Phytotoxic and Enzymatic Study of Philodendron meridionale on seeds of Lactuca sativa L. Estudo fitotóxico e enzimático de Philodendron meridionale em sementes de Lactuca sativa L. Estudio fitotóxico y enzimático de Philodendron meridionale en semillas de Lactuca sativa L

In an effort to identify novel biopesticides, the present study aimed to assess the effects of Philodendron meridionale (Buturi & Sakur) stem and leaf ketonic and ethanolic extracts (SKE, SEE, LKE, and LEE, respectively) on the germination, growth, root respiration, and enzymatic activities of Lactuca sativa L. seeds, and to measure the Research, Society and Development, v. 10, n. 1, e5610111336, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i1.11336 2 associated saponins, phenolics, and flavonoids that may possess herbicidal, pharmaceutical, or pesticidal activities. The extracts were obtained using a modified Soxhlet apparatus and methanolic extracts of 0.1, 0.25, 0.5, 0.75 and 1.0 mg.mL were tested, with water and methanol as a control. The presence of saponins and the concentration of phenolic compounds were determined. Allelopathic activity was evaluated in tests of germination, growth, cellular respiration and enzymatic activity. The presence of saponins and the concentration of phenolic compounds equivalent to 225.12 for LKE, 240.45 for LEE, 193.28 for SKE, and 265.14 for SEE (mg·g.gallic acid), and flavonoids 52.74 for LKE, 54.31 for LEE, 72.74 for SKE, and 67.21 for SEE (mg.g.quercetin) were determined. The allelopathy of the P. meridionale extracts against L. sativa was confirmed through negative effects on L. sativa seed germination, radical growth and morphology, dry mass, and the concentrations of α-amylase (EC 3.2.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), and polyphenol oxidase (EC 1.10.3.1). It was likely that the allelopathic action of the P. meridionale extracts was related to its effects on the membrane permeability and oxidative stress of the treated L. sativa seeds. The P. meridionale extracts contained saponins, calcium oxalate crystals, and flavonoids, including phenolic compounds, which are known allelochemicals with herbicidal activities.


Introduction
Knowledge about the mechanisms of action caused by secondary metabolites, physical-chemical characterization and biological tests of botanical products facilitated the development and industrial use of products, including medicines, cosmetics, foods, insecticides and herbicides, generated from of native species (Pino et al., 2013).
Philodendron meridionale (Buturi & Sakur) is a recently described and non-threatened Brazilian endemic, found in the states of Paraná and Santa Catarina (Buturi et al., 2014). The species is a hemiepiphyte and, so far, has not been the focus of any investigative research. However, other species of Philodendron have been reported to contain substances with biological activities, including anti-hemorrhagic (Moura et al., 2015), cytotoxic (Hassanein et al., 2011;Ghareeb et al., 2015), insecticide (Santiago et al., 2014) and larvicidal (Alliance et al., 2017).
Allelopathy is the influence, beneficial or harmful, of one species of plant on another (Zimdahl, 2018) and occurs when plants release substances (ie, allelochemicals), usually secondary metabolites, which affect the growth and survival of the surrounding vegetation (Bogatek & Gniazdowska, 2007). Substances can work by affecting plant respiration, photosynthesis, enzymatic activity, water relations, stomatal opening, phytohormone levels, mineral availability, cell growth and the structure and permeability of cell membranes and walls (Rezende et al., 2003).
The resistance or tolerance of secondary metabolites that serve as allelochemicals is becoming more and more defined and is found in more sensitive species than others, such as Lactuca sativa L. (lettuce) (Ferreira & Aquila, 2000;Reigosa et al., 2013). This is the most common plant used as a target species among hydrophytes, due to the short time required for germination (24 to 48 h) and growth (Elakovich, 1999, Gatto et al., 2020.
The aim of the present study was to evaluate the effects of ketone and ethanolic extracts of P. meridionale stems and leaves on germination, growth, root respiration and enzymatic activities of L. sativa seeds and to analyze saponins, phenolics and associated flavonoids that may have herbicides, pharmaceutical products or pesticides.

Collection of botanical material
Philodendron meridionale specimens were collected at the Botanical Garden Campus of Federal University of Paraná, Brazil (25°26′58′′ S, 49°37′12′′ W -906 m altitude). The herbarium specimens were deposited in the collection of the Botanical Museum of Curitiba, under registration number 390207, and were identified by a Philodendron specialist, Dr.

Mônica Cássia Sakuragui.
Access to the botanical material was permitted and licensed by Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SISGEN) and was registered under number A0EB51A as required by the Brazilian legislation.

Preparation of ketonic na ethanolic extracts
To prepare leaf ketonic extracts (LKE) and stem ketonic extracts (SKE), P. meridionale leaves and stems were separately dried, crushed in cutting and hammer mills, and extracted using a Soxhlet apparatus with propanone. The remaining residues were extracted using 99.9% ethanol in a modified Soxhlet apparatus (Carvalho et al., 2009) to obtain leaf ethanolic extracts (LEE) and stem ethanolic extracts (SEE). For each extraction procedure, the extracts were filtered, using a Büchner funnel, concentrated, and then refrigerated.

Measurement of flavonoid contents
Flavonoid contents were measured using the guidelines of Chang et al. (2002) and expressed in mg equivalents of quercetin per g crude extract (y = 0.0313x -0.016, R² = 0.9997).

Saponin screening
Three assay tubes, each with equal volumes of aqueous extract (0.02 g of all extracts were diluted in 5 mL of water, in triplicate), were shaken vigorously for 5 min and then allowed to rest for 30 min. The presence of saponins was then confirmed by the persistence of >1 cm foam in the tube (Reginatto, 2017).

Preparation of the allelopathic test
Six milliliters of the methanolic solution (LKE, SKE, LEE, and SEE) of various concentrations (0.1, 0.25, 0.5, 0.75, and 1.0 mg.mL -1 ) were added to boxes (i.e., gerbox) that contained two sheets of Whatmann® filter paper (1.0) that had been autoclaved at 120°C for 20 min. For preparation, the extracts were diluted and kept at 30°C for 24 h to allow evaporation of the solvent. They were then resuspended using the autoclaved filter paper pre-moistened with 6 mL distilled water. A similar procedure was also performed using methanol (solvent control) and water (negative control). The experiment was conducted in quadruplicate, and the gerbox with L. sativa seeds were incubated in a germination chamber (Bio-Oxygen Demand incubator), with a relative humidity of ~80% and constant temperature of 25C (Brasil, 2009;Krause et al., 2015;Merino et al., 2018;Gatto et al., 2020).

Germination experiment
Germination of the seeds was checked over a period of 7 days, and germinated seeds were removed from the gerbox.
Seeds were considered to have germinated when the radical appeared to protrude through the tegument, and germination was measured in terms of rate (speed and percentage; (Laboriau, 1983) and germination velocity index (GVI; Maguire, 1962). The GVI was calculated for each replicate, according to the following equation: GVI: G1/N1 + G2/N2 + Gn/Nn, where GVI represents the germination speed index.G1, G2, ..., Gn represent the number of seeds that germinated on each day, and N1, N2, ..., Nn represent the number of days from sowing until the seventh day, using the sum of the germinated seed quantity ratio and the day of germination (Krause et al., 2015;Merino et al., 2018).
The natural germination rate (percentage) of the L. sativa seeds was assessed in a parallel test, in which one hundred L. sativa seeds (≤ 25 per gerbox) were treated with water.

Growth experiment
On the last day of the germination test (i.e., 7th day), the hypocotyl and radical lengths were measured using a pachymeter, and to measure dry mass, the seedlings were dried in an oven at 60°C until reaching a constant weight (Macias et al., 2000;Merino et al., 2018)

Root respiration experiment
Root respiration capacity was measured according to Steponkus and Lanphear (1967); Krause et al. (2015) and Merino et al. (2018). Briefly, 1.0 cm pieces were cut from the ends of 10 roots and transferred into test tubes with 5 mL 0.6% (m/v) triphenyl tetrazolium hydrochloride and 1 mL sodium phosphate (mono and dibasic) buffer (0.05 mol.L -1 pH 7.0). The tubes were incubated at room temperature for 2 h and then transferred to a water bath (30 °C). After 15 h, the solutions were drained, the roots were rinsed with distilled water, 7 mL 95% (v/v) ethanol was added, and the tubes were heated in a boiling water bath (~100 °C) for 15 min. After cooling to room temperature, 10 mL 95% (v/v) ethanol was added to each tube, and the absorbance of each tube at 530 nm was measured, using 95% (v/v) ethanol as a blank.

Enzyme activity experiment
Methanolic extracts of 1.0 mg.mL -1 concentration were prepared at the end of the determined germination period and growth of the seedlings exposed to P. meridionale extracts. The seedlings were triturated using liquid nitrogen and then frozen (-20°C). Subsequently, 10 mL potassium phosphate buffer (0.2 mol.L -1 , pH 7.0) was added, and the samples were centrifuged (2500 rpm for 20 min at 4°C). The resulting supernatants were kept refrigerated (4°C) and used as enzymatic extracts (Putter, 1974) for the following assays.

Analysis and Statistics
The analyses were conducted in triplicate and the readings were completed in spectrophotometer UV/Vis SHIMADZU-1601. Research, Society and Development, v. 10, n. 1, e5610111336, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i1.11336 6 For the germination and growth bioassays, the experimental delineation was completely randomized, with six treatments (solvent control, negative control, and four extracts) and four replicates per treatment, and a total of twenty L. sativa seeds were used for each treatment. The germination and growth data were subject to variance analysis (ANOVA), using Origin 9.0 (OriginLab, Northampton, MA), and the means of each variable were compared using the Tukey test, at p= 0.05.

Results and Discussion
Most of the control L. sativa seeds germinated within 24 h, whereas most of the seeds treated with high extract concentrations (0.5, 0.75, and 1.0 mg·mL -1 ) germinated between 24 and 48 h; the effect of the extracts increased as the treatment concentration increased. The treatment had no effect on GVI value.
The extract treatments inhibited root growth, regardless of concentration, but did not affect hypocotyl growth ( Figure   1). Interference with the primary root growth of treated plants, as observed in this study, is one of the main indicators used for the study of extracts with allelopathic potential (Souza-Filho et al., 1997).  Research, Society andDevelopment, v. 10, n. 1, e5610111336, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i1.11336 8 The extract treatments also affected seedling morphology, for example, causing roots to become atrophied and disproportionate ( Figure 2). When compared with the roots of the control seedlings, the roots of the extract-treated seedlings were also thicker, and either hairier or hairless.
Several researchers have described effects that are similar to those observed in the present study and suggested that roots are damaged more than hypocotyls, mostly likely as a result of the direct contact of roots with the treatment solutions (Cruz-Ortega et al. 1998;Chung et al. 2001;Aumonde et al. 2012;Sousa et al. 2020;Torquato et al. 2020). Munns and Tester (2008) reported that root growth can be restricted by water deficit stress and ionic toxicity, both of which can cause metabolic and physiological damage. However, Marenco and Lopes (2005) noted that, although shoot length was unaffected, the damage of plants from water deficit could be discounted, as there was evidence that the extracts had toxic effects on plant growth factors. Extract treatment also increased the concentrations α-amylase, APX, CAT, and PPO (Table 1) and, regardless of concentration, reduced the dry mass of the L. sativa seedlings, although not by more than 43% (Table 1).  Hoffmann et al. (2007) also reported that plant extracts reduced seedling mass. An alternative explanation is that the seedlings accumulated less water as a result of the action of the extracts, which would thereby inhibit growth and potentially alter cell membrane permeability. These processes also reflect the increased production and accumulation of free-radicals, which could indirectly affect the translocation and allocation of assimilates by affecting enzyme activities (Aumonde et al., 2013).
In the present study, the observed increases in CAT, APX, and PPO activities suggested that the plant defense systems were activated, perhaps in response to allelopathy, and that this caused oxidative stress in the treated seedlings. Meanwhile, the elevated α-amylase activity indicated that the plant energy system was weakened. According to Almeida et al. (2008), these factors result from changes in seedling growth.
CAT and APX are the two most important contributors to H2O2 detoxification (Bhatt & Tripathi, 2011). Furthermore, cell wall-bound APX catalyzes the polymerization of phenols to form the lignins that function as mechanical barriers to prevent the entry of substances (Kirkby & Römheld, 2007;Viecelli et al., 2010). It is likely that the contribution of PPO to disease resistance is related to the oxidization of phenolic and quinone compounds; like polymers, these can form protein complexes that act as physical barriers against the penetration of pathogens (Campos et al., 2004). The elevated levels of PPO observed in the present study suggested that phenolic compounds might act as allelochemicals. The elevated α-amylase activity observed in the present study could be attributed to defense-related increases in cellular energy expenditure.
Allelochemicals can also alter the cellular respiration of plants that are exposed to botanical extracts (Carmo et al., 2007). In the present study, we observed alterations in the membranes of treated L. sativa seeds, especially in their permeability. However, the P. meridionale extracts did not affect seedling respiration (Table 1).
In the present study, phenolic and flavonoid compounds were dosed in extracts of P. meridionale; the concentration of phenolic compounds were equivalent to mg. quercetin, respectively. The observation of persistent froth also suggested the presence of saponins in the extracts.
Saponins, tannins, and flavonoids are among the allelochemicals that are often considered to have direct or indirect effects and can be used under natural conditions owing to their hydrosolubility (Rice 1984;Ferreira & Aquila, 2000). Saponins may exert phytotoxicity through their effects on membrane lipids or through effects on specific enzymes (Duke & Oliva, 2005). Einhellig (2005); Maraschin- Silva and Aquila (2006) reported that flavonoids and phenolic acids altered the permeability of cell membranes. Lucchesi and Oliveira (1988) and Hoffmann et al., (2007) noted that crystals, such as calcium oxalate raphides, and natural polymers, such as tannins, lignins, and resins, can also have allelopathic effects. Lignins, resins, druses and raphides of calcium oxalate have been reported in P. meridionale (Swiech et al., 2016) and the present study confirmed the presence of saponins and quantified phenolic compounds, including flavonoids. These substances might be responsible for the observed effects, either through isolated actions or a synergic mechanism.
The phytotoxicity of many products probably results from a general disturbance to the cell growth of treated plants, rather than from any specific mechanism (Einhellig, 1994;Borella & Pastorini, 2009). Furthermore, as allelochemicals belong to a variety of chemical classes, it is unlikely that they will share modes of action. As suggested by Reigosa et al., (1999), this might also explain the paucity of described dose-dependent relationships, as in the present study.

Conclusions
In the present study, we found that extracts of Philodendron meridionale affect the germination rate, enzymatic processes and morphology of Lactuca sativa seeds. These findings demonstrate effects on membrane permeability and oxidative stress. The combined action and potential synergy of the metabolites found in the extracts of P. meridionale make the species suitable for use in cultures (mainly organic) and the metabolic actions characterized in this model can be correlated in drug therapies.
Further studies are needed in planting houses and in the field to observe if the P. meridionale extracts induce allelopathic action, given that climatic conditions and plant-soil-microorganism interactions can interfere with growth and germination conditions.