The Callistemon viminalis (Myrtaceae) blossoms were collected on the campus of the Federal University of Lavras (UFLA) in Lavras, MG, Brazil, in March 2012 in the morning on a day with mild temperatures and no precipitation. Lavras is located in the South of Minas Gerais, Brazil, latitude 21˚14'S, longitude 45˚00'W Gr and at an altitude of 918 m. The species was identified in the Department of Biology at the Federal University of Lavras, and its voucher specimen is found recorded in the ESAL Herbarium of the Federal University of Lavras (record number 26,624).

The essential oil from fresh flowers (300 g) was extacted by hydrodistillation over a 2 h period in a modified Clevenger apparatus (Pharmacopoeia, 2010) [10] . After extraction, the hydrolact was centrifuged at 965.36 g for 5 min to obtain a better separation of the essential oil from the water. The essential oil was stored refrigerated in an amber bottle. Together with the extraction, the moisture content was determined according to the method of Pimentel et al. [11] . The yield of the essential oil was calculated and expressed in weight of oil per unit weight of plant material on a Moisture-Free Basis (% w/w MFB).

Chemical analysis of the essential oil from Callistemon viminalis blossoms was performed in the Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Portugal. Analyses by gas-liquid chromatography (GLC) were performed on a Perkin Elmer 8700 gas chromatograph equipped with two flame ionization detectors (FID), and a data processing system. Two columns of different polarities were installed: a DB-1 fused silica column with a methyl silicone stationary phase (30 m × 0.25 mm ID, film thickness, 0.25 mm; J & W Scientific Inc.) and a DB-17HT fused silica column (30 m × 0.25 mm, film thickness 0.25 mm; J & W Scientific Inc.). The oven temperature was programmed from 45˚C to 175˚C in increments of 3˚C/min and, subsequently, from 175˚C to 300˚C at 15˚C/min. The temperature was maintained at 300˚C for 10 min. The temperatures of the injector and detector were 290˚C and 280˚C, respectively. Hydrogen, adjusted to a linear velocity of 30 cm/s, was used as the carrier gas. The split ratio was 1:50. The percentage composition of the essential oils was determined by integration of peak areas without the use of correction factors. The values given represent the average of two injections.

In the gas-liquid chromatography/mass spectrometric (GLC/MS) analysis, an Autosystem XL chromatograph equipped with a fused silica column DB-1 (30 m × 0.25 mm ID, film thickness 0.25 mM one; J & W Scientific Inc.) and connected to a Perkin-Elmer Turbo Mass (program version 4.1) spectrometer was used. The oven temperature was programmed from 45˚C to 175˚C at a rate of 3˚C/min and, subsequently, at 15˚C/min to 300˚C, where the temperature was maintained for 10 min. The temperature of the transfer line was 280˚C; the ionization chamber temperature was 220˚C; the carrier gas was helium, adjusted to a linear velocity of 30 cm/s. The split ratio was 1: 40; the ionization energy, 70 eV; ionization current, 60 μA; mass range, 40 - 300 U; and the scan time, 1 s. The identity of the compounds was determined by comparison of their retention indices with respect to the C9-C16 n-alkanes. The mass spectra were compared with those of standard commercial and reference compounds present in essential oils existing the laboratory and with a mass spectral library developed in the laboratory [12] .

The germination and growth bioassays were conducted in the UFLA Seed Testing Laboratory. For the preparation of the stock solution, 0.250 mL of the essential oil emulsified in Tween 80 was used in a 1:1 (v/v) ratio and dissolved in distilled water to give a 1% stock solution. The remaining concentrations (0.1 and 0.01 v/v) were prepared by dilution, and a 1.0% v/v solution of Tween 80 in water was used as a control. The test solutions were added only at the start of the bioassays, and, when necessary, only distilled water was added [5] -[13] .

Four replicates containing 50 seeds of each species, totaling 200 seeds per treatment, were used for the germination test. The seeds were placed in gerbox-type germination boxes containing two sheets of sterilized blotting paper, which were soaked with solutions containing different concentrations of the essential oil, whose volumes were equivalent to 2.5 times the mass of the dry paper. The germination boxes were maintained in a BOD at 20˚C using a 12 h photoperiod [14] . Seed germination was monitored for seven days, with daily counts of lettuce seedlings until the seventh day of sowing and assessment of normal seedlings [14] .

The verification of the effect of essential oils from the flowers of Callistemon viminalis was performed by the following tests using four concentrations:

First germination count: count of normal seedlings germinated (that presented the perfect vegetative structures) on the fourth day after sowing [14] .

Germination Speed Index (GSI): determined according to the formula given by Maguire (1962), 16 GVI = N1/N2 + 1/2 + ··· + Nn/n where Nn = number of germinated seeds observed on the first, second and nth days after planting the seeds, respectively. Thus, the GSI may range from zero, if no seed germinated, to a hundred, if all the seeds germinated on the first day.

Average length of root and hypocotyl: at the end of the germination test, normal seedlings had their aereal parts and root parts measured using a millimetric ruler and the average results were expressed in centimeters per seedling [14] .

Determination of dry matter: after performing the measurement of the seedlings, they were packaged in paper bags and dried in an oven at 60˚C to constant weight. The means were expressed in grams [15] .

A completely randomized design (CRD) with four replications for the variables—first germination, germination speed index (GSI), length of root, shoot length and dry weight—was employed. The results were subjected to analysis of variance, and quantitative variables that were significant according to the F test were subjected to regression analysis. Data were analyzed using the Analysis of Variance for Balanced Data System (Sisvar) statistical program, according to Ferreira (2011) [16] .

The average essential oil content found in the flowers of C. viminalis was 0.24% [w/w, Moisture-Free Basis (MFB)]. In agreement with the data observed in this study, Pires et al. (2013) [17] in studies with leaves and flowers of C. viminalis observed that these showed yields of essential oils from 1.42% to 0.30% for leaves and flowers, a value close to that obtained in this study compared with the flowers.

The chemical constituents present in the essential oil extracted from the flowers of C. viminalis, followed by their retention indices and their concentrations, are presented in Table 1. Compounds belonging to the monoterpene class have been identified of in various concentrations. The principal compounds were 1, 8-cineole

*RI = Calculated retention index relative to C₉-C₁₆ n-alkanes on the DB-1 column, **t = trace (<0.05%).

(66.93%), α-pinene (16.00%) and limonene (10 04%). Silva et al. (2010) evaluated the chemical composition of the essential oils from the leaves of C. viminalis collected in Brazil and found 1, 8-cineole (65.0%), α-pinene (12.0%) and α-terpineol (13.0%) to be the principal compounds. They found α-terpineol in much smaller amounts (0.22%) in the essential oil from the flowers, a result that disagrees with the findings of the present study. According Gobbo-Neto and Lopes (2007) [18] , climatic factors, the age and development of the plant, and the different plant organs can influence not only the total amount of metabolites produced, but also the relative proportions of the constituents in the mixture.

There was a dose-dependent effect Figure 1 for the variables—dry mass, GSI, shoot length and length of the

roots—that is, their values were inversely proportional to the concentration of the essential oil. The different concentrations of the essential oil did not affect the first count and the germination of the lettuce seeds.

Singh et al. (2005) [19] detected a high toxicity of the essential oil from eucalyptus species (Eucalyptus citriodora), which belong to the same family as C. viminalis (Myrtaceae), against the weed Parthenium hysterophorus. The authors observed that the seedling length decreased with increasing concentration of the eucalyptus oils (0.2 - 5.0 μL·mL⁻¹). Their result corroborated that observed in the present study. The germination was completely inhibited at 5.0 μL·mL⁻¹. This result substantiates the bioherbicidal effect of the essential oil on the germination and initial growth of weeds and differs from the results obtained in this study. Similarly, Yamaguchi et al. (2011) [20] studied aqueous extracts of eucalyptus (Eucalyptus globulus Labill.), which belongs to the Myrtaceae family, and found that the percentage of germination within seven days of all cultivated species decreased with increasing concentrations of the extract. A greater decrease (24%, 12% and 7% germination of lettuce seeds) was observed with the application of higher concentrations of the extract (70%, 90% and 100%, respectively).

All the concentrations of the essential oil from the flowers caused a significant decrease in the GSI. A reduction in germination rate occurred because of the delay in seed germination during the first three days of the experiment Figure 1. The essential oil also affected the growth of lettuce seedlings and caused a reduction in the length of shoots and the root system, this decrease being proportional to the concentration of the essential oil Figure 1. Similar results were found by Verdeguer et al (2009) [21] , who observed a decrease in the rate of of germination and growth of Amaranthus hybridus and Portulaca oleracea with increasing concentrations of 0.125.; 0.5 and 1 μL·mL⁻¹ when essential oils from Eucalyptus (Eucalyptus camaldulensis) were used.

The decrease in the dry weight of the plants was proportional to the concentration of the essential oil applied Figure 1. This observation could be confirmed by the results for the length of shoots and root parts because they demonstrate the manner in which the plant grows and the amount of dry mass is directly linked to these parameters.

According to Rodrigues, Rodrigues and Reis (1999) [22] , allelopathic compounds inhibit germination and growth by interfering with cell division, membrane permeability and the activation of enzymes. This allelopathic effect presented by the essential oil from C. viminalis may be due to the presence of the principal constituents 1, 8 cineole, α-pinene and limonene or to chemical synergism between the compounds in the essential oil.