Scientia Horticulturae 122 (2009) 96–101

Phytotoxic properties of Drosophyllum lusitanicum leaf extracts and its maincompound plumbagin

Sandra Goncalves a,b, Marco Ferraz a,b, Anabela Romano a,b,*a Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugalb IBB/CGBUTAD, 5001-801 Vila Real, Portugal

A R T I C L E I N F O

Article history:

Received 12 November 2008

Received in revised form 5 February 2009

Accepted 30 March 2009

Keywords:

Bread wheat

Growth and germination inhibition

Lettuce

Natural plant products

Phytotoxic bioassays

A B S T R A C T

The aim of this work was to evaluate the phytotoxic properties of aqueous and hexane extracts from the

insectivorous plant Drosophyllum lusitanicum (L.) Link using lettuce and bread wheat as model species.

The results obtained confirmed that both germination and seedling growth bioassays were sensitive and

able to detect the heterotoxicity potential of D. lusitanicum extracts. Aqueous and hexane extracts at

several concentrations significantly inhibited the seed germination of lettuce and wheat, although wheat

was less sensitive. The inhibitory effects of plumbagin, the major compound found in D. lusitanicum

hexane extracts, were also evaluated. Comparing the results of the assays obtained with extracts and

plumbagin it was postulated that plumbagin is the principal compound responsible for the phytotoxic

effects of the extracts on lettuce but not on wheat. Therefore, although the phytotoxic potential of D.

lusitanicum was demonstrated, further studies are needed to clearly specify the compounds responsible

for the inhibitory effects and to ensure if the results obtained with the model species are reproducible to

weed species in field conditions.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

Secondary metabolites produced and accumulated by plantscan induce both inhibitory and stimulatory effects on organismsand may play roles in shaping plant and microbial communities(Pennacchio et al., 2005). Germination inhibitory compounds andallelochemicals are phytotoxic compounds produced by plantsthat aid them in both interspecific and intraspecific competitions(Meyer et al., 2007). The search for allelochemicals/phytotoxins is agrowing research field, because these compounds have a greatpotential for controlling noxious weeds and could be used asherbicides in agriculture (Singh et al., 2003a).

Concerns about ecological, environmental, and health problemspossibly associated with synthetic pesticides have increasedinterest in the development of new classes of environmentallysafe herbicides (Dayan et al., 1999). The ability of a plant species toinhibit the germination of other plants is an untapped resource forweed control in crops that could revolutionize organic cropproduction. The study of the phytotoxic potential offers usefulclues in the investigation of new models of natural herbicides that

* Corresponding author at: Faculty of Sciences and Technology, University of

Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal.

Tel.: +351 289800910; fax: +351 289818419.

E-mail address: aromano@ualg.pt (A. Romano).

0304-4238/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2009.03.028

could be more specific and less harmful than the syntheticsubstances used in agriculture (Singh et al., 2003a; Bogatek et al.,2006; Hachinohe and Matsumoto, 2007). The natural plantproducts have a number of advantages over synthetic herbicidesas they usually possess complex structures, exhibit structuraldiversity and hence are invaluable sources of lead compounds;have more chiral centers; have high molecular weight with no orlow amount of halogens or heavy atoms; are environmentallybenign as they degrade rapidly in the environment; and have noveltarget sites of action different from the synthetic herbicides (Dayanet al., 1999; Duke et al., 2000, 2002; Singh et al., 2003a).

Strategies for the discovery of compounds with phytotoxicproperties are analogous to those for the discovery of compoundsin the pharmaceutical industry and involve the screening of crudeextracts and purified compounds for biological activity (Vyvyan,2002). These initial bioassays must be quick, economical, andrelevant to the system in question. The most widely used biologicalassays are seed germination and seedling growth studies (Vyvyan,2002). The most commonly used species for the bioassays isLactuca sativa L., a readily available sensitive species thatgerminates rapidly and uniformly. However, according to Dayanet al. (2000) both mono- and dicotyledons species should be usedin assays to determine the potential selectivity of the agent.

Naphthoquinones and other related quinonoid compounds areone of the major natural product classes with varied biologicalactivities (Akendengue et al., 1999). The naphthoquinone plum-

S. Goncalves et al. / Scientia Horticulturae 122 (2009) 96–101 97

bagin (5-hydroxy-2-methyl-1,4-naphthoquinone), which occurs inplants from the Plumbaginaceae and Droseraceae families, hasbeen shown to exert anticarcinogenic (Sugie et al., 1998; Srinivaset al., 2004; Hsu et al., 2006; Kuo et al., 2006), antiatherosclerotic(Ding et al., 2005), antimicrobial (Lim et al., 2007) and insecticidaleffects (Ganapaty et al., 2004; Goncalves et al., 2008). Moreover,the antifeedant (Tokunaga et al., 2004) and allelochemicalpotentials of plumbagin were also described (Spencer et al.,1986; Kocacaliskan and Terzi, 2001; Rischer et al., 2002; Meyeret al., 2007).

Leaves of Drosophyllum lusitanicum (L.) Link (Drosophyllaceae),an carnivorous plant endemic to the western Iberian Peninsula andnorthwest Morocco, contain flavonoids (luteolin, leucocyanidin,leucodelphinidin), phenolic compounds and large amounts ofplumbagin (Nahalka et al., 1998; Budzianowski et al., 2002;Grevenstuk et al., 2008). The insecticidal (Goncalves et al., 2008)and antimicrobial (Goncalves et al., 2009) activities of D.

lusitanicum extracts have previously been described by our group,however, the phytotoxicity of D. lusitanicum extracts has not beenevaluated yet. Thus, the present study describes the inhibitoryeffects of aqueous and hexane extracts from D. lusitanicum, and itsmain compound, plumbagin, on seed germination and seedlinggrowth of two model species, lettuce (L. sativa) and wheat (Triticum

aestivum L.).

2. Materials and methods

2.1. Plant material and extracts preparation

Healthy mature leaves of D. lusitanicum were collected in March2005 from a population located in Algarve region (Portugal). Theplant material was authenticated by Dr. A.I. Correia from theBotanical Garden of the University of Lisbon (Lisboa, Portugal)where a voucher specimen was deposited under the number LISU206396.

Samples of fresh and dried (at 45 8C in a ventilated drying oven)plant material were used to prepare aqueous extracts. Freshmaterial was divided into small pieces (�1.5 cm) and dried materialwas powdered using a blender. The extracts were obtained byextracting different quantities of fresh (2.5, 5 and 10 g) or dried (2.5, 5,10, 20 and 30 g) plant material with 100 ml of distilled water in ashaker over 24 h at room temperature. The mixtures were thenvacuum filtered (Whatman filter paper No. 1). Aliquots of the extractsobtained from fresh and dried plant material at different concentra-tions (w/v) were stored at �20 8C until tested. The concentrations ofaqueous extracts are presented as a percentage (%, w/v) of the plantmaterial weight per water volume used for extraction.

Fresh plant material of D. lusitanicum was extracted with n-hexane (Riedel-de Haen, Buchs, Switzerland) in a Soxhletapparatus over 8 h. The extract obtained was concentrated in arotary-evaporator under reduced pressure at 50 8C until dry. Thestock solution of the hexane extract (100%) was obtained byresuspending the crude extract in n-hexane (10 mg ml�1). Thissolution was stored at �20 8C and was later diluted with n-hexaneto obtain the different concentrations to be tested.

2.2. Phytotoxic effects of the plant extracts

Seeds of L. sativa cv. Capitata and T. aestivum cv. Tamega weresurface-sterilized for 20 min in 20% (v/v) sodium hypochlorite(NaClO) solution, and rinsed several times with sterile distilledwater. To test the phytotoxicity, 500 ml of the extract at differentconcentrations were added separately over Whatman filter papersNo. 1 in Petri dishes (9 cm Ø). Then filter papers were moistenedwith 2 or 2.5 ml (for lettuce or wheat, respectively) of distilledwater, and 20 seeds were evenly placed into the treated filter

paper. For the hexane extract the filter paper was only moistenedafter the evaporation of the solvent. Controls were prepared in asimilar way with pure solvent, allowing evaporation, or distilledwater only. Seeds were incubated at 25 � 2 8C in darkness.

In the tests with lettuce seeds, three concentrations (2.5, 5 and10%, w/v) of aqueous extract prepared with fresh and dried plantmaterial and nine concentrations of hexane extract (0.5, 1, 2.5, 5,10, 25, 50, 75 and 100%, v/v) were assayed. For wheat seeds, threeconcentrations (10, 20 and 30%, w/v) of aqueous extract preparedwith dried plant material and five concentrations of hexane extract(10, 25, 50, 75 and 100%, v/v) were assayed. These concentrationswere selected from preliminary assays. For each extract andconcentration 10 repetitions with 20 seeds were performed.

In another experiment seeds of lettuce and wheat were exposedto hexane extract at 25 or 100%, respectively, for 3, 6 and 24 h andthen transferred under sterile conditions onto filter paperscontaining only distilled water where they remained until theend of germination (5 days). The experimental conditions were likethose mentioned above, and they were repeated 5 times for eachspecies.

2.3. Phytotoxic effects of plumbagin

The quantification of plumbagin in the hexane extract (1.0 ml)was performed by the external standard methodology on anAgilent (Agilent Technologies, Little Falls, DE, USA) 6890 Series gaschromatograph interfaced to an Agilent 5973 N mass selectivedetector as described by Grevenstuk et al. (2008). The content ofplumbagin in each concentration of the hexane extract tested wascalculated. Then solutions of plumbagin (from Plumbago indica,Sigma, Steinheim, Germany) in n-hexane, in a range of concentra-tions (from 0.02 to 10.63 mM) close to those observed in theextracts were prepared. The effect of those solutions on lettuce(0.02, 0.04, 0.08, 0.17, 0.33, 0.66, 1.33, 2.66, 5.31, 7.97 and10.63 mM) and wheat seeds (0.66, 1.33, 2.66, 5.31, 7.97 and10.63 mM) was evaluated as described for the hexane extract. Ascontrol n-hexane only was used. For each plumbagin concentrationand species 5 repetitions with 20 seeds were tested.

2.4. Data collection and statistical analysis

Seed germination was assessed daily, and seedling (root andshoot) growth and biomass were measured after 5 days. Data weresubjected to one-way ANOVA followed by the comparison ofmultiple treatment levels with the control applying the Dunnetttest at 5% level. For the germination results the ANOVA wasperformed for data at 5 days only. The analysis was performedusing the SPSS statistical package for Windows (release 15.0; SPSSInc., Chicago, IL, USA). The values presented in the figures andtables are the means of the repeated experiments.

3. Results and discussion

Although the use of solvent extractions for an increased yield ofphytochemicals from plant tissue extracts is common in theliterature, certain extracted chemicals may not be released into anatural environmental under average field conditions. However,water-soluble allelochemicals such as phenolics have beenidentified in several species (Singh et al., 2003b; Batish et al.,2006a, 2007). Therefore, in this work, both hexane and aqueousextracts from D. lusitanicum were evaluated for their phytotoxicproperties under laboratorial conditions.

Aqueous extracts obtained from both fresh and dried plantmaterial had an inhibitory effect on germination of lettuce seeds,although the dried extract was more effective (Fig. 1A). The freshextract did significantly reduce the germination of lettuce seeds at

Fig. 1. Effect of aqueous extracts of D. lusitanicum on the germination of lettuce (A)

and wheat (B) seeds. Each value is the mean � S.D. of 10 repetitions with 20 seeds.

*Represents significant difference from control at P < 0.05 applying Dunnett test.

Table 1Effects of D. lusitanicum extracts on growth and biomass of lettuce seedlings.

Extract Concentration Root length Shoot leng

% mm I (%) mm

Control – 18.00 � 1.44 0 18.00 � 1.1

Hexane 0.5 16.60 � 1.26 7.78 15.47 � 1.4

1 12.93 � 1.35 28.15 14.60 � 1.4

2.5 9.71 � 0.95* 46.03 16.21 � 1.5

5 4.80 � 1.29* 73.33 9.00 � 1.4

10 0* 100 7.00 � 2.5

Aqueous fresh 2.5 16.00 � 3.28 11.11 18.00 � 2.1

5 13.40 � 3.90 25.56 12.27 � 1.5

10 0* 100 8.36 � 2.1

Aqueous dried 2.5 0* 100 3.75 � 0.5

5 0* 100 0*

10 0* 100 4.00 � 1.1

I – inhibition. Seedling (root and shoot) growth and biomass were measured 5 days af* Represents significant difference from control at P < 0.05 applying Dunnett test.

Table 2Effects of D. lusitanicum extracts on growth and biomass of wheat seedlings.

Extract Concentration Root length Shoot leng

% cm I (%) cm

Control – 5.73 � 0.54 0 3.31 � 0.1

Hexane 10 4.13 � 0.26* 27.94 2.19 � 0.1

25 2.09 � 0.27* 63.45 1.21 � 0.1

50 0.97 � 0.16* 83.06 0.47 � 0.0

Aqueous dried 10 2.47 � 0.49* 56.81 1.99 � 0.1

20 2.53 � 0.45* 55.88 1.65 � 0.2

30 0.86 � 0.13* 85.03 0.81 � 0.1

I – inhibition. Seedling (root and shoot) growth and biomass were measured 5 days af* Represents significant difference from control at P < 0.05 applying Dunnett test.

S. Goncalves et al. / Scientia Horticulturae 122 (2009) 96–10198

the concentrations of 5 and 10% (76 and 22% germination,respectively) (P < 0.05). The dried extract completely inhibitedseed germination at those concentrations and significantly reduced(P < 0.05) germination at 2.5% (w/v). Moreover, an inhibition ofradicle expansion was observed in the few seeds germinated incontact with all dried extracts and fresh at 10% (Table 1).Furthermore, some concentrations of aqueous extracts induced asignificant reduction in the root and shoot length, and biomass(Table 1). The germination of wheat seeds was less affected. In fact,fresh extract did not affect the germination in this species (data notshown) and dried extract only reduced germination significantly(P < 0.05) at the highest concentrations (20 and 30%) (Fig. 1B).However, some seeds presented morphological abnormalities. Thegrowth and biomass of wheat seedlings treated with dried extracts(at 10, 20 and 30%) were lower as compared with the control(P < 0.05) (Table 2). Besides inhibiting radicle and hypocotylselongation other morphological abnormalities occurred in thepresence of the extracts. Compared to the controls, the roots oftreated plants were thicker and brownish in color. These resultssupport the use of seedling biomass as a screening tool for toleranceor sensitivity of a species to the phytotoxic effects of another speciesas previously observed by several authors (Ben-Hamouda et al.,2001; Batish et al., 2006b). Batish et al. (2007) observed thatallelochemicals from Chenopodium murale induced a significantreduction in root and shoot length, dry matter accumulation,chlorophyll content and amount of protein and carbohydrates in twolegume crops.

Seed germination of lettuce was reduced when treated withhexane extract at the concentrations 5 and 10% (v/v) (59 and 17%germination, respectively), differences tested at 5 days compared

th Fresh root weight Fresh shoot weight

I (%) mg I (%) mg I (%)

2 0 1.82 � 0.12 0 8.33 � 0.60 0

6 14.07 1.73 � 0.14 4.76 6.96 � 0.48 16.41

1 18.89 1.83 � 0.19 0 7.26 � 0.50 12.81

1 9.92 1.61 � 0.12 11.70 6.47 � 0.54 22.30

1* 50.00 1.04 � 0.27 42.86 5.77 � 0.67* 30.74

2* 61.11 0* 100 5.43 � 1.69* 34.79

5 100 2.24 � 0.46 0 9.03 � 1.15 0

8* 31.85 2.34 � 0.37 0 8.03 � 0.64 3.6

6* 53.54 0* 100 6.03 � 0.62 27.6

5* 79.17 0* 100 6.63 � 1.27 20.44

100 0* 100 0* 100

5* 77.78 0* 100 2.90 � 0.36* 65.17

ter treatment. Each value is the mean � S.D. of 10 repetitions with 20 seeds.

th Fresh root weight Fresh shoot weight

I (%) mg I (%) mg I (%)

3 0 7.13 � 0.64 0 36.23 � 1.96 0

6* 34.00 4.27 � 0.48* 40.04 20.79 � 2.04* 42.63

9* 63.38 4.85 � 0.53* 31.90 14.21 � 2.27* 60.79

6* 85.81 2.79 � 0.24* 60.85 6.65 � 0.91* 81.65

5* 39.84 4.17 � 0.48* 41.53 21.77 � 2.08* 39.91

3* 50.30 5.33 � 0.97* 25.26 16.19 � 2.75* 55.31

3* 75.64 2.37 � 0.22* 66.72 9.21 � 1.37* 74.59

ter treatment. Each value is the mean � S.D. of 10 repetitions with 20 seeds.

Fig. 2. Effect of D. lusitanicum hexane extract on the germination of lettuce (A) and

wheat (B) seeds. Each value is the mean � S.D. of 10 repetitions with 20 seeds.

*Represents significant difference from control at P < 0.05 applying Dunnett test.

Fig. 3. Effect of different exposure periods to D. lusitanicum hexane extract, on the

germination of lettuce and wheat seeds. The extract was applied at 25 and 100% for

lettuce and wheat, respectively. Each value is the mean � S.D. of 5 repetitions with 20

seeds. *Represents significant difference from control at P < 0.05 applying Dunnett test.

Fig. 4. Comparison of germination rate (5 days after treatment) of lettuce (A) and

wheat (B) seeds after 5 days of treatment with D. lusitanicum hexane extract or

plumbagin. Each value is the mean � S.D. of 5 repetitions with 20 seeds.

S. Goncalves et al. / Scientia Horticulturae 122 (2009) 96–101 99

to control being significant (P < 0.05), and was completelyinhibited by concentrations equal or higher than 25% (Fig. 2A).In addition, at higher concentrations hexane extract significantlyreduced growth and biomass of the seedlings (Table 1).

The germination rate of wheat seeds was significantly reduced(P < 0.05) by hexane extract at concentrations equal or higher than25% (Fig. 2B). In addition, those concentrations reduced growthand biomass (Table 2) affecting both radicle and coleoptile. Asobserved in the assays with aqueous extracts, wheat seeds wereless sensitive to hexane extract. In wheat only the pure extract(100%) inhibited germination greatly. The differences betweenboth species were evident through a comparison of the germina-tion rates of seeds treated with the same concentration of extract(Fig. 2).

Based on the results shown in Fig. 2, the lowest hexane extractconcentration with inhibitory effect (25 and 100% for lettuce andwheat, respectively) was adopted to evaluate the effect ofdifferent exposure periods (3, 6 and 24 h). It was observed thatfor both species the inhibition of germination increased with alengthened period of exposure to the extract (Fig. 3). A 3 hexposure was sufficient to cause a significant reduction (P < 0.05)in germination rate, as compared with the control. In the case oflettuce, 24 h of exposure completely inhibited germination.These results show that the inhibition caused by D. lusitanicum

extract was rapid, apparently irreversible, and cumulative as itincreased with increasing time of exposure. Similar results wereobtained on durum wheat seeds exposed to coumarins (Abena-voli et al., 2006).

Plants contain thousands of natural products, but not all arephytotoxic. In the process of discovering phytotoxic agents in plantextracts or residues, the identification and quantification of thecontained compounds is an indispensable step. It was previouslydemonstrated that D. lusitanicum extracts contain large amounts ofplumbagin (Grevenstuk et al., 2008) that has allelochemicalpotential (Meyer et al., 2007). Thus, the content of plumbagin in

the hexane extracts used in this work was quantified andcorresponding concentrations of the commercial compoundtested. Fig. 4 shows the effect of plumbagin in comparison tohexane extract on lettuce and wheat germination. As was observedfor D. lusitanicum extracts, wheat was less sensitive to the effect ofplumbagin than lettuce. By analysing the obtained curves, somedifferences between the two species can be appointed (Fig. 4). Inthe case of lettuce, the curves obtained are similar and parallel, so itseems that plumbagin is the principal compound responsible for

Table 3Effects of plumbagin on growth and biomass of lettuce seedlings.

Plumbagin Root length Shoot length Fresh root weight Fresh shoot weight

mM mm I (%) mm I (%) mg I (%) mg I (%)

0 29.60 � 1.3 0 21.20 � 0.63 0 2.87 � 0.15 0 8.50 � 0.39 0

0.02 26.73 � 2.67 9.68 21.80 � 0.82 0 2.93 � 0.16 0 9.27 � 0.56 0

0.04 24.20 � 2.30 18.24 20.33 � 0.33 4.09 2.63 � 0.14b 8.14 7.64 � 0.22 10.12

0.08 29.53 � 1.66 0.23 21.40 � 0.94 0 2.88 � 0.23 0 8.54 � 0.47 0

0.17 27.13 � 2.20 8.33 20.27 � 0.51 4.40 2.61 � 0.15 9.07 8.11 � 0.31 4.55

0.33 24.13 � 1.77 18.47 21.60 � 0.69 0 2.40 � 0.21 16.28 8.33 � 0.42 2.04

0.66 19.93 � 1.37* 32.66 19.47 � 1.48 8.18 2.22 � 0.14* 22.56 7.73 � 0.37 9.10

1.33 9.60 � 1.70* 67.57 13.60 � 1.76* 35.85 1.79 � 0.23* 37.67 4.46 � 0.47* 47.53

I – inhibition. Seedling (root and shoot) growth and biomass were measured 5 days after treatment. Each value is the mean � S.D. of 10 repetitions with 20 seeds.* Represents significant difference from control at P < 0.05 applying Dunnett test.

Table 4Effects of plumbagin on growth and biomass of wheat seedlings.

Plumbagin Root length Shoot length Fresh root weight Fresh shoot weight

mM mm I (%) mm I (%) mg I (%) mg I (%)

0 6.10 � 0.45 0 2.66 � 0.26 0 9.10 � 1.36 0 27.10 � 3.24 0

0.66 5.66 � 0.28 7.29 2.37 � 0.19 10.88 6.12 � 0.47 32.72 20.54 � 3.49 24.19

1.33 5.17 � 0.28 15.30 2.69 � 0.16 0 8.29 � 0.72 8.91 26.33 � 2.42 2.83

2.66 4.16 � 0.23* 31.88 2.19 � 0.22 17.57 6.97 � 1.20 33.33 19.30 � 3.12 11.07

5.31 3.43 � 0.38* 43.72 1.77 � 0.21* 33.47 6.07 � 0.72 23.44 19.30 � 3.12 28.78

7.97 1.67 � 0.22* 72.68 0.81 � 0.15* 69.46 4.03 � 0.25* 55.68 8.02 � 1.07* 70.40

10.63 1.53 � 0.20* 74.86 0.42 � 0.08* 84.12 5.58 � 1.56* 38.71 5.28 � 0.99* 80.52

13.29 0.84 � 0.13* 86.16 0.29 � 0.03* 89.12 2.73 � 0.46* 69.96 3.57 � 0.36* 86.84

I – inhibition. Seedling (root and shoot) growth and biomass were measured 5 days after treatment. Each value is the mean � S.D. of 10 repetitions with 20 seeds.* Represents significant difference from control at P < 0.05 applying Dunnett test.

S. Goncalves et al. / Scientia Horticulturae 122 (2009) 96–101100

the allelopathic effects (Fig. 4A). In wheat seeds the differentresponses observed (Fig. 4B) may indicate that other unknownextract components may be responsible for inhibition. Plumbaginat various concentrations also affected root and shoot growth, andbiomass in both lettuce and wheat seedlings (Tables 3 and 4).

The most widely used bioassays for the action of phytotoxinsare seed germination and seedling growth studies. The delay andreduction of seed germination and/or inhibition of root and shootgrowth are usually the first symptoms of phytotoxic stress (Dayanet al., 2000). In the present work these bioassays demonstrated thephytotoxicity of aqueous and hexane D. lusitanicum extracts. Theresults obtained with aqueous extracts prove the presence ofwater-soluble compounds with phytotoxic properties in thisspecies. Further work for the identification and quantification ofthe compounds present in the aqueous extracts is required. Thedried extract was more effective than the fresh one, possibly due tothe methodologies used to prepare each extract. When preparingthe fresh extract, and in order to simulate the natural conditions inthe field, the plant material was neither dried nor powdered.Moreover, it is important to point out that during the dried extractpreparation the plant material was weighed after dehydrationmaking this extract more concentrated.

Seed size influences the concentration of compounds necessaryto produce an effect on seed germination, and consequentlysmaller seeds are generally more sensitive to phytotoxic com-pounds (Perez, 1990; Machado, 2007). This is probably the reasonwhy wheat seeds were less sensitive than lettuce to the D.

lusitanicum extracts. The different effects on germination observedin lettuce and wheat seeds correspond with other authors’observations, namely that there are differences among speciesand also among varieties (Perez-Leal et al., 2005). While wheatgermination was less affected by D. lusitanicum extracts, seedlings’growth was significantly affected; this finding suggests that theextracts could be used for post-emergence weed control. However,it is not clear if the reduction in root and shoot growth of wheat

seedlings is a direct effect of D. lusitanicum extract or rather areflection of delayed germination (Figs. 1B and 2B).

According to Meyer et al. (2007) if a plant extract inhibitsgermination at a low concentration, it is often an indication thatphytotoxic compounds might be present in the extract. In fact, theresults obtained, particularly for lettuce, show that D. lusitanicum

hexane extract reduced germination rate at lower concentrations(5% corresponding to 0.5 mg ml�1 of crude extract). The literaturepoints out that physical factors could interact synergistically withphytotoxic compounds producing more complex interactions. Inthis work these arguments can be discarded, since the environ-mental conditions were controlled in all the assays. However, toevaluate in detail the phytotoxic properties of D. lusitanicum extractsstudies involving the incorporation of plant residues into the soil andthe evaluation of the effects on soil nutrient dynamics, pH, electricalconductivity and nutrient availability must be conducted.

In conclusion, the results of the present work show that D.

lusitanicum extracts have potent phytotoxic properties. However, itis not clear if those properties are entirely related with plumbagin.Thus, further studies are needed to clearly specify the compoundsresponsible for that activity. Moreover, supplementary assaysmost be conducted to ensure if the results obtained with the twomodel species (lettuce and wheat) are reproducible for the controlof weed species in field conditions.

Acknowledgement

Sandra Goncalves acknowledges a grant from Fundacao para aCiencia e a Tecnologia (Grant SFRH/BPD/31534/2006).

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