Allelopathic Effects of Psychotria viridis Ruiz & Pavon on the Germination and Initial Growth of Lactuca sativa L .

The effects of aqueous and ethanol extracts and leaf fractions of Psychotria viridis Ruiz & Pavon (chacrona) at different concentrations on the germination and initial growth of Lactuca sativa L. were tested, and the phenolic and flavonoid compounds of these extracts and fractions were assessed. The bioassays consisted of the following treatments: crude aqueous extract (CAE) at 25, 50, 75 and 100% concentration, crude ethanol extract (CEE) and ethyl acetate, dichloromethane and methanol fractions at 6.25, 12.5, 25, 50 and 100% concentration and a control group. All treatments consisted of five replicates. The CAE, CEE and the ethyl acetate fraction of P. viridis caused both positive and negative effects on the seeds and seedlings of L. sativa. By contrast, the dichloromethane and methanol fractions only caused negative effects on L. sativa. The following compounds were identified in the extracts and fractions: gallic acid, chlorogenic acid, caffeic acid, ellagic acid, catechin, orientin, vitexin, quercetin, apigenin, rutin and luteolin, and the presence of the alkaloid N,N-dimethyltryptamine (DMT) has also been reported in the literature. P. viridis had allelopathic effects in all types of plant extracts and fractions tested, and one of these compounds or their combined action may account for these effects.

Certain plant species produce chemical substances that positively or negatively affect the germination and/or growth of other plants when released into the environment.This phenomenon is known as allelopathy, and its main function is to decrease or eliminate competition (Rice, 1984;Ferreira & Borghetti, 2004;Fujii & Hiradate, 2007).Bagchi, Jain, and Kumar (1997) considered allelochemicals as a resource for the development of natural herbicides to be used in organic farming to minimize the environmental impact caused by commercial herbicides or as plant growth stimulants given the variety of secondary metabolite activities, especially allelopathic activity.Accordingly, the present study aimed to evaluate the allelopathic effects of leaf extracts of P. viridis on the germination and growth of Lactuca sativa L. and to chemically identify the phenolic and flavonoid compounds present therein given the presence of the alkaloid N,N-dimethyltryptamine (DMT) in P. viridis leaves (Quinteiro, Teixeira, Moraes, & Silva, 2006) and the reports of alkaloids, flavonoids and phenolic compounds with allelopathic action, and because the occurrence of alkaloids has already been previously studied.

Collection and Identification of Botanical Material
P. viridis leaves were collected in the morning period in a planting area belonging to the União do Vegetal Beneficent Spiritualist Center (Centro Espirita Beneficente União do Vegetal-CEBUDV), Highway Vicente Teles s/n, District of Santa Fé, municipality of Crato, Ceará state (CE), Brazil, located at 7°11′S and 39°27′W.
The plant material was collected and treated according to the usual plant collection methods, identified and sent for confirmation.The voucher specimens were deposited in the Dárdano de Andrade-Lima Herbarium of the Regional University of Cariri (Herbário Caririense Dárdano de Andrade-Lima da Universidade Regional do Cariri, HCDAL-URCA) under the registration number 6159.

Preparation of Extracts
A total of 200 g of fresh leaves of P. viridis was blended in 427 ml of distilled water using an industrial blender to prepare the crude aqueous extract (CAE).The volume of distilled water used was set based on the ratio between the fresh matter weight (FMW) and dry matter weight (DMW) (Medeiros, 1989).
For the crude ethanol extract (CEE) preparation, 500 g of fresh leaves of P. viridis was ground and soaked in 3L of ethanol P.A. (99.3%) and stored in glass containers for seven days.That mixture was then filtered and the solvent was evaporated using a rotary vacuum evaporator and concentrated using a water bath.The ethanol extracte was vaporated fully.
The extract was fractionated by vacuum filtration using solvents of increasing polarity to prepare the fractions.Thus, 10.9 g of ethanol extract from P. viridis leaves was used for this purpose generating the following yields: hexane fraction: 0.42 g; dichloromethane fraction: 1.03 g; ethyl acetate fraction: 1.25 g and methanol fraction: 7.01.The hexane fraction was discarded because it failed to show sufficient yield to perform the bioassays.The dichloromethane (DCMF), ethyl acetate (EAF), and methanol (MF) fractions were used in the bioassays.
The (CEE) and fractions were dissolved in 66% ethanol in a ratio of 1:1, where 100 mg of the crude ethanol extract and 100 ml of ethanol were obtained, thus obtaining the stock solution of 100%, while the concentrations of 6.25, 12.5, 25, 50% were obtained by dilution (Mazzafera, 2003).

Bioassays
The aqueous extract bioassays consisted of four treatments at the concentrations of 100, 75, 50 and 25% and one control group at 0% (distilled water).The ethanol extract and the dichloromethane, ethyl acetate and methanol fractions were at concentrations of 100, 50, 25, 12.5 and 6.25%.Each treatment consisted of five replicates with 20 L. sativa seeds.The experimental design used was completely randomized (CRD).
According to Souza, Cattelan, Vargas, Piana, Bobrowski, and Rocha, (2005), the main advantage of using lettuce as the target of allelopathic studies lies in the sensitivity of the seeds of the species, therefore, even in low concentrations of allelochemicals its process of Germination can be compromised.In addition, germination is rapid in approximately 24 h, has linear growth, is insensitive to pH differencesIn wide range of variation and the osmotic potentials of the solutions (RICE, 1984).
The experiments were conducted in Petri dishes with two filter paper disks moistened with 3 ml of the extract and fractions in different extract concentrations.Conversely, the control was moistened in 3 ml of distilled water.The experiments were conducted in a biological oxygen demand (BOD) seed germination chamber at a temperature of 25 °C and photoperiod of 12 hours for seven days.The plates were left open for 48 hours to evaporate the alcohol completely for the bioassays with ethanol extract and fractions (Mazzafera, 2003).
The pH of the extracts and fractions at different concentrations was analyzed using a pH meter.The osmolarity of the crude aqueous extract at different concentrations was also analyzed using an osmometer.Solutions with pH higher than 6.0, which is the range considered optimal by Macias, Gallindo, and Molinillo (2000), were adjusted using 0.1 N KOH and 5% HCl solutions.

Parameters Assessed
The following parameters were assessed: germination percentage (GP), germination speed index (GSI; assessed every 24 hours) and hypocotyl and radicle length (evaluated after seven days of sowing).Five seedlings were used per replicate to measure the length of L. sativa hypocotyls and radicles, totaling 25 seedlings per treatment.jas.ccsenet.

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Seedling       Macias et al. (2000) recommend adjusting the pH of aqueous extracts to 6.0 because this is the optimal pH range for seed germination and observation of allelopathic effects.Both seedling germination and growth are affected when the pH is extremely alkaline or extremely acid (Roy, 1986), with deleterious effects observed under pH conditions below 4 and above 10 (Eberlein, 1987).An extract may contain solutes, including sugars, amino acids and organic acids, that may mask the allelopathic effect of the extract because they affect the pH according to Ferreira and Áquila (2000).
The osmotic potential of the aqueous extract was -0.06, -0.09, -0.14 and -0.18 MPa at concentrations of 25, 50, 75 and 100%, respectively.Such parameters constitute acceptable standards for seedling germination and growth in tests with potential allelopathics.Research studies, including those conducted by Mano (2006) and Gatti, Perez & Lima, (2004), have shown that those values are acceptable for allelopathic tests with seed germination.

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Table 2 .
pH values according to the concentration of the ethanol extract and fresh leaf fractions of P. viridis