Book HB 39_4

RP Soratto et al. tuber yield, with values of 46.8% and Proportion of tuber yield >4.5 cm (%) Tuber yield > 4.5 cm (Mg ha-1) 50.0 B. de Campos-2013 45.0%, respectively, which were higher Itaí-2014 than that in BC-2013 (Table 3). (A) There was no effect of the studied 40.0 Aa Aa Aa Aa Aa factors on tuber mean weight (Table 3). However, there was an interaction 30.0 Aa between fertilizer management and site-year on the tuber yield of the special Bb Bab Bab Bab class (diameter >4.5 cm), as well as on the proportion of tuber yield contained 20.0 in that class. In BC-2013, T5 treatment resulted in tuber yield of the special 0.0 Aa Ab Aa Aa Aa class 27.1% (5.8 t ha-1) higher than that 90.0 Ab Abc Ba of the T1 treatment (Figure 2A), due to the higher proportion of special size (B) tubers (Figure 2B), as no differences 80.0 were observed in the total tuber yield (Table 3). On the other hand, in Itaí- Aa 2014 there was no difference between treatments for tuber yield and proportion 70.0 Bc of tubers in the special class (Figure 2). Only in the T5 treatment, there was 60.0 no difference in the tuber yield of the special class between site-years, due to 50.0 the high proportion of tubers of special 0.0 size obtained in BC-2013. However, in the other treatments, tuber yield of T1 T2 T3 T4 T5 the special class in Itaí-2014 was, on average, 56.4% (12.8 t ha-1) higher than Fertilization management* in BC-2013 (Figure 2A). Only in the T1 treatment, the percentage of tubers of the Figure 2. Yield of tubers >4.5 cm (A) and proportion of tuber yield >4.5 cm (B) of potato special class in BC-2013 was lower than cultivar Agata as affected by fertilization management in two site-years. *Description of in Itaí-2014 (Figure 2B). fertilization treatments is shown in Table 1. Different lowercase letters indicate a significant difference among fertilization managements in the same site-year, whereas different uppercase The results indicate that the exclusive letters indicate a significant difference among site-year in the same fertilization management, use of MAP and the total removal of K at p≤0.05 according to LSD test. Botucatu, UNESP, 2013-2014. fertilizer (KCl) from the potato planting furrow presents itself as a management replacement of 420 kg ha-1 P2O5 using of the special class of ‘Agata’ potato alternative, with maintenance of tuber the formula NPK 4-14-8 with MAP compared to the application of the same yield of the cultivar Agata even in fertilizer did not alter the marketable K rate totally in the planting furrow. This soil with medium K concentration, tuber yield; however, in soil with low P was attributed to the slow diffusion of corroborating the results obtained by and medium K concentrations, there was K in the soil after application before Panique et al. (1997). Evaluating the a reduction of 6.6 t ha-1 (18.5%) in the hilling and to the nutritional imbalance sources and rates of K in the planting tuber yield of the special class. Lower observed in the concentrations of K, furrow with a fixed rate of MAP, rates of P and N at planting furrow and Ca, and Mg in the leaves. Thus, with they observed that K fertilization the redirection of K from planting to the K application in the post-planting increased the total tuber yield only in post-planting and sidedressing showed phase, there was possibly enough time 5 out of 11 evaluated site-years, which greater yield of larger tubers in the year for its uptake before the maximum was attributed particularly to water with less water availability in the initial demand period, which is from 45 to limitations and high K concentrations stage of crop development (Figure 2). 56 DAP (Fernandes et al., 2011), as in the studied soils. In addition, the This can be explained by the lower evidenced by the absence of differences results obtained with half P rate as MAP saline effect of MAP at planting due between leaf K concentrations in the confirm the inefficiency of applying to its greater solubility (Raij, 1997; treatments (Table 2). Thus, under these more than 250 kg ha-1 P2O5 during Nascimento et al., 2018), associated conditions of lower water availability, planting of ‘Agata’ potato grown in with the decrease in N supply and the the split application of K possibly soil with medium P concentration absence of KCl in the planting furrow. favored its adequate function in the (Fernandes & Soratto, 2016a). Yagi translocation of carbohydrates to the et al. (2017) found that, in soil with In the same condition of the BC-2013 tubers (Westermann, 2005), providing high concentrations of P and K, the experiment (adjacent area), Job et al. larger and heavier tubers (Karam et al., (2019) did not find an effect of the split 2009), and increasing the tuber yield of 394 application of K in the planting furrow the special class (Figure 2A). and in the sidedressing for tuber yield Horticultura Brasileira 39 (4) October - December, 2021

Fertilization management strategies for ‘Agata’ potato production Table 4. Fertilizer rate, information on the fertilizer application capacity and operation, and estimated operational yield of fertilizer application at potato planting according to fertilization management. Botucatu, UNESP, 2013-2014. Information Treatments grouped by planting fertilization T1(1) T2 and T3 T4 and T5 Fertilizer rate (kg ha-1) 1700 1275 638 Fertilizer applicator capacity (kg) 2000 2000 2000 Working speed of fertilizer applicator (km h-1) 10 10 10 Area fertilized with each fertilizer refilling (ha) 1.18 1.57 3.13 Fertilizer refilling time (min) 5.8 5.8 5.8 Fertilizer refilling time per working hour (min) 15.7 11.8 5.9 Area fertilized per working hour (ha) 2.36 2.57 2.88 Relative operational yield (%) 100 109 122 (1)Description of fertilization managements is shown in Table 1. Considering only planting this, in BC-2013, this fertilization 1961. Yield, quality, and phosphorus uptake fertilization, the non-application of strategy increased the tuber yield of of potatoes as influenced by placement and K fertilizer, due to the use of MAP special class compared to the standard composition of potassium fertilizers. American (formula more concentrated in P), NPK fertilization, but without changing Potato Journal 38: 272-285. increased the operational yield by 9%, this variable in Itaí-2014 (Figure 2A). compared to standard NPK fertilization Furthermore, the total tuber yield and CARDOSO, AD; ALVARENGA, MAR; MELO, (Table 4). The reduction in the P rate also the percentage of tuber DM were not TL; VIANA, AES. 2007. Produtividade e increased the efficiency of the fertilizer affected by the exclusive use of MAP qualidade de tubérculos de batata em função de application capacity in the planting at planting, regardless of the form of doses e parcelamentos de nitrogênio e potássio. furrow. This is due to the need for less KCl application (Table 3). Thus, the Ciência e Agrotecnologia 31: 1729-1736. downtime for refilling the fertilizer application of 255 kg ha-1 P2O5 as the applicator when lower rates of fertilizer MAP at planting and the transfer of DELEO, JPB; BOTEON, M. 2020. Especial are used per area. It is important that K from planting to applications in the batata: gestão sustentável. Hortifruti Brasil 19: the exclusive use of formula with only post-planting phase or in post-planting 12-27. Available at https://www.hfbrasil.org. N and P at planting, such as MAP, may phase and at hilling, increases the br/br/revista/acessar/completo/bataticultura- require complementary fertilization to operational yield of planting fertilization se-capitaliza-em-plena-pandemia.aspx. supply other nutrients, such as Ca and S. and maintains total tuber yield. It also Accessed October 23, 2020. increases the tuber yield of the special The studied factors did not affect class of ‘Agata’ potato under conditions FERNANDES, AM; SORATTO, RP. 2016a. the percentage of DM in the tubers, of lower water availability in the Response of potato cultivars to phosphate which was on average 14.8% (Table vegetative stages of the crop grown in fertilization in tropical soils with different 3). Fernandes et al. (2015) also found soils with mean availability of P and K phosphorus availabilities. Potato Research no changes in the percentage of DM (Figure 2A; Tables 3 and 4). 59: 259-278. in the tubers of the cultivar Agata as a function of the rates of P applied in soil ACKNOWLEDGMENTS FERNANDES, AM; SORATTO, RP. 2016b. with medium nutrient concentration, Phosphorus fertilizer rate for fresh market even with a marked response in the tuber The authors thank to the potato potato cultivars grown in tropical soil with low yield. Job et al. (2019) did not verify the grower (Grupo Ioshida), who provided phosphorus availability. American Journal of effect of splitting K fertilization on the the area and logistical support for the Potato Research 93: 404-414. specific gravity of the ‘Agata’ potato. experiments, and to Conselho Nacional de Desenvolvimento Científico e F E R N A N D E S , A M ; S O R AT TO , R P ; The phosphate nutrition of potato Tecnológico (CNPq), for granting an GONSALES, JR. 2014. Root morphology and cultivar Agata was favored by the award for excellence in research to the phosphorus uptake by potato cultivars grown exclusive use of MAP in the planting first and fourth authors. under deficient and sufficient phosphorus and KCl in the post-planting phase in supply. Scientia Horticulturae 180: 190-198. Itaí-2014, with greater water availability REFERENCES until leaf sampling. This was observed FERNANDES, AM; SORATTO, RP; MORENO, despite the leaf P concentrations being BERGER, KC; POTTERTON, PE; HOBSON, EL. LA; EVANGELISTA, RM. 2015. Effect of slightly reduced by treatments with half phosphorus nutrition on quality of fresh tuber the P rate at planting with MAP and with of potato cultivars. Bragantia 74: 102-109. K at post-planting and sidedressing, in both site-years (Figure 1). Contrasting FERNANDES, AM; SORATTO, RP; SILVA, BL. 2011. Extração e exportação de nutrientes Horticultura Brasileira 39 (4) October - December, 2021 em cultivares de batata: I - Macronutrientes. Revista Brasileira de Ciência do Solo 35: 2039-2056. FIXEN, PE; BRUULSEMA, TW. 2014. Potato management challenges created by phosphorus chemistry and plant roots. American Journal of Potato Research 91: 121-131. HAILU, G; NIGUSSIE, D; ALI, M; DERBEW, B. 2017. Nitrogen and phosphorus use efficiency in improved potato (Solanum tuberosum L.) cultivars in southern Ethiopia. American Journal Potato Research 94: 617-631. 395

RP Soratto et al. JADOSKI, SO; SUCHORONCZEK,A; SANTOS, LA; VITTI, GC. 2018. Phosphorus mobility regimes and phosphorus rates on water and J. 2017. Efeito de deficiência hídrica no and behavior in soils treated with calcium, phosphorus use efficiencies in potato. Scientia desenvolvimento vegetativo, produção e ammonium, and magnesium phosphates. Horticulturae 190: 64-69. distúrbios fisiológicos em tubérculos da Soil Science Society of America Journal 82: batata Ágata. Brazilian Journal of Applied 622-631. TREHAN, SP; CLAASSEN, N. 1998. External K Technology for Agricultural Science 10: requirement of young plants of potato, sugar 97-107. OLIVEIRA, JPM; OLIVEIRA FILHO, beet and wheat in flowing solution culture LCI; POCOJESKI, E. 2017. A aplicação resulting from different internal requirements JOB, ALG; SORATTO, RP; FERNANDES, AM; localizada de monoamônio fosfato favorece a and uptake efficiency. Potato Research 41: ASSUNÇÃO, NS; FERNANDES, FM; YAGI, disponibilidade de P no solo e sua absorção. 229-237. R. 2019. Potassium fertilization for fresh Scientia Agraria 18: 12-19. market potato production in tropical soils. WESTERMANN, DT. 2005. Nutritional Agronomy Journal 111: 3351-3362. PANIQUE, E; KELLING, KA; SCHULTE, EE; requirements of potatoes. American Journal HERO, DE; STEVENSON, WR; JAMES, RV. Potato Research 82: 301-307. KARAM, F, ROUPHAEL, Y; LAHOUD, R; 1997. Potassium rate and source effects on BREIDI, J; COLLA, G. 2009. Influence of potato yield, quality, and disease interaction. YAGI, R; NAZARENO, NRX; CHORNOBAY, genotypes and potassium application rates on American Potato Journal 74: 379-398. AK; CAMPOS, JF; DZIERWA, A. 2017. yield and potassium use efficiency of potato. Mistura de NPK 4-14-8 com MAP como Journal of Agronomy 8: 27-32. PAULETTI, V; MENARIM, E. 2004. Época de alternativa para a adubação de plantio da batata aplicação, fontes e doses de potássio na cultura ‘Atlantic’. Revista Brasileira de Tecnologia LORENZI, JO; MONTEIRO, DA; MIRANDA da batata. Scientia Agraria 5: 15-20. Aplicada nas Ciências Agrárias 10: 23-32. FILHO, HS; VAN RAIJ B. Raízes e tubérculos, p.221-229. In: VAN RAIJ, B; CANTARELLA, RAIJ, BV. 1997. Adubação fosfatada. In: RAIJ, YAGI, R; NAZARENO, NRX; SORATTO, RP. H; QUAGGIO, JA; FURLANI, AMC. (eds). B; CANTARELLA, H; QUAGGIO, JA; 2020. Agronomic and economic interactions Recomendações de adubação e calagem para FURLANI, ACM (eds). Recomendações between sidedressed nitrogen and potassium o Estado de São Paulo, 2nd ed. Boletim Técnico de adubação e calagem para o Estado de fertilizations on ‘Atlantic’ potato. Brazilian 100. Inst. Agron. Campinas, Campinas, 1997. São Paulo. 2nd ed. Bol. Tecn. 100. Instituto Archives of Biology and Technology 63: p.285. Agronômico, Campinas, SP, Brazil. p. 8-13. e20190640. MALAVOLTA, E; VITTI, GC; OLIVEIRA, REIS JUNIOR., RA; FONTES, PCR; NEVES, YAGI, R; SORATTO, RP; NAZARENO, NRX; SA. 1997. Avaliação do estado nutricional JCL; SANTOS, NT. 1999. Total soil electrical SILVA, HL; DZIERWA, AC. 2019. Tuber das plantas: princípios e aplicações. 2. ed. conductivity and critical soil K+ to Ca2+ and yield and economic result of ‘Atlantic’ potato POTAFOS, Piracicaba, Brasil, 319p. Mg2+ ratio for potato crops. Scientia Agricola. in response to NPK fertilizer formulas. 56: 993-997. Horticultura Brasileira 37: 379-383. NASCIMENTO, CAC; PAGLIARI, PH; FARIA, SUN, Y; CUI, X; LIU, F. 2015. Effect of irrigation 396 Horticultura Brasileira 39 (4) October - December, 2021

Research HERCOS, GFL; BELISÁRIO, CM; ALVES, AES; MAIA, GPAG; CAVALCANTE, MD. 2021. Physicochemical characterization, bioactive compounds and antioxidant capacity of bitter melon. Horticultura Brasileira 39: 397-403. DOI: http://dx.doi.org/10.1590/s0102-0536-20210408 Physicochemical characterization, bioactive compounds and antioxidant capacity of bitter melon Guilherme F de L Hercos 1 ;ID Celso M Belisário 1 ;ID Anny E de S Alves 1 ;ID Geisa Priscilla AG Maia 2 ;ID Maísa D Cavalcante 3ID 1Instituto Federal de Educação, Ciência e tecnologia Goiano (IF Goiano), Rio Verde-GO, Brasil; [email protected]; celso. [email protected]; [email protected]; 2Universidade Estadual de Campinas (Unicamp), Campinas-SP, Brasil; [email protected]; 3Universidade Estadual Paulista (Unesp), São José do Rio Preto-SP, Brasil; [email protected] ABSTRACT RESUMO The bitter melon (Momordica charantia) is an exotic fruit that Caracterização físico-química, compostos bioativos e has adapted very well to the Brazilian soils, and can be found on capacidade antioxidante do melão de São Caetano practically all regions. Several advantages related to consumption of this fruit can be reported, highlighting its antifungal activity, O melão de São Caetano (Momordica charantia) é um fruto reduction of glycemic indexes, and also due to the content of natural exótico que se adaptou muito bem aos solos do Brasil, podendo ser pigments, such as lycopene and β-carotene, making it promising as encontrado em praticamente todas as regiões. Diversas vantagens a functional food. Based on the demand for foods of this nature, this relacionadas com o consumo desse fruto podem ser reportadas, research aimed at the biometric characterization, physicochemical, destacando-se sua atividade antifúngica, redução de índices bioactive compounds and the pulp and seeds’ antioxidant capacity of glicêmicos, e também pelo conteúdo de pigmentos naturais, como bitter melon, collected in the southwest region of Goias. The fruits o licopeno e o β-caroteno, tornando-o promissor como alimento were collected from plants inside a native vegetation area on Campus funcional. Partindo da demanda por alimentos dessa natureza, esta Rio Verde at the Instituto Federal Goiano, and the evaluations were pesquisa objetivou a caracterização biométrica, físico-química, carried out from October 2020 to April 2021. The fruits showed compostos bioativos e capacidade antioxidante da polpa e sementes high variability in dimensions and weight, high concentrations of do melão de São Caetano, coletados na região sudoeste do estado flavonoids, tannins, carotenoids, total phenolics and antioxidant de Goiás. Os frutos foram coletados de plantas localizadas em uma capacity. According to results, the bitter melon can be used as a raw área de vegetação nativa do Campus Rio Verde do Instituto Federal material to develop food formulations with functional characteristics Goiano, e as avaliações ocorreram de outubro de 2020 a abril de and drugs potentially effective in the treatment of diabetes and 2021. Observou-se elevada variabilidade de dimensões e massas, diseases caused by oxidative processes. altas concentrações de flavonoides, taninos, carotenoides, fenólicos totais e capacidade antioxidante. De acordo com os resultados, o melão de São Caetano pode ser utilizado como matéria prima para o desenvolvimento de formulações alimentícias com características funcionais e de fármacos potencialmente eficazes no tratamento de diabetes e de doenças causadas por processos oxidativos. Keywords: Momordica charantia, flavonoids, tannins, carotenoids, Palavras-chave: Momordica charantia, flavonoides, taninos, vitamin A, antioxidant capacity. carotenoides, vitamina A, capacidade antioxidante. Received on May 26, 2021; accepted on August 11, 2021 Among the various plant species orange (Assis et al., 2015). be associated with natural pigments from Brazilian Cerrado, there is This fruit is mainly consumed for contents, such as lycopene and the bitter melon (Momordica charantia) β-carotene, in addition to other phenolic (Figure 1), a plant of the Curcubitaceae therapeutic purposes, related in studies compounds, which are also responsible family, monoecious, originating from about antifungal properties (Celoto et for inhibition of oxidative processes eastern India and southern China. It al., 2011) and for treatment of diabetes (Macedo et al., 2017). presents isolated yellow flowers in mellitus (Kim et al., 2020), among the leaf axils, oblong fruits, similar to others, such as cancer prevention, Being seasonal fruits, with great cucumber, with variable length and antiviral, antimicrobial, analgesic and variability, its biometric characterization weight dimensions. The fruit is green anti-inflammatory action (Tan et al., is essential, especially for future studies when immature and mature fruits are 2016; Bortolotti et al., 2019; Sur & about improvement of the cultivar Ray, 2020). Many of its potentials may (Assis et al., 2015). Furthermore, its Horticultura Brasileira 39 (4) October - December, 2021 397

GFL Hercos et al. fruits are consumed for being rich in dried and tared porcelain capsules. L-1); bioactive compounds and antioxidant Then, samples were taken to an oven at M= sample weight (g). capacity. Consequently, studies about 105°C for 24 h. flavonoids content (Madala et al., pH measurement 2016), total phenolics (Chung et al., Lipids The pH was measured by previously 2016), ascorbic acid (Ng & Kuppusamy, The lipids content was measured by calibrated bench pHmeter. For this, 2 2019), among others, can validate such Bligh & Dyer method. About 3.0 g were g of sample were placed in a beaker properties and provide high additive weighed in a 100 mL beaker, then 10 containing 30 mL water. Measurements value to the product, which is also part mL of chloroform, 20 mL of methanol were taken after shaking the system with of the income of families of small rural and 8 mL of distilled water were added, a glass stick. producers. capped and rotary stirred on a magnetic stirrer for 30 minutes. After stirring, 10 Determination of total titratable Considering other researches about mL of chloroform and 10 mL of aqueous acidity the fruit, which has many potentials, this sodium sulphate 1.5% (w/v) were added, research evaluated the biometric and stirred for another 2 minutes and the About 5 g of sample were macerated, physicochemical data, the total phenolic layers separated in a separatory funnel. transferred to a 100 mL flask, and diluted compounds, vitamin C, flavonoids, Approximately 15 mL of the lower layer with distilled water. The titrant solution carotenoids and antioxidant capacity of were removed, 1 g of anhydrous sodium was 0.1 M sodium hydroxide, and the bitter melon pulp and seeds, obtained in sulphate was added, capped and shaken indicator was 1% (m/v) phenolphthalein native vegetation in the Southwest of the by hand for 2 minutes. 5 mL of filtrate in an ethanolic solution. (Zenebon et state of Goiás. were filtered and transferred to an oven al., 2008). at 80°C for 30 minutes to evaporate the MATERIAL AND METHODS solvent. It was cooled in a desiccator Phenolic total and tannin contents and weighed again. The phenolic total content was About 3 kg of fruits were harvested obtained by Folin-Ciocalteu method in October 2020, from plants in an area Proteins as described by Chung et al. (2016). of native vegetation on Campus Rio Approximately 0.5 g of sample Approximately 3 g of sample were Verde of the Instituto Federal Goiano, at was weighed on tissue paper and the added to a 100 mL aqueous-alcoholic Rodovia Sul Goiana km 01, Rural Zone, sample transferred to a Kjeldahl flask. solution 50% (v/v). The system was Rio Verde-GO. After collected, the fruits Afterward, 7 mL of sulfuric acid and agitated for 24 hours, then filtrated, were sanitized and stored in a freezer about 2.5 g of catalytic mixture (90% and finally supernatant was collected at -18ºC until the moment of analysis. potassium sulphate + 10% copper and transferred to 100 mL flasks and All physicochemical and antioxidant sulphate) were added. It was heated on the volume was filled with aqueous- capacity evaluations were carried out an electric hotplate until the solution alcoholic solution. with fruit pulp and seeds, in triplicate. turned blue-green and free of undigested The tannin contents (%) were material. The tube containing the determined by calibration curve using Ascertainment of size, weight and digested sample was connected to the tannic acid standard, ranging from 20 pulp yield distillation set, with the tapered end to 100 mg L-1. Absobance readings of the soft drink dipped in 20 mL of were 760 nm in spectrophotometer UV/ Biometric parameters were 4% boric acid with 5 drops of mixed Vis (Makkar, 2000). In test-tube were determined according to norms of indicator (methyl red + bromocresol added 8.2 mL distilled water, 0.3 mL Instituto Adolfo Lutz, and presented green). 40% sodium hydroxide solution extract, 0.5 mL Folin-Denis’s reagent similarly to the research by Assis et was added to the tube containing the and 1 mL aqueous sodium carbonate al. (2015). Using precision scale and digested sample, using a funnel with a solution at 7.5% (w/v). After agitation digital caliper, measurements were taken stopcock, until a slight excess of base and subsequent rest for 30 minutes, from 150 fruits, measuring longitudinal was ensured. It was heated to boiling and absorbance readings were performed axis or length (mm), equatorial axis or distilled to obtain about 75 mL distillate. in spectrophotometer at 760 nm. The width (mm), fruit weight with seed (g), Excess sulfuric acid was titrated with a phenolic total contents (mg GAE 100 pulp weight without seed (g) and pulp standardized 0.1 mol L-1 hydrochloric g-1) were determined by calibration yield (%). To measure the weight of the acid solution. To determine the protein curve using gallic acid standard, ranging fruits without seeds, they were opened content, the values obtained in the from 0 to 500 mg L-1. manually, using a previously sanitized analysis were substituted in equation 1 laboratory spatula. (Zenebon et al., 2008). Flavonoid contents Total flavonoids (mg of pyrocatechin Moisture content Protein (%) = V x N x 0,014 x 100 x 6,38 100 g-1) were quantified by colorimetric The moisture content (% w.b.) m method (Thiruvengadam et al., 2014), was determined according to Instituto using Pyrocatechin as an analytical Adolfo Lutz (Zenebon et al., 2008). Where: standard instead of Quercetin. In Approximately 2.5 g of the sample were test-tube were added 0.5 mL extract, weighed and transferred to previously V= volume of hydrochloric acid 0.3 mL NaNO2 (aq) at 5% (w/v), 0.3 mL AlCl3 (methanolic) at 10% (w/v) 398 consumed (mL); N= hydrochloric acid solution (mol Horticultura Brasileira 39 (4) October - December, 2021

Physicochemical characterization, bioactive compounds and antioxidant capacity of bitter melon and 2 mL NaOH (aq) at 1 mol L-1. and 1 mL DPPH. After preparation, the UV/Vis spectrometer, for equation 4. After shaking, each tube was left to Abs*Vol* 104 rest for 10 minutes, next, absorbance solutions were stored for 30 minutes in CT= E11%cm*P readings were performed at 510 nm in spectrophotometer, previously tared the dark, then the absorbance was read Where: with aqueous-alcoholic solution 50% in a spectrophotometer at 518 nm. The CT= total carotenoids (μg g-1); (v/v). The flavonoid contents were determined by calibration curve using antioxidant capacity was calculated Abs= maximum absorbance; pyrocatechin standard, ranging from 10 from equation 3. Vol= dilution volume (mL); to 50 mg L-1. AA (%)=100- �AAbsb(ss(anmepglaet)iv-eAcbos(nbtrlaonl)k)� *100 = molar extinction coefficient petroleum ether (β-carotene: 2592); Where: in P= sample weight (g). Abs(sample) = sample absorbance; Vitamin C Abs(blank) = blank absorbance; About 5 g of sample was weighed and transferred to a 300 mL Erlenmeyer Abs(negative control) = negative Statistical analysis flask with 50 mL distilled water. Then, 10 mL of sulfuric acid solution at 20% control absorbance. Biometric evaluations were was added. After homogenization, 1 mL of aqueous potassium iodide solution at To calculate the antioxidant activity performed from 150 fruits, and the 10% (w/v) and 1 mL of aqueous starch solution at 1% (w/v) were added. The of trolox, used as positive control, means of the parameters presented resulting solution was titrated with potassium iodate solution at 0.002 M, the Abs (sample) was replaced by the followed by their respective standard until blue color was observed. The amount of vitamin C (mg 100 g-1) was positive control absorbance. deviations. In addition, the results were calculated using equation 2 (Zenebon et al., 2008). Total carotenoids submitted to descriptive analysis of For determination of the carotenoid frequency distribution and estimate of content (μg g-1), the methodology Pearson’s correlation coefficient. The described by Souza et al. (2012) was means of physicochemical variables, utilized. About 5 g of the sample carotenoid content and antioxidant from each treatment in triplicates was capacity were presented followed by macerated with about 30 mL cold their respective standard deviations. 100*v*F acetone and 5 g of Hyflo Supercel for 2 p vit C= minutes, followed by vacuum filtration. RESULTS AND DISCUSSION The extraction was repeated six times Where: until the filtrate became as clear as Ascertainment of size, weight and V= potassium iodate solution consumed possible. pulp yield (mL); The filtrate was then transferred to Table 1 shows the biometric a 250 mL separation funnel containing parameters evaluated. The fruits had F= 0.8806; 50 mL cold petroleum ether. Three an average weight of 5.30±2.37 g, with consecutive washes were performed a high coefficient of variation. The P= sample weight (g). with 100 mL distilled water to remove calculated value was similar, but lower acetone. The ether-dissolved phase than that obtained by Assis et al. (2015) Antioxidant activity by DPPH was transferred to an amber flask; then, of 6.38 g. The minimum weight value radical assay the same volume of 10% (w/v) KOH obtained was 1.35 g, and the maximum methanolic solution was added, and the was 3.99 g. The antioxidant capacity was mixture was stored at room temperature measured according to Mensor et al. for 24 h for saponification of lipids and The high coefficients of variation for (2001), using a blend (1:1) methyl chlorophyll. the biometric parameters of bitter melon alcohol aqueous solution at 50% and can be attributed to genetic variability, acetone aqueous solution at 70% as This mixture was then transferred environmental factors (biotic and extracting solution. The capacity of to a separation funnel containing 50 abiotic), and the genotype-environment the extract to reduce the DPPH radical mL cold petroleum ether, followed interaction. Research on tropical fruit (2,2-diphenyl-1-picrylhydrazyl) 0.30 by washing with 50 mL aliquots of species shows high variability in fruit mmol L-1 was compared with the distilled water until the pH of the ether size, seed, volume and weight of fruits activity of the synthetic antioxidant phase was near neutral. About 3 g of and seeds (Zuffo et al., 2014). trolox (6-hydroxy-2,5,7,8- anhydrous sodium sulfate were added, tetramethylchroman-2-carboxylic acid) at 100 mg L-1, used as positive control. the mixture filtered, and the ether extract This low uniformity of biometric 2.5 mL of extract was added to concentrated on a rotary evaporator parameters may also be due to the 1 mL DPPH solution, and sample blanks consisted of 2.5 mL of extract (35°C and 90 rpm). The concentrate fact that the fruits were collected from and 1 mL of extraction solution. For positive control, 2.5 mL trolox and 1 was transferred to a 50 mL balloon plants in an area of native vegetation. mL DPPH were used, and its blank consisted of 2.5 mL trolox and 1 mL and diluted to the desired volume with These plants were not inserted in that ethanol. The negative control of the samples and the positive control were petroleum ether. environment in a planned way, because, prepared with 2.5 mL ethanol solution Total carotenoid quantification was according to Assis et al. (2015), this performed using the scanning method species is commonly known as a weed, with readings from 300 to 700 nm in which develops in different places, Horticultura Brasileira 39 (4) October - December, 2021 399

GFL Hercos et al. without its propagation being controlled. the final product influences the level of ranged from 11.6 to 27.8 mm, with an There was a relationship between acceptability by end users. In addition, average very near to highest frequency industries prefer rounded fruits due to found by Santana et al. (2013), from 17 the longitudinal axis and the equatorial their ease of cleaning and processing to 19 mm. The coefficients of variation axis (LA/EA) with an amplitude varying (Chitarra & Chitarra, 2005). of the longitudinal axis and equatorial from 1.36 to 2.99, and an overall mean axis of fruits were 22.59% and 18.40%, of 2.04±0.28. From this relationship Figure 2 shows the normal respectively, values similar to those it is possible to indicate the shape of distribution fit, as well as the number obtained by Assis et al. (2015), of the fruits, if the relationship between of observations of the longitudinal axis, 18.08% and 13.16%, respectively. the axes is greater than or equal to 1, equatorial axis, fruit weight and pulp it indicates elongated and cylindrical yield of bitter melon. The mean pulp yield was 62.02%. fruits, that is, the longitudinal diameter Carvalho & Muller (2005) classified is greater than the equatorial diameter The longitudinal axis of bitter melon the pulp yield of native fruits as very (Silva et al., 2016). had a mean of 39.61 mm, with amplitude low (less than 20%), low (from 21 to ranging from 21.7 to 61.1 mm. In 40%), medium (from 41 to 60%); high The characterization of the fruit’s general, the values obtained were lower (from 61 to 80%) and very high (more shape is very important for post-harvest than results presented by Santana et al. than 81%). The high pulp yield of bitter handling, especially in the case of (2013), that most fruits present a length melon demonstrates great feasibility fruits destined for agro-industrial of 49 to 57 mm. Regarding the equatorial of applying it in the development of processing, because the visual aspect of axis of the fruits, the values obtained products in the food and pharmaceutical industries. Figure 1. Overview of the bitter melon. A= inflorescence; B= immature fruit; C= mature fruit, and D= fruit seeds. Photography= authors. Rio Verde, IFGoiano, 2020. From the Pearson’s correlation coefficients (R) the correlation of the Table 1. Biometric data of bitter melon. Rio Verde, IFGoiano, 2020. different physical characteristics of the fruits was determined as positively high Parameters Minimum Mean Maximum SD CV (%) (from 0.7 to 0.9) (Table 2). Longitudinal axis (mm) 21.7 39.61 61.1 8.95 22.59 The parameter that presented the highest correlation with the fruit pulp Equatorial axis (mm) 11.6 19.41 27.8 3.56 18.40 yield was the fruit weight (0.983), followed by equatorial axis (0.879) and Fruit weight (g) 1.56 5.30 12.44 2.37 44.78 the longitudinal axis (0.859). Pedron et al. (2004), analyzing the biometric Pulp yield (%) 50.05 62.02 77.71 5.54 8.95 parameters of butiazeiro fruits, also observed a high correlation between LA/EA 1.36 2.04 2.99 0.28 14.10 weight and pulp yield. LA/EA= ratio (longitudinal axis/equatorial axis); SD= standard deviation; CV (%)= Physicochemical parameters, coefficient of variation. bioactive compounds and antioxidante activity The moisture content of the pulp was 86.50±1.27% (w.b.) and of seeds 56.06±2.16% (w.b.). Knowing that the pulp and the seeds are used as unconventional foods, data about their initial moisture contents are important, especially for planning the elaboration of products that depend on the drying process. The proteins content of the pulp was 0.90±0.06%. Poovitha & Parani (2017) observed that protein extracts from M. charantia pulp significantly reduced blood glucose levels and improved islet morphology in diabetic rats. Momordica charantia lectin (MCL), a protein present in the fruit, has some toxicity, but it is an excellent antitumor agent that acts by inhibiting cell viability, inducing autophagy, apoptosis and cell 400 Horticultura Brasileira 39 (4) October - December, 2021

Physicochemical characterization, bioactive compounds and antioxidant capacity of bitter melon cycle arrest in some cancer lines (Fang product (Ferreira & Aquila, 2000). According to UV/Vis spectra (Figure et al., 2019). The vitamin C content found in the 3), of readings from 400 to 600 nm, it was possible to identify maximum The lipid content of the pulp was fruit pulp was lower than found by Ng absorptions in the pulp ether extract at 0.87±0.06%; Yuwai et al. (1991), & Kuppusamy (2019), of 20 mg 100 445 and 474 nm, and in the seeds ether through spectroscopy studies, confirmed g-1 (d.b.) using the methodology of extract at 445, 470 and 499 nm. Pacheco the presence of eleostaric acid in L-ascorbic acid oxidase and analytical et al. (2012), extracting lycopene from the fruits of Momordica charantia. curve in HPLC. This difference in arils of bitter melon seeds, observed Eleostaric acid is a fatty acid that has content may be due to the way in which maximum absorptions at 445, 472 and shown inhibitory effects on breast the result is expressed, as we use the 503 nm. cancer cells (Grossmann et al., 2009). unit of measurement on a wet basis, and if converted to a dry basis, the The lycopene content was lower The results regarding concentration would be similar. than that found in the cited research. physicochemical parameters, bioactive This difference can be explained by the compounds and antioxidant activity are The high content of vitamin C, in geografic location of fruit harvest or by presented in Table 3. addition to indicating the importance the efficiency of the extraction process, of the fruit as a supplier of an important as they used column chromatography The fruit pulp of bitter melon had a nutritional compound as described by in order to produce standards with high pH of 5.46, and the seed 6.34. Acidity Ng & Kupussamy (2019), can also be purity, while in this work, the extraction is associated to pH, and the verification considered a reducer of reactive oxygen had no such objective, dispensing the of these physicochemical factors is species, as the antioxidant activity is chromatography. important, as it indicates a relationship directly related with the prevention of with the balance between sugars, amino several types of cancer, as reported by About the value of β-carotene acids and organic acids, and has an Sur & Ray (2020). found in the fruit pulp extract, it is influence on the quality of the final estimated around 180 μg of retinol per 100 g, because 1 μg of β-carotene Figure 2. Number of observations and normal distribution of the longitudinal axis (A), being equivalent to 0.167 μg of retinol equatorial axis (B), fruit weight (C), and pulp yield (D) of the bitter melon. Rio Verde, (BRASIL, 2005). The bitter melon IFGoiano, 2020. showed a higher β-carotene content than that found by Khoo et al. (2011) Table 2. Pearson’s correlation coefficient (R) from biometrics data of bitter melon. Rio in mango and pineapple, showing its Verde, IFGoiano, 2020. potential to offer this vitamin when consumed. Comparisons R Longitudinal axis x Equatorial axis 0.804 The antioxidant activities of pulp Longitudinal axis x Pulp yield 0.859 and seeds were different, with 75% in Longitudinal axis x Fruit weight 0.860 the seed. Yoshime et al. (2019) also Equatorial axis x Pulp yield 0.879 obtained antioxidant activity values Equatorial axis x Fruit weight 0.880 greater than 50% in the methods used, Fruit weight x Pulp yield 0.983 and attributed this high capacity to the presence of tocopherol and other bioactive compounds present in the bitter melon seed oil. The phenolic compounds content and flavonoids were similar to those found by Chung et al. (2016), in root extracts from the same plant. Despite being from different parts of the plant, it is an important attribute to explain the high antioxidant capacity. Thus, the antioxidant potential of the seeds of this fruit can be attributed to the high concentrations of flavonoids, tannins and total phenolic compounds. The antioxidant capacity of bitter melon fruit extracts is associated to levels of flavonoids, carotenoids and total phenolic compounds. Furthermore, the high levels of tannins and flavonoids are associated with a reduction in Horticultura Brasileira 39 (4) October - December, 2021 401

GFL Hercos et al. Table 3. Physicochemical caracterization, bioactive compounds and antioxidante activity of bitter melon pulp and seeds. Rio Verde, IFGoiano, 2020. pH ATT Vit C CT CFT T AAT F P 5.46±0.05 2.85±0.36 11.57±2.99 21.49±0.03 31.02±0.95 0.54±0.15 9.06±0.34 223.46±5.33 S 6.34±0.05 2.04±0.46 10.42±1.54 10.54±0.05 55.75±1.90 1.24±0.25 75.89±3.48 256.79±7.40 P= pulp; S= seed; ATT= total titratable acidity (%); Vit C= vitamin C (mg 100 g-1); CT= total carotenoids (μg g-1); T= tannins (%); CFT= phenolic total compounds (mg GAE 100 g-1); AAT= antioxidant activity (%); F= flavonoids (mg of pyrocatechin 100 g-1). Figure 3. UV/vis absorbance spectra of pulp (A) and seeds (B) of bitter melon in petroleum ether with readings from 400 to 600 nm. Rio Verde, IFGoiano, 2020. inflammatory reactions, as reported by REFERENCES Elicitation enhanced the production of phenolic Bortolotti et al. (2019). compounds and biological activities in hairy ASSIS, JP; SOUSA, RP; LINHARES, PCF; root cultures of bitter melon (Momordica From the context presented, some PEREIRA, MFS; MOREIRA, JC. 2015. charantia L.). Brazilian Archives of Biology final considerations can be made. As the Avaliação biométrica de caracteres do melão and Technology 59: 1-10. plants used for collection were native, de São Caetano (Momordica charantia L). not having been developed with specific Revista Brasileira de Plantas Medicinais 17: DAVIES, BH. Carotenoids. In: GOODWIN, management or genetic improvement, 505-514. TW (ed). 1976. Chemistry and biochemistry the biometric characteristics showed of plant pigments. London, UK: Academic high variability. However, the pulp yield BORTOLOTTI, M; MERCATELLI, D; POLITO, press. p.38-65. and the physicochemical characteristics L. 2019. Momordica charantia, a nutraceutical of the fruit showed viability for its approach for inflammatory related disease. FANG, EF; FROETSCHER, L; SCHEIBYE- application in industrial scale. Frontiers in Pharmacology 10: 1-9. KNUDSEN, M; BOHR, VA; WONG, JH; NG, TB. 2019. Atividades antitumorais emergentes The high bioactive compounds BRASIL. Agência Nacional de Vigilância do melão amargo (Momordica charantia). content, as total phenolics, flavonoids, Sanitária (Anvisa). 2005. Resolução da Current Protein and Peptide Science 20: carotenoids and vitamin A, gave Diretoria Colegiada – RDC N° 269 de 22 de 296-301. Momordica charantia high antioxidant setembro de 2005. Available: http://www. activity, which correlated with its anvisa.gov.br/alimentos/legis/especifica/ FERREIRA, AG; AQUILA, MEA. 2000. protein and lipid content, indicate the regutec.htm Alelopatia: uma área emergente da fruit as a promising raw material for ecofisiologia. Revista Brasileira de Fisiologia the development of food formulations CARVALHO, JEU; MULLER, CH. 2005. Vegetal 12: 175-204. characterized as functional foods Biometria e rendimento percentual de polpa and potentially effective drugs in the de frutas nativas da Amazônia. EMBRAPA. GROSSMANN, ME; MIZUNO, NK; DAMMEN, treatment of diabetes and degenerative Amazônia Oriental-Comunicado Técnico ML; SCHUSTER, T; RAY, A; CLEARY, MP. diseases such as cancer. (INFOTECA-E). Availble: http://www. 2009. Eleostearic acid inhibits breast cancer infoteca.cnptia.embrapa.br/infoteca/handle/ proliferation by means of an oxidation- ACKNOWLEDGMENTS doc/404792 dependent mechanism. Cancer Prevention Research 2: 879-886. The authors acknowledge the CELOTO, MIB; PAPA, MFS; SACRAMENTO, support from Instituto Federal Goiano, LVS; CELOTO, FJ. 2011. Atividade KHOO, HE; PRASAD, KN; KONG, KW; phytochemistry laboratory and CNPq. antifúngica de extratos de Momordica JIANG, Y; ISMAIL, A. 2011. Carotenoids charantia L. sobre Colletotrichum musae. and their isomers: color pigments in fruits and Revista Brasileira de Plantas Medicinais 13: vegetables. Molecules 16: 1710-1738. 337-341. KIM, SK; JUNG, JJ; JUNG, HJ; YOON, CHITARRA, MIF; CHITARRA, AB. 2005. Pós- N; KANG, SS; ROH, GS; HAHM, JR. colheita de frutos e hortaliças: fisiologia e 2020. Hypoglycemic efficacy and safety of manuseio. Lavras, BR: FAEPE. 785p. Momordica charantia (bitter melon) in patients with type 2 diabetes mellitus. Complementary CHUNG, Ill-M; THIRUVENGADAM, M; Therapies in Medicine 52: e102524. REKHA, K; RAJAKUMAR, G. 2016. MACEDO, IYL; GARCIA, LF; NETO, JRO; LEITE, KCS; FERREIRA, VS; GHEDINI, 402 Horticultura Brasileira 39 (4) October - December, 2021

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Research KOEFENDER, J; MANFIO, CE; CAMERA, JN; SCHOFFEL, A; GOLLE, DP. 2021. Micropropagation of lavender: a protocol for production of plantlets. Horticultura Brasileira 39: 404-410. DOI: http://dx.doi.org/10.1590/s0102-0536-20210409 Micropropagation of lavender: a protocol for production of plantlets Jana Koefender 1* ;ID Candida Elisa Manfio 2 ;ID Juliane N Camera 1ID; André Schoffel 1 ;ID Diego Pascoal Golle 1ID 1Universidade de Cruz Alta (Unicruz), Cruz Alta-RS, Brasil; [email protected] (author for correspondence); ju_camera@yahoo. com.br; [email protected]; [email protected]; 2Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Epagri-SC), Ituporanga-SC, Brasil; [email protected] ABSTRACT RESUMO Lavender (Lavandula dentata) has several medicinal applications, Micropropagação de lavanda: um protocolo para produção both in the fresh form and after industrial processing. Productivity de mudas increase could be obtained through the mass propagation, expanding the possibility of obtaining raw material for production of essential A lavanda (Lavandula dentata) possui diversas aplicações oil. The objective of this work was to develop a micropropagation medicinais, tanto in natura como após processamento industrial. O protocol from explants obtained from seeds. Aspects of in vitro incremento à produtividade pode ser obtido por meio da propagação germination, multiplication, elongation and rooting were evaluated massal, ampliando a possibilidade de obtenção de matéria prima para a and, in sequence, the acclimatization process as well. The in vitro produção de óleo essencial. O objetivo deste trabalho foi desenvolver germination of lavender seeds can be obtained using a medium um protocolo de micropropagação a partir de explantes obtidos de composed of agar and water. For multiplication, the use of MS medium sementes. Foram avaliados aspectos da germinação, multiplicação, (Murashige & Skoog, 1962) added with 5.0 µM 6-benzylaminopurine alongamento e enraizamento in vitro e, em seguida, o processo de to the nutrient medium is the most appropriate. For elongation and aclimatização das mudas. A germinação in vitro de lavanda pôde ser rooting, the use of growth regulators is unnecessary. Acclimatization obtida apenas com a utilização do meio composto por ágar e água. can be obtained directly by transferring the explant to plastic bags with Para a multiplicação, o uso de meio MS (Murashige & Skoog, 1962) substrate, kept in a greenhouse. It was possible to develop a protocol acrescido de 5,0 µM de 6-benzilaminopurina ao meio nutritivo é o for the in vitro cultivation (establishment, multiplication, elongation, mais apropriado. Para o alongamento e enraizamento, a utilização rooting and acclimatization) of lavender from explants of seed origin. de reguladores de crescimento é dispensável. A aclimatização pode ser obtida diretamente com a transferência do explante para sacos plásticos com substrato, mantidos em casa de vegetação. Foi possível desenvolver um protocolo para o cultivo in vitro (estabelecimento, multiplicação, alongamento, enraizamento e aclimatização) de lavanda a partir de explantes de sementes. Keywords: Lavandula dentata, in vitro cultivation, essential oils, Palavras-chave: Lavandula dentata, cultivo in vitro, óleos essenciais, mass multiplication. multiplicação massal. Received on February 9, 2021; accepted on August 19, 2021 Over 25,000 plant species with al., 2018). The genus Lavandula has is a natural preservative (tested on medicinal properties are used essential oils of medicinal properties several microorganisms), antimicrobial, worldwide. Those uses include related to the control of acne, cramps, antioxidant, anti-inflammatory and pest everything from ethnobotanical practices flatulence, in addition to the relaxing and control (Wells et al., 2018). to the production of medicines. Currently, also antibacterial actions. Its application the herbal medicine production chain in perfumery is relevant, as well as the In Brazil, lavender (Lavandula grows more (around 15%) than the landscape use of Lavandula spp. for the dentate) is the most cultivated species production of synthetic medicines (4%) beauty of the plantations and its pleasant and like other species of the genus, it is (Rodrigues, 2016). The global market aroma. The leading world producers known for its medicinal and aromatic for vegetable essential oils grew by are France, Bulgaria, Great Britain, potential. Studies have demonstrated 7.5 billion dollars a year through 2018. Australia and Russia (Adamuchio et al., the application of hydroalcoholic extract In addition, lavender oils represent an 2017). Lavender essential oil has more of this species with anti-inflammatory important share of this market, with than 100 constituents (Devasigamani et properties (Algieri et al., 2016). 1,500 tons of oil per year (Wells et al., 2020) and, among other properties, Nevertheless, the hepatoprotective and antioxidant action of lavender has been 404 Horticultura Brasileira 39 (4) October - December, 2021

Micropropagation of lavender: a protocol for production of plantlets demonstrated (Pereira et al., 2019). increase in the demand for new plant units were kept in a growth room (the Martins et al. (2019) identified more products, tissue culture can contribute to characteristics of the growth room will than 20 compounds in the essential oil of obtain bioactive compounds of interest be described in a specific topic). At 15 this species, which confirms its potential (Dias et al., 2016) from the in vitro days, the germination percentage was for the industry. Among the compounds, cultures. evaluated. fenchone, eucalyptol and camphor stand out (Martins et al., 2019). The objective of this work Multiplication was to develop a protocol for the For the in vitro multiplication, The Brazilian demand for Lavandula micropropagation of lavender from the experiment was carried out in a spp. has aroused the interest of explants of seed origin. Here, we have completely randomized design, using farmers, as there is a market, but developed a protocol involving the a 2 x 3, bifactorial scheme, where few commercial crops in the country. stages of germination, establishment, levels of “A” factor was represented The seminal propagation is difficult multiplication, elongation and by the presence or absence of 1-alpha- especially because of the high cost acclimatization. naphthalenoacetic acid (NAA, 0 and of importing seeds, but also because 1.0 µM) and the levels of “B” factor of the frequent need of a previous MATERIAL AND METHODS were represented by three different treatment to overcome dormancy. concentrations of cytokinin (BAP, Seeds of the lavender genus have slow The experiment was conducted at 6-benzylaminopurine): 0; 5.0; 10.0 germination rates and heterogeneous the Plant Tissue Culture Laboratory µM; totaling six treatments and ten growth, which is a problem to improve at the University of Cruz Alta (Cruz replicates, each composed of a 200 mL industrial production with homogeneity Alta-RS), from February to December flask containing two explants (nodal in the composition of oils. As a result, 2019. The explants are originated from segments) and 20 mL culture medium. lavender growers around the world show commercial seeds (Feltrin®) obtained Thus, a total of 60 experimental units preference for vegetative propagation from an agricultural store. The steps of and 120 explants were analyzed. After (clones), material for uniform production the experiments are described below. 60 days of cultivation, the following (Wells et al., 2018; Rodrigues et al., variables were evaluated: number of 2020). In vitro germination shoots per explant, shoot length (cm), For the in vitro germination, leaf number, hyperhydricity (%) and Some researches about the in vitro an experiment was carried out in a callus formation (%). cultivation of Lavandula spp. have been completely randomized design, with carried out, to produce homogeneous four treatments and 20 replicates. The Elongation and rooting plant material. Studies with L. dentata use of different culture media was Regarding the in vitro elongation were carried out, but with different evaluated, where one was composed and rooting of the explants in vitro (1 explant sources when compared to only of water/agar (7 g L-1); and the cm long nodal segments) as well as the our study. Jordan et al. (1998) and other of MS medium (Murashige & acclimatization, the experiments were Echeverrigaray et al. (2005) obtained Skoog, 1962) at the concentrations of conducted in a completely randomized micropropagation using nodal segments 25%, 50% and 100% (in which the agar design. Two growth regulators, IAA of adult plants as explants. Machado was added in the same concentration as (indoleacetic acid) and GA3 (gibberellic et al. (2011) developed a protocol mentioned above). The experimental acid) were tested at concentrations for the regeneration of L. dentata by unit consisted of 300 mL flasks, of 0, 5,0 and 10,0 µM, totaling nine shoot tips. With other species of the containing 40 mL nutrient medium and treatments, with 10 replicates, each Lavandula genus, a protocol for L. five lavender seeds (Lavandula dentata) composed of a 200 mL flask containing angustifolia in vitro cultivation from (Feltrin® commercial seeds), totaling 80 two explants and 20 mL culture medium. young segments was possible from experimental units and 400 seeds. Prior For the elongation, after 60 days in seeds’ germination. Different light to inoculation, the seeds were soaked vitro cultivation, root formation (%), sources and nutritional media were in a solution of gibberellic acid (GA3) hyperhydricity (%), callus formation used (Machado et al., 2013; Oliveira at 1.5 mg L-1 for 48 hours to increase (%), leaf number, shoot length (cm) et al., 2019). Also, for L. angustifolia, and accelerate germination according and number of shoots per explant were cultivation was possible from leaf to reports for the genus (Aoyama et evaluated. explants, via indirect organogenesis al., 1996; Oliveira et al., 2019). Next, (Devasigamani et al., 2020). So, the seeds went through a disinfestation Acclimatization in vitro production can contribute as process, which occurred in a laminar In relation to the acclimatization it allows the multiplication to obtain flow cabinet in the following sequence: phase, four treatments were tested, in plantlets that make the culture feasible 1 minute in 70% alcohol, 20 minutes which the experimental units remained (Adamuchio et al., 2017). Guo et al. in 4% sodium hypochlorite, and triple in the growth room for the first 10 days, (2014) report the importance of the washing with sterile distilled water. namely: permanence in the cultivation micropropagation technique for mass After disinfestation and inoculation in flask (in culture medium) sealed with production of plants that may be of the nutrient medium, the experimental aluminum foil; permanence in a culture industrial interest. In addition, with the flask (in culture medium) sealed with plastic film; maintain in the cultivation Horticultura Brasileira 39 (4) October - December, 2021 405

J Koefender et al. flask (in culture medium) without stage for being more economical. Also, (a species of the same genus) with the the average germination obtained was use of GA3 for in vitro germination have sealing; direct transplant to substrate 80%: medium water-agar (81%), MS also shown satisfactory results, but, still, medium at 25% (80%), 50% (79%) slightly less than 80% (Oliveira et al., packed in plastic bags (the substrate and 100% (80%). Considering that the 2019). literature reports that lavender seeds used was Carolina Soil® class XVI). have a slow germination process, and Multiplication a low germination percentage in tissue Regarding multiplication, for all Afterwards, regardless of the previous culture (Panizza & Tognoni, 1991), evaluated variables in this experiment, the results in this study demonstrated interaction was observed between the treatments, all explants were transferred a good germination percentage and the different concentrations of NAA and in vitro establishment of the species. BAP used. In relation to number of to disposable plastic cups containing In addition, studies of culture media shoots per explant, the greatest increase that promote in vitro germination are in multiplication occurred when 5,0 µM autoclaved substrate, kept in a growth important, to maximize the germination BAP was used in the absence of NAA, rate (Rosa et al., 2012). In culture media with the formation of 9.05 shoots per room for one week and, subsequently where the concentration of nutrients is explant. A study with the species of reduced, the responses tend to be of the same genus Lavandula angustifolia transferred to 300-mL plastic bags lesser magnitude compared to those obtained rates of 1.9 to 3.3 sprouts per in which there is no such reduction. In explant (in the LS medium with 1.0 µM with substrate containing one plant this study, it appears that the nutrients BAP), lower than that obtained in this absence did not harm the germination of study (Machado et al., 2013). and transferred to greenhouse with lavender seeds, which can directly result In relation to the shoot length, in the minimization of cultivation costs. the best result was observed when controlled temperature and irrigation. 30 Results of studies with L. angustifolia growth regulators (2.29 cm) were not used, possibly because the days after transferring the explants to the greenhouse, the survival (%) and length (cm) of the plantlets were evaluated. In experiments in which the MS medium was used, 100 mg L-1 of inositol, 30 g L-1 of sucrose and 7 g L-1 of agar were added (except in the germination experiment with agar-water treatment). The pH of the media was adjusted to 5.8 and sterilized for 20 minutes at 121°C and 1.5 atm pressure. The conditions of the growth room (all experiments) were characterized by 25±3ºC temperature and 16 h photoperiod, obtained with Table 1. Number of shoots per explant, shoot length (cm), leaf number, hyperhidricity (%) and callus formation (%) of lavender plants (Lavandula dentata) submitted to different BAP daylight-type fluorescent lamps, with and NAA concentrations in vitro cultivation. Cruz Alta, Unicruz, 2015. 40 µmol m-2 s-1 light intensity. Statistical analysis BAP 0 µM 5.0 µM 10.0 µM Means The normality of the errors was Evaluated variables NAA tested by the Kolmogorov-Smirnov test and the homogeneity of variances Shoots per explant (no) 0.0 µM 2.00 a B 9.05 a A 4.70 a B 5.25 1.0 µM 2.10 a A 3.95 b A 1.45 b B 2.50 by the test of Bartlett. When necessary, for analysis purposes, the data were Means 2.05 6.50 3.07 transformed by the function, CV (%) 38 however, the exposed data were the 0 µM 2.29 a A 0.75 a B 1.31 a AB 1.45 1.0 µM 1.30 a A 1.07 a A 1.04 a A 1.14 originals. Afterwards, the results were Shoot length (cm) submitted to Analysis of Variance and when the “F” value was significant, the Means 1.79 0.91 1.17 multiple comparison of the means was CV (%) 26 performed using the Tukey test at 5% 0 µM 13,5 a AB 10,1 b B 15,5 a A 13,03 1.0 µM 11,7 a B 16,2 a A 13,9 a AB 13,93 probability of error. The analyses were Leaf (nº) performed with the aid of the SISVAR® Means 12,6 13,15 14,7 statistical software (Ferreira, 2011). CV (%) 14 RESULTS AND DISCUSSION Hyperhidricity (%) 0 µM 1.0 a A 7.0 a B 9.0 a B 5.67 4.33 Germination 1.0 µM 1.0 a A 6.0 b B 6.0 b B For the lavender seed germination experiment, no differences were Means 1.0 6.5 7.5 observed among the tested treatments; the use of nutritive medium (MS CV (%) 73 medium at 25, 50 or 100%) or just water-agar was indifferent. Therefore, Callus formation (%) 0 µM 10 a A 90 a B 50.2 a AB 50.06 the use of water-agar is indicated at this 1.0 µM 60 b A 60 a A 80 a A 66.6 Means 35 75 65.1   CV (%) 67 *Means followed by uppercase letter in the line and lowercase letter in the column are not different from each other by the Tukey test, p<0.05. 406 Horticultura Brasileira 39 (4) October - December, 2021

Micropropagation of lavender: a protocol for production of plantlets Figure 1. Overall aspect of in vitro multiplication of Lavandula dentata. The treatments refer to combinations between NAA and BAP (µM) namely: a) 0-0; b) 1.0-0; c) 0-5.0; d) 1- 5.0; e) 0-10.0; f) 1-10.0. In figure “c”, the arrows point to the shoots that appear at the base of the explant. In figures “d” and “e”, the arrows show the hyperhydricity aspect of the treatments. Side bars = 1.0 cm. Cruz Alta, Unicruz, 2015. Figure 2. Overall aspect of the elongation of lavender plants (Lavandula dentata) (a) in which the arrows point to the root formation; from the beginning of the acclimatization process in the cultivation room (b); development in the greenhouse (c). Bar = 1.0 cm. Cruz Alta, Unicruz, 2015. Horticultura Brasileira 39 (4) October - December, 2021 407

J Koefender et al. Table 2. Number of shoots per explant, shoot length (cm), leaf number, callus formation (%), be seen in Figure 1. hyperhidricity (%) and root formation (%) of lavender plants (Lavandula dentata) submitted It can be considered that the data to different concentrations of GA3 and IAA in vitro cultivation. Cruz Alta, Unicruz, 2015. obtained in the experiment show the Evaluated variables IAA 0 µM GA3 adequate in vitro multiplication rates 5.0 µM 10.0 µM Means obtained as this is the most relevant data in this step of the study, mainly due 0.0 µM 2.00 Aa 2.45 Aa 3.77 Aa 2.74 to the multiplication rate. The highest rate was 9.05 shoots per explant. Some Shoots per explant (no) 0.5 µM 1.10 Ba 1.55 ABa 1.85 Aa 1.50 studies with species of the same genus have also obtained favorable results 1.0 µM 1.95 Aa 2.11 Aa 2.08 Aa 2.05 with the use of cytokinins, however, not approaching the average obtained in Means 1.68 2.04 2.57 this study. In L. coronopifolia, the use of BAP allowed to obtain 3.6 shoots per   CV (%) 26 explant (Khateeb et al., 2017). On the other hand, for L. angustifolia, also with 0.0 µM 1.85 Bb 3.00 Aa 3.59 Aa 2.81 cytokinin supplementation (BAP), Vinh et al. (2017) obtained 3.06 shoots per Shoot length (cm) 0.5 µM 2.10 Ab 2.65 Aa 2.40 Aab 2.38 explant. It is important to mention that the calluses observed in this treatment 1.0 µM 3.09 Aa 2.67 Aa 2.92 Ab 2.89 were small and did not compromise multiplication; likewise, hyperhydricity Means 2.35 2.77 2.97 only in the most basal part of the explant did not compromise the transfer to the CV (%) 29 culture media of the next stages and development. 0.0 µM 13.50 Aab 15.90 Aa 17.27 Aa 15.56 Elongation and rooting Leaf (no) 0.5 µM 12.40 Ab 14.60 Aa 16.20 Aa 14.40 On the elongation phase, there was interaction between the different 1.0 µM 16.91 Aa 16.22 Aa 16.33 Aa 16.49 concentrations of IAA and GA3 for number of shoots per explant, shoot Means 14.27 15.57 16.60 length, leaf number and root formation (Table 2). For the number of shoots, CV (%) 24 using 0 and 1,0 µM of IAA did not show any differences in the different 0.0 µM 10.00 0.50 0.73 3.74 A concentrations of GA3 (Table 2), ranging the number of shoots per explant from Callus formation (%) 0.5 µM 0.30 0.40 0.60 0.43 A 1.10 to 3.77. The use of IAA at 0,5 µM, alone or with 5.0 µM GA3, reduced the 1.0 µM 0.55 0.22 0.67 0.48 A number of shoots. Means 3.62 A 0.37 A 0.66 A For shoot length, at 0 µM of IAA and 10.0 µM of GA3, a length of 3.59 CV (%) 25 cm was achieved. The number of leaves in lavender was not influenced by the 0.0 µM 10.00 0.20 0.27 3.49 different concentrations of GA3. An increase in the number of leaves was Hyperhidricity (%) 0.5 µM 0.10 0.20 0.20 0.17 observed at concentrations 0 µM of GA3, as the concentrations of IAA were 1.0 µM 0.00 0.22 0.33 0.19 incremented. There was no difference between the treatments tested for Means 3.37 0.21 0.27 callus formation (Table 2). The highest percentage of lavender root formation   CV (%) 30 was obtained at the concentration of 0 µM IAA and 0 µM GA3. The greatest 0.0 µM 20.00 Aa 0.30 Ba 0.18 Ba 6.83 hyperhydricity was observed in the absence of growth regulators, however, Root formation (%) 0.5 µM 0.40 Ab 0.30 Aa 0.30 Aa 0.33 in the best treatment for elongation (10 1.0 µM 0.09 Ab 0.33 Aa 0.33 Aa 0.25 Means 6.83 0.31 0.27   CV (%) 30 *Means followed by the same uppercase letter in the line and the same lowercase letter in the column are not different from each other by the Tukey test, p<0.05. morphogenic routes are not channeled low lignification, impairing stomatal towards multiplication, but towards function. It can reduce the mechanical the development of the explant; the strength and survival of explants number of leaves was higher when (Machado et al., 2013). using the combination of 1,0 µM NAA and 5,0 µM BAP. Hyperhydricity The use of BAP (mainly in the 5.0 was observed as the concentration of µM concentration) increases the variable BAP increased, but in the later stages callus formation, considering just callus it did not compromise development formed at the base of the explants (Table and acclimatization, nor did it affect 1). However, this number only reflects the multiplication of the explants. small calluses formed at the base of the Hyperhydricity is a physiological explants, which did not interfere with problem of excessive hydration and the development and later stages of this research. The multiplication aspect can 408 Horticultura Brasileira 39 (4) October - December, 2021

Micropropagation of lavender: a protocol for production of plantlets Table 3. Survival and plant height of lavender plants (Lavandula dentata) in different ways MACHADO, MP. 2017. Aspectos gerais sobre for acclimatization (different cultivation flasks seals with subsequent passage to plastic cups a cultura da Lavanda (Lavandula spp.). Revista or direct passage to bags). Cruz Alta, Unicruz, 2015. Brasileira de Plantas Medicinais 19: 483-490. Treatments Survival Plant height ALGIERI, F; RODRIGUEZ-NOGALES, A; (%) (cm) VEZZA, T; GARRIDO-MESA, J; GARRIDO- Cultivation flask sealed with aluminum foil MESA, N; UTRILLA, MP; GONZÁLEZ- Cultivation flask sealed with plastic film 46.15 b* 2.23 a TEJERO, MR; CASARES-PORCEL, M; Cultivation flask without sealing 93.33 a 3.96 a MOLERO-MEZA, J; CONTRERAS, MDM; Plastic bag for seedlings direct transplant SEGURA-CARRETERO, A; PÉREZ- 80.00 ab 3.45 a PALACIO, J; DIAZ, C; VERGARA, N; 83.33 ab 3.20 a VICENTE, F; RODRIGUEZ-CABEZAS, ME; GALVEZ, J. 2016. Anti inflamatory activity of CV (%) 15 36 hidroalcoholic extracts of Lavandula dentata L. and Lavandula stoechas L. Journal of *Means followed by the same letter are not different from each other by the Tukey test, p<0.05. Ethnopharmacology 190: 142-158. µM of GA3) it was only 0.27%(Table 2). calluses in some shoots but however, still AOYAMA, EM; ONO, EO; FURLAN, MR. 1996. Works carried out by Machado et al. offering the highest rate of propagation. Estudo da germinação de sementes de lavanda It should be mentioned that additional (Lavandula angustifolia Miller). Scientia (2013) demonstrate that on the rooting studies should be carried out in order to Agricola 53: 2-3. percentage of lavender plants, the reduce hyperhydricity. It is important to number of main roots per micro cuttings mention that the observed calluses were DEVASIGAMANI, L; DEVARAJAN, R; was higher when auxin was not added small and at the base of the explants, LOGANATHAN, R; RAFATH, H; PADMAN, to the culture medium, thus, a reduction not preventing the fragmentation for M; MV, GR; GIRIDHAR, L; HC, C; KUPPAN, in the number of roots in the presence subculture in the elongation experiments; N. 2020. Lavandula angustifolia L. plants of IBA and NAA was observed. In in the same way, hyperhydricity was regeneration from in vitro leaf explants- preliminary studies, different responses in the basal portion of the explants, derived callus as conservation strategy. were also observed in the induction of barely noticeable, not preventing the Biotecnología 20: 76-82. in vitro rooting of lavender plants with continuation of cultivation either. For the addition of plant regulators. The better acclimatization, the process DIAS, MI; SOUSA, MJ; ALVES, RC; overall aspect of the elongation and should be started in cultivation flasks FERREIRA, ICFR. 2016. Exploring plant acclimatization can be seen in Figure 2. sealed with plastic film, and only tissue culture to improve the production of later transferred to the greenhouse. phenolic compounds: a review. Industrial Acclimatization It was possible to obtain a complete Crops and Products 82: 9-22. In acclimatization process of protocol for the micropropagation of lavender plants, no statistical difference Lavandula dentata from explants of ECHEVERRIGARAY, S; BASSO, R;ANDRADE, was observed on the height of the seed origin, from in vitro cultivation to LB. 2005. Micropropagation of Lavandula plants, testing different treatments. acclimatization. dentata from axillary buds of field-grown However, the treatments differed in adult plants. Biologia Plantarum: 49: 439-334. terms of survival. So, all evaluated ACKNOWLEDGEMENTS treatments were recommended, except FERREIRA, DF. 2011. Sisvar: a computer the use of cultivation flasks wrapped To the National Council for Scientific statistical analysis system. Ciência e with aluminum foil. We observed that, and Technological Development for Agrotecnologia 35: 1039-1042. although the height of the plants that financing the scholarship (CNPq). To survived showed no statistical difference Economics Developing, Science and GUO, L; XUE, F; GUO, J; NA, R. 2014. Plant between the treatments tested, the Technology General Office (SICT- tissue culture: a recent progress and potential percentage of survival was higher when RS) and World Bank (BIRD), to the applications. Journal of Agricultural Science the cultivation flasks were wrapped with Coordination for the Improvement of and Technology 15: 2088-2095. plastic film, however it was indifferent Higher Education Personnel (CAPES), whether we used a plastic cup or plastic to Research Support Foundation of JORDAN, AM; CALVO, MC; SEGURA, J. 1998. bag (Table 3). the State of Rio Grande do Sul for the Micropropagation of adult Lavandula dentata For the in vitro cultivation of scholarships (FAPERGS). plants. The Journal of Horticultural Science lavender from explants of seed origin, the and Biotechnology 73: 93-96. germination process can be performed REFERENCES using a medium composed only of KHATEEB, WA; KANAAN, R; EL-ELIMAT, T; agar and water. For the multiplication, ADAMUCHIO, LG; DESCHAMPS, C; ALU’DATT, M; LAHHAM, J; EL-OQLAH, the use of MS medium added with 5,0 A. 2017. In vitro propagation, genetic stability µM BAP allows to achieve a greater and secondary metabolite analysis of wild number of shoots, therefore showing lavender (Lavandula coronopifolia Poir). the occurrence of hyperhydricity and Horticulture, Environment, and Biotechnology 58: 393-405. Horticultura Brasileira 39 (4) October - December, 2021 MACHADO, MP; CIOTTA, MN; DESCHAMPS, C; ZANETTE, F; CÔCCO, LC; BIASI, LA. 2013. Propagação in vitro e caracterização química do óleo essencial de Lavandula angustifolia cultivada no Sul do Brasil. Ciência Rural 43: 283-289. MACHADO, MP; SILVA, ALL; BIASI, LA. 2011. Effect of plant growth regulators on in vitro regeneration of Lavandula dentata L. shoot tips. Journal of Biotechnology and Biodiversity 2: 28-31. MARTINS, RP; GOMES, RAS; MALPASS, ACG; OKURA, MH. 2019. Chemical characterization of Lavandula dentata L. essential oils grown in Uberaba-MG. Ciência Rural 49: 1-7. MURASHIGE, T; SKOOG, F. 1962. A revised 409

J Koefender et al. medium for rapid growth and bioassays with MRM; MARIN, JJG; CARDOSO, SM. 2019. MUNIZ, MFB; CURTI, AR. 2012. Superação tobacco tissue culture. Physiologia Plantarum Hepatoprotection of Mentha aquatica L., da dormência e germinação in vitro de 15: 473-497. Lavandula dentata L., and Leonurus cardiaca sementes de bracatinga (Mimosa scabrella L. Antioxidants 267:1-13. Bentham). Semina: Ciências Agrárias 33: OLIVEIRA, RC; ASMAR, SA; SILVA, HFJ; 1021-1026. MORAIS, TP; LUZ, JMQ. 2019. Regulators, RODRIGUES, WC. 2016. Competitividade e VINH, DT; HOA, MT; KHAI, PC; MINH, culture media and types of lights in vitro mudança institucional na cadeia produtiva TV. 2017. Micropropagation of lavender lavander culture. Ciência Rural 49: 1-7. de plantas medicinais no Brasil. Interações (Lavandula angustifolia). Journal of 17: 267-277. Innovations in Pharmaceutical and Biological PANIZZA, M; TOGNONI, F. 1991. Sciences 4: 7-11. Micropropagation of Lavandin (Lavandula RODRIGUES, DB; RADKE, AK; SOMMER, WELLS, R; TRUONG, F; ADAL, AM. 2018. officinalis Chaix x Lavandula latifolia LR; ROSA, DSB; SCHUCH, MW; ASSIS, Lavandula essential oils: a current review of Villars cv. ‘Grosso’). In: BAJAJ, YPS (ed). AM. 2020. Quality of light and indolbutiric applications in medicinal, food and cosmetic Biotechnology in Agriculture and Forestry. acid in vitro rooting of lavander. Ornamental industries of lavender. NPC – Natural Product New York: Spring. p. 295-305. Horticulture 26: 89-94. Communications 13: 1403-1417. PEREIRA, OR; MACIAS, RIR; DOMINGUES, ROSA, FC; REINIGER, LRS; GOLLE, DP; 410 Horticultura Brasileira 39 (4) October - December, 2021

Research LOPES, CA; CARVALHO, ADF; PEREIRA, AS; AZEVEDO, FQ; CASTRO, CM; EMYGDIO, BM; SILVA, GO. 2021. Performance of Solanum phureja- derived bacterial-wilt resistant potato clones in a field naturally infested with Ralstonia solanacearum in Central Brazil. Horticultura Brasileira 39: 411-416. DOI: http://dx.doi.org/10.1590/s0102-0536-20210410 Performance of Solanum phureja-derived bacterial-wilt resistant potato clones in a field naturally infested with Ralstonia solanacearum in Central Brazil Carlos A Lopes 1 ;ID Agnaldo DF Carvalho 1 ;ID Arione S Pereira 2 ;ID Fernanda Q Azevedo 2 ;ID Caroline M Castro 2 ;ID Beatriz M Emygdio 2 ;ID Giovani O Silva 1ID 1Embrapa Hortaliças, Brasília-DF, Brasil; [email protected]; [email protected]; [email protected];2Embrapa Clima Temperado, Pelotas-RS, Brasil; [email protected]; [email protected]; [email protected]; beatriz. [email protected] ABSTRACT RESUMO Bacterial wilt (BW), or brown rot, caused by the soil and Comportamento de clones de batata resistentes à murcha seed borne bacterium Ralstonia solanacearum, is one of the most bacteriana, derivados de Solanum phureja, em campo devastating diseases of potatoes cultivated in warmer regions of naturalmente infestado com Ralstonia solanacearum no Brasil the world. There are no potato cultivars with a desirable level of Central BW resistance, although it has been recognized that resistance can be an outstanding component for disease management. However, A murcha bacteriana (MB), ou murchadeira, causada pela bactéria the sources of resistance available lack agronomic traits required Ralstonia solanacearum e transmitida pelo solo e pela semente, é by potato growers, therefore being of little interest to breeders. uma das doenças mais devastadoras da batata cultivada nas regiões The objective of this work was to evaluate the performance of 11 mais quentes do mundo. Seu controle é muito difícil e não existem clones selected for BW resistance and improved for tuber traits cultivares resistentes a ela, embora a resistência seja reconhecida upon selection in the last two decades. The clones under test were como importante componente para o manejo integrado da doença. compared with susceptible and resistant clones and cultivars, in a No entanto, as fontes de resistência à MB disponíveis até então completely randomized blocks design with three replications of single apresentam muitas características indesejáveis, sendo, por isso, de lines of 10 plants, in a field naturally infested with race 1, biovar 1, pouco interesse aos melhoristas que têm o foco no desenvolvimento phylotype II of R. solanacearum. BW incidence was assessed 60-70 de genótipos de interesse comercial. O objetivo deste trabalho foi de days after planting and total tuber yield in each plot was recorded avaliar 11 clones selecionados nas duas últimas décadas na Embrapa 110 days after planting. All the evaluated clones presented higher Hortaliças para resistência à MB e, ao mesmo tempo, com seleção levels of resistance to BW compared with the commercial varieties, leve para algumas características comerciais durante os ciclos de not differing from the resistant, not commercial, controls. In a next seleção. O nível de resistência dos clones foi avaliado por quatro step, these clones will be characterized for other desirable traits anos consecutivos em área experimental naturalmente infestada com and those which combine high level of resistance and commercial a raça 1, biovar 1, filotipo II de R. solanacearum. Como testemunhas, characteristics will be recommended for breeders for enriching the foram usados clones ou cultivares previamente identificados como genotypic background in the search for commercial varieties. We resistentes ou suscetíveis. O delineamento experimental foi de blocos also confirmed that the cultivar BRSIPR Bel displays an intermediate ao acaso com três repetições e parcelas de 10 plantas, em linha level of resistance, what makes it an interesting genitor for its good única. A avaliação foi feita pela incidência da doença no período agronomic characteristics. The findings of this work demonstrate de 60 a 70 dias após o plantio e a produção total de tubérculos nas that the improved potato clones selected under tropical conditions in parcelas registrada 110 dias após o plantio. Todos os clones avaliados the Embrapa’s pre-breeding project possess high and stable levels of apresentaram níveis de resistência superiores aos das cultivares mais resistance to bacterial wilt, being a valuable resource for breeders. plantadas no Brasil e não diferiram das testemunhas resistentes. Como somente a resistência não basta para ser um bom progenitor, esses clones serão avaliados para as características comerciais de maior relevância aos programas de melhoramento. Em adição, foi confirmada a resistência intermediária da cultivar BRSIPR Bel, o que a torna um genitor de interesse pelas suas ótimas características comerciais. Este trabalho demonstrou que clones selecionados na Embrapa para resistência à MB em condições tropicais apresentam alto e estável grau de resistência e podem vir a ser usados, de imediato ou após novos ciclos de seleção, em programas de melhoramento visando ao desenvolvimento de cultivares. Keywords: Solanum tuberosum, brown rot, resistance. Palavras-chave: Solanum tuberosum, murchadeira, resistência. Received on May 24, 2021; accepted on October 21, 2021 Horticultura Brasileira 39 (4) October - December, 2021 411

CA Lopes et al. Growers worldwide live with an agronomically advanced materials The BW-resistant potato clones, already existing risk of cultivating (Iqbal et al., 2013). MB54-2, MBF21-05, MBF3-03, MBF3- potatoes (Solanum tuberosum) in 06, MBF6-03, MBF6-07, MBF9-01, regions and seasons with environmental Resistance is usually linked with MBF9-02, MB195-10, MB57-32, and conditions which deviate from the ideal undesirable traits in wild potato MB9846-01 were obtained from true for the crop. This is especially relevant species and on Cruza 148, the most seeds of populations derived from in tropical and subtropical regions known genotype in the world for being crosses involving S. tuberosum cultivars like those that occur in Brazil. These cultivated in some African countries (or advanced clones from Brazilian conditions favor the establishment and mainly because of its resistance to BW Breeding Program) and BW-resistant the intensity of diseases like bacterial and late blight (Phythophtora infestans) clones which resistance was derived wilt (BW), or brown rot, caused by (Schmiediche & Martin, 1986; Tung et from S. phureja. These clones are the soil and seed borne bacterium al., 1993; Patil et al., 2012; Gutarra et result of a pre-breeding project for BW Ralstonia solanacearum, one of the al., 2015). resistance started at Embrapa Hortaliças most devastating diseases of potatoes in the 1980’s in close cooperation with cultivated in warmer regions of the Systematic efforts to obtain BW- the International Potato Center (CIP), world (Lopes et al., 2011; Yuliar & resistant cultivars have been carried Lima, Peru, following a selection Toyota, 2015; Lopes & Rossato, 2018; out the last five decades, after high protocol developed and adjusted to meet Elphinstone, 2020). levels of resistance were identified the demands of the Brazilian market of in Solanum phureja diploid clones fresh and processing potatoes (Lopes BW induces foliage wilt followed (Sequeira & Rowe, 1969; Schmiediche, et al., 2018). This protocol consists by plant death and tuber rotting, 1986). This source of resistance was of crossing BW resistant clones with resulting in yield losses that can reach found to be dominant and readily cultivars or advanced clones and the up to 80% in a susceptible cultivar transferred to commercial cultivars of S. progeny challenged at the seedling whenever environmental conditions tuberosum without carrying some of the stage with artificial inoculation with are favorable for disease onset and undesirable traits found in wild species R. solanacearum under greenhouse. spread (Champoiseau et al., 2009). But (Schmiediche & Martin, 1986; French The surviving putative resistant clones economic losses can be even greater, et al., 1998). are then exposed in a field naturally since the bacterium infests the soil, infested with race 1, biovar 1, phylotype making it impossible to grow many In order to improve the agronomic II (R1B1P2) of R. solanacearum (Lopes susceptible Solanaceae species for years characteristics of resistant clones, et al., 2018), where, besides evaluated (Elphinstone, 2020). Embrapa’s pre-breeding program for for resistance, they are selected (visual BW resistance started in the early light selection) especially for yield and Although cultural practices, such 1980’s in a close collaboration with the tuber type. A new cycle of selection is as crop rotation, planting season and International Potato Center (CIP) and started, crossing the best selected clones use of disease-free seeds are control relied on crosses of commercial cultivars with different commercial variety or measures that significantly contribute of S. tuberosum mainly with clones of S. advanced clone in order to avoid genetic to reduce bacterial wilt losses, host phureja. Resistant clones were selected depression by endogamy (Mihovilovich plant resistance can be an additional following a protocol that started from et al., 2017; Lopes et al., 2018). effective strategy for fighting the disease artificial inoculation of seedlings in With this methodology, besides BW (Mihovilovich et al., 2017; Muthoni et a greenhouse followed by a series of resistance, we performed selection for al., 2020). However, there are no major greenhouse and field multiplications tuber commercial traits in conformity potato cultivars worldwide reliably and selection (Mihovilovich et al., with the goals established in Embrapa’s resistant to BW, even though different 2017; Lopes et al., 2018). This protocol potato breeding project coordinated by responses on susceptibility degrees are allowed the combination of BW Embrapa Clima Temperado. observed. Unfortunately, the available resistance with good agronomic and partial resistance is not enough to quality traits; thus, the advanced clones Seed production protect the crop in excessively hot or can be used by breeders in new crossings To avoid latent infection which could wet conditions (Carvalho et al., 2017; in selection cycles or eventually be interfere with disease onset, tubers Elphinstone, 2020; Muthoni et al., released as cultivars. used as seeds for the experiments were 2020). produced in the previous years in a field The objective of this work was to at Embrapa Hortaliças without recent The genetic control of BW resistance evaluate the BW field resistance of 11 history of bacterial wilt. The exception is particularly complex in tetraploid clones selected for BW resistance and was in 2020, when apparently healthy species like potato, where resistance commercial traits in the last two decades tubers of ‘BRSPR Bel’ and the resistant genes are found in wild species usually in a pre-breeding section of Embrapa’s controls ‘MB-03’ and ‘Cruza 148’ were linked to undesirable traits (Schmiediche potato breeding program. saved from the infested plot in the same & Martin, 1986; Tung et al., 1993; Patil period of 2019. Tubers were harvested et al., 2012; Muthoni et al., 2020). This MATERIAL AND METHODS in August/September and maintained in linkage usually makes breeders to avoid wild species as genitors, preferring Tested clones 412 Horticultura Brasileira 39 (4) October - December, 2021

Performance of Solanum phureja-derived bacterial-wilt resistant potato clones in a field naturally infested with Ralstonia solanacearum in Central Brazil a cold chamber (3-10ºC). v. 8.2 software (Muller & Fetterman, commercial susceptible controls, 2003). The single effects of the variable ‘Asterix’, ‘Agata’, and ‘Monalisa’ - the Assessment of bacterial wilt clones, year of evaluation and the latter a commercial susceptible cultivar resistance interactions, whenever significant, were replaced by ‘Agata’, and did not differ split and grouped by the Scott-Knott test from the resistant controls, ‘Cruza 148’ The advanced 11 resistant clones at 5% probability level, using the Genes and ‘MB 03’ (Table 1). were evaluated at Embrapa Hortaliças, v. 1990.2019.120 software (Cruz, 2013). Brasília, DF, Brazil (15°56’S, 48°08’W, All these BW resistant clones 998 m altitude) in the period of May- RESULTS AND DISCUSSION presented also higher total tuber yield August of 2017 to 2020 in a field than the susceptible cultivars, and as naturally infested with R1B1P2 of Typical BW symptoms developed a result of more plant survival in the R. solanacearum, which is prevalent in the R. solanacearum infested area field, highlighting for this trait the in warmer regions/countries (Lopes in all four years of testing, therefore clones MB54-02, MBF3-06, MB195- et al., 2018, Muthoni et al., 2020). allowing the observation of differences 10, MB57-32, MB9846-01, and the The experiments consisted of plots in resistance levels among the potato standard BW resistant control MB-03 of 10-plant single lines with three genotypes. Even though the disease (Table 2). replications in a RCBD. The susceptible intensity varied among years, due to controls were ‘Agata’ and ‘Asterix’, differences in temperature and late These results, therefore, indicate Dutch cultivars most planted in Brazil rainfalls which affected disease onset striking differences observed between for fresh market and processing, and development, genotype reactions the susceptible controls, displaying respectively, and ‘Monalisa’, that was were consistent (Table 1). All clones high disease incidences and practically widely cultivated in Brazil in the past previously selected for BW resistance no yield, and the resistant clones, with and was replaced by other cultivars were significantly different from the yields close or higher than 1 kg/plant partly due to its high susceptibility to due to high plant survival (Tables 1 BW (Silveira et al., 2007; Lopes et al., and 2). 2018). Resistant controls consisted of clones MB-03, selected at Embrapa, The Brazilian cultivar BRSIPR Bel and Cruza 148, provided by CIP as an international resistant control. Both Table 1. Means of bacterial wilt incidence (%) of potato clones and cultivars cultivated for clones are highly resistant to BW, four consecutive years in a field plot infested with Ralstonia solanacearum in Brasilia, DF. but lack important commercial traits Brasilia, Embrapa Hortaliças, 2017-2020. (Silveira et al., 2007; Lopes et al., 2018). The Brazilian potato cultivar Clone/cultivar Year BRSIPR Bel, which displayed an intermediate level of resistance to BW 2017 2018 2019 2020 in a preliminary study (Carvalho et al., 2017), was also used as control. MB54-02 - 5.24 Bd 17.12 Ac 12.97 Ac The plants were grown according Agata* 73.02 Aa 100.00 Aa 100.00 Aa 75.21 Aa to local standard fertilization and foliar pest control procedures in order Asterix* 59.72 Bb 96.61 Aa 100.00 Aa 71.91 Aa to obtain acceptable yield. Disease incidence (% of wilted plants) was BRSIPR Bel 48.87 Bb 41.37 Bb - 93.09 Aa measured 60 to 70 days after planting, depending on the year, as soon as at least Cruza 148** 21.83 Ac 10.09 Bd 5.24 Bd 16.42 Ac 80% of the plants of susceptible controls were wilted, occasion that clearly MBF21-05 - 49.81 Ab 56.52 Ab 16.30 Bc distinguished resistance levels among the genotypes. Plants were harvested MBF3-03 14.85 Bc 42.78 Ab 65.64 Ab - 110 days after planting, 10 days after a desiccant application, and the total tuber MBF3-06 3.42 Cd 17.83 Bc 39.58 Ab 12.36 Bc yield in each plot was recorded (Lopes et al., 2018). MBF6-03 1.91 Cd 36.11 Ab 19.13 Bc 10.09 Cd The data of percentage of incidence MBF6-07 1.91 Bd 12.97 Ac 7.48 Bd 12.97 Ac of BW and of total tuber yield per plot of each year were individually submitted MBF9-01 1.91 Bd 19.13 Ac 10.00 Bd 12.36 Ac to the analysis of variance, after tested for normality, and transformed by MBF9-02 24.93 Bc 36.11 Ab 57.72 Ab 17.12 Bc when needed. A joint analysis of the four years was performed using the SAS MB-03** 0.00 Bd 5.24 Bd 20.00 Ac 7.48 Bd MB195-10 1.91 Bd 14.45 Ac 7.48 Bd 7.48 Bd MB57-32 21.69 Ac - 28.52 Ac - MB9846-01 22.18 Ac 9.46 Bd 23.11 Ac 25.92 Ac Monalisa* 100.00 Aa 100.00 Aa 100.00 Aa - General mean 26.54 37.33 41.40 27.98 CV (%) 34.49 24.07 25.17 41.23 Same capital letter in the line and small letter in the column belong to the same group (Scott- Knott 5%); -= Not evaluated; *Susceptible controls; **Resistant controls. Horticultura Brasileira 39 (4) October - December, 2021 413

CA Lopes et al. confirmed its intermediate resistance Table 2. Means of total tuber yield (kg/10 plants) of potato clones and cultivars cultivated level in two of the three years. The for four consecutive years in a field plot infested with Ralstonia solanacearum in Brasilia, high incidence of the disease in 2020 DF. Brasilia, Embrapa Hortaliças, 2017-2020. is explained by the presence of latent infection in the seeds, since the tubers Clone/cultivar 2017 2018 Year 2020 available for planting this year were 2019 saved from a plot where BW occurred (Ciampi et al., 1980). This cultivar MB54-02 - 11.47 Aa 6.67 Aa 8.33 Aa has oblong tuber format, good tuber appearance and high values of tuber Agata* 0.54 Bd 0.00 Bd 0.45 Bd 1.63 Ac yield and specific gravity or tuber dry matter content, and low level of sugars, Asterix* 0.88 Bd 0.43 Bd 0.12 Bd 1.65 Ac being released as a cultivar mainly for frying chips (Pereira et al., 2015). BRSIPR Bel 2.52 Bc 4.00 Ab - 0.30 Cd It was not selected for BW resistance in its development process, but has Cruza 148** 2.47 Cc 7.83 Aa 7.09 Aa 5.39 Bb consistently shown some tolerance to the disease possibly due to a fortuitous gene MBF21-05 - 6.19 Ab 3.97 Ab 5.86 Ab combination (Carvalho et al., 2017). This result reinforces the importance of MBF3-03 4.80 Ab 3.61 Ab 2.52 Bc 0.00 Cd using “clean” seeds in potato production and the “zero tolerance” for BW in MBF3-06 12.46 Aa 6.84 Aa 9.47 Aa 9.30 Aa the seed certification process (MAPA, 2021). MBF6-03 4.77 Ab 3.81 Ab 4.05 Ab 3.21 Ab It has been reported that the MBF6-07 7.83 Aa 5.94 Bb 5.90 Bb 5.48 Bb genetic background of the commercial genitor used in the recurrent selection MBF9-01 7.41 Aa 5.73 Bb 3.66 Bb 3.94 Bb contributes with the level of resistance of the progenies (Tung et al., 1990). For MBF9-02 7.08 Aa 5.89 Bb 2.60 Cc 7.43 Aa example, when a BW-resistant clone is crossed with a cultivar selected under MB-03** 10.31 Aa 11.38 Aa 6.21 Bb 10.06 Aa tropical conditions and, additionally, displays an intermediate level of BW MB195-10 7.68 Aa 6.96 Aa 7.27 Aa 6.69 Aa resistance, such as ‘BRSIPR Bel’, the family yields higher numbers of MB57-32 8.53 Aa - 7.76 Aa - clones which survive on soils infested with R. solanacearum (Carvalho et al., MB984601 9.04 Aa 7.28 Aa 9.78 Aa 9.28 Aa 2017). That is why ‘BRSIPR Bel’ has been one of the main progenitors for Monalisa* 0.00 Ad 0.00 Ad 0.00 Ad - BW resistance used at the Embrapa’s breeding program since it was released, General mean 5.75 5.46 4.85 5.24 highlighting the performance of the clone MB 54-02 in this study. CV (%) 13.49 18.52 20.50 23.74 Partial resistance has been reported Same capital letter in the line and small letter in the column belong to the same group (Scott- in commercially acceptable S. Knott 5%); -= Not evaluated; *Susceptible controls; **Resistant controls. tuberosum genotypes not bred for BW resistance, such as ‘Ontario’and ‘Prisca’ in the development of BW-resistant cultivars in a soil naturally infested in Papua New Guinea and Madagascar, potato cultivars is that the control of the with R. solanacearum race 3, biovar respectively (Nielsen & Haynes, 1960; resistance is polygenic, what constraints 2, phylotype II in Rio Grande do Sul French, 1994; Norman et al., 2020); the transfer of the identified quantitative state in the South of the country. Cruza ‘Araucaria’, ‘Achat’ and ‘Granola’ trait loci into desirable genotypes 148, of unknown origin, probably from (Lopes & Quezado-Soares, 1995; especially in tetraploid species. In a cross between S. tuberosum and S. Quezado-Duval & Lopes, 1999; Silveira addition, these loci are usually linked demissum (Schmiediche, 1986; Muthoni et al., 2007), ‘Désirée’, ‘Kennebec’, to undesirable traits found in the wild et al., 2020) is the most known BW- ‘Red Norland’, and ‘Red Pontiac’ in the species used as sources of resistance, resistant variety available in the world USA (Norman et al., 2020). such as glycoalkaloid content, stolon (Gutarra et al., 2015), being cultivated length, eye depth, and tuber shape in some African countries and having The main reason for the slow advance (Schmiediche & Martin, 1986; French resistance also to late blight caused by et al., 1998; Patil et al., 2012; Lopes et Phythophtora infestans (Wulff et al., 414 al., 2018; Muthoni et al., 2020). 2007). Unfortunately, it also carries many undesirable genes that makes it Even though the resistant clones an unacceptable genitor in breeding selected in Brasilia have not been programs (Tung et al., 1990). tested in different potato-producing regions, we believe that the resistant Worldwide, BW pre-breeding from S. phureja, like the one found programs have not reported advanced in clone MB-03, can withstand other promising resistant genitors suitable variants of the pathogen. Silveira et for cultivar development programs due al. (2007) evaluated the clones Cruza to unstable resistance and undesirable 148 and MB-03 and reported that they tuber traits, what frustrate breeders who were significantly more resistant to search for introgressing BW resistance in bacterial wilt than other clones and tropical-adapted commercial genotypes Horticultura Brasileira 39 (4) October - December, 2021

Performance of Solanum phureja-derived bacterial-wilt resistant potato clones in a field naturally infested with Ralstonia solanacearum in Central Brazil (Lopes et al., 2018; Muthoni et al., Online. Plant Health Progress. American insumos-agropecuarios/insumos-agricolas/ 2020; Norman et al., 2020). In the Phytopathological Society. Madison, WI, sementes-e-mudas/publicacoes-sementes-e- present work, we observed good levels USA. Available at: <https://apsjournals.apsnet. mudas/INN32de20denovembrode2012.pdf> of resistance to BW, not differing from org/doi/abs/10.1094/PHP-2009-0313-01-RV>. Accessed on January 12, 2021. the resistant controls ‘Cruza 148’ and Accessed on March 18, 2021. ‘MB-03’, mainly the clones MB54- MIHOVILOVICH, E; LOPES, C; GUTARRA, 02, MBF3-06, MB1950-10, MB57- CIAMPI, L; SEQUEIRA, L; FRENCH, ER. L; LINDQVIST-KREUZE, H; ALEY, P; 32, MB9846-01, and the standard 1980. Latent infection of potato tubers by PRIOU, S; BONIERBALE, M. 2017. Protocol resistant control ‘MB-03’. Resistance Pseudomonas solanacearum. American Potato for assessing bacterial wilt resistance in on all of them is derived from S. Journal 57: 377-386. greenhouse and field conditions. International phureja, mainly present on ‘MB9846- Potato Center. Available at: <https://cgspace. 01’, which consistently performed CRUZ, CD. 2013. GENES - a software package cgiar.org/bitstream/handle/10568/83310/CIP- as a good genitor for BW breeding for analysis in experimental statistics and Protocol-assessing-bacterial-wilt resistance. programs. These resistant clones will quantitative genetics. Acta Scientiarum 35: pdf;jsessionid=7ADEE4308F1DF840EFC9 be further characterized for the most 271-276. D793F7472B89?sequence=6>. ISBN 978- relevant commercial traits, and those 92-9060- 214-9. 35 p. Accessed on November which combine high resistance with ELPHINSTONE, JG. s/d. Estimating yield 10, 2020. desirable traits, will be recommended loss in potato due to bacterial wilt caused to breeders for the next cycles of by Pseudomonas solanacearum. Available MULLER, KE; FETTERMAN, BA. 2003. selection and for widening the genetic at: <https://www.jircas.go.jp/sites/default/ Regression and ANOVA: An integrated background of the genitors. We also files/publication/tars/tars22-_120-130.pdf>. approach using SAS Software. New York: John confirmed that the cultivar BRSIPR Accessed on Abril 15, 2020. Wiley & Sons. 592p. Bel displays an intermediate level of resistance, being also an interesting FRENCH, ER. 1994. Strategies for integrated MUTHONI, J; SHIMELIS, H; MELIS, R. parental clone as it has good agronomic control of bacterial wilt of potatoes. Pages 2020. Conventional breeding of potatoes characteristics. The findings of this work 199-207 In: HAYWARD, AC; HARTMAN, for resistance to bacterial wilt (Ralstonia demonstrate that the improved potato GL (eds). Bacterial wilt: the disease and its solanacearum): Any light in the horizon? clones selected under tropical conditions causative agent, Pseudomonas solanacearum. Australian Journal of Crop Science 14: in the Embrapa’s pre-breeding project in Wallingford: CAB International, 1994. 485-494. the last decades can be used in breeding programs as sources of high and stable FRENCH, ER; ANGUIZ, R; ALEY, P. 1998. The NIELSEN, LW; HAYNES, FL. 1960. Resistance resistance to BW under field conditions. usefulness of potato resistance to Ralstonia in Solanum tuberosum to Pseudomonas solanacearum for the integrated control of solanacearum. American Potato Journal 37: ACKNOWLEDGMENTS bacterial wilt. p.381-385 IN: PRIOR, PH; 260-267. ALLEN C; ELPHINSTONE J (eds). Bacterial To the Brazilian National Council wilt disease: Molecular and ecological NORMAN, DJ; YUEN, JMF; BOCSANCZY, for Scientific and Technological aspects. Report of the Second International AM. 2020. Threat of brown rot of potato Development (CNPq), for financial Wilt Symposium, Gosier, Guadeloupe, France. and existing resistance. American Journal support; To the International Potato Springer-Verlag, Berlin, Germany. of Potato Research 97: 272-277. https://doi. Center, for providing germplasm and org/10.1007/s12230-020 -09775-3 technical support; To the Research and GUTARRA, L; KREUZE, J.; LINDQVIST- Rural Extension Company of Santa KREUZE, H; MENDIBURU, F. 2015. PATIL, VU; GOPAL, J; SINGH BP. 2012. Catarina (EPAGRI) for collaboration Variation of resistance to different strains of Improvement for bacterial wilt resistance in on the first crossings. Ralstonia solanacearum in highland tropics potato by conventional and biotechnological adapted potato genotypes. American Journal approaches. Agricultural Research 1: 299-316. REFERENCES of Potato Research 92: 258-265. PEREIRA, AS; NAZARENO, NR; SILVA, CARVALHO, ADF; LOPES, CA; RAGASSI, IQBAL, AM; LONE, AA; WANI, SA; WANI, GO; BERTONCINI, O; CASTRO, CM; CF. 2017. Desempenho de cultivares de SH; NEHVI, FA. 2013. Pre-breeding and HIRANO, E; ANTONIO C BORTOLETTO, batata sob diferentes espaçamentos em population improvement. LS - An International AC; TREPTOW, RO; DUTRA, LF; LIMA, solo naturalmente infestado com Ralstonia Journal of Life Sciences 2: 188-197. DOI: MF; GOMES, CB; KROLOW, A.C.R; solanacearum. Horticultura Brasileira 35: 10.5958/j.2319-1198.2.2.012 MEDEIROS, CAB; CASTRO, LAS; 507-511. DOI - http://dx.doi.org/10.1590/ SUINAGA, FA; LOPES, CA; MELO, PE. S0102-053620170406 LOPES, CA; QUEZADO-SOARES, AM. 1995. 2015. BRSIPR Bel: Cultivar de batata para Estabilidade da resistência da batata ‘Achat’ chips com tubérculos de boa aparência. CHAMPOISEAU, PG; JONES, JB; ALLEN C. à murcha-bacteriana. Horticultura Brasileira Horticultura Brasileira 33:135-139. 2009. 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CA Lopes et al. SILVEIRA, JRP; DUARTE, V; MORAES, MG; Pseudomonas solanacearum in the potato: I. GA. 2007. Identification of stable resistance to LOPES, CA; FERNANDES, JM; BARNI, Effects of sources of resistance and adaptation. Phytophthora infestans in potato genotypes V; MACIEL, JLN. 2007. Epidemiological Euphytica 45: 203-210. evaluated in field experiments in Peru. analysis of clones and cultivars of potato Experimental Agriculture 43: 353-363. https:// in soil naturally infested with Ralstonia TUNG, PX; HERMSEN, JG; VANDER, PZ; doi.org/10.1017/S0014479707004991 solanacearum biovar 2. Fitopatologia SCHMIEDICHE, PE. 1993. Inheritance of YULIAR, Y; TOYOTA, K. 2015. Recent trends Brasileira 32: 181-188. Resistance to Pseudomonas solanacearum in in control methods for bacterial wilt diseases tetraploid potato. Plant Breeding 111: 23-30. caused by Ralstonia solanacearum. Microbes TUNG, PX.; RASCO, ET; ZAAG, PV; and Environments 30: 1-11. SCHMIEDICHE, P. 1990. Resistance to WULFF, EG; PÉREZ, W; NELSON, RJ; BONIERBALE, M; LANDEO JA; FORBES, 416 Horticultura Brasileira 39 (4) October - December, 2021

Research MIAN, S; CONSTANTINO, LV; NUNES, MP; VENTURA, MU; SPINOSA, WA; HATA, NNY; SPAGNUOLO, FA; OLIVEIRA, C; GONÇALVES, LSA. 2021. Post-harvest quality and sensory acceptance of Italian tomatoes grown under organic, integrated and conventional management. Horticultura Brasileira 39: 417-424. DOI: http://dx.doi.org/10.1590/s0102-0536-20210411 Post-harvest quality and sensory acceptance of Italian tomatoes grown under organic, integrated and conventional management Silas Mian 1 ;ID Leonel Vinicius Constantino1 ;ID Maria Paula Nunes 1 ;ID Mauricio U Ventura 1 ;ID Wilma A Spinosa 2 ;ID Natalia NY Hata 2 ;ID Felipe A Spagnuolo 3 ;ID Claudia Oliveira 4 ;ID Leandro SA Gonçalves 1ID 1Universidade Estadual de Londrina, Depto. Agronomia (UEL), Londrina-PR, Brasil; [email protected]; [email protected]; mpnunes@ uel.br; [email protected]; [email protected]; 2Universidade Estadual de Londrina, Depto. Ciência e Tecnologia de Alimentos (UEL), Londrina-PR, Brasil; [email protected]; [email protected]; 3Instituto de Desenvolvimento Rural do Paraná, Iapar-Emater, Cornélio Procópio-PR, Brasil; [email protected]; 4Centro Universitário Filadélfia (UNIFIL), Londrina-PR, Brasil; [email protected] ABSTRACT RESUMO Some studies comparing the quality of tomatoes under organic Qualidade pós-colheita e aceitação sensorial de tomates and conventional management can be found in literature; they italianos produzidos sob manejos orgânico, integrado e are still inconclusive and many of them do not address integrated convencional management, though. The aim of this study was to evaluate physical, biochemical and sensory traits of Italian tomatoes under organic, Há estudos que comparam a qualidade do tomate em manejo conventional and Hightech integrated managements. Physical orgânico e convencional, mas ainda são inconclusivos e muitos (firmness, length, width, mesocarp thickness, mass and volume) deles não abordam o manejo integrado. Objetivou-se avaliar and biochemical traits (titratable acidity, sugar content, soluble as características físicas, bioquímicas e sensoriais dos tomates solids content, vitamin C content, lycopene content, beta-carotene italianos produzidos em manejos orgânico e convencional e no content and antioxidant capacity) were evaluated, besides the sensory manejo integrado Hightech. Foram avaliadas características físicas attributes as appearance, aroma, flavor and texture evaluated by 105 (firmeza, comprimento, largura, espessura do mesocarpo, massa consumers. The organic fruits showed greater width, fresh mass and fresca e volume), bioquímicas (acidez titulável, teores de açúcares, pericarp thickness, glucose and vitamin C. The fruits grown under de sólidos solúveis, de vitamina C, de licopeno, de betacaroteno e integrated management stood out for higher levels of beta-carotene, capacidade antioxidante) e a análise sensorial dos frutos, baseada na whereas the fruits grown under conventional management showed aparência, aroma, sabor e textura, avaliados por 105 consumidores. greater acidity and higher lycopene content, as well as less firmness. Os frutos orgânicos apresentaram maior largura, massa fresca e No difference in relation to the acceptance of tomatoes under the espessura do pericarpo, glicose e vitamina C. Os frutos do manejo different managements used was noticed, although most consumers integrado destacaram-se por níveis mais elevados de betacaroteno, preferred organic tomatoes, which were the largest and sweetest. No enquanto os frutos do manejo convencional apresentaram maior difference in relation to the acceptance of tomatoes produced under acidez e maior teor de licopeno, bem como menor firmeza. Não different managements was noticed, despite the discrimination in houve diferença na aceitação do tomate nos diferentes manejos, physical and biochemical traits. Hence, the managements affected embora a maioria dos consumidores preferiram os tomates orgânicos, only the physical and biochemical traits of Italian tomatoes. que eram os maiores e os mais doces. Não houve diferença na aceitação dos tomates produzidos em diferentes manejos, apesar da discriminação em características físicas e bioquímicas. O manejo afetou as características físicas e bioquímicas dos tomates italianos. Keywords: Solanum lycopersicum, production management, post- Palavras-chave: Solanum lycopersicum, manejo de produção, harvest, consumer test. horticultura, pós-colheita, teste com consumidores. Received on November 13, 2020; accepted on July 8, 2021 Tomato is one of the most consumed production of 4.1 million tons in 57.1 phytosanitary products and soluble vegetables worldwide, with an thousand hectares (FAO, 2020). Family fertilizers can be used (Silva et al., annual production of just about 182.3 farmers are responsible for most of the 2011). However, family farmers tend million tons in an area of approximately production in the country, making it to prefer organic management, as it 4.8 million hectares. Its production an important income source for small includes environmentally sustainable totaled 64.7 million dollars (FAO, producers (Neto et al., 2018). practices, since consumers are getting 2020). Brazil is the third largest tomato more and more interested in pesticide- producer in the American Continent and The Brazilian tomato culture mostly free vegetables. Organic management is the tenth in worldwide ranking, with employs conventional production characterized by the use of biofertilizers management, in which synthetic 417 Horticultura Brasileira 39 (4) October - December, 2021

S Mian et al. like tanned manure and pesticides which managements. However, considering climate). are alternative to synthetic products that tomato is a very popular food in The harvested fruits were mixed to such as Bordeaux mixture, natural oils Brazilian cuisine, as well as, in the and extracts, use of insect traps and worldwide cuisine, it is essential to verify make one sample. The tomatoes grown strategies for the biological control of the consumer's preference and attest to under the conventional management diseases and pests (Muñoz et al., 2016). what level agricultural management were grown in a property in Uraí- affects the quality of the fruit, evaluating PR. The tomato production under In Brazil, many tomato growers its physical, biochemical and sensory Hightech integrated management was adopt cultural practices aiming to attributes (Stone et al., 2012). Borguini carried out on Flor do Café farm, reduce the use of synthetic crop & Silva (2009) reported consumer's located in Londrina-PR (23°17’34’’S, protection products, searching for a preference for the flavor of the tomato 51°10’24’’W, 55 altitude, humid more sustainable agricultural practice cultivars Carmen and Débora produced subtropical climate), using pots with and production cost reduction, using in conventional management, whereas commercial substrate Carolina Soil®. soluble fertilizers for nutritional Rodríguez et al. (2001) verified no supplementation via fertigation. This difference in relation to consumer's The fruits were harvested at breaker management uses conservation practices acceptability for aroma and flavor stage/phase (fruits showing yellow- and good agricultural practices, for among field-grown tomatoes, under reddish color up to 20% of the surface) an efficient use of the soil and an non-certified organic, conventional and and, then, stored in paper bags at 25ºC appropriate environmental management integrated managements, characterized to standardize maturation up to stage 6 of pests and diseases (Fadini & Antonio, by the minimum use of chemical (totally red fruits). 2004). One of the models for this products. management is called Hightech, which Phytometric analysis uses high technology for an automatic Considering the above, this Length, width and fruit mesocarp control of the greenhouse, rationally study aimed to evaluate physical and thickness were measured with the aid applying fertilizers, regulated by light biochemical traits, related to consumer's of a digital caliper (EC799, Starrett®), intensity temperature and humidity, acceptance and preference. expressed in mm. The fresh mass was using grafted seedlings and biological expressed in g, determined using a pesticides, which guarantee the ideal MATERIAL AND METHODS bench-top semi-analytical balance. To conditions for the development of the determine the volume, the fruits were crop in a protected environment. Tomato seedlings of cultivar immersed in graduated cylinder with a Grazianni® were grown on substrate capacity of 2000±20 mL, with distilled Some studies can be found in Carolina Soil® by Hidroceres. The water, in order to calculate the density, literature comparing the quality of seedlings were grafted onto the cultivar mass and volume ratio, expressed in g cherry, Santa Cruz and long shelf-life Emperador® RZ-F1 (RijkZwaan), cm-3. tomatoes grown under organic and which presents high resistance to conventional managements (Borguini & ToMV Tomato mosaic vírus (ToMV), Firmness Silva, 2009; Hallmann, 2012; Vieira et TSWV (Tomato Spotted Wilt Virus); Five fruits were evaluated and al., 2014). Most of them are inconclusive Ff (Fulvia fulvum); Fol (Fusarium wilt, firmness was determined in newtons to report which management enables Fusarium oxysporum f. sp. lycopersici); (N), using compression and puncture fruits with better quality, though. Va (Verticilliun Albo-artrum); Vd tests with a bench-top texturometer Moreover, studies which subjected (Verticillium dahliae). The seedlings (Model TA.XT Plus, Stable Micro tomatoes to sensory evaluation using a were transplanted 28 days after sowing, System, the United Kingdom). For consumer taste panel, which represents in areas of commercial production under the compression tests, a flat aluminum the last link in the vegetable marketing protected environment. probe 35 mm diameter, with 0.5 mm chain, are rare. s-1 speed was used, until deforming the The experimental design used was fruit surface 2 mm (Arazuri et al., 2007). Many studies on tomato randomized block, with three treatments physicochemical quality in relation to and six replicates; the treatments were Titratable acidity the type of management adopted are the three production managements: Titratable acidity was quantified controversial. For instance, Chassy organic, integrated and conventional. by titration with a standardized 0.1N et al. (2006) observed higher soluble The plants grown under organic NaOH solution up to pH 8.2, according solid content in tomatoes under organic management were cultivated in two to method No. 942.15 of AOAC (1997), management, whereas Borguini & certificated properties, one located in the results were expressed in grams of Silva (2009) verified higher soluble Uraí-PR (23º12’5”S, 50º47’43”W, 436 citric acid equivalent/100 g sample. solid content in cultivar Sweet Grape m altitude, humid subtropical climate) tomatoes grown under conventional and the other one in São Jerônimo da Sucrose, fructose and glucose management. Furthermore, Juroszek et Serra-PR (23°42’53”S, 50°43’46”W, contents al. (2009) did not verify any difference 938 m altitude, humid subtropical between soluble solid content in fruits The extract for sugar quantification grown under these two production was adapted from Constantino et al. (2020) method, using 1.0 g of the sample suspension in 10.0 mL distilled water, shaken for two hours (Orbital-Nova 418 Horticultura Brasileira 39 (4) October - December, 2021

Post-harvest quality and sensory acceptance of Italian tomatoes grown under organic, integrated and conventional management Orgânica) at room temperature. The and at 470 nm for lycopene, expressed and ends, proposed by Villanueva et al. suspension was centrifugated at 1013xg in mg kg-1. (2005). For the ranking preference test, (Excelsa 2 Fanem model 205N) for five the samples were served simultaneously minutes and the supernatant filtered Antioxidant capacity by DPPH and the panelists ranked them in using 0.45 µm pore size cellulose acetate assay ascending order according to preference, membrane filter (Millipore®). indicating the most and least appreciated To determine antioxidant activity attributes, in addition to a structured The sugars were separated using using 2.2-diphenyl-1-picryl-hydrazyl questionnaire to assess the panelist’s DIONEX AS-AP chromatographic radical scavenging (DPPH•), 50.0 profile. For purchase intent of the system (Thermo Scientific) Dionex™ μL of 70% (v/v) ethanol extract were samples, a 5-point scale was used (5 = CarboPac™ PA10 4250 mm and mixed with 1.0 mL of 100 mM acetate would certainly buy up to 1 = certainly electrochemical detector (ICS-5000). buffer (pH 5.5), 1.0 mL ethanol and would not buy). The design adopted was The mobile phase consisted of NaOH 0.5 mL of 250.0 μM DPPH ethanolic a randomized complete block in which (20 mmol L-1) from 0 to 52 minutes solution. The tubes were kept at room the samples were the treatments and the for separation, NaOH (200 mmol L-1) temperature for 15 min in the dark panelists were the blocks. from 52 to 63 minutes for column and the absorbance of the remaining regeneration, and NaOH (20 mmol DPPH• radical was measured using a Statistical analysis L-1) from 63 to 80 minutes to return to spectrophotometer (Thermo-Genesys) Data showed normality and working condition. The injected sample 517 nm wavelengths. Trolox (6-hydroxy- homogeneity, tested by Shapiro-Wilk volume was 10 µL and flow rate kept 2,5,7,8-tetramethylchroman-2- and Barlett tests, respectively. Data were at 1.0 mL min-1. To quantify, we used carboxylic acid) was adopted as submitted to F test (P>0.05) through the external standardization: sucrose, standard for quantification and the variance analysis and the averages fructose and glucose (Sigma Aldrich, results expressed in µmol of trolox compared by Tukey test (P<0.05). purity >99%) and the results were equivalent antioxidant capacity (TEAC) Additionally, for grouping analysis, we expressed in g 100 g-1 or % (m/m). 100 g-1 (Brand-Williams et al., 1995). used Euclidean distance and Ward’s method applied to the standardized Soluble solid contents Antioxidant capacity determined averages, and viewed in the heat map, The juice of five fruits was extracted by FRAP assay to verify the groups formed among and soluble solid content was determined the samples. R software was used to using direct reading with the aid of FRAP assay (ferric reducing perform the statistics analysis, using Atago digital bench-top refractometer, antioxidant power) was adapted by packages ExpDes.pt (Ferreira et al., model RX-5000α-Plus, with automatic Benzie & Strain (1999), using 50.0 μL 2018) and pheatmap (Kolder, 2018). temperature adjustment to 25oC, and the ethanol extract and 2.5 mL of FRAP results expressed in oBrix. reagent. The results were expressed in RESULTS AND DISCUSSION Ascorbic acid content (Vitamin C) μmol of antioxidant capacity equivalent Vitamin C content was quantified to trolox 100 g-1. Using the variance analysis, we using AOAC standard method (1984) observed a significant effect for most modified by Benassi & Antunes (1988), Acceptance and sensory preference physical and biochemical traits of the using 10.0 g of the sample and 40 mL of fruits fruits, except for length and density, of oxalic acid (Synth) at 2% (m/v). antioxidant activity, soluble solid After extraction, the suspensions were The acceptance test was carried content and ratio. The fruits produced filtered and the supernatant titrated out in the Laboratório de Análise under organic management showed with 2.6-dichlorophenol-indophenol Sensorial do Departamento de Ciência e higher values of width (55.62 mm) and solution. The method was applied using Tecnologia deAlimentos at Universidade pericarp thickness (9.22 mm), whereas five replicates and the results expressed Estadual de Londrina (UEL) and in the for fresh mass, the value was statistically in mg of ascorbic acid 100 g-1. Laboratório de Gastronomia do Centro the same compared with the integrated Universitário Filadélfia (UNIFIL), management and, both were superior Lycopene and beta-carotene applied in only one section with 105 comparing with the conventional fruits content panelists: consumers and gastronomy (Table 1). The highest values of these professionals. attributes verified in organic fruits can Beta-carotene and lycopene be explained considering richness of extractions adapted from Adalid et For the sensory analysis, the organic substances and greater water al. (2010). were prepared with 0.5 tomatoes of each treatment were cut retention capacity in soils cultivated mg of a macerated fruit sample with into 4.0-cm-thick pieces, discarding under organic management (Stertz et 5.0 mL ethanol and hexane solution the fruit ends. The samples were coded al., 2005). (3:2, v/v). Lycopene was quantified with three random digits and served on according to Rodriguez-Amaya (2001) disposable plates, along with a paper Stertz et al. (2005) observed that the and Rodriguez-Amaya & Kimura napkin and a glass with drinking water. organic fruits showed greater diameter, (2004); hexane fraction was read with the aid of a spectrophotometer (Genesys For the acceptance test, samples 419 10, Thermo) at 450 nm for beta-carotene were served one at a time with an individual form to assess size, shape, color, aroma, flavor, texture and global acceptance, using the 10 cm hybrid hedonic scale anchored in the center Horticultura Brasileira 39 (4) October - December, 2021

S Mian et al. Table 1. Physical and biochemical traits of Italian tomatoes grown under organic, conventional average levels of vitamin C (19.40 mg and integrated managements. Londrina, UEL, 2019. 100 g-1), whereas the conventional fruits showed lower average (12.96 mg 100 Traits Management systems g-1), not differing from the integrated Organic Integrated Conventional CV (%) management which showed an average of 11.96 mg 100 g-1. According to Pék Physical et al. (2010), a great sunlight incidence may result in fruits containing higher Length (mm) 81.82ns 76.60ns 76.57ns 5.15 concentrations of vitamin C, which can justify the lowest contents of this Width (mm) 55.62 a 51.50 b 51.15 b 4.39 attribute in fruits grown under integrated managements, considering that the solar Fresh mass (g) 141.48 a 110.30 ab 93.10 b 17.99 incidence is smaller in this management using high tech. Hallmann (2012) also Density (g cm-3) 1.16ns 1.05ns 1.00ns 18.75 verified higher vitamin C concentration in organic tomatoes than in tomatoes Pericarp thickness (mm) 9.22 a 7.46 b 6.00c 4.68 under conventional management, in two years of cultivation, and highlighted that Biochemical the concentration of this compost can be influenced by the type of nitrogen Lycopene (µg 100 g-1) 6.34 a 5.43 b 6.56 a 5.40 used. This can also justify the highest content of this vitamin in organic fruits Beta carotene (µg 100 2.86 c 8.38 a 4.39 b 10.08 in relation to the other fruits evaluated g-1) in this study, since the nitrogen used in organic management shows slow FRAP 94.16ns 86.06ns 91.57ns 12.21 release, compared to the conventional and integrated managements. DPPH 842.74ns 835.22ns 845.09ns 1.82 In conventional fertilization, nitrogen SST (°Brix) 2.96ns 2.86ns 2.88ns 2.67 is available in large quantities for the plant, resulting in an increase in protein ATT (% citric acid) 0.26 b 0.22 b 1.22 a 7.50 synthesis and decrease in carbohydrate synthesis. However, nitrogen availability Ratio 11.64ns 12.96 ns 13.56 ns 7.98 in organic fertilizers is limited for the plant, as its release is slow, Vitamin C (mg 100 g-1) 19.40 a 12.33 b 11.96 b 9.08 resulting in an increased carbohydrate metabolism. This fact can contribute Glucose (g 100 g-1) 1.85 a 0.06 b 0.06 b 5.01 to a greater production of vitamin C, since this vitamin is metabolized from Fructose (g 100 g-1) 1.92 a 1.85 a 1.55 b 8.11 carbohydrates (Worthington, 2001), which corroborates a higher vitamin Means followed by the same letter in the column did not differ significantly from each other by C concentration in organic fruits as Tukey test (p<0.05); Antioxidant activity by FRAP assay (µmol 100 g-1); antioxidant activity by verified in this study. DPPH assay (µmol 100 g-1); SST= total soluble solids;ATT= titratable acidity; Ratio= SST/ATT. Soluble solid contents did not differ fresh mass and density, comparing with these managements was more intense among the managements and presented the conventional management. Vieira et when comparing to the integrated 2.9°Brix average value (Table 1), similar al. (2014), comparing the post-harvest management. Tomatoes showing higher value to the one verified by Ferreira et quality of minitomatoes cultivar Sweet lycopene content are desirable, as this al. (2010) who suggested that the type Grape, produced under conventional carotenoid is an antioxidant compound of management does not interfere in this and organic management, verified known to prevent many types of cancer; attribute. However, the results found greater fresh mass in conventional it represents approximately 90% of this by Chassy et al. (2006) are different. fruits, showing the same diameter and content in tomatoes, which contributes These results showed that organic fruits length, though. Riahi et al. (2009) for the functional characteristic of the present higher contents of soluble solids did not observe any difference for fruit (Navarro-González & Periago, compared to fruits under conventional fresh mass of the fruits of different 2016). management. According to Kader et al. tomato cultivars produced under two (1978), tomatoes which show soluble managements in Tunisia, the production Antioxidant activity both by FRAP solids and acidity ratio higher than 10 yield was greater under conventional method and DPPH did not present are considered of good flavor, and in this management, though. any statistical difference among the study the values ranged from 11.64 to treatments (Table 1), even having The highest beta carotene different contents for bioactive concentrations were verified in fruits compounds like vitamin C, lycopene and under integrated management, whereas beta carotene. Juroszek et al. (2009) did the lowest lycopene concentrations were not verify any difference for antioxidant also verified in these fruits (Table 1). activity between fruits produced under According to Dumas et al. (2002), light organic and conventional managements intensity favors carotenoid production, either. On the other hand, Mergawi & mainly lycopene, which corroborates the Al-Redhaiman (2010) and Pascale et results of this study, in which lycopene al. (2016) verified superior antioxidant content was greater in fruits under activity in organic fruits comparing with conventional and organic managements, the conventional ones. considering that the solar irradiation in The organic tomatoes showed higher 420 Horticultura Brasileira 39 (4) October - December, 2021

Post-harvest quality and sensory acceptance of Italian tomatoes grown under organic, integrated and conventional management 13.56. The authors concluded that the in conventional fertilization stresses the fruit firmness in the puncture test tomatoes under the three production out the plant, stimulating the sugar (Figure 2A). The fruits which were managements showed desirable balance production. grown under integrated management between sugars and acids. showed greater firmness, requiring a The grouping analysis using the heat force of 0.118 N for exocarp rupture Glucose concentration was 96.8% map (Figure 1) showed higher similarity and greater force for mesocarp and higher in organic fruits comparing between the integrated and conventional endocarp perforation. Firmness of with the others, whereas the highest managements, as the tomatoes grown organic tomatoes was intermediate, fructose concentrations were verified under these managements present requiring a force of 0.088 N for exocarp in fruits under organic and integrated similar fresh mass, pericarp thickness, rupture. The conventional-management managements (Table 2). According density, width and vitamin C content. fruits required a force of 0.062 N for to Leiva-Brondo et al. (2015), The fertilization may be the principal exocarp rupture, as well as longer organic tomatoes have contents of responsible factor, considering that time to penetrate the mesocarp, which glicose and fructose 7.7 and 10.4% soluble fertilizers were used in these suggests a more flexible shell. higher than in conventional-grown two managements. tomatoes. However, Gilsenan et al. In compression test (Figure 2B), (2012) observed higher concentrations Firmness is a very important trait, the organic tomatoes showed superior of glicose and fructose in tomatoes considering the shelf-life trait of the firmness, 7.09 N for 2 mm fruit surface under conventional management, and product, as well as for marketing deformation. The tomatoes grown justify that the high salt availability chain to the final destination. Some under integrated management were differences were verified considering intermediate, 6.08 N for deformation, even though up to 1.5 seconds of Figure 1. Grouping analysis and heat map of the physical and biochemical traits of Italian compression, the force exerted was tomatoes under different production systems. Londrina, UEL, 2019. greater than in organic fruits. The conventional management resulted in less firm fruits, requiring 3.83 N to deform the fruit surface. The puncture test can be associated with the canine biting, which ruptures the exocarp and penetrates the mesocarp. The compression test is related to the bite force with the molar teeth pressing the fruit into the bite; this test is associated with actions like squeezing the fruit by consumers (Lucas et al., 2004; Mioche et al., 2004). Fruit firmness responds negatively to an exaggerated increased Figure 2. Texturograms of Italian tomatoes under different production systems by the puncture (A) and compression (B) assays. Londrina, UEL, 2019. Horticultura Brasileira 39 (4) October - December, 2021 421

S Mian et al. Consumers' acceptance Figure 3. Boxplot of sensory acceptance scores of the tomatoes under different production systems. Londrina, UEL, 2019. availability of nitrogen in the form of the fruit firmness. being weekly consumers, more than ammonia and potassium for plants, as Riahi et al. (2009) verified some 20% daily consumers, approximately this trait is directly related to cell turgor 10% infrequent consumers, less than pressure (Knee, 2002). In this case, organic tomatoes firmer than the 10% consume the fruit every fifteen days the high fertilizer concentration in the conventional ones for just one of the and the minority consume it monthly. conventional production management cultivars among the four ones evaluated. may decrease firmness, as it was verified According to Caris-Veyrat et al. (2004), As for the fruit preference itself, in this study. the firmness of the conventional concerning the different production tomatoes decreased 16% during storage managements, 42% of consumers In the integrated management, the time, whereas the decrease in organic preferred the organic ones, 30% tomatoes composition of the nutrient solution fruits was only 7%. Polat et al. (2010) grown under integrated management and is controlled according to the plant found no difference in firmness among 22% under conventional management. demand and its phenological stage, tomatoes grown under organic and Appearance was the most appreciated made available, automatically, under conventional managements in Turkey. trait in organic tomatoes and flavor the command of a software. The organic for tomatoes under integrated and farmers, who took part in this study, More than 50% of the panelists conventional managements, whereas have plenty of experience in growing were women, approximately 40% the least appreciated traits were texture tomatoes under this management. In were men, 70% of the total were for organic and integrated tomatoes, and relation to the conventional management, up to 25 years old. Regarding to appearance for conventional ones. great part of producers overuse high occupation, 80% of the consumers solubility chemical fertilizers (Rahman were students, 5% public servants, 3% No difference was verified in relation & Zhang, 2018), influencing, negatively, tradepeople and approximately 2% to consumers' acceptance regarding to small producers, in which 60% reported tomatoes under the three production 422 Horticultura Brasileira 39 (4) October - December, 2021

Post-harvest quality and sensory acceptance of Italian tomatoes grown under organic, integrated and conventional management managements (Figure 3). On the other did not differ. Even so, an expressive Características físico-químicas e sensorias do hand, Borguini & Silva (2009) reported consumer's preference for organic fruits tomate (Lycopersicon esculentum) produzido that regarding to the evaluated sensory was verified. por cultivo orgânico em comparação attributes, difference in flavor was ao convencional. Alimentos e Nutrição verified between fruits grown under Considering what was observed in Araraquara 16: 355-361. organic and conventional managements. this study, we could conclude that some However, Ferreira et al. (2010) reported difference can be noticed in relation BRAND-WILLIAMS, W; CUVELIER, ME; that organic tomatoes showed to be to physical and biochemical quality BERSET, C. 1995. Use of a free radical firmer according to the consumer’s in tomatoes grown under different method to evaluate antioxidant activity. LWT evaluation, whereas conventional fruits managements, but no sensory difference 28: 25-30. were better in relation to flavor and was verified, though. The cultivation of presented greater global acceptance, tomato under the integrated management C A R I S - V E Y R AT, C ; A M I O T, M J ; in this study different cultivars were was similar to that of the conventional TYSSANDIER, V; GRASSELLY, D; BURET, used in each production management, management as they presented tomatoes M; MIKOLAJCZAK, M; GUILLAND, JC; though. with similar fresh mass, pericarp BOUTELOUP-DEMANGE, C; BOREL, P. thickness, density, width and vitamin 2004. Influence of organic versus conventional Concerning the consumer’s purchase C content. Organic tomatoes proved to agricultural practice on the antioxidant intention, it is possible to say that 70% be of better quality, as they had greater microconstituent content of tomatoes and of them would buy the fruits grown width, fresh mass, pericarp thickness, derived purees; consequences on antioxidant under organic management, whereas vitamin C content and glucose content. plasma status in humans. Journal of 54 and 53% would buy the fruits Agricultural and Food Chemistry 52: 6503- under integrated and conventional ACKNOWLEDGMENTS 6509. https://doi.org/10.1021/jf0346861 managements, respectively. Thus, we conclude that although sensory To farmers who took part in the CHASSY, AW; BUI, L; RENAUD, ENC; VAN, acceptance did not differ for tomatoes, experiment, to the technicians Ernestina HM; MITCHELL, AE. 2006. 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Research ANTUNES, PS; ERPEN-DALLA CORTE, L; BUENO, JCM; SPINOSA, WA; RESENDE, JTV; HATA, FT; CABRERA, LC; ZEFFA, DM; GONÇALVES, LSA; CONSTANTINO, LV. 2021. Firmness and biochemical composition of Shitake and Shimeji commercialized in natura and consumers' opinion survey. Horticultura Brasileira 39: 425-431. DOI: http://dx.doi.org/10.1590/s0102-0536-20210412 Firmness and biochemical composition of Shitake and Shimeji commercialized in natura and consumers' opinion survey Pamela dos S Antunes 1 ;ID Lígia Erpen-Dalla Corte 1 ;ID Janaina CM Bueno 2 ;ID Wilma Aparecida Spinosa 1 ;ID Juliano Tadeu V Resende 1 ;ID Fernando T Hata 1 ;ID Lilian C Cabrera 3 ;ID Douglas M Zeffa 4 ;ID Leandro SA Gonçalves 1 ;ID Leonel Vinícius Constantino 1ID 1Universidade Estadual de Londrina (UEL), Centro de Ciências Agrárias, Londrina-PR, Brasil; [email protected]; ligiacorte@uel. br; [email protected]; [email protected]; [email protected]; [email protected]; 2Universidade Estadual do Centro-Oeste (UNICENTRO), Pós-graduação em Agronomia, Guarapuava-PR, Brasil; [email protected]; 3Universidade Estadual de Londrina (UEL), Centro de Estudos Sociais Aplicados, Londrina-PR, Brasil; [email protected]; 4Universidade Estadual de Maringá (UEM), Pós- graduação em Genética e Melhoramento, Maringá-PR, Brasil; [email protected] ABSTRACT RESUMO Mushroom production has been growing in Brazil due to the Firmeza e composição bioquímica de Shitake e Shimeji culinary versatility of this food and its high nutritional value. comercializados in natura e pesquisa de opinião com consumidores However, few studies associating the aspects of its quality with the profile and preference of its consumers can be found in literature. A produção de cogumelos vem crescendo no Brasil, devido à Thus, in this study, we aimed to evaluate the firmness and biochemical versatilidade culinária desse alimento e alto valor nutricional. Ainda composition of Shimeji and Shitake, as well as, the consumers’profile são escassos estudos que associam os aspectos de sua qualidade com and preference for purchasing these mushrooms. The mushrooms o perfil e preferência dos seus consumidores. Assim, nesse trabalho were characterized by firmness, protein and lipid content, total foi comparada a firmeza e composição bioquímica de Shimeji e phenolic compounds, total flavonoids and antioxidation activity. Shitake, e verificado o perfil e a preferência dos consumidores para In addition we carried out a survey on opinion of 308 mushroom compra. Os cogumelos foram caraterizados pela firmeza, teores de consumers. The two mushrooms contain high protein content and proteínas, lipídios, compostos fenólicos totais, flavonoides totais e low lipid content, and also expressive concentrations of bioactive atividade antioxidade, além da realização de pesquisa de opinião com compounds and antioxidant activity, suitable for these demanding 308 consumidores de cogumelos. Os dois cogumelos contêm alto teor functional food consumers. Shitake is approximately 4 times firmer de proteínas e baixo de lipídios, além de concentrações expressivas than Shimeji and the highest levels for all analyzed biochemical de compostos bioativos e de atividade antioxidante, desejáveis attributes did not differ between the two studied mushrooms, except aos consumidores exigentes em alimentos funcionais. O Shitake é for protein content. The opinion survey indicated that young and aproximadamente 4 vezes mais firme quer o Shimeji e os maiores adult women consume more mushrooms than men. Most of them buy teores para todos os atributos bioquímicos analisados não diferiram mushrooms in supermarkets, and they are mostly higher educated entre os dois cogumelos, exceto para o teor de proteínas. A pesquisa people. No significant higher consumer preference for one of the de opinião indicou que mulheres jovens e adultas consomem mais two mushrooms analyzed was verified; however, most participants cogumelos que os homens. A maioria deles adquire os cogumelos em reported that they prioritize mushrooms which have characteristic supermercados, e são pessoas com escolaridade, majoritariamente, de aroma and flavor, smooth and juicy texture, being a convenient nível superior. Não foi constatada significativa maior preferência dos ingredient for a quick-cooking meal. consumidores por um dos dois cogumelos analisados, mas a maioria dos participantes relataram que na compra priorizam cogumelos com aroma e sabor característicos, textura macia e suculenta, e que permita rapidez no preparo de refeições. Keywords: Lentinula edodes, Pleurotus ostreatus, edible mushrooms, Palavras-chave: Lentinula edodes, Pleurotus ostreatus, cogumelos firmness, fungiculture, sensory analysis. comestíveis, firmeza, fungicultura, análise sensorial. Received on February 22, 2021; accepted on October 6, 2021 Edible mushroom market has grown Brazilian cuisine, due to an increasing Mushrooms have been used in human significantly in recent years, due number of vegan people, since these diet for many years. They have a high to an increasing demand for high mushrooms are considered an excellent content of protein and fiber, low content nutritional value foods. Moreover, source of protein, alternative to those of of fat, around 2% to 6%, vitamins A, we highlight the Asian influence in animal origin. K and B and minerals like potassium, Horticultura Brasileira 39 (4) October - December, 2021 425

PS Antunes et al. calcium and iron (Valverde et al., 2014; opinion survey which was carried out & Hillis, 1959). Chun et al., 2020). Mushrooms also have with mushroom consumers. For quantifying the total flavonoids, antimicrobial activities (Alves et al., 2012), anticancer activities (Finimundy In natura mushrooms, Shitake and ethanolic extract was mixed with a 5.0% et al., 2013), compounds that protect Shimeji, were bought at supermarkets in aluminum chloride solution in a basic against inflammatory processes (Yu et the city of Londrina-PR, commercialized medium, reading at 425 nm. Quercetin al., 2009) and cardiovascular diseases in 200-g trays, covered by transparent was used as analytical standard and the (Rahman et al., 2018). plastic film and stored at 10ºC. The mean of five replicates was expressed in samples consisted of five trays of each mg of quercetin equivalents (QE)/100 Mushroom production has grown kind of mushroom, selected from simple g of the sample (Gurnani et al., 2016). worldwide. In 2013, per capita random sampling. To perform the consumption exceeded in 4.7 kg per analysis, the samples were kept under To determine antioxidant activity, year, representing more than 30 times refrigeration (8ºC) for up to three days we used 2.2-diphenyl-1-picryl-hydrazyl since 1978 (Royse et al., 2017). The and, for each kind of mushroom, all radical scavenging (DPPH•) adapted possibility of reusing agro-industrial trays were mixed to obtain a compost from Brand-Williams et al. (1995). waste as a substrate is another factor sample. Absorbance was recorded at 517 nm which contributes to the increase in and analytical curve was performed mushroom production (Paula et al., Firmness using Trolox (6-hydroxy-2,5,7,8- 2001). Firmness was expressed in N and tetramethylchroman-2-carboxylic measured according to Arazuri et al. acid) as standard. The mean of five In Brazil, most of the production (2007), using a bench texturometer replicates was expressed in % free is carried out by small and medium (Model TA.XT Plus, Stable Micro radical scavenging. Additionally, FRAP family farming producers (Cabrera System, The United Kingdom) from the assay (Benzie & Strain, 1996) was also et al., 2020), which represent 80% compressive strength of the cap (upper applied to determine extract reducing of producers and provide about 12 part of the basidiomycete), applying a 35 power, with reading at 517 nm. The thousand tons of mushrooms per year mm diameter aluminum probe at 0.5 mm mean of the five replicates was expressed to the market (APTA, 2019). Most s-1 speed. Firmness mean was obtained as μmol of trolox equivalent antioxidant consumers are high-income people using the analysis of ten mushrooms. capacity (TEAC)/100 g of sample. (Boin & Nunes, 2018). Protein and lipid contents Profile and preference of The most commonly-consumed consumers edible mushrooms are Champignon Crude protein content was quantified de Paris (Agaricus bisporus), canned by nitrogen distillation (Kjeldahl Opinion survey to verify the form, followed by Shimeji (Pleurotus method), corrected using a factor of preference and profile of mushroom ostreatus) and Shitake (Lentinula 4.38 and, to quantify lipids, Soxhlet consumers consisted of an online edodes), both in natura (ANPC, 2019), extraction method (AOAC, 2012) was questionnaire, using the Google Form whose aroma, flavor and texture stood used. Both attributes were expressed in tool, which required adherence to out, comparing to their dehydrated and % (m/m), using ten replicates for each the Informed Consent Form. The canned forms. mushroom. questionnaire link was posted on social media to reach as many people as In spite of the fact that some studies Bioactive compounds and possible from different parts of Brazil. addressing the biochemical composition antioxidant activity The individuals who participated of mushrooms can be found in literature, are mushroom consumers over the just few studies addressing the profile To quantify total phenolic age of 17: being the criteria used for of their consumers were carried out. compounds, flavonoids and antioxidation recruitment. The research was approved Thus, this study evaluated firmness and activity, a suspension was prepared by the Ethics Committee for Research biochemical composition of Shitake and using 1.00 g of fresh samples with Involving Human Beings at UEL, under Shimeji marketed in natura, and the 10.0 mL ethanol at 70% (v/v), under No. 13651019.0.0000.5231. profile and preference of consumers, agitation for 30 min. Afterwards, the aiming at directing the fungiculture samples were centrifuged at 1013 x g The survey included 20 questions, of production chain. (Excelsa 2 Fanem modelo 205N) for 5 which nine were related to consumers' minutes and supernatant was separated profile and 11 to consumers' preferences MATERIAL AND METHODS for analysis (Vázquez et al., 2008). (Appendix). The questions were about: region of the country where the Samples Content of phenolic compounds was consumer lived, sex, age, profession, This study was carried out in two quantified through spectrophotometric education level, monthly income, stages: the first consisted of analyzing method using the Folin-Ciocalteu consumption frequency, choice criteria, firmness and biochemical traits of reagent. Gallic acid was adopted as the consumed species, place of purchase, Shitake and Shimeji commercialized analytical standard and readings were preference in relation to Shitake and in natura, and the second stage was an taken spectrophotometrically at 765 Shimeji, traits which they like the most, nm. The mean of the five replicates was the least pleasing trait, the way in which expressed in mg equivalent of gallic acid the mushroom is acquired, the form of equivalents per 100 g of sample (Swain 426 Horticultura Brasileira 39 (4) October - December, 2021

Firmness and biochemical composition of Shitake and Shimeji commercialized in natura and consumers' opinion survey consumption, the purpose of use and the Shimeji comparing with other species are natural antioxidants and have factor which would make the consumer of macrofungi were also reported several therapeutic properties, such eat more of the product. in literature. Palacios et al. (2011) as inhibition of lipid peroxidation, reported that the mushroom species antimicrobial activity, tumor Statistical analysis Calocybe gambosa, Cantharellus activities, chemopreventive and anti- The means of the biochemical cibarius, Craterellus cornucopioides, inflammatory activities (Tanase et al., characterization of the mushrooms were Hygrophorus marzuolus, Lactarius 2014; Muszyńska et al., 2018). Greater compared through t test (p<0.05), using deliciosus and Pleurotus ostreatus antioxidant activities of Shitake were R software. Participant profiles were contain lower levels of phenolic also observed, double the capacity to plotted in graphs, and the consumers' compounds and they also mentioned that capture free radicals (75.51%) when preference submitted to Qui-squared the species Boletus edulis has almost compared with Shimeji (34.89%). test (p<0.05). three times more phenolic compounds Shitake's antioxidant activity is superior than Shimeji, and Agaricus bisporus to that of common foods in Brazilian RESULTS AND DISCUSSION two times more. diet, such as snap beans and kale (Rigueira et al., 2016; Fukuji et al., Biochemical composition Boonsong et al. (2016) found a mean 2019). The highest contents of phenolic of 24.25±0.49 for phenolic compounds compounds, flavonoids, antioxidant in Shitake (Lentinus edodes), low value Antioxidant activity involves the activity and lipids were found in Shitake comparing with the one found in this interaction of different components mushrooms, whereas the protein content study. Gaitán-Hernández et al. (2020) in food, because it depends on the did not differ for both mushrooms (Table proved that the fungus strain and the concentration and action of carotenoids, 1). The variation found for the evaluated substrate significantly interfere in the vitamin C, vitamin E and phenolic traits can be attributed to the differences content of this attribute, reporting values compounds. Thus, the different in growing conditions, fact which was from 1.59 to 2.71 mg 100 g-1 of fresh concentrations and composition of not investigated in this study, as well Shitake, using as substrate vineyard antioxidants could clarify the difference as the intrinsic genetic traits of each pruning, sorghum stubble, sugarcane found in the contents of antioxidant species. bagasse and oak. activities of Shitake and Shimeji (Table Shitake showed four times higher 1), since in 100 grams of Shitake, 2.1 compression strength comparing Contents of phenolic compounds and mg of vitamin C can be found, whereas with Shimeji (Table 1), which can be antioxidant activity in mushroom cap 1.6 mg can be found in Shimeji (Mattila associated with a greater amount of can be reduced at temperatures above et al., 2001). pectin, cellulose and hemicellulose 25.55ºC and in relation to the exposure present in Shitake cell wall, since these time (Liaotrakoon & Liaotrakoon, Crude protein content was the only structural components are related to 2018). Shitake mushrooms grow at low biochemical attribute which did not the mechanical resistance and tissue temperatures, from 15 to 20ºC (Maki et differ significantly among mushrooms cohesiveness (Zhongdong et al., 2006). al., 2001), whereas Shimeji mushrooms (Table 1), showing that both are Firmness is directly related to the grow at moderate temperatures, which interesting for people who adopt a mushroom texture, implying its sensory range from 18 to 30°C (Jaramillo et al., high-protein diet. However, Andrade acceptability and form of preparation for 2013), which can explain the lowest et al. (2008) reported that the crude consumption. contents of phenolic compounds found protein contents, as well as the ether Another fact which can influence on in Shimeji in this study. extract, ash and Shitake fibers differed a greater firmness of Shitake, is a larger from the other evaluated mushrooms, fruiting body compared to Shimeji. This High contents of total phenolic justified by the effect of the macrofungus relationship was already observed in compounds are desirable, since they cherry tomatoes: larger fruits showed higher firmness when comparing with Table 1. Mean (±standard deviation) of firmness and biochemical attributes of Shitake and medium and small-sized fruits (Islam et Shimeji mushrooms. Londrina, UEL, 2020. al., 2019). Li et al. (2017) observed that the fungus Volvariella volvacea showed Attribute Mushroom firmness of 2.71 N and the largest fruiting body among the evaluated Shimeji Shitake mushrooms. In Table 1, we noticed that the Firmness (N) 1.44±0.32 5.52±0.27* content of total phenolic compounds in Shitake mushroom was approximately Content of total phenolic compounds (mg 100 34.73±5.07 193.80±12.16* four times higher than in Shimeji. Lower g-1) contents of total phenolic compounds in Content of total flavonoids (mg 100 g-1) 109.90±6.70 271.0±14.88* Antioxidation activity - DPPH (%) 34.89±1.72 75.51±2.13* Antioxidation activity - FRAP (µmol 100 g-1) 12.48±1.62 152.40±19.81* Crude protein content (%) 26.64±0.56 27.09±3.23 Ethereal extract content - lipids (%) 0.47±0.08 1.26±0.04 * Mean followed by asterisk is significantly higher by t-Student test (P<0.05). Horticultura Brasileira 39 (4) October - December, 2021 427

PS Antunes et al. lineage interaction with the eucalyptus increase the nitrogen content in edible Champignon (5.42%). substrate. Furlani & Godoy (2007) mushrooms (Ajonina & Tatah, 2012). reported that differences in biochemical That is the reason why, studies on the Market survey of mushroom compounds of the mushrooms may chemical composition of substrates for consumers show differences among the evaluated growing mushrooms, relating to readily lots of the same species, showing that available forms of nitrogen, are relevant. Atotal of 308 individuals participated the differences are related to the region in the survey, the majority being women where the macrofungus was cultivated The low lipid content is another (64%) living in the Southern region of and the substrate used in this cultivation. prominent factor for mushrooms, even Brazil. The authors noticed that 64% of though a significant difference for this the consumers are young and adults up Carbon and nitrogen ratio (C/N) of attribute was noticed (Table 1). Shitake to 35 years old, with high scholar degree the substrate is an important fact, which showed almost three times the mean (64% with complete higher education). can determine the protein content in lipid content (1.26%), comparing with We highlight that mainly young and mushrooms. This is due to the fact that Shimeji (0.47%). According to Furlani adult Brazilians are concerned about these macrofungi have a high rate of & Godoy (2007), Shitake has higher a healthy eating habit, in addition to assimilation of this nitrogen (Ghosal lipid content (4.39%) compared with confirming that people with a higher et al., 2016). Thus, substrates with low Shimeji (4.30%), although the contents level of education opt for healthier food C/N ratio are preferably indicated to of both are lower than that of the choices (Finger et al., 2013). Also, thirty nine people, working in the food sector, Figure 1. Factors related to purchasing and consuming of mushrooms. Londrina, UEL, 2020. 428 Horticultura Brasileira 39 (4) October - December, 2021

Firmness and biochemical composition of Shitake and Shimeji commercialized in natura and consumers' opinion survey and three fungi farmers took part in the Trends, 2020). regular frequency can be justified by survey. These people form an important A third of participants reported that the awareness of the health benefits of group for this evaluation since they mushrooms and the increase in Brazilian are directly related to the market and they consume mushrooms weekly and income since the 1990s (Royse, 2014). production chain, respectively. Among a minority of them reported consuming these participants, 40% reported a mushrooms daily (Figure 1). The Boin & Nunes (2018) also reported monthly income of 4 or more minimum frequency of mushroom consumption that the frequency in mushroom salaries, similar to the US scenario by Brazilians is similar to that found consumption is influenced by the where most consumers have an income in other countries, such as China (Wen educational level, gender and family of more than $100,000 per year (Fresh et al., 2016) and Portugal (Boin & size, considering that the gender and Nunes, 2018), mostly weekly. This size of the family have a negative effect Figure 2. Consumers' preference in relation to form of consumption and preparation of Shitake and Shimeji mushrooms. Londrina, UEL, 2020. Figure 3. Sensory attributes that impact consumers' preference for Shitake and Shimeji. Londrina, UEL, 2020. 429 Horticultura Brasileira 39 (4) October - December, 2021

PS Antunes et al. on the frequency of consumption, while consumed macrofungus (Shirur et al., mushrooms, in order to ensure longer the level of education has a positive 2014), whereas the oyster mushroom shelf life. Nevertheless, in Brazil no effect. Men from families with more (Pleurotus spp.) is the most popular in updated information on volume and than four people tend to consume less Ghana (Kortei et al., 2018). distribution of mushrooms as well as mushrooms, whereas consumers with the main forms of commercialization higher education levels and smaller Sautéed mushrooms in butter, olive (fresh, dehydrated and preserved) can families consume mushrooms more oil or oil are the most popular form of be found. This fact greatly affects the often. preparation reported by participants decision-making in relation to the (Figure 2). As for consumption, most national fungiculture production chain. According to the reports, the most participants (53%) prefer mushrooms consumed mushrooms are Shitake, as meal accompaniment, and the second No difference was observed between Shimeji, Champignon and Portobello form as a main dish (38%). This eating Shitake and Shimeji (Figure 3), even (Figure 1), whereas Hiratake is the least habit is similar to the one of American considering that the firmer texture consumed mushroom. These results are people, 75% use the mushrooms as and darker color of Shitake is not similar to the ones found for worldwide ingredients in dishes and 37% as the appreciated by most consumers. Thus, consumption, and showed that the main dish for meals (Fresh Trends, Shitake is as important as Shimeji for the most consumed species are the ones 2020). Brazilian market. Nevertheless, to meet belonging to the genera Lentinula spp., the consumers' demand, investment on Pleurotus spp., Auricularia spp. and The determinant attributes for researches and technical assistance for Agaricus spp., 22%, 19%, 18% and 15% purchasing Shitake and Shimeji are the sector is necessary, for longer shelf at worldwide level, respectively (Royse flavor and aroma (Figure 3). The life, production cost optimization and et al., 2017). consumers also reported that they formal organization of the production buy the product according to texture, chain. The consumers generally buy soft and juicy, and if they are good mushrooms at the supermarket, ingredients for a quick-made dish. These REFERENCES according to the Brazilian participants. results show that Brazilians have criteria In Turkey, the product is also purchased which are different from Indians when AJONINA, AS; TATAH, LE. 2012. 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Research LINO, VAS; BEZERRA NETO, F; LIMA, JSS; SANTOS, EC; NUNES, RLC; GUERRA, NM; LINO, FKKS; SÁ, JM; SILVA, JN. 2021. Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages. Horticultura Brasileira 39: 432-443. DOI: http://dx.doi. org/10.1590/s0102-0536-20210413 Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages Vitor AS Lino 1 ;ID Francisco Bezerra Neto 1ID; Jailma SS Lima 1ID; Elizangela C Santos 1 ;ID Renato LC Nunes 2ID; Natan M Guerra 1 ;ID Francisca Karla KS Lino 1 ;ID Jolinda M Sá 3 ;ID Josimar N Silva 1ID 1Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró-RN, Brasil; [email protected]; [email protected]; jailma@ufersa. edu.br; [email protected]; ntnguerra@ gmail.com; [email protected]; [email protected]; 2Instituto Federal do Ceará (IFCE), Limoeiro do Norte-CE, Brasil, [email protected]; 3Universidade Federal de Campina Grande (UFCG), Pombal-PB, Brasil; [email protected] ABSTRACT RESUMO One of the biggest challenges in the intercropping system of two Consórcio de beterraba e rúcula sob adubação verde e crops is to obtain the optimal dose of green manure and the adequate densidade de plantio levam a vantagens agroeconômicas population density of the crops. So, the objective of this work was to evaluate the performance of beet and arugula intercropping, Um dos maiores desafios no sistema consorciado de duas culturas influenced by green manuring with Merremia aegyptia and Calotropis é obter a dose ótima de adubos verdes e a densidade populacional procera and arugula population densities in two cultivation years, adequada das culturas. Assim, o objetivo deste trabalho foi avaliar o in semi-arid environment. The experimental design used was in desempenho da beterraba e rúcula em consórcio quando influenciadas randomized complete blocks, with the treatments arranged in a 4 x pela adubação verde com Merremia aegyptia e Calotropis procera 4 factorial scheme, with 4 repetitions. The first factor of this scheme e densidades populacionais de rúcula, em dois anos de cultivo, em consisted of equitable amounts of M. aegyptia and C. procera ambiente semiárido. O delineamento experimental utilizado foi em biomass (20, 35, 50 and 65 t ha-1 on a dry basis) and the second blocos completos casualizados, com os tratamentos arranjados em factor, by arugula population densities (40, 60, 80 and 100% of the esquema fatorial 4 x 4, com 4 repetições. O primeiro fator desse recommended density for single cropping, corresponding to 400, 600, esquema foi constituído de quantidades equitativas de misturas de 800 and 1,000 thousand arugula plants ha-1). The production and its biomassa de M. aegyptia e C. procera (20, 35, 50 e 65 t ha-1 em base components were evaluated on beet and arugula. In addition to these seca) e, o segundo fator, de densidades populacionais de rúcula (40, characteristics, the following agro-economic indicators were also 60, 80 e 100% da densidade recomendada para o cultivo solteiro, determined for each treatment: system productivity index (SPI), land correspondendo a 400, 600, 800 e 1.000 mil plantas de rúcula ha-1). equivalent coefficient (LEC) and monetary equivalent ratio (MER). A produção e seus componentes foram avaliados na beterraba e na The greatest agro-economic advantages of the beet with arugula rúcula. Além dessas características, também foram determinados intercropping were achieved with a system productivity index (SPI) of os seguintes indicadores agroeconômicos para cada tratamento: 53.47 t ha-1, land equivalent coefficient (LEC) of 0.84 and a monetary índice de produtividade do sistema (SPI), coeficiente equivalente equivalent ratio (MER) of 1.56, respectively, combining 65 t ha-1 of M. de terra (LEC) e razão equivalente monetária (MER). As maiores aegyptia and C. procera biomass with the arugula population density vantagens agroeconômicas do consórcio de beterraba com rúcula of 1,000 thousand plants ha-1. The maximum optimized commercial foram alcançadas com um índice de produtividade do sistema (SPI) productivity of beetroots in the system intercropped with arugula de 53.47 t ha-1, coeficiente equivalente da terra (LEC) de 0.84 e uma was 23.20 t ha-1 using 65 t ha-1 of M. aegyptia and C. procera and in razão equivalente monetária (MER) de 1.56, respectivamente, na the arugula population density of 1,000 thousand plants ha-1, while combinação de 65 t ha-1 de biomassa de M. aegyptia e C. procera the maximum arugula productivity intercropped with beet was 9.65 com a densidade populacional de 1 milhão de plantas de rúcula por t ha-1, in the same combination of green manures amount and arugula hectare. A produtividade comercial máxima otimizada de raízes de population density. beterraba no sistema consorciado com rúcula foi de 23,20 t ha-1 na quantidade de 65 t ha-1 de M. aegyptia e C. procera e na densidade populacional de 1 milhão de plantas de rúcula por hectare, enquanto a máxima produtividade de rúcula, em consórcio com beterraba, foi de 9,65 t ha-1, na mesma combinação de quantidade de adubos verdes e densidade populacional de rúcula. Keywords: Beta vulgaris, Eruca sativa, Merremia aegyptia, Palavras-chave: Beta vulgaris, Eruca sativa, Merremia aegyptia, Calotropis procera, mixed-cropping economic returns. Calotropis procera, retornos econômicos em culturas consorciadas. Received on July 26, 2021; accepted on October 28, 2021 432 Horticultura Brasileira 39 (4) October - December, 2021

Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages Beet and arugula are vegetables biomass. dry season, which usually occurs from whose cultivation is growing in Another production factor that affects June to January and a rainy season, from the semi-arid region of northeastern February to May (Alvares et al., 2014). Brazil, as they are economically viable intercropped vegetable production During the experimental periods, the crops and offer healthy products to systems is the population density of recorded average values for minimum, consumers, although their consumption component crops, as it induces a series mean and maximum temperatures, is still low (Andrade Filho et al., 2020). of changes in the plant growth and relative humidity and precipitation for In view of the population’s search for development and needs to be known the cropping years 2018 and 2019 were healthy food, the demand for these in greater detail to determine yield and respectively: 23.68, 28.44 and 33.2ºC; vegetables is increasing and so their production efficiency of the system 22.61, 27.79 and 32.98ºC; 66.74 and production systems need scientific and (Bezerra Neto et al., 2005). 67.39%, and 0.4 and 4.4 mm (INMET, technological information. 2019). It is known that the increase in The production systems with these population density can influence the The soils in the experimental areas vegetables in the semi-arid region have quality of tuberous roots, as the carrot were classified as typical Argisol Red been through intercropping cultures, increasing the number of fine roots and Yellow Dystrophic with a sandy-loam where the cultures are able to coexist reducing the average root size, due texture (Santos et al., 2018). In each together efficiently, in order to increase to the greater competition for water experimental area, simple soil samples the aggregated production per unit of and nutrients imposed on plants. The of the 0-20 cm surface layer were input, guarantee against breaking crop population increase also interferes in the collected, homogenized to obtain a and market fluctuations, meet food aerial part of the plants, increasing the composite sample, representative of preference and/or cultural demands, production of branches and decreasing the area, whose results in cultivation in and increase producer's income (Gebru, their diameter (Oliveira et al., 2017). 2018 were: pH (water)= 8.10; EC= 0.24 2015). This cultivation practice allows dS m-1; O.M.= 4.97 g kg-1; N= 0.35 g optimizing the use of environmental Batista et al. (2016), studying the kg-1; P= 22.80 mg dm-3; K= 64.70 mg resources, such as nutrients, water and intercropping of carrot with arugula dm-3; Ca= 3.28 cmolc dm-3; Mg= 0.78 solar radiation, since the plant species manured with M. aegyptia in different cmolc dm-3; Na= 32.70 mg dm-3; Cu= have different growth cycles. population densities, obtained greater 0.10 mg dm-3; Fe= 1.91 mg dm-3; Mn= productive and economic efficiency of 11.67 mg dm-3; Zn= 2.63 mg dm-3. In The cultivation of a tuberous the intercropped system when using 2019 cultivation the results were: pH root with a broadleaf vegetable in an the carrot population density of 200 (water)= 7.10; EC= 0.10 dS m-1; O.M.= intercropped system requires large thousand plants ha-1 and of 1,000 5.27 g kg-1; N= 0.28 g kg-1; P= 22.00 amounts of nutrients, mainly due to thousand arugula plants ha-1. mg dm-3; K= 69.47 mg dm-3; Ca= 2.70 its short period of development and cmolc dm-3; Mg= 0.50 cmolc dm-3; Na= growth (Silva et al., 2018). One of Aiming to provide greater subsidies 26.70 mg dm-3; Cu= 0.24 mg dm-3; Fe= the ways to meet this requirement is for the development of technologies 2.71 mg dm-3; Mn= 12.17 mg dm-3; Zn= through the use of green manuring with for the production of beet tuberous 5.27 mg dm-3. mixtures of biomass from spontaneous vegetable intercropped with the arugula species in the Caatinga biome, such leafy crop, this work evaluated the The experimental design used was in as Merremia aegyptia and Calotropis performance of beet and arugula, complete randomized blocks, with the procera. These species, according to influenced by green manuring of M. treatments arranged in a 4 x 4 factorial Linhares et al. (2012), contain a good aegyptia and C. procera and arugula scheme, with 4 replicates, where the first supply of nutrients, suitable biomass population densities in two cropping factor consisted of equitable amounts production and low C/N ratio, which years in a semi-arid environment. of M. aegyptia and C. procera biomass provides the decomposition and faster mixtures (20, 35, 50 and 65 t ha-1 on a release of nutrients for plants. MATERIAL AND METHODS dry basis) and, the second factor were arugula population densities (40, 60, 80 Moraes et al. (2019), using C. Two field experiments were and 100% of the recommended density procera as a fertilization source in the conducted at the Experimental Farm for single cropping, corresponding cowpea intercropped with beet, obtained ‘Rafael Fernandes’ of the Universidade to 400, 600, 800 and 1,000 thousand greater productive efficiency of the Federal Rural do Semi-Árido (UFERSA), arugula plants). The recommended intercropped system, incorporating 65 located in the district of Lagoinha, 20 km population densities for single croppings t ha-1 biomass of this green manure into from the municipality of Mossoró, RN of beet and arugula in the region are the soil. In turn, Silva et al. (2018), (5°03’37”S, 37°23’50”W, 80 m altitude), 500 and 1,000 thousand plants ha-1, manuring the intercropping of beet and being the first experiment from October respectively (Silva et al., 2011; Oliveira lettuce with M. aegyptia, obtained the to December 2018 and the second from et al., 2015a). In each block, beet and highest agro-economic performance of September to November 2019. The arugula single plots were planted with the intercropping of these vegetables climate of the region, according to the biomass equitable amounts of M. with the incorporation of approximately Köppen’s classification, is ‘BShw’, aegyptia and C. procera optimized by 35.30 t ha-1 of this green manure dry and very hot, with two seasons: a Horticultura Brasileira 39 (4) October - December, 2021 433

VAS Lino et al. the research to obtain the agronomic and then, subjected to laboratory analysis, method was necessary. economic indexes of the intercropped whose chemical compositions were, in The arugula and beet, in the two systems. The spacings used in each 2018: N= 11.40 g kg-1; P= 2.36 g kg-1; population are described in Table 1. K= 2.20 g kg-1; Mg= 9.75 g kg-1 and Ca= cropping years, were harvested at 30 8.30 g kg-1, for hairy woodrose and N= and 70 day after planting, continuing The intercropping of beet with 18.40 g kg-1; P= 3.14 g kg-1; K= 4.50 g with evaluations. For the arugula crop, arugula was established in alternating kg-1; Mg= 13.35 g kg-1 and Ca= 16.30 g plant height, number of leaves per plant, strips in the proportion of 50% of the kg-1, for roostertree. In 2019: N= 16.60 green mass yield and dry mass of shoots area cultivated with beet and 50% of g kg-1; P= 2.79 g kg-1; K= 47.80 g kg-1; were determined. the area cultivated with arugula. In Mg= 7.07 g kg-1 and Ca= 19.35 g kg-1, each experimental plot, the alternating for hairy woodrose and N= 21.90 g For the beet crop we evaluated plant strips consisted of four rows, flanked kg-1; P= 1.92 g kg-1; K= 20.90 g kg-1; height, number of leaves per plant, by two rows of arugula on one side and Mg= 9.22 g kg-1 and Ca= 17.00 g kg-1 productivity of commercial roots, dry two rows of beet on the other side, used for roostertree. mass of shoots, and productivity of as borders. The total area of each plot classified roots according to diameter, in was 2.88 m2 (2.40 x 1.20 m), with a The cultivars of ‘Early Wonder’ large (>7 cm), extra AA (≥6 and <7 cm), useful area of 1.60 m² (1.60 x 1.00 m), beet and ‘Cultivated’ arugula were extra A (≥5 and <6 cm), and extra (>4 where this area consisted of two central sown in October in the first cropping, and <5 cm) (Horta et al., 2001). strips of plants, excluding the two final and in November in the second arugula plants of each row used as borders. In cropping of the year 2018 and in In the intercropped system of beet this cultivation system, the same beet September in the first cultivation, and arugula, the evaluated agronomic population density of the single crop and in October in the second arugula and economic indexes were: was used. cropping in the year 2019, in holes approximately 3 cm deep, with three to a) The system productivity index Single vegetable crops were four seeds per hole, and covered with (SPI), calculated by the expression established in a total area of 1.44 m2 commercial substrate. After thinning, (Oseni & Aliyu, 2010): SPI = [(Yb/Ya) (1.20 x 1.20 m), with a useful area of two plants were left per hole for arugula x Yab] + Yba, where: Yb represents the 0.80 m2 (0.80 x 1.00 m), where beet and one plant per hole for beet in the commercial productivity of beetroots was planted in the spacing 0.20 x 0.10 intercropped system, and in single and Ya the green mass yield of arugula m and the arugula in the spacing 0.20 cropping only one plant per hole for in single cropping; Yab is the green x 0.05 m. Plants were harvested in the both crops. mass yield of arugula in intercropped 4 central cultivation lines, excluding system with beet; Yba is the commercial the sidelines, as well as the first and Irrigations were daily, using a micro productivity of beetroots in intercropping last plants of each cultivation line, sprinkler system, partitioned into two with arugula. The advantage of this SPI considered borders. irrigations (morning and evening). is that it standardizes the productivity The amount of supplied water was of the secondary crop (arugula) based The soil preparation consisted of determined from the coefficient of the on the main crop (beet). The higher the plowing and harrowing, followed by beet crop (average Kc: 0.83) (Oliveira value of this index, the more efficient raising the beds with the aid of a soil Neto et al., 2011), with irrigation depth, the intercropped system is. grower. After soil preparation, a pre- when necessary, of approximately 8 mm planting solarization was carried out for day-1. Weeds were controlled whenever b) The land equivalent coefficient 30 days with 30 μm transparent plastic necessary, by manually removing them. (LEC) determined by the formula used (Vulca Brilho Bril Fles) following No chemical pest or disease control by Diniz et al. (2017): LEC = LERb x the methodology recommended by LERa, where: LERb and LERa represent Silva et al. (2006), in order to reduce the partial land equivalent ratios of the population of phytopathogenic beet and arugula land, respectively. organisms present in the soil, which could potentially harm crop productivity. Table 1. Description of the population densities of beet and arugula used in the intercropping systems and in the single crops, with their respective spacing. Mossoró, UFERSA, 2018-2019. The materials used as green manures were hairy woodrose (Merremia Population densities of intercropping Spacings (m) aegyptia) and roostertree (Calotropis crops (thousand plants ha-1) procera), collected from native vegetation in several locations in Beet Arugula Beet Arugula the rural area of the municipality of 0.20 x 0.05 0.20 x 0.100 (2 pls)† Mossoró, RN, before the beginning of 500 400 0.20 x 0.05 0.20 x 0.075 (2 pls) flowering. After collecting the green 0.20 x 0.05 0.20 x 0.060 (2 pls) manures, these were crushed into two 500 600 0.20 x 0.05 0.20 x 0.050 (2 pls) to three centimeters fragments. These were dehydrated at room temperature 500 800 until reaching 10% moisture content and 500 1,000 Population densities of the crops in monocropping (thousand plants ha-1) Beet 500 0.20 x 0.10 Arugula 1,000 0.20 x 0.05 † 2pls - Means two plants per hole. 434 Horticultura Brasileira 39 (4) October - December, 2021

Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages For the intercropped system, the indices and indicators, homogeneity A response surface was adjusted for minimum value of LEC is 0.25; with of variances was observed between all characteristics of the arugula as a that, the intercropped system shows the the cropping years; so, an average of function of the treatment factors. The advantage of production of the index, these indices or indicators between the maximum values of plant height (PH) when it exceeds the value of 0.25. cropping years was made. After that, obtained in 2018 and 2019 were 15.47 a regression analysis was performed and 17.86 cm in the arugula densities c) The monetary equivalent ratio for all variables, where a procedure of 600 and 1,000 thousand plants ha-1; (MER) was determined by the formula for adjusting a response surface was the number of leaves per plant (NLP) used by Afe & Atanda (2015): MER performed as a function of the equitable was 8.5 in the arugula density of 400 = (GIba + GIab)/GIb, where: GIba is the amounts of M. aegyptia and C. procera thousand plants ha-1, both characteristics gross income of beet in intercropping biomass incorporated into the soil and manured in the equitable amount of 65 with arugula. GIab is the gross income arugula population densities, through t ha-1 of M. aegyptia and C. procera of arugula in intercropping with beet; the Table Curve 3D software (Systat biomass (Figures 1A, 1B and 1C). This GIb is the highest gross income of Software, 2021). higher result in the height of arugula beet in single cropping, compared to plants in 2019 and in the population the arugula. This index measures the RESULTS AND DISCUSSION density of 1,000 thousand plants ha-1 economic superiority, or not, of the may be related to the morphological intercropping over single cropping of Arugula crop behavior of arugula growth caused by the more economical crop. The higher There was no significant interaction the milder temperatures and higher value of this index, the more viable is between the production factors, equitable relative humidity compared to the year the cropping system. amounts of M. aegyptia and C. procera 2018. Silva et al. (2021) state that both biomass, arugula population densities air temperature and soil temperature A joint analysis of variance of the and cropping years in the evaluated affect crop growth and development two growing years was performed for all characteristics of the arugula. However, processes. variables of the crops in intercropping, a significant interaction between the using the Sisvar software (Ferreira, cropping years and arugula population In addition, these results show the 2011), observing the fulfillment of the densities was recorded only for the plant use of environmental resources by assumption that the ratio of the mean height (Table 2). arugula plants, where even in different squares of the errors of the two cropping population densities, the amount of years, should not be greater than 7. the green manures incorporated into In the analysis of the agro-economic Table 2. F values for plant height (PH), number of leaves per plant (NLP), green mass yield (GMY) and dry mass of shoots (DMS) of arugula in bi-cropping intercropped with beet in different equitable amounts of M. aegyptia and C. procera biomass and population densities of arugula in the cropping years 2018 and 2019. Mossoró, UFERSA, 2018-2019. Sources of variation PH NLP GMY DMS Blocks (cropping years) 3.16** 8.36** 2.74** 4.11** Cropping years (Y) 182.46** 60.11** 74.77** 11.13** Amounts of M. aegyptia and C. procera biomass (A) 58.46** 25.36** 52.14** 21.95** Population densities of arugula (D) 2.12ns 11.26** 26.66** 10.95** YxA 1.48ns 1.09ns 0.01ns 0.11ns YxD 3.21* 1.34ns 0.82ns 0.54ns AxD 1.80ns 1.08ns 1.23ns 0.84ns YxAxD 1.14ns 0.59ns 0.59ns 0.84ns Monocropping (M) x Intercropping (I) 2.21ns 4.71* 24.51** 281.67** Y x M vs I 0.04ns 0.13ns 20.90** 1.27ns CV (%) 7.51 9.43 18.75 18.75 Cropping years 2018 13.40B 6.90B 2018 2019 0.10B 2019 15.94A 7.84A 10.05aA 7.93bA 0.12A Cropping systems 5.61bB 7.76aA Intercropping 14.08A 7.92A 0.29A Monocropping 14.67A 7.36A 0.12B ** = P < 0.01; * = P < 0.05; ns = P > 0.05. † Means followed by different lowercase letters in the row or uppercase in the column differ statistically from each other by the F test at the 5% probability level. Horticultura Brasileira 39 (4) October - December, 2021 435

VAS Lino et al. the soil provided sufficient nutrients and dry mass of shoots (DMS), the Regardless of the competition provided for a good growth and development maximum values obtained were 9.65 by the arugula population densities, of the crop, even though there is a and 0.16 t ha-1 in the densities of 1,000 the amount of 65 t ha-1 of the green different intraspecific and interspecific and 400 thousand arugula plants ha-1 manures biomass was responsible for competition. in the amount of 65 t ha-1 of the green providing a high yield of arugula leaves manures biomass (Figures 1D and 1E). when intercropped with beet, expressed For the green mass yield (GMY) z = 5.96* +637.87*/x -19022.12*/x2 +0.064**y z = 17.26** +0.024**x -116.98**/y R2 = 0.82** R2 = 0.96** A-2018 B-2019 20.54 20.54 18.32 Plant heigth (cm) 18.32 Plant heigth (cm) 16.10 16.10 13.88 13.88 11.66 11.66 65 100 65 40 100 CA.mprooucnetrsa5o0bf iMom. aae3sgs5y(pt thiaa-2a10n)d 40 Adr6euz0ngs=uilta6ie.p6s8o3(0p%*u*lRa-1tDi.o1Sn3C.)10-4**x2 +0CA..0mp3ro2ou*cn*etyrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d Adr6eun0gsuiltaieps8o(p0%ulRatDioSnC) R2 = 0.91** C 10 Number of leaves per plant 9 8 7 6 65 100 z = 5.96** -177.41**/x +0.C0A8.m4p*roo*ucynetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n) d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) R2 = 0.95** D z = 0.02* +2.5**/x +1.23.10-3**y E R2 = 0.91** 12.06 Green mass yield (t ha-1) 0.22 Dry mass of shoots (t ha-1) 9.65 0.18 7.24 0.13 4.83 0.09 2.42 0.04 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aa3esgs5y(pt thiaa-2a10n)d 40 Ad6reu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adre6un0gsuiltaiepso8(p%0ulRatDioSnC) Figure 1. Plant height (A and B), number of leaves per plant (C), green mass yield (D) and dry mass of shoots (E) of arugula in bi-cropping intercropped with beet in different combinations of equitable amounts of M. aegyptia and C. procera biomass and arugula population densities in the cropping years 2018 and 2019. Mossoró, UFERSA, 2018-2019. 436 Horticultura Brasileira 39 (4) October - December, 2021

Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages in the growth and development of the and the green mass yield of the plants organisms. crop. Filgueira (2013) reports that the due to the increase in the availability Significant interaction was recorded efficiency of the use of organic fertilizers of nutrients and favoring the physical is related to the increase in the aerial part properties and the activities of the soil between the cropping years and cropping systems only in green mass z = 28.26** -128.31*/x +0.646**y -0.0076**y2 z = 37.79** +0.047*x +0.088**y R2 = 0.76** R2 = 0.66** A-2018 B-2019 54,72 54,72 48,52 Plant heigth (cm) 48,52 Plant heigth (cm) 42,32 42,32 36,12 36,12 29,92 29,92 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aae3sgs5y(pt thiaa-2a10n)d 40 Adr6eu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaieps8o(p0%ulRatDioSnC) z = 26.41** -280.63*/x -166.13**/y C-2018 z = 34.63** -0.048*x -240.27**/y D-2019 R2 = 0.64** R2 = 0.81** 38,16 Productivity of commercial 38,16 Productivity of commercial roots (t ha-1) roots (t ha-1) 31,24 31,24 24,31 24,31 17,39 17,39 10,46 10,46 65 100 65 100 CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) z = 0.46** -2.86*/x -1.84**/y E-2018 z = 0.62** +3.79*/x -5.87**/y F-2019 R2 = 0.66** R2 = 0.90** 0,74 Dry mass of shoots (t ha-1) 0,74 Dry mass of shoots (t ha-1) 0,63 0,63 0,51 0,51 0,40 0,40 0,28 0,28 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aa3esgs5y(pt thiaa-2a10n) d 40 Ad6reu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adre6un0gsuiltaiepso8(p%0ulRatDioSnC) Figure 2. Plant height (A and B), productivity of commercial roots (C and D) and dry mass of shoots (E and F) of beet intercropped with arugula in bi-cropping in different equitable amounts of M. aegyptia and C. procera biomass and population densities of arugula in the cropping years 2018 and 2019. Mossoró, UFERSA, 2018-2019. Horticultura Brasileira 39 (4) October - December, 2021 437

VAS Lino et al. yield (Table 2). Studying the cropping mass of shoots stood out from those of due to the incorporation of the green years within each cropping system, the year 2018. In relation to cropping manures, thus reflecting in greater the year 2018 within the intercropped systems, the intercropping stood out agronomic efficiency of the intercropped system stood out from 2019 in terms of from monocropping in terms of dry system. It is known that green manuring, green mass yield, whereas the yield in mass of arugula shoots. In terms of in addition to improving soil fertility, 2019 differed from that of 2018 in the plant height and number of leaves per increases the organic matter content, monocropping (Table 2). On the other plant the cropping systems were similar decreases erosion rates, favors soil hand, analyzing the cropping systems (Table 2). water retention and increases soil within each year, in 2018 the arugula microbiota activity, increasing the green mass yield in the intercropping Nutritional support and the adequate availability of nutrients and reducing stood out from that of monocropping, number of plants per area in intercropped the number of invasive plants (Graham while in 2019 these systems behaved system are essential for good growth and & Haynes, 2006). On the other hand, similarly in terms of green mass yield. development of crops, thus resulting morphophysiological differences in The climatic differences between 2018 in productive gains. However, it is crops and treatment factors, such as (higher temperatures and lower relative known that balanced amounts of population density, proportion of crops humidity) and 2019 (lower temperatures nutrients combined with a not too high in intercropping and application of and higher relative humidity), together cultivation density results in an efficient fertilizers, regulate competition between with the population densities tested, and productive cropping system with component crops for growth limiting were the main responsible for these products of good commercial quality. factors (Morgado & Willey, 2008). differences in green biomass production Therefore, in view of this, the greater of arugula due to intra- and interspecific These results obtained from the total absorption of nutrients and the competition of the intercropped crop in arugula productivity and its components increase in the population density of relation to the single crop. in the intercropped system indicate the component crops in the intercropped that there was maximization in the use system were the main cause of obtaining In 2019, the plant height, number of environmental resources, due to an advantage in the intercropping. of leaves per plant and arugula dry improvements in the physical, chemical and biological characteristics of the soil Table 3. F values for plant height (PH), dry mass of shoots (DMS), productivity of commercial roots (PCR) and productivities of large (PLR), extra AA (PEAAR), extra A (PEAR) and extra (PER) roots of beet intercropped with arugula in different equitable amounts of M. aegyptia and C. procera biomass and population densities of arugula in the cropping years 2018 and 2019. Mossoró, UFERSA, 2018-2019. Sources of variation PH DMS PCR PLR PEAAR PEAR PER Blocks (cropping 2.19* 2.30* 2.41* 0.90ns 1.09ns 1.31ns 0.47ns years) Cropping years (Y) 120.74** 71.23** 138.57** 194.91** 28.24** 0.07ns 23.86** Amounts of M. 4.52** 9.80** 35.05** 19.86** 7.21** 1.22ns 0.84ns aegyptia and C. procera biomass (A) Population densities 3.65* 0.63ns 1.79ns 0.23ns 0.25ns 2.26ns 0.90ns of arugula (D) YxA 5.47** 3.70** 5.03** 5.74** 1.49ns 1.03ns 4.38** YxD 0.21ns 1.89ns 0.41ns 2.09ns 0.99ns 0.11ns 1.44ns AxD 0.38ns 1.45ns 0.26ns 0.68ns 0.89ns 0.49ns 1.50ns YxAxD 0.58ns 0.80ns 0.89ns 0.82ns 0.52ns 0.58ns 1.00ns Monocropping (M) x 8.10** 1.24ns 43.43** 3.33* 9.27** 7.75** 6.43* Intercropping (I) Y x M vs I 8.98** 1.88ns 23.12** 0.06ns 0.93ns 11.61** 14.16** CV (%) 8.90 23.35 14.93 45.87 35.20 39.25 28.53 Cropping years 2018 37.77B 0.36B 17.17B 3.62B 4.97B 4.20A 4.38A 2019 46.80A 0.52A 24.43A 10.08A 7.02A 4.03A 3.30B Cropping systems 2018 2019 2018 2019 2018 2019 2018 2019 Intercropping 37.77bB 44.80aA 0.44A 17.33bB 26.24aA 5.16B 5.99B 4.20aA 4.03aB 4.38aA 3.30bA Monocropping 45.21aA 44.61bA 0.48A 32.79aA 25.94bA 7.28A 8.39A 3.82bA 7.76aA 1.90bB 3.78aA ** = P < 0.01; * = P < 0.05; ns = P > 0.05. † Means followed by different lowercase letters in the row or uppercase in the column differ statistically from each other by the F test at the 5% probability level. 438 Horticultura Brasileira 39 (4) October - December, 2021

Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages Oliveira et al. (2015b), working intercropping under different amounts since the best productive performances with the agronomic viability of of C. procera biomass in different of intercropped systems were observed arugula and lettuce bi-cropping in population densities, reported a low in the highest population densities, intercropping with carrot in strip- interspecific competition between crops, due to the better use of environmental lnz = 0.46* +1.344.10-3**y2 -1.67.10-5**y3 z = 8.22** -0.06**x +0.18**y R2 = 0.79** R2 = 0.89** A-2018 B-2019 20.16 20.16 15.63 Productivity of large 15.63 Productivity of large roots (t ha-1) roots (t ha-1) 11.09 11.09 6.56 6.56 2.02 2.02 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aae3sgs5y(pt thiaa -2a10n)d 40 Adr6eu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaieps8o(p0%ulRatDioSnC) z = 6.33** -1.30.1017*e-x -9.83.108**e-y z = 3.15** +93.87**/x -3.16.1017**e-x -2.18.106**y3 R2 = 0.87** R2 = 0.75** C D 7.92 Productivity of extra AA 6.18 Productivity of extra A roots (t ha-1) roots (t ha-1) 6.64 5.29 5.37 4.40 4.09 3.51 2.82 2.62 65 100 65 100 CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) z = 5.34** -0.011*x -0.014*y z = 3.75** -1.52.1017*e-x -3.22.10-6*y3 R2 = 0.48* R2 = 0.54** E-2018 F-2019 5.28 5.28 4.43 Productivity of extra 4.43 Productivity of extra roots (t ha-1) roots (t ha-1) 3.58 3.58 2.73 2.73 1.88 1.88 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aa3esg5sy(pt thiaa2-a10n)d 40 Ad6reu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adre6un0gsuiltaiepso8(p%0ulRatDioSnC) Figure 3. Productivity of large (A and B), extra AA (C), extra A (D) and extra (E and F) beetroots intercropped with arugula in bi-cropping in different equitable amounts of M. aegyptia and C. procera biomass and population densities of arugula in the cropping years 2018 and 2019. Mossoró, UFERSA, 2018-2019. Horticultura Brasileira 39 (4) October - December, 2021 439

VAS Lino et al. resources by the component crops. clearly seen that the best combination the production of dry mass, as it causes between the biomass amounts of the a deficiency in the translocation of Beet crop mixtures of the green manures and photoassimilates, as reported by Paciullo There was no significant interaction the arugula population densities in et al. (2011). Therefore, the maximum between production factors, biomass the cropping years, was registered in dry mass production of the beet was equitable amounts of M. aegyptia and the biomass amount of 65 t ha-1 of M. achieved in the lowest arugula density C. procera, arugula population densities aegyptia and C. procera independently tested. In intercropped systems, where and cropping years in the evaluated of the population density used in the nutritional conditions of the soil are characteristics of beet. However, agronomic variables: plant height, suitable for cultivation, competition significant interactions between commercial productivity, dry mass for light can be more intense, and the cropping years and equitable amounts of shoots and productivity of large use of higher densities can increase of M. aegyptia and C. procera biomass roots. These results are related to the competition for this natural resource were recorded only for the agronomic nutritional contribution, due to the (Gebru, 2015; Nascimento et al., 2018). variables: plant height, shoot dry matter, efficiency of the highest dose of green productivity of commercial roots, and manures, which promoted the good The increase in productivity with productivities of large and extra roots development of the crops. However, it the equitable amounts of the green (Table 3). is known that the adequate supply of manures is due, in part, to the greater A response surface was adjusted for nutrients incorporated into the soil, can availability of nutrients, mainly N, P and each interaction, where the maximum promote the good growth and vegetative K released by fertilizers (Batista et al., values reached in these agronomic development of the crops, the expansion 2016), as well as by the improvement variables were, respectively, 40.68 and of the photosynthetic area and the in the chemical and physico-chemical 48.23 cm (PH); 21.04 and 29.01 t ha-1 activation and increase of the production characteristics of the soil obtained by (PCR); 0.41 and 0.62 t ha-1 (DMS); 4.27 potential (Favacho et al., 2017). adding these increasing amounts of and 16.69 t ha-1 (PLR) and 4.60 and green manures. According to Fontanétti 3.73 t ha-1 (PER) in the combinations of The results obtained from the height et al. (2006), the absorption of nutrients equitable amounts of M. aegyptia and C. of beet plants may also be associated resulting from the mineralization of procera biomass and arugula population with the intense competition for light, these green manures by vegetables density of 42 and 1,000 thousand and due to the increase in the population depends, to a large extent, on the 65 and 1,000 thousand; 65 and 1,000 density of the arugula, which probably synchrony between the decomposition thousand and 65 and 400 thousand; promoted the growth of the beet. With and mineralization of plant residues and 65 and 1,000 thousand and 65 and 400 less space between component crops of the moment of greatest demand for thousand; 65 and 1,000 thousand and 65 of the intercropping, beet plants grew the crop. and 400 thousand; 20 and 400 thousand in search of light, the main climatic and 20 t ha-1 and 900 thousand plants element that determines their growth, A response surface was adjusted for ha-1 in the cropping years 2018 and 2019 in addition to the water and nutrients the productivities of extra AA and extra (Figures 2A, 2B, 2C, 2D, 2E, 2F, 3A, available in the soil solution, as reported A roots, where the maximum values 3B, 3E and 3F). The climatic differences by Taiz & Zeiger (2013). obtained in these variables were 6.33 between 2018 (higher temperatures and 5.25 t ha-1 in the combinations of and lower relative humidity) and For the dry mass of beet shoots in green manures biomass amounts and 2019 (lower temperatures and higher 2019, it can be inferred that the greater arugula population densities of 65 t relative humidity), together with the shading imposed by the increase in the ha-1 and 830 thousand, and 20 t ha-1 and population densities tested, were the number of arugula plants in the area, 430 thousand plants ha-1, respectively main responsibles for these differences negatively impacted photosynthesis (Figures 3C and 3D). This increase in in the evaluated characteristics. and, consequently, allowed the dry mass productivity is also due to the increase In view of these results, it can be of beet shoots reach the maximum in in population density up to a certain a lowest population. Shading reduces density, where there is a decrease in those productivities (extra AA and Table 4. F values for system productivity index (SPI), land equivalent coefficient (LEC) and extra A roots), due to the excessive monetary equivalent ratio (MER) of beet intercropped with arugula in different equitable competition that is established between amounts of M. aegyptia and C. procera biomass and population densities of arugula. Mossoró, the crop plants of the cultivation system. UFERSA, 2018-2019. There were significant interactions Sources of variation SPI LEC MER between crop systems and cropping Blocks 0.45ns 1.33ns 0.81ns years for the agronomic characteristics Amounts of M. aegyptia and C. procera biomass (A) 21.47** 14.33** 16.02** evaluated in the beet except for the dry Population densities of arugula (D) 84.72** 77.60** 90.66** mass of shoots, productivity of large AxD 1.23ns 1.41ns 1.32ns roots and productivity of extra AA roots CV (%) 10.67 (Table 3). Studying the cropping years ** = P < 0.01; ns = P > 0.05. 15.70 7.92 within the intercropping, 2019 stood out from 2018 in terms of plant height and commercial productivity, while 440 Horticultura Brasileira 39 (4) October - December, 2021

Beet-arugula intercropping under green manuring and planting density induce to agro-economic advantages in the case of extra root productivity monocropping in the plant’s height, in must be evidenced by the nutritional the behavior was reverse. This result the commercial productivity and in the complementarity provided by the is possibly related to the sharing productivity of extra beetroots; however, mixture of the two species through the of environmental resources by the the productivity of extra A roots, was cycling of nutrients (Favacho et al., intercropping system in the year where surpassed by monoculture (Table 3). In 2017). The highest productivity of large the weather conditions were not the 2018, monocropping stood out from the roots was achieved by combining the most favorable, consequently producing intercropping in terms of plant height highest amount of biomass mixture of smaller sized roots of beetroot, as and commercial root productivity, while green manures, with the highest arugula extra sized roots. No differences were in the case of productivity of extra roots, population density tested in 2018, and observed between the cropping years in the behavior was reverse. There was no with the lowest arugula density in the productivity of extra A roots. On the significant difference between cropping 2019. This shows that the interspecific other hand, within the monocropping, systems in the productivity of extra A competition was not affected by the year 2018 differed from 2019 in roots in that year. population density, thus allowing the plant height, commercial productivity, highest productivity of large roots to while in the productivities of roots extra In the productivity of large and be achieved in the greatest amount of A, and extra the behavior was reverse extra AA roots, the monocropping stood the green manures incorporated. This (Table 3). out from the intercropping; however, result was due to the climatic differences in the dry mass of shoots there was between 2018 (higher temperatures Studying the cropping systems no significant difference between the and lower relative humidity) and 2019 within cropping years, it can be seen that cropping systems (Table 3). (lower temperatures and higher relative within the year 2019, the intercropped humidity). system did not differ from the The increase in productivity with the addition of the green manures z = 68.03** -629.25**/x -537.09**/y z = 1.17** -13.18**/x -12.99**/y R2 = 0.96** R2 = 0.93** A B 61.43 Sistem productivity 0.98 index (t ha-1) 47.86 0.77 Land equivalent coefficient 34.29 0.56 20.72 0.35 7.15 0.14 65 100 65 100 CA.mprooucnetrsa5o0bf iMom. aae3sgs5y(pt thiaa-2a10n)d 40 Adr6eu0ngsuiltaieps8o(0p%ulRatDioSnC) CA.mprooucnetrs5ao0bf iMom. aa3es5gs y(pt thiaa2-a10n) d 40 Adr6eun0gsuiltaieps8o(p0%ulRatDioSnC) z = 1.95** -15.13**/x -15.41**/y C R2 = 0.95** 1.94 Monetary equivalent ratio 1.61 1.28 0.95 0.62 65 100 CA.mprooucnetrs5ao0bf iMom. aa3es5gsy(pt thiaa2-a10n)d 40 Adr6eun0gsuiltaiepso8(p0%ulRatDioSnC) Figure 4. System productivity index (A), land equivalent coefficient (B) and monetary equivalent ratio (C) of beet intercropped with arugula in bi-cropping in different equitable amounts of M. aegyptia and C. procera biomass and population densities of arugula. Mossoró, UFERSA, 2018-2019. Horticultura Brasileira 39 (4) October - December, 2021 441

VAS Lino et al. Agro-economic indexes manures can also provide nutrient the intercropped systems. Significant interactions between the cycling in the soil, bringing nutrients The maximum optimized factors of production studied, equitable that are in greater depth to the surface. amounts of M. aegyptia and C. procera commercial production of beetroots in biomass and population densities of The high SPI value (53.47 t ha-1) the intercropped system with arugula arugula were recorded for the agro- obtained in the combination of 65 t ha-1 was 23.20 t ha-1 using 65 t ha-1 of M. economic indices: system productivity of the mixture of the green manures aegyptia and C. procera biomass in index (SPI), land equivalent coefficient with 1,000 thousand arugula plants ha-1 a density of 1,000 thousand arugula (LEC), and monetary equivalent ratio demonstrates the agronomic efficiency plants ha-1. Maximum productivity of (MER) (Table 4). of the intercropped system of beet arugula intercropped with beet was 9.65 However, a response surface was with arugula in this combination in t ha-1, in the same combination of green adjusted for each agro-economic index relation to the monocropping system manures amount and population density (Figure 4). The maximum values of these cultures. Likewise, the high of arugula. The maximum optimized reached for SPI, LEC and MER were LEC value obtained (0.84), expressed productivities for large roots, extra AA 53.47 t ha-1, 0.84 and 1.56 in the this same agronomic efficiency of the and extra were 10.28; 6.33 and 4.12 t ha-1 combination of equitable amount of intercropping of these two vegetables obtained using 65 t ha-1 green manure the green manures biomass and arugula in this same combination of treatment biomass and in the arugula population population densities of 65 t ha-1 and factors. According to Diniz et al. (2017), densities of 850; 820 and 400 thousands 1,000 thousand plants ha-1, respectively when the LEC value is greater than plants ha-1, respectively. (Figures 4A, 4B and 4C). 0.25 the intercropped system shows an These results also indicate that advantage of production in relation to REFERENCES the use of high population densities monocropping of the cultures. of the arugula crop (secondary crop), AFE, AI; ATANDA, S. 2015. Percentage did not result in a negative effect In addition, it is possible to observe yield difference, an index for evaluating on the system intercropped with by the obtained value of MER (1.56) that intercropping efficiency. American Journal of beet, mainly in terms of competitive the agronomic efficiency of the beet and Experimental Agriculture 5: 459-465. pressure by solar radiation, nutrients arugula intercropping was translated into and other environmental resources. It economic terms by MER. 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