Ecotoxicology https://doi.org/10.1007/s10646-019-02133-y Secondary metabolites that could contribute to the monodominance of Erythrina fusca in the Brazilian Pantanal 1 2 3 4 Darlene Gris ● Amanda Galdi Boaretto ● Maria Rita Marques ● Geraldo Alves Damasceno-Junior ● Carlos Alexandre Carollo 5 Accepted: 2 November 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Erythrina fusca is a dominant species in the Brazilian Pantanal. We hypothesized that E. fusca possess allelopathic potential and we evaluated effects of extracts on germination and development of Lactuca sativa, a bioindicator species. We tested the effect of leaves, bark, roots, and seeds extracts of E. fusca on germination and speed index, using high, moderate and low concentration (0.2, 1 and 5 mg mL–1). To evaluate effects on development, we subjected seedlings of L. sativa to the same treatments and measured root and aerial part length. High concentration of extracts reduced L. sativa germination; leaves extract caused the maximum reduction on germination of L. sativa, similar to 2,4-Dichlorophenoxyacetic acid (2,4-D); this fl 1234567890();,: 1234567890();,: extract has avonoids and saponins as main compounds, classes that also occur in the bark and roots extracts in lower concentrations; bark and roots (5 mg mL–1), leaves and roots (1 mg mL–1) decreased these traits as well, but in lower magnitude. A significant reduction in root length was induced by highest concentration of all extracts (5 mg mL–1); the results suggest that erythrinic alkaloids should interfere in the root length once the seeds accumulate almost exclusively this class of compounds. Our results showed that all parts of E. fusca had adverse effects on germination or development of L. sativa, showing that different class of compounds secondary metabolites is involved in this activity. Possibly, this phytotoxicity influences monodominance of E. fusca in Pantanal, but studies are essential to evaluate effects of it on other native species. Keywords Allelopathy ● Flavonoids ● Erythrinic alkaloids ● Tree dominance ● Tropical wetland Introduction Supplementary information The online version of this article (https:// doi.org/10.1007/s10646-019-02133-y) contains supplementary material, which is available to authorized users. Erythrina fusca Lour. (Fabaceae) is a monodominant tree species found in the Brazilian Pantanal and one of the most * Darlene Gris widespread species of the genus (Russo and Baguinon [email protected] 1997; Pott et al. 2011). E. fusca occurs in riparian forests, 1 Programa de Pós-Graduação em Ecologia e Conservação, mainly in the Amazon region and Pantanal, and is mono- Universidade Federal de Mato Grosso do Sul (UFMS), dominant only in the plains of the northern Paraguay River, Campo Grande, Mato Grosso do Sul 79070-900, Brazil in the Cáceres sub-region of the Pantanal (Lorenzi 1998; 2 Laboratório de Produtos Naturais e Espectrometria de Massas, Pott et al. 2011). Universidade Federal de Mato Grosso do Sul (UFMS), Monodominant species can grow aggregate and may Campo Grande, Mato Grosso do Sul 79070-900, Brazil represent more than half of the total number of trees from a 3 Departamento de Bioquímica, Instituto de Biociências, plant community (Connell and Lowman 1989; Hart et al. Universidade Federal de Mato Grosso do Sul (UFMS), 1989). One mechanism that may influence the dominance of Campo Grande, Mato Grosso do Sul 79070-900, Brazil some plant species is allelopathy since this process can 4 Departamento de Botânica, Instituto de Biociências, Universidade affect plant-plant and plant-environment interactions (Hart Federal de Mato Grosso do Sul (UFMS), Campo Grande, Mato Grosso do Sul 79070-900, Brazil 1990; Macías et al. 2008). According to Rice (1984), allelopathy refers to both 5 Laboratório de Produtos Naturais e Espectrometria de Massas, Universidade Federal de Mato Grosso do Sul (UFMS), inhibitory as well as stimulatory effect, direct or indirect, Campo Grande, Mato Grosso do Sul 79070-900, Brazil which one plant species performs on another may have D. Gris et al. inhibitory impacts, mediated by secondary metabolites such Materials and methods as flavonoids, saponins, and alkaloids. Thus, allelopathy is the capacity to accumulate metabolites that affect germi- Plant material and study area nation, development and/or reproduction of other organisms (Ooka and Owens 2018). E. fusca is a deciduous species that blooms from May to The family Fabaceae frequently showed allelopathic September with fructification occurring in November potential (Oliveira et al. 2008; Cândido et al. 2010; Aguilera (Lorenzi 1998). This species is monodominant in the Pan- et al. 2015;Idetal.2015). Allelopathy is also common in tanal sub-region of Cáceres, more specifically in the region the genus Erythrina (Soares et al. 2002; Centenaro et al. of Taiamã Ecological Station (Pott et al. 2011), where this 2009; Oliveira et al. 2012, 2013); this genus is rich in vegetation type occupies 16% of the total area (Frota et al. unusual secondary metabolites, such as tetracyclic alkaloids 2017). This vegetation is characterized by low-density (erythrinic alkaloids), terpenoids, flavonoids (especially vegetation predominating with arboreal individuals of E. pterocarpans and C-hexoside), coumarins, and saponins fusca, as well as few individuals of other arboreal species. A (Yenesew et al. 2003; Juma and Majinda 2004; Dao et al. previous study showed that E. fusca represents 77% of the 2009; Pérez et al. 2015). Studies of metabolites from E. total of individuals in these stands (Gris et al. unpubl. res.). fusca confirmed the presence of flavonoids, such as pter- The herbaceous stratum is very homogeneous, dominated ocarpans (Innok et al. 2009, 2010). Despite the mono- by grasses, with soil covered by a histosol layer with leaf dominance and the presence of phytotoxicity metabolites, litter, primarily composed by E. fusca leaves (Gris et al. no study has covered the allelopathic effects of E. fusca, not unpubl. res.). We collected the material only from adult even on a bioindicator species. plants: mature leaves and seeds, without predator attack; The use of the bioindicator Lactuca sativa L. (Aster- branches of secondary roots and bark slices of the basal aceae) is a usual method for testing allelopathic potential trunk region. since it has a rapid life cycle and is highly sensitive to the We performed the fieldwork during the dry season, in action of allelochemicals (even in low concentrations) and November 2013, in these monodominant stands at Taiamã also has as all stages of development well known (Ferreira Ecological Station, between the coordinates: 16°50′58.2″S and Aquila 2000). Meanwhile, the germination and devel- 57°28′25.7″W and 16°52′57.4″S 57°30′22.2″W. The opment patterns are not known for many wild species, and it regional climate is Aw (with dry winter) according to the is challenging to infer about the potential allelopathic Köppen classification (Alvares et al. 2013), with two sea- effects on these wild plant species. Therefore, the results of sons: dry season from May to September and rainy season the experiment using L. sativa are quick and easy to from October to April. The average precipitation is understand, because if we regulate all external factors and 1227 mm, and the average annual temperatures are around compare percentages of germination and/or development of 26 °C, we calculated these values from the data obtained L. sativa with controls (negative and positive), any change from National Institute of Meteorology (INMET 2019). in this trait could be attributed to the adverse potential of the We prepared a fertile sample using herbarium techniques tested extracts/compounds. Several recent studies use L. (Mori et al. 1989; Bridson and Forman 2004) and the sativa as a plant model in allelopathy experiments (e.g., voucher was deposited in the CGMS Herbarium under Wang et al. 2016, 2019; Fernandes et al. 2018; Carvalho registration CGMS 40967. et al. 2019; Scrivanti and Anton 2019; Silva et al. 2019). Furthermore, considering that E. fusca affects the devel- Plant extraction opment of L. sativa seeds, we can infer about its probable effect on wild eudicots. We dehydrated plant material in an air-dry oven at 50 °C for Therefore, we believe that E. fusca has allelopathic 24 h. We briefly ground and homogenized each plant part potential and, we hypothesized that: (1) extracts from separately in a Willey-type mill. The seed extract was different parts of the plant retard or inhibit seed germi- prepared through percolation, using ethanol:water (7:3), 20 nation and development of the bioindicator species (L. drops per minute during three days of extraction. Other sativa); (2) inhibitory activity increase with increasing plant tissues were submitted to a pressurized fluid extractor concentration of each extract. Thus, to get an initial idea (DIONEX®—ASE 150), using a mixture of ethanol:water about this, we aimed, in this study, to evaluate the effects (7:3), with temperature of 130 °C, static extraction time of of E. fusca leaves, bark, root, and seed extracts on ger- 4 min, 150% volume wash, five cycles of extraction and mination and development of the bioindicator L. sativa 100 s of purge. The extracts were concentrated in a rotary and identify the secondary metabolites of different evaporator, lyophilized and maintained at −20 °C until tests tested parts. were performed. Secondary metabolites that could contribute to the monodominance of Erythrina fusca in the Brazilian. Bioassay and analysis The analyses were done in the negative and positive ionization mode, but we only depicted the chromatogram For the germination experiment, we used Petri dishes (9 cm at positive mode (m/z 120–1200). The UV wavelength was in diameter) containing two sheets of filter paper, previously monitoring between 240 and 800 nm. The capillary vol- autoclaved. We solubilized all extracts in MES buffer (2- tage applied was 4500 Kv, and Nitrogen was used as the morpholinoethanesulfonic acid) 10 mM, pH 6.0.