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Industrial Crops and Products 32 (2010) 674–677

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l homepage: www.e lsev ier .com/ locate / indcrop

hort communication

creening for natural inhibitors of germination and seedling growth in nativelants from Central Argentina

ara M. Palaciosa,∗,1, Soledad del Corrala, María C. Carpinellaa,1, Gustavo Ruizb

Fine Chemical and Natural Products Laboratory, School of Chemistry, Catholic University of Córdoba, Cno a Alta Gracia Km 10, Córdoba, ArgentinaHerbarium Marcelino Sayago, School of Agricultural Science, Catholic University of Córdoba, Córdoba, Argentina

r t i c l e i n f o

rticle history:eceived 10 December 2009eceived in revised form 4 May 2010ccepted 10 May 2010

eywords:ative plantsermination inhibitionaccharis salicifoliaphryosporus charua

a b s t r a c t

Ethanol extracts obtained from aerial parts of 71 native plants from Central Argentina were tested fortheir herbicidal activity in germination assays on Avena sativa and Raphanus sativus. Extracts derivedfrom Angelphytum aspilioides, Baccharis salicifolia, Cortaderia rudiuscula, Eupatorium hookerianum andMandevilla laxa, showed 100% inhibition of the germination of A. sativa at 10 mg/ml. In the case of R. sativus,extracts from Achyrocline tomentosa, Angelphytum aspilioides, B. salicifolia, Melissa officinalis, Minthostachysverticillata, Ophryosporus charua and Podranea ricasoliana, applied at 10 mg/ml, showed 100% germinationinhibition. For each extract, the mean effective concentrations that inhibit germination (ECg50), root(ECr50) and shoot (ECs50) growth were determined. According to these values and the extract yield,an index was calculated in order to establish a ranking of the most active plants. For inhibition of A.sativa, the ranking was B. salicifolia > A. aspilioides > C. rudiuscula > M. laxa > E. hookerianum. The ECg50,

vena sativaaphanus sativus

ECr50 and ECs50 of B. salicifolia against A. sativa were 0.36, 0.88 and 0.91 mg/ml, respectively, showingmore activity than that of 2,4-D as a germination inhibitor and 44 and 1.1 times less active than 2,4-Das a root and shoot inhibitor, respectively. The ranking for the inhibition of R. sativus was O. charua > A.aspilioides > P. ricasoliana > B. salicifolia > A. tomentosa > M. officinalis > M. verticillata. The O. charua extractpresented ECg50, ECr50 and ECs50 of 1.04, 1.04 and 1.49 mg/ml, respectively. According to the obtainedresults, the extracts of B. salicifolia and of A. aspilioides were the only ones capable of inhibiting the

of bo

germination and growth

. Introduction

Increasing concern about the risks to the environment anduman health deriving from the use of synthetic pesticides has

ed to the current major trend in pest management involvingore environmentally friendly agricultural techniques and the

earch for less hazardous chemicals or biologically based prod-cts (Isman, 2006; Hong et al., 2003). Today approximately 0.7%f the agricultural area in Latin America is managed organically,hich represents 6.4 million ha under organic cultivation, of which

.7 million ha are in Argentina alone (Willer and Klicher, 2009).ost organic production in Argentina is of cereals and meat and is

estined for export.Farmland used to grow organic crops must not have been treated

ith synthetic pesticides and herbicides for at least three yearsrior to the organic harvest, and weed control is thus based pri-

∗ Corresponding author. Tel.: +54 351 4938000x611; fax: +54 351 4938000x611.E-mail address: sarapalacios@ucc.edu.ar (S.M. Palacios).

1 These authors are members of the National Research Council of ArgentinaCONICET).

926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2010.05.004

th test species.© 2010 Elsevier B.V. All rights reserved.

marily on the use of agricultural techniques (Turner et al., 2007).A more rational way of controlling them may result from identi-fying natural substances that can control the emergence of weeds(Lin et al., 2006; Xuan et al., 2005). Products of plant origin arefrequently based on secondary metabolites, which, in many cases,have a defensive purpose in nature, especially in plants which havefew options other than chemical ones for avoiding and combat-ing pests or competitive plant species. Thus, evaluation of plantsfor pesticide activities is a highly viable strategy for pesticide dis-covery (Cantrell et al., 2007; Carpinella et al., 1999, 2003, 2005;Palacios et al., 2007; Diaz Napal et al., 2009). In this report, we exam-ine the germination inhibitory properties of 71 plants from CentralArgentina against Avena sativa and Raphanus sativus, as part of aprogram for the selection of highly active plant species for herbicidedevelopment.

2. Materials and methods

2.1. Plant material

Plants were collected in the hills of Córdoba Province, Argentina,from November 2006 to December 2008. Voucher specimens have

S.M. Palacios et al. / Industrial Crops and Products 32 (2010) 674–677 675

Table 1Germination inhibitory effects of extracts from native plants from Central Argentina.

Plant species Family Yield (%)b IG (%)a

A. sativa R. sativus

Acacia aroma Gillies ex Hook. & Arn. Fabaceae 29.6 12.36 3.37Achyrocline satureioides (Lam.) DC. Asteraceae 4.5 62 1.2Achyrocline tomentosa Rusby Asteraceae 4.3 4.28 100Aloysia citriodora Palau Verbenaceae – 81 9Aloysia gratissima (Gill. & Hook.) Tronc. Verbenaceae 1.7 17.64 7.78Ambrosia elatior L. Asteraceae 2.2 44.18 59.55Amphilophium cynanchoides (DC.) L.G. Lohmann Bignoniaceae 7.4 13.79 60.5Anemia tomentosa (Savigny) Sw. Schizaceae 1.9 63.64 2.25Angelphytum aspilioides (Griseb.) H. Rob. Asteraceae 6.8 100 100Argemone subfusiformis G. B. Ownbey Papaveraceae 14.4 13.79 0Artemisia verlotiorum Lamotte Asteraceae 3.5 78.16 42.05Baccharis artemisioides Hook. & Arn. Asteraceae 2.8 81 29Baccharis coridifolia DC. Asteraceae 2.2 81 2Baccharis flabellata Hook. & Arn. Asteraceae 7.5 8.04 12.36Baccharis salicifolia (Ruiz & Pav.) Pers. Asteraceae 10.2 100 100Bidens pilosa L. Asteraceae 4.8 5.8 25.58Capparis atamisquea Kuntze Capparaceae 3.15 47.98 42.7Condalia microphylla Cav. Rhamnaceae 1.6 40.85 5.62Cortaderia rudiuscula Stapf Poaceae 3.5 100 14Cotoneaster glaucophylla Franch. Rosaceae 9.1 42.8 1.2Croton lachnostachyus Baill. Euphorbiaceae 3.5 22 0Cuphea glutinosa Cham. & Schltdl. Lythraceae – 1.15 21.35Cynoglosum amabile Stapf & J.R. Drumm. Boraginaceae 2.7 15.56 20Dipsacus fullonum L. Dipsacaceae 5.2 31.03 79.02Dolichandra unguis-cati (L.) L.G. Lohmann Bignoniaceae 9.9 14.94 66.7Dysphania ambrosioides (L.) Mosyakin & Clemants Chenopodiaceae 3.0 76.2 53Elaphoglosum lorentzii (Hieron.) H. Christ Lomariopsidaceae 10.5 87.5 4.71Eryngium horridum Malme Apiaceae 4.7 57.35 39.08Eupatorium buniifolium Hook. & Arn. Asteraceae 5.2 40 23.3Eupatorium hookerianum Griseb. Asteraceae 3.4 100 1.11Eupatorium viscidum Hook. & Arn. Asteraceae 2.9 9.19 74.16Flourensia campestris Griseb. Asteraceae 11.8 5.8 11.63Gomphrena pulchella Mart. Amaranthaceae 3.2 62 5.56Grindelia pulchella Dunal Asteraceae 6.7 25.56 6.67Jarava ichu Ruiz & Pav. Poaceae 1.5 82.93 17Jodina rhombifolia (Hook. & Arn.) Reissek Santalaceae 5.0 77.7 0Lepechinia meyenii (Walp.) Epling Lamiaceae 3.8 37.16 17.65Ligaria cuneifolia (Ruiz & Pav.) Tiegh. Loranthaceae 4.4 1.14 1.14Lippia turbinata Griseb. Verbenaceae 4.8 13.79 29.21Lithrea molleoides (Vell.) Engl. Anacardiaceae 7.2 33 30Mandevilla laxa (Ruiz & Pav.) Woodson Apocynaceae 2.4 100 58.8Marrubium vulgare L. Lamiaceae 3.2 56.97 11.9Melinis repens (Willd.) Zizka Poaceae 1.2 52.22 5.95Melissa officinalis L. Lamiaceae 4.3 87.5 100Microliabum candidum (Griseb.) H. Rob. Asteraceae 3.7 56.52 69.77Minthostachys verticillata (Griseb.) Epling Lamiaceae 3.6 81.25 100Morrenia brachystephana Griseb. Asclepiadaceae – 5.74 7.86Ophryosporus charua (Griseb.) Hieron. Asteraceae 13.6 20.83 100Pavonia aurigloba Krapov. & Cristóbal Malvaceae 2.3 45.71 1.11Podranea ricasoliana (Tanfani) Sprague Bignoniaceae 4.5 11.49 100Porlieria microphylla (Baill.) Descole, OıDonell & Lourteig Zygophyllaceae 1.1 85.7 38.89Pterocaulon alopecuroides (Lam.) DC. Asteraceae 5.5 76.39 33.71Pyrostegia venusta (Ker Gawl.) Miers Bignonaceae 0.9 14.28 3.41Ruprechtia apetala Wedd. Polygonaceae 1.9 80 11.1Schizachyrium condensatum (HBK) Nees Poaceae 1.9 46.51 7Senecio madagascariensis Poir. Asteraceae 4.9 31.76 4.71Senecio vira-vira Hieron. Asteraceae 3.9 28.17 44.94Senna aphylla (Cav.) H.S. Irwin & Barneby Fabaceae 5.1 42.5 17.04Sida rhombifolia L. Malvaceae 2.2 21.34 30.68Solanum argentinum Bitter & Lillo Solanaceae 5.4 61.97 19.1Solanum palinacanthum Dunal Solanaceae 3.7 5.55 4.44Solanum sisymbriifolium Lam. Solanaceae 2.5 1.41 46.07Sphaeralcea cordobensis Krapov. Malvaceae 3.1 73.35 31.39Spharalcea cordobensis Krapov. (mutant) Malvaceae 1.9 32.94 22.35Tagetes minuta L. Asteraceae 2.5 16.85 0Tripodanthus flagellaris (Cham. & Schltdl.) Tiegh. Loranthaceae – – 3.41Vernonia mollissima Hook. & Arn. Asteraceae 2.0 32.39 16.85Vernonia nudiflora Less. Asteraceae 7.2 33.33 7.78Viguiera tucumanensis (Hook. & Arn.) Griseb. Asteraceae 13.6 64.79 39.32Wedelia glauca (Ortega) Hicken Asteraceae 6.4 23.6 25.58Zanthoxylum coco Hook. f. & Arn. Rutaceae 1.7 10 88.24

a Data represent the mean of three replicates.b Yield of extract per 100 g of plant material.

676 S.M. Palacios et al. / Industrial Crops and Products 32 (2010) 674–677

Table 2Most active plants against Avena sativa.

Specie ECg50 ECr50 ECs50 Rank

Values in mg/ml(lower, upper)

Angelphytum aspilioides 0.92 (0.74–1.17) 1.51 (1.31–1.74) 1.88 (1.58–2.23) 2Baccharis salicifolia 0.36 (0.19–0.65) 0.88 (0.76–1.02) 0.91 (0.74–1.13) 1Cortaderia rudiuscula 0.90 (0.76–1.07) 1.85 (1.46–2.33) 1.27 (0.97–1.66) 3Eupatorium hookerianum 2.44 (1.89–3.16) 5.92 (4.09–8.58) 1.97 (1.32–2.96) 5Mandevilla laxa 1.03 (0.82–1.29) 3.36 (2.33–4.87) 1.93 (0.93–4.05) 42,4-D 1.22 (0.97–1.55) 0.02 (0.01–0.03) 0.82 (0.53–1.29)

Table 3Most active plants against Raphanus sativus.

Specie ECg50 ECr50 ECs50 Rank

Values in mg/ml (lower, upper)

Angelphytum aspilioides 2.02 (1.75–2.33) 0.98 (0.77–1.24) 1.16 (0.91–1.47) 2Achyrocline tomentosa 1.71 (1.33–2.21) 1.81 (1.46–2.23) 2.24 (1.81–2.78) 5Baccharis salicifolia 2.32 (1.99–2.70) 1.39 (1.16–1.65) 1.58 (1.31–1.90) 4Melissa officinalis 1.47 (1.19–1.82) 3.49 (2.97–4.10) 3.44 (2.73–4.32) 6

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Minthostachys verticillata 2.64 (2.10–3.32) 3.97 (3.Ophryosporus charua 1.04 (0.82–1.30) 1.04 (0.Podranea ricasoliana 1.15 (0.83–1.59) 1.14 (0.2,4-D 0.33 (0.23–0.46) 3 × 10−

een deposited in the “Marcelino Sayago” Herbarium of the Schoolf Agricultural Science, Catholic University of Córdoba and wereuthenticated by the botanist, Gustavo Ruiz. Plants were selectedccording to their availability, accessibility and especially to theack of scientific information about their activity and/or chemicalattern. The vegetable material was air-dried at room tempera-ure, crushed and extracted by 48 h maceration with ethanol. Yieldsf each viscous extract, obtained after solvent removal, expresseds percentage weight of air-dried plant material, are shown inable 1.

.2. Seeds

Common oat Avena sativa and wild radish Raphanus sativus seedssed for herbicide testing, were purchased from Semillería Florensa

n Córdoba.

.3. Germination bioassay

Thirty seeds of A. sativa or R. sativus were placed in a 9-cmetri dish lined with filter paper, treated with 2 ml of ethanolxtract solution at 10 mg/ml and 4 ml of distilled water after ethanolas evaporated. Controls received ethanol (2 ml), evaporated andml of distilled water. Each treatment was replicated three times.he dishes were placed in a growth chamber (25 ± 1 ◦C, 70–75%elative humidity, with a photoperiod of 16:8 light–dark cycle).fter 2 and 7 days for R. sativus and A. sativa, respectively, ger-ination was assessed and the germination inhibition index was

alculated as IG% = [1 − (T/C)] × 100, where T and C are the numberf seeds germinated in the treatment and the control, respec-ively. 2,4-D (Atanor S.A., Argentina) was used as a positive control.hose extracts with highest activity and 2,4-D were evaluated at.0001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 2 mg/ml and the num-er of germinated seeds, and seedling root and shoot length wereecorded. Effective doses capable of inhibiting 50% of germination,

oot growth or shoot growth inhibition were calculated as ECg50,Cr50 and ECs50, respectively. The results were analyzed by t-testp < 0.05) and ECg50, ECr50 and ECs50 values were calculated by Pro-it analysis based on percent of germination inhibition or root orhoot growth inhibition.

5) 2.64 (2.10–3.32) 78) 1.49 (1.23–1.81) 12) 1.52 (1.17–1.97) 3

10−6–2 × 10−4) 3 × 10−3 (1 × 10−3–4 × 10−3)

A rank for each of the most active plants was calculated consid-ering an index (In) calculated for the most active plant extracts, bythe following equation for each seed tested:

In = y−1n .ECg50n(germination).ECr50n(root).ECs50n(shoot)

where In is the rank of the species n, yn is the yield extract of plantn relative to 100 gr of plant material, and the ECg50n, ECr50n andECs50n are the concentration of extract of plant n that inhibits 50% ofgermination, root and shoot growth, respectively. The plant extractwith lowest In is the most active one and consequently received thelowest rank number (Rank = 1) with higher In receiving consecutiverank numbers (Rank > 1).

3. Results and discussion

The germination inhibitory properties of each extract obtainedfrom the 71 plant species were evaluated in a germination bioassayagainst two species, Avena sativa and Raphanus sativus, represent-ing mono- and dicotyledonous plants, respectively. The results ofthis screening are presented in Table 1. Most of the species werenative to our environment although some of them (Cotoneasterglaucophylla, Dipsacus fullonum, Marrubium vulgare, Melissa offic-inalis and Podranea ricasoliana) are actually naturalized. Manyplant extracts (19/70 = 27%) showed germination inhibition of A.sativa with an IG of 70% or higher at 10 mg/ml, while only 14%(10/71) of the tested extracts were effective against R. sativuswith an IG > 70% at the same concentration. Fourteen extracts pos-sessed an IG of between 80 and 100% against A. sativa, and 8against R. sativus. Only five extracts (Angelphytum aspilioides, Bac-charis salicifolia, Cortaderia rudiuscula, Eupatorium hookerianum andMandevilla laxa) showed complete inhibition of the germinationof A. sativa at 10 mg/ml, representing 7% of the plants studied.In the case of R. sativus, seven plant extracts (10%) (Achyroclinetomentosa, Angelphytum aspilioides, B. salicifolia, Melissa officinalis,Minthostachys verticillata, Ophryosporus charua and Podranea ric-

asoliana) showed an IG of 100%. These highly effective extractswere again assessed on A. sativa and R. sativus, in order todetermine their mean effective concentration that inhibits germi-nation (ECg50) and root (ECr50) and shoot (ECs50) growth. Withthese values and the extract yield, an index was calculated for

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S.M. Palacios et al. / Industrial C

ach of the most active plants, to establish a ranking betweenhem.

For the inhibition of A. sativa, the ranking in order of importanceas B. salicifolia > A. aspilioides > C. rudiuscula > M. laxa > E. hookeri-

num (Table 2). The ECg50, ECr50 and ECs50, of B. salicifolia againsthe test monocotyledonous species were 0.36, 0.88 and 0.91 mg/ml,espectively (Table 2) while for A. aspilioides extract they were 0.92,.51 and 1.88 mg/ml, respectively (Table 2). The extract derivedrom B. salicifolia was 3.4 times more active than 2,4-D as a germi-ation inhibitor and 44 and 1.1 times less active than 2,4-D, as aoot and shoot inhibitor, respectively, against A. sativa.

The ranking for the most active plants against R. sativus was. charua > A. aspilioides > P. ricasoliana > B. salicifolia > A. tomen-

osa > M. officinalis > M. verticillata (Table 3). O. charua extractresented ECg50, ECr50 and ECs50 of 1.04, 1.04 and 1.49 mg/ml,espectively and the values for A. aspilioides extract against R.ativus were 2.02, 0.98 and 1.16 mg/ml (Table 3), respectively. O.harua extract showed lower activity (3.1 times) than 2,4-D forermination inhibition of R. sativus, and 34,000 and 496 times lessctive than the reference herbicide for root and shoot inhibition,espectively.

From these analyses, it is evident that the most promisinglants are B. salicifolia, O. charua and A. aspilioides due to theiremarkable potency, their regularity in inhibiting both germina-ion and seedling growth, and also to the high extract yield whichs an indicator of the efficiency per biomass unit. The extractsf B. salicifolia and of A. aspilioides were the only ones capablef inhibiting the germination and growth of both test species,nderscoring their potential as broad-spectrum natural herbi-ides.

B. salicifolia is a resinous medicinal plant (Scarpa, 2004) thatrows as a shrub up to 2 m, is very resistant to extreme climate con-itions and is widely distributed in Central Argentina. A. aspilioides

s a suffrutex of small dimensions (40 cm in height), with attrac-ive yellow inflorescence. No medicinal uses have been describedor this plant. Finally, O. charua, less frequent in our region, is a tallhrub, up to 1.2 m high, with a small white capitulum and medic-nal uses described as an antisyphilitic and astringent (Barboza etl., 2006).

The ratio of activity between B. salicifolia or O. charua extract

nd 2,4-D, suggests that the extract of these plants could yield purective compounds with a potency equal or close to that of the ref-rence herbicide, and enable the development of new substanceshat could be effective for weed control, as germination and shootrowth inhibitors.

nd Products 32 (2010) 674–677 677

In conclusion, the plants with a good ranking, especially B. sali-cifolia, O. charua and A. aspilioides, may be considered as potentialweed control materials in organic agriculture, due to their potenciesand yields. Also it could be profitable to study the allelochemicalsproduced by these plants and their mechanism of action.

Acknowledgements

This work was supported by the Catholic University of Córdoba,FONCYT, Grant Numbers: BID 1728 OC/AR, PICT 33593 and PICTOCRUP 6-31396. SDC gratefully acknowledges receipt of a fellow-ship from FONCYT. We thank Joss Heywood for revising the Englishlanguage.

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