Journal of Seed Science

Online version: ISSN 2317-1545 JOURNAL OF SEED SCIENCE

Continuation of the “Revista Brasileira de Sementes / Brazilian Seed Journal”

Volume 42

Collection of published articles from January to March, 2020

This Journal’s articles are indexed in: - BINAGRI / Ministério da Agricultura, Pecuária e Abastecimento www.agricultura.gov.br - SCIELO (Scientific Eletronic Library on line) www.scielo.br - SCOPUS - AGROBASE - AGRIS Coordenadoria Geral e Informação Documental Agrícola BINAGRI/MAPA - EBSCO Information Services

Associação Brasileira de Tecnologia de Sementes Brazilian Association of Seed Technology GENERAL INFORMATION

The Journal of Seed Science (JSS) is the official publication of the Brazilian Association of Seed Technology, and publishes original articles and revisions in the field of Seed Science and Technology and related areas of the agricultural services. The JSS is a publication in continuation of the “Revista Brasileira de Sementes” (Brazilian Seed Journal), from January/2013. The mission of JSS is to publish scientific papers in the area of Seed Science and Technology, providing to the national and international agricultural sectors the knowledge for producing high quality seeds and the benefits from its use. Besides, JSS aims to contribute for the development and improvement of technologies that would aid in the economic and social improvement of the population, guaranteeing the basic input of the agricultural production and the preservation of the vegetable species. The JSS is published four times a year, although special numbers can be published. The Editorial Board is composed by one or more editors, Associated Editors and a Scientists Committee formed by scientists working with Seed Science and Technology. The annual subscription rate of JSS is US$ 200.00 abroad. The price of each number is US$ 60.00. Subscriptions and or individual number should be requested by sending a money order to ABRATES, at the following address:

JOURNAL OF SEED SCIENCE Universidade Federal de Viçosa - Departamento de Agronomia Avenida PH Rolfs, s/n°, 36570-900, Viçosa - MG – Brasil Fone: +055(31) 3612-4490 / Email: [email protected] ABRATES Londrina - Fone/FAX: +055(43) 3025-5120 Email: [email protected]

INFORMAÇÕES GERAIS

A revista ”Journal of Seed Science” (JSS) é uma publicação oficial da Associação Brasileira de Tecnologia de Sementes (ABRATES) e destina-se a divulgar trabalhos científicos originais sobre Ciência e Tecnologia de Sementes e áreas correlatas. O JSS é a publicação que dará continuidade à Revista Brasileira de Sementes (Brasilian Seed Journal) a partir de Janeiro de 2013. A missão do JSS é publicar trabalhos científicos na área de Ciência e Tecnologia de Sementes, divulgando ao setor agrícola nacional e internacionals avanços do conhecimento para a obtenção de sementes de alta qualidade e informações relativas aos benefícios resultantes da sua utilização. Além disso, contribuir para o desenvolvimento e aprimoramento de tecnologias que auxiliem no desenvolvimento econômico e social da população, garantindo o insumo básico da produção agrícola e a preservação das espécies vegetais. A periodicidade do JSS é trimestral, podendo, no entanto, serem publicadas edições especiais. O Comitê Editorial é composto por um ou mais Editores, por Editores Associados e um corpo de Assessores Científicos, formado por cientistas que trabalham em Tecnologia de Sementes. O preço da assinatura, ao ano, é de US$ 85,00 (oitenta e cinco dólares americanos) para o território nacional e de US$ 200,00 (duzentos dólares americanos) para o exterior. Os pedidos acompanhados de cheque nominal à ABRATES deverão ser encaminhados à Diretoria Técnica e de Divulgação no seguinte endereço:

Home Page www.abrates.org.br/revista e-mail: [email protected]

Layout Editor and Design: Jéssica Akemi Ychisawa ASSOCIAÇÃO BRASILEIRA DE TECNOLOGIA DE SEMENTES (BRAZILIAN ASSOCIATION OF SEED TECHNOLOGY) EXECUTIVE BOARD - 2017/2020

President: Francisco Carlos Krzyzanowski (EMBRAPA SOJA) 1st Vice-President: Fernando Augusto Henning (EMBRAPA SOJA) 2nd Vice-President: Maria Laene Moreira de Carvalho (UFLA) Financial Director: José de Barros França Neto (EMBRAPA SOJA) Vice-Financial Director: Alessandro Lucca Braccini (UEM) Technical and Communication Director: Denise Cunha Fernandes dos Santos Dias (UFV) Vice-Technical and Communication Director: Gilda Pizzolante de Pádua (EMBRAPA)

Supervisory Board - holders: Francisco Guilhien Gomes Junior (USP/ESALQ) Luiz Eduardo Panozzo (UFPel)

Alternates: Claudio José Barbedo (Instituto de Botânica, SP) Géri Eduardo Meneghello (UFPel) Laércio Junio da Silva (UFV)

JOURNAL OF SEED SCIENCE

EDITORS Denise Cunha Fernandes dos Santos Dias - Universidade Federal de Viçosa Gilda Pizzolante de Pádua - Empresa Brasileira de Pesquisa Agropecuária

Associated Editors: Alek Sandro Dutra (UFC) José de Barros França-Neto (EMBRAPA SOJA) Alessandro Lucca Braccini (UEM) José da Cruz Machado (UFLA) Augusto César Pereira Goulart (EMBRAPA) Julio Marcos-Filho (USP/ESALQ) Ceci Castilho Custódio (UNOESTE) João Almir Oliveira (UFLA) Claudemir Zucareli (UEL) Laércio Junio da Silva (UFV) Claudio José Barbedo (Instituto de Botânica) Luciana Magda de Oliveira (UDESC) Denise Cunha Fernandes dos Santos Dias (UFV) Marcela Carlota Nery (UFVJM) Eduardo Euclydes de Lima e Borges (UFV) Maria Laene Moreira de Carvalho (UFLA) Eduardo Fontes Araújo (UFV) Maria Luiza Nunes Costa (UFMS) Elisa Serra Negra Vieira (EMBRAPA FLORESTAS) Norimar D’Ávila Denardin (UPF) Fatima Conceição Márquez Piña Rodrigues (UFSCar) Raquel Maria de Oliveira Pires (UFLA) Fernando Augusto Henning (EMBRAPA SOJA) Renato Delmondez de Castro (UFBA) Francisco Carlos Krzyzanowski (EMBRAPA SOJA) Salvador Barros Torres (EMPARN) Francisco Guilhien Gomes Júnior (USP/ESALQ) Silvio Moure e Cícero (USP/ESALQ) Gilda Pizzolante de Pádua (EMBRAPA/EPAMIG) Sttela Dellyzete Veiga Franco da Rosa (EMBRAPA CAFÉ) Heloísa Oliveira Santos (UFLA) Warley Marcos Nascimento (EMBRAPA HORTALIÇAS) International Associated Editors: Peter Toorop (Kew Garden/England) Omar Bazzigalupi (EEA INTA/Argentina)

English Language Reviewers: Editora Genesis Infoservice Ltda. Lloyd John Friedrich Fausto Bahia Teixeira

Reviewers Alexandre Duarte Fábio Oliveira Diniz Alexandre Silva Fatima Conceição Márquez Pina Rodrigues Ana Lúcia Pereira Kikuti Fernanda Brunetta Godinho Anghinoni Ana Paula Silva Francisco Guilhien Gomes Junior André Dantas de Medeiros Hugo Tiago Ribeiro Amaro André Luiz dos Santos Jaqueline Malagutti Corsato Andréa Santos Oliveira José de Barros França-Neto Augusto César Pereira Goulart Julio Marcos-Filho Bárbara França Dantas Leonardo Gonçalves Bastos Bruno Gomes de Noronha Lilian Gomes de Moraes Dan Ceci Castilho Custódio Maria Luiza Nunes Costa Cileide Maria Coelho Marília Lazarotto Daniel Teixeira Pinheiro Maristela Panobianco Denise Cunha Fernandes dos Santos Dias Mauro Pacheco Edgard Augusto Toledo Picoli Renata Silva-Mann Eduardo Euclydes de Lima e Borges Renato Delmondez Castro Eduardo Santos Barboza da Silva Robson Celestino Meireles Everson Reis Carvalho Stefânia Vilas Boas Coelho JOURNAL OF SEED SCIENCE ISSN 2317-1545 v. 42, Jan./Mar. 2020

CONTENTS

ARTICLES

Physiological performance of Garcinia gardneriana (Planch. & Triana) Zappi: a species with recalcitrant and dormant seeds. Willian Goudinho Viana, Ana Paula Lando, Rosa Angelica da Silva, Cláudia Dias da Costa, Pedro Henrique Mastriane Vieira, Neusa Steiner.

Physiological response of soybean seeds to spray volumes of industrial chemical treatment and storage in different environments. Julia Abati, Cristian Rafael Brzezinski, Elieges Carina Bertuzzi, Fernando Augusto Henning, Claudemir Zucareli.

Germination and seed ecology ofBuchenavia tomentosa Eichler (Combretaceae). Amanda Ribeiro Correa, Ana Mayra Pereira da Silva, Vitor Sthevan Mendes da Silva, Elisangela Clarete Camili, Antonio Renan Berchol da Silva, Maria de Fátima Barbosa Coelho.

Effect of reduced water potential on seed germination of a forest tree: a hydrotime approach. Luís Felipe Daibes, Victor J.M. Cardoso.

Digital image processing of coated perennial-soybean seeds and correlation with physiological attributes. Amanda Justino Acha, Henrique Duarte Vieira.

Enzyme activity in the micropylar region ofMelanoxylon brauna Schott seeds during germination under heat stress conditions. Marcone Moreira Santos, Eduardo Euclydes de Lima e Borges, Glauciana da Mata Ataíde, Raquel Maria de Oliveira Pires, Debora Kelli Rocha.

Vigor test of (strong) normal intact Amburana cearensis (Allemão) A.C. Smith seedling. Josenilda Aprígio Dantas de Medeiros, Sarah Patrícia Lima Nunes, Francival Cardoso Félix, Cibele dos Santos Ferrari, Mauro Vasconcelos Pacheco, Salvador Barros Torres.

X-ray imaging and digital processing application in non-destructive assessing of melon seed quality. André Dantas de Medeiros, Maycon Silva Martins, Laércio Junio da Silva, Márcio Dias Pereira, Manuel Jesús Zavala León, Denise Cunha Fernandes dos Santos Dias.

Simulated drift of dicamba: effect on the physiological quality of soybean seeds. Estevam Matheus Costa, Jacson Zuchi, Matheus Vinícius Abadia Ventura, Leandro Spíndola Pereira, Geovani Borges Caetano, Adriano Jakelaitis.

Chemical treatment and size of corn seed on physiological and sanitary quality during storage. Karen Marcelle de Jesus Silva, Renzo Garcia Von Pinho, Édila Vilela de Resende Von Pinho, Renato Mendes de Oliveira, Heloísa Oliveira dos Santos, Thomas Simas Silva. Physiological and antioxidant changes in sunflower seeds under water restriction. Thais de Castro Morais, Daniel Teixeira Pinheiro, Paola Andrea Hormaza Martinez, Fernando Luiz Finger, Denise Cunha Fernandes dos Santos Dias.

Improvement of the methodology of the tetrazolium test using different pretreatments in seeds of the genus Epidendrum (Orchidaceae). Seir Antonio Salazar Mercado, Jesús David Quintero Caleño, Laura Yolima Moreno Rozo.

NOTES

Accelerated aging for evaluation of vigor inBrachiaria brizantha ‘Xaraés’ seeds. Ariadne Morbeck Santos Oliveira, Marcela Carlota Nery, Karina Guimarães Ribeiro, Adriana Souza Rocha, Priscila Torres Cunha.

Specificity and sensibility of primer pair in the detection ofColletotrichum gossypii var. cephalosporioides in cotton seeds by PCR technique. Mirella Figueiró de Almeida, Sarah da Silva Costa, Iara Eleutéria Dias, Carolina da Silva Siqueira, José da Cruz Machado. JOURNAL OF SEED SCIENCE ISSN 2317-1545 v. 42, Jan./Mar. 2020

CONTEÚDO

ARTIGOS

Desempenho fisiológico de sementes deGarcinia gardneriana (Planch. & Triana) Zappi: uma espécie com sementes recalcitrantes e dormentes. Willian Goudinho Viana, Ana Paula Lando, Rosa Angelica da Silva,Cláudia Dias da Costa, Pedro Henrique Mastriane Vieira, Neusa Steiner.

Resposta fisiológica de sementes de soja a volumes de calda do tratamento químico industrial e armazenamento em diferentes ambientes. Julia Abati, Cristian Rafael Brzezinski, Elieges Carina Bertuzzi, Fernando Augusto Henning, Claudemir Zucareli.

Germinação e ecologia de sementes de Buchenavia tomentosa Eichler (Combretaceae). Amanda Ribeiro Correa, Ana Mayra Pereira da Silva, Vitor Sthevan Mendes da Silva, Elisangela Clarete Camili, Antonio Renan Berchol da Silva, Maria de Fátima Barbosa Coelho.

Efeito da redução do potencial hídrico na germinação de sementes de uma árvore tropical: uma abordagem do tempo hídrico. Luís Felipe Daibes, Victor J.M. Cardoso.

Processamento digital de imagens de sementes de soja perene revestidas e correlação com atributos fisiológicos. Amanda Justino Acha, Henrique Duarte Vieira.

Atividade enzimática na região micropilar de sementes de Melanoxylon brauna Schott durante a germinação sob estresse térmico. Marcone Moreira Santos, Eduardo Euclydes de Lima e Borges, Glauciana da Mata Ataíde, Raquel Maria de Oliveira Pires, Debora Kelli Rocha.

Teste de vigor de plântulas normais intactas (fortes) de Amburana cearensis (Allemão) A.C. Smith. Josenilda Aprígio Dantas de Medeiros, Sarah Patrícia Lima Nunes, Francival Cardoso Félix, Cibele dos Santos Ferrari, Mauro Vasconcelos Pacheco, Salvador Barros Torres.

Aplicação do teste de raios-X e processamento digital na avaliação não-destrutiva da qualidade de sementes de melão. André Dantas de Medeiros, Maycon Silva Martins, Laércio Junio da Silva, Márcio Dias Pereira, Manuel Jesús Zavala León, Denise Cunha Fernandes dos Santos Dias.

Deriva simulada de dicamba: efeitos sobre a qualidade fisiológica de sementes de soja. Estevam Matheus Costa, Jacson Zuchi, Matheus Vinícius Abadia Ventura, Leandro Spíndola Pereira, Geovani Borges Caetano, Adriano Jakelaitis. Tratamento químico e tamanho da semente de milho na qualidade fisiológica e sanitária durante o armazenamento. Karen Marcelle de Jesus Silva, Renzo Garcia Von Pinho, Édila Vilela de Resende Von Pinho, Renato Mendes de Oliveira, Heloísa Oliveira dos Santos, Thomas Simas Silva.1

Alterações fisiológicas e antioxidativas em sementes de girassol submetidas à restrição hídrica. Thais de Castro Morais, Daniel Teixeira Pinheiro, Paola Andrea Hormaza Martinez, Fernando Luiz Finger, Denise Cunha Fernandes dos Santos Dias.

Aprimoramento da metodologia do teste de tetrazólio utilizando diferentes pré-tratamentos em sementes do gênero Epidendrum (Orchidaceae). Seir Antonio Salazar Mercado, Jesús David Quintero Caleño, Laura Yolima Moreno Rozo.

NOTAS

Envelhecimento acelerado para avaliação do vigor de sementes de Brachiaria brizantha cv Xaraés. Ariadne Morbeck Santos Oliveira, Marcela Carlota Nery, Karina Guimarães Ribeiro, Adriana Souza Rocha, Priscila Torres Cunha.

Especificidade e sensibilidade de um par de primer na detecção de Colletotrichum gossypii var. cephalosporioides em sementes de algodão pela técnica de PCR. Mirella Figueiró de Almeida, Sarah da Silva Costa, Iara Eleutéria Dias, Carolina da Silva Siqueira, José da Cruz Machado. Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Physiological performance of Garcinia gardneriana (Planch. Journal of Seed Science, v.42, e202042001, 2020 & Triana) Zappi: a species with recalcitrant and dormant seeds http://dx.doi.org/10.1590/ 2317-1545v42222357 Willian Goudinho Viana1 , Ana Paula Lando1 , Rosa Angelica da Silva1 , Cláudia Dias da Costa1 , Pedro Henrique Mastriane Vieira1 , Neusa Steiner1*

ABSTRACT: Garcinia gardneriana (Planch. & Triana) Zappi is a native species widely distributed in Brazil. It has ornamental features, edible fruits, and its leaves have medicinal properties; however, its potential has not been explored. The aim of this study was to evaluate seed physiological performance due to dormancy, desiccation and storage tolerance. Only decoated seeds -1 germinated. Seeds had an initial water content of 1.07 g H2O.g dw and final germination of 95%. -1 Both desiccation to 0.67 g H2O.g dw and storage at 25 °C for ninety days resulted in decreased -1 germination, 43 and 41%, respectively. Desiccation below 0.25 g H2O.g dw and storage for ninety days at 5 °C were lethal. A rapid decrease in enzymic protection by superoxide dismutase and ascorbate peroxidase was correlated to desiccation sensitivity. Total polyamines content was higher in fresh seeds and markedly decreased with desiccation. The decrease in enzyme activity and polyamines content seems to be associated with seed viability loss. In sum, G. gardneriana seeds have a low tolerance to desiccation and are sensitive to chilling. Therefore, the seeds can be categorized as recalcitrant and dormant, a rare combination in terms of seed biology.

Index terms: antioxidant enzymes, desiccation tolerance, physical dormancy, polyamines, germination.

Desempenho fisiológico de sementes de Garcinia gardneriana (Planch. & Triana) Zappi: uma espécie com sementes recalcitrantes e dormentes

RESUMO: Garcinia gardneriana (Planch. & Triana) Zappi é uma espécie nativa amplamente distribuída no Brasil. Tem características ornamentais, frutos comestíveis e folhas com propriedades medicinais. No entanto, seu potencial ainda não é explorado. O objetivo deste estudo foi avaliar o desempenho fisiológico das sementes devido à dormência, dessecação e tolerância ao armazenamento. Apenas sementes sem tegumento germinaram. As sementes -1 apresentaram um conteúdo inicial de água de 1,07 g H2O.g dw e germinação final de 95%. Ambos, dessecação para 0,67 g H O.g-1 dw e armazenamento a 25 °C por noventa dias, resultaram 2 *Corresponding author em decréscimo na germinação, 43 e 41%, respectivamente. Dessecação abaixo de 0,25 g H O.g-1 2 E-mail: [email protected] dw e armazenamento por noventa dias a 5 °C foram letais. Uma rápida diminuição na proteção enzimática pela superóxido dismutase e ascorbato peroxidase foi correlacionada à intolerância Received: 4/6/2019. à dessecação. O conteúdo total de poliaminas foi maior nas sementes frescas e diminuiu com a Accepted: 11/12/2019. dessecação. A diminuição da atividade enzimática e do conteúdo de poliaminas está associada à perda de viabilidade de sementes. Em suma, as sementes de G. gardneriana têm baixa tolerância à dessecação e são sensíveis à refrigeração. Portanto, as sementes podem ser categorizadas 1Laboratório de Fisiologia Vegetal, como recalcitrantes e dormentes, uma combinação rara em termos de biologia de sementes. Departamento de Botânica, Universidade Federal de Santa Termos para indexação: enzimas antioxidantes, tolerância à dessecação, dormência física, Catarina, 88040-900 – Florianópolis, poliaminas, germinação. SC, Brasil.

Journal of Seed Science, v.42, e202042001, 2020 2 W. G. Viana et al.

INTRODUCTION

Garcinia gardneriana (Planch. & Triana) Zappi, known as bakupari, is a fruit species from the Clusiaceae family, which comprises 27 genera and 1.090 species (Stevens, 2007). The species is widely distributed in Brazil, occurring in the Amazon Rainforest, Caatinga, Central Brazilian Savanna and Atlantic Rainforest (Bittrich et al., 2015). It is a tree- sized plant with bright yellow fruits, containing one to three seeds. Although not domesticated, this species has high economic potential. Its morphological structure makes it very suitable to be used as an ornamental plant, its fruits can be consumed in natura, and its leaves are used in the traditional medicine to treat inflammations and infections (Cechinel Filho et al., 2000). Nevertheless, little is known about the seed germination process and the influence the environment has on it. According to Bewley et al. (2013), seed germination begins with water absorption by the seed and ends with radicle protrusion, followed by the emergence of the embryonic axis through the surrounding structures. It is a physiological process that requires favorable environmental conditions of temperature, oxygen, humidity and light. However, when one or more environmental conditions are unfavorable, the germination does not occur. Furthermore, characteristics of the seed itself, or of the dispersion unit, may preclude germination, giving it a state of dormancy (Baskin and Baskin, 2014). Physical dormancy is a state where the seed fails to germinate due to an impermeable structure, which prevents water from being absorbed (Baskin et al., 2000). Water content (WC) is correlated not only to seed germination but also to storage capacity, and this is directly associated with desiccation tolerance. In the 1970s, seeds were divided into two categories: orthodox and recalcitrant (Barbedo et al., 2013). According to this classification, desiccation-tolerant -1 seeds (orthodox) survive desiccation to very low WC, below 0.1 g H2O.g dw, and withstand dry storage for long periods without a significant loss in viability (Marques et al., 2018). On the other hand, desiccation-sensitive seeds (recalcitrant) -1 often have high WC and do not tolerate desiccation, losing viability quickly below 0.25 g H2O.g dw. This dichotomy, however, is now only appropriate for technological purposes, given the vast range of different behaviors that can be found within these two groups (Barbedo et al., 2013; Barbedo, 2018). For example, some species of the genus Garcinia were classified as being recalcitrant and dormant (Liu et al., 2005; Anegbeh et al., 2006). This combination in seeds is quite rare; in a survey of 886 species, only 1.4% were recalcitrant and had physical dormancy (Tweddle et al., 2003). Vázquez-Yanes and Orozco-Segovia (1993) state that very little is known about seeds that possess this combination, thus evidencing the importance of this species in studies about seed physiology behavior and viability. Seed viability loss is dependent on a plethora of factors, among them genetics, mechanical damage, temperature and humidity, and seed WC (McDonald, 2004). Physiological deterioration can be seen as a result of the loss of membrane integrity, electrolyte leakage, reactive oxygen species (ROS) production, lipid peroxidation, changes in enzymatic activity etc. (Goel and Sheoran, 2003). The cellular damage caused by these free radicals is usually reduced or prevented by a protective mechanism involving peroxidase-scavenging enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX) (Goel et al., 2003; McDonald, 2004). Together with antioxidant enzymes, polyamines (PAs) have also been suggested to play a role in free radical scavenging (Drolet et al., 1986). According to Flores and Galston (1984), under water deficit, there is an accumulation of PAs that could act directly or indirectly as a free radical scavenger. Therefore, to study how these substances change and link this with seed physiological performance is of primary importance to understand the G. gardneriana seeds physiology. Considering the current global concern on strategies for plant conservation of recalcitrant seeds and the lack of literature on the storage, dormancy-breaking and germination requirements of seeds from forest species, this study was developed. To the best of our knowledge, no studies have investigated that this species is indeed dormant and recalcitrant, neither was the relationship of this characteristic with the seed metabolism and dehydration. Thus, physical and physiological aspects such as the effects of the seed coat, desiccation and storage tolerance of G. gardneriana seeds were investigated. Additionally, PAs contents and enzyme activities were quantified over these effects, looking to correlate its contents to the physiological seed performance of this species. The results of this study improve

Journal of Seed Science, v.42, e202042001, 2020 Physiological performance of Garcinia gardneriana (Planch. & Triana) Zappi seeds 3 the ecological understanding of seed physiology performance, and lead to practical applications in crops and seed conservation.

MATERIAL AND METHODS

Fruit harvesting and seed processing Mature fruits of G. gardneriana were harvested from a population located in Içara, Santa Catarina. The seed processing consisted of the removal of the mucilaginous mesocarp by friction, followed by washing in water and drying in a paper towel. Seeds with visible mechanical damage or very small were discarded.

Imbibition curve and effect of the seed coat on water uptake Intact (i.e., with seed coat) and decoated seeds were placed in germination boxes containing vermiculite substrate saturated with sterilized distilled water. The boxes with seeds were arranged in growth chambers at 25 ± 2 °C and photoperiod of twelve hours (142 μmol. m-2.s-1). Seeds were weighed before imbibition, at two-hour-intervals for the first twelve hours, and at twenty-four-hour-intervals until 50% of seed germination. A seed was considered germinated when it produced radicle and epicotyl (normal seedling). The percentage of mass increment over time, as a function of initial seed mass, was calculated as , where Mi = initial fresh mass of the sample, and Mt = mass sample (𝑀𝑀𝑀𝑀 − 𝑀𝑀𝑀𝑀) at the time of harvesting (Justo% et= al.,[ 2007). To] ∗determine100 whether water was absorbed or retained in the seed coat, ten 𝑀𝑀𝑀𝑀 intact seeds and ten decoated seeds were imbibed in methylene blue solution for fifteen days (based on the imbibition curve), then cross-sectioned and evaluated in a stereo microscope for the absorption of the solution (Liu et al., 2005).

Water content determination Seeds were dried at 105 ± 3 °C for 24 h (Brasil, 2009). Four replicates of five intact seeds were cut into small pieces, -1 weighed initially and after the drying period. The WC was expressed on a dry weight basis (g H2O.g dw) (Black and Pritchard, 2002).

Desiccation tolerance To evaluate desiccation tolerance, fresh seeds (FS) were dried in hermetically sealed containers using silica gel at room temperature (average of 27 °C). The seeds were desiccated for 5, 10, 15 and 20 days, using as reference the WC determined for FS. Silica gel was used in a ratio of 2:1 and was replaced every 24 h (Zhang and Tao, 1989). Every day seeds were weighed, and water loss was calculated according to Hong and Ellis (1996).

Tetrazolium test Four replicates of eight seeds were used for all treatments. A longitudinal cut was made in the seed, and the embryos were immersed in 1% 2,3,5-triphenyltetrazolium chloride solution at 30 °C for two hours (Brasil, 2009). The embryos were classified as viable or unviable, according to Brasil (2009).

Electrolyte leakage Electrolyte leakage was estimated according to Marcos-Filho et al. (1987). Four replicates of eight seeds were used. The seeds were weighed and imbibed in 100 mL beakers containing 50 mL deionized water and kept in a growth chamber at 25 °C for different imbibition times (2, 4, 6, 8, 10, 12 and 24 h). After each imbibition period, the electrolyte leakage was evaluated using a conductivity sensor (SD201, Saiv A/S, Norway). The results were expressed in μS. cm-1.g-1.

Journal of Seed Science, v.42, e202042001, 2020 4 W. G. Viana et al.

Germination and seedling evaluation For the germination test, seeds were previously disinfested with 70% ethanol for one minute, and sodium hypochlorite solution (2%, v/v) for five minutes. Germination counts were made daily. Germination criteria were the same used for the imbibition curve (normal seedling). The viability of the non-germinated seeds was verified through the tetrazolium test. At the end of the germination tests, the mean germination time (MGT) was calculated according to Edwards (1934).

Storage tolerance Tolerance to storage was evaluated by storing intact FS at 25 °C and 5 °C for ninety days. After the storage period, the seeds were submitted to the germination test, MGT and seedling evaluation, as described previously.

Polyamines quantification For PAs determination, three samples (200 mg) of embryos of fresh and desiccated seeds were ground in 1.6 mL of 5% (v/v) perchloric acid. Free and conjugated PAs were extracted, dansylated and quantified, according to Steiner et al. (2007), with modifications. PAs concentration was determined using a fluorescence detector at 340 nm (excitation) and 510 nm (emission). Peak areas and retention times were measured by comparison with standard PAs: putrescine, spermidine and spermine. The 1,7-diaminoheptane (DAH) was used as the internal standard.

Enzyme extraction and assays Three samples (300 mg) of embryos of fresh and desiccated seeds were homogenized on ice with 1 mL of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 15000 g for twenty minutes at 4 °C. Protein content was determined according to Bradford (1976). The resulting supernatant was filtered and used for the enzyme assays. Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured by monitoring the inhibition of photochemical reduction of NBT, according to Giannopolitis and Ries (1977).

Catalase (CAT; EC 1.11.1.6) activity was determined by following the consumption of hydrogen peroxide (H2O2) (Peixoto et al., 1999). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was determined by following the decrease in A290 (Koshiba, 1993). Glutathione reductase (GR; EC 1.6.4.2) activity was determined by following the oxidation of NADPH (Bailly and Kranner, 2011). SOD, CAT, APX and GR activities of each extract were measured three times, and the results correspond to the means ± SD of the values obtained with three different extracts and three measurements per extract (i.e., nine measurements).

Statistical procedures The design was completely randomized in all tests unless stated otherwise. Each treatment was composed of four replications of fourteen seeds each. Data normality was evaluated using the Shapiro-Wilk test and analyzed using an analysis of variance (ANOVA), followed by a Student-Newman-Keuls posthoc test (p < 0.05). Statistical procedures were carried out with R 3.4.4 programming environment (R Core Team).

RESULTS AND DISCUSSION

Decoated seeds absorbed water slowly and started germinating twelve days after sowing (DAS) (Figure 1a). Removal of the seed coat resulted in a mass increment of 11% throughout the imbibition test. On the other hand, intact seeds showed a rapid increase in mass in the first hour, reaching 5% in only six hours of imbibition. However, after this point, water absorption plateaued; between 8 and 360 h, there was a mass increase of only 1%, thus showing that water absorption became nearly stable. Intact seeds failed to germinate by the end of the imbibition test. The imbibition test with methylene blue solution showed that only decoated seeds absorbed the solution (Figure 1b).

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Data are means ± SD. Bar: 1 mm. Star: beginning of germination. Figure 1. Imbibition curve of G. gardneriana seeds and the effects of the seed coat in water uptake. (a) Dynamics of increment on initial fresh mass in function of time (%). Details of water uptake in the first 24 h indicated on the right lower corner. (b) Cross-section of seeds imbibed in methylene blue solution. Intact seed showing no absorption and decoated seed with visible absorption of the solution.

According to Hidayati et al. (2001), the increase in mass does not necessarily indicate that water has surpassed the seed coat; instead, it can be true that water was absorbed just on the seed coat itself. The data presented in this work corroborate with this statement since the seeds could not overcome the physical barrier imposed by the seed coat, resulting in the near stabilization of water absorption. Conversely, decoated seeds absorbed water slowly but throughout until seed germination occurred. According to Liu et al. (2005), this slow absorption rate is due to the seeds having a high initial WC. Nevertheless, 50% of the seeds germinated at the end of the test in a relatively short period. These results indicate that the seed coat of G. gardneriana confers physical dormancy on the seeds, which prevents water uptake and quick germination. In nature, these structures that preclude germination become permeable through the action of moist and dry heat, strong acids, coat piercing, or even by going through an animal gut (Vázquez-Yanes and Orozco-Segovia, 1993). This means that, in nature, G. gardneriana seeds could have different germination times, depending on the speed on which these factors act on the seed coat, which is a good strategy for germination and seedling establishment success on a wild ecosystem. In this sense, because the seed coat of G. gardneriana prevents germination, to propagate this species in practical application or experimental research, the removal of the seed coat is recommended to obtain more immediate and uniform germination. The lifespan of recalcitrant seeds is significantly affected by temperature, humidity and the WC of the seeds (Marques -1 et al., 2018). G. gardneriana FS were shed at a high WC (1.07 g H2O.g dw), showed high viability (Figure 2b), the lowest electrolyte leakage rate (Figure 2c) and fastest MGT and highest normal seedling production (Table 1). The seeds lost -1 water at a rate of 4% per day until the end of the desiccation period (Figure 2a). Reduction in WC to 0.67 g H2O.g dw had an adverse effect on the seeds; germination was reduced by 57% (Figure 2d). This discrepancy suggested that this was -1 the critical WC for these seeds. Desiccating seeds to 0.43 g H2O.g dw was very detrimental. All tests indicate that seeds -1 do not tolerate this WC loss; only 8% of seeds germinated (Figure 2d). Desiccation below 0.25 g H2O.g dw was lethal. Our results, together with the fact that a few Garcinia species were classified as having recalcitrant seeds (Liu et al., 2005; Malik et al., 2005), suggest that G. gardneriana also has this behavior, since its seeds are highly sensitive to desiccation. Seeds of G. gardneriana also showed to be very sensitive to storage and chilling. FS were capable of retaining viability for -1 ninety days at 25 °C to the same level as seeds desiccated to 0.67 g H2O. g dw (Table 2). One possible explanation for this may be that these seeds lost water slowly during storage due to the high temperature. Therefore, these seeds may also have

Journal of Seed Science, v.42, e202042001, 2020 6 W. G. Viana et al. suffered the same damages as the desiccated seeds, which caused the loss of viability. Seeds stored for ninety days at 5 °C did not germinate (Figure 2d), thus indicating that this species is very sensitive to chilling.

Abbreviation: FS = fresh seeds. Means followed by the same letters do not show significant differences according to the SNK test (p < 0.05). Bar: 1 cm. Figure 2. Physiological performance of the seeds due to desiccation. (a) Water content of G. gardneriana seeds as a function of desiccation time (days). (b) Tetrazolium test with G. gardneriana seeds of different WC. On the right, Percentage of positive reaction of G. gardneriana seeds to 1% 2,3,5-triphenyltetrazolium chloride (Tetrazolium). CV = 9.58%. On the left, the effect of desiccation on the Tetrazolium salt reaction with the embryo tissues. Seeds -1 that reacted positively to tetrazolium: FS, 0.67 and 0.43 g H2O.g dw. Seeds that did not react to tetrazolium: -1 -1 -1 0.25 and 0.14 g H2O.g dw. (c) Effects of desiccation on electrolyte leakage (μS. cm .g ) in G. gardneriana seeds. -1 CV = 11.73%. (d) Germination curve of G. gardneriana seeds with different WC (g H2O.g dw). CV = 6.29%. Journal of Seed Science, v.42, e202042001, 2020 Physiological performance of Garcinia gardneriana (Planch. & Triana) Zappi seeds 7

Table 1. Mean germination time (MGT) and germination of G. gardneriana seeds with different water content.

Germination (%) Treatment MGT (days) Normal seedling Abnormal seedling Dead seed Fresh seeds 18 ± 0.3 c 95 ± 3 a 5 ± 3 c 0 d -1 0.67 g H2O.g dw 22 ± 0.2 b 43 ± 5 b 39 ± 4 a 19 ± 3 c -1 0.43 g H2O.g dw 24 ± 1 a 8 ± 3 c 14 b 78 b -1 0.25 g H2O.g dw 0 d 0 d 0 d 100 a -1 0.14 g H2O.g dw 0 d 0 d 0 d 100 a Data are mean ± SD. Means followed by the same letters do not show significant differences according to the SNK test (p < 0.05).

Table 2. Mean germination time (MGT) and germination of G. gardneriana seeds with different storage conditions.

Germination (%) Treatment MGT (days) Normal seedlings Abnormal seedlings Dead seeds Fresh seeds 18 ± 0.3 b 95 ± 3 a 5 ± 3 b 0 c Stored at 25 ºC 21 ± 2 a 41 ± 3 b 39 ± 4 a 20 ± 3 b Stored at 5 ºC 0 c 0 c 0 c 100 a Data are mean ± SD. Means followed by the same letters do not show significant differences according to the SNK test (p < 0.05).

According to Pammenter and Berjak (1999), the loss of germination capacity is associated with a lack of mechanisms related to the acquisition of tolerance to desiccation. Among these mechanisms are protective proteins and metabolites (LEAs, antioxidant enzymes, sugars, polyamines), hormones (ABA accumulation, GA reduction), transcription factors and physiological processes (DNA and protein repair, cytoplasm vitrification, membrane stabilization) (Marques et al., 2018). However, maintaining this machinery comes with a high cost; hence, several species have lost tolerance to desiccation or storage, depending on the environment they are found. Seed longevity, desiccation tolerance and dormancy are very important traits for seeds that are found in a heterogeneous environment because they enhance the ability to survive in adverse conditions (Marques et al., 2018). On the other hand, most species that evolved in relatively stable environments, such as ecosystems with typical rainy seasons, do not need these mechanisms since the environment they are found favors faster germination (Souza et al., 2015). Barbedo et al. (2013) define seed longevity as an interaction among genetic information, reduction factors (WC, temperature, degree of maturation at shedding) and improvement factors (dormancy). These factors seem to be established in a dynamic way, varying in intensity based on what is more advantageous to the adaptation of the species. Therefore, according to their model, the paradoxical combination of physical dormancy and desiccation sensitivity in G. gardneriana can be explained. To shed light on the metabolism of these seeds, antioxidant enzymes and polyamines as biomolecules involved in the seed physiology performance were also studied. G. gardneriana FS showed high activities of SOD, CAT, APX and GR (Figure 3), indicating that the antioxidant system was operating efficiently in the removal of ROS, since seeds were the most viable. Several studies have investigated the relationship between loss of viability and the oxidative stress in recalcitrant seeds (Li and Sun, 1999; Varghese and Naithani, 2002; Cheng and Song, 2008). ROS play an important role in seed physiology, acting as signaling of cellular pathways, but also as toxic products that accumulate under stress conditions, such as desiccation (Jeevan Kumar et al., 2015). For the successful destruction of superoxide radicals and H2O2, scavenging enzymes need to work together. Superoxide radicals produced by biochemical reactions in plant cells are rapidly converted to H2O2 by SOD, and then CAT converts H2O2 to H2O and oxygen (Cheng and

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Song, 2008). APX and GR are closely correlated in the ascorbate-glutathione cycle and are known for scavenging H2O2 directly and indirectly (Bailly, 2004). Comparatively to G. gardneriana FS, desiccation resulted in a decrease in SOD and APX activity (Figures 3a and 3b), with no difference among the desiccated treatments. CAT activity showed an inverse pattern, increasing significantly (Figure 3c). GR activity did not differ among fresh and desiccated seeds (Figure 3d). According to Bailly et al. (2001), loss of SOD and APX activity leads to an accumulation of ROS, which can be compensated by the increase in CAT activity, who also scavenges H2O2. This seems to be the case for G. gardneriana seeds, where CAT activity increased in desiccated seeds, as a result of impairment of SOD and APX activities. However, it was not enough to keep the seeds viable. Even though GR is known for contributing to the regeneration of ascorbate and participates indirectly in scavenging of H2O2 (Tommasi et al., 2001), in G. gardneriana desiccated seeds, this enzyme does not seem to have played a significant role, because APX activity decreased and viability decreased as well. The results indicate that the significant decreases in SOD and APX may be correlated to the desiccation sensitivity of the seeds. These results corroborate with the study of Li and Sun (1999), who found a correlation between the rapid decrease in enzymatic protection by SOD and APX against

Abbreviations: APX = ascorbate peroxidase; CAT = catalase; GR = glutathione reductase; SOD = superoxide dismutase; FS = fresh seeds. CV SOD: 19.5%; CV APX: 7%; CV CAT: 7.5%; CV GR: 13%. Bar: germination percentage. Enzyme activity means followed by the same letters do not show significant difference according to the SNK test (p < 0.05).

Figure 3. Effects of desiccation of G. gardneriana seeds on SOD (a), APX (b), CAT (c) and GR (d) activities.

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oxidative stress and desiccation sensitivity of cocoa (Theobroma cacao) axes and cotyledons. Conversely, Varghese and Naithani (2002) found that SOD increased markedly with desiccation of neem (Azadirachta indica A. Juss) seeds; however, an impairment in CAT leads to ROS production, thus resulting ultimately in the loss of viability. This contrast in enzyme activities serves as evidence that an entirely functional and cooperating antioxidant system is required to retain seed viability. Together with antioxidant enzymes, the role of PAs regarding stress tolerance has been widely investigated. One of the many roles PAs play is in the scavenging of ROS by influencing the activity of antioxidant enzymes (Li et al., 2015b; Juzoń et al., 2017). In general, plants are characterized for possessing a high capacity of increasing endogenous contents of PAs in response to stress (Juzoń et al., 2017). In G. gardneriana seeds, free PAs content was higher in FS for PUT and SPD (Figures 4a and 4b). After desiccation, the content of these two PAs significantly decreased. Conversely, -1 SPM content increased when seeds were desiccated to 0.67 g H2O.g dw, but decreased when seeds were desiccated further (Figure 4c). These results corroborate with the fact that PUT is used for the synthesis of SPD, which is then converted into SPM (Liu et al., 2015).

Abbreviations: PAs = polyamines; PUT = putrescine; SPD = spermidine; SPM = spermine; FS = fresh seeds. CV free PUT: 23%; CV free SPD: 11%; CV free SPM: 26%; CV Total PAs: 18%. Enzyme activity means followed by the same letters indicate a significant difference between treatments at p < 0.05 according to the SNK test. Figure 4. Endogenous contents (nmol.g-1 fw) of free polyamines: PUT (a), SPD (b), SPM (c) and total PAs (free + conjugated). (d) In mature seeds of G. gardneriana under different data are means of three replicates ± SD.

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It seems that for G. gardneriana, PUT and SPD was converted to SPM in an attempt to stop seed deterioration caused by desiccation. SPM is involved in membrane stability and osmotic adjustment, both important processes during the desiccation phase of seeds (Li et al., 2015b). Furthermore, several studies found that the exogenous application of SPM increased drought tolerance by elevating the activity of antioxidant enzymes, such as SOD and CAT, which then resulted in a decrease in ROS activity (Shi et al., 2010; Li et al., 2015a), indicating the effective role of this PA against abiotic stress. These findings corroborate with the SPM contents found for G. gardneriana seeds (Figure 4c). -1 Desiccation to 0.67 g H2O.g dw resulted in an increase in SPM content; however, the action of SPM in membrane stabilization and increasing the activity of antioxidant enzymes was not enough to prevent seed deterioration and, thus, the seed viability was lost. The high content of total PAs (Figure 4D) for G. gardneriana FS, and the markedly decrease after desiccation and germination indicate a positive correlation in seed viability and PAs in this species. These data are considered to be a starting point for understanding and establishing the main biological criteria for better management of this species seeds in its wild ecosystem, considering its economic, medicinal and ecological potential.

CONCLUSIONS

The results indicate that the seed coat in this species precludes imbibition and, consequently, germination. The seeds do not tolerate desiccation and storage; therefore, this species is categorized as having highly recalcitrant seeds.

ACKNOWLEDGMENTS

The authors thank the staff of the Plant Physiology Laboratory and Plant Developmental Physiology and Genetics Laboratory (LFDGV) of the Universidade Federal de Santa Catarina, Brazil.

REFERENCES

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Journal of Seed Science, v.42, e202042001, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Physiological response of soybean seeds to spray volumes of Journal of Seed Science, v.42, industrial chemical treatment and storage in different environments e202042002, 2020 http://dx.doi.org/10.1590/2317- 1545v42221062 Julia Abati1 , Cristian Rafael Brzezinski1 , Elieges Carina Bertuzzi*1 , Fernando Augusto Henning2 , Claudemir Zucareli1

ABSTRACT: The increase in spray volumes in industrial seed treatments may increase the deterioration and reduce the physiological potential of seeds, especially when stored in unfavorable environments. The aim of this study was to evaluate the effect of spray volumes obtained by the combination of different chemical products, via industrial treatment, on the physiological potential of soybean seeds during storage in different environments. A completely randomized experimental design was used in a 5 × 3 factorial arrangement, with four replications. The factors were five spray volumes (0, 600, 1200, 1800 and 2400 mL.100 kg-1 of seeds), obtained from the combination of different products in commercial use, and three storage periods (0, 60 and 120 days), evaluated separately in two environments (storage without climate control and cold storage). The following variables were evaluated: germination, first germination count, seedling emergence in sand, emergence speed index, seedling length (total, shoot, and root) and dry matter (shoot and root). The physiological potential of soybean seeds is reduced by increasing the spray volume used in the industrial treatment and by prolonging the storage period. However, this effect is mitigated by the controlled conditions of cold storage.

Index terms: Glycine max (L.) Merrill, germination, vigor, storage, physiological seed quality.

Resposta fisiológica de sementes de soja a volumes de calda do tratamento químico industrial e armazenamento em diferentes ambientes

RESUMO: O aumento dos volumes de calda no tratamento industrial de sementes pode aumentar a deterioração e reduzir o potencial fisiológico das sementes, principalmente quando armazenadas em ambientes desfavoráveis. Assim, objetivou-se avaliar o efeito de volumes de calda, obtidos pela combinação de diferentes produtos químicos, via tratamento industrial, sobre o potencial fisiológico de sementes de soja ao longo do armazenamento em diferentes ambientes. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 5 x 3, com quatro repetições. Os fatores foram cinco volumes de calda (0, 600, 1200, 1800 e 2400 mL.100 kg-1 de sementes), obtidos a partir da combinação de *Corresponding author diferentes produtos comercialmente utilizados, e três períodos de armazenamento (0, E-mail: [email protected] 60 e 120 dias), avaliados separadamente em dois ambientes (armazém não controlado e câmara fria). As variáveis avaliadas foram: germinação, primeira contagem de germinação, emergência de plântulas em areia, índice de velocidade de emergência, comprimento de Received: 3/10/2019. plântula (total, parte aérea e raiz) e massa seca (parte aérea e raiz). O potencial fisiológico Accepted: 10/21/2019. de sementes de soja é reduzido com o aumento do volume de calda utilizado no tratamento industrial e com o prolongamento do período de armazenamento, no entanto, esse efeito é mitigado pelo armazenamento em condições controladas de câmara fria. 1Departamento de Agronomia, Termos para indexação: Glycine max (L.) Merrill, germinação, vigor, armazenagem, qualidade Universidade Estadual de Londrina fisiológica de sementes. (UEL), Caixa Postal 6001, 86057-970 – Londrina, PR, Brasil.

2Embrapa Soja, Caixa Postal 231, 86001-970 – Londrina, PR, Brasil.

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INTRODUCTION

Soybean holds the position of the most important crop in Brazilian agribusiness with prominence in planted area, production, and yield (CONAB, 2018). This creates the need for investments in research, especially to increase availability of cultivars with high yield potential and of quality seeds (Krzyzanowski et al., 2018). The growth of soybean production in Brazil was preceded by scientific and technological advances. Techniques of production, quality control, storage, and seed treatment have been refined in recent years and have contributed to this rise in the production sector (Krzyzanowski et al., 2018). Due to new technologies linked to seed production, the value of this raw material has increased, with a significant effect on production costs. Consequently, the seed market has been ever more demanding in regard to high quality seeds that perform their function: becoming adult plants with high vigor and yield (Peske, 2017). In this context, seed treatment has evolved over the past decade (Brzezinski et al., 2017); it has become industrialized and directed to high performance, refining the quality of the seed finish through use of polymers and dry powder that ensures protection of the active ingredient and makes for better flow at the time of sowing (Tonin et al., 2014). This process precedes sowing and involves the technique of application of chemical and/or biological products aiming to ensure seed health in the beginning stages of development to promote higher crop yield (Scheeren et al., 2010; Mattioni et al., 2012; Cantarelli et al., 2015). Seed treatment is a widespread practice in Brazilian agriculture – nearly 98% of soybean and maize seeds are treated with fungicides and/or insecticides to garantee seedling emergence and better crop stand (Nunes, 2016). There is a range of products available that can be added to the seed through industrial treatments, such as fungicides, insecticides, nematicides, micronutrients, stimulants, inoculants, biological products, and others that provide the seed and plant protection in the initial phase of development (Avelar et al., 2011; Parisi and Medina, 2013; Bertuzzi et al., 2017). However, in some situations, some active ingredients or interactions among products can lead to reduced germination and vigor and, consequently, reduced seedling establishment as a result of phytotoxicity in the seeds (Taylor and Salanenka, 2012; Alves et al., 2017). In addition, little is known regarding the spray volume applied and its negative effect on the physiological potential over the storage period, since this process normally uses liquid products. An increase in seed moisture content accentuates the deterioration rate, increasing hydrolysis reactions, which may worsen through an increase in temperature, a factor related to the storage environment, which acts directly on intensification of the chemical reactions (Marcos-Filho, 2015; Meneghello, 2014). Therefore, storage in a controlled environment may provide better storability of chemically treated seeds, facilitating conservation and trade logistics. Conservation of the physiological potential of seeds is vital for the practice and success of seed treatment. The variety of product options with different purposes for use in industrial seed treatment makes this a complex step, and it increases the spray volume necessary to contain all the molecules and adjuvants offered by the seed industry (Nunes, 2016). Currently, with the increasing demand for industrial seed treatment, many seed companies have worked in an intense way in the short period of time that precedes sowing. This creates the need for results that provide indications of the best spray volume, storage period and environment to better plan this step of production, as well as efforts to reduce costs (Pereira et al., 2018). Therefore, the aim of this study was to evaluate the effect of spray volumes obtained through the combination of different chemical products, via industrial seed treatment, on the physiological potential of soybean seeds over the storage period in different environments.

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MATERIAL AND METHODS

The experiment was conducted in the Seed and Grain Technology Center of the Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Embrapa Soja, Londrina, PR, Brazil, in the Seed Physiology and Technology Laboratories on the soybean cultivar BRS 359 RR. This genotype is stable, and does not have a genetic trait of rupture in the seed coat, such as rips or microfissures. The initial quality of the seed lot was 89% germination, 96% emergence in sand, and emergence speed index of 25.6. A completely randomized experimental design was used in a 5 × 3 factorial arrangement, with four replications. The factors consisted of five spray volumes used in industrial seed treatment (0, 600, 1200, 1800 and 2400 mL.100 kg-1 of seeds), obtained from the combination of different products in commercial use, and three storage periods (0, 60 and 120 days), evaluated separately in two environments (storage without climate control and cold storage). During the experiment, temperature and relative humidity (RH) were monitored in the storage environment without climate control with the Data Logger HT-500 device (Figure 1). In cold storage, the temperature and RH were programmed and maintained at 10 ºC and 50%, respectively. To obtain different spray volumes, first the maximum volume to be used was determined and, based on that value, the other volumes were obtained through reduction in the doses of biostimulant, polymer, inoculant and micronutrient (Table 1). The products used were F: fungicides (carbendazim + thiram); I: insecticides (imidacloprid + thiodicarb); N: nematicide (abamectin); M: micronutrients (cobalt, molybdenum and zinc); P: polymer (Peridiam™); B: biostimulant (kinetin + gibberellic acid, such as GA3 + 4-indol-3-ylbutyric acid) and IN: inoculant (Bradyrhizobium japonicum). Seeds were treated with the assistance of a Batch Modular Coater (BMC) machine, similar to those used in industrial seed treatment (IST), though projected for small seed quantities. After the treatment and storage, the physiological potential of the seeds was determined by the following evaluations: Germination(G): performed with a hundred seeds per replication, two subsamples of fifty seeds, for a total of four hundred seeds per treatment. The seeds were distributed in rolls of germitest paper towel, moistened with distilled water in the amount of 2.5 times the dry weight of the substrate. After setting them up, the rolls were placed in a seed

Figure 1. Daily mean of the temperature (ºC) and relative humidity (%) throughout the storage period of soybean seeds in an environment without climate control. Journal of Seed Science, v.42, e202042002, 2020 4 J. Abati et al.

Table 1. Products used in treatment of soybean seeds and respective doses to obtain different spray volumes. Fungicide (F), insecticide (I), nematicide (N), biostimulant (B), micronutrient (M), polymer (P), and inoculant (IN).

F I N M P B IN Total Treatment Dose mL.100 kg-1 1 – – – – – – – 0 2 200 300 100 – – – – 600 3 200 300 100 400 200 – – 1200 4 200 300 100 400 200 600 – 1800 5 200 300 100 400 200 800 400 2400 germinator under the temperature of 25 ºC for a period of eight days, at which time the percentage of normal seedlings was calculated (Brasil, 2009). First germination count(FGC): performed together with the germination test, with evaluation made five days after setting up the test, calculating the percentage of normal seedlings (Brasil, 2009). Seedling emergence in sand (SE): performed with four hundred seeds per treatment, divided into a-hundred-seed subsamples for each replication. The seeds were sown in plastic trays containing sand into which seeds were placed at a depth of three centimeters. The test was conducted under greenhouse conditions and moisture was maintained through irrigation, according to seedling needs. Final evaluation of the number of normal seedlings emerged was made on day twelve, and the results were expressed in percentage. Seedling emergence speed index (ESI): performed together with the test of seedling emergence in sand. Evaluations were made daily as of the beginning of emergence, registering the number of seedlings emerged up to day twelve following sowing. Calculation of the emergence speed index was through the equation suggested by Popinigis (1977): ESI = N1/D1 + N2/D2 + Nn/Dn, where N1 = number of emerged seedlings on the first day; Nn = accumulated number of emerged seedlings; D1 = first day of counting and Dn = number of days counted after sowing. Total seedling length (SL), shoot length (ShL) and root length (RL): five subsamples of twenty seeds each were used, for a total of a hundred seeds per treatment. The seeds were distributed in rolls of paper towel moistened with distilled water in the amount of 2.5 times the weight of the dry paper and then kept in a seed germinator at 25 °C for five days (Nakagawa, 1999). After that, the total length, shoot length and root length of the normal seedlings were determined with the assistance of a millimeter ruler, and results were expressed in cm per seedling. Shoot dry matter(SDM) and root dry matter(RDM): performed with the normal seedlings obtained in the seedling length test, removing the remaining part of the seed and separating the shoot and the root. After that, these parts were placed in paper bags and then in a forced air circulation laboratory oven, where they remained for 24 hours at a temperature of 80 ºC (Nakagawa, 1999). At the end of this period, dry matter was evaluated on a scale with precision of 0.0001 g, and results were expressed in grams. The data obtained were analyzed in regard to normality and homoscedasticity using the Shapiro-Wilk and Hartley tests, respectively, which indicated no need for transformation. Analysis of variance was performed and the mean values for storage periods and spray volume were compared by the Tukey test at 5% probability separately for each storage environment. Analysis were done by the computational program Sistema para Análise de Variância – SISVAR (System for Analysis of Variance) (Ferreira, 2011).

RESULTS AND DISCUSSION

The results of analysis of variance indicated a significant effect for the interaction between the storage period and spray volume. There was a different response between the environments evaluated: in the environment of cold

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storage, there was significance in analysis of G, ESI, SE, ShL, and SDM. In the storage environment without climate control, there was significance in all of the tests, except for SDM and RDM (Table 2). In relation to effects in an isolated manner, spray volume had a similar response between the storage environments. A significant effect was found for first germination count (FGC), germination (G), emergence speed index (ESI), seedling emergence in sand (SE), and shoot length (ShL) for both environments. Analysis of root length (RL), shoot dry matter (SDM), and root dry matter (RDM) did not exhibit change between the two storage situations. Seedling length (SL) in the cold storage was significantly affected by the spray volume. For the isolated effect of the storage period, the effect was significant for all the parameters evaluated, both in the storage environment without climate control and in cold storage (Table 2). In the cold storage environment, there was no reduction in the values of germination up to sixty days, except for the highest spray volume (Table 3). At the end of 120 days, there were no significant differences only for the control and for the volume of 1800 mL.100 kg-1 of seeds. Comparing spray volumes within each storage period, for all the periods, the control showed better germination results. The percentage of germination decreased according to the increase in the spray volume and over the storage period. This same response is observed for the seedling performance tests (ESI, SE, ShL and SDM), in which the results were also significant for the interaction between spray volume and the storage period (Tables 3, 4 and 5). Brzezinski et al. (2015) tested early application of the industrial chemical treatment of soybean seeds with the main commercial brands used separately and in combinations of fungicides, insecticides, and nematicides, and they concluded that early treatment of soybean seeds, 240 days before sowing, affects crop establishment, a-thousand-seed weight, and grain yield in relation to treatment in pre-sowing. In addition, the authors found that the chemical treatments tested containing fungicides and insecticides in association improve crop establishment; however, they do not change the yield performance of soybean when used in pre-sowing. This meets the basic aim of the chemical seed treatment, which is to protect seed and seedlings (Scheeren et al., 2010; Krzyzanowski et al., 2018). For the storage environment without climate control, germination varied over the storage period and among the spray volumes. In the control, germination decreased significantly at 120 days. However, for the treated seeds, as the spray volume increased, reduction intensified, with significant reduction in germination already at sixty days.

Table 2. Values of mean square of analysis of variance of the traits of physiological potential of seeds of the soybean cultivar BRS 359 RR in different environments, storage periods, and spray volumes used in industrial seed treatment. Cold storage Traits Source of variation FGC G ESI SE SL ShL RL SDM RDM Period (P) 339.45* 729.65* 114.49* 894.86* 367.86* 609.04* 524.86* 1.48* 0.30* Volume (V) 3160.35* 2147.60* 126.57* 398.90* 7.72 2.71* 2.60 0.04 0.06 P*V 54.47 84.73* 12.91* 55.26* 6.07 3.31* 5.16 0.07* 0.03 CV (%) 11.71 9.45 6.84 3.64 6.20 6.34 10.90 9.59 26.28 Storage without climate control Period (P) 11069.21* 12865.26* 1497.36* 17654.83* 734.60* 580.56* 601.12* 2.28* 0.47* Volume (V) 3932.37* 4562.14* 199.51* 1704.09* 22.04* 11.24* 3.28 0.02 0.01 P*V 194.55* 150.82* 24.39* 400.59* 13.77* 4.97* 6.57* 0.01 0.01 CV (%) 11.61 11.43 8.79 5.87 5.69 7.06 9.30 8.30 16.04 *: significant at 5% probability by the F test; CV: coefficient of variation; FGC: first germination count; G: germination; ESI: emergence speed index; SE: seedling emergence in sand; SL: seedling length; ShL: shoot length; RL: root length; SDM: shoot dry matter; RDM: root dry matter.

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Table 3. Germination of soybean seeds of the cultivar BRS 359 RR in different environments, storage periods, and spray volumes used in industrial seed treatment.

Cold storage Storage period (days) Spray volume (mL) 0 60 120 0 89 Aa 87 Aa 80 Aa 600 72 Ab 80 Aa 59 Bb 1200 72 Ab 66 ABb 58 Bb 1800 62 Abc 54 Ac 54 Abc 2400 60 Ac 47 Bc 45 Bc CV (%) 9.45 Storage without climate control 0 89 Aa 91 Aa 53 Ba 600 72 Ab 70 Ab 21 Bb 1200 57 Acd 60 Ab 11 Bbc 1800 62 Abc 45 Bc 8 Cc 2400 48 Ad 30 Bd 1 Cc CV (%) 11.43 Mean values followed by the same uppercase letter in the row and lowercase letter in the column do not differ from each other by the Tukey test at 5% probability.

Table 4. Emergence speed index of soybean seedlings of the cultivar BRS 359 RR in different storage periods and spray volumes used in industrial seed treatment.

Cold storage Storage period (days) Spray volume (mL) 0 60 120 0 25.50 Aa 26.82 Aa 19.13 Bab 600 25.00 Aa 20.08 Bb 19.64 Ba 1200 19.75 Ab 16.95 Bc 15.29 Bc 1800 19.75 Ab 16.10 Bc 16.72 Bbc 2400 19.25 Ab 14.62 Bc 14.74 Bc CV (%) 6.84 Storage without climate control 0 25.61 Aa 26.71 Aa 12.23 Ba 600 22.78 Ab 21.74 Ab 4.75 Bb 1200 20.03 Acd 17.32 Bc 3.81 Cb 1800 22.01 Abc 17.56 Bc 3.18 Cb 2400 18.64 Ad 9.51 Bd 3.37 Cb CV (%) 8.79 Mean values followed by the same uppercase letter in the row and lowercase letter in the column do not differ from each other by the Tukey test at 5% probability.

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Table 5. Soybean seedling emergence in sand of the cultivar BRS 359 RR in different storage periods and spray volumes used in industrial seed treatment.

Cold storage Storage period (days) Spray volume (mL) 0 60 120 0 96 Aa 92 Aa 94 Aab 600 96 Aa 83 Bb 95 Aa 1200 94 Aab 78 Cb 84 Bcd 1800 88 Ab 72 Bc 88 Abc 2400 89 Ab 72 Cc 80 Bd CV (%) 3.64 Storage without climate control 0 96 Aa 91 Aa 68 Ba 600 95 Aa 85 Bab 33 Cb 1200 92 Aab 79 Bb 28 Cbc 1800 90 Aab 81 Bb 23 Cc 2400 84 Ab 52 Bc 20 Cc CV (%) 5.87 Mean values followed by the same uppercase letter in the row and lowercase letter in the column do not differ from each other by the Tukey test at 5% probability.

A study with dynamic cooling and storage in a refrigerated and non-refrigerated environment showed that soybean seeds decrease or increase the speed and the intensity of deterioration according to the environment to which they were exposed during storage. The best results for preservation of physiological quality were in an environment at 13 °C for up to 225 days, which was the longest period evaluated (Ferreira et al., 2017). In the present study, in the storage environment without climate control, there were temperature oscillations, with minimum readings of 21 °C and maximum readings of approximately 30 °C, with a mean temperature over the storage period of 25 °C. Relative humidity varied from 58% to 91%, with a mean value of 75% (Figure 1). These conditions explain the greater reduction in seed germination in the storage environment without climate control, especially for the higher spray volumes, in relation to cold storage. In general, the same response was observed for the tests of FGC, ESI (Table 4), SE (Table 5), SL and ShL (Table 6). For root length for the zero period, a result inferior to the sixty-day period was obtained, but it was equal to or greater than the 120-day period (Table 7). In addition to the storage conditions, the speed of deterioration is affected by genetic factors, the history of seed formation and maturation and post-harvest handling, including storage (Baudet and Villela, 2012; Marcos-Filho, 2015). Other authors concluded that the chemical treatment should be applied near the time of sowing because storage had a negative effect on the physiological quality of maize seeds (Bittencourt et al., 2000; Fessel et al., 2003). The results obtained may be associated with possible oscillation in the moisture content in accordance with the differences in spray volume of each treatment, which, in general, acted negatively on seed physiological potential. Ludwig et al. (2011) found that after the soybean seed treatments, the moisture content increased a mean of 1% and that throughout storage without climate control, this parameter oscillated 2.6% downward in sixty days, rose 0.9% in 120 days, and once more declined 0.7% in 180 days of storage. This shows that there is a tendency for seeds, even with seed treatment, to reach hygroscopic equilibrium according to variations in the environment in which they are stored (Baudet and Villela, 2012). Furthermore, for soybean seeds treated with a single spray volume of 600 mL.100 kg-¹ of seeds, however, with

Journal of Seed Science, v.42, e202042002, 2020 8 J. Abati et al. a different active ingredient and mode of action, in storage without control of temperature and relative humidity conditions, a negative effect on seed quality was observed (Ludwig et al., 2011). The same authors found variation among cultivars and among treatment combinations since some interactions led to better seed physiological quality. Segalin et al. (2013) concluded that it is possible to use the treatment in spray volumes of up to 1400 mL.100 kg-1 of seeds, without causing physical and physiological damage to the soybean seeds, regardless of the cultivar or the size of the seed. However, they did not evaluate storage time. For maize seeds, Fessel et al. (2003) found that the treatment with some insecticides reduced germination, and this effect intensified as the storage period went on. Other authors concluded that the reduction in physiological quality varied according to the hybrid tested, the active ingredient of the product used, and the period that the seeds remained in storage (Bittencourt et al., 2000; Bertuzzi et al., 2017). Furthermore, for the storage environment without climate control, there was interaction between the spray volume and the storage period in the tests of FGC, SL and RL (Table 7), in which the same response described for the other tests predominated. Smaniotto et al. (2014) observed reduction in the physiological potential of seeds stored in an environment without climate control in accordance with the initial moisture content. This helps explain the reduction in physiological potential over the storage period and the drastic reduction in this attribute with the increase in spray volume. Dan et al. (2010) found that soybean seeds treated with insecticides and stored in an environment without climate control had loss of physiological quality as the storage period increased, in agreement with the results obtained in this study. Tonin et al. (2014) also observed that for maize, the effect of application of insecticide varied in accordance with the genetic characteristics of the hybrid and with the storage conditions, and they highlighted that control of temperature and humidity are crucial to maintain the physiological quality of treated seeds. Zorato and Henning (2001) obtained a positive effect from fungicide treatment on the quality of soybean seeds during and after the period of ninety days of storage. Dias et al. (2018) evaluated spray volume in soybean seed lots with

Table 6. Shoot length of soybean seedlings of the cultivar BRS 359 RR in different storage periods and spray volumes used in industrial seed treatment.

Cold storage Storage period (days) Spray volume (mL) 0 60 120 0 20.50 Aab 11.11 Ba 10.56 Ba 600 19.50 Abc 9.59 Bab 10.92 Ba 1200 18.25 Ac 9.96 Bab 10.84 Ba 1800 21.50 Aa 9.10 Bb 10.32 Ba 2400 19.00 Abc 9.66 Bab 9.99 Ba CV (%) 6.34 Storage without climate control 0 20.42 Aa 10.91 Ba 10.42 Ba 600 20.60 Aa 10.53 Bab 8.46 Cbc 1200 17.06 Ab 10.25 Bab 8.05 Cc 1800 16.69 Ab 10.01 Bab 8.23 Cc 2400 19.68 Aa 9.05 Bb 10.16 Bab CV (%) 7.06 Mean values followed by the same uppercase letter in the row and lowercase letter in the column do not differ from each other by the Tukey test at 5% probability.

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different moisture contents and concluded that seeds with low moisture are affected by the treatment, regardless of the spray volume, and that seeds with intermediate moisture are affected by higher spray volumes. According to Taylor and Salanenka (2012), coating soybean seeds with polymers, alone or in association with fungicide and insecticide, does not delay the speed of imbibition of the seeds. For the cultivar BRS 359 RR, regardless of the spray volume of the industrial treatment of soybean seeds, there is loss of quality in storage, especially after sixty days. However, with the increase in spray volume, there is a tendency of reducing physiological potential even more, and this may be associated with seed moistening, an increase in metabolic activity and acceleration of deterioration, since the lot used is considered to be of intermediate quality. In general, in the storage environment without climate control, the higher temperature and variations in relative humidity intensify the process of reduction in quality, especially in seeds treated with a higher spray volume. These results suggest that the investment in storage with climate control and adjustments in spray volume can enhance the storage period, attenuating the damage to physiological quality of the seed lots.

Table 7. First germination count of seeds, seedling length, and seedling root length of the soybean cultivar BRS 359 RR in different periods of storage and spray volumes used in industrial seed treatment in a storage environment without climate control.

First germination count of seeds Storage period (days) Spray volume (mL) 0 60 120 0 79 Aa 86 Aa 37 Ba 600 59 Ab 57 Ab 12 Bb 1200 45 Acd 49 Abc 5 Bbc 1800 50 Abc 41 Bc 4 Cbc 2400 38 Ad 18 Bd 0 Cd CV (%) 11.61 Seedling length 0 33.16 Aab 33.51 Aa 25.76 Ba 600 34.25 Aa 32.89 Aa 19.42 Bc 1200 29.84 Bc 33.10 Aa 20.88 Cbc 1800 30.68 Abc 31.98 Aa 19.14 Bc 2400 32.29 Aabc 31.53 Aa 23.98 Bab CV (%) 5.69 Seedling root length 0 12.74 Ca 22.60 Aa 15.34 Ba 600 13.64 Ba 22.36 Aa 10.96 Cb 1200 12.78 Ba 22.85 Aa 12.83 Bab 1800 13.99 Ba 21.97 Aa 10.91 Cb 2400 12.61 Ba 22.48 Aa 13.82 Bab CV (%) 9.30 Mean values followed by the same uppercase letter in the row and lowercase letter in the column do not differ from each other by the Tukey test at 5% probability.

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CONCLUSIONS

The physiological potential of soybean seeds generally decreases with an increase in the spray volume used in the industrial treatment and as the storage period goes on. The negative effects of the increase in spray volume used in the industrial treatment are mitigated by storage in the climate-controlled environment of cold storage compared to storage without climate control, emphasizing that these results may vary in other genotypes not tested in this study and also depend on the initial physiological quality of the seed lot.

ACKNOWLEDGMENTS

Our thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for granting a scholarship to the first author. Thanks also to the Universidade Estadual de Londrina and to the Empresa Brasileira de Pesquisa Agropecuária (Embrapa Soja) for its structure and financial support in developing this study.

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Journal of Seed Science, v.42, e202042002, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Germination and seed ecology ofBuchenavia tomentosa Journal of Seed Science, v.42, e202042007, 2020 Eichler (Combretaceae) http://dx.doi.org/10.1590/2317- 1545v42223782 Amanda Ribeiro Correa1* , Ana Mayra Pereira da Silva1 , Vitor Sthevan Mendes da Silva1 , Elisangela Clarete Camili1 , Antonio Renan Berchol da Silva2 , Maria de Fátima Barbosa Coelho1

ABSTRACT: Buchenavia tomentosa produces fruits with ecological function for Cerrado’s fauna. The aims of this paper were to quantify seed germination and behavior on thermal conditions and explain about water absorption of dispersal structure in B. tomentosa seeds. Ripe fruits were pulped, the endocarp removed, and seeds used in the germination tests at temperatures of 10 to 45 °C. Seeds were placed in rolls of filter paper and then placed in germination chambers, at twelve hours of photoperiod. Germination models at sub and supra optimal temperatures were made from the germination rate (Tg), from the time to germination of 50% of the seeds

(t50). Germination speed index (GSI), measurements of shoot and root lengths and dry mass at each temperature were obtained. The water imbibition curve of seed with or without endocarp adhered and scarified or not was made and other samples were tested for emergence in sand.

Cardinal temperatures were: base temperature (Tb) of 9.23 °C; maximum temperature (Tmax) of 44.6 °C; optimum temperature (To) of 29.24 °C and thermal time of 89.71 °C.days. Seedlings showed higher GSI, root and aerial part length and higher root dry mass at the optimal temperature. The endocarp retards, but does not prevent water absorption and emergence.

Index terms: mirindiba-do-Cerrado, boca-boa, thermal time model, germination model, endocarp.

Germinação e ecologia de sementes de Buchenavia tomentosa Eichler (Combretaceae)

RESUMO: Buchenavia tomentosa produz frutos com função ecológica para a fauna do Cerrado. Objetivou-se, neste trabalho, quantificar a germinação e o comportamento das sementes em condições térmicas e esclarecer a absorção de água pela estrutura de dispersão de B. tomentosa. Frutos maduros foram despolpados, o endocarpo removido, e as sementes utilizadas nos testes *Corresponding author de germinação, às temperaturas de 10 a 45 °C. As sementes foram colocadas em rolos de papel e E-mail: [email protected] em seguida em câmaras de germinação sob doze horas de fotoperíodo. Modelos de germinação em temperaturas sub e supra ótimas foram obtidos por meio da taxa de germinação (T ), a partir Received: 9/5/2019. g Accepted: 10/24/2019. do tempo para germinação de 50% das sementes (t50). Índice de velocidade de germinação (GSI), medidas dos comprimentos e massas secas da parte aérea e raiz das plântulas em cada temperatura foram obtidos. A curva de embebição de água das sementes com ou sem endocarpo aderido foi avaliada, e outras amostras testadas quanto à emergência em areia. As temperaturas 1Departamento de Fitotecnia e cardinais foram: temperatura base (Tb) de 9,23 °C; máxima (Tmax) de 44,6 °C; ótima (To) de 29,24 Fitossanidade, Universidade Federal °C e soma térmica de 89,71 °C.days. As plântulas apresentaram maior comprimento das raízes e de Mato Grosso (UFMT), 78060-900 parte aérea e maior massa seca das raízes na temperatura ideal. O endocarpo retarda, mas não – Cuiabá, MT, Brasil. impede a absorção de água e emergência das sementes. 2Departamento de Solos e Engenharia Rural, Universidade Termos para indexação: mirindiba-do-Cerrado, boca-boa, modelo térmico, modelos de Federal de Mato Grosso (UFMT), germinação, endocarpo. 78060-900 – Cuiabá, MT, Brasil.

Journal of Seed Science, v.42, e202042007, 2020 2 A. R. Correa et al.

INTRODUCTION

Germination occurs at a minimum cardinal temperature range (Tb), below which the germination process does not occur; a maximum temperature (Tmax), above which the germination is not complete; and, an optimum temperature

(To), in which the germination speed is faster. Cardinal temperatures serve as basis of predictive models, due to the frequently linear relationship found between germination rate (Tg) and temperature, in sub and supra-optimal models (Bradford, 2002). The relationship of linearity makes possible to estimate thermal models for the time required, in °C days (°C.days) until the conclusion of germination (Bradford, 2002) as in Silybum marianum seeds (Parmoon et al., 2015), crop species (Tribouillois et al., 2016) and spontaneous species (Hardegree, 2006). Some thermal studies based on germinations models were carried with seeds of tropical forest (Mattana et al., 2018; Lamarca et al., 2011; Cardoso and Pereira, 2009). These studies can be useful to understand ecological aspects related to seed germination, such as the basis and optimal temperature for seed germination and vigor. These factors can support models for the seedlings’ establishment in some areas, as well as resilience of plant communities on habitat. Another factor related to germination are adaptive seed dispersal structures, important for the survival of some species in the habitat, such the protection of embryo against the fire passage, as in macaw palm (Bicalho et al., 2016). The endocarp still helps to maintain the moisture content for complete seed germination and establishment (Mattana et al., 2018). However, dispersal structures interfere on germinative aspects, such as water absorption, dormancy induction and time to germination in optimum conditions, as in Byrsonima basiloba (Silveira et al., 2012) and Empetrum hermaphroditum (Baskin et al., 2002). These diaspores may present palisade layers responsible for the physical barrier of water entry and induce physical seed dormancy (Baskin and Baskin, 2014), or increase the time to germination. Buchenavia tomentosa Eichler (Combretaceae) is an important species which occurs in Cerradão, semideciduous forest, ciliary forest, gallery forest, dry and Cerrado forest (Mendonça et al., 2008). It’s known as mirindiba-do-Cerrado, mirindiba and boca-boa, with fruits elliptic or with spherical drupes ranging from 2 to 5 cm in diameter, with yellow pericarp and stony endocarp (Campos Filho and Sartorelli, 2015). The species presents stone endocarp adhered in their seeds. B. tomentosa presents intense fruit production, important for the maintenance of Cerrado’s flora and fauna, and the fruits are efficiently propagated by several animals, such as ants, deer and mainly by tapir, which consume the pulp. On the tapir’s case, the seeds pass through the digestive tract, favors the removal of pulp adhered to the fruits and does not affect the germination potential (Farias et al., 2015). The dispersers can still contribute to the endocarp’s remover and faster species populations’ formation at Cerrado. For B. tomentosa, thermal time models to seed germination and the study based on ecology of dispersal structure contribute to the understand the species’ evolution at Cerrado areas and is able to provides data to help predict seed germination in future scenarios of temperature increase. In addition, B. tomentosa seed ecology studies may predict the competition of seed germination and maintenance against other natives and exotic plants and animal predation. The aims of this study were to quantify germination and seed behavior on thermal conditions and water absorption of dispersal structure in B. tomentosa seeds.

MATERIAL AND METHODS

Ripe and yellow fruits of B. tomentosa were harvested from plants at Cerrado, their native habitat, after natural dispersion of matrices located in Cuiabá, MT (15°61’S, 56°65’W). The annual average temperature is 28 °C, and maximum and minimum average air temperature are 34 and 22 °C, respectively (Figure 1). Every year, maximum air temperatures of the region reach around 40 °C on many days during spring and summer (INMET, 2018), periods that coincide with wet seasons. Cerrado is characterized by a dry season from April to September (from mid-autumn to early spring) and a wet season from October to March (from mid-spring to early autumn) (Reys et al., 2013). The fruits were pulped, washed, and the seeds with endocarp adhered were placed to dry on filter paper for 24 Journal of Seed Science, v.42, e202042007, 2020 Germination of B. tomentosa Eichler seeds 3 Rainfall (mm) Temperature (°C) Temperature

Month Downloaded data from INMET (http://www.inmet.gov.br/projetos/rede/pesquisa/). Figure 1. Maximum, average and minimum air temperature (max. temp., ave. temp. and min. temp., respectively) and rainfall of region in the last twenty years. hours at 27.65 ± 3 °C and 54.39 ± 3% of relative humidity (RH). Then, the seeds were submitted to drying, to determine water content, at 105 ± 3 °C for 24 hours (Brasil, 2009). The water content was 11%. They were then stored in a refrigerated chamber at 18 ± 2 °C, and RH of 63 ± 4%, for two weeks until the tests. The seeds were submitted to germination test from 10 to 45 °C, at intervals of 5 °C, with photoperiod of twelve hours, in germination chambers. Due to damaging fungal interference and for higher accuracy determination of cardinal temperatures, the endocarp was mechanically removed from seeds using a hydraulic press at 0.8 t, and the seeds used in the thermal parameters. The seeds were placed to germinate on a filter paper substrate, moistened with distilled water in the proportion of 2.5 the mass of the dry paper, with four replications of 25 seeds per temperature. The water replacement was performed daily to maintain always the initial proportion of water in both temperatures. The seeds were considered as germinated after emission of 2 mm of primary root, and germinations were counted daily for one month. The results were expressed as percentage of germination (Brasil, 2009). For seedling behavior evaluation, germination speed index (GSI) was determined based on Maguire (1962). Also, ten seedlings were randomly chosen of each replication of the germination test, in each temperature. Root and shoot length were evaluated at fifteen days of the beginning of the germination test. The same ten seedlings were weighed on an analytical balance (accuracy of ± 0.01 g) and then placed in aluminum capsules for drying at 80 ± 1 °C for 24 hours, to determine the dried root masses and aerial part (Nakagawa, 1999). In order to simulate differences in hydration by seeds, curves of water absorption were made for seeds with intact endocarps, seeds with scarified endocarps (with the aid of an emery) and seeds without endocarp (removed mechanically, as in the thermal models experiment). The seeds’ water content in all treatment before water absorption was determined by the drying method at 105 ± 3 °C, for 24 hours, with three replicates of fifteen seeds; and the results expressed as in percentage (%) of wet mass basis (Brasil, 2009). For the water absorption curve, five replicates of ten seeds in each treatment were placed to soak on germination paper in an incubator at 30 °C, temperature close to the optimum determined by thermal models. Periodic, weighing was done every hour until the primary root emission with 2 mm, in 50% of the seeds for replication. The water uptake by the seeds monitored at each weighing was transformed into water content by the Hampton and Tekrony (1995) formula:

𝑊𝑊1(100 − 𝐴𝐴) 𝐵𝐵 = 100 − [ 2 ] Where: B: water content at the time of evaluation (%); W1: initial𝑊𝑊 mass (g); A: initial water content on wet basis (%);

W2: mass at the time of evaluation (g). Differences in seed emergence were tested through the seeds’ burial with intact endocarp adhered, scarified endocarp and without endocarp. Five replications of ten seeds per treatment were buried in sand boxes moistened to Journal of Seed Science, v.42, e202042007, 2020 4 A. R. Correa et al.

60% of the sand’s water retention capacity, at 3 cm depth, and the emergence counted daily. The seeds were placed on the stand, in laboratory condition, at 25 ± 3 °C and 50 ± 6% of temperature and relative air humidity, respectively, and maintained for twelve hours of photoperiod.

Statistical analyzes

In the germination experiment, cardinal temperatures were evaluated by determining the germination rate (R50), defined as the inverse of the time required to reach 50% germination (t50) at each temperature tested, considering the phase of germinations’ linear response by the time. To estimate the R50 parameters, t50 was determined in each replicate, by equations of germinations’ regression according to the time, and a final mean was established with the standard deviations. R50 data were regressed with temperatures, using linear models to estimate the base (Tb) and maximum (Tmax) temperature, in which R50 is equal to zero; and the optimum temperature (To), when R50 is faster, using sub and supra optimal models.

Optimum temperature (To) was calculated considering the maximum germination rate to reach t50, using the intercept of the sub and supra optimal models, according to Mattana et al. (2018). A thermal time model (°C.days) was adjusted using the following equation at sub (Eq.1) and supra-optimal (Eq.2) temperatures (Bradford, 2002):

(Eq. 1)

𝑇𝑇𝑠𝑠𝑠𝑠𝑠𝑠 = (𝑇𝑇 − 𝑇𝑇𝑏𝑏)𝑡𝑡50 (Eq. 2) 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑚𝑚𝑚𝑚𝑚𝑚 50 Tsub is the thermal time to germination at sub-optimal𝑇𝑇 = (𝑇𝑇 temperatures;− 𝑇𝑇)𝑡𝑡 T is the germination temperature; Tb is the base temperature; Tsupra is the time to germination at supra-optimal temperatures; Tmax is the germination maximum temperature.

The time to emergence of 50% (te50) of the seedlings was calculated similarly to the thermal time experiment, by equations of regression of emergences as a function of time (days). Parameters of thermal time to germination, final percentage of germination and seedling emergence were estimated by Excel software; and t50 data, final germination percentage, GSI, seedling length and mass were submitted to normality test and compared by Anova (p < 0,05) and Tukey test (p < 0,05) on the SISVAR software.

RESULTS AND DISCUSSION

Dynamics of cumulative germination by the time of B. tomentosa seeds without endocarp adhered varied at different temperatures. The time to reach t50 was faster at 30 °C, with 4.5 ± 0.45 days, despite at 25 °C the t50 did not differ statistically (Figure 2). The t50 was slower at 15 and 40 °C; at 20 or 35 °C the seed behavior was also similar (Figure 2). These results show that changes in temperatures below 25 °C and above 30 °C become effective in reducing the germination time of 50% of B. tomentosa seeds, and this reduction is quantitatively similar at both sub and supra optimal temperatures.

By using equations of the time to reach 50% of germination (t50) (Table 1), it was possible to estimate germination rate (R50) and model’s parameters to the cardinal temperatures in suboptimal and supra-optimal temperatures (Figure 3). The basis temperatures estimated by germination models (Table 2) for B. tomentosa is very close to the observed in the experiment, showing the efficacy of the models to predict thermal sum for germination. These values coincide with the ones obtained for other tropical species, especially for Anogeissus leiocarpa (Tb of 9.65 °C,

Tmax of 40.5 °C), a Combretaceae family’s species occurring at African Savanna (Mattana et al., 2018). The species

Melanoxylon brauna (Tb of 12.1 to 12.6 °C, Tmax of 42.4 to 43.0 °C) (Flores et al., 2014), Lippia javanica (Tb of 7 °C)

(Mattana et al., 2017) and Ocotea pulchella (Tb of 11 °C and Tmax of 33 to 42 °C) (Pires et al., 2009) also had cardinal temperatures similar as the one determined in this work for B. tomentosa.

In relation to To, Brancalion et al. (2010) found that temperatures close to 25 °C would be considered optimal for Cerrado’s species, based on a database of 95 native species, which was also found for Anogeissus leiocarpa (To of 24.9 °C) (Mattana et al., 2018). However, To of 30-35.8 °C for other native species, such as Melanoxylon brauna Journal of Seed Science, v.42, e202042007, 2020 Germination of B. tomentosa Eichler seeds 5

(Flores et al., 2014); To of 30 to 35 °C for Tabebuia aurea (Pacheco et al., 2008), and To of 25 to 30 °C for Eugenia sp.

(Lamarca et al., 2011) were found close to To (29.24 °C) for B. tomentosa. (days) 50 t

Temperature (°C) Averages followed by the same letters do not differ by Tukey test (p < 0.05). Error bars represent standard errors.

Figure 2. Germination of Buchenavia tomentosa Eichler seeds without endocarp adhered, based on t50 values at different incubation temperatures.

Table 1. Equations estimated for seed germination of Buchenavia tomentosa Eichler at constant temperatures, according to the time (days).

Temperature (°C) Equation R2 15 y = -1.2262x2 + 38.917x – 262.07 0.93 20 y = -0.9508x2 + 26.084x – 88.879 0.99 25 y = -1.75x2 + 34.8x – 89.383 0.99 30 y = -1.4745x2 + 30.765x – 63.2 0.91 35 y = -0.6926x2 + 21.682x – 75.623 0.99 40 y = -0.0699x2 + 6.2118x – 27.146 0.94 R2: Coefficient of determination of the regression. ) 50 Germination rate (1/t

Temperature (°C) Error bars represent standard errors. Figure 3. Germination rate of B. tomentosa Eichler seeds according to temperature in sub and supra optimal conditions.

Journal of Seed Science, v.42, e202042007, 2020 6 A. R. Correa et al.

Table 2. Parameters of the sub and supra optimal models for seed germination of Buchenavia tomentosa Eichler.

2 Model Equation R Tb (°C) To (°C) Tmax (°C) TS (°C.days)

Sub optimal y1 = 0.0112x – 0.1034 0.99 9.23 – – 89.71

Supra optimal y2 = -0.0146x + 0.6512 0.98 – – 44.6

y1 = y2 – – – 29.24 – 2 R is the coefficient of determination of regression; Tb, To and Tmax is the base, optimum e maximum germination temperature, respectively. TS: the thermal sum to germination on temperatures.

The germination in a wide temperature range presented by B. tomentosa seeds, from 15 to 40 °C, and thermal requirement for maximum speed germination estimated on 29 °C, are high temperature prerequisites compared to species from non-tropical areas (Cochrane et al., 2014; Trudgill et al., 2000). It’s usually explained for adaptation and thermal characteristics acquired at native regions (Trudgill et al., 2000). B. tomentosa is native from Cerrado and Amazon Rainforest. In the first biome, the trees begin to fruit in March, and the dispersal starts in June (Farias et al., 2015); whereas in the second one, the fructification begins in September, with dispersion in May (Camargo et al., 2008). At Cerrado, due to the occurrence of a well-defined dry season and short water during a long part of year (Figure 1), the seeds are able to germinate only at the time of water availability, which coincides with the period of high average temperatures (Brancalion et al., 2010). Optimum germination temperatures can be determined for seed adaptation to the environment. A recent study showed significant relationship of To and annual average temperature of the occurrence region species (Cochrane et al., 2014). The average temperature of the studied region in the last twenty years was 28°C (Figure 1), close to To for B. tomentosa (29 °C). In this way, the environment may have determined the thermal requirement of high temperatures for seed germination. However, other specific studies are necessary to infer about the influence of the other climatic condition on the thermal requirement for B. tomentosa seeds. Thermal time model (TT) to germination of 50% of the seeds was average 89.71 °C.days (Table 2). Compared to TT of 196 °C.days for Anogeissus leiocarpa (Mattana et al., 2018), it is a lower thermal requirement, however, the endocarp removal and the faster water absorption in B. tomentosa are considered, with consequently less time to complete the germination process. However, in the tropical Verbenaceae species of Lippia genus, which do not present barriers to water absorption, values close to those required by B. tomentosa, from 69 to 84 °C.days to reach 50% of germination, were observed between treated and non-treated seeds with gibberellic acid (GA3) (Mattana et al., 2017). The temperature is still influenced by the final germination percentage; the regression model estimated 80% of germination at 19.7 °C and maximum percentage at 28.9 °C (Figure 4A). Next to the Tb and Tmax, at 15 and 40°C, the final germination was smaller and differed significantly to the others temperatures (Figure 4B). There was no germination at 10 and 45 °C. Likewise, highest GSI estimated by model occurred at 28.8 °C (Figure 4C), with significant statistical reduction above or below 30 °C (Figure 4D), similar to the value obtained in the optimum thermal condition for germination (29.24 °C).

Although t50 at 30 °C did not differed from 25 °C, when considering germination speed of 100% of the seed population, the differences between the same temperatures become significant. Around to the optimum temperature (30 °C), seedlings presented a greater length and root dry mass; whereas at aerial part only the length was significantly higher at the optimal temperature (Figures 5A and 5B). At the optimal condition, the root growth rate was 0.74 cm.day-1, faster than other temperatures in the same time period. The higher speed of radicle elongation favors the establishment of seedlings in the environment. When conditions of water availability are not limiting, the seeds can germinate and establish faster in optimal temperatures. Despite the reduction of final germination percentage only at 15 and 40 °C, the lowest germination speed, the length and root mass of B. tomentosa seeds above or below 30 °C may be significant for seedling survival. Slow- germinating seeds, in non-optimal conditions, unlike faster-germinating seeds, can be predated prior to emergence and subject to competition with other plants for moisture and nutrients (Norden et al., 2009).

Journal of Seed Science, v.42, e202042007, 2020 Germination of B. tomentosa Eichler seeds 7 Final germination (%) Final germination (%)

Temperature (°C) Temperature (°C)

5 5 4.5 4.5 4 4 3.5 3.5 3 3 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 Germinaton speed index Germinaton speed index 0

Temperature (°C) Temperature (°C) Averages followed by the same letters do not differ by Tukey test (p < 0.05). Error bars represent standard errors. Figure 4. Final germination percentage (A and B) and germination speed index (C and D) of Buchenavia tomentosa Eichler seeds without endocarp adhered at different incubation temperatures.

Root lenght (cm) 2 Aerial part lenght (cm) 0

Temperature (°C)

2.4 2.0 1.6 1.2 0.8

Root dry mass (g) 0.4

0 Aerial part dry mass (g) Temperature (°C) Averages followed by the same letters do not differ by Tukey test (p < 0.05). Error bars represent standard errors. Figure 5. Length (A) and dry mass (B) of the roots and aerial part of Buchenavia tomentosa Eichler seedlings at different incubation temperatures.

Journal of Seed Science, v.42, e202042007, 2020 8 A. R. Correa et al.

An important fact is that the seed behavior in supra-optimal temperatures around 40 °C, where the seedlings survival is substantially impaired, is relevant in years of elevated temperatures in wet season, condition often found at Cerrado. As well, in future scenarios of temperature increase, the elevations of average daily temperatures and prolonged days with high temperatures delays the B. tomentosa seedlings’ development, hinders the establishment and competition. Above 40 °C it is not completed. Regarding the absorption of water by the seeds without endocarp adhered and with endocarp scarified or not, there was difference in the absorption pattern and in the time to complete the germination. The three phases of water absorption were observed only for seeds without endocarp (Figures 6A and 6B), as reported by Bewley (1997), where the imbibition of most seeds follows a three-phase pattern. Seeds without endocarp took 36 hours to complete the phase 1 of water absorption (Figure 6A), and they obtained an increase in the wet mass of 23.1% in relation to the initial water content. In the case of seeds with scarified or non- scarified endocarp adhered, they required 48 hours to complete the same phase (Figure 6B). At this initial period, there is intense absorption of water by the seeds (Bewley and Black, 1994). The acceleration in the imbibition process of seeds without endocarp still occurs in Anogeissus leiocarpa (Mattana et al., 2018) and Byrsonima basiloba (Silveira et al., 2012), due to the remove of the seeds’ external mechanic barrier, facilitating the inflow of water during the hydration process. Despite the absence of the endocarp to benefit the speed of the water imbibition process, there is no physical dormancy in the B. tomentosa seeds, since there is no total impediment of water inflow during hydration (Baskin and Baskin, 2004). According to Mattana et al. (2018), the importance of the endocarp is linked to the relatively slow water uptake by seeds, which represents adaptation to the low soil moisture, once the imbibition has begun. When the availability of water in the soil is enough to completely soak the seeds, the endocarp’s humidity can guarantee the germination and, later, the seedlings’ establishment in the case of possible lack of water. Another important relevance to dispersal structure in B. tomentosa is the protection against fire passage, which prevents the embryo damage, as in macaw palm (Bicalho et al., 2016). These facts have ecological significance for maintenance of this species at Cerrado. Seeds without endocarp adhered started the emergence at 25 days, ten days less than others treatment, caused by the removal of the mechanical barrier to water absorption, in comparison with seeds with scarified or non-scarified endocarp adhered (Figure 7), as observed in the absorption curve (Figure 6A).

According to the estimates of the regression models (Figure 7), the time to reach 50% of seedlings emergence (te50) was 29 days in seeds without endocarp, whereas the other treatments did not reach te50. The presence of damaging fungi in the endocarp may have prevented the seeds from reaching te50 on laboratory conditions; however, in nature, the balance between beneficial and damaging microorganisms plays a fundamental role in the seed germination. Moisture content (%) Moisture content (%)

Time (h) Time (h) Error bars represent standard errors. Figure 6. Water absorption curve in Buchenavia tomentosa Eichler seeds (A), in seeds with scarified endocarp adhered (Se) and intact endocarp (Ie) (B).

Journal of Seed Science, v.42, e202042007, 2020 Germination of B. tomentosa Eichler seeds 9 Seedling emergency (%) Seedling emergency

Time (days) Figure 7. Percentage of seedlings emergence of Buchenavia tomentosa Eichler from seeds without endocarp adhered (Swe), seed with scarified endocarp (Se) and seed with intact endocarp (Ie).

The endocarp can be removed in the field for dispersers. When it happens, they contribute to accelerate the germination and seed emergence due to the faster water absorption. On the other hand, in scenarios with reduced rainfall, the structure acts in the maintenance of the moisture until the seeds complete the germination process, showing their importance for B. tomentosa.

CONCLUSIONS

The basis temperatures determined by models to reach 50% of B. tomentosa Eichler germination are: Tb of 9,23 °C and Tmax of 44,6 °C; and the seeds without endocarp adhered needs average 89.71 °C.days to germinate. Around the optimal temperature of 30 °C, the seedlings showed higher root and aerial part length and higher root dry mass. The endocarp adhered to the seeds retard but does not prevent water absorption in B. tomentosa seeds.

ACKNOWLEDGEMENT

The authors thank the Programa de Pós-graduação em Agricultura Tropical of the Universidade Federal de Mato Grosso for the infrastructure available, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support to the first author.

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Journal of Seed Science, v.42, e202042007, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Effect of reduced water potential on seed germination of a Journal of Seed Science, v.42, e202042003, 2020 forest tree: a hydrotime approach http://dx.doi.org/10.1590/2317- 1545v42224519 Luís Felipe Daibes*1 , Victor J.M. Cardoso1

ABSTRACT: Hydrotime (θH) models provide information on seed tolerance to low water potential and time to germination under different conditions. Here it was evaluated the capacity of graphic and probit model to describe germination parameters and germination times (t) in a tropical legume (Peltophorum dubium). Germination tests were conducted under reduced water potentials (polyethylene glycol solutions from 0 to -1.2 MPa) at 25 °C. Regression lines were applied to investigate the relationship between germination rates (1/t) and water potential for different germination percentages (fractions 10, 30, 50 and 70%). Those regressions were used in the graphic model to calculate θH (1/slope) and determine the base water potential (Ψb) as the point which the line intercepts the x-axis (G% = 0). In the probit model, germination percentages were transformed to probit units and plotted against Ψb-values to describe germination response under a single regression line. Values for θH varied from 1.8 to 2.0 MPa day in both models, and Ψb showed a normal distribution, as presupposed by the probit model. Predicted germination times (t10 and t50) mostly fell within observed times, thus showing biological relevance of the models to describe the effects of water potential on seed germination.

Index terms: osmotic potential, Peltophorum, probit, psi-base, hydrotime model.

Efeito da redução do potencial hídrico na germinação de sementes de uma árvore tropical: uma abordagem do tempo hídrico

RESUMO: Modelos do tempo hídrico (θH) explicam a tolerância das sementes a potenciais hídricos reduzidos. Neste estudo, foi avaliada a capacidade dos modelos gráfico e probit para descrever parâmetros germinativos e tempos de germinação em uma leguminosa tropical (Peltophorum dubium). Foram conduzidos testes de germinação sob potenciais hídricos reduzidos (0 a -1.2 MPa) a 25 °C e utilizadas regressões para investigar a relação entre a taxa de germinação (1/t) e potencial hídrico para diferentes frações (10, 30, 50 e 70% de germinação).

As regressões foram utilizadas no modelo gráfico para calcular θH (1/inclinação da reta entre taxa de germinação e potencial hídrico) e determinar os valores de potencial hídrico base (Ψb), ponto *Corresponding author no qual a reta intercepta o eixo x (G% = 0). No modelo de probit, as porcentagens de germinação E-mail: [email protected] foram transformadas em probit e plotadas contra os valores de Ψb para descrever a germinação em uma única reta. Os valores de θ variaram de 1.8 a 2.0 MPa dia em ambos os modelos, e Received: 5/26/2019. H Accepted: 11/12/2019. Ψb apresentou distribuição normal, conforme pressuposto pelo modelo probit. A maioria dos tempos de germinação (t10 e t50) preditos ficou dentro dos tempos observados, demonstrando relevância biológica dos modelos na descrição dos efeitos do potencial hídrico na germinação. 1Universidade Estadual Paulista Termos para indexação: potencial osmótico, Peltophorum, probit, psi-base, modelo de (UNESP), Instituto de Biociências, tempo hídrico. Departamento de Botânica, 13506- 900 – Rio Claro, SP, Brasil.

Journal of Seed Science, v.42, e202042003, 2020 2 L. F. Daibes and V. J. M. Cardoso

INTRODUCTION

Water uptake is the principal factor starting the germination process, promoting cell respiration, DNA synthesis and growth (Bewley et al., 2013). Therefore, water potential (Ψ) strongly drives seed germination, by regulating the amount of water able to realize work in a solution. In a physiological sense, the decrease of water potential reduces germination capacity (G%), as well as the rate of the germination process (Gummerson, 1986; Bradford, 1995). Therefore, the germination rate (GR, inverse of germination time, t) seems to linearly decrease with negative Ψ-values, until the point at which seeds stop the germination process due to low water potential (base water potential, or Ψb). Because seeds can only accomplish germination above a Ψb-value, threshold models can be developed to describe seed germination responses to water potential (Bradford, 1990; Alvarado and Bradford, 2002; Finch-Savage, 2004).

Above Ψb, seeds require an accumulated Ψ (MPa) through time (hydrotime, or θH) to germinate and, therefore, hydrotime models were used to describe germination responses to reduced water potentials, mostly in crops (e.g. Gummerson, 1986; Dahal and Bradford, 1994; Windauer et al., 2007). Concerning native species, few studies in current literature investigated such threshold models regarding water relations and seed germination of tropical trees (Daws et al., 2008). Some studies evaluated the role of Ψ on seed germination of Brazilian species (Botelho and Perez, 2001; Fonseca and Perez, 2003; Rego et al., 2007), but rarely explaining whether germination would fit the presupposes of hydrotime models (Cardoso and Pereira, 2008; Oliveira et al., 2019). Most attention regards to the role of temperature, rather than water potential, as described by thermal time models in neotropical species (Cardoso and Pereira, 2009; Pires et al., 2009; Daibes and Cardoso, 2018; Duarte et al., 2018). Furthermore, seed germination can be expressed in different germination fractions (percentages), given the distribution and variation of time to germinate within seeds in a population (Garcia-Huidobro et al., 1982; Gummerson, 1986; Alvarado and Bradford, 2002). Hence, GR (germination rate) decreases with Ψ, but also varies according with the percentiles of seeds (i.e. seeds within the 10% percentile do germinate faster than the seeds within the 50 or

70% percentile). Likewise, θH requirements are related to GR and different Ψb-values might be expected for different fractions of seed germination (Bradford, 1990; Alvarado and Bradford, 2002). This might be important to understand the proportion of seeds which can be recruited in seasonal environments, where seeds are subjected to desiccation during the dry season, facing low water potentials in the soil seedbanks (Cavallaro et al., 2016). Therefore, this study aimed to investigate the role of water potential in seed germination of Peltophorum dubium, a tropical tree legume typically occurring in South American seasonal forests. Specifically, it was addressed how hydrotime models (graphic model and probit model) would describe seed germination of the species, explaining hydrotime requirements and Ψb. Using non-dormant seeds (previously alleviated from physical dormancy), it was hypothesized that seed germination would behave such as predicted by the model, following presupposes of graphical model (linear relationship of germination rate with decreasing of Ψ-values) and repeated probit model (normal distribution of Ψb with a single value of hydrotime requirement).

MATERIAL AND METHODS

Seed harvesting Seeds were obtained from a certified producer located in the municipality of Porto Ferreira (21°3’S; 47°2’W; state of São Paulo, Brazil). The harvesting site shows average temperatures from 19 to 25 °C and mean annual precipitation of 1500 mm (Daibes and Cardoso, 2018). The species has a widespread distribution throughout South America, mostly occurring in seasonal forests from the Paraguai-Paraná Basin to the state of Bahia (Barneby, 1996). The harvesting was performed in May 2011, in different mother plants and seeds kept stored within the pods (indehiscent fruit) inside paper bags under low temperatures (~5 °C) until their use in the experiments, few months later. Dispersal period may range from April to December, and ripened fruits remain attached to the trees for several months (Carvalho, 2002). The total

Journal of Seed Science, v.42, e202042003, 2020 Hydrotime and germination of a forest tree 3

viability of seeds was high by the beginning of germination trials (~90%) and no light requirement was detected in P. dubium seeds (Perez et al., 2001).

Germination trials To conduct germination tests under different water potentials, seeds were removed from the pods and carefully screened to remove malformed and/or predated seeds. Then, seeds were individually scarified with a sandpaper to overcome physical dormancy. Once scarified, seeds were expected to behave as non-dormant seeds in the germination trials (Daibes and Cardoso, 2018). Different water potentials were obtained by aqueous solutions of PEG 6000 (polyethylene glycol), ranging from zero (distilled water) to -1.5 MPa (0; -0.3; -0.5; -0.7; -0.9; -1.1; -1.3; -1.5). PEG solutions were prepared according to Villela et al. (1991), as adapted from Michel and Kaufmann (1973). Seeds were then set to germinate on a double layer of filter paper soaked in at least 6 mL of the corresponding PEG solution in Petri dishes (90 mm), sealed with plastic film to prevent evaporation of the solution. Three replicates of twenty seeds were used in each water potential treatment, and germination tests were conducted in germination chambers under the constant temperature of 25 °C, considered within the range of optimal conditions for seed germination of the species (Daibes and Cardoso, 2018). Because P. dubium seeds are non-photoblastic, all germination trials were conducted in the dark. Seed germination (radicle protrusion) was daily counted for one month or until the germination of all seeds in the plate. At each counting, Petri dishes were carefully re-wrapped again in the plastic film. By the end of the trials, remaining seeds were visually inspected to attest viability and scored as dead.

Data analysis and hydrotime modeling Prior to hydrotime modeling, the germination capacity (%) and time (t) of germination through the different water potential treatments were evaluated. Germination capacity was statistically compared using GLMs with a binomial distribution in lme4 package (Bates et al., 2015) in R software (R Core Team, 2018), considering distilled water (Ψ = 0) as the control (baseline) in the analysis. Observed t was calculated to different germination percentiles (10, 30, 50 and 70%) by linear interpolation of two nearest points in the germination curves, then obtaining the x-axis interception to the corresponding fraction (Steinmaus et al., 2000). Model parameters were obtained for two hydrotime models: graphic model and probit model. In the graphic model, the germination rates for the different

germination fractions (GR(g) = 1/t) were regressed against the treatments to assess the linearity among GR and water potential (Bradford, 1990). Base water potential (Ψb) was estimated as the point which the regression lines

intercepted the x-axis (GR = 0), while hydrotime (θH) was obtained as the inverse of regression line slope (θH = 1/ slope), according to Gummerson (1986). In the probit model, germination percentages were transformed to probit units and plotted as function of Ψb. Linear regression was used to evaluate the relationship between observed germination and the predicted line. The

value of θH was probed repeated times and considered the best-fitting values which showed higher R² and least residual model (Dahal and Bradford, 1994; Bradford, 1995). Predicted germination curve was derived from the original probit regression line (Table 1) using a normal distribution in Excel® (Cardoso, 2011). Once obtained hydrotime parameters of θ both models, germination times were estimated by rearranging the basic equation: H = (Ψ–Ψb).t(g), thus considering

t(g) = θH/(Ψ–Ψb). Hence, the model-predicted germination times were calculated for the 10 and 50% percentiles (t10 and t50) and compared to observed t-values in relation to confidence intervals (95%). Because Ψb is plotted in the

x-axis in the probit model, the following equation was considered: t(g) = θH/(Ψ–((probitG–a) / b), where “a” is the intercept, and “b” the slope of the probit regression.

Journal of Seed Science, v.42, e202042003, 2020 4 L. F. Daibes and V. J. M. Cardoso

Table 1. Parameters of hydrotime model regression lines: equation and R2, base water potential (Ψb, MPa) and

hydrotime values (θH) for seed germination of Peltophorum dubium.

Equation R² Ψ-base θH Graphic model 10% y = 0.559x + 0.526 0.98 -0.94 1.8 Graphic model 30% y = 0.488x + 0.435 0.97 -0.89 2.0 Graphic model 50% y = 0.480x + 0.390 0.95 -0.81 2.1 Graphic model 70% y = 0.448x + 0.328 0.99 -0.73 2.2 Probit model y = 5.480x + 3.866 0.88 -0.7 ± 0.18 1.8 The graphic model was performed for different germination fractions (10, 30, 50 and 70%), while Ψb of probit model regards to 50% of seed germination ± standard deviation.

RESULTS AND DISCUSSION

Seeds showed high germination capacity (90%) in water potentials of 0 and -0.3 MPa, with a significant reduction to ~70% at -0.5 MPa, decreasing to ≤ 10% at -0.7 and -0.9 MPa (Figure 1). Germination was null under the treatments of -1.1, -1.3 and -1.5 MPa. Germination rates showed a linear relationship with water potential, decreasing GR according to the reduction of Ψ and showing a general parallel pattern among the different germination fractions (10, 30, 50 and 70%; Figure 2). Therefore, the interception points in the x-axis showed Ψb-values varying from -0.7 to -0.9 MPa among percentiles in the graphic model (Table 1). Regression lines for the different germination fractions in the graphic model showed slope ranging from 0.448 to 0.559 and θH values (1/slope) around 2.0 MPa day (Table 1). Because the parallel lines show a relatively similar slope (i.e., a similar hydrotime for different germination percentiles), it is the Ψb which drives germination parameters of the germination fractions. In the probit model, it was possible to clump the different germination curves (Figure 3A) into a single curve of germination percentages as function of Ψb (Figure 3B). Base water potential followed a normal distribution in the probit model, which described 88% of germination parameters, showing θH = 1.8 MPa day (Table 1). On the other hand, probit model might underestimate seed tolerance to low water potentials (Ψb(50) = -0.7 MPa), whereas graphic model predicts a little more negative Ψb value for the 50% fraction (-0.8 MPa; Table 1). Nevertheless, predicted times

P value = 0.003 under Ψ of -0.5 MPa in relation to control (distilled water 0 MPa), and P < 0.001 under Ψ ≤ -0.7 MPa compared to control. Figure 1. Germination capacity (%, mean ± SD) of Peltophorum dubium seed germination under different water potentials (Ψ, MPa), showing significant decrease under reduced water potentials.

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Continuous lines are linear regressions predicted by graphic model. Figure 2. Relationships of germination rate (GR) under different water potentials for different germination fractions (10, 30, 50 and 70%).

Figure 3. (A) Seed germination curves (cumulative germination percentage vs. time, in days) under different water potentials of 0, -0.3, -0.5 and -0.7 MPa. (B) Germination curve as function of Ψb showing a normal distribution pattern (continuous line) predicted by probit model.

Journal of Seed Science, v.42, e202042003, 2020 6 L. F. Daibes and V. J. M. Cardoso of germination (t10 and t50) under different water potentials mostly fell within the confidence intervals of observed times, both for graphic and probit models (Table 2). Whether falling outside the confidence intervals, the error in germination time predictions never exceed one day more than the observed times. Germination times to 10% of seed germination (t10) were around two days under distilled water (0 MPa) and slowed to ~ eight days under -0.7 MPa. Similarly, t50 ranged from 2.5 to 8.8 days under 0 to -0.5 MPa, and such fraction was never reached under water potentials ≤ -0.7 MPa (Table 2). Seeds of P. dubium fitted hydrotime presupposes, following patterns predicted by graphic and probit models. Models described seed germination parameters and predicted germination times similarly to found in crops throughout the world (Gummerson, 1986; Dahal and Bradford, 1994; Windauer et al., 2007; Patanè et al., 2009). Fitting hydrotime models in native trees may thus aid in seed testing analysis by describing the potential of a given species or population to tolerate moisture stress (Bradford and Still, 2004). Moreover, θH values provide information on time to emergence, predicting germination under continuous variables. Among forest trees, changing in germination times and Ψb seems to explain germination responses of pioneer species in tropical ecosystems (Daws et al., 2008). In our study species, seed tolerance to lower Ψ was relatively sensitive compared to weed species, for instance, which may tolerate values < -1.0 MPa (Boddy et al., 2012). Some species from desert communities may also germinate under lower Ψ-values, such as Erodium texanum, which shows Ψb of -2.32 MPa (Huang et al., 2016), while our study species never germinated under values lower than -0.9 MPa. Therefore, moisture stress seems to be a constraining factor for seed germination of P. dubium seeds, which might be recruited during less stressful conditions during the rainy season. Likewise, seeds of Senna spectabilis were relatively sensitive to Ψ, but tolerance to moisture stress seems to be increased when seeds are subjected to dehydration cycles, thus helping their survival under drought conditions (Lima et al., 2018). Moreover, we argue threshold models can be used to compare germination requirements among species and/or populations in contrasting environments (Rosbakh and Poschlod, 2015; Tudela-Isanta et al., 2018; Picciau et al., 2019) thus providing insights on community assembly rules (Poschlod et al., 2013). Seeds of seasonal environments may face drought stress which may hamper germination and establishment (Cavallaro et al., 2016), and seed modeling may help to choose species for reforestation programs. Therefore, the hydrotime approach is an underexplored tool in the ecological management of tropical plant communities (Bradford, 2005).

Table 2. Observed (mean ± 95% confidence interval) and predicted germination times (graphic model and probit model) for 10 and 50% of seed germination (t10 and t50) of Peltophorum dubium seeds in different water potentials.

Ψ (MPa) Observed Graphic model Probit model t10 0 1.97 ± 0.31 2.17 1.92 -0.3 2.61 ± 0.39 3.18 2.82 -0.5 4.55 ± 1.43 4.63 4.10 -0.7 8.0 ± 3.20 8.49 7.53 -0.9 – – – t50 0 2.67 ± 0.33 2.51 2.55 -0.3 3.61 ± 0.17 3.97 4.44 -0.5 8.28 ± 3.65 6.51 8.76 -0.7 – – – -0.9 – – – The germination was null under water potentials ≤ -0.9 MPa.

Journal of Seed Science, v.42, e202042003, 2020 Hydrotime and germination of a forest tree 7

The results provide a preliminary approach on hydrotime models, by testing the basic assumptions of the models and linear relationships with water potential (Bradford, 1995). Likewise, most model presupposes were followed by P. dubium seeds under different temperatures, described by thermal time models (Daibes and Cardoso, 2018). Temperature and water potential show important interactions to describe germination parameters under laboratory conditions (Alvarado and Bradford, 2002). However, few studies were accounted for such interactions (hydrothermal time) in Brazilian species (Simão et al., 2010; Oliveira et al., 2019). Seedling emergence from soil seedbanks should also be examined, in order to validate such models under field conditions (Forcella et al., 2000). Some critics were made to probit analysis due the lack of independency among germination counting through the days. Therefore, cumulative germination percentages would be temporally dependent, breaking the principle of independency among samples in a regression analysis (Hay et al., 2014). Solving this issue would require a considerable higher amount of seeds, often impossible to achieve from native populations. Analysis derived from semi-parametric distributions (survival analysis, for instance) could help us to fix such problems by taking in account the probability of individual seeds to germinate or fail (Onofri et al., 2010; McNair et al., 2012). Despite such relatively recent approaches proposed, their connection to hydrotime assumptions and usage in statistical software remains a matter of inquiry (Cao et al., 2013). Nevertheless, the predictions of germination times from linear models showed to be useful, showing biological relevance (Bradford, 1995; Bradford and Still, 2004). Therefore, germination parameters may be drawn from relatively simpler equations, and the advantage of probit model is to derive predictions of germination time from a single line. The graphic model, on the other hand, requires different regression lines according to the desired germination fraction wished to describe (Daibes and Cardoso, 2018). Either way, the application of a model does not exclude the use of other methods to predict seed germination. Linear relationship of GR and water potential may be helpful to achieve general patterns of model presupposes and can serve as a support to evaluate germination behavior before running probit analysis.

CONCLUSIONS

Hydrotime models describe germination of a forest tree and the applicability of such models for seed testing and/or ecological purposes is still underestimated. Future studies should account for the variation in seed germination within and among species and the interactions of water potential with temperature. Field experiments would be warranted for validation of model descriptions under natural variation.

ACKNOWLEDGEMENTS

This study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001, and the authors received grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Journal of Seed Science, v.42, e202042003, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Digital image processing of coated perennial-soybean seeds Journal of Seed Science, v.42, e202042004, 2020 and correlation with physiological attributes http://dx.doi.org/10.1590/2317- 1545v42227516 Amanda Justino Acha1* , Henrique Duarte Vieira1

ABSTRACT: Perennial soybean (Neonotonia wightii) is a Fabaceae with potential to be used in consortium with Poaceae plants to improve pasture quality. In order to add value to perennial soybean seeds and improve their seed distribution, seeds coated with different materials in coating machine were characterized by digital image analysis and physical attributes related to physiological attributes in order to define the ideal amount and material to be used in the coating. Different material quantities were tested, 150 g, 200 g and 250 g, divided into layers, namely: sand, calcium silicate + sand and limestone + sand. Coating promoted maximum increments of approximately 350% in seed mass and significant increases of up to 230% in area, 154% in maximum diameter, 162% in minimum diameter, 167% in contained diameter and 152% in perimeter. The coating was also efficient in reducing the moisture of the pellets by increasing the layers that cover the seeds. The sand + limestone combination resulted in the largest pellets. The combination of sand + silicate did not interfere with plant speed and formation. Thus, it was considered the appropriate material for the coating of perennial soybean seeds.

Index terms: seed coating, silicate, pellets, digital image analysis, Neonotonia wightii.

Processamento digital de imagens de sementes de soja perene revestidas e correlação com atributos fisiológicos

RESUMO: A soja perene (Neonotonia wightii) é uma Fabaceae com potencial para ser utilizada em consórcio com plantas da família Poaceae, a fim de melhorar a qualidade das pastagens. Na busca por agregar valor às sementes de soja perene e melhorar a sua distribuição na semeadura, sementes revestidas com diferentes materiais foram caracterizadas via análise de *Corresponding author imagem digital e os atributos físicos relacionados com atributos fisiológicos, a fim de definir E-mail: [email protected] a quantidade e o material ideais a serem utilizados no revestimento. Foram testados 150 g, Received: 8/16/2019. 200 g e 250 g de material divididos em camadas, sendo eles: areia, silicato de cálcio + areia e Accepted: 11/14/2019. calcário + areia. O recobrimento promoveu incrementos máximos de aproximadamente 350% à massa das sementes e aumentos significativos de até 230% na área, 154% no diâmetro máximo, 162% no diâmetro mínimo, 167% no diâmetro contido e 152% no perímetro. O recobrimento foi eficiente, também, em reduzir a umidade dos péletes com o aumento das 1Universidade Estadual do Norte camadas que recobrem as sementes. A combinação de areia + calcário resultou nos maiores Fluminense (UENF), Departamento péletes. A combinação de areia + silicato não interferiu na velocidade e formação de plantas, de Produção Vegetal, 28013-602 – Campos dos Goytacazes, RJ, Brasil. sendo considerado o material adequado para o recobrimento de sementes de soja perene. This article is part of the first author’s Termos para indexação: recobrimento, silicato, péletes, análise digital de imagens, doctoral thesis, which was defended Neonotonia wightii. at the Universidade Estadual do Norte Fluminense Darcy Ribeiro.

Journal of Seed Science, v.42, e202042004, 2020 2 A. J. Acha and H. D. Vieira

INTRODUCTION

In pasture management, the practice of intercropping plants with forages of the family Fabaceae has generated increased yields and contributed to the sustainability of the system, as it elevates the protein content of forage, improves nitrogen incorporation into the soil and increases protection against erosion. Additionally, this practice favors pest control and the maintenance of rest areas (Tambara et al., 2017). According to Gama et al. (2013), the perennial soybean (Neonotonia wightii (Am.) Lackey) has great potential for use in intercropping due to its ability to incorporate 150 to 300 kg of N ha-1.yr-1, in addition to producing an average of 20 to 30 t.ha-1 of fresh matter and 6 to 8 t.ha-1 of dry matter yearly. This Fabaceae species can help in the recovery of degraded pastures in addition to attaining high yields, deep rooting and high defoliation resistance. Constant improvement of seed-processing technologies is fundamental for the agricultural sector, since the current market is demanding and aware of the importance of quality (Melo et al., 2016). Seed coating is a technique capable of altering the physical traits of seeds which allows the seed producer to add mineral salts, fungicides or insecticides as necessary, thereby adding value to the seed for marketing (Derré et al., 2013). For high-quality seeds to be supplied to the producer at a fair price, research must be conducted to define the methodology, type and concentration of materials capable of providing high-quality coating without compromising the seed. Many problems have been reported regarding the quality of coated seeds for pastures, which are due mainly to the use of inadequate practices; e.g. uneven coating, contamination by other species and coating that reduces seed germination. Divergences may exist between studies on seed coating due to the material used and thickness of the layer deposited on the seeds (Somrat et al., 2018; Xavier and Vieira, 2018). Image analysis is a tool used in basic and applied studies that aims at elucidating various aspects of seed behavior and the improvement of methodologies for the evaluation of different seed-quality attributes. This method proposes to increase efficiency and reliability of results, as it reduces human error (Medeiros et al., 2018). The technique consists of obtaining information of recorded objects based on physical traits like color, texture, geometry etc. Because it is a non-destructive method, the seeds can be set to germinate after the image is captured. Thereafter, possible relationships between the physical and physiological traits can be defined based on the analyses (Guedes et al., 2011; Zang et al., 2018). However, information on the use of image analysis in the evaluation of the quality of coated seeds and the quality of the coating process is still limited, considering the broad diversity of existing species and the specific characteristics of each one. Thus, studies are warranted to improve this technique (Silva et al., 2014). On these bases, the present study proposes to characterize, by digital image analysis, the physical attributes of coated perennial soybean seeds and evaluate their correlation with physiological attributes in order to define the optimum quantity and material to be used in coating.

MATERIAL AND METHODS

The Neonotonia wightii seeds were acquired from the BRSeeds® company and subjected to chemical scarification with concentrated sulfuric acid for twenty minutes, except for seeds for intact control treatment. Subsequently, they were washed in abundant water and left to dry at room temperature prior to being coated.

Seed coating To coat the seeds, it was refined the technique employed by Acha et al. (2016), using a bench-top seed coating machine (N10 Newpack®) with the pan rotating at a speed of 64.5 rpm and the adhesive solution applied at a pressure of four bar, for two seconds. Next, the hot-air blower was activated at a temperature of 50 ºC, for three minutes. The seed coating process consists of the formation of layers, which are composed of two portions of 12.5 g of filler material and two jets of adhesive material (Cascorez® extra glue, based on polyvinyl acetate [PVA] diluted in deionized water previously heated to 70 ºC, at the 1:2 [v/v] ratio, respectively).

Journal of Seed Science, v.42, e202042004, 2020 Digital image and physiological attributes of coated perennial-soybean seeds 3

One hundred grams of perennial-soybean seeds which had been previously scarified were placed in the coating pan alongside a portion of filler material (12.5 g). Next, a jet of adhesive solution was applied, and the seed mass was tumbled in the pan for one minute. Afterwards, another jet of glue and another 12.5 g portion of filler material were applied. For the following layers, a jet of adhesive solution was immediately applied, followed by a portion of filler material; then another jet of adhesive solution and then the second portion of the filler material. Subsequently, the hot-air blower was activated, finishing layer formation. This procedure was repeated until the programmed layers were complete.

Treatments The following materials or combinations were tested as fillers: fine sand – A (sieved through a 0.35 mm square mesh); sand + calcium silicate – AS; and sand + dolomitic limestone – AC (0.25 mm), with the number of layers (grams of material) varying between 6 (150 g), 8 (200 g) and 10 (250 g). The filler materials were mixed at the 1:1 ratio before being deposited in the pan. Material density: fine sand: 2.91 g/cm³; calcium silicate: 2.66 g/cm³; dolomitic limestone: 2.86 g/cm³.

Tests and analysis After coating, the seeds were evaluated for the physical and physiological traits. Laboratory – The following variables were analyzed, following the Rules for Seed Testing (Brasil, 2009): one- thousand-seed weight, water content, between-paper germination test (% G) and germination speed index (GSI) (Maguire, 1962). Greenhouse – The following variables were analyzed: emergence test (% E), performed in trays with coarse sand previously washed in abundant water, and emergence speed index (ESI), which was evaluated over thirty days (Maguire, 1962). Subsequently, at the end of ninety days, shoot and root length and dry matter were measured following the methodology adopted by Acha et al. (2018). Digital analysis of the seeds – It was performed using GroundEye® S120 software, formerly known as SAS®, a semi- automated seed analysis tool which extracts numerous data from the capture of a high-resolution two-dimensional image. The system provides individual information of each seed and groups (the data into color, texture, shape, morphology and uniformity), providing over three hundred traits. After the variables were refined based on definition and importance, those which best met the needs for the evaluation of coating quality were selected (Table 1), namely,

Table 1. Variables obtained in the digital image analysis using GroundEye®.

Variable Definition Formula It is a process of reducing the amount of pixels in an image, which consists in removing all redundant pixels Where A is the area, p is the perimeter Fine-tuning producing a new simplified image. (Gonzalez and and 4π is the normalization factor Woods, 2008) T = Where P stands for perimeter Irregular contour Sets the level of tuning of the object and PC stands for convex perimeter Detects and counts the amount of “gaps” between Contour deformation the convex perimeter and the original perimeter of an – object Corresponding to the amount of space an object’s Where p represents one image pixel Area (cm²) surface has and R represents object pixels Maximum diameter (cm) It is the longest line that goes through the centroid – Minimum diameter (cm) It is the shortest line that goes through the centroid – It is the largest diameter of the circumference that fits Contained diameter (cm) within the object – It is the measure of the contour of a two-dimensional 2 (bh) – rectangle Perimeter (cm) object, that is, the sum of all sides of a geometric figure 2r – circle

Journal of Seed Science, v.42, e202042004, 2020 4 A. J. Acha and H. D. Vieira fine-tuning, irregular contour and contour deformation, which relate to the contour; and area (cm2), maximum diameter (cm), minimum diameter (cm), contained diameter (cm) and perimeter (cm), which pertain to seed size (TBIT, 2014).

Statistical procedure The seed-coating experiment was undertaken as a completely randomized design with nine treatments in four replicates with 100 g of seed per treatment. The laboratory tests also followed a completely randomized design, whereas a randomized-block design was adopted for the greenhouse part of the experiment. Nine coating treatments and two control treatments (no scarification or scarified) were tested in the laboratory and in the greenhouse, in four replicates per environment, with fifty seeds each. The data collected after the evaluations were subjected to the Shapiro-Wilk normality test, with no need for transformation and homoscedasticity of the variances evaluated by Bartlett’s test. Analysis of variance was performed, and means were compared by Tukey’s test at the 5% probability level, using Sisvar® statistical software. Pearson’s correlation coefficient (r) was also calculated between the evaluated physical and physiological variables. For all analyzed variables, the control treatments were not included in analysis of variance and test of means; thus, for them, only descriptive analysis was performed.

RESULTS AND DISCUSSION

In the intact and scarified control treatments, one-thousand-seed weight was 5.82 and 5.55 g, respectively. For the different coatings tested, the coating methodology employed was efficient in providing increases of approximately 350, 280 and 200% in seed weight in the treatments with ten layers of sand + limestone, ten layers of sand + silicate and ten layers of sand, respectively, compared to the scarified control treatment (Figure 1). These values are certainly related to the density of materials and their combination; i.e., even though fine sand has the highest density (2.91 g/cm3) among the tested materials, when combined with dolomitic limestone (2.86 g/cm3) and calcium silicate (2.66 g/cm3), it heightens their adherence to the seed, consequently providing higher seed weights. In terms of seeding uniformity, the increase in seed weight is a positive factor, as it favors precision seeding, allowing the seeds to be used by most mechanical seeders. However, even though the increase in seed weight is an important criterion in the evaluation of coating quality, this criterion should not be evaluated individually, since an increase in seed weight is directly linked to the final cost of the product and to the physiological quality of the seed (Acha et al., 2016). The water content of the coated seeds is of fundamental importance for the evaluation of their quality and proper storage (Marcos-Filho, 2015). The water content of seeds of the control treatments were 8.69% and 11.59%, for intact and scarified seed, respectively. After being coated, the seed moisture content was reduced in all treatments, being progressive with the increase of the number of layers. The lowest moisture content (4.33%) was observed in the treatment with ten layers of silicate + sand. Thus, it can be stated that the time and temperature for seed drying during coating was efficient in all treatments tested.

Figure 1. Perennial-soybean seeds. A) Intact seed. B) Sand coating – ten layers. C) Silicate + sand coating – ten layers. D) Limestone + sand coating – ten layers.

Journal of Seed Science, v.42, e202042004, 2020 Digital image and physiological attributes of coated perennial-soybean seeds 5

Among the variables provided by GroundEye® within the “shape” group, it was selected those that least met the coating-quality criteria referring to the geometric traits, which are important for evaluating, classifying and standardizing the pellets formed by the different treatments. Area, maximum, minimum and contained diameters and perimeter are the variables referring to seed geometry and which are responsible for describing the region occupied by the seed in the image plane. Guedes et al. (2011) evaluated the area, perimeter, maximum and minimum diameters, circularity and roundness of soybean seeds and concluded that digital analysis is valid to determine the physical quality of seeds when compared to manual evaluation methods. All treatments were able to translate the increases provided in seed weight during coating into pellet size (Table 2). However, the treatments involving eight and ten layers of sand + limestone stood out with the highest means for the selected variables, demonstrating the adhesion power of the sand + limestone + PVA glue combination. As seen for one-thousand-seed weight, the weight of coated seeds increased by 230, 180 and 190% in the sand + limestone, sand + silicate and sand treatments, respectively, when compared to the uncoated seeds, whose area was 0.036 cm2. These gains were obtained in the treatments with ten coating layers. Coating also increased the diameters and the perimeter as the number of layers was increased. Thus, the increases provided to seed weight after coating were reflected on pellet size (Table 2). As coating thickness is increased, the perennial-soybean seeds gain protection against the attack of pests, both in the field and in storage, slowing the seed deterioration process (Gardarin et al., 2010). Thickness is an important factor also when aiming to add fertilizers, fungicides and other agricultural additives, which should be added at a certain distance from the embryo to prevent toxicity. However, it is not a coating criterion to be evaluated separately, and thus other parameters should be used as well. Considering that the area variable refers to the amount of space occupied by the object’s surface, that the diameters are based on the object’s circumference and that perimeter is the sum of the entire contour of the two-dimensional object (TBIT, 2014), these variables are directly linked and are positively influenced by one-thousand-seed weight (r > 0.81). GroundEye® was efficient in determining the maximum and minimum diameters of coated Stylosanthes sp. and perennial-soybean seeds in the studies of Acha et al. (2016), Silva et al. (2017), Acha et al. (2018) and Xavier and Vieira (2018). These authors obtained significant increases for those variables with the different coating methodologies

Table 2. Digital analysis of coated perennial-soybean seeds by GroundEye® software. Area, maximum diameter (MAXD), minimum diameter (MIND), contained diameter (CONT) and perimeter.

Treatments* Area (cm²) MAXD (cm) MIND (cm) CONT (cm) Perimeter (cm) A6 0.051 d¹ 0.288 e 0.218 e 0.210 e 0.873 d A8 0.062 bc 0.320 cd 0.238 cd 0.228 cd 0.967 c A10 0.070 b 0.343 b 0.245 c 0.236 c 1.037 b AS6 0.053 d 0.299 de 0.220 e 0.213 de 0.880 d AS8 0.065 bc 0.327 bc 0.243 cd 0.239 bc 0.965 c AS10 0.066 bc 0.328 bc 0.251 bc 0.246 bc 0.970 c AC6 0.058 cd 0.308 cde 0.229 de 0.218 de 0.928 cd AC8 0.079 a 0.365 a 0.263 ab 0.255 ab 1.087 ab AC10 0.083 a 0.374 a 0.272 a 0.265 a 1.112 a Intact seed 0.036 0.243 0.168 0.159 0.732 Scarified seed 0.036 0.250 0.163 0.156 0.736 CV% 5.54 2.78 2.78 3.04 2.68 *Control treatments: intact and scarified seeds. Scarified seeds with six, eight and ten layers of sand (A6, A8 and A10); with six, eight and ten layers of sand + calcium silicate (AS6, AS8 and AS10); and with six, eight and ten layers of sand + limestone (AC6, AC8 and AC10). ¹Means followed by the same letter in the column do not differ significantly between each other according to Tukey’s test at the 5% probability level.

Journal of Seed Science, v.42, e202042004, 2020 6 A. J. Acha and H. D. Vieira applied, which served as base for refining the technique applied in this experiment. Correlation analysis between the variables area, maximum, minimum and contained diameter, perimeter and one- thousand-seed weight provides precise information about the efficiency of the methodology in producing high-quality coating, which makes it possible to identify the best material and the proportion for adhesion. The analysis in GroundEye® indicates whether coating was able to change the initial seed shape, besides the possibility of rapidly and efficiently classifying and standardizing the pellets regarding their size, reducing the chances of errors made by the evaluator. Accordingly, interpreting these traits is essential for perfecting the coating technique, and this may result in the discard of treatments that do not meet the main objectives of seed coating, which are to change the size, shape and density of seeds. For a reliable digital analysis of the traits represented by an image, one must make use of techniques that treat and eliminate false aspects that might be erroneously detected and interpreted. Among them, the fine-tuning variable is employed to reduce undesirable pixels; i.e., it is a process designed to reduce the form into a more simplified version (skeleton) while maintaining the essential characteristics of the original object, considering even small imperfections (Gonzalez and Woods, 2008; Artero and Tommsselli, 2009; Russi et al., 2017). It is a variable that indicates how many adjustments were necessary to prepare the image of the object for a perfect analysis. Fine-tuning is negatively correlated (p < 0.05) with contour irregularity (r = -0.97) and positively correlated with contour deformation (r = 0.87). This result was observed when correlating the data of the thinning variable with contour irregularity and contour deformation in all treatments tested. The data presented in Table 3 confirm this correlation, as was observed that the treatments with eight and ten layers of sand + silicate provided significantly higher fine-tuning values. These treatments led to lower contour irregularity and, consequently, a higher deformation index, when compared to the other treatments. It is believed that the high thinning index for silicate + sand treatments is associated with the fact that it has low fixation compared to the other materials, probably due to the difference in particle size between silicate and sand, resulting in a higher number of “blurs” in the image. Considering that contour irregularity defines the level of fine-tuning of the analyzed object (TBIT, 2014), when was interpreted the indices obtained by digital image analysis through GroundEye®, it was observed an inverse relationship between contour irregularity and the fine-tuning values. Thus, lower the contour irregularity values mean a greater need

Table 3. Digital analysis of coated perennial-soybean seeds by GroundEye® software, describing fine-tuning, contour irregularity and contour deformation.

Treatments* Fine-tuning Contour irregularity Contour deformation A6 0.840 cd¹ 0.029 ab 26.340 e A8 0.838 cd 0.029 ab 27.816 de A10 0.817 d 0.032 a 27.655 de AS6 0.869 ab 0.023 c 33.349 bc AS8 0.874 a 0.023 c 36.165 ab AS10 0.880 a 0.023 c 36.665 a AC6 0.839 cd 0.028 b 28.390 de AC8 0.836 cd 0.028 b 30.550 cd AC10 0.845 bc 0.027 b 31.736 c Intact seeds 0.840 0.29 30.260 Scarified seeds 0.840 0.28 31.162 CV% 1.21 5.03 4.48 *Control treatments: intact and scarified seeds. Scarified seeds with six, eight and ten layers of sand (A6, A8 and A10); with six, eight and ten layers of sand + calcium silicate (AS6, AS8 and AS10); and with six, eight and ten layers of sand + limestone (AC6, AC8 and AC10). ¹Means followed by the same letter in the column do not differ significantly between each other according to Tukey’s test at the 5% probability level (p < 0.05).

Journal of Seed Science, v.42, e202042004, 2020 Digital image and physiological attributes of coated perennial-soybean seeds 7 to adjust the object represented in the image. It indirectly assists the evaluation of coating quality, as it is associated with the thinning variable. In terms of contour deformation, the difference in particle size between fine sand and calcium silicate possibly provided a coating in which the materials did not fit perfectly, requiring for greater image corrections. Given this information, in the treatments in which only sand and the sand + limestone combination was used, contour uniformity was superior to that obtained with sand + silicate, suggesting that these particles fit better during coating. This is because sand and limestone share the same particle size (0.25 mm), thus needing less fine-tuning to be better interpreted by the software and forming a coating with fewer “gaps” between the convex perimeter and the original perimeter. The results obtained for fine-tuning, contour irregularity and contour deformation demonstrate that it is possible to evaluate the aesthetic result of the combinatory or non-combinatory action of the materials tested in coating. These are important criteria to be adopted when aiming at improved pellet finishing quality. Attesting the quality of the lot of perennial-soybean seeds used, a GSI of 2.5 and a germination percentage of 25.5% were detected in laboratory conditions for the intact seeds. For the seeds scarified in sulfuric acid, GSI was 11.4, and germination percentage was 59.5%. In the greenhouse, the seeds achieved an ESI of 1.7% and an emergence percentage 26.5% (intact seeds); and an ESI of 4.1% and 51.5% emergence (scarified seeds). In both environmental conditions, chemical scarification showed to be efficient, breaking the integument dormancy specific to seeds of Neonotonia wightii (Acha et al., 2016). In examining the influence of the treatments on the speed and formation of normal perennial-soybean seedlings in the laboratory and in the greenhouse (Figures 1 and 2), it was observed that, in laboratory conditions, the maximum GSI and germination values achieved were 9.2 and 66%, respectively. In the greenhouse, the observed ESI and emergence percentage were 4.13 and 56%, respectively. For both conditions, those values refer to the treatment with six layers of sand. The seeds coated with six layers were the lightest and smallest for all tested filler materials (Table 2). Thus, they possess a smaller barrier to be overcome during germination in comparison to the other treatment groups. In the specific case of treatment A6 (six layers of sand), the layer was more easily broken due to the particle size of sand, which reduces the adhesion of this material when in low amounts, especially in the scarified seeds, which have a smoother surface. For this reason, the A6 treatment resulted in coating with many imperfections and, consequently, a product of lower quality that did not meet the main purpose of seed coating, which is to increase its size. Seed scarification is known to accelerate the soaking process, which at one point may cause alterations in the conformation and structure of the membrane system, influencing germination (Marcos-Filho, 2015). Considering that the A6 treatment numerically exceeded (G = 66% and E = 56%) the values of normal seedlings achieved by the scarified- control seeds (G = 59.5% and E = 51.5%), it is believe that the treatment with six layers of sand provided an external protection to the seeds by controlling the water input, preventing the scarified seeds from suffering physiological damage that would be reflected in the formation of normal seedlings with the rapid water absorption. The treatments of perennial-soybean seeds with sand + silicate stood out positively for germination (GSI) and formation of seedlings in laboratory conditions (% G), not differing statistically from the treatment with six layers of sand, which achieved the highest numerical values (Figures 2 and 3). Thus, the sand + silicate combination provided a coating in which the seeds managed to break the created barrier with greater ease, regardless of the amount of material tested in this study. It is noteworthy that coating should not be an obstacle to root development or to the plant shoots, but rather be water-soluble and allow the passage of oxygen for natural embryo development (Santos et al., 2010). As observed by Xavier et al. (2015) in seeds of Stylosanthes cv. Campo Grande, the coating formed by layers of calcium silicate + sand in this study, in Neonotonia wightiicv. Comum, easily came apart when in contact with water, regardless of the number of layers tested. As such, this coating stood out positively. Despite using larger proportions of filler materials and mechanically scarified seeds, unlike the methodology applied in the current study, Xavier and Vieira (2018) also observed positive results in the germination of perennial-soybean seeds coated with calcium silicate alone, combined with sand and combined with activated coal. The authors found no statistical differences between the treatments. Journal of Seed Science, v.42, e202042004, 2020 8 A. J. Acha and H. D. Vieira

10 A GSI ESI

8 AB AB AB AB 6 BC a

GSI / ESI 4 ab ab abc abc C bc C 2 C c c c 0 A6 A8 A10 AS6 AS8 AS10 AC6 AC8 AC10 Figure 2. Germination speed index (GSI) of coated perennial-soybean seeds and seedling emergence speed index (ESI).

Scarified seeds with six, eight and ten layers of sand (A6, A8 and A10); with six, eight and ten layers of sand + calcium silicate (AS6, AS8 and AS10); and with six, eight and ten layers of sand + limestone (AC6, AC8 and AC10).

80 Germination Emergence A

60 a

AB AB AB BC ab BCD 40 abc abc bc bc 20 CD CD D bc c bc Germination / Emergence (%) / Emergence Germination

0 A6 A8 A10 AS6 AS8 AS10 AC6 AC8 AC10 Figure 3. Germination percentage of coated perennial-soybean seeds and seedling emergence. Scarified seeds with

six, eight and ten layers of sand (A6, A8 and A10); with six, eight and ten layers of sand + calcium silicate (AS6, AS8 and AS10); and with six, eight and ten layers of sand + limestone (AC6, AC8 and AC10).

The sand + limestone combination used in coating provided greater resistance to the pellets. Regardless the number of layers, this treatment tended to generate the lowest GSI, ESI and germination and emergence percentages (Figures 2 and 3), though not differing statistically from the treatments with ten layers of sand. Considering that the portion and type of material used influence the diffusion of gases and water between the seed and the external environment (Nascimento et al., 2009), it can be stated that because sand and limestone share the same particle size (0.25 mm) and are denser, the increase in the number of sand + limestone layers also causes an increase in particle aggregation capacity and, with their perfect fit, a higher coating resistance is obtained. This makes it difficult for gas exchange. Therefore, the sand + limestone combination (AC6, AC8 and AC10) and the use of 250 g of sand (A10) cause an impairment in water uptake. Consequently, low percentages of formed seedlings were achieved in comparison to the other treatments (Figures 2 and 3). These results were also observed by Xavier et al. (2015) and Santos et al. (2010) in seeds of Stylosanthes and Brachiaria grasses, respectively. The values achieved in the coating treatments under different environmental conditions reveal a numerical decrease in the speed and formation of normal seedlings in the greenhouse compared to the number attained in laboratory. Given the reduced environmental control in a greenhouse (i.e. temperature and luminosity fluctuations, compared to the test conducted in laboratory, and how the emergence test was conducted [in trays with sand as a substrate]), it is believed that these variations delayed germination and seedling formation, since sudden changes in environmental Journal of Seed Science, v.42, e202042004, 2020 Digital image and physiological attributes of coated perennial-soybean seeds 9 conditions tend to deregulate the seed metabolism (Marcos-Filho, 2015), and the use of sand as a substrate is another barrier to be overcome by the seedling. The amount and material of filler used in seed coating did not significantly influence (p < 0.05) the growth of seedlings formed in the greenhouse after ninety days of sowing (Table 4). However, the treatment with six layers of sand stood out for the numerical gains obtained in shoot dry matter (2.91 g/pl) and root dry matter (5.54 g/pl) in relation to scarified-control treatment (1.36 g/pl and 4.30 g/pl, respectively for shoot and root dry matter). Thus, once again, the protective action of the seed coating is highlighted, where the six layers of sand promote a slower soak to the seed, reducing damage to the cell membrane and thus favoring the emergence and formation of plants. Despite not significantly differing from the other treatments, the combination of ten layers of sand + limestone provided a 15% gain in shoot length and a 52% gain in root length when compared to the scarified-control treatment, which provided 1.36 cm and 16.97 cm long shoots and roots, respectively. This result is believed to be related to the action of calcium on the plant metabolism, where it acts as an essential element that plays an important role in the division and elongation of plant cells (Ahmad et al., 2016). It should be emphasized that all coating treatments resulted in higher root length values than the scarified-control treatment (16.97 cm) (Table 4). Xavier et al. (2015) examined the growth of Stylosanthes cv. Campo Grande plants originating from seeds coated with different materials, including the sand + calcium silicate and sand + limestone combinations, and observed no significant effects of the tested treatments. However, seed coating provided numerical gains in plant growth, when compared to control treatment. To classify the coating quality, the evaluation criteria must be associated so that decisions are not made based solely on only one trait. It is emphasized that these criteria should represent the important physical and physiological traits for the evaluation of seed quality. Therefore, the geometric traits (area, maximum, minimum and corrected diameters and perimeter) pertaining to pellet size obtained by GroundEye® were correlated (r) with the physiological variables (germination, GSI, emergence and ESI) evaluated in laboratory and greenhouse conditions (Table 5). There was an inverse relationship between the physical and physiological variables; i.e., as the area, maximum, minimum and corrected diameter and perimeter increased, there was a significant decrease in germination and emergence percentages as well as in the speed of plant formation in laboratory (GSI) and greenhouse (ESI), for the treatments in which the seeds were coated with sand and sand + limestone. In the treatments involving sand + silicate, however, regardless of pellet size, there was no significant effect on the speed and formation of normal seedlings; i.e.,

Table 4. Plant growth parameters under greenhouse conditions, evaluated ninety days after sowing.

Treatments* Shoot length (cm) Dry shoot mass (g/pl) Root length (cm) Root dry mass (g/pl) A6 1.42 ± 0.381 2.91 ± 2.44 18.45 ± 0.97 5.54 ± 5.46 A8 1.28 ± 0.32 0.65 ± 0.31 18.59 ± 1.06 1.20 ± 0.68 A10 0.99 ± 0.21 0.20 ± 0.13 21.19 ± 2.74 0.32 ± 0.26 AS6 1.26 ± 0.32 1.86 ± 0.50 18.80 ± 0.79 3.49 ± 2.40 AS8 1.22 ± 0.39 2.27 ± 1.51 20.17 ± 3.93 2.12 ± 1.54 AS10 1.21 ± 0.29 1.42 ± 1.04 19.52 ± 1.58 2.28 ± 1.88 AC6 0.95 ± 0.66 2.22 ± 2.42 19.21 ± 1.70 2.89 ± 3.00 AC8 1.04 ±0.31 0.28 ± 0.10 17.23 ± 4.95 0.52 ± 0.26 AC10 1.56 ± 0.53 0.69 ± 0.69 25.74 ± 8.27 0.78 ± 0.75 Intact seeds 1.09 ± 0.40 1.36 ± 0.55 18.06 ± 1.34 1.74 ± 0.92 Scarified seeds 1.36 ± 0.32 2.87 ± 1.71 16.97 ± 1.35 4.30 ± 2.27 *Control treatments: intact and scarified seeds. Scarified seeds with six, eight and ten layers of sand (A6, A8 and A10); with six, eight and ten layers of sand + calcium silicate (AS6, AS8 and AS10); and with six, eight and ten layers of sand + limestone (AC6, AC8 and AC10). 1Means ± standard deviation (n = 4).

Journal of Seed Science, v.42, e202042004, 2020 10 A. J. Acha and H. D. Vieira sand + calcium silicate and the tested amounts of this combination did not significantly influence the physiological quality of the seeds when compared to the other treatments. Despite not being an element essential to plants, calcium silicate is known to cause alterations in chemical composition, cell mechanical resistance, tolerance to abiotic stresses and attack of pathogens and pests, making the seed more vigorous (Rodrigues et al., 2011). Therefore, silicate coating has great potential to be the base for the inclusion of fertilizers, insecticides and/or fungicides, thereby adding greater qualitative value to the seeds. In coated seeds, the increase in seed size leads to reduced germination and emergence speed because the material deposited on the seed becomes a physical barrier that needs to be overcome (Acha et al., 2016). Thus, in the present study, GroundEye® was efficient in evaluating the quality of the cover through physical evaluations related to the characteristics of the contour and seed size. These variables were used to estimate the physiological quality of perennial soybean seeds coated with the different materials tested. As such, it is a satisfactory piece of equipment for researchers seeking to improve coating techniques and large-scale companies, as it provides high accuracy and speed assessments and is not destructive. Upon the conclusion of assessments, it is observed that the combination of ten layers of sand + silicate provided satisfactory results for seed coating quality, considering the 280% increase in seed weight, facilitating mechanical sowing, and the increase in seed thickness, with gains in area, diameters and perimeter, which increase seed protection and facilitate the application of agricultural additives. This treatment is thus of great relevance, as it did not significantly interfere with the speed and formation of seedlings in laboratory and greenhouse conditions, in addition to had provided numerical gains in root growth, when compared to the other coating treatments tested.

Table 5. Correlation analysis Person’s (r) of the different tested materials, between the physical variables area, maximum diameter (MAXD), minimum diameter (MIND), corrected diameter (CORD) and perimeter; and the physiological variables germination (G), germination speed index (GSI), emergence (E) and emergence speed index (ESI).

Sand G GSI E ESI Area -0.870* -0.855* -0.778* -0.814* MAXD -0.869* -0.857* -0.788* -0.818* MIND -0.846* -0.816* -0.727* -0.768* CORD -0.885* -0.870* -0.701* -0.739* Perimeter -0.882* -0.864* -0.789* -0.823* Silicate + sand G GSI E ESI Area 0.215 0.175 -0.329 -0.393 MAXD 0.185 0.123 -0.301 -0.375 MIND 0.271 0.231 -0.294 -0.350 CORD 0.271 0.219 -0.295 -0.351 Perimeter 0.208 0.173 -0.331 -0.396 Limestone + sand G GSI E ESI Area -0.839* -0.807* -0.597* -0.652* MAXD -0.842* -0.813* -0.597* -0.651* MIND -0.831* -0.785* -0.586* -0.659* CORD -0.815* -0.773* -0.588* -0.666* Perimeter -0.832* -0.800* -0.605* -0.657*

*Significant according to Pearson’s (r) test at the 5% probability level.

Journal of Seed Science, v.42, e202042004, 2020 Digital image and physiological attributes of coated perennial-soybean seeds 11

CONCLUSIONS

Digital image analysis is efficient in precisely determining the physical traits of coated seeds, regardless of the material used. The correlation between the physical and physiological variables reveals that the progressive increase in the size of seeds coated with sand and sand + limestone negatively influences the speed and formation of normal seedlings in laboratory and greenhouse conditions. The sand + calcium silicate combination in the amount of 250 g, split into ten layers, is the most recommended material/quantity to coat seeds of perennial soybean.

ACKNOWLEDGMENTS

CAPES, FAPERJ, UENF.

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Journal of Seed Science, v.42, e202042004, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Enzyme activity in the micropylar region ofMelanoxylon brauna Journal of Seed Science, v.42, e202042009, 2020 Schott seeds during germination under heat stress conditions http://dx.doi.org/10.1590/2317- 1545v42229988 Marcone Moreira Santos*1 , Eduardo Euclydes de Lima e Borges2 , Glauciana da Mata Ataíde3 , Raquel Maria de Oliveira Pires4 , Debora Kelli Rocha4

ABSTRACT: Recent studies indicate that global temperatures will rise substantially in the 21st century, leading to the extinction of several plant species, as plant metabolism and germination are greatly affected by temperature. Melanoxylon brauna, a tree species native to the Atlantic Forest that occurs from northeastern to southeastern Brazil, is one of the many species threatened by global warming. Despite the economic and ecological importance of M. brauna, studies investigating the influence of heat stress on seed germination and biochemical responses are still incipient. This study aimed to evaluate enzyme activity in the micropylar region of M. brauna seeds during germination under heat stress conditions. Endo- β-mannanase, α-galactosidase, polygalacturonase, pectin methylesterase, pectin lyase, total cellulase, 1,3-β-glucosidase, and 1,4-β-glucosidase activities were determined in micropyles of seeds imbibed for 24, 48 and 72 h at 25, 35 and 45 °C. Seed germination was highest at 25 °C. Endo-β-mannanase activity was not detected under any of the experimental conditions, but imbibition temperature had a significant effect on the activity of all other enzymes.

Index terms: cell wall, enzyme, germination, temperature.

*Corresponding author Melanoxylon E-mail: marconemoreirasantos@ Atividade enzimática na região micropilar de sementes de hotmail.com brauna Schott durante a germinação sob estresse térmico Received: 10/14/2019. Accepted: 1/10/2020. RESUMO: Estudos recentes indicam alta na temperatura global no decorrer desse século, fato que pode ocasionar a extinção de diversas espécies vegetais, visto que processos como a germinação são influenciados pela temperatura. Dentre as espécies com risco de extinção está a Melanoxylon brauna, árvore nativa da Mata Atlântica de ocorrência nas regiões Nordeste 1Departamento de Ciência e Sudeste. Mesmo diante de sua importância econômica e ecológica, estudos referentes à Florestal, Universidade Federal germinação em condições de estresse térmico, bem como suas consequências bioquímicas Rural de Pernambuco, 52171-900 – nas diferentes partes das sementes, ainda são incipientes. Diante disso, objetivou-se avaliar a Recife, PE, Brasil. germinação e a atividade enzimática na região micropilar de sementes de M. brauna durante 2 a germinação sob estresse térmico. Avaliou-se germinação nas temperaturas de 25, 35 e Departamento de Engenharia Florestal, Universidade Federal de 45 °C. As atividades das enzimas endo-β-mannanase, α- galactosidase, polygalacturonase, Viçosa, 36570-900 – Viçosa, MG, Brasil. pectinametilesterase, pectinaliase, celulases totais, β-1,3- e β-1,4-glucosidases foram avaliadas em micrópilas embebidas por 0, 24, 48 e 72 h a 25, 35 e 45 °C. Houve maior germinação a 3Departamento de Ciências Agrárias, 25 °C. Não foi detectada atividade da enzima endo-β-mananase em nenhuma das condições Universidade Federal de São João avaliadas. A temperatura de embebição influenciou a atividade das demais enzimas. Del-Rei, 36307-352 – São João Del- Rei, MG, Brasil. Termos para indexação: parede celular, enzima, germinação, temperatura. 4Departamento de Sementes, Universidade Federal de Lavras, 37200-000 – Lavras, MG, Brasil.

Journal of Seed Science, v.42, e202042009, 2020 2 M. M. Santos et al.

INTRODUCTION

Melanoxylon brauna Schott is a tree native to the Brazilian Atlantic Forest. It occurs mainly in the southeast and northeast regions of the country. The tree has excellent wood properties, and its bark and sap are widely used in traditional medicine (Lorenzi, 2008). However, because of the overexploitation of its timber, the species is currently classified as vulnerable in the official list of endangered flora of Brazil (Brasil, 2014). Information on seed germination may contribute to the development of conservation strategies for M. brauna. A factor that may greatly affect the conservation status of M. brauna is climate change. Studies showed that global temperatures will rise by 1 to 5 °C over this century (PBMC, 2013). Will the species be able to adapt to the effects of global warming? What can be done to prevent M. brauna and other vulnerable tree species from becoming extinct? To answer these questions, it is fundamental to increase the knowledge about the influence of temperature on the different physiological processes involved in seed germination. Germination is mediated by a series of complex physical, physiological and biochemical processes. To emerge, the radicle must pass through the micropyle. Radicle emergence is preceded by the weakening of the micropylar endosperm and elongation of the embryonic axis (Yan et al., 2014). Although an essential part of seed germination, the mechanisms involved in the rupture of the micropylar endosperm, especially tissue weakening, are still little understood (Nonogaki et al., 2010). Such process is known to involve the loss of cell wall integrity by the action of hydrolases, transglycosylases, cellulases, hemicellulases, and reactive oxygen species (Borges et al., 2015; Koen et al., 2017; Singh et al., 2017). Enzymes activated at the initial stages of imbibition, such as β-mannanase, α-galactosidase (α-Gal), polygalacturonase (PG), pectin methylesterase (PME), pectin lyase (PL) and cellulase, are responsible for the degradation of cell wall polysaccharides and, consequently, the weakening of the micropylar endosperm, contributing to radicle emergence (Betts et al., 2017; Mascher et al., 2017). Temperature is a major factor influencing enzyme activity during germination. It can decrease, enhance, or even inhibit enzymatic processes and, therefore, accelerate or slow down seed metabolism (Laghmouchi et al., 2017). It may also influence the imbibition rate, altering the speed of chemical reactions that promote the mobilization of reserves and synthesis of necessary compounds for seedling growth. Thus, temperature is a determining factor for the occurrence of a species at a given locality (Medina et al., 2016). Considering the importance of M. brauna, the predictions of climate change, the influence of temperature on germination, and the lack of information about biochemical processes occurring in the micropylar region, this study aimed to assess the activity of endo-β-mannanase, α-Gal, PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase in the micropylar endosperm of M. brauna seeds during germination under heat stress conditions.

MATERIAL AND METHODS

Sample harvesting and preparation Fruits of M. brauna were harvested in Leopoldina (21°31’55”S 42°38’35”W), Minas Gerais, southeastern Brazil, in September 2015. The pods were sun dried, manually threshed, and the seeds cleaned. Empty or damaged seeds and debris were discarded. Healthy, intact seeds were selected and stored in a cold chamber at 5 °C and 60% relative humidity until use. The experiments were conducted between February and August 2016. A completely randomized design with five replications of twenty seeds per treatment was used.

Germination test Seeds were placed between two sheets of germination paper in petri dishes, moistened with distilled water, and incubated for 24, 48 and 72 h at 25, 35 or 45 °C in BOD incubators. Seeds were considered germinated upon radicle emergence.

Journal of Seed Science, v.42, e202042009, 2020 Enzyme activity in of Melanoxylon brauna seeds 3

Determination of enzyme activity Enzyme activity was determined in the micropylar region of dry seeds and seeds imbibed for 24, 48 and 72 h at 25, 35 and 45 °C. Micropyles were extracted (Figure 1), subjected to the same conditions as described above, and analyzed for enzyme activity. Endo-β-mannanase activity was determined by the gel-diffusion assay of Downie et al. (1994), with modifications. The gel was first washed with distilled water, then incubated with buffer solution for thirty minutes, and washed once more with distilled water. Congo red dye (0.5% w/v) was added, the gel was incubated for thirty minutes, washed with ethanol for ten minutes, and rinsed with distilled water. NaCl solution (1 M) was added until a white halo was observed where the samples were pipetted. α-Gal activity was quantified according to Borges et al., 2004. One unit of enzyme activity was defined as the amount of protein that releases 1 nmol of p‐nitrophenol per minute under the assay conditions. PG activity was determined by the 3,5-dinitrosalicylic acid (DNS) method as adapted by Miller (1959). One unit of PG activity was defined as the amount of protein that produces 1 μmol of galacturonic acid per minute of reaction. PME was extracted according to Pinto et al. (2011), and its activity was quantified according to Grsic-Rausch and Rausch (2004). One unit of PME activity is equivalent to the amount of enzyme required to produce 1 μmol of NADPH per minute of reaction at 25 °C and pH 7.5. PL activity was determined by the spectrophotometric method of Albersheim and Kilias (1962). Absorbance was read at 235 nm. Enzyme concentration was calculated using a molar absorption coefficient of 5550 L mol−1.cm−1 (Albersheim et al., 1996). Total cellulase activity was measured by the filter paper assay (Ghose, 1987). A 6 cm2 strip of filter paper was placed in a test tube containing 0.5 mL of sample (seed incubation solution) and 1.0 mL of 50 mM sodium acetate buffer pH 5.0. The reaction was interrupted by the addition of 1 mL of DNS. The concentration of reducing sugars was determined spectrophotometrically at 540 nm. 1,3-β-Glucosidase activity was determined using p-nitrophenyl β-d-glucopyranoside as substrate, and 1,4-β-glucosidase activity was determined using carboxymethyl cellulose as substrate. Reactions were conducted in 50 mM phosphate-buffered saline pH 6.0 (Singhania et al., 2013). Enzyme activity was expressed as units per gram of substrate. Proteins were quantified by the Bradford method (1976) using a standard curve (2.5–50 µg) of bovine serum albumin.

Figure 1. Representative photograph of a Melanoxylon brauna seed (A) and its micropylar region (B).

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Statistical analysis Data were subjected to analysis of variance followed by Tukey’s test at p < 0.05. Relationships between enzyme activity and germination conditions were investigated by regression analysis. Statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA).

RESULTS AND DISCUSSION

Radicle emergence was observed in seeds imbibed for 72 h. Germination was highest (83%) at 25 °C, decreasing to about 55% at 35 °C. Imbibition for 72 h at 45 °C led to complete loss of seed viability (Table 1). Temperature exerts a great influence on germination, as it regulates water absorption and biochemical reactions involved in seed metabolism (Bewley et al., 2013; Graeber et al., 2014). In general, seeds of subtropical and tropical species germinate at 20–30 °C (Oliveira et al., 2016; Silva et al., 2016). The germination behavior of M. brauna seeds observed in the current study agrees with that reported in the literature. Flores et al. (2014) found that M. brauna seeds were able to germinate between 12.3 and 42.5 °C, but not at 45 °C; the optimum temperature was 27 °C. Imbibition at 45 °C for 72 h was shown to cause irreversible damage to M. brauna seeds, impairing their germination, even when seeds were later transferred to 25 °C (Santos et al., 2017). Radicle emergence depends not only on the growth potential of the embryo, but also on the reduction in mechanical resistance in the micropylar region (Bewley et al., 2013). Although the physical and biochemical aspects involved in this process are not fully understood, it is known that hydrogen peroxide production, hydrolases, and cellulases have an important role in weakening the cell wall (Zhang et al., 2014; Santos et al., 2017). Enzyme activity was not observed in isolated micropyles imbibed for up to 72 h. These results suggest that hydrolases and cellulases are produced in the embryonic axis and transferred to the micropylar region during germination, which reinforces the hypothesis that these enzymes contribute to radicle emergence by weakening the micropylar region. Endo-β-mannanase activity was not detected under any of the evaluated conditions. Previous reports showed that its activity is intensified at the end of the germination process, mainly during seedling formation (Ferreira et al., 2018). The activity of all other enzymes differed significantly with temperature. α-Gal activity was highest at 25 and 35 °C (Figure 2A). PG, PME and PL showed higher activities after 72 h of imbibition at 45 °C (Figures 2B, 2C and 2D). These enzymes are crucial for cell wall degradation and radicle emergence (Borges et al., 2015; Bicalho et al., 2016). α-Gal activity depends on environmental conditions, including temperature (Coffigniez et al., 2018). In M. brauna seeds, the highest activity occurred at the optimal germination temperature (25 °C). In addition to metabolizing carbohydrate reserves, α-Gal hydrolyzes cell wall polysaccharides and raffinose family oligosaccharides, thereby providing energy for germination (Bicalho et al., 2016; Farias et al., 2015). The low enzyme activity found at 45 °C shows that α-Gal is sensitive to heat stress. Similar to the observed in the current study, α-Gal activity was highest at 25 °C in Dalbergia nigra seeds (Ataíde et al., 2016). PG is essential for the germination of Schizolobium parahyba and Arabidopsis sp. seeds (Magalhães et al., 2009; Han and Yang, 2015; Scheler et al., 2015). The enzyme catalyzes the hydrolysis of 1,4-α-glycosidic bonds between galacturonic acid residues in the pectin chain. In the present study, PG activity was higher at 45 °C for all imbibition

Table 1. Germination percentage of Melanoxylon brauna seeds at different temperatures.

Temperature (ºC) Germination (%) 25 83 a 35 55 b 45 0 c Means followed by different letters differ significantly by Tukey’s test at p < 0.05.

Journal of Seed Science, v.42, e202042009, 2020 Enzyme activity in of Melanoxylon brauna seeds 5 times. The optimum germination temperature does not always coincide with the optimum temperature for enzyme activity. In D. nigra seeds, PG activity peaked at 40, 45 and 60 °C, but germination percentage was highest at 25 °C (Ataíde et al., 2016). In fruits of Uapaca kirkiana, Ziziphus mauritiana, Tamarindus indica and Berchemia, the optimum temperature range for PG activity was shown to be 25 to 37 °C (Muchuweti et al., 2005). In Prunus persica fruits, PG activity was highest during imbibition at 35 °C (Sainz and Vendrusculo, 2015). PME and PG have related functions. PME catalyzes the de-esterification of pectic substances by hydrolyzing methyl ester groups, producing pectin with a lower degree of methylation, which is then used as a substrate by PG (Sainz and Vendrusculo, 2015). PME activity increased after 24 h of imbibition at 25, 35 and 45 °C, but decreased after 48 h at 35 °C. At the basis of these results, it is possible to infer that the enzyme is produced before germination and is associated with the weakening of the seed coat and the degradation of the micropylar endosperm. These results agree with those obtained by Borges et al. (2015). The authors reported an increase in PME activity during imbibition of M. brauna seeds at 30 °C. PME activity was detected in Lepidium sativum seeds, suggesting that the enzyme plays an important role in testa rupture during radicle emergence (Scheler et al., 2015). PL activity was highest at 45 °C and increased with imbibition time at all temperatures. PL breaks down oligogalacturonides of the cell wall, deteriorating the lateral endosperm. The enzyme also induces the synthesis of expansins, which are mediators of the germination process (Zhao et al., 2008; Cao, 2012; Sainz and Vendrusculo, 2015).

Asterisks (*) indicate statistically significant differences between means. Vertical bars represent the standard error of the mean (n = 5). Figure 2. α-Galactosidase (A), polygalacturonase (B), pectin methylesterase (C) and pectin lyase (D) activities in the micropylar region of Melanoxylon brauna seeds during imbibition at 25, 35 and 45 °C.

Journal of Seed Science, v.42, e202042009, 2020 6 M. M. Santos et al.

Although hydrolases play a fundamental role in M. brauna seed germination, under heat stress (45 °C), high PG, PME and PL activities may contribute to seed deterioration. It is possible that an excessive increase in enzyme activity enhanced reserve degradation, accelerating the loss of cell wall integrity and increasing damage to cell membranes (Santos et al., 2017). Total cellulase and 1,4-β-glucosidase activities were highest at 35 °C after 48 h of imbibition (Figures 3A and 3C). This temperature also favored 1,3-β-glucosidase activity, which was found to increase with imbibition time (Figure 3B). At 45 °C, 1,3-β- and 1,4-β-glucosidase activities were lowest after 72 h of imbibition. This result is likely due to the loss

Asterisks (*) indicate statistically significant differences between means. Vertical bars represent the standard error of the mean (n = 5). Figure 3. Total cellulase (A), 1,3-β-glucosidase (B) and 1,4-β-glucosidase (C) activities in the micropylar region of Melanoxylon brauna seeds during imbibition at 25, 35 and 45 °C. Journal of Seed Science, v.42, e202042009, 2020 Enzyme activity in of Melanoxylon brauna seeds 7 of seed vigor caused by heat stress, resulting in protein denaturation and loss of enzyme activity (Santos et al., 2017). Cellulases are responsible for the degradation of cellulose, a major component of the plant cell wall. β-Glucosidases break the chemical bond between the glucose units of cellobiose, releasing free glucose. By doing so, they contribute to the weakening of the micropylar endosperm and provide energy for radicle emergence, as observed in seeds of Coffea arabica (Castro and Pereira, 2010), Lactuca sativa (Chen et al., 2016) and L. sativum (Ogórek, 2016). Enzyme activity was related to germination percentage, but was less affected by high temperatures. The increase in enzyme activity in heat-stressed seeds probably contributed to cell wall degradation, leading to the accumulation of reactive oxygen species and membrane damage (Santos et al., 2017). The results show that PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase activities can be used to assess the physiological quality of M. brauna seeds.

CONCLUSIONS

M. brauna seeds showed optimal germination at 25 °C. Imbibition at 45 °C for 72 h resulted in the death of all seeds. Endo-β-mannanase activity was not detected after 72 h of imbibition at any of the tested temperatures. α-Gal activity was highest after 48 h of imbibition at 25 °C and lowest after 24 h at 45 °C. PG activity was highest after 48 h of imbibition at 45 °C and lowest after imbibition at 25 °C. PME and PL activities increased during 72 h of imbibition at 45 °C, but decreased during imbibition at 25 °C. Total cellulase, 1,3-β-glucosidase, and 1,4-β-glucosidase activities where highest during the first hours of imbibition at 45 °C, but decreased markedly after 48 h. High PG, PME, PL, total cellulase, 1,3-β-glucosidase and 1,4-β-glucosidase activities during imbibition indicate the occurrence of heat stress in M. brauna seeds.

ACKNOWLEDGMENTS

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Pró-Amazonas Project, nº 52), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for their financial support.

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Journal of Seed Science, v.42, e202042009, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Vigor test of (strong) normal intact Amburana cearensis Journal of Seed Science, v.42, e202042011, 2020 (Allemão) A.C. Smith seedlings http://dx.doi.org/10.1590/2317- 1545v42221611 Josenilda Aprígio Dantas de Medeiros1 , Sarah Patrícia Lima Nunes1 , Francival Cardoso Félix1 , Cibele dos Santos Ferrari1 , Mauro Vasconcelos Pacheco1 , Salvador Barros Torres2*

ABSTRACT: The aim of this study was to adapt the vigor test methodology of (strong) normal intact seedlings of Amburana cearensis and evaluate efficiency in physiological classification of seed lots. The study was conducted in two stages: morphological characterization of seedlings and physiological analysis of seed lots. To do so, the following tests were carried out: (strong) normal seedlings, germination, emergence, first count, germination speed index, tetrazolium, length of strong normal seedlings, length and dry mass of seedlings. The experimental design was completely randomized. The Tukey and Kruskal-Wallis tests were used to compare the results, and the correlation between the variables was analyzed by Spearman and Pearson coefficients. The seedlings of A. cearensis are semi-hypogeal phanerocotylar, with development of normal seedlings on the ninth day after sowing. The vigor test of strong normal seedlings, length of strong normal seedlings, dry matter, and tetrazolium led to physiological classification into different vigor levels. The vigor test of (strong) normal seedlings, the length of strong normal seedlings, and tetrazolium (vigor) were effective for vigor classification of A. cearensis seeds.

Index terms: Fabaceae, seed quality, forest seed.

Teste de vigor de plântulas normais intactas (fortes) de Amburana cearensis (Allemão) A.C. Smith

RESUMO: Objetivou-se adequar a metodologia do teste de vigor de plântulas normais intactas (fortes) de Amburana cearensis e avaliar a eficiência na classificação fisiológica de *Corresponding author lotes de sementes desta espécie. O estudo foi conduzido em duas etapas: caracterização E-mail: [email protected] morfológica de plântulas e análise fisiológica dos lotes de sementes. Para isso, realizaram-se Received: 3/22/2019. os testes de vigor de plântulas normais fortes, germinação, emergência, primeira contagem, Accepted: 11/5/2019. índice de velocidade de germinação, tetrazólio, comprimento de plântulas normais fortes, comprimento e massa seca de plântulas. O delineamento experimental utilizado foi o inteiramente casualizado. Para comparação dos resultados foram realizados os testes de Tukey e Kruskal-Wallis, sendo a correlação entre as variáveis analisadas pelos coeficientes de 1Unidade Acadêmica Especializada Spearman e Pearson. As plântulas de A. cearensis são do tipo semi-hipógea fanerocotiledonar, em Ciências Agrárias, Universidade com presença de plântulas normais ao nono dia após a semeadura. O teste de vigor de Federal do Rio Grande do Norte, Caixa Postal 07, 59280-000 – plântulas normais fortes, comprimento de plântulas normais fortes, massa seca e tetrazólio Macaíba, RN, Brasil. proporcionaram classificação fisiológica em diferentes níveis de vigor. O teste de vigor de plântulas normais fortes, o comprimento de plântulas normais fortes e o teste de tetrazólio 2Departamento de Ciências (vigor) são eficientes para a classificação do vigor de sementes de A. cearensis. Agronômicas e Florestais, Universidade Federal Rural do Semi-Árido, 59625- Termos para indexação: Fabaceae, qualidade de sementes, sementes florestais. 900 – Mossoró, RN, Brasil.

Journal of Seed Science, v.42, e202042011, 2020 2 J. A. D. Medeiros et al.

INTRODUCTION

A plant native to the Caatinga (xeric shrubland) ecosystem, Amburana cearensis (Fabaceae) is known as cumaru or amburana-de-cheiro (Almeida et al., 2017). The diverse marketable products obtained from this species come from predatory extractivism (Campos et al., 2013). A. cearensis has been used for food purposes and in creation of soaps and perfumes, but it is also the object of pharmacological studies, which have confirmed the effectiveness of bark extracts as antiseptics against bacteria (Sá et al., 2014) and for anti-inflammatory activity (Lopes et al., 2013). Morphological characteristics of seedlings can provide important information on the quality of seed lots, since the seedling represents the initial phase of embryo development (Souza et al., 2018). Seedling vigor tests are promising for evaluating the physiological quality of seed lots, for they are able to predict the development and establishment of seedlings in the field. Normal seedlings are those that have all their essential structures (root system and shoots) with potential for continuing their development and giving rise to normal plants. Normal seedlings can be classified as intact seedlings, seedlings with small defects, and seedlings with secondary infection. Intact normal seedlings can be defined as those that have developed, complete, and healthy essential structures (Brasil, 2013), and as such can also be considered “strong normal seedlings”, whereas seedlings with small defects and/or secondary infection can be classified as “weak normal seedlings” (Krzyzanowski and Nakagawa, 1999). Thus, these authors also affirm that seed lots that have a higher percentage of intact normal seedlings have greater chances of emerging and producing normal plants under adverse field conditions. Characterization of seedlings presented in the Rules for Seed Testing is generalized (Brasil, 2009), since there is no definition of parameters for specific description of the morphology of strong (intact) normal seedlings, weak normal seedlings (with small defects, with secondary infection), and abnormal morphology for native forest species. Therefore, parameters that describe seedling morphology in a detailed manner are necessary with the aim of increasing the effectiveness of the seedling vigor classification test for forest species. Seedling classification as an evaluative indicator of vigor is a parameter in which seed lots resistant to adversities in the field are detected. It is also an even more specific criterion that is in accordance with the morphological characterization of each species, making vigor classification even more reliable and rigorous (Oliveira et al., 2009). Thus, the aim of this study was to adapt the methodology of the seedling vigor test of Amburana cearensis and evaluate its effectiveness in physiological classification of seed lots.

MATERIAL AND METHODS

The study was developed with lots of A. cearensis seeds coming from the municipalities of Soledade, PB (7°3’25’’ S and 36°21’46’’ W; altitude: 521 m) and Petrolina, PE (9°23’34’’ S and 40°30’ 8’’ W; altitude 376 m). In the first stage of experiment, morphological study of the seedlings was performed, analyzing the structures with the aid of a magnifying glass and Munsell Color Chart (Munsell Color, 1963) for determination of color. The seeds were disinfested in 2.5% sodium hypochlorite for two minutes (Brasil, 2013) and treated with the fungicide Dithane NT (4 g.kg-1 of seed). After that, a hundred seeds were sown in sheets of paper towel (Germitest®), organized in rolls, and placed in a Biochemical Oxygen Demand (B.O.D.) germination chamber at 25 °C with a 12-h photoperiod. Morphological description of the seed germination process proceeded through daily observation and recording of images, from primary root emergence to formation of the first leaves. Formation of a normal seedling was the criterion of germination. Morphological classification of the seedlings was made in accordance with Miquel (1987), considering the position of the cotyledons in germination, the type of cotyledon, and the persistence of seed coat. For morphometry, all the parts that compose the seedling (root, hypocotyl, epicotyl, cotyledons and eophiles) were measured with a millimeter ruler, aiming to create an evaluation chart for seedling classification. In the second stage of this study, seeds from five A. cearensis seed lots were used for evaluation of physiological quality. The moisture content of the seeds from each seed lot was determined from two replications of 4.5 g through Journal of Seed Science, v.42, e202042011, 2020 Seedling vigor test in Amburana cearensis 3 the laboratory oven method at 105 °C/24 h (Brasil, 2009) and results were obtained in percentage. To carry out the germination tests, two hundred seeds (8 x 25) from each lot were incubated in a B.O.D. germinator at 25 °C and 12-h photoperiod over thirty days, and the results were expressed in percentage of normal seedlings (strong and weak). For the field emergence test, two hundred (4 x 50) seeds from each seed lot were distributed in a seed bed in full sun, with subsoil as a substrate. On the thirtieth day after sowing, the percentages of emerged normal seedlings (strong and weak) were calculated (Brasil, 2013). The germination speed index (GSI) was elaborated from daily counting of normal seedlings, and results were calculated according to the equation proposed by Maguire (1962). Regarding first germination count, seeds that gave rise to normal (strong and weak) seedlings were counted on the 11th day after sowing, and results were expressed in percentage. For seedling length and dry matter, the seedlings were measured with a millimeter ruler, and results were expressed in cm.seedling-1. The seedlings without the cotyledons were placed to dry in a forced air ventilation oven at 65 °C for 48 hours. For the tetrazolium test, the seeds were pre-hydrated in distilled water at 35 °C for 24 hours in order to remove the integument and facilitate staining of the seeds. After that, four replications of 25 seeds were submersed in a 0.075% tetrazolium solution for three hours at 25 °C (Guedes et al., 2010). Then the seeds were classified in four classes, according to França-Neto et al. (1998): class 1 (viable – highly vigorous) – pink colored seeds; class 2 (viable – vigorous) – seeds that have up to 40% of the cotyledons with strong carmine red or milky white color and the central cylinder with pink color; class 3 (viable – not vigorous) – more than 40% strong carmine red or milky white color in the cotyledons and around the central cylinder, which remains pink; and class 4 (not viable) – strong carmine red and milky white color in the central cylinder. For evaluation of viability, the percentage of seeds in classes 1, 2 and 3 was considered; whereas for vigor classification, the percentage in classes 1 and 2 was considered (Figure 1). Seedlings were classified with the aid of an evaluation chart elaborated from the morphometry of the seedlings at the end of the germination test. The chart was organized on the following basis: adaptation of the methodology of Krzyzanowski and Nakagawa (1999), who considered the terminologies of strong and weak normal seedlings for seeds of cultivated plants; the criteria of normal seedlings (intact, with small defects, and with secondary infection) and abnormal seedlings described in the Rules for Seed Testing (Brasil, 2009); and the morphological description and morphometry of seedlings of the species. From morphological and morphometric characterization of the seedlings, with the assistance of the chart, the seedlings were classified as strong normal seedlings, weak normal seedlings, and abnormal seedlings. The morphometry of strong normal seedlings consisted of measuring each component part of the seedling (root, hypocotyl, epicotyl, eophiles, and cotyledons), and the evaluation chart was elaborated based on these data. The classification of strong normal seedlings was given to all seedlings that exhibited proportionality and intact essential structures according to the selection criteria of the evaluation chart, such as root length within the interval of 5-12.8 cm, based on morphometry, and mean seedling length greater than or equal to nineteen cm.

Class 1 – pink color; class 2 – up to 40% of the cotyledons with strong carmine red or milky white color and the center cylinder with pink color; class 3 – more than 40% strong carmine red or milky white color in the cotyledons and around the center cylinder, which remains pink; class 4 – strong carmine red and milky white color in the center cylinder. Figure 1. Classification of Amburana cearensis seeds for evaluation of viability and vigor through the tetrazolium test.

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Seedlings that had “small defects”, such as necrosis spots on the roots and/or eophiles, mild twisting in hypocotyl, three cotyledons, root length less than five cm, and total seedling length less than 19 cm, were considered weak normal seedlings for the species under study. In seedling morphology, abnormal seedlings were those that exhibited twisting in the root collar and root, spiral shape, necrosis and albinism. Following the morphometric chart, seedlings classified as strong normal (intact plants) were counted for each lot, and data were expressed in percentage. After that, a complementary variable was joined with the strong normal seedling vigor test (SV), the length of strong normal seedlings (L-NS), in which the seedlings classified as strong normal seedlings were measured, and the result was expressed in cm.seedling-1. The test of strong normal seedling vigor (SV) and length of strong normal seedlings (L-NS) were correlated with the other vigor tests (first germination count, GSI, emergence, tetrazolium, seedling length, and seedling dry matter). Descriptive statistical analysis and frequency distribution were performed on the data regarding morphometry of the seedlings (all the parts that compose the seedling). For physiological characterization of the seeds of each seed lot, a completely randomized experimental design was used, with comparative tests (Tukey test at 1% probability) for parametric data and the Kruskal-Wallis test at 5% probability for non-parametric data, applied to the mean values of the lots for each variable separately. Due to the aim of adapting the methodology of the test of strong normal seedling vigor, correlation was considered only for the variables “strong normal seedlings” and “length of strong normal seedlings” with the “other vigor tests” using the Spearman and Pearson coefficients to check the levels of physiological quality of the seeds of each lot. For the correlation classification criteria, a “positive” or “negative” value of the r coefficient will indicate a positive or negative association, respectively, between the variables. The greater the absolute value of the coefficient, the stronger the relationship between the variables – when the r value was from 0.0 to 0.39, the correlation was considered “weak”; from 0.4 to 0.69, “moderate”; and from 0.7 to 1, “strong”. The statistical software with Bioestat 5.0 was used in all the analyses (Ayres et al., 2007).

RESULTS AND DISCUSSION

Germination of A. cearensis seeds is in accordance with the semi-hypogeal phanerocotylar type, with primary root emergence on the fourth day and formation of normal seedlings on the ninth day after sowing (Figure 2). The root is cylindrical and initially of light gray color (10Y 8/8), later turning pale yellow (5Y 8/3); furthermore, it has a thin root cap of copper color (5YR 8/6) that is difficult to see with the naked eye (Figure 3A). This detail of the structure was not found by Cunha and Ferreira (2003); however, Loureiro et al. (2013) described it. The hypocotyl is short and of yellowish green color (5GY 7/4), with lenticels across its entire length and rapid growth from the fifth to the ninth day after sowing (Figure 2). The epicotyl is initially of light gray color (10Y 8/8), turning to yellowish green (5GY 7/4), with lenticels in the basal region and cataphylls in the intermediate region and/or near the eophiles. The cataphylls are specialized leaves and, in this case, they protect the lateral buds (Figure 3B). The cotyledons are opposite each other, isophyllous, and fleshy, with an oblong shape, and petiolate, with striation along the petiole (Figure 3C). They are initially of light gray color (10Y 8/8) and, after that, yellowish green (5GY 7/4), findings similar to those described by Loureiro et al. (2013). The eophilesare imparipinnate, with five to seven leaflets with a short petiole (Figure 3D). They initially develop folded over, giving the impression of a pointed tip, which explains the difference from the morphological description made by Cunha and Ferreira (2003), because when the leaflets are fully open, they have an elliptical shape with a rounded base and tip and brochidodromous venation, yellowish green color (5GY 7/4), and short hairs spaced out on the abaxial surface (Figure 3E). These observations show that there is divergence from the description made by Loureiro et al. (2013), who indicate leaves with a sharp tip and penniveined venation. Seedling morphometry shows only the epicotyl near zero in asymmetry and kurtosis simultaneously (Table 1). Journal of Seed Science, v.42, e202042011, 2020 Seedling vigor test in Amburana cearensis 5

ca – cataphyll, co – cotyledons, eo – eophiles, ep – epicotyl, hy – hypocotyl, rt – root. Figure 2. Germination process of a normal seedling of Amburana cearensis.

rc – root cap, ca – cataphyll, ab – axillary bud. Figure 3. Primary root of Amburana cearensis with root cap (A), cataphyll (B), cotyledon (C), eophiles in development (D), and venation of leaflet (E).

Table 1. Descriptive statistics of morphometry of strong normal seedlings of Amburana cearensis. Min. Max. Mean ± Error Variable Variance Standard CV (%) Asymmetry (G1) Kurtosis (G2) (cm) Deviation Root 5.0 12.8 7.78 ± 0.31 2.6 1.6164 21 1.0966 2.0741 Hypocotyl 0.5 1.3 0.91 ± 0.05 0.1 0.2825 31 0.0453 -1.467 Epicotyl 6.4 14.0 10.30 ± 0.35 3.5 1.8826 18 0.0910 -0.1767 Eophiles 0.2 3.0 0.93 ± 0.05 0.2 0.4669 51 1.6307 4.1869 Cotyledon length 1.4 2.9 2.11 ± 0.06 0.1 0.3202 15 0.0006 0.6583 Cotyledon width 0.9 1.4 1.06 ± 0.02 0.0 0.1160 11 0.8427 1.1003

Asymmetry in all the variables proved to be positive, and kurtosis is leptokurtic for the hypocotyl and epicotyl and platykurtic for roots, eophiles, and length and width of the cotyledons (Table 1). In statistical distribution, asymmetry determines the frequency of the distribution between values of 1 and -1, in which deviation from the median and Journal of Seed Science, v.42, e202042011, 2020 6 J. A. D. Medeiros et al. mode is observed in relation to the mean. Kurtosis measures the dispersion of the values, characterizing the degree of flatness of the frequency distribution (Acchile et al., 2017). Roots were considered an important factor for differentiation between strong and weak normal seedlings, and their platykurtic distribution indicates low concentration of length values in the center (medial axis), with significant distribution below and above the mean, which allowed seedlings with root length from 5 to 12.8 cm to be classified as normal. The hypocotyl, however, was not significant for differentiation between strong and weak normal seedlings since its leptokurtic distribution indicates high concentration of values in the center and tail curve, i.e., low variance in length, making it impossible to classify seedlings by the criterion of hypocotyl length. Strong normal seedlings of A. cearensis had proportional structures, with isophyllous cotyledons, in which pairs of them have the same length and width. Just as the cotyledons, the eophiles and the hypocotyl had a high coefficient of variation (51 and 31%, respectively). This fact is probably due to the small number of structures evaluated, since, in the species studied, investment of energy expenditure apparent in primary growth of the root and later in the epicotyl is observed, and eophiles develop from half of the growth process of the seedling on. The most recurrent weak normal seedlings of A. cearensis exhibited a slight twist in the collar, the region of transition of the hypocotyl and root; necrosis spots on the root, leading to its bifurcation; and development of axillary buds at the point of connection of the cotyledons to the stem when the epicotyl and/or cataphyll were damaged, leading to the formation of two new epicotyls identical to the epicotyl of seminal origin (Figure 4). This development of buds is not reported in the Rules for Seed Testing (Brasil, 2009) and, as this case was monitored after the germination test, it was found that individuals generated healthy and well-developed seedlings. Thus, these individuals can be classified as weak normal seedlings. The most frequent abnormalities in the seedlings were those arising from necrosis and accentuated twisting of the collar and of the roots. Necrosis in only the eophiles did not affect epicotyl growth, and, in addition, there was growth of new eophiles from the bud protected by the cataphyll of the epicotyl of seminal origin; these were characterized as weak normal seedlings. Necrosis was also found on the epicotyls arising from the axillary buds, still in initial growth, characterizing them as abnormal seedlings. The moisture contents of the seed lots were similar (≤ 0.8% among lots), (Table 2). This nearness in moisture content is essential so that there is no interference in the result of the tests of physiological quality, recommending that the difference in these values be less than 2% (Marcos-Filho, 2015; Javorski et al., 2018).

bi – bifurcation, ne – necrosis, tw – twisting. Figure 4. Classification of Amburana cearensis seedlings into strong normal (SD-SN), weak normal (SD-WN), and abnormal (SD-AB) categories.

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Table 2. Moisture content (MC), germination (G), emergence (E), seed viability and vigor by tetrazolium (T-VB and T-VG), first germination count (FGC), strong normal seedling vigor (SV), germination speed index (GSI), seedling length (SD-L), length of strong normal seedlings (L-NS), and seedling dry matter (SD-DM) of Amburana cearensis.

Seed lot Variable 1 2 3 4 5 MC (%) 7.2 6.7 6.9 6.4 6.8 G* (%) 82 ± 1.7 ab 70 ± 3.8 b 83 ± 5.4 ab 87 ± 2.6 a 79 ± 2.9 ab E (%) 79 ± 2.2 a 78 ± 8.8 a 83 ± 4.4 a 81 ± 3.3 a 72 ± 5.1 a T-VB* 86 ± 2.6 a 93 ± 3.4 a 92 ± 3.2 a 98 ± 1.2 a 92 ± 3.2 a T-VG ** 69 ± 3.8 b 78 ± 3.8 ab 69 ± 3.4 b 92 ± 3.3 a 69 ± 5 b FGC* (%) 4 ± 1.6 ab 10 ± 2.6 a 1 ± 0.5 b 4 ± 1.2 ab 3 ± 1.5 ab SV** (%) 45 ± 5.7 b 51 ± 4.6 ab 29 ± 3.0 b 65 ± 3.5 a 51 ± 5.8 ab GSI** 1.06 ± 0.0 ab 1.22 ± 0.1 a 0.90 ± 0.1 b 1.18 ± 0.0 a 1.15 ± 0.0 a SD-L** (cm.seedling-1) 15.9 ± 0.6 ab 15.6 ± 1.0 ab 14.7 ± 1.1 b 19.9 ± 0.8 a 16.4 ± 0.9 ab L-NS** (cm.seedling-1) 9.4 ± 1.6 b 11.9 ± 1.2 ab 7.0 ± 1.2 b 15.5 ± 1.0 a 11.1 ± 1.4 ab SD-DM** (mg.seedling-1) 72.9 ± 2.0 b 73.6 ± 3.4 ab 72.5 ± 4.6 b 88.6 ± 4.6 a 78.4 ± 1.4 ab *Mean values followed by the same letters in the row do not differ statistically from each other by the Kruskal-Wallis Test at 5% probability. **Mean values followed by the same letters in the row do not differ statistically from each other by the Tukey Test at 1% probability.

Germination showed a statistical difference only between lots 2 and 4, in which lot 2 expressed low viability in relation to lot 4, without statistical differences being observed (at 5% probability) of these lots compared to lots 1, 3 and 5 (Table 2). In the germination test, hard and dormant seeds were not observed, and the ungerminated seeds were dead and/or in an advanced state of contamination by microorganisms. Seedling emergence did not show a statistical difference at 5% probability among the seed lots, while first germination count and the germination speed index (GSI) were statistically similar to the results of lot 4, in spite of the inferior result of germination for the seeds of lot 2 (Table 2). The GSI of the A. cearensis seeds ranged from 0.9 to 1.22, indicating that the values near zero emphasize the need for a minimum of thirty days for final evaluation of germination. In spite of that, the GSI was able to differentiate physiological quality, showing that the seeds of lots 1, 2, 4 and 5 have higher speeds of germination than those of lot 3 (Table 2). In first germination count, difference was found only between lots 2 and 3 (10% and 1%, respectively). The low germination percentage in the first count is characteristic of the species, which has a slow and uneven germination process, as observed in the results of germination speed (Table 2). The first count and GSI tests are interdependent because the greater percentage of seedlings in the first evaluation means seed germination speed was rapid in a determined lot (Nakagawa, 1994). This is confirmed in the results of this study, in which vigor levels obtained in the first germination count and in GSI of seeds from the different lots were similar (Table 2). The length of seedlings coming from seeds of lot 4 was significantly greater than that of seedlings coming from lot 3. It was found that the length of strong normal seedlings and dry matter had similar rankings, with the seeds from lot 4 resulting in greater length and dry matter (15.5 cm.seedling-1 and 88.6 mg.seedling-1, respectively) of seedlings compared to those of lots 1 and 3 (9.4 and 7 cm.seedling-1; 72.9 and 72.5 mg.seedling-1) (Table 2). In the seedling length and dry matter tests, it should be considered that seeds from the most vigorous lots give rise to seedlings of greater length and dry matter, due to greater capacity for transformation of plant tissues and translocation of reserves (Amaro et al., 2015) from the cotyledons or from the endosperm for growth of the embryonic axis. Viability by the tetrazolium test, which considered all the seeds from classes 1, 2 and 3, was also not effective for evaluating quality differences among the seed lots (Table 2). Nevertheless, in evaluation of vigor by the tetrazolium Journal of Seed Science, v.42, e202042011, 2020 8 J. A. D. Medeiros et al. test, in which the total percentage of seeds of only classes 1 and 2 was considered, physiological ranking was found in three vigor levels: lot 4 was considered to be of high vigor because it was superior to lots 1, 3 and 5 (lots of low vigor), but did not differ from the seeds of lot 2, whose quality was considered to be intermediate (Table 2). In the seedling vigor test, the seeds of lot 4 gave rise to 65% strong normal seedlings, which was superior to the seeds of lots 1 (45%) and 3 (29%), which did not differ from each other and were considered to be of low vigor. In contrast, the seeds from lot 2 and 5 (both with 51% strong normal seedlings) did not differ from each other and were classified as medium vigor (Table 2). Tests based on seedling performance have been increasingly studied for evaluation of seed physiological quality. These tests are not simply complementary to the germination test, but protagonistic, in light of their speed and effectiveness, especially the seedling length test (Sena et al., 2015). The test of primary root length of A. cearensis seedlings was recommended over other vigor tests for evaluation of physiological quality due to its sensitivity for classification in vigor levels (Guedes et al., 2015). Correlations among the physiological vigor tests (first germination count, tetrazolium, seedling length, and seedling dry matter) and the strong normal seedling vigor test were significant only for seedling length, length of strong normal seedlings, and GSI. The two variables for length had strong positive correlation, in which strong normal seedling length had greater correlation (r = 0.9275) (Table 3). Therefore, the variables analyzed are proportional. Length of strong normal seedlings had correlation with the other variables only for total seedling length, vigor of strong normal seedlings, and GSI (Table 3). The fact that the correlation is positive implies that the greater the length of the strong normal seedlings, the greater seedling length, strong normal seedling vigor, and GSI will be. The Spearman and Pearson correlation coefficient ranges from -1 to 1; the nearer 1 or -1, the stronger the correlation, and proximity to zero shows that correlation is weak. Correlation for purposes of evaluation of the efficacy of vigor tests can lead to incomplete information since its significance indicates similarity of variation between two variables, but not necessarily their precision in estimation of

Table 3. Correlation between strong normal seedling vigor of Amburana cearensis (SV) and length of strong normal seedlings (L-NS) and the other variables analyzed.

Correlation Coefficient (r) P Correlation SV (%) X L-NS (cm.seedling-1) 0.9275* < 0.0001 Strongly positive L-NS (cm.seedling-1) X SD-L (cm.seedling-1) 0.7918* < 0.0001 Strongly positive SV (%) X SD-L (cm.seedling-1) 0.7857* < 0.0001 Strongly positive GSI X L-NS (cm.seedling-1) 0.5816* < 0.0001 Moderately Positive GSI X SV (%) 0.5646* < 0.0001 Moderately Positive SV (%) X FGC 0.1475* 0.3638 Ns SV (%) X SD-DM (mg.seedling-1) 0.3349* 0.0346 Ns SV (%) X E (%) 0.1805** 0.4463 Ns SV (%) X T-VG (%) -0.052** 0.8276 Ns L-NS (cm.seedling-1) X FGC (%) 0.1561* 0.3362 Ns L-NS (cm.seedling-1) X SD-DM (mg.seedling-1) 0.2555* 0.1114 Ns L-NS (cm.seedling-1) X E (%) -0.1673* 0.302 Ns L-NS (cm.seedling-1) X T-VG (%) -0.2705* 0.0913 Ns FGC: first germination count; GSI: germination speed index; SD-L: seedling length; SD-DM: seedling dry matter; E: emergence; T-VG: tetrazolium test in vigor classification. *Spearman coefficient. **Pearson coefficient. Ns: not significant.

Journal of Seed Science, v.42, e202042011, 2020 Seedling vigor test in Amburana cearensis 9 the physiological quality of seeds themselves. Therefore, correlation should be applied as a complementary statistical model to understand the variations among the tests evaluated in a determined study (Sena et al., 2015). In the present study, correlation was not carried out between the viability (germination) test and the vigor tests, because the intent was to check if the physiological ranking of the “strong normal seedling vigor” (SV) and “length of strong normal seedling” (L-NS) tests led to similarities with the vigor levels obtained in the other vigor tests. In addition, comparing viability tests (such as the germination test) in which the germination percentage of seeds from the different lots was similar is not justifiable (Table 2). The effectiveness of the strong normal seedling vigor test was confirmed because it led to the same physiological ranking as the length of strong normal seedling and seedling dry matter tests, classifying the lots in three vigor levels (high, medium and low). Similar results were also obtained by the tetrazolium seed vigor test. Thus, lot 4 was established as more vigorous than lots 1 and 3, whereas lots 2 and 5 were considered to be of intermediate physiological quality.

CONCLUSIONS

From morphological classification of seedlings into strong normal, weak, and abnormal seedlings, it is possible to adapt the normal intact (strong) seedling vigor test of A. cearensis. The vigor test based on identification and quantification of strong normal seedlings, evaluation of length of strong normal seedlings, and application of the seed vigor test through tetrazolium were effective for classification of vigor of A. cearensis seed lots.

ACKNOWLEDGMENTS

The present study was carried out with support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Funding Code 001. Our thanks also for support from the Agrarian Sciences Specialized Academic Unit of the Universidade Federal do Rio Grande do Norte, Macaíba, RN, where the study was conducted.

REFERENCES

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CUNHA, M.C.L.; FERREIRA, R.A. Aspectos morfológicos da semente e do desenvolvimento da planta jovem de Amburana cearensis (Arr. Cam.) A.C. Smith – cumaru – Leguminosae Papilionoideae. Revista Brasileira de Sementes, v.25, n.2, p.89-96, 2003. http:// www.scielo.br/pdf/%0D/rbs/v25n2/19654.pdf FRANÇA-NETO, J.B.; KRZYZANOWSKI, F.C.; COSTA, N.P. O teste de tetrazólio em sementes de soja. Londrina: EMBRAPA-CNPSo, 1998. 72p. GUEDES, R.S.; ALVES, E.U.; GONÇALVES, E.P.; VIANA, J.S.; SILVA, K.B.; GOMES, M.S.S. Metodologia para teste de tetrazólio em sementes de Amburana cearensis (Allemão) A.C. Smith. Revista Brasileira de Plantas Medicinais, v.12, n.1, p.120-126, 2010. http:// www.scielo.br/pdf/rbpm/v12n1/v12n1a17.pdf GUEDES, R.S.; ALVES, E.U.; SANTOS-MOURA, S.S.; GALINDO, E.A. Teste de comprimento de plântula na avaliação da qualidade fisiológica de sementes de Amburana cearensis (Allemão) A.C. Smith. Semina: Ciências Agrárias, v.36, n.4, p.2373-2382, 2015. https://www.researchgate.net/publication/282423364_Teste_de_comprimento_de_plantula_na_avaliacao_da_qualidade_ fisiologica_de_sementes_de_Amburana_cearensis_Allemao_AC_Smith JAVORSKI, M.; CASTAN, D.O.C.; SILVA, S.S.; GOMES-JUNIOR, F.G.; CICERO, S.M. Image analysis to evaluate the physiological potential and morphology of pearl millet seeds. Journal of Seed Science, v.40, n.2, p.127-134, 2018. http://www.scielo.br/pdf/jss/ v40n2/2317-1545-jss-40-02-127.pdf KRZYZANOWSKI, F.C.; NAKAGAWA, J. Teste de vigor baseados no desempenho das plântulas. In: KRZYZANOSWKI, F.C.; VIEIRA, R.D.; FRANÇA-NETO. J.B. Vigor de sementes: conceitos e testes. Londrina: ABRATES, 1999. 218p. LOPES, A.A.; MAGALHÃES, T.R.; UCHÔA, D.E.A.; SILVEIRA, E.R.; AZZOLINI, A.E.C.S.; KABEYA, L.M.; LUCISANO-VALIM, Y.M.; VASCONCELOS, S.M.M.; VIANA, G.S.B.; LEAL, L.K.A.M. Afrormosin, an isoflavonoid from Amburana cearensis A. C. Smith, modulates the inflammatory response of stimulated human neutrophils. Basic & Clinical Pharmacology & Toxicology, v.113, n.6, p.363-369, 2013. https://onlinelibrary.wiley.com/doi/full/10.1111/bcpt.12106 LOUREIRO, M.B.; TELES, C.A.S.; VIRGENS, I.O.; ARAÚJO, B.R.N.; FRENANDEZ, L.G.; CASTRO, R.D. Aspectos morfoanatômicos e fisi- ológicos de sementes e plântulas de Amburana cearensis (Fr. All.) A.C. Smith (Leguminosae - Papilionoideae). Revista Árvore, v.37, n.4, p.679-689, 2013. http://www.scielo.br/pdf/rarv/v37n4/11.pdf MAGUIRE, J.D. Speeds of germination-aid selection and evaluation for seedling emergence and vigor. Crop Science, v.2, p.176-177, 1962. MARCOS-FILHO, J. Seed vigor testing: an overview of the past, present and future perspective. Scientia Agricola, v.72, n.4, p.363- 374, 2015. http://www.scielo.br/pdf/sa/v72n4/0103-9016-sa-72-4-0363.pdf MIQUEL, S. Morphologie fonctionelle de plantules d’espèces forestières du Gabon. Bulletin du Muséum d’Histoire Naturelle, v.9, n.4, p.101-121, 1987. MUNSELL COLOR (FIRM). Munsell Color Charts for Plant Tissues. 2. ed. Baltimore, 1963. NAKAGAWA, J. Testes de vigor baseados no crescimento de plântulas. In: VIEIRA, R.D.; CARVALHO, N.M. Testes de vigor em sementes. Jaboticabal: FUNEP, 1994. 164p. SÁ, M.B.; RALPH, M.T.; NASCIMENTO, D.C.O.; RAMOS, C.S.; BARBOSA, I.M.S.; SÁ, F.B.; LIMA-FILHO, J.V. Phytochemistry and preliminary assessment of the antibacterial activity of chloroform extract of Amburana cearensis (Allemão) A.C. Sm. against Klebsiella pneumoniae carbapenemase-producing strains. Evidence-Based Complementary and Alternative Medicine, v.2014, 2014. http:// downloads.hindawi.com/journals/ecam/2014/786586.pdf OLIVEIRA, A.C.S.; MARTINS, G.N.; SILVA, R.F.; VIEIRA, H.D. Testes de vigor em sementes baseados no desempenho de plântulas. Revista Cientifica Internacional, v.1, n.4, p.1-21, 2009. http://www.interscienceplace.org/isp/index.php/isp/article/view/35/34 SENA, D.V.A.; ALVES, E.U.; MEDEIROS, D.S. Vigor de sementes de milho cv. “Sertanejo” por testes baseados no desempenho de plântulas. Ciência Rural, v.45, n.11, p.1910-1916, 2015. http://www.scielo.br/pdf/cr/v45n11/1678-4596-cr-45-11-01910.pdf SOUZA, T.S.; SOUZA, T. M.; PANOBIANCO, M. Morphological characterization of fruit, seed and seedling, and seed germination test of Campomanesia guazumifolia. Journal of Seed Science, v.40, n.1, p.75-81, 2018. http://www.scielo.br/pdf/jss/v40n1/2317-1537- jss-40-01-75.pdf

Journal of Seed Science, v.42, e202042011, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

X-ray imaging and digital processing application in Journal of Seed Science, v.42, non-destructive assessing of melon seed quality e202042005, 2020 http://dx.doi.org/10.1590/2317- 1545v42229761 André Dantas de Medeiros1* , Maycon Silva Martins1 , Laércio Junio da Silva1 , Márcio Dias Pereira2 , Manuel Jesús Zavala León1 , Denise Cunha Fernandes dos Santos Dias1

ABSTRACT: Non-destructive and high throughput methods have been developed for seed quality evaluation. The aim of this study was to relate parameters obtained from the free and automated analysis of digital radiographs of hybrid melons’ seeds to their seeds’ physiological potential. Seeds of three hybrid melon (Cucumis melo L.) cultivars from commercial lot samples were used. Radiographic images of the seeds were obtained, from which area, perimeter, circularity, relative density, integrated density and seed filling measurements were generated by means of a macro (PhenoXray) developed for ImageJ® software. After the X-ray test, seed samples were submitted to the germination test, from which variables related to the physiological quality of the seeds were obtained. Variability between lots was observed for both physical and physiological characteristics. Results showed that the use of the PhenoXray macro allows large- scale phenotyping of seed radiographs in a simple, fast, consistent and completely free way. The methodology is efficient in obtaining morphometric and tissue integrity data of melon seeds and the generated parameters are closely related to physiological attributes of seed quality.

Index terms: automated image analysis, Cucumis melo L., seed radiography, relative density.

Aplicação do teste de raios-X e processamento digital na avaliação não-destrutiva da qualidade de sementes de melão

RESUMO: Métodos não-destrutivos e de alto desempenho têm sido desenvolvidos para avaliação da qualidade de sementes. O objetivo deste estudo foi relacionar parâmetros obtidos a partir da análise gratuita e automatizada de radiografias digitais de sementes de melão híbrido com o seu potencial fisiológico. Foram utilizadas amostras de sementes comerciais de três cultivares *Corresponding author híbridas de melão (Cucumis melo L.), cada uma representada por três lotes. Foram obtidas E-mail: [email protected] imagens radiográficas das sementes, das quais foram geradas determinações de área, perímetro, circularidade, densidade relativa, densidade integrada e preenchimento da cavidade interna de Received: 10/9/2019. sementes, por meio de uma macro (PhenoxXray) desenvolvida para o software ImageJ®. Após o Accepted: 1/8/2020. teste de raios-X, as sementes foram submetidas ao teste de germinação, a partir do qual foram obtidas variáveis relacionadas à qualidade fisiológica. Observou-se variabilidade entre lotes para as características físicas e fisiológicas. Os resultados demonstraram que o uso da macro 1 PhenoXray permite a fenotipagem em larga escala das radiografias de sementes de maneira Departamento de Agronomia, Universidade Federal de Viçosa, simples, rápida, consistente e totalmente gratuita. A metodologia é eficiente na obtenção de 36570-900 – Viçosa, MG, Brasil. dados morfométricos e de integridade tecidual em sementes de melão, e os parâmetros gerados apresentam estreita relação com atributos fisiológicos da qualidade das sementes. 2Departamento de Agropecuária, Universidade Federal do Rio Termos para indexação: análise automática de imagens, Cucumis melo L., radiografia de Grande do Norte, 59280-000 – sementes, densidade relativa. Macaíba, RN, Brasil.

Journal of Seed Science, v.42, e202042005, 2020 2 A. D. Medeiros et al.

INTRODUCTION

The introduction of modern and high-performance techniques capable of generating consistent and fast results, less subjectively and non-destructively, presents great potential for the safe evaluation of the quality of agricultural products (Mahajan et al., 2015). Among the techniques that reach this proposal is the analysis based on electromagnetic X-ray radiation. This technique, predominantly used in medical applications, has been used in several areas, including seed quality inspection (Rahman and Cho, 2016). However, the visual analysis of the radiographic images is time-consuming and can generate reading errors due to subjective interpretations (Medeiros et al., 2018). The current challenge is to develop methods capable to produce relevant information from the radiographs of seeds, on a large scale in a fast and consistent manner. Recent advances in the field of computer-aided digital image processing have contributed to making feasible this type of approach. Public domain software such as ImageJ® has provided promising perspectives to address this purpose and provide automation of analyses related to seed radiographs. ImageJ® is presented as the world’s fastest imaging software (Miart et al., 2018; Schindelin et al., 2012). Semi-automated applications with the use of this software in the analysis of radiographic images of seeds has demonstrated potential for seed analysis (Abud et al., 2018; Medeiros et al., 2018). However, while this type of analysis allows for greater interaction and autonomy of the analyst in the adjustments, it makes the process slow, time-consuming, and often compromises the standardization of analyses on a large scale. Recently, a macro for ImageJ®, called PhenoXray (Medeiros et al., 2019), was developed. This macro allowed large-scale phenotyping of brachiaria grass seeds radiographs, obtaining parameters related to their physiological quality. The application of these technologies to high value-added seeds, such as hybrid melons, could be of interest to the seed industry, since it allows the evaluation of seed quality in a non-destructive, fast and standardized way. Also, it would be an alternative to be used in quality control programs during seed production, being important for the decision making regarding the approval or disposal of seed lots, which would mean saving time and resources. Considering that the rapid technological development indicates greater opportunities for X-ray inspection in the agricultural sector, especially in seed technology, this research had the aim to relate parameters obtained from the free and automated analysis of digital radiographs of hybrid melons’ seeds to their physiological potential.

MATERIAL AND METHODS

Plant material Seeds of three hybrids of melon were used, Bazuca F1 (American cantaloupe, lots 1 to 10), Goldex F1 (Yellow, lots 11 to 20), and Pampa F1 (Italian cantaloupe, lots 21 to 30), from lot samples from the 2017 crop season, each represented by ten seed lots. The seeds were submitted to the following analyses:

Physical analyses Seed water content: the seeds were initially submitted to the determination of the water content by the oven method at 105 ± 3 °C for 24 hours, based on the Rules for Seed Testing (Brasil, 2009); for this purpose, two subsamples of 5 g of seeds were used for each lot. Results were expressed as a percentage (wet basis). X-ray test: for the analysis of the internal morphology of the seeds, five replicates of twenty seeds each of each lot were used. Seed samples were positioned with the embryonic axis facing down and adhesive-tape fixed in an orderly manner on adhesive paper, to allow subsequent individual identification in the posterior analyses. Seeds were then placed inside the Faxitron digital X-ray equipment, model MX-20 (Faxitron X-ray Corp. Wheeling, IL, U.S.A). To generate the radiographic images, the equipment was configured with the radiation exposure time of ten seconds, 23 kV voltage, 41.6 cm focal length and contrast of the calibrated image in 16383 (width) x 3124 (center). The digital images generated

Journal of Seed Science, v.42, e202042005, 2020 X-ray analysis of melon seeds 3 were saved in computer in TIFF format, following their processing and analysis. Automated analysis of the radiographs: for the automated image analysis, the PhenoXray macro (https://sites. google.com/ufv.br/phenoxray) (Medeiros et al., 2019) developed for ImageJ® software was used. The macro was developed to process images automatically, being necessary only to indicate the folder containing all the images, properly identified (lot and replication), in the ImageJ® software. The analysis started with the segmentation of the images using the ImageJ® Threshold mode. The threshold was automatically set using Yen’s automatic multilevel thresholding method to segment the images in the regions corresponding to the melon seeds. The regions of interest were then analyzed from the analyze particles command, which integrates the software ImageJ®. Only particles with a surface area between 594 and 2.376 pixels2 were considered, which was efficient to select the areas corresponding to the seeds and to ignore areas of noises in the images. The parameters obtained from the images were: area – selection area obtained in square pixels, and later converted into units of square millimeters (mm2); perimeter – the length in millimeters of the outer limit of the selection; circularity area – obtained by the equation: C = 4π * ; relative density (average gray) – defined as the sum of the gray values (perimeter)2 of all pixels in the selected area divided by the number of pixels in the selection, expressed in gray.pixel-1; integrated density – the sum of the pixel values in the image or selection, that is equivalent to the product of area and relative density, expressed in gray.mm2.pixel-1; filling – percentage of seed area effectively filled by high density material. After, the images were processed and the measurement results were automatically saved to a TXT file. In addition, files in the JPEG format corresponding to the images’ analysis were created in the same directory, which allowed to check the performance of the macro in the processing of the images.

Physiological analysis Germination test: conducted on rolls of paper towel for germination humidified with water equivalent to 2.5 times the dry paper mass and kept in a germinator at a constant temperature of 25 °C (Brasil, 2009). The same seeds submitted to the X-ray test were used, maintaining the same seed arrangement originally used in the previous test. Daily counts of the number of seeds exhibiting primary root protrusion and germinated seeds (normal seedlings) were carried out until the eighth day after sowing. From these data, the speed of germination index (GSI), the speed of primary root protrusion (RPS) and the synchrony were calculated, according to the formulas described in Silva et al. (2019). All the variables obtained based on the germination test were calculated using the Germcalc function, contained in the package SeedCalc of the R software. Seedling analysis: after the final count of the germination test, the seedlings and non-germinated seeds of each replicate were scanned, and the generated images were evaluated in ImageJ® software. The images were used to measure the length of the shoot and the primary root of the seedlings, expressed in mm.seedling-1. The length data were processed with the package SeedCalc of the R software, through the Plantcalc function, and the parameters were calculated: seedling total length and uniformity indexes, vigor and corrected vigor.

Experimental design and statistical analysis The experiment was conducted in a completely randomized experimental design, with four replications. Data were submitted to analysis of variance (ANOVA). After confirming the normal distribution of errors by the Shapiro-Wilk test and the homogeneity of variances by the Bartlett test, the averages were grouped by the Scott-Knott test (p ≤ 0.05). Subsequently, the Pearson (r) linear correlation coefficients were calculated for all combinations between the physiological and physical quality tests of the seeds, where the significance of the r values was determined by the t test (p ≤ 0.05). Principal component analysis was also performed. The software used in the statistical analysis was R, version 3.5.1.

Journal of Seed Science, v.42, e202042005, 2020 4 A. D. Medeiros et al. RESULTS AND DISCUSSION

The X-ray equipment configurations adopted in this study, combined with the low variation of water content among the evaluated seed lots of each cultivar (cv. Bazuca F1: 8.1 to 8.8 %; cv. Goldex F1: 8.0 to 8.9 %; cv. Pampa F1: 7.8 to 8.7 %), allowed a clear visualization of the main internal structure of the seeds, as well as the identification of embryonic malformation and physical damages (Figure 1). The water content of the seeds has a high relation with the optical density of the radiography (Simak, 1991). Therefore, the uniformity of seed water content between seed lots is necessary to compare parameters related to tissue density, as gray levels of the radiographs. Studies carried out with X-ray analysis on pumpkin seeds (Silva et al., 2014), watermelon and melon (Gomes-Junior et al., 2012) demonstrated the possibility of adequate visualization of the internal morphology of seeds under water contents between 6.5% and 12%. Besides the water content, the level of detail of the internal seed parts of the radiographic image can be affected by other intrinsic factors of each species, such as its chemical composition (Simak, 1991). In sesame seeds, the X-ray test did not allow the determination of the level of development of internal seed structure, which according to the authors was due to its high oil content, typical of oilseeds (Nogueira-Filho et al., 2017). However, as shown in Figure 1, in melon seeds, whose oil content is within the range of 25.2 to 44.8% (Ibeto et al., 2012), it was possible the visualization with a high degree of detail of the internal seed morphology, with easy visual identification of seeds with incomplete filling and embryonic malformation (Figure 1B), as well as physical damage caused by predation of insects and cracks, possibly during handling or drying processes (Figure 1C). Thus, it is possible to verify that the X-ray test itself, i.e, the visual analysis of the radiographs, is a suitable tool for studies with oilseeds, such as melon, allowing an accurate analysis of the seed physical quality by simple visual evaluation. Although the evaluation of the X-ray by the analyst is the simplest and most sensitive approach, it is a laborious, time-consuming process as well as subject to errors inherent in its subjectivity.

Figure 1. Radiographic images of melon seeds with indications of the internal parts of a well-formed seed (a), seeds with visible embryonic malformation (b) and with physical damages (c). Journal of Seed Science, v.42, e202042005, 2020 X-ray analysis of melon seeds 5

Table 1 shows the variables obtained with the use of the PhenoXray macro from ImageJ®, which allowed the complete automation of the analysis of seed radiographs of the thirty seed lots of hybrid melon. Statistical differences were observed for most of the analyzed variables, except for relative density and perimeter, between the seed lots of the cultivars Goldex F1 and Pampa F1, respectively. From the analysis of the size and shape (area, perimeter and circularity), it was evident the different characteristics observed between seed lots of the same cultivar, which was confirmed by the significant difference between the values obtained. For the cultivar Bazuca F1, the values of area, perimeter and circularity ranged from 30.2 to 39.5 mm2, 29.7 to 42.4 mm and 0.30 to 0.50, respectively. Seeds of “Goldex F1” showed larger areas in absolute value in relation to the other cultivars, with variation observed from 41.7 to 43.9 mm2; the perimeter and circularity, in turn, ranged from 35.8 to 53.6 mm2, 0.23 to 0.45, respectively. Finally, the cultivar Pampa F1 showed a variation of 29.2 to 38.0 mm2 of area, 34.4 to 40.6 mm of perimeter (the lots did not differ in relation to this parameter) and 0.29 to 0.41 of circularity. According to Tanabata et al. (2012), information on the quantitative evaluation of seeds’ shape and size, when obtained by reliable and high throughput methodology, can benefit several fields of plant research, such as plant breeding programs, acting together with functional analysis studies and improvement of genomic assisted crops. Moreover, studies have shown a close relationship between these characteristics and seedling vigor (Abud et al., 2018; Javorski et al., 2018; Medeiros et al., 2018), since the variability of seed size and shape may be related to the environment during the maturation process. The variables relative density, integrated density and filling allowed to stratify from two to four groups the ten seed lots of each cultivar. The relative and integrated densities are variables only recently reported in seed research, and with great potential for seed lots’ evaluation (Abud et al., 2018; Medeiros et al. 2018). On the other hand, the filling variable has already been evaluated in a larger number of studies (Gomes-Junior et al., 2013), but not fully automated using ImageJ® software. These variables, calculated by means of the gray values of each pixel in the image, give an idea of the resistance that a given tissue presents to the passage of X-rays, since the photons in an X-ray beam can be transmitted, scattered (Compton dispersion) or absorbed (photoelectric collision) when they collide with an object (Kotwaliwale et al., 2014). Thus, higher gray densities indicate denser tissues, that is, there is greater impediment to X-ray passages. Several authors pointed out that the evaluation of aspects such as the internal cavity occupied by the embryo, the presence of mechanical damages, stains that indicate tissue deterioration or seed malformations is necessary in studies with X-rays in seeds (Borges et al., 2019; Gomes-Junior et al., 2013). However, according to Medeiros et al. (2018), all these characteristics are in a way represented by the relative density, since seeds deteriorated or with less filling show lower gray levels in the radiographic image. Thus, the image presents lower levels of radiopacity (light) and higher levels of radioluminescence (dark), which can be quantified by means of relative density. It is important to emphasize that for a comparison between seed relative densities from different radiographs, the contrast used by the equipment in calibrating the image (width and center adjustment) must be standardized in all of them. In addition, the relative density is influenced by other objects with higher density that are inserted with the seeds in the acquisition of the image, such as fruit remnants, stones and other types of impurities. Another important point is that different X-ray systems, even using analogous configurations of energy and electrical current may not produce similar results, because of the types of X-ray detectors used (Kotwaliwale et al., 2014). With the filling variable, which represents the percentage of area of the seed effectively filled by high density material, we noticed that the lowest observed averages value was 93.6%, which indicates high uniformity of embryonic filling perceived in the seed lots studied. Borges et al. (2019) also used the filling variable to quantify the empty seed area of tomato, and observed a close relationship between seed empty space and its physiological quality. Although the physical parameters generated from X-ray image analysis are initially interesting and efficient in differentiating seed lots, it is necessary to check the physiological quality data from the seeds of the respective lots to make a precise inference about a possible relation of these variables with the seed physiological potential.

Journal of Seed Science, v.42, e202042005, 2020 6 A. D. Medeiros et al.

Table 1. Average values of the physical parameters obtained through the automated analysis of radiographic images of 30 lots of hybrid melon seeds using the PhenoXray macro.

Area Per. Rel. dens. Int. dens. Filling Circ. Seed lot mm2 mm gray.pixel-1 gray.mm2.pixel-1 % Cultivar Bazuca F1 1 34.56 b 29.69 d 0.496 a 94.29 a 3261 b 99 a 2 35.29 b 31.28 c 0.460 b 92.15 a 3254 b 98 a 3 30.18 c 31.92 c 0.401 c 88.63 b 2678 c 98 a 4 30.79 c 39.14 b 0.295 d 85.73 b 2637 c 96 b 5 39.50 a 32.50 c 0.474 b 97.04 a 3833 a 98 a 6 38.12 a 41.35 a 0.309 d 87.27 b 3328 b 95 c 7 38.05 a 42.38 a 0.302 d 85.72 b 3263 b 95 c 8 37.34 a 40.07 b 0.317 d 89.58 b 3344 b 95 c 9 38.35 a 41.05 a 0.314 d 88.61 b 3399 b 95 c 10 30.97 c 33.11 c 0.389 c 80.00 c 2479 c 94 d Fc 46.60* 52.08* 65.51* 10.01* 28.60* 78.53* CV (%) 3.28 4.23 5.78 3.83 5.55 0.49 Cultivar Goldex F1 12 43.11 a 37.04 c 0.408 a 95.1 4102 a 98 a 12 41.67 b 38.18 c 0.395 a 88.76 3706 b 97 a 13 43.50 a 36.67 c 0.420 a 91.19 3970 a 97 a 14 43.37 a 35.84 c 0.441 a 93.69 4062 a 98 a 15 43.80 a 36.07 c 0.445 a 89.28 3914 a 98 a 16 42.50 b 49.80 b 0.254 b 88.31 3762 b 94 c 17 41.93 b 50.32 b 0.243 b 86.32 3622 b 93 c 18 42.46 b 49.88 b 0.266 b 87.01 3707 b 93 c 19 43.89 a 46.89 b 0.287 b 92.89 4081 a 96 b 20 42.51 b 53.60 a 0.234 b 84.54 3603 b 93 c Fc 4.11* 30.40* 25.24* 1.50ns 2.27* 84.26* CV (%) 1.99 6.76 11.71 6.95 7.5 0.52 Cultivar Pampa F1 21 29.19 d 37.17 0.349 b 90.78 b 2665 b 95 a 22 34.26 b 37.38 0.333 b 91.43 b 3136 a 94 b 23 32.52 c 34.67 0.356 b 97.97 a 3195 a 95 a 24 34.25 b 34.4 0.376 a 102.15 a 3502 a 96 a 25 35.17 b 35.03 0.382 a 99.89 a 3531 a 96 a 26 37.31 a 35.5 0.399 a 88.69 b 3313 a 96 a 27 36.67 a 34.59 0.409 a 89.66 b 3291 a 96 a 28 37.27 a 35.13 0.403 a 89.01 b 3324 a 96 a 29 38.00 a 35.9 0.399 a 89.40 b 3402 a 96 a 30 32.10 c 40.6 0.288 b 78.80 c 2532 b 95 b Fc 42.50* 1.04ns 3.69* 4.54* 9.54* 5.39* CV (%) 2.77 11.58 12.06 7.7 7.62 0.82

Lower case = comparison within each column for each evaluation by the Scott-Knott test (p < 0.05); *, ns = significant and not significant by the F test (p < 0.05), respectively; Fc = F calculated; CV = coefficient of variation; Per. = perimeter; Circ. = circularity; Rel. dens. = relative density; Int. dens. = integrated density. Journal of Seed Science, v.42, e202042005, 2020 X-ray analysis of melon seeds 7

Table 2 presents the variables obtained through the evaluation of the physiological potential of melon seeds. Statistical differences were detected for most of the variables, except for the germination synchrony variable, among the lots of the cultivar Pampa F1. Percentage of germination representing the percentage of normal seedlings at eight days after sowing, showed that all seed lots, except lot 10, produced values greater than 80% of germination, which is the minimum value established for the marketing of melon seeds in Brazil, indicating high viability of the seeds. For the primary root protrusion, the same pattern was observed for the germination data; however, the values were generally higher because of the evaluation considering only the emission of the primary root (> 2 mm), not necessarily culminating in the formation of a normal seedling. From the data of the speed of germination index (GSI), speed of primary root protrusion (RPS) and synchrony, it can be observed grouping in up to four levels for cultivar Bazuca F1, five levels for Goldex F1 and four levels for Pampa F1. These results indicate significant differences in germination timing and in the rates of germination and primary root protrusion between the seed lots. According to Finch-Savage and Bassel (2016), this is an important information, since the irregular seedling emergence of seedlings can lead to phenological delays and variations in plant growth in further phenological stages, also affecting harvesting. Seedling image analysis using ImageJ® software allowed the identification of differences in seedling growth and uniformity of the different seed lots, expressed by means of seedling length and uniformity, vigor and corrected vigor indexes (Table 2). For the cultivars Bazuca F1 and Pampa F1, the variable length of seedling was more sensitive than the germination test to detect differences between the lots. It was also observed that for some seed lots that presented a high percentage of germination (for example, lots 1, 2, 3, 5), smaller values of average seedling length were observed, which shows a high viability of the seeds, however this lower growth can be an indication of low vigor. By means of the seedling uniformity index (Table 2), some lots with lower performance in the average seedling length showed high uniformity in their development, or vice versa, that is, Lot 1 and Lot 6, respectively. Other authors have pointed out that the uniformity of seedling development is an index that should be taken into account in the evaluation of seed lots, since it can provide useful information on the degree of deterioration, initial growth potential and seedling emergence uniformity (Leão-Araújo et al., 2019). For the vigor index, which considers the growth rate and seedling uniformity, the previous results were confirmed, highlighting differences of physiological potential in at least two levels to cultivar Goldex F1 and in three levels for the other cultivars. The corrected vigor index, which consists of the vigor index product with the germination percentage, was efficient to detect differences in at least four vigor levels among the seed lots of the three cultivars. For Medeiros and Pereira (2018), the corrected vigor index makes a more efficient beaconing of the results of the vigor index, since it performs an adjustment based on germination, offering a more representative result of the physiological potential of the seeds. In general, the data obtained for the different seed lots and cultivars indicate differences in seed physiological quality (Table 2). Significant differences were also observed between the lots of the three cultivars when considering the physical parameters obtained from the X-ray images, such as relative density, integrated density and filling (Table 1). However, to verify possible relationships between the physical variables, obtained by the automated analysis of radiographs, with the variables of physiological potencial, obtained through the test of germination and seedling growth, a correlation analysis was performed (Figure 2). Significant correlations (p < 0.05) of some descriptors of size and shape with physiological quality were observed. For the cultivar Bazuca F1, the seed area showed correlation with seedling length (r = 0.71), vigor index (r = 0.77) and corrected vigor (r = 0.67). The perimeter and the circularity were correlated only with the germination synchrony (r = -0.65 and r = 0.67, respectively). For the cultivar Goldex F1, only the perimeter and the circularity presented correlations with the variables of physiological quality (for example, germination: r = -0.92 and r = 0.87; GSI: r = -0.82 and r = 0.75; RPS: r = -0.91 and r = 0.88; uniformity: r = -0.86 and r = 0.80; vigor: r = -0.81 and r = 0.76; corrected vigor: r = -0.89 and r = 0.85,

Journal of Seed Science, v.42, e202042005, 2020 8 A. D. Medeiros et al.

Table 2. Average values of the data obtained in the evaluation of the physiological potential of thirty lots of hybrid melon seeds.

Germ. Rad. pro. SL Unif. Vigor Corr. vigor GSI RPS Sync Seed lot % mm Index Cultivar Bazuca F1 1 95 a 100 a 6.33 a 11.8 b 1.00 a 9.24 c 892 a 770 b 731 b 2 94 a 100 a 6.27 a 14.2 a 1.00 a 9.50 c 871 a 768 b 722 b 3 95 a 97 a 6.18 a 12.0 b 0.90 b 9.41 c 868 a 763 b 725 b 4 86 b 90 b 5.54 b 10.9 b 0.84 b 8.08 d 790 b 678 c 583 c 5 97 a 98 a 6.30 a 11.4 b 0.88 b 9.47 c 882 a 772 b 749 b 6 94 a 99 a 5.97 a 11.2 b 0.69 c 12.56 a 835 b 857 a 806 a 7 85 b 100 a 5.23 b 10.6 b 0.60 c 11.45 b 817 b 809 b 688 b 8 95 a 99 a 5.90 a 10.0 b 0.61 c 12.35 b 856 a 860 a 817 a 9 93 a 99 a 5.57 a 11.0 b 0.54 d 11.86 a 866 a 848 a 789 a 10 64 c 76 c 3.75 c 7.4 c 0.47 d 8.96 c 660 c 644 c 412 d Fc 9.15* 23.38* 16.44* 13.73* 17.67* 28.30* 12.46* 14.05* 52.51* CV (%) 8.15 3.65 7.55 9.3 13.8 6.53 5.2 5.58 5.36 Cultivar Goldex F1 11 98 a 100 a 6.45 a 19.3 a 0.91 a 13.36 a 864 a 899 a 881 a 12 98 a 99 a 6.14 a 18.5 a 0.67 b 10.83 b 864 a 811 b 795 b 13 96 a 100 a 6.19 a 17.4 b 0.82 a 12.41 a 857 a 863 a 828 b 14 99 a 100 a 6.26 a 18.2 a 0.69 b 12.66 a 874 a 880 a 871 a 15 93 a 98 a 5.77 b 17.4 b 0.60 b 12.00 b 816 b 828 b 770 b 16 89 b 99 a 5.62 b 14.1 c 0.65 b 11.36 b 795 b 795 b 708 c 17 89 b 98 a 5.75 b 12.4 d 0.79 a 11.79 b 813 b 819 b 729 c 18 85 b 100 a 5.47 b 14.6 c 0.75 b 11.23 b 748 c 767 b 652 d 19 91 b 97 a 6.02 a 12.0 d 0.94 a 11.93 b 806 b 820 b 747 c 20 84 b 93 b 5.30 b 9.1 e 0.65 b 10.84 b 747 c 753 b 633 d Fc 4.18* 3.89* 4.48* 57.22* 4.88* 3.45* 4.66* 3.87* 15.54* CV (%) 7.11 2.26 7.31 6.51 15.49 8.28 5.86 6.44 6.32 Cultivar Pampa F1 21 96 a 100 a 6.24 a 19.4 a 0.88 11.77 c 849 b 836 b 803 b 22 94 a 97 b 5.98 b 18.4 b 0.78 11.50 d 809 b 807 c 759 c 23 97 a 100 a 6.29 a 19.7 a 0.86 12.96 b 816 b 861 b 836 b 24 100 a 100 a 6.61 a 20.0 a 0.94 14.54 a 917 a 967 a 967 a 25 98 a 100 a 6.44 a 18.0 b 0.92 13.93 a 898 a 936 a 918 a 26 91 b 99 a 5.88 b 19.0 a 0.78 12.11 c 778 c 813 c 740 c 27 87 b 99 a 5.55 b 19.2 a 0.71 11.41 d 758 c 778 c 677 d 28 97 a 100 a 6.28 a 18.8 a 0.79 12.43 c 825 b 848 b 822 b 29 97 a 100 a 6.35 a 17.6 b 0.87 12.25 c 840 b 849 b 823 b 30 92.5 b 100 a 6.00 b 10.7 c 0.81 10.70 d 815 b 782 c 719 c Fc 4.05* 2.69* 4.93* 62.49* 1.83ns 11.93* 6.40* 9.32* 21.18* CV (%) 4.51 1.33 5.05 4.22 14.07 6.14 5.2 5.35 5.34 Lowercase = comparison within each column for each evaluation by the Scott-Knott test (p < 0.05); *, ns = significant and not significant by the F test (p < 0.05); Fc = F calculated; CV = coefficient of variation; Germ. = germination; Rad. pro. = radicle protrusion; GSI = germination speed index; RPS = root protrusion speed; sync = synchrony; SL = seedlings length; Unif = Uniformity; Corr. vigor = corrected vigor. Journal of Seed Science, v.42, e202042005, 2020 X-ray analysis of melon seeds 9 respectively). Finally, for the cultivar Pampa F1, there was a significant correlation only of the RPS with the perimeter and the circularity (r = -0.86 and r = 0.68, respectively). There are also strong and significant correlations between the variables related to tissue density and filling of the seeds with the variables of physiological quality (Figure 2). In general, the responses of the cultivars did not follow the same trend, which can be explained by the history of the lots and the different characteristics of the seed of each cultivar. For the cultivar Pampa F1, for example, the lots showed narrow variation in the percentage of germination and a wide variation in vigor, observed by more evident differences in the length of seedlings, which may have led to a lower correlation of X-ray variables with germination (r < 0.60) and higher correlations with variables related to seedling length (for example, relative density: r = 0.90). The high correlations observed for the parameters: relative density, integrated density and filling with the physiological variables are important indicators for the validation of the methodology of automated analysis of X-ray images to evaluate seed quality. Abud et al. (2018) and Medeiros et al. (2018) also observed high correlations between the relative density variable, obtained from radiographs of broccoli and leucena seeds, respectively, with variables of physiological quality, especially the seedling length. This indicate that the relative density parameter is promising to estimate the physiological potential of these seeds. In Figure 3 it is possible to identify the association between physical characteristics and the physiological quality of seeds. It was observed that tissues with high integrity generated more opaque images, with zones of high density (shown in green, yellow and red), as the resistance to the passage of X-rays was greater. When these high-density zones were uniform throughout the seed, the relative density remained high and culminated in the emergence of more vigorous

Figure 2. Pearson correlation represented by heatmap (a) and by curves (b) between the generated variables of the automated analysis of the radiographs and the evaluation of germination and seedling length for each hybrid melon cultivar. Journal of Seed Science, v.42, e202042005, 2020 10 A. D. Medeiros et al. seedlings (Figure 3A). On the other hand, seeds with low integrity tissues possessed lower relative densities, due to the low resistance to X-rays, which generated more translucent or darkened zones in the radiography (represented in the colors blue and purple), and that may be related to the seedlings less developed (Figure 3B). In other cases, it is possible to detect physical damages, such as cracks or predation by insects, represented by bands or centers of low density that lead to the fall in the values of relative density. However, since this damage does not compromise the embryonic axis, germination can occur and lead to the development of normal seedlings (Figures 3C and 3D). According to some authors, such as Silva et al. (2014), this relationship observed in Figure 3 is not always true, since seeds classified as well formed by the X-ray technique can give rise to abnormal seedlings or dead seeds, and since the radiography allows to verify if there are or not tissue malformation, but does not necessarily establish a direct relationship with physiological processes. However, in this work, it was possible to suppose that even if the physical and physiological variables are not 100% correlated; there might be a strong association between them, which can lead to advances in procedures of pre-selection of seed lots by farmers and by the seed industry. It is important to note that the time spent in the analysis of a radiograph from twenty-seed samples each using the PhenoXray macro was only 0.5 seconds (using an Intel Core i5-4200U CPU 1.60GHz processor). Thus, it is a fast, reproducible, standardized, easy and inexpensive method to measure the physical characteristics of the seeds, which, according to Huang et al. (2015), contributes to guarantee quality seeds for sowing.

Figure 3. Radiographic images of melon seeds, combined with color representations (2D and 3D) of the density along the seed and its respective seedlings.

Journal of Seed Science, v.42, e202042005, 2020 X-ray analysis of melon seeds 11

From the multivariate principal component analysis (PCA), using the set of data obtained for the three cultivars and the fifteen characteristics evaluated, it can be noticed that the first two components (PC1 and PC2) accounted for 71.2% of the variability of the data. Thus, by means of several linear combinations, it was possible to reduce from fifteen dimensions to only two, which explained a significant percentage of the observations (Figure 4). It was observed that there was a greater dispersion of the seed lots of the cultivar Bazuca F1 by the central ordering diagram, indicating the high variability among the lots for the characteristics that constituted the PCA. In contrast, a larger grouping of the lots of the cultivar Pampa F1 was observed, confirming previous results related to the uniformity of the lots of this hybrid, as observed for the data obtained in the physical and physiological analyses (Tables 1 and 2). In general, the seed lots that were distant and opposite to the vectors of physical and physiological quality (represented in the circle of correlations centered on the right side of the central ordering diagram) were the ones that presented the lowest values for these characteristics. In the circle of correlations (Figure 4), the vectors that comprise the variables obtained from the automated analysis of the radiographs were close to the vectors of physiological quality, presenting, in turn, factorial loads with similar distribution in the components. These results indicate a high correlation between the characteristics, as already demonstrated in the correlation matrix (Figure 3). Thus, these variables were efficient to determine the level of physical integrity of the seeds, besides being associated to the physiological quality, such as viability and vigor. Therefore, the use of the PhenoXray macro, developed in ImageJ® for the automated analysis of seed radiographs, allows the large-scale analysis, in a fast and consistent way, with promising parameters related to the physical integrity of the seed and related to its physiological potential. The use of ImageJ® for algorithm development and subsequent creation of customizable macros has been successfully reported in other studies (Legland et al., 2017; Miart et al., 2018; Tello et al., 2018; Vasseur et al., 2018) and represents a significant advance for both the automation of analysis and the free diffusion of technologies at the global level, by the opensource nature of the software.

Figure 4. Biplot obtained by the linear combination of the variables related to the physical and physiological characteristics of thirty lots of hybrid melon seeds of three cultivars.

Journal of Seed Science, v.42, e202042005, 2020 12 A. D. Medeiros et al.

Moreover, the parameters: relative density, integrated density and filling, obtained from the automated analysis of radiographs of melon seeds, are promising to infer the physiological quality of the seeds and can be recommended for the preliminary evaluation and decision making regarding the disposal of seed lots, optimizing this process and reducing production costs.

CONCLUSIONS

The automated analysis of radiographic images allows a simple and fast way to obtain reliable information about the physical characteristics of the seeds and to generate parameters related to the physiological quality. The relative and integrated densities were, among the variables obtained from X-ray images, those that stood out to estimate the physiological potential of the seeds.

ACKNOWLEDGEMENTS

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial support.

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Journal of Seed Science, v.42, e202042005, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Simulated drift of dicamba: effect on the physiological Journal of Seed Science, v.42, e202042014, 2020 quality of soybean seeds http://dx.doi.org/10.1590/2317- 1545v42224236 Estevam Matheus Costa1* , Jacson Zuchi1 , Matheus Vinícius Abadia Ventura1 , Leandro Spíndola Pereira1 , Geovani Borges Caetano1 , Adriano Jakelaitis1

ABSTRACT: The release of soybean varieties resistant to dicamba herbicide leads to the possibility of dicamba herbicide drift into soybean seed production fields and reduction in the physiological quality of soybean seeds. The aim of this study was to evaluate the physiological quality of soybean seeds as a function of the application of reduced rates of dicamba in two phenological phases. A randomized block experimental design was used, with four replications. The 4 × 2 + 1 factorial arrangement consisted of four reduced rates -1 (0.028, 0.28, 2.8 and 28 g.ha ) of dicamba applied in the V4 and R2 stages + a control. The physiological quality of the seeds was evaluated after harvest and at six months after -1 storage at 20 °C. At the rate of 28 g.ha of dicamba applied in the V4 and R2 stages, seed germination declined by 15% and 42%, respectively. After storage, seeds under the 28 g.ha-1 dicamba treatment had 64% lower germination compared to the lowest application rate evaluated, and electrical conductivity doubled in relation to the control. The physiological quality of soybean seeds declines under these reduced rates of dicamba applied in the V4 and R2 stages, both before and after storage.

Index terms: application times, germination, Glycine max, reduced application rates, storage.

Deriva simulada de dicamba: efeitos sobre a qualidade fisiológica de sementes de soja

RESUMO: Com a inserção de variedades de soja resistentes ao herbicida dicamba, surge a possibilidade de deriva deste herbicida em campos de produção e a ocorrência de redução na qualidade fisiológica das sementes. Objetivou-se avaliar a qualidade fisiológica das sementes de soja em função da aplicação de subdoses de dicamba em duas fases fenológicas. O delineamento foi em blocos casualizados, com quatro repetições. O esquema fatorial 4 x 2 + -1 1 composto por quatro doses (0,028, 0,28, 2,8 e 28 g.ha ) de dicamba aplicados em V4 e R2 + testemunha. A qualidade fisiológica das sementes foi avaliada após a colheita e aos seis meses *Corresponding author -1 após armazenadas a 20 °C. Na dose de 28 g.ha de dicamba aplicados em V4 e R2, as sementes E-mail: [email protected] apresentaram redução da germinação em 15 e 42%, respectivamente. Após o armazenamento, as sementes do tratamento com 28 g.ha-1 de dicamba apresentaram germinação 64% menor Received: 5/20/2019. se comparado à menor dose avaliada e a condutividade elétrica duplicou em relação à Accepted: 10/25/2019. testemunha. As sementes de soja têm sua qualidade fisiológica reduzida por subdoses de dicamba aplicadas nos estádios V4 e R2 tanto antes quanto após o armazenamento.

1Instituto Federal Goiano, campus Termos para indexação: armazenamento, épocas de aplicação, germinação, Glycine max, subdoses. Rio Verde, 75901-970 – Rio Verde, Goiás, Brasil.

Journal of Seed Science, v.42, e202042014, 2020 2 E. M. Costa et al.

INTRODUCTION

The development of soybean varieties with tolerance to hormonal herbicides, such as 2,4-D and dicamba, has been adopted as part of a program for management of eudicot weeds that are tolerant or resistant to the herbicides commonly used in the crop, such as glyphosate (Silva et al., 2018). However, when this technology is not used within the principles of integrated weed management, it may lead to an increase in the application of these hybrids in soybean production areas (Solomon and Bradley, 2014). The quality of soybean seeds can be affected, in accordance with the genotype, the edaphic and climatic conditions, and biotic factors, and quality may deteriorate during storage under inadequate temperature and relative humidity conditions (Zuchi et al., 2013). Though deterioration occurs, it can be retarded, depending on the storage conditions and on the seed characteristics (Cardoso et al., 2012). Deterioration reduces the quality, viability, and vigor of seeds, due to aging or to the effects of adverse environmental factors (Siadat et al., 2012). Auxinic herbicides act in a similar way to indole-3-acetic acid (IAA); however, they are more persistent and active than IAA, damaging sensitive crops even at very low concentrations (Oliveira-Júnior, 2011), as occurs in cases of spray drift, which is deviation of particles applied in a certain area to an adjacent area, and contamination from spray equipment. The application of endogenous auxins on soybean seeds negatively regulates gibberellin biosynthesis and leads to an increase in the concentration of abscisic acid, resulting in secondary dormancy and reducing germination through delay in primary root emergence (Shuai et al., 2017). In addition, these hormones regulate fruit and seed formation (Ren and Wang, 2016). Hormonal herbicide drift can lead to injuries in parent plants, reduce yield, and affect the physiological quality of soybean seeds. The application of auxinic herbicides in the vegetative and reproductive stages of soybean reduces seed quality (Silva et al., 2018). Dicamba applied in the reproductive stage of the soybean crop affects the seed and, consequently, the plants of the following crop season (Barber et al., 2016; Miller and Norsworthy, 2018). Application of dicamba reduced the germination and vigor of soybean seeds (Silva et al., 2018). Robinson et al. (2013) observed changes in the composition of seeds, with reductions in lipid contents in soybean seeds when the plants were treated with dicamba in the vegetative and reproductive stages, while the protein content decreased when dicamba applications occurred in the V2 and V5 stages and increased under applications made in R2. The aim of this study was to evaluate the physiological quality of soybean seeds as a function of application of reduced rates of dicamba in two phenological phases (V4 and R2) of the crop.

MATERIAL AND METHODS

The trial was set up in Rio Verde, GO, at 17°48’67”S and 50°54’18”W, and altitude of 720 m. Climate in the region according to the Köppen-Geiger climate classification is Aw (tropical), with rainfall in October to April and a dry period from May to September. Soil in the location, of clayey texture (64.5%), had the following traits at the depth of 0 to

20 cm: pH (CaCl2) of 5.4, organic matter content of 3.9%, and a base saturation index of 71%. During the period of conducting the experiment in the field, the rainfall registered was 147, 244, 267, 136, and 20 mm, while the mean temperature was 25, 24.4, 24.8, 24.9, and 26.3 °C for the months of November and December (2017) and January, February, and March (2018), respectively. For the field experiment, a randomized block experimental design was used, with four replications. The factorial arrangement adopted was 4 × 2 + 1, with four reduced application rates (0.028, 0.28, 2.8 and 28 g of acid equivalent per hectare) of dicamba applied in two phenological stages of the soybean crop (V4 and R2) + an additional treatment without application of the herbicide. The V4 stage is characterized by development of the second trifoliate leaf on 2 the main stem, and the R2 stage is characterized by full flowering of the soybean plants. The plots were 25.2 m , with eight rows of 7 m length, spaced at 0.45 m. The area used for data collection was the five central meters of five center rows of each plot. Journal of Seed Science, v.42, e202042014, 2020 Effect of dicamba drift on soybean seeds 3

The soybean variety used was ADV 4672 IPRO and was sown mechanically in a no-till system, with eighteen seeds per linear meter. Seed treatment, crop management practices, and plant health management were carried out in accordance with EMBRAPA (2013). Dicamba drift was simulated using a backpack sprayer under CO2 pressure, regulated to obtain a constant pressure of 1.5 bar and spray volume of 170 L.ha-1. Model XR Teejet 8002VB extended range flat spray tips were used. Harvest was performed manually in the R8 stage (full maturity – 95% of the pods with mature color), and plants were mechanically threshed. After threshing, the seeds were manually cleaned and then dried in a forced-air circulation oven at a temperature of 25 °C until the seeds reached 10.5% moisture; the drying process took approximately ten hours. After drying, part of the seeds was stored in a BOD type chamber in polyethylene bags at a constant temperature of 20 °C for six months. Relative humidity (RH) and temperature were recorded by a digital data logger (precision: 0.1 °C; 5.0% RH). The seeds were placed in BOD under controlled conditions for a period of six months. Moisture content was not affected; the moisture level was 9.92% before storage and 10.64% after six months of storage. The traits described below were evaluated through duplicate samples of fifty seeds for each one of four replications, following the same experimental design used in the field: Moisture content: determined using a sample with weight from 4.5 to 5 g by the laboratory oven method at 105 ± 3 °C for 24 hours (Brasil, 2009). Electrical conductivity (EC): Each replication of fifty soybean seeds was first weighed (precision of 0.001 g), recording the weight for use in calculations, and then placed to soak in plastic cups (200 mL) containing 75 mL of deionized water and kept at 25 °C for 24 h (Hampton and Tekrony, 1995; Vieira and Krzyzanowski, 1999). At the end of the soaking period, electrical conductivity was read, using a digital conductivity meter Technal, model TEC-4MP. Results were expressed in micro Siemens per centimeter per gram (µS cm-1.g-1). Germination (G): Before sowing, seeds were treated with carbendazim + thiram at the rate of 0.3 and 0.7 g of active ingredient per kilogram of seed. The seeds were then placed to germinate in a germination paper substrate (Germitest®) moistened with water in the amount of 2.5 times the weight of the dry Germitest® paper, at 25 °C. Evaluations were made at five days (first germination count) and eight days after sowing, and results were expressed in percentage of normal seedlings (Brasil, 2009). First germination count (FGC): Counting and recording the number of normal seedlings, performed on the fifth day after setting up the germination test (Brasil, 2009). Emergence speed index (ESI) and emergence (E): Tests were performed through sowing in seed beds containing sand substrate, with four replications of fifty seeds. Sowing depth was 2.5 cm. The emerged seedlings were counted daily up to stabilization of emergence. Seedlings with cotyledons in the horizontal position were considered as emerged. Results of the emergence speed index were calculated according to Maguire (1962). Accelerated aging (AA): “gerbox” boxes were used, containing 40 mL of distilled water. Two hundred seeds of each treatment were distributed on an internal metallic screen in each gerbox, and the gerboxes were incubated at 41 °C for 48 hours (Marcos-Filho, 1999). After that period, the germination test was conducted on the seeds, under the conditions described above. Seedling length (SL): ten seedlings coming from the germination test of each experimental unit were used. Evaluation was made on the eighth day after setting up the germination test, selecting seedlings classified as normal in the germination test of the seeds that did not pass through the accelerated aging process. Total seedling length was determined from the tip of the main root to the cotyledonary node (connection of cotyledon to the stem) with the assistance of a millimeter ruler at eight days after sowing (Krzyzanowiski et al., 1999). The Shapiro-Wilk normality test (p ≤ 0.05) was used on the results, and analysis of variance (p ≤ 0.05) on those meeting the presuppositions. When significant, the results were subjected to the Dunnett test for contrasts of the treatments with the non-treated control, and to Tukey’s test (p ≤ 0.05) for contrast of the mean values between reduced application rates and herbicide application times. Data regarding germination from the accelerated aging test

Journal of Seed Science, v.42, e202042014, 2020 4 E. M. Costa et al. were transformed into arcsin of the square root of x/100, in which x was the value in percentage. Analyses were made through the software ASSISTAT (Silva and Azevedo, 2002).

RESULTS AND DISCUSSION

There were significant interactions between the reduced application rates of dicamba (0.028, 0.28, 2.8 and 28 -1 g.ha ) and the phenological stages (V4 and R2 - in which the herbicide was applied) on germination percentage (G). There was also significant interaction between the factors and the control treatment in the evaluations made soon after harvest (Table 1). Seed germination evaluated after soybean harvest was lowest for the highest application rate -1 of dicamba, especially when applied in the R2 stage. The application rate of 28 g.ha , i.e., 5.8% of the application rate ® recommended on the label of Dicamax , was enough to reduce germination by 14% in V4 and 39% in R2 in relation to the control (Table 1).

Table 1. Germination percentage (G) and first germination count (FGC) of soybean seeds under reduced rates of

dicamba applied in the V4 and R2 stages in evaluations made after crop harvest and after six months of storage. G (%) FGC (%) Mean Mean Reduced application rate V R V R (g a.e. ha-1) 4 2 4 2 Seeds obtained after harvest 0.028 84 abA 90 aA 87 92 97 94 a 0.28 93 aA 94 aA 93 94 97 95 a 2.8 90 aA 92 aA 91 93 96 94 a 28 77 bA (-) 52 bB (-) 64 74 (-) 67 (-) 71 b Mean 86 82 – 88 89 – Control 91 97 CV (%) 6.84 – 5.83 –

FA 42.6077* 42.8049* ns ns FB 3.8257 0.2257 ns FAxB 11.3549* 2.3962

FTreatxAddit. 4.6642* 6.7670* Seeds stored for six months after storage 0.028 77 83 80 a 91 92 92 a 0.28 87 82 84 a 95 91 93 a 2.8 82 70 76 a 93 80 87 a 28 34 23 29 b 63 71 67 b Mean 70 64 – 85 84 – Control 62 93 CV (%) 23.90 – 13.99 –

FA 21.2300* 8.3836* ns ns FB 0.9986 0.1970 ns ns FAxB 0.5459 1.0906 ns ns FTreatxAddit. 0.3775 1.5939 ns *Significant by the F test (p > 0.05); : not significant by the F test; FA = application rate factor; FB = soybean phenological stage factor; FAXB = interaction; FTreatxAddit. = treatments x additional. Mean values followed by different lowercase letters in the columns or different uppercase letters in the rows differ from each other by Tukey’s test (p < 0.05). Mean values followed by (-) were less than the control by the Dunnett test (p < 0.05).

Journal of Seed Science, v.42, e202042014, 2020 Effect of dicamba drift on soybean seeds 5

Dicamba applied at flowering reduced germination of soybean seeds (Wax et al., 1969). Use of dicamba in the V5 stage reduced soybean seed germination an average of 14% (Silva et al., 2018). Miller and Norsworthy (2018) observed -1 reduction of 5% in germination of soybean seeds treated with 28 g.ha of dicamba applied in R2, whereas at the application rate of 3.5 g.ha-1, there was no reduction. These authors also observed reduction of 69% in germination -1 with 28 g.ha of dicamba applied in the R5 stage. In the stored seeds, no interaction was observed between the reduced application rates and the times of application of the herbicide for seed germination percentage (Table 1). Nevertheless, a significant effect was found for the reduced application rates, which decreased the germination percentage at the highest rate applied. The rate of 28 g.ha-1 of dicamba reduced germination by 64% in relation to the lowest rate tested (Table 1). Germination of the treatments with the reduced application rate of 28 g.ha-1 of dicamba did not achieve 80% germination, the minimum required for sale of soybean seeds in Brazil (Brasil, 2009). In relation to FGC of the seeds after soybean harvest, no effect of the interaction between the reduced application rate and herbicide application time factors was observed, only the effect of the reduced application rates, with reduction of 24.5% at the rate of 28 g.ha-1 of dicamba in relation to the lowest rate applied (Table 1). For the reduced application rate of 28 g.ha-1, at both application times, the values of FGC were below that of the non-treated control. After storage, there was no significant interaction between the factors; nevertheless, the reduced application rates of dicamba caused a reduction in FGC. At the reduced rate of 28 g.ha-1 of dicamba, germination was 67%, while in the other treatments, the FGC was between 93% and 87% (Table 1). There was reduction of 19% in FGC due to applications -1 of 29.8 g.ha of dicamba in R2 and of 8% for applications in V5 (Silva et al., 2018), showing that the reduced application rates of dicamba lowered seed vigor in a more expressive way in applications in R2. Thus, drift events that occur in that stage make production of soybean seeds inviable. In the accelerated aging test of the seeds after soybean harvest, no significant interaction was observed between the reduced rates of dicamba factor and plant stage factor, but only the effect of application of reduced rates was observed, with the lowest values in the AA test at the application rate of 28 g.ha-1 of dicamba (Table 2). Considering the effects of treatments in relation to the control, germination for the reduced application rate of 28 g.ha-1 of dicamba applied in V4 and in R2 decreased soybean seed germination by 38% and 40%, respectively. After storage, there was no significant interaction between the reduced application rates of dicamba and the plant stages at dicamba application. Nevertheless, there was a significant effect from the reduced application rates of dicamba; the germination percentage in accelerated aging was 5% at the highest application rate evaluated, whereas in the other reduced application rates, germination ranged from 30% to 38% (Table 2). These data corroborate those obtained by Miller and Norsworthy (2018), who observed reductions of 18% and 45% in seed germination in the -1 accelerated aging test in the treatments with 28 g.ha of dicamba in the R2 and R3 stages, respectively. For the reduced application rate of 3.5 g.ha-1 of dicamba, these same authors did not observe reductions as a result of the applications made in the R1, R2, and R3 stages. Accumulation of dicamba can reduce seed germination (Auch and Arnold, 1978). Dicamba that is not metabolized in the plant is transported to the seed during the stages of seed filling (Thompson and Egli, 1973). Thus, reduction in germination due to the reduced application rates of dicamba tested indicates that the soybean seeds were not able to metabolize this herbicide during the storage period, which reduced germination. In length of seedlings from seeds after soybean harvest and from stored seeds, there was no interaction between the reduced application rates of dicamba and the plant stages at which application was made; nevertheless, seedling length declined as a function of the reduced application rates of dicamba (Table 2). In the newly harvested seeds, length declined by around 43% at the reduced application rate of 28 g.ha-1 compared to the treatment with 2.8 g.ha-1 and the treatment of 0.28 g.ha1 (Table 2). After storage, seedling length declined by 33% at the application rate of 28 g.ha-1 in relation to the application rate -1 of 0.28 g.ha . Seedling length was also less when dicamba had been applied in R2. In that stage, there was reduction

Journal of Seed Science, v.42, e202042014, 2020 6 E. M. Costa et al. of 2.3 cm compared to the V4 stage. The study in reference to length and dry matter of seedlings or of their parts is effective in detecting subtle differences in seed vigor (Vanzolini et al., 2007). The reduced application rates of dicamba aiming to simulate drift reduces the physiological quality of soybean seeds, affecting their vigor, since there was lower transfer of nutrients from the seed to the seedling in the treatments with 28 g a.e ha-1. In the emergence speed index (ESI) of the newly harvested seeds, there was no significant interaction between the factors of reduced dicamba application rate and the stage of the plant at the time of dicamba application. Nevertheless, in the seeds evaluated after six months of storage, there was significant interaction between the factors tested. The ESI was lower in the seeds under the higher application rate of dicamba, both in the newly harvested seeds and in the stored seeds (Table 3). Slow, reduced, or uneven emergence can result in gaps in stand, delay in development, problems for weed control, and interference in plant characteristics related to harvest (Marcos-Filho, 2013). -1 The ESI of the stored seeds in the treatments with the application rate of 28 g.ha of dicamba, applied in V4 and R2 was 3.1 and 6.9, respectively. At the rates lower than this, the ESI ranged from 8.3 to 10.2, with reduction of up to 70%

Table 2. Accelerated aging (AA) and seedling length (SL) of soybean seeds under reduced rates of dicamba applied in

the V4 and R2 stages in evaluations made after crop harvest and after six months of storage.

AA (%) SL (cm) Mean Mean Reduced application rate V R V R (g a.e. ha-1) 4 2 4 2 Seeds obtained after harvest 0.028 78 79 78 a 8.8 10.3 9.5 a 0.28 80 68 74 a 11.7 11.1 11.4 a 2.8 75 79 77 a 11.3 11.2 11.2 a 28 51(-) 49(-) 50 b 6.8 6.3 6.5 b Mean 71 69 – 9.6 9.7 – Control 81 9.2 CV (%) 12.26 – 14.79 –

FA 19.4619** 19.9520* ns ns FB 0.5071 0.0271 ns ns FAxB 1.3217 1.0389 ns FTreatxAddit. 6.3989* 0.3269 Seeds stored for six months after harvest 0.028 25 37 31 ab 15.4 14.3 14.8 ab 0.28 56 20 38 a 16.1 16.0 16.0 a 2.8 34 26 30 ab 15.4 11.2 13.3 ab 28 3 6 5 b 12.8 8.9 10.8 b Mean 30 22 – 14.9 A 12.6 B – Control 35 12.2 CV (%) 49.36 – 22.06 –

FA 3.3352* 4.5233* ns FB 0.8250 4.9096* ns FAxB 1.6132 0.9666ns ns FTreatxAddit. 0.5987 0.9360ns ns *Significant by the F test (p > 0.05); : not significant by the F test; FA = application rate factor; FB = soybean phenological stage factor; FAXB = interaction; FTreatxAddit. = treatments x additional. Mean values followed by different lowercase letters in the columns or different uppercase letters in the rows differ from each other by Tukey’s test (p < 0.05). Mean values followed by (-) were less than the control by the Dunnett test (p < 0.05).

Journal of Seed Science, v.42, e202042014, 2020 Effect of dicamba drift on soybean seeds 7 in the ESI (Table 3). Speed of emergence is fundamental for rapid establishment of seedlings. Thus, greater ESI results in better performance and greater capacity for resisting stresses that may interfere in the growth and development of the plant (Dan et al., 2010). The occurrence of dicamba drift at the reduced rate of 28 g.ha-1, 5.8% of the rate recommended on the label, in a soybean seed production field can result in damage to the seeds produced from the soybean plants that were contaminated, since these reduced rates of dicamba decrease the vigor of soybean seeds. The emergence percentage in sand after harvest of the seeds was not affected by interaction between application rates and the time periods of application, whereas in the stored seeds, there was significant interaction between these factors (Table 3). In the seeds evaluated after harvest, the emergence percentage was lower at the application rate of 28 g.ha-1 of dicamba, with 78% emergence, regardless of the phenological stage in which it was applied. After storage, -1 at the highest application rate of dicamba (28 g.ha ), there was greater reduction when application was made in R2 than in V4 (Table 3). Auch and Arnold (1978) observed reductions in soybean seedling emergence due to the treatment with 11 and 56 g.ha-1 of dicamba.

Table 3. Emergence speed index (ESI) and emergence percentage (E) of soybean seeds under reduced rates of dicamba

applied in the V4 and R2 stages in evaluations made after crop harvest and after six months of storage.

ESI E (%) Mean Mean Reduced application rate V R V R (g a.e. ha-1) 4 2 4 2 Seeds obtained after harvest 0.028 9.6 8.9 9.3 a 96 92 94 a 0.28 9.7 9.0 9.4 a 97 92 94 a 2.8 9.3 9.5 9.4 a 93 93 93 a 28 8.3 7.1 7.7 b 86 71 78 b Mean 9.2 8.6 – 93 A 87 B – Control 9.7 95 CV (%) 9.29 – 8.28 –

FA 7.4815* 8.3180* ns FB 4.0471 5.2527* ns ns FAxB 0.8851 1.4788 ns ns FTreatxAddit. 3.0507 1.5682 Seeds stored for six months after harvest 0.028 8.3 aA 9.1 aA 8.7 80 aA 85 aA 82 0.28 10.2 aA 8.4 abB 9.3 91 aA 86 aA 88 2.8 8.8 aA 8.8 abA 8.8 83 aA 83 aA 83 28 3.1 bB 6.9 bA 5.0 37 bB 64 bA 50 Mean 7.6 8.3 – 73 79 – Control 8.8 81 CV (%) 12.76 – 11.54 –

FA 29.3740* 30.6646* ns FB 3.9951 4.6684*

FAxB 10.2919* 5.0964* ns ns FTreatxAddit. 2.6923 1.1385 ns *Significant by the F test (p > 0.05); : not significant by the F test; FA = application rate factor; FB = soybean phenological stage factor; FAXB = interaction; FTreatxAddit. = treatments x additional. Mean values followed by different lowercase letters in the columns or different uppercase letters in the rows differ from each other by Tukey’s test (p < 0.05).

Journal of Seed Science, v.42, e202042014, 2020 8 E. M. Costa et al.

Severe reductions in the speed and percentage of seedling emergence generally result in problems during plant development (Marcos-Filho, 2013), such as reduction in the ability of the crop to compete with weeds and more susceptibility to attack from pathogens. Therefore, dicamba drift can potentially reduce crop yield in the event of use of seeds that have been contaminated by this herbicide, since reduction in seed physiological potential indirectly affects agricultural production, due to its reflections on initial plant stand (Marcos-Filho, 2005; Marcos-Filho, 2013). In electrical conductivity, there was significant interaction between the application rate and the time period of application in the seeds evaluated after harvest, whereas for the stored seeds, there was no significant interaction (Table 4). After harvest, electrical conductivity of the seeds was higher in the treatments with 28 g.ha-1 of dicamba applied at R2. At that application rate, electrical conductivity was two times greater than in the control treatment and higher than in the other treatments. After storage, electrical conductivity of the seeds coming from soybean plants treated with 28 g.ha-1 of dicamba applied in R2 was double the electrical conductivity values of the control treatment, an effect similar to that obtained

Table 4. Electrical conductivity (EC) of soybean seeds under reduced rates of dicamba applied in the V4 and R2 stages in evaluations made after crop harvest and after six months of storage.

EC (µS cm-1 g-1) Mean Reduced application rate V R (g a.e. ha-1) 4 2 Seeds obtained after harvest 0.028 81 aA 70 aA 76 0.28 73 aA 73 aA 73 2.8 86 aA 80 aA 83 28 92 aA 140 bB (-) 116 Mean 83 91 – Control 71 CV (%) 14.83 –

FA 20.3090* ns FB 3.1322

FAxB 9.1157*

FTreatxAddit. 5.4284* Seeds stored for six months after harvest 0.028 111 121 116 a 0.28 106 115 110 a 2.8 124 121 123 a 28 143 196 (-) 169 b Mean 121 138 – Control 95 CV (%) 21.81 –

FA 7.7299* ns FB 3.2206 ns FAxB 1.5916

FTreatxAddit. 5.5371*

ns *Significant by the F test (p > 0.05); : not significant by the F test; FA = application rate factor; FB = soybean phenological stage factor; FAXB = interaction; FTreatxAddit. = treatments x additional. Mean values followed by different lowercase letters in the columns or different uppercase letters in the rows differ from each other by Tukey’s test (p < 0.05). Mean values followed by (-) were less than the control by the Dunnett test (p < 0.05).

Journal of Seed Science, v.42, e202042014, 2020 Effect of dicamba drift on soybean seeds 9 in the newly harvested seeds. After storage, electrical conductivity of the control was 95.4 µS cm-1.g-1, and at the -1 -1 -1 application rate of 28 g.ha , it reached 142.8 and 195.9 µS cm .g for application made in the V4 and R2 stages, respectively (Table 4). For high vigor seeds, electrical conductivity should be between 70 and 80 µS cm-1.g-1 (Vieira and Krzyzanowiski, 1999). The increase in electrical conductivity shows that there was damage in the cell membrane system (Marcos-Filho et al., 1987). This damage results in leaching of sugars, amino acids, electrolytes, and other water soluble substances (Heydecker, 1974), leading to reduction in the vigor of seeds from plants reached by dicamba drift. During deterioration, the first events that occur are disorganization and loss of control of the permeability of seed membranes, and this situation results in reduced germination and in embryo death (Delouche and Baskin, 1973).

CONCLUSIONS

Reduced rates of dicamba applied in the V4 and R2 stages of the soybean crop reduce seed physiological quality after crop harvest and after six months of storage. The application rate of 28 g.ha-1 more expressively reduces germination, first germination count, emergence speed index and emergence percentage, seedling length, and electrical conductivity of soybean seeds, especially after a period of storage.

ACKNOWLEDGMENTS

This study was conducted with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) under funding code 001 and of the Instituto Federal Goiano, Rio Verde campus.

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Journal of Seed Science, v.42, e202042014, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Chemical treatment and size of corn seed on physiological Journal of Seed Science, v.42, e202042010, 2020 and sanitary quality during storage http://dx.doi.org/10.1590/2317- 1545v42219569 Karen Marcelle de Jesus Silva1* , Renzo Garcia Von Pinho1 , Édila Vilela de Resende Von Pinho1 , Renato Mendes de Oliveira2 , Heloísa Oliveira dos Santos1 , Thomas Simas Silva1

ABSTRACT: The aim of this study was to investigate the effect of treatment with insecticides and fungicides on the physiological quality of corn seeds, classified in sizes, in different periods of storage. Seeds of the hybrid BM915 PRO, classified in two sizes (CH20/64 and CH24/64), were treated with a mixture of carbendazim + thiram (Derosal Plus®), pirimiphos methyl (Actellic®), deltamethrin (K-obiol®), and water (standard treatment). In addition to the standard treatment adopted by the company, treatments with clothianidin (Poncho®), thiamethoxam (Cruizer®), and fipronil (Shelter®) were added to the spray mixture. The seeds were stored in a non-climate-controlled environment in multi-ply paper packages. The physiological quality of the seeds was evaluated every ninety days over a period of 270 days by the germination test, cold test, and accelerated aging. Sanitary quality was also evaluated through the Blotter test. The physiological quality of seeds of the hybrid BM915 PRO is maintained up to ninety days of storage, regardless of the chemical treatment used and the size of the seeds. The addition of the insecticide to the standard treatment used in chemical seed treatment does not affect the action of the fungicides on the fungi in the seeds.

Index terms: Zea mays, seed vigor, conservation, sanitary treatment, industrial treatment.

Tratamento químico e tamanho da semente de milho na qualidade fisiológica e sanitária durante o armazenamento

RESUMO: Objetivou-se verificar a interferência do tratamento com inseticidas e fungicidas sobre a qualidade fisiológica de sementes de milho, classificadas em dois tamanhos, em diferentes períodos de armazenamento. Sementes do híbrido BM915 PRO, classificadas em dois tamanhos (CH20/64 e CH24/64), foram tratadas com calda caracterizada pela mistura de Carbendazim + Thiram (Derosal Plus®), Deltametrina (Actellic®), Pirimifós metílico (K-obiol®) e água (tratamento padrão). Além do tratamento padrão adotado pela empresa, foram acrescentados à mistura da calda tratamentos com clotianidina (Poncho®), tiametoxam (Cruizer®) e fipronil (Shelter®). As sementes foram armazenadas em ambiente não climatizado *Corresponding author em embalagens de papel multifoliado. A qualidade fisiológica das sementes foi avaliada, a E-mail: [email protected] cada noventa dias, por um período de 270 dias por meio do teste de germinação, teste de frio e envelhecimento acelerado. Foi avaliada, ainda, a qualidade sanitária por meio do Blotter Received: 2/6/2019. Accepted: 11/26/2019. test. Concluiu-se que a qualidade fisiológica das sementes do híbrido BM915 PRO é mantida até os noventa dias de armazenamento, independente do tratamento químico utilizado e do tamanho das sementes. A adição do inseticida ao tratamento padrão utilizado no tratamento químico das sementes não afeta a ação dos fungicidas sobre os fungos presentes nestas. 1Universidade Federal de Lavras.

Termos para indexação: Zea mays, vigor de sementes, conservação, tratamento sanitário, 2Universidade Estadual de Montes tratamento industrial. Claros.

Journal of Seed Science, v.42, e202042010, 2020 2 K. M. J. Silva et al.

INTRODUCTION

Seed is one of the main inputs in agriculture, and its quality is one of the primary factors for establishing any crop (Nunes et al., 2009). Seed quality can be affected by different biotic and abiotic factors, such as chemical treatment and storage conditions. Seed treatment has been used as a tool for protecting seeds in the field and in storage for the purpose of maintaining physiological quality, and it is a valuable method for controlling and/or preventing pest and pathogen attacks. Lack of this initial protection can have a direct impact on yield. Chemical treatment consists of incorporating artificially developed chemical products on the seeds. This modality has been increasingly adopted by farmers since it is easy to perform and can be practiced in a controlled environment through the ease of uniformly distributing small amounts of products in growing areas, through reduced need for complementary applications of pesticides on developing crops, and through its low relative cost, which, even so, provides significant increases in final yield. Some factors affect the performance of the seed chemical treatment, such as type of seed, physical and physiological condition of the seed lot to be treated, seed size, product formulation, active ingredient, and application rate of the product (Machado, 2000). Inadequate application of chemical products on seeds can increase risks of deterioration of their physiological quality, due to possible phytotoxic effects. Salgado and Ximenes (2013) evaluated the effect of treatment of maize seeds with insecticides in different storage periods and concluded that seed treatment with insecticide and storage affect seed germination. According to these authors, the interaction of seed treatment and storage affect the number of ungerminated seeds. In contrast, the advantages of using seed with protection against external biological agents, such as fungi, insects, nematodes, etc. are well-known (Tonin et al., 2014). In Brazil, nearly 100% of hybrid maize seeds are treated with fungicides and insecticides in the seed industry for pest control in storage; 35% are treated with insecticides, and the rest receive insecticide application on the farm property itself (Nunes, 2016). The fact that chemical treatment of maize seeds is a widely used practice before storage and near the time of sowing highlights the importance of conducting studies on the chemical products used for treatment, as well as their effect on the quality of seeds under storage. Thus, the aim of this study was to investigate the effect of insecticide and fungicide treatment on the physiological quality of maize seeds classified in two sizes in different storage periods.

MATERIAL AND METHODS

The experiment was conducted in the Seed Sector of the Department of Agriculture of the Universidade Federal de Lavras (UFLA) in the municipality of Lavras, MG, Brazil. The seeds used were of the maize hybrid BM915 PRO, with a flat shape, classified in two sizes (CH20/64 and CH24/64), granted by the Hellix Sementes company, from the 2015/2016 crop season. The seeds were treated manually using plastic bags, in which the seeds and the chemical products were mixed in a uniform manner. The seeds were treated with a spray mixture composed of insecticides and fungicides adopted by the production company, characterized by a mixture of carbendazim + thiram (Derosal Plus®), pirimiphos methyl (Actellic®), deltamethrin (K-obiol®), and water (standard treatment) (Table 1). In addition to the standard treatment adopted by the company, the insecticides clothianidin (Poncho®), thiamethoxam (Cruizer®), and fipronil (Shelter®) were separately added to the spray mixture (Table 2). The spray volume used was the same for all the treatments. The insecticide application rates were those recommended by the manufacturers. After the treatments, the seeds were placed in multi-ply paper bags, similar to those used by the production company, kept on wooden pallets, and stored in a non-climate-controlled environment. The environment referred to is a shed covered with galvanized steel roofing that is closed, located in the Seed Sector of the Universidade Federal de Lavras. The Journal of Seed Science, v.42, e202042010, 2020 Quality of chemically treated and stored corn seeds 3

Table 1. Products used in composition of the standard treatment applied in the seed treatment and their respective application rates.

Commercial product Active ingredient Class Application rate of the active ingredient carbendazim Fungicide 2.04 g a.i./100 kg of seed Derosal Plus® thiram Fungicide 4.76 g a.i./100 kg of seed K-obiol® deltamethrin Insecticide 0.02 g a.i./100 kg of seed Actellic® pirimiphos methyl Insecticide 0.04 g a.i./100 kg of seed

Table 2. Insecticides and application rates used in the seed treatments.

Commercial product Active ingredient Application rate of the active ingredient Poncho® clothianidin 210 g a.i./100 kg of seed Cruizer® thiamethoxam 210 g a.i./100 kg of seed Shelter® fipronil 62.5 g a.i./100 kg of seed

storage period began on May 25th, 2016 and ended on February 16th, 2017. During this period, temperatures ranged from 18 °C to 25.9 °C, and relative humidity within the storage location oscillated from 44% to 77%. Seed quality was evaluated at the beginning of the storage period and every ninety days, over a period of 270 days, through the tests described as follows. Germination test: performed using germitest paper moistened with water in the amount of 2.5 times the dry weight of the paper, with four replications of fifty seeds in a germinator at 25 °C. The first germination count was performed at four days after setting up the test, and the last, at seven days. Classification was made of normal seedlings, abnormal seedlings, and dead seeds, and the result was expressed in percentage of abnormal seedlings (Brasil, 2009b). Cold test without soil: performed using germitest paper as a substrate, moistened with water in the amount of 2.5 times the dry weight of the paper, with four replications of fifty seeds in the form of rolls, similar to the germination test. The rolls were placed in plastic bags and kept in a chamber regulated to 10 °C for seven days. After that period, the rolls were removed from the plastic bags and kept in a seed germinator regulated to 25 °C, for four days (Krzyzanowski et al., 1999). After that, the seedlings were evaluated, considering only normal seedlings, following the criteria adopted for the germination test (Brasil, 2009b). Accelerated aging: gerbox transparent plastic boxes (11.5 × 11.5 × 3.5 cm) were used with screens inside, on which the seeds were distributed so as to form a uniform layer. At the bottom of each plastic box, 40 mL of distilled water was added, establishing an environment with 100% relative humidity. The boxed were closed and kept in an aging chamber (BOD type) at 42 °C for 72 hours. After that period, the seeds were removed from the chamber and the germination test was performed, evaluating normal seedlings on the fifth day after setting up the test. Seed health test: the sanitary quality of the seeds was determined through the filter paper method (blotter test), following the method of the Manual de Análise Sanitária de Sementes (Brasil, 2009a). Eight replications of fifty seeds were used per treatment. The seeds were placed on three sheets of previously sterilized and moistened filter paper in Petri dishes that were transparent and with lids. The samples were cooled in a freezer for 24 h. After that, the samples were incubated at 20 ± 2 °C for a period of ten days under a regime of twelve hours of light. At the end of this period, the seeds were individually examined under a stereomicroscope at a resolution of 30–80 X. Results were expressed in percentage of occurrence of fungi. The experiment was conducted in a completely randomized experimental design in a 2 × 4 × 4 factorial arrangement, with four replications, constituted by two classifications regarding seed size (CH20/64 and CH24/64), four chemical treatments, and four storage periods. Journal of Seed Science, v.42, e202042010, 2020 4 K. M. J. Silva et al.

Data were analyzed through the Sisvar statistical program (Ferreira, 2011). The Tukey test at 5% probability was used to compare the means, and a regression study was performed for the storage period factor.

RESULTS AND DISCUSSION

Germination test There was no difference among the chemical treatments used up to 180 days of storage for seeds of size CH24/64 (Table 3). Nevertheless, in seeds of size CH20/64 under the treatment with thiamethoxam at 0 days and clothianidin at ninety days of storage, results inferior to the other treatments were observed. From 180 days of storage on, seeds of size CH20/64 did not show statistical differences among the treatments. In spite of loss of quality over the storage period (Figure 1), seeds of the hybrid BM915 PRO maintained germination percentages above the minimum standard established by the Ministry of Agriculture for commercialization (85% germination). It is noteworthy that the use of the standard treatment resulted in smaller reductions in seed physiological quality in relation to the others. Similar results were observed by Rosa et al. (2012) and Tonin et al. (2014) upon using hybrid maize seeds treated with different insecticides. The authors found loss of physiological quality when the seeds were treated with thiamethoxam and stored in a natural environment for 180 and 270 days. Mariucci et al. (2018) observed reduction in germination of hybrid maize seeds under treatments with insecticides, fungicides, and different combinations of inoculants after 45 days of storage. Lorenzetti et al. (2014) found that maize seeds treated with fipronil did not lose physiological quality after a 42-day storage period, which corroborates the results obtained in the present study. For the results of germination, the addition of the active compound thiamethoxam to the standard treatment used by the production company resulted in loss of physiological quality of maize seeds. Various authors affirm that the quality of stored seeds of hybrid maize treated with insecticides is affected not only by the chemical product used in the seed treatment, but also by the genotype and by the conditions of the storage environment (Bittencourt et al., 2000; Marcos-Filho, 2005; Rosa et al., 2012; Tonin et al., 2014). These assertions corroborate the results of this study.

Table 3. Germination percentage of seeds of the maize hybrid BM915 PRO in accordance with storage periods (SP), seed size, and chemical treatments.

Treatment SP (days) Seed size *Standard treat. + *Standard treat. + *Standard treat.+ *Standard treat. clothianidin thiamethoxam fipronil CH 20/64 96 Aa 95 Aa 88 Bb 97 Aa 0 CH 24/64 97 Aa 98 Aa 97 Aa 99 Aa CH 20/64 96 Aa 90 Bb 97 Aa 95 Aa 90 CH 24/64 98 Aa 97 Aa 98 Aa 95 Aa CH 20/64 93 Ba 96 Aa 93 Aa 92 Ba 180 CH 24/64 98 Aa 95 Aa 96 Aa 96 Aa CH 20/64 90 Ba 91 Aa 89 Aa 89 Ba 270 CH 24/64 97 Aa 90 Ab 92 Aab 95 Aab CV (%) 3.09 Mean values followed by the same uppercase letter in the column and lowercase letter in the row within each storage period do not differ from each other by the Tukey test at 5% significance. *Standard treatment.

Journal of Seed Science, v.42, e202042010, 2020 Quality of chemically treated and stored corn seeds 5

Stantard treatment + thiamethoxan y = 89.075000 + 0.103611x 0.00036x2 R2 = 85.53% Stantard treatment + thiamethoxan y = 98.550000 - 0.018889x R2 = 78.11% R2 = 99.02%

Figure 1. Germination percentage of seeds of the maize hybrid BM915 PRO, sizes CH20/64 and CH24/64, in accordance with storage periods and chemical treatments.

Cold test without soil There were no significant effects of the treatments in this test. Bernardi (2015), working with other hybrid, found that treated and stored maize seeds for up to ninety days did not lose physiological performance under the cold test, and seed vigor remained equivalent to that at the beginning of the experimental period. Horri and Shetty (2007) found that decreases in the viability and vigor of seeds treated with insecticides are not only due to the metabolic changes caused by possible phytotoxic effects of the products, but may also be attributed to damage to cell membranes. Mariucci et al. (2018) likewise observed reduction in vigor of maize seeds treated with the combination of fungicides, insecticides, and inoculants and stored for ninety days. The query formulated by Hammann (2008) suggests that use of the cold test for vigor evaluation of chemically treated seeds is inappropriate, due to the high number of treated seeds placed per volume of substrate to carry out the test, whether this is conducted in paper or boxes with soil. This would result in a high concentration of active compounds in relation to the true potential for product dilution under field conditions. Nevertheless, Popinigis (1997) defends the use of the cold test to predict seed performance in the field or in storage, as well as to determine vigor among seed lots and evaluate the effect of fungicide seed treatment. This is because the combination of low temperatures and high humidity is used to allow survival of only the most vigorous seeds, since test conditions can reduce the speed of germination and favor the performance of harmful microorganisms (Marcos-Filho et al., 1987).

Accelerated aging There was difference among the chemical treatments at 0 days of storage; the lowest vigor values were observed in seeds under the standard treatment + thiamethoxam. At ninety days, there was no difference among treatments. At 180 and 270 days of storage, the lowest results were attributed to the standard treatment + clothianidin and to the standard treatment + fipronil, respectively (Table 4). There was no statistical difference in relation to seed size at zero and ninety days of storage (Table 5). In general, the physiological quality of the seeds declined over the storage period, given the loss of vigor observed in the accelerated aging test, regardless of the chemical treatment used and the size of the seeds (Figures 2 and 3). In this test, high temperature and high humidity conditions prevail, which are severe for the seeds and favorable to development of storage fungi, such as Penicillium and Aspergillus, thus leading to a more rapid decline in vigor. A greater reduction in percentage of germination was observed in seeds of size CH20/64 compared to those of size CH24/64 (Figure 2).

Journal of Seed Science, v.42, e202042010, 2020 6 K. M. J. Silva et al.

Table 4. Germination percentage of seeds of the maize hybrid BM915 PRO under accelerated aging in accordance with chemical treatments and storage periods.

Storage period (days) Treatment 0 90 180 270 *Standard treat. 91 AB 93 A 84 A 76 AB *Standard treat + clothianidin 96 A 92 A 46 C 85 A *Standard treat + thiamethoxam 85 B 86 A 73 B 79 AB *Standard treat + fipronil 94 AB 92 A 73 B 72 B CV (%) 9.07 Mean values followed by the same uppercase letter in the column do not differ from each other by the Tukey test at 5% significance. *Standard treatment.

Table 5. Germination percentage of seeds of hybrid maize BM915 PRO under accelerated aging in accordance with seed size and storage periods.

Storage period (days) Seed size 0 90 180 270 CH 20/64 92 A 89 A 76 A 73 B CH 24/64 91 A 92 A 61 B 83 A CV (%) 9.07 Mean values followed by the same uppercase letter in the column do not differ from each other by the Tukey test at 5% significance.

Stantard treatment + thiamethoxan y = 81

Figure 2. Germination percentage of seeds of the maize Figure 3. Germination percentage of seeds of the maize hybrid BM915 PRO under accelerated aging in hybrid BM915 PRO under accelerated aging in accordance with storage periods and seed size. accordance with storage periods and chemical treatments.

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Seed health In addition to the effects on physiological quality, the standard treatment adopted by the company and the addition of other compounds to the seed chemical treatment did not completely inhibit the occurrence of microorganisms, regardless of the chemical treatment and seed size, and this occurrence was variable throughout the storage period (Table 6). Fusarium was found in greater quantity in seeds compared to the fungi characteristic of stored grains in the initial period of the experiment (from zero to ninety days). Up to ninety days of storage, there was an increase in the occurrence of the fungi Fusarium, Aspergillus flavus, and Penicillium spp. in the seeds, in all the hybrids used. From that period on, a reduction in these pathogens was observed, even coming to lack of detection in some treatments. The same response was found both for seeds of size CH20/64 and for those of size CH24/64. However, for the seeds of size CH24/64, the reduction in the quantity of pathogens was slower. Lasca et al. (2005) evaluated the occurrence of fungi in seeds of two maize hybrids under chemical treatment and found greater occurrence of Fusarium verticillioides in seeds of the hybrid Z 8392 (84.75%), and the second greatest

Table 6. Mean percentage values of the occurrence of fungi in seeds of the maize hybrid BM915 PRO, sizes CH20/64 and CH24/64, in accordance with chemical treatments at 0, 90, 180, and 270 days of storage.

**Fungus (%)

Days of FU AF PE AN AO Treatment storage CH20/64 CH24/64 CH20/64 CH24/64 CH20/64 CH24/64 CH20/64 CH24/64 CH20/64 CH24/64

*Standard treat. 77.5 85.5 3 0.5 19.5 33 0 0.5 1 0 *Standard treat. 39 48 3.5 2.5 14.5 20 1 1 0.5 0.5 + clothianidin 0 *Standard treat. 63 53.5 1.5 0 17 20.5 0.5 0.5 3 1.5 + thiamethoxam *Standard treat. 20.5 48.5 4 0 18.5 19.5 0 0 1.5 0.5 + fipronil *Standard treat. 79 48.5 3.5 1.5 40.5 41.5 0 0.5 3.5 1.5 *Standard treat. 38.5 47.5 3.5 0.5 52.5 52.5 0.5 0.5 0.5 0 + clothianidin 90 *Standard treat. 30 54 4 2 36 59.5 0 0 3.5 1.5 + thiamethoxam *Standard treat. 75 98 6.5 1 47 45.5 2.5 0 3.5 3.5 + fipronil *Standard treat. 0 0 5 2 8.5 7 0 0 0.5 0 *Standard treat. 0 0 6 5 6.5 9 0 0 1 0 + clothianidin 180 *Standard treat. 0 0 4.5 3 9 13.5 0 0 1.5 0 + thiamethoxam *Standard treat. 0 0 1.5 1 2 4.5 0 1.5 0 0.5 + fipronil *Standard treat. 0.5 0 3.5 0 0 0.5 0 2 0 0 *Standard treat. 0 0 1 0.5 0.5 2 0 0 0 0 + clothianidin 270 *Standard treat. 0 0 1.5 0.5 0 0.5 0 2 0 0 + thiamethoxam *Standard treat. 0 1 0 0 13 6.5 0 1 0.5 0 + fipronil **FU – Fusarium; AF – Aspergillus flavus; PE – Penicillium spp.; AN – Aspergillus niger; AO – Aspergillus ochraceus. *Standard treatment.

Journal of Seed Science, v.42, e202042010, 2020 8 K. M. J. Silva et al. in the hybrid DAS 9560 (61.50%). According to Galperin et al. (2003), this fungus is widely disseminated and can cause considerable damage to the maize crop. However, it has been shown that the fungi Fusarium verticillioides, Penicillium sp., and Aspergillus sp. found in the seeds used in this study generally do not affect germination of maize seeds under normal sowing conditions (Pinto, 1996). In general, it was observed that the mixture of insecticides with the standard treatment did not negatively affect the action of the fungicide on the pathogens.

CONCLUSIONS

The physiological quality of the maize hybrid seeds BM915 PRO is maintained up to ninety days of storage, regardless of the chemical treatment used and the size of the seeds. Addition of the insecticide to the standard treatment used in seed chemical treatment does not affect the action of the fungicides on the fungi present in the seeds.

ACKNOWLEDGMENTS

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for granting scholarships.

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LORENZETTI, E.R.; RUTZEN, E.R.; NUNES, J.; CREPALLI, M.S.; LIMA, P.H.P.; MALFATO, R.A.; OLIVEIRA, W.C. Influência de inseticidas sobre a germinação e vigor de sementes de milho após armazenamento. Cultivando o Saber, v.7, n.1, p.14-23, 2014. https://www. fag.edu.br/upload/revista/cultivando_o_saber/5399b51186157.pdf

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NUNES, J.R.G.; MENEZES, N.L.; CARGNELUTTI-FILHO, A. Qualidade fisiológica de sementes de sorgo silageiro submetidas a diferentes sequências de beneficiamento. Pesquisa Agropecuária Gaúcha, v.15, n.1, p.21-28, 2009. http://www.fepagro.rs.gov.br/ upload/1398783827_art03.pdf

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POPINIGIS, F. Fisiologia da semente. Brasília: AGIPLAN, 1997. 287p.

ROSA, K.C.; MENEGHELLO, G.E.; QUEIROZ, E.S.; VILLELA, F.A. Armazenamento de sementes de milho híbrido tratadas com tiametoxam. Informativo ABRATES, v.22, n.3, p.60-65, 2012.

SALGADO, F.H.M.; XIMENES, P.A. Germinação de sementes de milho tratadas com inseticidas. Journal of Biotechnology and Biodiversity, v.4, n.1, p.49-54, 2013. https://www.researchgate.net/publication/250613046_Germinacao_de_sementes_de_ milho_tratadas_com_inseticidas

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Journal of Seed Science, v.42, e202042010, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Physiological and antioxidant changes in sunflower seeds Journal of Seed Science, v.42, under water restriction e202042008, 2020 http://dx.doi.org/10.1590/2317- 1545v42225777 Thais de Castro Morais1 , Daniel Teixeira Pinheiro1* , Paola Andrea Hormaza Martinez1 , Fernando Luiz Finger1 , Denise Cunha Fernandes dos Santos Dias1

ABSTRACT: Seed vigor may be determinant for field performance, especially under water restriction conditions. Sunflower is a crop subject to these conditions in the field and, therefore, the aim of this study was to evaluate the physiological and antioxidant changes in sunflower seeds under water restriction. Two lots of sunflower seeds (cv. Hélio 253) with different vigor levels were used. After initial characterization, seeds were placed to germinate under water potentials of 0.0, -0.2, -0.4, -0.6 and -0.8 MPa and evaluated by tests of germination, first germination count, shoot length and primary root length. The activities of the antioxidant enzymes SOD, CAT, POX and APX were also evaluated at 0, 2, 4 and 6 days after sowing. Water restriction led to a decrease in germination and slower seedling growth, regardless of seed vigor level. SOD activity was similar in the two lots, with reduction in activity four days after sowing. CAT activity was affected differently during germination in the two lots, and it was generally higher in the most vigorous lot. In higher vigor seeds, there was lower POX activity in water restriction treatments compared to the control. In general, seeds of lower vigor have lower capacity for activation of antioxidant enzymes, especially peroxidases.

Index terms: enzyme analysis, water deficit, germination, Helianthus annuus L., vigor.

Alterações fisiológicas e antioxidativas em sementes de girassol submetidas à restrição hídrica

RESUMO: O vigor das sementes pode ser determinante para o seu desempenho em campo, especialmente sob condições de restrição hídrica. O girassol é uma cultura sujeita as essas condições no campo, e assim, o objetivo do trabalho foi avaliar as alterações fisiológicas e antioxidativas em sementes de girassol submetidas à restrição hídrica. Foram utilizados dois lotes da cv. Hélio 253 diferindo quanto ao vigor. Após a caracterização inicial, as sementes foram colocadas para germinar sob os potenciais de 0,0; -0,2; -0,4; -0,6 e -0,8 MPa e avaliadas quanto a germinação, primeira contagem e comprimento de parte aérea e raiz primária. Foram avaliadas *Corresponding author também as atividades das enzimas antioxidativas SOD, CAT, POX e APX aos 0, 2, 4 e 6 dias após E-mail: [email protected] a semeadura. A restrição hídrica provocou decréscimo na germinação e menor crescimento das plântulas independentemente do nível de vigor das sementes. A atividade da SOD foi semelhante Received: 7/9/2019. para os dois lotes, com redução aos quatro dias após a semeadura. A atividade da CAT foi afetada Accepted: 10/22/2019. de modo diferente ao longo da germinação dos dois lotes sendo, em geral, mais alta no lote de maior vigor. Nas sementes de maior vigor, houve menor atividade da POX nos tratamentos de restrição hídrica em relação ao controle. Em geral, sementes de menor vigor possuem menor 1Universidade Federal de capacidade de ativação de enzimas antioxidativas, principalmente as peroxidases. Viçosa (UFV), Departamento de Agronomia, 36570-900 – Viçosa, Termos para indexação: análise enzimática, déficit hídrico, germinação, Helianthus annuus L., vigor. MG, Brasil.

Journal of Seed Science, v.42, e202042008, 2020 2 T. C. Morais et al.

INTRODUCTION

The expressive increase in planted area of sunflower (Helianthus annuus L.) has led to an increase in the demand for high quality seeds. Sunflower is grown in most Brazilian states and is concentrated especially in the Cerrado region (Brazilian tropical savanna) between the soybean and maize crop seasons. It is of fundamental importance in rotation systems (CONAB, 2017). The crop is subject to variations in edaphic and climatic conditions, mainly in regard to soil water availability in the seedling emergence phase (Backes et al., 2008). Water restriction in the soil at the time of sowing reduces the emergence and development of seedlings due to interference in the water uptake and cell elongation processes (Finch-Savage and Bassel, 2016; Marcos-Filho, 2015). Under water stress conditions, field emergence and initial seedling development depend on the level of seed vigor. Albuquerque and Carvalho (2003) found that the effect of seed vigor in sunflower on reduction of field emergence is associated with stress conditions at the time of sowing. These authors found that under water restriction at -1.1 MPa, obtained by moisture control in the soil, there was reduction in seedling emergence even for higher vigor seed lots. In a study carried out with two sunflower cultivars under water stress, Carneiro et al. (2011) observed a reduction in germination, length, and dry matter of seedlings, above all at the water potential of -0.8 MPa with the use of polyethylene glycol (PEG 6000). Similar results were observed by Luan et al. (2014), using the osmotic agents PEG 6000 and sodium chloride (NaCl). Water restriction during the germination process can lead to oxidative stress in seeds and increase the production - - of reactive oxygen species (ROSs), such as the superoxide radical (O2 ), the hydroxyl radical (OH ), hydrogen peroxide 1 (H2O2) and singlet oxygen ( O2) (Jaleel et al., 2007; Mouradi et al., 2016; Nguyen et al., 2019). The intensity of cell damage is determined by the capacity of seeds to eliminate these free radicals through defense systems, including the action of antioxidant enzymes, which promote control of the intracellular concentration of ROSs (Kapoor et al., 2015). The inner content of these compounds and the activation of the antioxidant defense system are associated with successful germination, especially in situations of abiotic stresses (Chen and Arora, 2013; Jisha et al., 2013; Savvides et al., 2016). Among the main antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) are constantly regulated in the process of neutralization of excessive ROSs at the cell level (Bailly, 2004; Del Río et al., 2018; Groß et al., 2013; Kibinza et al., 2011). However, this regulation may be inefficient if the stress is more accentuated, with an increase in free radical production. In sunflower seeds, antioxidant enzyme activity and seed physiological potential were not affected when under water stress and salt stress up to the water potential of -0.4 MPa, but under the potential of -0.8 MPa, antioxidant capacity during germination was reduced, affecting seedling growth (Carneiro et al., 2011). Low vigor seeds are less efficient in neutralizing ROSs and, thus, oxidative stress increases ROS production and, consequently, reduces germination (Bailly, 2004). In oil seeds such as sunflower, the effects of oxidative stress are mainly related to peroxidation of lipids and oxidation of proteins and nucleic acids (Bailly et al., 2008; Xin et al., 2014; Yin et al., 2015). In general, studies on water stress in sunflower seeds do not consider the level of seed vigor and mainly evaluate aspects related to germination, emergence and seedling growth (Albuquerque and Carvalho, 2003; Carneiro et al., 2011; Luan et al., 2014). Therefore, the aim of this study was to evaluate the physiological potential and changes in the antioxidant enzyme system of sunflower seeds, with different vigor levels, when under water restriction.

MATERIAL AND METHODS

This study was conducted in the Seed Laboratory of the Plant Science Department of the Universidade Federal de Viçosa, Viçosa, MG, Brazil. Two seed lots of sunflower of the cultivar Hélio 253 were used, collected in the 2014 crop season and supplied by the HELIAGRO company. First, the seeds from each lot were evaluated in regard to physiological quality by the following tests: Journal of Seed Science, v.42, e202042008, 2020 Evaluation of sunflower seeds under water restriction 3

Germination: This was conducted with eight replications of 25 seeds, following the method described in the Rules for Seed Testing (Brasil, 2009). The seeds were sown in paper moistened with water in the amount of 2.5 times the weight of the dry paper. Rolls were formed and they were kept in a seed germinator at 25 °C, with an eight-hour photoperiod. The number of normal seedlings were counted ten days after sowing, and results were expressed in percentage (Brasil, 2009). First germination count: This was conducted together with the germination test, calculating the percentage of normal seedlings obtained on the fourth day after sowing (Brasil, 2009). Shoot length and root length: Four replications of ten seeds were sown at an equal distance on a line drawn on the upper third of the rolls of paper towel moistened to 2.5 times the weight of the dry substrate and kept at 25 ºC with an eight-hour photoperiod (Nakagawa et al., 1999). On the tenth day after sowing, shoot length and root length of the seedlings were measured through use of a ruler, and the results were expressed in cm.seedling-1. Seedling emergence: This was carried out in a greenhouse in trays containing a substrate of soil and sand in the proportion of 2:1. Four replications of fifty seeds were sown at a depth of 1 cm, and daily counts were made until stabilization of the number of seedlings to calculate the percentage of seedling emergence and the emergence speed index (ESI) (Maguire, 1962). Accelerated aging: This was carried out with 250 seeds, distributed over a screen within a “Gerbox” plastic box containing 40 mL of distilled water at the bottom. The boxes were closed with a lid, enclosed in plastic bags, and kept in a BOD incubator at 41 °C for 48 hours. After that period, the seeds were placed to germinate as described for the germination test. The percentage of normal seedlings was evaluated at four days after sowing. Electrical conductivity:two hundred seeds, subdivided into four replications of fifty seeds from each lot and previously weighed, were placed in plastic cups containing 75 mL of distilled water and kept in a seed germinator at the temperature of 25 °C for 24 hours. After that period, the electrical conductivity of the solution was measured with a conductivity meter, and results were expressed in μS cm-1.g-1. Seeds from each lot were subjected to water restriction. For that purpose, they were placed to germinate as described above for the germination test using paper moistened with PEG 6000 solutions at the following potentials: 0.0 (control), -0.2, -0.4, -0.6 and -0.8 MPa, obtained according to Villela et al. (1991). Germination was evaluated at four and ten days after sowing, and results were expressed in percentage of normal seedlings (Brasil, 2009). Root length and shoot length were measured as described for characterization of the lots. For evaluation of antioxidant enzyme activity, seeds were used at 0 days (twelve hours of imbibition in paper towel moistened with water; control) and after 2, 4 and 6 days of germination for all treatments under water restriction. To obtain the extracts used in determinations of the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX), the plant material was frozen in liquid nitrogen and kept at -80 °C. After that, around 0.3 g of plant matter was macerated and 2 mL of extraction medium, potassium phosphate buffer (0.1M, pH 6.8) was added, containing 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% polyvinylpolypyrrolidone (PVPP) (w/v) (Peixoto et al., 1999). After that, the material was homogenized and then centrifuged twice at 15,000 xg for fifteen minutes at 4 °C to remove the oil layer from the supernatant. Superoxide dismutase (SOD): The method proposed by Del Longo et al. (1993) was used, adjusted for sunflower seeds, through addition of 30 µL of crude enzyme extract to 2.97 mL of the reaction medium sodium phosphate buffer (50 mM, pH 7.8), containing 13 mM methionine, 75 µM nitroblue tetrazolium (NBT), 0.1 mM EDTA and 2 µM riboflavin. The reaction was conducted at 25 °C for five minutes in a reaction chamber lighted with 15 W fluorescent bulbs. The blank was obtained under the same conditions, however, in the absence of light. Thus, the photoreduction of the NBT was determined by measuring absorbance at 560 nm (Giannopolitis and Ries, 1977). A unit of SOD was defined as the amount of enzyme able to inhibit 50% of the photoreduction of the NBT (Beauchamp and Fridovich, 1971). Catalase (CAT): This was determined according to the protocol proposed by Havir and McHale (1987) through addition of 30 µL of the crude enzyme extract in 2.97 mL of the reaction medium potassium phosphate buffer (50 mM,

Journal of Seed Science, v.42, e202042008, 2020 4 T. C. Morais et al. pH 7.0 and 12.5 mM H2O2). Enzyme activity was obtained based on reading in a spectrophotometer at the wavelength of 240 nm during the first minute of the reaction at 25 °C, and then calculated using the molar extinction coefficient of 36 M-1.cm-1 (Anderson et al., 1995). The results were expressed in µmol.min-1.mg-1 of protein. Peroxidase (POX): This was determined by the addition of 50 μL of the crude enzyme extract to 2.95 mL of the reaction medium potassium phosphate buffer (25 mM, pH 6.8), 20 mM pyrogallol and 20 mM hydrogen peroxide. During the first minute of reaction, the increase in absorbance was observed at the wavelength of 420 nm at 25 °C. Enzyme activity was calculated using the molar extinction coefficient of 2.47 mM.L-1.cm-1 (Chance and Maehly, 1955) and expressed in μmol.min1.mg-1 of protein. Ascorbate peroxidase (APX): This was determined through addition of 50 μL of the crude enzyme extract in 2.95 mL of the reaction medium potassium phosphate buffer (50 mM, pH 7.8), containing 0.25 mM ascorbic acid, 0.1 mM

EDTA and 0.3 mM H2O2. The decrease in absorbance at 290 nm was observed during the first minute at 25 °C. Enzyme activity was calculated using the molar extinction coefficient 2.8 mM-1.cm-1 (Nakano and Asada, 1981), and the result was expressed in µmol.min-1.mg-1 of protein. Protein content: This was determined by the method of Bradford (1976), using BSA as a standard. The quantity of 100 μL of the enzyme extract was used, adding 1 mL of the Bradford reagent, followed by shaking. After twenty minutes, absorbance of the sample was read in a spectrophotometer at 595 nm. The data were used for calculations of antioxidant enzyme activity. Experimental design and statistical analysis: The experiment was set up in a completely randomized design with four replications. The data obtained in the tests of germination, first germination count, plant length and plant dry matter were analyzed in a 2 (lots) × 5 (osmotic potentials) factorial arrangement, and then by regression analysis. The data on enzyme activity, lipid peroxidation (MDA) and protein content were analyzed in a 2 (lots) × 5 (osmotic potentials) × 4 (days after sowing – DAS) factorial arrangement and were represented by the mean value, with the respective standard deviation.

RESULTS AND DISCUSSION

The two sunflower seed lots had similar germination percentages. Nevertheless, they differed in regard to vigor by the tests of first germination count, accelerated aging, seedling emergence, emergence speed index and electrical conductivity, with greater vigor for the seeds of lot 1 (Table 1). There was reduction in germination and in first germination count in both seed lots with the decrease in osmotic potential (Figure 1). The increase in osmotic concentration of the PEG 6000 solution reduces water absorption by

Table 1. Characterization of the physiological potential of seeds of lots 1 and 2 of sunflower, cultivar Hélio 253.

Germination First germination Accelerated aging SL RL Lot (%) count (%) (%) (cm.seedling-1) (cm.seedling-1) 1 95 A 91 A 92 A 11.9 A 13.7 A 2 91 A 80 B 73 B 10.1 A 12.4 A CV (%) 3.0 5.5 11.3 18.0 24.3

SDM RDM Emergence Electrical Lot -1 -1 ESI conductivity (mg.seedling ) (mg.seedling ) (%) (μS.cm-1.g-1) 1 45.0 A 15.9 A 100 A 11.1 A 65.2 A 2 44.8 A 12.4 A 86 B 8.9 B 75.7 B CV (%) 17.7 17.8 4.5 9.4 3.4 SL = shoot length; RL = root length; SDM = shoot dry matter; RDM = root dry matter; ESI = emergence speed index. Mean values followed by the same letter in the row do not differ from each other by the F test (p < 0.05).

Journal of Seed Science, v.42, e202042008, 2020 Evaluation of sunflower seeds under water restriction 5

seeds, causing reduction in germination percentage (Lewandrowski et al., 2017). Generally, the germination percentage decreases along with the decrease in water potential, but for each species, there is a potential at which there is no germination (Ávila et al., 2007). Results of the germination test showed that the two seed lots had linear reduction under water restriction. However, lot 1 (greater vigor) had higher germination than lot 2 (lower vigor), regardless of the potential tested (Figure 1A). Considering first germination count (Figure 1B), the most drastic water restrictions (-0.6 and -0.8 MPa) had a similar effect for the two seed lots tested, with a percentage of normal seedlings near 0% (Figure 1B). In a study carried out with sunflower genotypes, González-Belo et al. (2014) observed that reductions in germination at the potentials -0.3, -0.6, -0.9 and -1.2 MPa and different temperatures were correlated with linoleic acid contents in the seeds. According to Barros and Rossetto (2009), reduction in sunflower seed germination occurs beginning at the water potential of -0.3 MPa, and total inhibition of germination occurs at -0.9 MPa. In the present study, the germination of lot 2 seeds reached levels near 0 at the potential of -0.8 MPa and of approximately 20% in lot 1 seeds (greater vigor) (Figure 1). It has already been observed that water stress in sunflower seeds brings about irregular germination and uneven establishment of seedlings (Albuquerque and Carvalho, 2003), and it reduces germination speed, as was observed in the first germination count test (Figure 1B). Water restriction affected the initial development of the sunflower seedlings, with reduction in shoot length and root length as water restriction increased in the two seed lots (Figure 2). In general, water restriction affects germination and seedling development due to lower digestion and distribution of assimilates, and it limits diverse metabolic processes involved in the formation of new plant tissues and cell elongation (Bewley et al., 2013; Finch- Savage and Bassel, 2016). Similar results were obtained by Carneiro et al. (2011), where the shoot length of sunflower seedlings declined in a linear manner as the PEG 6000 concentration increased, with the lowest values at the potential of -0.8 MPa. These authors reported that the root length at the potentials of -0.2 and 0.4 MPa was greater than the control, decreasing from this water potential on, which was also observed in the present study (Figure 2B). These results can be explained by the adaptation mechanisms of the seedlings when they were under water stress, such as directing photoassimilates to greater root growth in an attempt to increase water uptake. Another possible effect on reduction of sunflower seedling growth under water restriction is lower chlorophyll production, as observed by Singh et al. (2015) and Manivannan et al. (2015). Such reductions were also observed by Germination (%) First germination count (%)

Osmotic potential (MPa) Osmotic potential (MPa) **: significant at 5% by the T-test. Figure 1. Germination (A) and first germination count (B) of seeds from two sunflower seed lots, cultivar Hélio 253, under water restriction in PEG 6000 solutions. Journal of Seed Science, v.42, e202042008, 2020 6 T. C. Morais et al.

Farjzadeh et al. (2017) in different sunflower lines under water stress, and they associated these results to damage to the chloroplasts, caused by ROSs. In addition, the biosynthesis of chlorophyll precursors may be compromised under osmotic stress (Moharramnejad et al., 2015). Thus, reduction in osmotic potential led to a reduction in the germination percentage of the lots, regardless of seed vigor (Figure 1), associated with lower seedling growth, with reduction in shoot and root length (Figure 2). In general, SOD activity was similar for the two seed lots tested, observing little change up to 4 days after sowing. In lot 1 (greater vigor), a slight reduction in activity was observed after four days for the potentials -0.2 and -0.8 MPa (Figure 3A). In lot 2 (lower vigor), this reduction was observed at all the osmotic potentials (Figure 3B). SOD − constitutes a group of metalloenzymes that catalyze the dismutation or disproportionation of superoxide radicals (O2 ) ⋅ into molecular oxygen (O2) and hydrogen peroxide (H2O2) and act in the first line of antioxidant defense (Del Río et al., 2018; Wang et al., 2016). Consequently, the increase in SOD activity is known to confer tolerance to oxidative stress caused by adverse environmental conditions (Jaleel et al., 2007). In a different way than observed in the present study, Fernández-Ocaña et al. (2011) observed a significant increase in SOD activity in sunflower seedlings under stress from low temperatures, even without apparent oxidative stress. These authors associate these observations to a genetic expression that activates this enzyme to prevent potential oxidative stress. In lot 2 (lower vigor), the increase in SOD activity regardless of the potential, in relation to the control, was not observed, and was greatest at the potential of -0.8 MPa in all the times analyzed (Figure 3B). According to Bailly (2004), low vigor seeds have lower efficiency in elimination of ROSs, generating oxidative stress. In this lot, reduction in SOD activity for all the potentials tested from the fourth day on confirms the lower capacity of activation of this enzyme in lower vigor seeds (Figure 3B). CAT is present in glyoxysomes and peroxisomes and, together with the peroxidases, it is responsible for conversion of H2O2 into O2 (Kibinza et al., 2011; Willekens et al., 1995). The activity of this enzyme was affected differently over the germination period in lots 1 and 2, and was generally higher in lot 1 (Figures 4A and B). There was variation in CAT activity over the germination period in the two lots tested. In lot 1 (greater vigor), an increase was observed at the potential of -0.6 MPa up to four days, decreasing at six days. At the potentials of -0.2 and -0.4 MPa, the response was different, with reduction in activity from 0 to four days and increase at six days. Comparing the different potentials, at six days, differences among the potentials 0, -0.2 and -0.8 MPa were not observed, and they were significantly higher at the potentials -0.4 and -0.6 MPa (Figure 4A). In lot 2 (lower vigor), in general, a reduction was observed in activity ) ) -1 -1 Shoot lenght (cm.seedling Root lenght (cm.seedling

Osmotic potential (MPa) Osmotic potential (MPa) *, **: significant at 1% and 5% by the T-test, respectively. Figure 2. Shoot length (A) and root length (B) of sunflower seedlings from lots 1 and 2, cultivar Hélio 253, under water restriction in PEG 6000 solutions.

Journal of Seed Science, v.42, e202042008, 2020 Evaluation of sunflower seeds under water restriction 7 up to two days, followed by an increase on the fourth day, and once more a decrease at six days (Figure 4B). Reduction in CAT activity with the decrease in osmotic potential coincide with reduction in vigor observed by the tests of first germination count and shoot and root length (Figures 1 and 2). Carneiro et al. (2011) evaluated sunflower seedlings and found reduction in CAT activity at the water potential of -0.8 MPa, also induced by PEG 6000. In contrast, Naderi et al. (2014) observed an increase in CAT activity in wheat seedlings under the potentials of -0.4 and -0.8 MPa for five days.

Just like CAT, POX oxidizes organic substrates, with H2O2 as the electron receptor molecule, resulting in release of

H2O and O2 (Mittler, 2002). POX activity was lower in the treatments with water stress in relation to the control (0 MPa) for the two lots evaluated, and it was more evident in lot 1 (higher vigor) (Figure 5). For the two lots, the potentials of protein) -1 .mg -1 SOD (U.min

Time (days after sowing) Time (days after sowing)

Bars: standard deviation. Figure 3. Activity of the enzyme superoxide dismutase (SOD) determined at 0, 2, 4 and 6 days after sowing for seedlings from lots 1 (A) and 2 (B) of sunflower, cultivar Hélio 253, under water restriction in PEG 6000 solutions. protein) - .mg -1 .min -1 CAT (µmol CAT

Time (days after sowing) Time (days after sowing)

Bars: standard deviation. Figure 4. Activity of the enzyme catalase (CAT) determined at 0, 2, 4 and 6 days after sowing for seedlings from lots 1 (A) and 2 (B) of sunflower, cultivar Hélio 253, under water restriction in PEG 6000 solutions. Journal of Seed Science, v.42, e202042008, 2020 8 T. C. Morais et al.

0 and -0.2 MPa resulted in higher POX activity, regardless of the exposure time. In lot 1 (higher vigor), this increase was more accentuated from the second day on, with an even more significant increase from the fourth day on (Figure 5A). In lot 2 (lower vigor), this increase was less accentuated, especially for the potentials of -0.4, -0.6 and -0.8 MPa (Figure 5B). These results may be due to the delay in seedling development and to the sensitivity of the less vigorous lots to stress. However, as CAT activity was greater (Figures 4A and B) and these enzymes exercise similar functions, such observations may also be associated with equilibrium of activity of these enzymes, especially in relation to lot 2. Similar to the present study, Manivannan et al. (2014) reported increases in POX activity in roots, stems and leaves of five sunflower cultivars under water stress.

The APX enzyme participates in conversion of H2O2 into O2 through a series of oxidations in the glutathione/ ascorbate cycle (Caverzan et al., 2012). Similar to POX, APX activity was more accentuated for the higher vigor lot (Figures 6A and B). These results are in agreement with germination percentage and shoot and root length, which protein) - .mg -1 .min -1 POX (µmol

Time (days after sowing) Time (days after sowing)

Bars: standard deviation. Figure 5. Activity of the enzyme peroxidase (POX) determined at 0, 2, 4 and 6 days after sowing for seedlings from lots 1 (A) and 2 (B) of sunflower, cultivar Hélio 253, under water restriction in PEG 6000 solutions. protein) - .mg -1 .min -1 APX (µmol

Time (days after sowing) Time (days after sowing)

Bars: standard deviation. Figure 6. Activity of the enzyme ascorbate peroxidase (APX) determined at 0, 2, 4 and 6 days after sowing for seedlings from lots 1 (A) and 2 (B) of sunflower, cultivar Hélio 253, under water restriction in PEG 6000 solutions. Journal of Seed Science, v.42, e202042008, 2020 Evaluation of sunflower seeds under water restriction 9 were lower in the lower quality lot when the seeds were placed under water restriction (Figures 1 and 2). In lot 1, there was an increase in APX activity on the second day in all the treatments, and the greatest activity was found at the potential of -0.6 MPa and the lowest activity in the control treatment. From the fourth day on, APX activity at the water potential of -0.8 MPa declined significantly, while in the treatments with a lower level of stress, activity declined especially from the second day on (Figure 6A). In a way similar to lot 1, lot 2 (lower vigor) generally had greater APX activity in relation to the control in all the periods evaluated, especially from the second day on (Figure 6B). Chakraborty and Pradhan (2012) found an increase in the activity of this enzyme in wheat seedlings on the third day of germination under water stress. Baloğlu et al. (2012) conducted a study on the effect of water stress in sunflower seedlings and concluded that APX is important for protection of root tissues in this species under more severe stress conditions. Locato et al. (2010) affirm that some enzymes of the peroxidase family are expressed in a constitutive manner. Others may be induced by environmental stresses, such that under more intense stress situations, there is greater activity of the APX enzyme. It is important to emphasize that the ROSs are predominantly beneficial to plant metabolism, performing diverse functions, from germination to cell signaling (Mittler, 2017). In addition, Manivannan et al. (2014) state that the level of enzyme activity of the antioxidant system depends not only on the species and vigor, but also on the duration and intensity of the stress, which was generally observed in this study. In general, considering the enzymes that act as peroxidases (CAT, POX, and APX), an increase can be seen in the activity with the longer time of exposure to water restriction, especially for the seeds with highest vigor (lot 1).

CONCLUSIONS

Water restriction led to a decrease in germination and lower growth of seedlings, regardless of the vigor level of the seeds. Water restriction affects antioxidant enzyme activity in sunflower seeds, especially beginning at two days of exposure to stress. Lower vigor seeds have lower capacity for activation of antioxidant enzymes, especially the peroxidases.

ACKNOWLEDGMENTS

Our thanks to the Plant Science Department of the Universidade Federal de Viçosa (UFV). Thanks also to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), finance code 001, and to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for funding.

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Journal of Seed Science, v.42, e202042008, 2020 Journal of Seed Science ISSN 2317-1545 ARTICLE www.abrates.org.br/revista

Improvement of the methodology of the tetrazolium Journal of Seed Science, v.42, test using different pretreatments in seeds of the genus e202042013, 2020 http://dx.doi.org/10.1590/2317- Epidendrum (Orchidaceae) 1545v42231028

Seir Antonio Salazar Mercado1* , Jesús David Quintero Caleño2 , Laura Yolima Moreno Rozo1

ABSTRACT: The aim of the present study was to determine the most suitable pretreatment to enhance the tetrazolium test in seeds of the Epidendrum genus. Initially, mature capsules were harvested at El Escorial village, in the municipality of Pamplona, Colombia. Subsequently, the seeds were subjected to five pretreatments: deionized water, 0.5% NaClO, 1.0% NaClO, 10.0% sucrose and a control group. Using the syringe method with cloth filter, the seeds were rinsed with distilled water and subjected to two concentrations of tetrazolium solution (0.25%, 1.0%) and three exposure times (6 h, 24 h and 48 h). Finally, the tetrazolium viability test results were correlated with the in vitro germination test. It was found that the use of deionized water improves the efficiency of the tetrazolium test in seeds of Epidendrum fimbriatum and E. microtum; as in seeds of E. elongatum when using 1.0% tetrazolium for 24 h. Similarly, a high correlation was found between viability and germination, using deionized water and 10.0% sucrose, with homogeneous results with each other.

Index terms: orchid, sodium hypochlorite, sucrose, seed viability.

Aprimoramento da metodologia do teste de tetrazólio utilizando diferentes pré-tratamentos em sementes do gênero Epidendrum (Orchidaceae)

RESUMO: O objetivo do presente estudo foi determinar o pré-tratamento mais adequado para aprimorar a metodologia do teste de tetrazólio em sementes do gênero Epidendrum. *Corresponding author Inicialmente, cápsulas maduras foram coletadas na vila de El Escorial, no município de E-mail: [email protected] Pamplona, Colômbia. Em seguida, as sementes foram submetidas a cinco pré-tratamentos: água deionizada, NaClO a 0,5%, NaClO a 1,0%, sacarose a 10,0% e um controle. Utilizando Received: 11/13/2019. o método de seringa com filtro de pano, as sementes foram lavadas com água destilada e Accepted: 2/17/2020. submetidas a duas concentrações de solução de tetrazólio (0,25% e 1,0%) e três tempos de exposição (6 h, 24 h e 48 h). Finalmente, os resultados do teste de viabilidade do tetrazólio foram comparados ao teste de germinação in vitro. Verificou-se que o uso de 1Department of Biology, Universidad água deionizada melhora a eficiência do teste de tetrazólio em sementes de Epidendrum Francisco de Paula Santander, San fimbriatum e de E. microtum, assim como em sementes de E. elongatum ao usar 1,0% de José de Cúcuta, Norte de Santander, tetrazólio por 24 h. Da mesma maneira, foi encontrada alta correlação entre viabilidade e Postal Code 540003, Colombia. germinação, utilizando água deionizada e 10,0% de sacarose, com resultados homogêneos. 2Department of Agricultural Sciences, Universidad Francisco Termos para indexação: orquidácea, hipoclorito de sódio, sacarose, viabilidade de sementes. de Paula Santander, San José de Cúcuta, Norte de Santander, Postal Code 540003, Colombia.

Journal of Seed Science, v.42, e202042013, 2020 2 S. A. S. Mercado et al.

INTRODUCTION

Orchidaceae is the largest family of flowering plants (Arditti and Ghani, 2000). It makes up one of the most diverse taxa among the phanerogams, with 30,000 recognized species, distributed in 899 genus (Ekmekçigil et al., 2019). They are present on all continents, except Antarctica (Hosomi et al., 2017). They are among the most threatened plants in the world. Colombia is considered one of the richest countries in the diversity of orchids; it is estimated that it has 4,270 species classified in 274 genera (Betancur et al., 2015). Having about 1,500 species (Cardoso-Gustavson et al., 2018) distributed in Latin America, the neotropical genus Epidendrum stands out, as it is one of the most expressive ecologically and horticulturally (Cavalcante et al., 2018). With great diversity, it has flowers characterized by a lip with frills or fringes, being widely cultivated as an ornamental plant (Sangma et al., 2018). In Colombia there are 527 species belonging to this genus (Betancur et al., 2015). Epidendrum is often considered to secrete a rewarding nectar (Pansarin and Pansarin, 2014). However, recent studies concluded that many of these species produce a secretion similar to nectar, as a pollinator’s deterrent, which is considered by many authors as a disguise (Cardoso-Gustavson et al., 2018). Such strategy indicates the deceptive presence of food (Cozzolino and Widmer, 2005), leading to short visits by pollinators, who abandon the inflorescence after visiting a single flower, resulting in decrease of pollination and consequent reduced fruiting (Cardoso-Gustavson et al., 2018). In addition, Epidendrum producers encounter difficulties in the crop development, such as high costs, very slow sexual and vegetative propagation rate for large-scale production (Cavalcante et al., 2018). It is necessary to highlight that most studies in the genus focus on ecology and the symbiosis with endophytic fungi (Gamboa-Gaitán and Otero-Ospina, 2016). For this reason, it is necessary to establish methods that provide information on the seed viability and physiological potential, to ensure their rapid germination and uniformity in establishing in vitro plants, with reduced waste of culture media. With the definition of these methods, the conservation of genetic variability and the preservation of germplasm and clonal propagation material will be improved in germoplasm banks. Improved methods for determining seed viability might also improve propagation of these species, since seeds are their main organ of propagation, which might directly affect the crop productivity (Doria, 2010). Currently, there are several assays to evaluate the physiological potential of seeds, which require short periods of time and are related to the integrity of cell membranes and respiratory activity. Germination and tetrazolium tests are among the main methods to evaluate the seed physiological potential (Salazar-Mercado and Vega-Contreras, 2017). The correct application of these methods is very important to generate satisfactory results (Hosomi et al., 2017). Therefore, it is necessary to identify and control aspects such as pretreatments of the seeds and tetrazolium concentrations in order to improve the methodology for performing the tetrazolium test. The 2,3,5-triphenyl tetrazolium chloride assay is qualitative for tissues and large organs when observed under a microscope. Although it seems to be destructive, the test is often used for embryos and embryonic axes, for a rapid assessment of viability (Salazar and Gélvez, 2015). The use of pretreatments such as scarification, soaking in water and cutting, improves the diffusion of the tetrazolium solution into seed tissues, being successfully used in several species. Hosomi et al. (2011) compared in vitro germination rates of seeds of several species from the Cattleya genus with the results of viability obtained by the tetrazolium test on seeds subjected to different pretreatments. They observed that the pretreatments implementation influences the effectiveness of the tetrazolium test in seeds of this important genus from the orchid family. Consequently, the aim of the present study is to evaluate the effect of four pretreatments used in the tetrazolium test in seeds of Epidendrum microtum, E. elongatum and E. fimbriatum.

MATERIAL AND METHODS

Plant material: the experiment was carried out in the Biology Laboratory of the Universidad Francisco de Paula Santander. Mature and naturally pollinated capsules were used (Figure 1), harvested at El Escorial village, in Pamplona Journal of Seed Science, v.42, e202042013, 2020 Improvement of the methodology of the tetrazolium test in orchid seeds 3 municipality, Colombia: Epidendrum microtum Lindl (N 07°34’41” W 072°64’155”, altitude: 2,870 masl), Epidendrum elongatum Jacq. (N 07°34’78” W 072°64’083”, altitude: 2,754 masl) and Epidendrum fimbriatum Lindl (N 07°34’476” W 072°64’08”, altitude: 2,765 masl); harvested after the dehiscence (48 h later, approximately). The seeds were rinsed with distilled water for the pretreatment application to assure the same conditions for all seeds (Table 1). Pretreatment´s application and tetrazolium test:a small portion of seeds was introduced into a sterile 5 mL syringe with a cloth filter, applying each of the pretreatments (chlorine 0.5%, chlorine 1.0%, deionized water, sucrose 10.0% and a control without any substance) for a period of ten minutes each. Subsequently, three rinses were made with deionized water and the tetrazolium test was performed. Subsequently, the tetrazolium (2,3,5-triphenyl tetrazolium chloride) solutions were prepared to 0.25% and 1.0%. Immediately, the seeds were immersed in the tetrazolium solution in total darkness for 6, 24 and 48 h. At the end of the set time, the seeds were rinsed three times with distilled water and examined in the stereoscope microscope (Leica EZ4). The seed viability evaluation was performed considering the red staining pattern due to the reduction of the tetrazolium solution in the living respiring cells of the seed tissues (Salazar-Mercado and Vega-Contreras, 2017; Salazar and Delgado, 2018). Seeds were considered as viable when more than half of the seed tissue were stained and non-viable when no red staining was developed on the seeds (Figure 2). Viability was presented as percentage and evaluated for each of the replications of every treatment applied.

Figure 1. Flowers and capsules of orchid species of Epidendrum genus. (A) Epidendrum microtum flowers; (B) Capsule of Epidendrum fimbriatum; (C) Epidendrum fimbriatum flower; (D) Epidendrum elongatum flower.

Table 1. Epidendrum elongatum seed viability, as determined by the tetrazolium test at different solution concentrations and exposure periods.

Seed viability (%): different tetrazolium concentrations and exposure periods

Pretreatments 0.25%-6h 0.25%-24h 0.25%-48h 1.0%-6h 1.0%-24h 1.0%-48h Control 17.0 ab 16 a 14.6 a 4.0 a 9.3 a 14.6 a Chlorine 0.5% 58.6 b 56 b 44.0 b 53.3 bc 53.3 b 2.6 a Chlorine 1% 1.3 a 1.3 a 0.0 c 25.3 ab 37.3 b 37.3 b Deionized water 5.3 a 100.0 c 96.0 d 16.0 a 86.6 c 58.6 c Sucrose 10% 14.6 ab 86.6 c 97.3 d 85.3 c 70.6 c 73.3 c Means within each column followed by the same letter do not differ by the Tukey test (p ≤ 0.05).

Journal of Seed Science, v.42, e202042013, 2020 4 S. A. S. Mercado et al.

Figure 2. Viability test using tetrazolium. (A) Epidendrum microtum non-viable seed; (B) E. microtum viable seed; (C) Epidendrum elongatum non-viable seed; (D) E. elongatum viable seed; (E) Epidendrum fimbriatum non-viable seed; (F) E. fimbriatum viable seed; (t): seed coat; (e): embryo.

In vitro germination test: with very few exceptions, in vitro germination of orchid seeds became increasingly popular for viability evaluation. The disinfection and sowing of the seeds were carried out according to the methodology described by Salazar-Mercado and Vega-Contreras (2017): initially, the seeds were immersed for one minute in 70% ethanol, then immersed in a solution with 0.75% sodium hypochlorite plus 0.1% Tween-20 for five minutes; subsequently, three rinses were performed with deionized water. After removing the filter from the syringe, a hundred seeds per plate were planted, distributing them evenly on the circumference of the plate with the help of a culture handle (sterilized), in five Petri dishes with 25 mL of MS basal medium composed of 100% macro and micronutrients (Murashige and Skoog, 1962), supplemented with 3 g.L-1 sucrose, 8 g.L-1 of agar and 1 g.L-1 of activated carbon. Next, the plates were sterilized at 15 pounds of pressure (Psi) at 121 °C for 25 minutes. The media were incubated under controlled conditions (26 ± 2 °C with a photoperiod of 16/8 h light/darkness) with a light intensity of 25 μmol.m-1 per second, provided by fluorescent light and 60% relative humidity. The germination percentage was obtained by observing a hundred seeds per treatment with the help of a stereoscope microscope (Leica EZ4). After twelve days, the seeds that had expanded embryos and testa rupture were considered as germinated (Salazar- Mercado and Vega-Contreras, 2017). Statistical analysis: for each treatment, ten replications with a hundred seeds each were used, with thirty treatments for each studied species. The data were randomly distributed and organized by means of an analysis of variance to determine the treatments’ effect on the amount of viable seeds observed. Germination (in vitro) and viability (tetrazolium test) were expressed in percentage. The data were analyzed using the ANOVA analysis of variance. The mean values were compared using Tukey’s test to determine the means with significant differences (p < 0.05).

RESULTS AND DISCUSSION

Epidendrum elongatum viability: in the results presented in Table 1, it can be observed that the treatment with 0.25% tetrazolium for 6 h resulted in a minimum viability of 1.3% when using the pretreatment of chlorine 1%, without significant differences with the values obtained by the deionized water pretreatment (5.3 %), similar to the results presented in seeds of Paphiopedilum SCBG Red Jewel seeds, where the use of 1% chlorine dramatically decreased tetrazolium staining, as reported by Fu et al. (2016). However, when using pretreatment with chlorine at 0.5%, the highest viability (58.6%) was obtained in this treatment (0.25% for 6 h). Next, it is observed that the use of 0.25% tetrazolium during 24 h resulted in a greater viability with the pretreatment of deionized water (100.0%); this can be explained by the fact that hydration of the seed tissues improves the absorption of tetrazolium and provides the activation of the enzymatic metabolism in the living cells (Carvalho et al., 2014). Similarly, it can be noticed that the treatment with 0.25% tetrazolium for 48 h resulted in 0.0% viability when using 1% chlorine as pretreatment, and the use of sucrose presented 97.3% viability, similar results to those obtained by Hosomi et al. (2011; 2017) in seeds of Cattleya species. This is because sucrose solution avoids possible imbibitional injuries, maintaining the balance between the seeds and their environment. With the 1.0% tetrazolium concentration for 6 h, the control pretreatment exhibited the Journal of Seed Science, v.42, e202042013, 2020 Improvement of the methodology of the tetrazolium test in orchid seeds 5 lowest viability (4.0%), contrasting with that obtained by Salazar and Gélvez (2015), where seeds without pretreatment of Epidendrum sp. and Epidendrum elongatum presented 85.4% and 89.6% viability respectively (using tetrazolium at 1%). However, pretreatment with sucrose produced the highest viability (85.3%), since the immersion of the seeds in sucrose activates the embryo metabolism, mainly related to the action of the dehydrogenase enzymes (Hosomi et al., 2011), which produces more reliable results. Likewise, the control pretreatment resulted in the lowest viability (9.3%), and the pretreatment with deionized water the highest (86.6%), when using 1% tetrazolium during 24 h. Epidendrum fimbriatum viability: according to the obtained results in the tetrazolium test, a marked tendency in the viability increase was observed with the use of sucrose, presenting five maximum viability values (Table 2): 46.6%, 98.6%, 97.3%, 80.0% and 94.6 % (0.25%-6 h, 0.25%-24 h, 0.25%-48 h, 1.0%-6 h and 1%-24 h, respectively). That is, the pretreatment with sucrose increased the viability in all the treatments except with the 1.0% treatment for 48 h. These results ratify what was presented by Suzuki et al. (2018), where previous use of sucrose increased viability (tetrazolium 1%) in Catasetum atratum cryopreserved seeds. However, it was demonstrated that the use of sucrose did not increase viability in seeds of Epidendrum secundum (69.15%), Epidendrum nocturnum (31.86%) and Epidendrum desciflorum (42.85%), when compared with in vitro germination. Similarly, the use of chlorine at 0.5% and 1.0% did not provide significant differences between them. In addition, they showed a very marked tendency to decrease viability, with the lowest values in all treatments except with the use of 1.0% tetrazolium for 6 h (2.6%). This is possibly due to the fact that chlorine readily penetrates the lipid cuticle and the lignified outer seed coat (Jevšnik and Luthar, 2015), which can cause damage to the embryo, since it is a strongly oxidizing compound (Jiang et al., 2017). Epidendrum microtum viability: according to the results shown in Table 3, it was observed that the use of sucrose produced the highest viability values in comparison with other treatments, except for the results with the use of deionized water and 1.0% chlorine in the tetrazolium treatments at 0.25% for 48 h and with the treatment of 1.0% for 6 h, respectively, which were 98.6% for each one. However, these results did not differ significantly from each other as compared to the use of sucrose (96.0% and 93.3%, respectively). Similar data were presented by Custódio et al. (2016), where preconditioning with 10.0% sucrose for five minutes significantly increased the staining in Dactylorhiza fuchsii seeds (85.0% viabiity). On the other hand, the use of 1.0% chlorine caused the lowest viability in the treatments with 0.25% for 6 h, 1.0% for 24 h and 1.0% for 48 h (34.6%, 76.0% and 36.0%, respectively), corroborating with the results presented by Custódio et al. (2016), who worked with seeds of Vanda curvifolia, where the increase in NaOCl concentration had a detrimental effect on viability. Other authors reported mixed results after scarification of orchid seeds with NaOCl, although this may be due to the use of high concentrations (1.0% and 5.0%) of NaOCl (Dowling and Jusaitis, 2012), which can interfere with the tetrazolium dehydrogenation (Lallana and García, 2013). In general, the sucrose pretreatment used showed that it works well for these species, representing a useful and basic protocol for a wide range of species (Hosomi et al., 2011).

Table 2. Epidendrum fimbriatum seed viability, as determined by the tetrazolium test at different solution concentrations and exposure periods.

Seed viability (%): different tetrazolium concentrations and exposure periods

Pretreatments 0.25%-6h 0.25%-24h 0.25%-48h 1.0%-6h 1.0%-24h 1.0%-48h Control 2.6 a 4.0 a 9.3 a 2.6 a 9.3 a 10.6 a Chlorine 0.5% 2.6 a 2.6 a 2.6 a 10.6 a 1.3 b 2.6 b Chlorine 1% 12.0 a 1.3 a 2.6 a 10.6 a 1.3 b 1.3 b Deionized water 46.6 b 94.6 b 96.0 b 80.0 b 98.6 c 13.3 ac Sucrose 10% 46.6 b 98.6 b 97.3 b 80.0 b 94.6 c 17.3 c Means within each column followed by the same letter do not differ by the Tukey test (p < 0.05).

Journal of Seed Science, v.42, e202042013, 2020 6 S. A. S. Mercado et al.

Table 3. Epidendrum microtum seed viability, as determined by the tetrazolium test at different solution concentrations and exposure periods.

Seed viability (%): different tetrazolium concentrations and exposure periods

Pretreatments 0.25%-6h 0.25%-24h 0.25%-48h 1.0%-6h 1.0%-24h 1.0%-48h

Control 81.3 a 84 ab 81.3 a 88.0 a 86.6 ab 89.3 ac Chlorine 0.5% 92.0 ac 96.0 b 72.0 a 90.6 a 94.6 ab 86.6 a Chlorine 1% 34.6 b 82.6 a 81.3 a 98.6 a 76.0 a 36.0 b Deionized water 96.0 c 78.6 a 98.6 a 90.6 a 96.0 ab 96.0 cd Sucrose 10% 96.0 c 96.0 b 96.0 a 93.3 a 98.6 b 97.3 d Means within each column followed by the same letter do not differ by the Tukey test (p < 0.05).

Germination in vitro: seed expansion was observed twelve days after sowing, three days before as reported by Salazar-Mercado and Vega-Contreras (2017). According to Sulong et al. (2016), the absorption of water and nutrients leads to a protocorm formation and later to rhizoids formation. In the case of Epidendrum elongatum Jacq., 90.0% of germination (in vitro) was obtained, data that correlates with the viability obtained with the use of 1.0% tetrazolium for 24 h, using distilled water as pretreatment (86.6%). Likewise, it correlates to a lesser degree with the 85.5% obtained in the treatment of 1.0% for 6 h, using sucrose (pretreatment) and moving further away from the viability obtained with the other treatments. This would imply in cost reduction of the procedure, since it is not necessary to use chlorine or sucrose as pretreatment in these species, resulting in a greater number of samples that can be evaluated (Carvalho et al., 2017). In the case of Epidendrum fimbriatum Lindl, 93.0% germination was obtained, statistically homogeneous data with 94.6% of viability presented in the treatments with tetrazolium at 0.25% for 24 h and deionized water (pretreatment) and 1.0% tetrazolium for 24 h and sucrose (pretreatment), placing the use of sucrose and deionized water as the best options among the pretreatments, independently of the concentration and time of exposure to tetrazolium. In the same way, 97.0% germination was obtained in seeds of Epidendrum microtum, homogeneous data within several results obtained with the tetrazolium test, such as when sucrose (pretreatment) was used in treatments with 0.25% tetrazolium for 6, 24 and 48 h and 1% tetrazolium for 24 and 48 h. Likewise, the use of deionized water as pretreatment in the tetrazolium test caused great homogeneity with the germination test (Table 3). Therefore, it can be inferred that regardless the concentration and time of exposure to the tetrazolium solution, the use of sucrose and distilled water increases the efficiency of the test in Epidendrum microtum seeds, reiterating that the use of deionized water represents a lower cost compared to the use of chlorine and sucrose. Regarding the use of chlorine as pretreatment, according to Salazar-Mercado et al. (2019) and Mercado and Bayona (2020), this chemical compound causes cell abnormalities at low concentrations, which could affect the viability and germination of orchid seeds. Therefore, the use of sodium hypochlorite for pretreatment of orchid seeds is not recommended. Additionally, it was observed that the exposure to tetrazolium solutions (for the two concentrations) at 24 h is homogeneous with the results obtained after 48 h; so it is recommended not to extend the test to 48 h in any of the three species tested on this research.

CONCLUSIONS

It was concluded that pretreatment with deionized water improves the efficiency of the tetrazolium test in Epidendrum elongatum Jacq. seeds when 1.0% tetrazolium concentration is used during 24 h. Likewise, the use of distilled water is seen as a viable and economic alternative to increase the efficiency of the tetrazolium test in Epidendrum fimbriatum Lindl seeds, showing a better correlation with the actual germination (in vitro). Likewise, although the use of distilled water and sucrose as pretreatment in the tetrazolium test in Epidendrum microtum seeds produced homogenous Journal of Seed Science, v.42, e202042013, 2020 Improvement of the methodology of the tetrazolium test in orchid seeds 7 results among them, the use of distilled water is recommended, due to the economic factor. Finally, it is important to mention that the use of chlorine (1.0 and 0.5%) in most cases decreases the effectiveness of the tetrazolium test.

ACKNOWLEDGEMENTS

To Universidad Francisco de Paula Santander (Cúcuta, Colombia) for their valuable collaboration and project financing (FINU–UFPS: 037-2019).

REFERENCES

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Journal of Seed Science, v.42, e202042013, 2020 Journal of Seed Science ISSN 2317-1545 NOTE www.abrates.org.br/revista

Accelerated aging for evaluation of vigor in Brachiaria Journal of Seed Science, v.42, e202042006, 2020 brizantha ‘Xaraés’ seeds1 http://dx.doi.org/10.1590/2317- 1545v42216691 Ariadne Morbeck Santos Oliveira1* , Marcela Carlota Nery2 , Karina Guimarães Ribeiro3 , Adriana Souza Rocha2 , Priscila Torres Cunha2

ABSTRACT: The aim of this study was to adjust the accelerated aging test to evaluate the physiological potential of seed lots of Brachiaria brizantha ‘Xaraés’, represented by four lots. Seeds were tested by traditional accelerated aging and with saturated NaCl solution in five aging periods: 0, 24, 48, 72 and 96 hours. The profile of the lots was determined by the following measures: moisture content, germination test, first germination count, germination speed index, initial stand, emergence, and emergence speed index. The accelerated aging test makes it possible to separate the lots by the method of saturated NaCl solution for 24 hours and it is appropriate for evaluation of seed physiological potential.

Index terms: physiological quality, accelerated aging, vigor.

Envelhecimento acelerado para avaliação do vigor de sementes de Brachiaria brizantha cv Xaraés

RESUMO: Objetivou-se, com esse estudo, adequar o teste de envelhecimento acelerado, para avaliar o potencial fisiológico de lotes de sementes de Brachiaria brizantha cv Xaraés representada por quatro lotes. As sementes foram submetidas ao envelhecimento acelerado tradicional e com solução saturada de NaCl, em cinco períodos de envelhecimento, 0, 24, 48, 72 e 96 horas. Para caracterização dos lotes, foram determinados o grau de umidade, o teste de germinação, primeira contagem da germinação, índice de velocidade de germinação, estande inicial, emergência e índice de velocidade de emergência. Conclui-se que o teste de envelhecimento acelerado possibilita a separação dos lotes pelo método com solução *Corresponding author saturada de NaCl por 24 horas, sendo apropriado para a avaliação do potencial fisiológico E-mail: ariadneoliveira86@gmail. das sementes. com

Termos para indexação: qualidade fisiológica, envelhecimento acelerado, vigor. Received: 11/20/2018. Accepted: 10/16/2019.

1Universidade Federal de Lavras (UFLA), 37200-000 – Lavras, MG, Brasil.

2Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM), 39100-000 – Diamantina, MG, Brasil.

3Universidade Federal de Viçosa (UFV), 36570-900 – Viçosa, MG, Brasil.

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INTRODUCTION

There are approximately 47 million hectares of natural pasture and 112 million hectares of planted pasture in Brazil. This total area of pasture is nearly three times greater than that used for grain production, which currently exceeds 56 million hectares (IBGE, 2018). Establishment and growth of pastures can occur through vegetative processes or, in the case of large properties with a high level of technology, through seeds. Thus, seed quality is fundamental for obtaining an adequate and uniform stand (Costa et al., 2011). For evaluation of this quality, the germination test is the official procedure that evaluates the capacity of seeds to produce normal seedlings under ideal conditions. Nevertheless, this test does not always reveal differences of performance between seed lots during storage or in the field (Carvalho and Nakagawa, 2012). Thus, it is important to evaluate seed vigor to complement the information provided by the germination test. Among the vigor tests developed, accelerated aging is one of the most studied for various species grown, and it is used in diverse quality control programs adopted in seed companies by exhibiting efficiency in comparison of vigor and in estimation of the storage potential of the seed lots. The accelerated aging test has shown a good relationship to the seedling emergence test in the field (Marcos-Filho, 2015a). Initially developed for the purpose of estimating the longevity of stored seeds (Delouche and Baskin, 1973), accelerated aging has been widely studied with the aim of standardizing it, for its use is quite promising. In this test, the seeds are subjected to simulated stress conditions through high temperatures and high relative humidity (100%), generating high respiration rates and consumption of seed reserves, causing degenerative changes in seed metabolism and changes in the antioxidant defense system and in the soluble sugar content in the embryo (Bijanzadeh et al., 2016; Barreto and Garcia, 2017; Moncaleano-Escandon et al., 2013; Das and Roychoudhury, 2014; Lehner et al., 2008). Thus, more vigorous seeds better tolerate this aging condition than those of lower vigor and produce a higher percentage of normal seedlings, evaluated by the germination test after aging (Baalbaki et al., 2009; Marcos-Filho, 2015b; Pereira et al., 2012; Silva et al., 2010; Moraes et al., 2016). Yet accelerated aging with water, which leads to relative humidity of 100%, can cause accentuated variations among the moisture contents of the samples, interfering in the results of the test. Thus, Jianhua and McDonald (1996) aimed to reduce the relative humidity during the test and proposed the use of a saturated NaCl saline solution, which leads to relative humidity of 76%, causing moderate aging, similar to the “natural aging” of the seeds, in contrast with the traditional procedure. Although the accelerated aging test has been widely studied for different species, the information available from it, aiming at evaluation of the vigor of forage seeds, is old or quite scarce. Accelerated aging at 43 ºC for 48 h was used by Dias et al. (2004) to monitor the physiological quality of Brachiaria brizantha cv Marandu seeds during storage. Cavalcante- Filho and Usberti (2008) used accelerated aging to break the dormancy of B. brizantha cv Marandu seeds and Mulato 1 and Mulato 2 hybrids. These authors concluded that the temperature of 43 °C for 72 h was effective in breaking dormancy. Usberti (1990) used accelerated aging to estimate storage of B. decumbens seeds and observed its effectiveness when conducted at 43 ºC and 100% RH for 36 and sixty hours. In this context, the aim of this study was to adjust the method of the accelerated aging test to evaluate the physiological quality of Brachiaria brizantha cv Xaraés seeds.

MATERIAL AND METHODS

The study was conducted in the Seed Analysis Laboratory of the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) in the city of Diamantina, MG. Four lots of Brachiaria brizantha cv. Xaraés seeds were used, which were evaluated regarding physiological quality through the following determinations and tests: Moisture content: this was determined by the laboratory oven method at 105 ºC for 24 hours (Brasil, 2009). Germination test: four replications of fifty seeds per lot, distributed on two sheets of blotting paper moistened with distilled Journal of Seed Science, v.42, e202042006, 2020 Vigor tests in Brachiaria brizantha ‘Xaraés’ seeds 3 water in the amount of 2.5 times the weight of the substrate, were placed in a plastic box and then in a B.O.D. type germinator at a temperature alternating between 20 ºC–35 ºC with constant lighting. Evaluations began at seven days (first germination count) and ended at 21 days after setting up the trials, based on the criteria recommended in Brasil (2009). The germination speed index (GSI) was performed together with the germination test, counting the number of germinated seeds daily until stabilization; calculations were made according to the formula of Maguire (1962). Seedling emergence test: four replications of fifty seeds were sown at 1 cm depth in a soil and sand substrate (1:2) in plastic trays. Moisture of the substrate was adjusted to 60% of water holding capacity. When emergence began, daily evaluations were made, computing the initial stand on the 7th day and the number of emerged seedlings until stabilization. The emergence speed index (ESI) was determined according to the formula of Maguire (1962). Accelerated aging (traditional procedure): two hundred seeds were placed on a metal screen in a plastic box containing 40 mL of distilled water at the bottom. The plastic boxes were placed in a B.O.D. type germination chamber for 24, 48, 72 and 96 hours, at the temperature of 45 °C. After passing through each period of accelerated aging, the seeds were placed for germination. The percentage of normal seedlings was evaluated on the 21st day after sowing (Brasil, 2009). Moisture content was also determined after each period of accelerated aging (Brasil, 2009). Accelerated aging (saturated saline solution): this was conducted as described for the traditional accelerated aging test; however, a saturated sodium chloride (NaCl) solution at the proportion of 40 g of NaCl to 100 mL of water was placed within each plastic box, which leads to relative humidity of 76% (Jianhua and McDonald, 1996). A completely randomized experimental design (CRD) was used, with four replications. A 4 × 5 factorial arrangement (four seed lots and five accelerated aging periods) was adopted for each method, both the traditional method and the method with saturated NaCl solution. Analysis of variance was used on the data and the mean values were clustered by the Scott-Knott test at 5% probability. The result of the accelerated aging test was correlated with those previously described through simple correlation.

RESULTS AND DISCUSSION

The moisture content of the seed lots evaluated was similar, ranging from 8.34% to 9.95%, i.e., there was a variation of less than one percentage point (pp) among the lots (Table 1). This indicates that the test was in the recommended range because, according to Marcos-Filho (2015a), the test should be set up with samples whose moisture content does not vary more than two percentage points. Variations in percentage points greater than this impede standardization of any method, as well as impede obtaining uniform results among laboratories or even within the same laboratory. The tests performed in the laboratory to obtain the initial quality of the seed lots (Table 1) did not stratify the lots, or when stratification occurred, it was only in two levels of quality, indicating similarity among the lots. This occurs because laboratory tests are performed under artificial ideal conditions, and the physiological potential of the seeds may be overestimated, which may limit ranking of the seed lots (Haesbaert et al., 2017).

Table 1. Results of normal seedlings in moisture content (MC), germination (G) (%), first germination count (FGC) (%), germination speed index (GSI), emergence (E) (%), initial stand (IS) (%) and emergence speed index (ESI), obtained from four lots of Brachiaria brizantha cv Xaraés seeds.

Lot MC G FGC GSI E IS ESI 1 8.34 64 a 19 b 3.25 b 53 b 25 a 3.11 b 2 8.31 70 a 32 a 4.23 a 63 a 23 a 3.74 a 3 9.95 64 a 13 b 3.25 b 43 c 18 b 2.53 c 4 9.94 66 a 16 b 3.54 b 31 d 14 b 1.86 d CV (%) – 8.70 22.99 6.36 8.65 13.21 7.18 Mean values followed by the same lowercase letter in the column do not differ from each other by the Scott-Knott test at 5% probability.

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In contrast, in the results of the tests related to seedling emergence, significant differences were observed among the lots; in the emergence test and the emergence speed index, the lots were ranked in four levels of quality. In these results, different responses of the lots were found when they were subjected to the different specific conditions of each test. In this respect, Marcos-Filho (2015a) highlights the need to use various tests to evaluate seed vigor, due to variation in the efficiency of the procedures currently available. Comparing exposure to traditional accelerated aging and the use of a saturated NaCl solution (Table 2), it was found that, regardless of the exposure period, the use of a saturated NaCl solution resulted in moisture contents that were considerably lower and more uniform than those observed for the seeds aged by the traditional procedure. This occurs because, according to Jianhua and McDonald (1996), in using the saturated NaCl solution, there will be a reduction in relative humidity within the plastic box, and the seed will absorb water at a slower rate, resulting in lower and more uniform moisture contents compared to traditional accelerated aging. This was also observed by Radke et al. (2018) in chia seeds. In addition, the use of saturated NaCl solution reduced fungal development during the test, due to restriction in relative humidity in the environment within the plastic boxes, which does not favor proliferation of microorganisms. In addition, it is probable that the salt solution releases chlorine and sodium ions to the medium and these chlorine ions have antifungal activity and contribute to a reduction in proliferation of fungi (Ávila et al., 2006). Various studies using salt solutions as an antifungal agent have proven to be effective in as in inhibition of conidia germination much as and inhibition of mycelium growth (Fallanaj et al., 2013; Youssef and Hussien, 2020). The absence of fungi during the accelerated aging test with saturated NaCl solution was also observed by Tunes et al. (2011) in ryegrass seeds. The results of normal seedlings after the traditional accelerated aging test (Table 3) show that there was little sensitivity in classification of the lots up to 72 hours of aging, with separation into only two levels of quality. Only after 96 hours of aging was it possible to observe differentiation of the lots into three levels of quality, such that lot 2 was of higher quality, lots 1 and 3 of intermediate quality, and lot 4 of lower quality. Nevertheless, in spite of this differentiation, for these periods of accelerated aging of 72 and 96 hours, the decline in germination was drastic, as also observed by Oliveira et al. (2014) in physic nut and by Silva et al. (2017) in rattlepod (Crotalaria). This may have occurred because, according to Marcos-Filho (2015a), long periods of traditional accelerated aging can bring about drastic conditions, making it difficult to detect significant differences in quality among seed lots. In addition, this reduction in germination may be related to the high moisture content of the seeds after aging, since seeds with a higher moisture content absorb water more rapidly, leading to deterioration, compared to seeds with lower moisture content (Morais and Rossetto, 2013; Tunes et al., 2012). Such a result can be attributed to the stress condition caused by accelerated aging, which triggers various metabolic reactions, producing what are known as reactive oxygen species (ROSs). An increase in production and accumulation of ROSs and their imbalance with antioxidant mechanisms characterize oxidative stress (Kibinza et al., 2011; Mittler, 2017). Various studies have shown the major damage that

Table 2. Moisture content of Brachiaria brizantha cv Xaraés seed lots under testing by traditional accelerated aging and accelerated aging with NaCl solution for 24, 48, 72 and 96 hours.

Treatments / Aging periods (hours) Lot Traditional NaCl 24 48 72 96 24 48 72 96 1 14.42 15.57 15.89 20.13 8.29 9.14 10.30 10.31 2 11.90 11.35 12.22 16.74 7.95 7.05 7.95 7.77 3 17.73 19.15 19.47 24.01 10.08 11.48 11.74 11.17 4 18.87 22.38 22.58 27.71 10.24 9.88 7.74 9.63

Journal of Seed Science, v.42, e202042006, 2020 Vigor tests in Brachiaria brizantha ‘Xaraés’ seeds 5 deterioration can cause in seeds through oxidative stress, including peroxidation and degradation of membrane lipids, oxidation of proteins, damage to nucleic acids, enzyme inhibition, and, at higher levels, cell death (Ahmad et al., 2012; Jeevan Kumar et al., 2015; Jyoti and Malik, 2013; Mishra et al., 2011). Through the method that uses saturated NaCl solution (Table 3), it was possible to distinguish the lots in four levels of quality after only 24 hours – lot 2 had higher quality, followed by lots 1, 3 and 4, thus coinciding with the results of emergence and ESI testing (Table 2). Use of the saturated NaCl solution led to lower seed deterioration, even after 96 hours of aging. This occurs because the use of this solution reduces the speed of water absorption by seeds, affecting seed vigor in a milder way, thus obtaining less drastic and more uniform results compared to the traditional method. Yet the effectiveness of the test is not diminished. Consistent results using this procedure for seed vigor evaluation were obtained by Tunes et al. (2013) in parsley seeds, by Lemes et al. (2015) in Bermuda grass, and Radke et al. (2016) in coriander. The effectiveness of the traditional accelerated aging test or the saturated saline solution test for seed vigor evaluation was also observed through the correlation between the results of the accelerated aging test and the results obtained in evaluation of seed lot quality, especially when correlated with the seedling emergence test (Figure 1). The correlation of data of a vigor test with seedling emergence in the field is crucial for the test to be considered effective, since a vigor test should not only classify lots into different vigor classes, but should also be as near as possible to results of seedling emergence in the field (Marcos-Filho, 2015a; Melo et al., 2017). Nevertheless, due to wide variation in the moisture content of the samples when using the traditional accelerated aging test, the accuracy of the results was negatively affected, advising against the use of this method for evaluation of seed physiological quality. This same result was observed by Gordin et al. (2015), who evaluated the physiological quality of Guizota abyssinica seeds and did not recommend the use of traditional accelerated aging, even when it stratified the lots in vigor levels or correlated with emergence; the author suggested using the saturated saline solution. In this respect, from the results observed in this study, it can be said that the method of accelerated aging with saturated NaCl solution was effective in detecting subtle differences among seed lots with similar germination. Thus, the period of 24 hours of aging with the use of a saturated NaCl solution proved to be more adequate since it can be performed in a shorter period, a desirable characteristic in a seed vigor test, as this allows energy savings in equipment and provides results in a faster way.

Table 3. Results of normal seedlings (%) obtained in the germination test by Brachiaria brizantha cv Xaraés seeds under different periods of traditional accelerated aging and accelerated aging with saturated NaCl solution.

Treatments / Aging periods (hours) Lot Traditional NaCl 24 48 72 96 24 48 72 96 1 52 b 49 a 37 a 27 b 51 b 55 a 58 a 40 b 2 63 a 46 a 44 a 45 a 62 a 57 a 58 a 52 a 3 51 b 26 b 25 b 22 b 42 c 27 b 34 b 30 c 4 36 c 27 b 26 b 15 c 31 d 21 b 18 c 22 c CV (%) 15.08 16.07 Mean values followed by the same letter in the column do not differ from each other by the Scott-Knott test at 5% probability.

Journal of Seed Science, v.42, e202042006, 2020 6 A. M. S. Oliveira et al.

FGC

Values marked with an “X” do not have significant correlation at 5% probability by the t-test. Figure 1. Simple Pearson correlation (r) between the variables of first germination count (FGC), germination speed index (GSI), germination (G), initial stand (IS), emergence speed index (ESI), emergence (E) and the different procedures of the accelerated aging test performed in Brachiaria brizantha cv Xaraés seeds.

CONCLUSIONS

The accelerated aging test allows separation of B. brizantha cv Xaraés seed lots by the method with saturated NaCl solution for 24 hours.

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Journal of Seed Science, v.42, e202042006, 2020 Journal of Seed Science ISSN 2317-1545 NOTE www.abrates.org.br/revista

Specificity and sensibility of primer pair in the detection Journal of Seed Science, v.42, of Colletotrichum gossypii var. cephalosporioides in cotton e202042012, 2020 http://dx.doi.org/10.1590/2317- seeds by PCR technique 1545v42229530

Mirella Figueiró de Almeida1* , Sarah da Silva Costa1, Iara Eleutéria Dias1, Carolina da Silva Siqueira1 , José da Cruz Machado1

ABSTRACT: Cotton Ramulosis (Gossypium hirsutum) is an important disease affecting cotton plantations in Brazil, and its causal agent, Colletotrichum gossypii var. cephalosporioides (Cgc), according to the Brazilian phytosanitary authority, was considered a regulated non quarantine pest. It makes this microorganism subject to standardization in seed certification programs. The current seed health testing for detecting that pathogen in seed samples does not provide reliable results for routine analysis. On this paper, attempts were made to design specific primers for detection of Cgc associated with cotton seed. Two primer sets were selected based on the analysis of a multiple alignment of gene’s sequence encoding the glyceraldehyde 3-phosphate dehydrogenase from Cgc, C. gossypii and reference strains of the C. gloeosporioides species complex. The conserved sites unique to Cgc strains were used to design specific fragment of 140 bp. The primer specificity was confirmed by using other fungi. The primers produced a detectable band of target DNA of Cgc in all inoculum potentials of the pathogen artificially inoculated by the water restriction technique. The developed primer pair represents, therefore, a reliable and rapid mean to diagnose the Ramulosis agent in cotton seed.

Index terms: Colletotrichum gossypii, Colletotrichum gloeosporioides, Ramulosis, water restriction.

Especificidade e sensibilidade de um par de primer na detecção de Colletotrichum gossypii var. cephalosporioides em sementes de algodão pela técnica de PCR

RESUMO: A ramulose do algodão (Gossypium hirsutum), causada por Colletotrichum gossypii var. cephalosporioides (Cgc), é uma doença importante que afeta as plantações de algodão no Brasil. De acordo com as autoridades fitossanitárias brasileiras, esse organismo tem sido considerado uma praga quarentenária não regulamentada, o que faz com que ela seja objeto de padronização em programas de certificação de sementes. Neste trabalho, um par de primer foi selecionado com base na análise de um alinhamento múltiplo de sequências do gene que codifica a gliceraldeído-3- *Corresponding author fosfato desidrogenase a partir de Cgc, C. gossypii e isolados de referência representantes de outras E-mail: [email protected] espécies do complexo C. gloeosporioides. Uma única região conservada de Cgc foi utilizada para desenhar um par de primer específico de 140 pb. A especificidade dos primers foi confirmada Received: 10/3/2019. pela utilização de outros fungos isolados de semente algodão. Os primers produziram uma banda Accepted: 2/5/2020. detectável de DNA de Cgc em todos os potenciais de inóculo artificialmente inoculados pela técnica de restrição hídrica. Os primers desenvolvidos representam, portanto, um meio confiável e rápido para diagnosticar Cgc em amostras de sementes de algodão. 1Departamento de Fitopatologia, Universidade Federal de Lavras, Termos para indexação: Colletotrichum gossypii, Colletotrichum gloeosporioides, ramulose, Caixa Postal 3037, 37200-000 – restrição hídrica. Lavras, MG, Brasil.

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INTRODUCTION

Ramulosis is one of the most prominent diseases in cotton (Gossypium hirsutum) in Brazil, and it is caused by Colletotrichum gossypii var. cephalosporioides A. S. Costa. This organism belongs to the Colletotrichum gloeosporioides species complex, as well as Colletotrichum gossypii South. (Cg), which causes Anthracnose in cotton (Salustiano et al., 2014). These fungi (C. gossypii var. cephalosporioides and C. gossypii) belong to the Ascomycota phylum, having as main feature the production of conidial mass with orange color in acervuli and conidia morphologically similar (Bailey et al., 1996). Both pathogens are transmitted by seeds and cause damages in cotton plants (Silva-Mann et al., 2005; Mehta and Mehta, 2010). Colletotrichum taxonomy was subject of extensive discussion by the variability of species classified in this genus; so, there are difficulties in the identification and separation of these organisms. Traditionally, the identification of that genus’ members was based on some morphological characteristics, with emphasis on morphometry of conidia, colony color, mycelial growth rate and pathogenicity (Bailey et al., 1996; Tozze-Júnior et al., 2006). Specifically for the Colletotrichum complex associated with cotton, it is not always possible to differ what are the pathogens involved in the symptomatology of Ramulosis and Anthracnose, as well as the different degrees of aggressiveness and symptoms (Carvalho et al., 2015). Within the seed pathology, the detection and differentiation between C. gossypii var. cephalosporioides and C. gossypii were carried out by using the “blotter” method, in which the assessment is based on mycelial growth habit of fungi developed in seeds after an incubation period (Tanaka et al., 1996). In this case, the high morphological similarities and isolate variability of these fungi make the results of such analysis questionable and not always consistent (Silva-Mann et al., 2002; Mehta and Mehta, 2010), determining the need to develop more accurate and reliable methods for this task. Accuracy in identification of C. gossypii var. cephalosporioides and C. gossypii is, thus, necessary and indispensable to diagnose and control the involved diseases, as well as demand for detection methods of these fungi in seed samples on laboratory routine activities (Carvalho et al., 2015). Molecular techniques and DNA sequence analysis were important to distinguish and identify populations of organisms at different levels. Currently, the PCR technique is used for direct detection of fungi and other organisms in association with seeds (Lee et al., 2002; Munkvold, 2009; Barrocas et al., 2012). This technology was successful in detecting, for example, Stenocarpella complex (S. maydis and S. macrospora) in maize (Romero and Wise, 2015), Fusarium oxysporum f.sp. phaseoli in bean seeds (Sousa et al., 2015), Sclerotinia sclerotiorum in soybean seeds (Botelho et al., 2015) and Corynespora cassiicola in soybean seeds (Sousa et al., 2016). This study aimed to design specific primer pair to detect Colletotrichum gossypii var. cephalosporioides in cotton seeds and establish a protocol for safer and more sensitive sanitary analysis in the detection of this pathogen by PCR, ensuring to the cotton producers a safer quality control and providing better protection for agricultural production environments in the country.

MATERIAL AND METHODS

Isolates obtention: Colletotrichum gossypii var. cephalosporioides isolates and other fungi species were obtained from the mycological collection of the Mycology Laboratory and of the Seed Pathology Laboratory of the Universidade Federal de Lavras (UFLA), in Lavras, MG, Brazil (Table 1). DNA extraction: genomic DNA was extracted from monosporic cultures of isolates grown on potato dextrose agar (PDA) for five days. The mycelium was scraped and homogenized in liquid nitrogen, and the extraction was performed using the Wizard®Genomic DNA purification kit (Promega, Madison, WI), according to the DNA extraction protocol recommended by the manufacturer. DNA concentrations were estimated using the NanoDrop 2000 instrument and visually in 1.2% agarose gel, by comparison of band intensity with a fragment size marker of 1 kb (Invitrogen).

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Table 1. Isolates of Colletotrichum gossypii var. cephalosporioides and others fungal species associated with cotton and others hosts used in the specificity test.

1 2 3 Specific Species CML Other code Geographic origine Host Primer4 C. gossypii var. cephalosporioides LAPS 22 Maracaju, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 23 Maracaju, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 24 Tangará da Serra, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 32 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2371 LAPS 259 Santa Helena de Goiás, GO Gossypium hirsutum + C. gossypii var. cephalosporioides 2372 LAPS 260 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2373 LAPS 261 Pedra Preta, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2374 LAPS 263 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2375 LAPS 264 Mineiros, GO Gossypium hirsutum + C. gossypii var. cephalosporioides 2376 LAPS 265 Campo Verde, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2377 LAPS 266 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2378 LAPS 267 Mineiros, GO Gossypium hirsutum + C. gossypii var. cephalosporioides 2379 LAPS 268 Mineiros, GO Gossypium hirsutum + C. gossypii var. cephalosporioides 2380 LAPS 269 Nova São Joaquim, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2381 LAPS 270 Nova São Joaquim, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2382 LAPS 271 Pedra Preta, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2383 LAPS 272 Itiquira, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2384 LAPS 273 Itiquira, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2386 LAPS 275 Itiquira, MT Gossypium hirsutum + C. gossypii var. cephalosporioides 2387 LAPS 276 Itiquira, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 277 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 392 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 393 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 396 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 397 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 398 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides LAPS 400 Primavera do Leste, MT Gossypium hirsutum + C. gossypii var. cephalosporioides CGCUber Uberlândia, MG Gossypium hirsutum + Colletotrichum gossypii 2327 CG3LEM Luís Eduardo Magalhães, BA Gossypium hirsutum -

Colletotrichum siamense sensu lato Anacardium 2884 CCJ73 Campo Grande, PB occidentale -

Colletotrichum tropicale Anacardium 2888 CCJ105 Fortaleza, CE occidentale -

Colletotrichum asianum Anacardium 2893 CCJ204 São Luís, MA occidentale -

Colletotrichum theobromicola Anacardium 2931 MT68 Pacajus, CE occidentale - Colletotrichum truncatum LAPS133 Rio Verde, GO Phaseolus vulgaris - Colletotrichum gloeosporioides CAA115/1 Acari, MG Annona reticulata - Colletotrichum fructicola CAA137 Acari, MG Annona crassiflora -

Continue...

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Table 1. Continuation.

1 2 3 Specific Species CML Other code Geographic origine Host Primer4 Colletotrichum karstii CAA81 Umbuzeiro, MG Annona crassiflora - Colletotrichum gigasporum 3316 LabioMMi3311 Brazil Piper aducum -

Aspergillus flavus Solo (Gossypium 2708 Montividiu, GO hirsutum) - Aspergillus clavatus 2734 Ibiá, MG Seed (Glycine max) -

Aspergillus chevalieri Seed (Phaseolus 2737 Lavras, MG vulgaris) - Bipolaris sorokiniana 3315 LabioMMi285 São Carlos, SP Piper aducum - Curvularia sp. CTC15 Pará - Alternaria alternata 3314 LabioMMi06 Brazil - Diaphorte sp. LAPS559 São Paulo, SP Glycine max - Phoma tarda 716 Campanha, MG Coffea arabica - Phoma exígua 940 Coromandel, MG Coffea arabica - Penicillium citrunum 3310 LabioMMi249 Teresina, PI - Penicillium terrigenum 1226 Montividiu, GO Gossypium hirsutum -

Fusarium oxysporum f. sp. Gossypium hirsutum vasinfectum 1119 Mato Grosso - Ascochyta sp. 361 Lavras, MG Baccharis sp. - Phomopsis sp. FEL89 Brazil - Clonostachys roseum CSO36 Brazil - Cercospora sp. LAPS255 Campo Verde, MT Glycine max - Fusarium paranaense 1830 Brazil Glycine max - Didymella sp. 193 Machado, MG Coffea arabica - Macrophomina sp. MA01 Primavera do Leste, MT Glycine max - Corynespora cassiicola LAPS467 São Paulo, SP Glycine max - Sclerotinia sp. LAPS242 Uberlândia, MG Glycine max - 1CML = mycological collection of the Plant Pathology Department, Universidade Federal de Lavras, Lavras, MG, Brazil. 2LAPS = mycological collection of the Seed Pathology Laboratory, Universidade Federal de Lavras, Lavras, MG, Brazil. 2LaBioMMi = Microorganisms Micromolecular Biochemistry Laboratory, Chemistry Department, Universidade Federal de São Carlos, São Carlos, SP, Brazil. 3States of Brazil: BA = Bahia; CE = Ceará; GO = Goiás; MG = Minas Gerais; MA = Maranhão; MT = Mato Grosso; PB = Paraíba; PI = Piauí; SP = São Paulo. 4Specific primer; (+) PCR amplification; (-) no PCR amplification.

Development of specific primers for detection and identification of C. gossypii var. cephalosporioides: alignments generated from the sequences of the work of Salustiano et al. (2014), using ClustalW implemented by MEGA5 (Tamura et al., 2011), were obtained for the partial DNA of glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Cgc isolates and other species from the C. gloesporioides species complex. Unique sites in the sequences of the Ramulosis’ etiologic agent were identified and used to design species-specific primers. The primer sequences were compared using the BLAST program in order to verify its homology with sequences previously deposited in GenBank (https:// www.ncbi.nlm.nih.gov/) (Table 2). The developed primer pair was analyzed for performance characteristics such as hairpin structure, potential self-dimer formation and stability of 3 termini, using OligoAnalyzer 3.1 integrated platform (https://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). The primers’ synthesis was performed by Sigma-

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Aldrich Brazil LTD. The genomic material isolated from C. gossypii var. cephalosporioides was subjected to PCR analysis. Determining primer specificity: the specificity of the primer pair was tested by PCR amplification of genomic DNA of 28 Cgc’s isolates, ten isolates of Colletotrichum’s other species and 21 isolates of other fungal species, which were reported in cotton seed and other host (Table 1). PCR was performed using 25 µL mix for PCR OneTaq (BioLabs), containing 10 pmol of forward and reverse primers and DNA 10 ng. The DNA amplification was performed under the following cycle conditions: 94 °C for four minutes (initial denaturation), 94 °C for 45 seconds (denaturation), 65 °C for 45 seconds (annealing), 72 °C for one minute (extension), and 34 cycles of 72 °C for ten minutes (final extension). To separate PCR products, an aliquot of 10 µL was used on 1.2% agarose gel, stained with GelRed® (Biotium®, Hayward, 95 CA, USA). The PCR products were observed in UV transilluminator, L-Pix HE equipament (Loccus Biotechnology, Brazil). Before using the specific primers, a PCR reaction was performed using universal GDF primers GDF (5´- GCCGTCAACGACCCCTTCATTGA- 3’) and universal GDR primers GDR (5´- GGGTGGAGTCGTACTTGAGCATGT- 3’) (Templeton et al., 1992), with the genomic DNA of all species used in this study to test if the genomic DNA was adequate for PCR amplification. The experiments were repeated at least two times. Sensivity evaluation of primers developed in seed samples:to evaluate the sensitivity of PCR reaction using primer pair, cotton seed with different infestation level inoculated with C. gossypii var. cephalosporioides was used, and a four-hundred-seed sample were prepared by mixing the artificially inoculated seeds with healthy seeds generating three infestation level (100%, 10% and 1%) per inoculum. For each infestation level of seeds, the test was performed in four replicates, and the experiment was repeated twice.

Table 2. GenBank accession numbers of Colletotrichum gossypii var. cephalosporioides and other species from the C. gloesporioides species complex used to obtain specific primer pair to Cgc.

1 2 3 GenBank Species CML Other code Host Origin number C. gossypii var. cephalosporioides 2373 LAPS 261 Gossypium hirsutum Pedra Preta, MT JX847009 C. gossypii var. cephalosporioides 2379 LAPS 268 Gossypium hirsutum Mineiros, GO JX847010 C. gossypii var. cephalosporioides 2384 LAPS 273 Gossypium hirsutum Itiquira, MT JX847011 C. gossypii var. cephalosporioides 2388 IAC 13350 Gossypium hirsutum Piracicaba, SP JX847012 C. gossypii var. cephalosporioides 2389 IAC 12405 Gossypium hirsutum Ituverava, SP JX847013 Colletotrichum gossypii 2324 IAC 1025 Gossypium hirsutum Campinas, SP JX847014

Colletotrichum gossypii Gossypium hirsutum Luis Eduardo Magalhães, 2325 CG 1 LEM BA JX847015

Colletotrichum gossypii Gossypium hirsutum Luis Eduardo Magalhães, 2327 CG 3 LEM BA JX847016 C. kahawae subsp. kahawae ICMP 17905 Coffea arabica Kenya JX010012 Colletotrichum gloeosporioides IMI 356878 Citrus sinensis Italy JX010056 Colletotrichum fructicola ICMP 18581 Coffea arabica Thailand JX010033 Colletotrichum siamense ICMP 18578 Coffea arabica Thailand JX009924 Colletotrichum asianum ICMP 18580 Coffea arabica Thailand JX010053 Colletotrichum theobromicola ICMP 17958 Stylosanthes guianensis Australia JX009948 Colletotrichum boninense CBS 112115 Leucospermum sp. Australia JQ005247 1CML = mycological collection of the Plant Pathology Department, Universidade Federal de Lavras, Lavras, MG, Brazil. 2LAPS = mycological collection of the Seed Pathology Laboratory, Universidade Federal de Lavras, Lavras, MG, Brazil. IAC = Campinas Agronomic Institute, Campinas, SP, Brazil. ICMP = International Collection of Microorganisms from Plants, New Zeland. CBS = Centralalbureau voor Schimmelcultures, Utrecht, The Netherlands. 3States of Brazil: BA = Bahia; GO = Goiás; MT = Mato Grosso; SP = São Paulo.

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Seed inoculation: cotton seeds CV delta opal susceptible to the Ramulosis’ etiologic agent were disinfected in 70% alcohol for one minute, followed by 1% of sodium hypochlorite solution for two minutes, then washed four times with autoclaved distilled water. The sterilized seeds were arranged in trays where they remained for 24 hours at room temperature to complete drying. After drying, it was used physiological conditioning method or water restriction for seed inoculation (Machado et al., 2012; Barrocas et al., 2014). Then, the seeds were artificially inoculated with the C. gossypii var. cephalosporioides strain CML2374 that growed in petri dishes with fifteen cm diameter containing PDA medium, modified by the addition of manitol adjusted with water potential of -1.0 MPa, as SPPM Software (computer program that relates solute potencial to solution composition). A sequence of data was generated over temperature, concentration, or potential ranges by specifying an initial value (Michel and Radcliffe, 1995), remaining seven days in BOD at 25 °C with a photoperiod of twelve hours. The seeds were placed in a single layer on the fungus colony, where they remained for 24 and 48 hours, being removed and placed in sterilized trays and dried in a laminar flow chamber for 24 hours. As controls, seeds were used without the fungus and with incubation in substrate with water restriction. DNA extraction of seed samples:the inoculated seed samples were macerated in mill (IKA® A11 analytical basic mill) with liquid nitrogen to obtain a thin powder. Samples with 0.04 g of this powder were placed in 1.5 mL microtubes in four replicates. The extraction was carried out with the use of Wizard®Genomic DNA purification kit (Promega, Madison, WI), according to the DNA extraction protocol recommended by the manufacturer. The PCR reaction and the cycle conditions were the same described for the specificity of the primer pair.

RESULTS AND DISCUSSION

Colletotrichum gossypii var. cephalosporioides specific primers designed from the GAPDH gene had the following sequences: CGC1F (5’- CAG ACT ACA AGG CCA ACG C- 3’) and CGC1R (5’- GAG TCG TAC TTG AGC ATG TAG- 3’). This primer pair amplifies a fragment of 140bp. This primers’ pair specifically amplified DNA of only its respective target, Cgc, in all reactions (Figure 1A). The primers did not cross-react with DNA of any other Colletotrichum species or other fungal species tested (Figure 1B and Table 1). The sensitivity of the primers’ pair may be considered high due to their capacity of detecting the pathogen in seed samples with minimal incidence of 1%, which was the limit used in this study. In the controls, there was no amplification of the genomic DNA from the causative agent of cotton Ramulosis (Figure 2). A PCR-based diagnostic assay using specific primers derived from the gene encoding the glyceraldehyde 3-phosphate dehydrogenase was developed for the Ramulosis’ causal agent from cotton, C. gossypii var. cephalosporioides. Furthermore, the primers were able to detect the pathogen in artificially infested cotton seeds. The PCR products obtained from the seeds showed characteristic bands, as observed in the pathogen’s DNA amplification in pure cultures. Thus, it was evident that the primer pair was effective in detecting the Ramulosis’ etiological agent in artificially infested cotton seeds, indicating no false positive result for contamination. These primer pair allowed the amplification of the genomic DNA samples from the C. gossypii var. cephalosporioides tested, being effective in detection of fungal incidences from 1 to 100% at different inoculum potential tested. In a study conducted by Guimarães et al. (2017), the pair of primers designed and described was used to quantify C. gossypii var. cephalosporioides in artificially inoculated cotton seeds by cPCR and qPCR techniques. The results showed that the primers used were reliable. Primers showed linearity in the standard curve generated by qPCR technique at each dilution level of Cgc DNA extracted from pure culture. The quantification of the inoculum potential by qPCR was 1.44 pg/ μL DNA at P24, which increases to 6.89 pg/ μL at P48 and 24.5 pg/ μL at P96. The authors concluded that there was proportionality between fungal DNA, inoculum potential, effects on germination and seed vigor. For other pathosystems, the sensitivity in detecting seeds’ pathogens is variable. For example, in a study conducted by Barrocas et al. (2012), Sternocarpella was detected in maize seeds infected with minimal incidence of 2% in the studied samples. In a study conducted by Sousa et al. (2015), Fusarium oxysporum f. sp. phaseoli fungus was detected Journal of Seed Science, v.42, e202042012, 2020 Specific primers to detection of Colletotrichum gossypii var. cephalosporioides 7 in lower levels of infection, and 0.25% incidence in beans seeds. One possibility of increasing the PCR sensitivity is prior incubation in favorable conditions for the development of fungi in seeds. Other example, cPCR and qPCR techniques were effective in detecting Colletotrichum lindemuthianum in beans seeds. It was possible using cPCR to detect the fungus in seed samples with 10% of incidence and with 0.25% incidence by qPCR technique (Gadaga et al., 2018).

A: lane M-100-bp marker (Axygen); lanes 1-16 (positive control): Colletotrichum gossypii var. cephalosporioides (LAPS22, LAPS23, LAPS32, CML2371, CML2372, CML2373, CML2374, CML2375, CML2376, CML2377, CML2378, CML2381, CML2382, CML2383, CML2384, CML2386); lanes 17-19 (negative control): lane 17: CML1119 (Fusarium oxysporum f. sp. vasinfectum); lane 18: MA01 (Macrophomina sp.); lane 19: water. B: lane M-100-bp marker (Axygen); lanes 1 and 2 (positive control): C. gossypii var. cephalosporioides (CML2374 e 2379); lanes 3-19 (negative control): lane 3: C. gossypii (CML2327); 4-12: other species of Colletotrichum (CML2884, CML2888, CML2893, CML2931, LAPS133, CAA115/1, CAA137, CAA81, CML3316); 13-19: other fungal species (CML2708, CML2734, CML2737, CML3315, CTC15, CML3314, LAPS559).

Figure 1. Specificity test of conventional PCR with primer pair CGC1F/ CGC1R.

Lane M: 50kb marker; lanes 1 and 2 - Cgc - CML2384 and 2374 isolates; lanes 3 to 6: 1% infection with seeds inoculated for 24 hours; lanes 7 to 10: 10% infection with seeds inoculated for 24 hours; lanes 11 to 14: 1% infection with seeds inoculated for 48 hours; lanes 15 to 18: 10% infection with seeds inoculated for 48 hours; lanes 19 to 22: control without fungus; lanes 23 to 26: 100% seeds infected with inoculation of 24 hours; lanes 27 to 30: 100% seeds infected with inoculation of 48 hours.

Figure 2. Sensitivity test of conventional PCR with primer pair CGC1F/ CGC1R in the detection of Colletotrichum gossypii var. cephalosporioides in samples of cotton seeds with different infection levels.

Journal of Seed Science, v.42, e202042012, 2020 8 M. F. Almeida et al.

CONCLUSIONS

The results of this study, which complement previous work done by the pathologist group involved in this project in order to detect the causal agent of cotton Ramulosis in seed samples, meet a long-year demand from seed producers in Brazil. This technology enables a sanitary quality control of cotton seeds with greater accuracy and speed, making health analysis of seeds, which is viable and extremely important for the cotton producers. It is also important to point out that, in practical terms, the health test protocol for the detection of C. gossypii var. cephalosporioides in cotton seed samples for quality certification programs can be made by implementing a health test by two methods, a molecular and a biological. In this case, samples would be initially subjected to PCR and subsequently applying the blotter test, as it was done by the current Rules for Seed Testing (Brasil, 2009a, b) for samples that had positive results in molecular testing. It is understood that combining these two methods makes the diagnosis of Ramulosis’ agent in cotton seed samples safer and feasible from an operational point of view on health routine analytical laboratories.

ACKNOWLEDGEMENTS

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing the scholarship for the first author. To the CNPq, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for funding and supporting for research.

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Journal of Seed Science, v.42, e202042012, 2020 INSTRUCTIONS TO AUTHORS

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(Short title) Storage of Euterpe oleracea Mart. seeds

Conservation of Euterpe oleracea Mart. Seeds1

Walnice Maria Oliveira do Nascimento2*, Sílvio Moure Cicero3, Ana Dionísia Luz Coelho Novembre3

ABSTRACT – text (200 words)

Index terms:

Conservação de sementes de açaí (Euterpe oleracea Mart.)1

RESUMO – texto (200 palavras)

Termos para indexação:

______1Submitted on______. Accepted for publication on ______. 2Embrapa Amazônia Oriental, Caixa Postal 48, 66095-100 – Belém, PA, Brasil. 3Departamento de ProduçãoVegetal, USP/ESALQ, Caixa Postal 9, 13418-900 – Piracicaba, SP, Brasil. *Corresponding author

Introduction text

Material and Methods text Results and Discussion text

Conclusions text Acknowledgement (optional) text References (Start in another page) Follow the ABNT NBR6023 as mentioned in the item References Model for tables presentation

Table 1. Relationship between seed quality and sowing densities on field seedling emergence and speed emergence index (SEI) in corn ‘BRS201’.

Number of seeds.ha-1 (1000) Seed quality (%) 50 60 70 Emergence (%) SEI Emergence (%) SEI Emergence (%) SEI Q1 (95.0) 94.8 a* 13.3 a* 95.1 a* 13.4 a* 97.0 a* 13.6 a* Q2 (90.0) 95.6 a 13.5 a 95.1 a 13.0 a 96.0 a 13.2 a Q3 (85.0) 84.2 b 10.9 b 83.7 b 10.6 b 82.0 b 10.7 b Q4 (75.0) 72.3 c 9.4 c 76.2 c 9.6 c 74.4 c 9.5 c Mean 86.7 11.8 87.5 11.6 87.4 11.8 *Means within each column followed by the same letter do not differ by Duncan test at 5% probability.

Model for figures presentation

100

40 Germination (%) Germination 20 (%) Germination

0 0 3 6 9 12 15 StorageStorage (days) (days) 2 3 2 4040 DAA: DDA: y =y 1.76= 1.76 - 7.26x – 7.26x + 1.88x + 1.88x-0.08x2 – 0.08x R =3 0.9637 R2 = 0.9637 2 2 5050 DAA: DDA: y =y 64.28= 64.28 + 5.24x + 5.25x - 0.238x – 0.238x R2 = 0.9163 R2 = 0.9637 2 3 2 6060 DAA: DDA: y =y 82.96= 82.96 + 3.15x + 3.15x - 0.38x + 0.38x + 0.013x2 – 0.013x R 3 = R0.93062 = 0.9305 7070 DAA: DDA: y =y 91.52= 91.52 - 3.48x + 3.48x - 0.68x + 0.68x2 - 0.029x2 – 0.029x3 R2 =3 0.9039 R2 = 0.9039

Figure 1. Germination of pepper seeds extracted from fruits harvested at 40, 50, 60 and 70 DAA and stored for 0, 3, 6, 9, 12 and 15 days. INSTRUÇÕES AOS AUTORES

1. ESCOPO E POLÍTICA A partir de 2017 a revista Journal of Seed Science (JSS) circulará apenas na versão on line. Serão aceitos para publicação artigos científicos originais e notas científicas, ainda não publicados, nem encaminhados a outra revista para o mesmo fim, em idioma português ou inglês. Para artigos submetidos em inglês, os autores deverão providenciar uma versão com qualidade. Todos os artigos serão publicados em inglês. A NOTA CIENTÍFICA é uma categoria de manuscrito científico que descreve uma técnica, uma nova espécie ou observações e levantamentos de resultados limitados. Tem o mesmo rigor científico dos “Artigos Científicos” e o mesmo valor como publicação. A classificação de um trabalho como NOTA CIENTÍFICA é baseada no seu conteúdo e mérito científico, mas pode tratar-se de um trabalho preliminar, simples e não definitivo sobre determinado assunto, com publicação justificada pelo seu ineditismo e contribuição para área. Os artigos serão publicados conforme a ordem de aprovação e relevância. Artigos de revisão sobre temas relevantes e atuais poderão ser publicados por autores convidados pela Editoria do JSS.

O JSS tem como objetivos: - Publicar artigos originais em áreas temáticas relevantes da Ciência e Tecnologia de Sementes; - Publicar artigos que representem contribuição significativa para o conhecimento da área, os quais deverão ter caráter científico e buscar abordar em profundidade temas e tendências no âmbito da Ciência e Tecnologia de Sementes; - Apresentar uma política rigorosa de avaliação dos artigos submetidos à publicação, com cada manuscrito sendo avaliado por dois revisores, criteriosamente selecionados na comunidade científica. A decisão de aceite para publicação pautar-se-á sempre na recomendação do corpo de editores e de revisores ad hoc; - Manter elevada conduta ética em relação à publicação e seus colaboradores; - Manter rigor com a qualidade dos artigos científicos a serem publicados.

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Alguns exemplos são apresentados a seguir:

Artigos de Periódicos:(não deverá ser mencionado o local de publicação do periódico). LIMA, L.B.; MARCOS-FILHO, J. Condicionamento fisiológico de sementes de pepino e germinação sob diferentes temperaturas. Revista Brasileira de Sementes, v.32, n.1, p.138-147, 2010. http://www.scielo.br/scielo.php?script=sci_ pdf&pid=S0101-31222010000100017&lng=en&nrm=iso&tlng=pt

Livros: MARCOS-FILHO, J. Fisiologia de sementes de plantas cultivadas. 2 ed. Londrina: ABRATES, 2015. 660p.

BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 395p. http:// www.agricultura. gov.br/arq_editor/file/2946_regras_analise__sementes.pdf

Capítulos de Livro: VIEIRA, R.D.; KRZYZANOWSKI, F.C. Teste de condutividade elétrica. In: KRZYZANOWSKI, F.C.; VIEIRA, R.D.; FRANÇA-NETO, J.B. (Ed.). Vigor de sementes: conceitos e testes. Londrina: ABRATES, 1999. p.4.1-4.26.

Leis, Decretos, Portarias: País ou Estado. Lei, Decreto, ou Portaria nº ..., de (dia) de (mês) de (ano). Diário Oficial da União, local de publicação, data mês e ano. Seção ..., p. ...

BRASIL. Medida provisória nº 1.569-9, de 11 de dezembro de 1997. Diário Oficial da República Federativa do Brasil, Poder Executivo, Brasília, DF, 14 dez. 1997. Seção I, p.29514.

Documentos Eletrônicos: BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. SNPC - Lista de cultivares protegidas. http://extranet. agricultura.gov.br/php/proton/cultivarweb/cultivares_protegidas.php Acesso em: 13 jan. 2010.

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Figuras As figuras (gráficos, desenhos, mapas ou fotografias) deverão ser numeradas em algarismos arábicos em programas compatíveis com o WORD FOR WINDOWS (TIFF 300 dpi) inseridas no texto preferencialmente como objeto. Os desenhos e as fotografias deverão ser digitalizados com alta qualidade (JPEG) e enviados no tamanho a ser publicado na revista. As legendas digitadas logo abaixo da figura e iniciadas com denominação de Figura, devem ser seguidas do respectivo número e texto, em letras minúsculas.

Unidades de medida Devem ser redigidas com espaço entre o valor numérico e a unidade. Ex: 10 ⁰C, 10 mL, µS.cm-1.g-1. O símbolo de percentagem deve ficar junto do algarismo, sem espaço. Ex: 10%. Utilizar o Sistema Internacional de Unidades em todo texto.

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Os autores de artigos publicados no JSS têm os seguintes benefícios: . O JSS é uma revista de acesso aberto . A tradução para o inglês dos artigos aprovados para publicação é custeada pela própria revista. . A submissão e revisão dos artigos é on line. . Rápida publicação: tempo médio de 7 meses entre submissão e publicação (2016). . Cópia dos artigos em pdf disponível sem taxas na Web. . JSS adota o Itheticate software contra plagiarismo. . Todos os artigos são publicados com DOI.

Envie seu artigo para http://www.scielo.br/scielo.php?script=sci_serial&pid=2317-1537&lng=pt clicando em Sub- missão on line

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Recomenda-se que as orientações explicitadas nestas instruções sejam seguidas plenamente pelo(s) autor(es), observando o modelo anexado a seguir. MODELO DE FORMATAÇÃO DE TRABALHO A SER ENCAMINHADO PARA O JSS:

(Título resumido) Armazenamento de sementes de açaí

Conservation of Euterpe oleracea Mart. Seeds1

Walnice Maria Oliveira do Nascimento2*, Sílvio Moure Cicero3, Ana Dionísia Luz Coelho Novembre3

ABSTRACT – text (200 words)

Index terms:

Conservação de sementes de açaí (Euterpe oleracea Mart.)1

RESUMO – texto (200 palavras)

Termos para indexação:

______1Submetido em______. Aceito para publicação em______. 2Embrapa Amazônia Oriental, Caixa Postal 48, 66095-100 – Belém, PA, Brasil. 3Departamento de Produção Vegetal, USP/ESALQ, Caixa Postal 9, 13418-900 – Piracicaba, SP, Brasil. *Autor correspondente

Introdução texto

Material e Métodos texto

Resultados e Discussão texto

Conclusões texto

Agradecimentos (opcional) texto Referências (iniciar em página separada) Seguir as normas da ABNT NBR6023 conforme já mencionado no item Referências. Modelo para apresentação de tabela

Tabela 1. Relação entre a qualidade de semente e a densidade de semeadura na emergência de plântulas em campo e o índice de velocidade de emergência em milho BRS 201.

Número de sementes.ha-1 (1000) Qualidade de semente 50 60 70 (%) Emergência (%) IVE Emergência (%) IVE Emergência (%) IVE Q1 (95,0) 94,8 a* 13,3 a* 95,1 a* 13,4 a* 97,0 a* 13,6 a* Q2 (90,0) 95,6 a 13,5 a 95,1 a 13,0 a 96,0 a 13,2 a Q3 (85,0) 84,2 b 10,9 b 83,7 b 10,6 b 82,0 b 10,7 b Q4 (75,0) 72,3 c 9,4 c 76,2 c 9,6 c 74,4 c 9,5 c Média 86,7 11,8 87,5 11,6 87,4 11,8 *Médias dentro de cada coluna seguidas da mesma letra não diferem entre si pelo teste de Duncan, a 5% de probabilidade,

Modelo para apresentação de figura

100

40 Germination (%) Germination

(%) Germinação 20

0 0 3 6 9 12 15 ArmazenamentoStorage (days) (dias) 2 3 2 4040 DAA: DDA: y =y 1.76= 1.76 - 7.26x – 7.26x + 1.88x + 1.88x-0.08x2 – 0.08x R =3 0.9637 R2 = 0.9637 5050 DAA: DDA: y =y 64.28= 64.28 + 5.24x + 5.25x - 0.238x – 0.238x2 R22 = 0.9163R2 = 0.9637 6060 DAA: DDA: y =y 82.96= 82.96Armazenamento + 3.15x + 3.15x - 0.38x + 0.38x2 + 0.013x2 (dias)– 0.013x3 R23 = R0.93062 = 0.9305 7070 DAA: DDA: y =y 91.52= 91.52 - 3.48x + 3.48x - 0.68x + 0.68x2 - 0.029x2 – 0.029x3 R2 3= 0.9039 R2 = 0.9039

Figura 1. Germinação de sementes de pimenta extraídas de frutos colhidos aos 40, 50, 60 e 70 DAA e armazenados por 0, 3, 6, 9, 12 e 15 dias.