrogen 2016. Londrina, fertilization. 2016. Londrina, 2016. Cultivar Cultivar Time (d) Time (d) Time (d) Cultivar 15 15 30 30 45 45 Baianinha Baianinha 0.0126 a A 15 0.0126 a A 0.0094 a B 30 0.0094 a B 0.0062 a45 C 0.0062 a C Caiuá BaianinhaCaiuá 0.00930.0126 b A a0.0093 A b A 0.00940.0094 a A a0.0094 B a A 0.00560.0062 a B a C 0.0056 a B *Means followed*MeansCaiuá by the followed same uppercase by the sameletters0.0093 uppercase in bthe A row letters and lowercasein the row0.0094 letterand a slowercase A in the column letter swere in 0.0056the not column significantly a B were not significantly different by Tukey’s test (p ≤ 0.05). different*Means by followedTukey’s bytest the (p same ≤ 0.05) uppercase. letters in the row and lowercase letters in the column were not significantly different by Tukey’s test (p ≤ 0.05).

Plant secondaryPlant compounds secondary arecompounds significantly are affectedsignificantly by theaffected fertilization, by the environmentalfertilization, environmental conditions, andconditions, degree of andmaturation degree of thematuration plant, in of addition the plant, to geneticin addition factors to genetic (RAMOS factors et al. (RAMOS, 2011). This et al. , 2011). This Plant secondary compounds are significantly regardless of the cultivar, exhibited an increasing was affectedthe case withwas by caffeicthe the case fertilization,acid, with which, caffeic regardless environmentalacid, which, of theregardless cultivar,linear responseof exhibited the cultivar, to anthe increasing exhibiteddoses of Nlinearan at increasing 15 response d after linear theto response to the dosesconditions, of Nthe atand 15doses degreed after of N ofthe at maturation application15 d after ofthe of the applicationN, plant,with a content ofapplication N, withpeak a ofat content aN, dose with peakof a content90 at kg a doseha peak-1. Noof at 90differencesa dose kg ha of-1 .90 No differences in addition to genetic factors (RAMOS et al., kg ha-1. No differences were observed between the 2011). This was the case with caffeic acid, which, remaining collection times (Figure 2). At 30 and 45

1020 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 through mineral fertilizers, which contributes to plant health and diversifies the production of active were observed substancesbetween the by remaining bacteria, such collection as phenols times and (Figure antibiotics 2). At (KIEHL, 30 and 452008). d, leaching of the nutrient Nitrogen fertilization in the contents of secondary compounds in cassava cultivars from the pots probably occurredRegarding because caffeic of acid, daily a significantirrigation, differencethereby making between the the added two Ncultivars unavailable was onlyto the observed at 15 d plants.d, leaching Similar of afterresults the nit nutrient wererogen obtained fertilization, from the by pots Malta with probably theet al. highest (2003) (2007) values in their assessedbeing study detected of the the relationships ineffects the leaves of sources betweenof Baianinha and dosesdoses (Table 3). In this occurred because of daily irrigation, thereby making of N and K, concentrations of phytochemical of N on the contentscultivar, ofthere chlorogenic was a decrease acids in caffeiccoffee beans.acid content According over timeto these, and authors,the lowest the value contents occurred of at 45 d after the added N unavailable to the plants. Similar results compounds, and attack by the cochineal insect chlorogenic acids (caffeic, p-coumaric, and ferulic acids) increased with increasing doses of N. However, were obtainedthe by application Malta et al. of (2003) N. In in the their Caiuá study culti ofvar, Coccus the contents viridis of caffeic Green acid (Hemiptera: were significantly Coccidae) higher in at 15 and 30 Fernandesthe effects (2007) ofd sources than assessed at and45 ddosesthe after relationships ofthe N application on the contentsbetween of N. dosesThecoffee effect of plants,N of and applying K, and concentrations reportedN was observed that of an phytochemicalin increasethe short - interm , and did not of chlorogenic acids in coffee beans. According the doses of N and K, 11 months after supplying compounds, andpersist attack in by the the long cochineal-term. This insect may Coccus be explained viridis Green by the (Hemiptera: way it was Coccidae)applied to inthe coffee pots, plantsbecause, although the to these authors, the contents of chlorogenic acids plants with a nutrient solution, directly increased N and reported that an increase in the doses of N and K, 11 months after supplying plants with a nutrient (caffeic, p-coumaric,irrigation andperformed ferulic after acids) the increased addition of contentN favored in the the leaves, availability which of in theturn nutrient led to a todecrease the plants during this solution,with increasing directlyperiod, increased doses daily of N irrigation N.content However, inmay the have Fernandesleaves, favored which leachingin in caffeic turn ledover acid to time. content.a decrease in caffeic acid content.

Figure 2. MeanFigure caffeic 1 acid. Ferulic contents acid in contents apical leaves in leaves of the of Baianinha the Caiuá and cassava Caiuá cultivarcassava cultivars submitted submitted to increasing doses of to Figureincreasing 2. Mean doses caffeic of nitrogen acid contents at different in apical times leaves of the of assessment. the Baianinha Londrina, and Caiuá 2016. cassava cultivars submitted to increasing dosesnitrogen of nitrogen at different at different times times of theof the assessment. assessment. Londrina, Londrina, 2016.2016.

0,018 0,033 y = 0.0072 + 0.00009x² 0,016

) ŷR =² =0,0306 0.9552 - 0,0007x + 0,000005x² ) 1 - 1 0,014 - 0,028 R² = 0,9308 0,012 0,01 µg g 0,023 0,008 0,006 0,018 0,004 0,013 erulic acid ( erulicacid

Caffeic (µg g acid Caffeic 0,002 F 0 0,008 0 0 30 30 60 60 90 90 Doses of N (kgDoses ha-1 )of N (kg ha-1) 15 (d) 30 (d) 4515 (d) (d) 4515 (d)dias 30

Table 3. Contents of caffeic acid in apical leaves of the cassava cultivars at distinct times after the The results that contradict those of the present study may be explained by differences-1 in assessment The resultsapplication that contradict of nitrogen those fertilization. of the present Londrina, dose) 2016. and at 30 d (at the 30 kg ha dose) (Table timesstudy or mayeven be byCultivar explained the use of by supplementation differences in assessment with K, in addition4). At theto N, 60 which kg ha was-1 dose,Time not (theperformedd) highest incontent the present was study.times The or evenhighest by thecontents use of of supplementation p-coumaric acid with in theK,15 Baianinhaobtained cultivar 15 d after were 30the observed application at 30 ofand N. 45 Ind after the45 in addition toBaianinha N, which was not performed 0.0126 in the a ACaiuá cultivar, no0.0094 changes a B were observed in0.0062 the a C the application of N (at the 0 kg ha-1 dose) and at 30 d (at the 30 kg ha-1 dose) (Table 4). At the 60 kg ha-1 present study.Caiuá The highest contents of p-coumaric0.0093 b Acontents of p-coumaric0.0094 aacid A between the different0.0056 a B dose,acid the in highestthe Baianinha*Means content followed cultivarwas obtained by were the same observed15 d uppercaseafter at the 30 applicationletters assessment in the of row N. times. andIn the lowercase Caiuá cultivar,letters in nothe changes column werewere not significantly different by Tukey’s test (p ≤ 0.05). -1 observedand 45 ind afterthe contents the application of p-coumaric of N (at acid the between 0 kg ha the different assessment times.

Table 4. Contents of p-cPlantoumaric secondary acid and rutincompounds in apical leavesare significantly of the Baianinha affected and Caiuáby the cassava fertilization, cultivars, environmental according to theconditions, different doses and degree of nitrogen of maturation and collection of the times. plant, Londrina, in addition 2016. to genetic factors (RAMOS et al., 2011). This Time p-Coumaric acid (d) was0 thekg hacase-1 with caffeic acid,30 kg which, ha-1 regardless of 60the kg cultivar, ha-1 exhibited an90 increasing kg ha-1 linear response to Baian. Caiuá Baian. Caiuá Baian. Caiuá Baian. Caiuá -1 15 0.015bAthe doses0 .of014aA N at 15 0d. 010bAafter the application0.010aA of 0N,.093aA with a content0.007aB peak at0 .a005aA dose of 900. 015aAkg ha . No differences 30 0.035aA 0.018aB 0.054aA 0.008aB 0.046bA 0.006aB 0.009aA 0.008aA 45 0.027aA 0.003aB 0.020bA 0.003aB 0.015cA 0.009aB 0.009aA 0.009aA 1021 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Gazola, D. et al.

Table 4. Contents of p-coumaric acid and rutin in apical leaves of the Baianinha and Caiuá cassava cultivars, according to the different doses of nitrogen and collection times. Londrina, 2016.

Time p-Coumaric acid (d) 0 kg ha-1 30 kg ha-1 60 kg ha-1 90 kg ha-1 Baian. Caiuá Baian. Caiuá Baian. Caiuá Baian. Caiuá 15 0.015bA 0.014aA 0.010bA 0.010aA 0.093aA 0.007aB 0.005aA 0.015aA 30 0.035aA 0.018aB 0.054aA 0.008aB 0.046bA 0.006aB 0.009aA 0.008aA 45 0.027aA 0.003aB 0.020bA 0.003aB 0.015cA 0.009aB 0.009aA 0.009aA Time Rutin (d) 0 kg ha-1 30 kg ha-1 60 kg ha-1 90 kg ha-1 Baian. Caiuá Baian. Caiuá Baian. Caiuá Baian. Caiuá 15 1.813aA 1.608aA 0.683bA 1.124aA 1.235aA 1.205aA 1.419aA 2.193aA 30 1.597aA 1.643aA 1.918aA 0.455bB 1.285aA 1.445aA 1.732aA 0.436bB 45 1.427aA 1.451aA 1.089bA 0.459bB 1.446aA 1.653aA 1.939aA 0.450Bb *Means followed by the same lowercase letters in the column and uppercase letters in the row were not significantly different by Tukey’s test (p ≤ 0.05)., between the cultivars and doses at different assessment times.

There were no statistical differences between and 45 d, there were no statistical differences at the the cultivars regarding p-coumaric acid at the doses doses of 0 and 60 kg ha-1. When using the 90 kg of 0, 30, and 90 kg ha-1 at 15 d post-fertilization. ha-1 dose, the Caiuá cultivar exhibited the highest At a dose of 60 kg ha-1, the Baianinha cultivar content of the compound at 15 d. The Baianinha had a higher concentration of this compound than cultivar exhibited the highest contents at 30 and 45 the Caiuá cultivar. In addition, at 30 and 45 d, the d and at the 30 and 60 kg ha-1 doses. These results Baianinha cultivar exhibited higher contents of the may be related to the genetic factors of the cultivars; compound when 0, 30, and 60 kg ha-1 of N were because the Caiuá cultivar is better adapted to applied. At the 90 kg ha-1 dose, the concentrations sandy soils, it exhibited a short-term response to of this acid did not differ between the two cultivars. the N supplied to the plants, whereas the Baianinha cultivar took a longer time to respond to nitrogen The concentration of rutin in the Baianinha fertilization. cultivar at the 0, 60, and 90 kg ha-1 doses of N did not differ between the times of assessment after the In the regression analysis of the Baianinha application of N (Table 4). At the 30 kg ha-1 dose, cultivar, the quadratic fit showed a maximum the highest content of the compound was obtained response of p-coumaric acid at 30 d, at the 27.5 kg ha-1 at 30 d after fertilization. In the Caiuá cultivar there dose, whereas at 45 d there was a decreasing linear was no difference between the assessment times fit. In the Caiuá cultivar, there was no significant fit when the 0 and 60 kg ha-1 doses were used. At the 30 in response to the different doses of N (Figure 3). and 90 kg ha-1 doses of N, the highest content of the p-Coumaric acid is associated with microorganisms flavonoid was observed at 15 d after the application the rumen, and can be toxic if high concentrations of N. are consumed (CARVALHO; PIRES, 2008). Similar data were obtained by Marschner (1995), who At 15 d, there were no statistical differences in concluded that high concentrations of N decreased the amounts of rutin between the cultivars when the the production of some phenolic compounds that used doses of N were 0, 30, and 60 kg ha-1. At 30 have a fungistatic action, including p-coumaric

1022 Semina: Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028, maio/jun. 2018 Nitrogen fertilization in the contents of secondary compounds in cassava cultivars

acid, and lignification of leaves, thereby reducing The decrease in the rutin content with the increase the resistance to obligate parasites while having no in the N dose was probably the result of nitrogen action on facultative parasites. Therefore, the use of metabolism, which is related to the carbon/nutrient cultivars with high contents of this compound may balance. The greater vegetative growth of the aerial be troublesome for cassava producers who use the part of the cassava plant with the production of new aerial part of the plant for feeding. leaves decreases the number of organic compounds designated to the synthesis of rutin. Moreover, N RegardingThe decrease rutin in in thethe Baianinharutin content cultivar, with the there increase in the N dose was probably the result of nitrogen directly alters the amount and quality of nutrients metabolism,was a quadratic which fitis relatedat 15 and to the45 d,carbon/nutrient with a minimum balance. The greater vegetative growth of the aerial part present in the plant. This effect occurs because it is response at doses of 43.75 kg ha-1 and 36.25 kg of the cassava plant with the production of new leavesmore decreases available the in the number phloem, of organic thus increasing compounds the ha-1 (0.9375 µg g-1 and 1.1000 µg g-1 of rutin, contents of free amino acids, soluble carbohydrates, desigrespectively).nated to the In synthesis the Caiuá of cultivar,rutin. Moreover, the quadratic N directly fit alters the amount and quality of nutrients present in and single-chain lipids, which are easily digestible thewas plant. obtained This effect at 15 occursd, with because a minimum it is morevalue availableat the in the phloem, thus increasing the contents of free food sources for phytophagous insects, such as P. 38.37 kg ha-1 dose (Figure 4). amino acids, soluble carbohydrates, and single-chain lipids,manihoti which (MATTSON, are easily 1980;digestible BUCHANAN food sources et al.,for phytophagous insects, such as P. manihoti (MATTSON,2000; 1980; FERNANDES, BUCHANAN 2007). et al., 2000; FERNANDES, 2007). FigureFigure 3. 3. pp-Coumaric-Coumaric acid acid contents contents in apical in apical leaves of leaves the Baianinha of the Baianinhacassava cultivar cassava submitted cultivar to increasing submitted doses to increasingof nitrogen doses at different of nitrogen times at of different the assessment. times ofLondrina, the assessment. 2016. Londrina, 2016.

0,06 y = 0.0354 + 0.0011x - 0.05x²

0,05 R² = 1 ) 1 -

g 0,04 (µg 0,03

acid 0,02

0,01 y = 0.0273 - 0.0002x R² = 0.9911 Coumaric 0 p - 0 30 60 90 Doses of N (kg ha-1) 15 (d) 30 (d) 45 (d)

Figure 4. Rutin contents in apical leaves of the Baianinha (A) and Caiuá (B) cassava cultivars submitted to increasing doses of nitrogen at different times of the assessment. Londrina, 2016.

Baianinha 2,5 y = 1.34994 - 0.0145x + 0.0002x2 R² = 0.9575

2 ) 1 - 1,5 µg g 1 y = 1.7112 - 0.035x + 0.0004x2 1023

Rutin( Semina:0,5 Ciências Agrárias, Londrina, v. 39, n. 3, p. 1015-1028,R² = maio/jun.0.6826 2018

0 0 30 60 90 -1 Doses of N (kg ha ) Gazola, D. et al.

FigureFigure 4. R 4.utin Rutin contents contents in in apical apical leavesleaves ofof the the Baianinha Baianinha (A) (A) and and Caiuá Caiuá (B) cassava (B) cassava cultivars cultivars submitted submitted to increasing to increasingdoses ofdoses nitrogen of nitrogen at different at differenttimes of the times assessment. of the assessment. Londrina, 2016. Londrina, 2016.

Baianinha 2,5 y = 1.34994 - 0.0145x + 0.0002x2 R² = 0.9575

2 ) 1 - 1,5 µg g 1 y = 1.7112 - 0.035x + 0.0004x2

Rutin( 0,5 R² = 0.6826

0 0 30 60 90 -1 Doses of N (kg ha )

Caiuá y = 1.6257 - 0.0307x + 0.0004x2 R² = 0.9918

1,85 ) 1 -

µg g 1,35 Rutin ( 0,85 0 30 60 90 Doses of N (kg ha-1) 15 (d) 30 (d) 45 (d)

*Means followed by the same lowercase letters in the column were not significantly different by Tukey’s test ( ≤ 0.05). Similar results were obtained by Malta et al. and the rigidity of the cell walls of these tissues, (2003), who observed a significant effect of the which hinder the insertion of the sucking mouthpart interaction between sources and doses of N on into the phloem. These morphological barriers are Similar results were obtained by Malta et al. (2003), who observed a significant effect of the total phenolic compounds. Ammonium nitrate and composed of waxes, complex carbohydrates, and interactionammonium between sulfate sources sources and produced doses of Nsimilar on total effects, phenolic long-chain compounds. proteins, Ammonium whose nitrate contents and inammonium the leaves sulfatewith sources a decrease produced in the similar concentration effects, of with total a phenolicdecrease in decreasethe concentration with the of applicationtotal phenolic of compounds high doses up of -1 compounds up to- 1doses of 161 kg-1 ha and 138 kg nitrogen during fertilization (MARSCHNER, 1995; to doses of 161 kg ha and 138 kg ha of N (total contents of phenolic compounds were 6.82% and 6.15%, ha-1 of N (total contents of phenolic compounds PHELAN et al., 1996; TAIZ; ZEIGER, 2010). respectively).were 6.82% and 6.15%, respectively). Hogendorp et al. (2006) reported that an increase in nitrogen fertilization led to an increase in the Moreover,Moreover, the the use use of high of high doses doses of N inof fertilization N in fertilization reduces the physical barriers to the feeding of attack frequency and reproduction of the citrus suckingreduces insects. the In physical leaves, thesebarriers barriers to theare thefeeding thickness of of the cuticle, epidermis, and parenchyma, and the mealybug, Planococcus citri (Risso) (Hemiptera: sucking insects. In leaves, these barriers are the rigidity of the cell walls of these tissues, which hinder Pseudococcidae),the insertion of the onsucking coleus, mouthpart Solenostemon into the thickness of the cuticle, epidermis, and parenchyma, phloem. These morphological barriers are composed of waxes, complex carbohydrates, and long-chain 1024 proteins, whose contentsSemina: in theCiências leaves Agrárias, decrease Londrina, with v. 39,the n.application 3, p. 1015-1028, of high maio/jun. doses 2018 of nitrogen during Nitrogen fertilization in the contents of secondary compounds in cassava cultivars scutellarioides. However, plants that are deficient Acknowledgments in nitrogen can allocate a higher amount of carbon We thank the Laboratory of Chemical Ecology for the production of plant defense secondary of Embrapa Soja for the technical and scientific compounds (HERMANS; MATTSON, 1992). support. The production of phenolic compounds is often associated with the response to plant–environment interaction mechanisms, which may be triggered References under stress conditions; this explains the data BELLOTTI, A. C.; SMITH, L.; LAPOINTE, S. L. Recent obtained in this study. Mineral nutrition is one of advances in cassava pest management. Annual Review of Entomology, New York, v. 44, n. 1, p. 343-370, 1999. the most important stress factors and, according to Freitas et al. (2008) and Leite et al. (2012), mineral BUCHANAN, B. B.; GRUISSEM, W.; JONES, R. L. Biochemistry and molecular biology of plants. Rockville: deficiency may lead to the increased or decreased American Society of Plant Physiologists, 2000. 1366 p. production of phenolic compounds. On the other BURR, I. W.; FOSTER, L. A. A test for equality of hand, the increase in phenolic compounds relative to variances. West Lafayette: University of Purdue, 1972. the availability of nutrients in the soil varies between 26 p. (Mimeo series, n. 282). plant species, and with the different biosynthesis CALATAYUD, P. A.; MÚNERA, D. F. Defensas naturales pathways of these compounds (HAUKIOJA et al., de la yuca a las plagas e artrópodos. In: OSPINA, B.; 1998). CEBALLOS, H. La yuca en el tercer milenio: sistemas modernos de producción, procesamiento, utilización y A balanced availability of nutrients is essential comercialización. Cali: CIAT, 2002. p. 250-254. for the plant to recover from attacks by pests CARVALHO, G. G. P.; PIRES, A. J. V. Organização dos through mechanisms of compensatory growth. tecidos de plantas forrageiras e suas implicações para os This compensation is related to the increase in ruminantes. Archivos de Zootecnia, Córdoba, v. 57, n. 1, photosynthesis as a result of due to increased p. 13-28, 2008. nitrogen fertilization, which allows the plant to CARVALHO, P. C. L.; FUKUDA, W. M. G. Estrutura better tolerate the attack (TRUMBLE et al., 1993). da planta e morfologia. In: SOUZA, L. de S. (Coord.). Aspectos socioeconômicos e agronômicos da mandioca. Thus, more studies with longer testing periods Cruz das Almas: EMBRAPA Mandioca e Fruticultura, are necessary to clarify the behavior of secondary 2006. 817 p. compounds in response to nitrogen fertilization in EKMEKCIOGLU, C.; FEYERTAG, J.; MARKTL, W. cassava cultivars, as well as their relationship with Cinnamic acid inhibits proliferation and modulates brush the plant’s development, defense mechanisms, and border membrane enzyme activities in Caco-2 cells. Cancer Lett, Heidelberg, v. 128, n. 2, p. 137-144, 1998. tolerance to attacks by pest insects. FERNANDES, F. L. Efeito de nitrogênio e de potássio na interação entre Coccus viridis e Coffea arábica. 2007. Dissertação (Mestrado em Agronomia) - Universidade Conclusion Federal de Viçosa, Viçosa, MG. FREITAS, M. S. M.; MONNERAT, P. H.; VIEIRA, I. J. The Caiuá cultivar accumulated a higher ferulic C. Mineral deficiency in passiflora alata curtis: vitexin acid content. The caffeic acid content decreased over bioproduction. 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