Short communicationInconsistency in the detection of phytotoxic effects: A test with Acacia dealbata extracts using two different methods
Graphical abstract
Introduction
The search for new bioactive compounds that can help to solve problems of different nature is a challenge for scientists. Active compounds can be obtained from several sources, being analyzing allelopathic plants – those that interfere with the growth of another by releasing natural chemicals into the environment (Rice, 1984) – the main way to discovering new effective natural chemicals. The interest in these natural substances is mainly focused on their biological activity as drugs in medicine or microbiology (Currais et al., 2014, Newman and Cragg, 2012, Sánchez et al., 2015), and as natural herbicides, pesticides or templates to synthesize new agrochemicals for reducing the usage of toxic products in agronomy (Dayan et al., 2009, Gerwick and Sparks, 2014, Lacret et al., 2012, Narwal, 2010, Varejão et al., 2013). Recently, natural compounds showing stimulatory mechanisms of adult stem cell and angiogenesis respectively are also of interest at least for medicine research (Morgan and Nigam, 2013, Pazhanisamy et al., 2012).
In general, the search for new natural compounds with biological activity is done using the standard bioassay-guided fractionation method (Dayan and Duke, 2006, Inderjit and Nilsen, 2003, Macías et al., 2008, Scognamiglio et al., 2013, Varejão et al., 2013). This method is a simple, repetitive, relevant and economic procedure (Dayan and Duke, 2006, Vyvyan, 2002). With this approach, plant materials are extracted with a solvent to obtain crude extracts that are subsequently fractionated with a set of solvents of increasing polarity. Fractions are then bioassayed and active ones are sub-fractionated and subjected to new bioassays until substances responsible for the activity are, if possible, identified (e.g., Macías et al., 2008, Rial et al., 2014). The most common biological assays for evaluating the phytotoxic activity of plant allelochemicals are germination and seedling growth studies using model-sensitive species (Dayan and Duke, 2006, Lotina-Hennsen et al., 2006), such as Lactuca sativa L. (Araniti et al., 2014, Macías et al., 2000, Omezzine et al., 2011, Omezzine et al., 2014). However, the bibliography shows less agreement about the dissolution process of chemicals used in the phytotoxic bioassays within the bio-guided fractionation procedure. There are two major types of methods for dissolving the compounds used in bioassays. Most commonly, the compounds are re-dissolved in the same organic solvents used for their fractionation. Then, solvents are left to evaporate and distilled water is added (e.g., Araniti et al., 2014, Dayan and Duke, 2006, Omezzine et al., 2011, Omezzine et al., 2014, Pereda-Miranda et al., 1993). Although water is the natural solvent, the actual concentration of natural compounds poorly soluble in water remains undetermined once organic solvents were evaporated (Dayan and Duke, 2006). The other type of methods includes phytotoxic bioassays where the compounds are re-dissolved in a minimum amount of DMSO and the solution is poured in buffered medium (Lacret et al., 2012, Macías et al., 2008, Novaes et al., 2013, Rial et al., 2014). In this case, DMSO is used to improve the dissolution of the extracted compounds (e.g., Macías et al., 2008, Macías et al., 2010). Both methods are currently used (e.g., Araniti et al., 2014, Macías et al., 2010, Omezzine et al., 2014, Rial et al., 2014), but generally the choice of one dissolution procedure over the other is not explained. Moreover, whether chemical compounds produce the same phytotoxic effect using one method or the other remains unexplored. Thus, we designed a study to test whether the dissolving method could affect the biological activity of natural compounds obtained with a bioassay-guided fractionation procedure. Since the dissolution of organic compounds extracted with solvents of low or intermediate polarity is expected to improve using DMSO as solvent (Macías et al., 2008), we hypothesized that bioassays using either organic solvents and water or DMSO and buffered medium might yield different phytotoxic effects for Acacia dealbata compounds. Particularly, we expect that hexane and ethyl acetate chemicals perform better in DMSO-buffer bioassays. In contrast, chemicals fractionated with water should not be affected by the type of bioassay or, if so, the effects would be higher when water is the solvent.
To do this, we selected A. dealbata Link, which is a woody legume native to Australia and invasive world-wide (Fuentes-Ramírez et al., 2011, Lazzaro et al., 2014, Lorenzo et al., 2010a, Lorenzo et al., 2012b, Richardson and Rejmánek, 2011). This species reduces the richness, diversity and cover of surrounding plants (Lorenzo et al., 2012b, Lazzaro et al., 2014), and modifies the community structure and functional diversity of soil microbes in its non-native ranges (Lorenzo et al., 2010c, Lorenzo et al., 2013, Lazzaro et al., 2014). The invasive success of A. dealbata has been partially related to the potential allelopathic interference on species in its vicinity, particularly during the flowering period (Lorenzo et al., 2008, Lorenzo et al., 2010b, Lorenzo et al., 2011, Lorenzo et al., 2013, Souza-Alonso et al., 2014). Natural compounds from different materials of A. dealbata (flowers, leaves and bark) were fractionated with solvents of increasing polarity (hexane, ethyl acetate and water) and the phytotoxic effect of these compounds was assessed on germination and seedling growth of L. sativa using guided bioassays with the two aforementioned procedures. Potential uses for the assayed chemicals according to the phytotoxic effects found are also discussed.
Section snippets
Sampling of plant material
Sampling of different parts of A. dealbata Link was conducted in February 2013 (flowers) and May 2013 (leaves and bark) in two invasive populations of this species in Galicia, Spain (40°09′00.33″N 8°39′41.21″W; 40°06′25.33″N 8°39′35.71″W), since the production of natural compounds is highly dependent on the plant part (Çirak et al., 2008, Aguilera et al., 2015). Flowers, leaves and bark were collected separately from 25 to 30 individuals of A. dealbata in each population. The same plant
Germination rate
The germination of L. sativa seeds was not affected by any of the tested chemical fractions in either bioassay (Table S2). Germination rates ranged from 91.4 to 104.3% in water bioassays and from 95.6 to 107.5% in buffer bioassays (Table S2).
Stem length
The stem length of L. sativa seedlings was significantly affected by chemicals extracted from flowers, leaves and bark tested in both water and buffer bioassays (Fig. 1, Table S3). For flowers, the ethyl acetate fraction had no effect in bioassays conducted
Discussion
In the present study, flowers, leaves and bark methanolic crude extracts were obtained from A. dealbata and were further fractionated with hexane, ethyl acetate and water. The resulting fractions were bioassayed at different concentrations on germination and seedling growth of L. sativa. We compared the phytotoxic effects of these fractions by using test solutions prepared either by re-dissolving each fraction in their own solvent or in DMSO-buffer. The pH of solutions tested in phytotoxic
Conclusions
We found that the phytotoxic effect of A. dealbata compounds varies depending on the dissolving method used and is higher in DMSO-Buffer Bioassays. In terms of the number of significant effects, chemical substances fractionated with hexane and ethyl acetate showed more phytotoxic effects in DMSO-Buffer than in Water Bioassays while compounds presents in water fractions showed the opposite trend. These results suggest that low- or intermediate-polar compounds perform better in buffered bioassays
Acknowledgments
This work was supported by the project CTQ2012-37734-C02-01 from funded by the “Ministerio de Economía y Competitividad (MEC)” of Spain and the European Social Fund (FEDER). PL is supported by a post-doctoral grant of the Portuguese Foundation for Science and Technology (FCT) (Reference: SFRH/BPD/88504/2012) cofunded by the European Social Fund. SRE is also supported by FCT with a Development Grant (IF/00462/2013).
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