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Weed Biology and Management 11, 1828 (2011)

RESEARCH PAPER

Allelopathic potential of Acacia melanoxylon on the germination and root growth of native species
wbm_401 18..28

MUHAMMAD I. HUSSAIN*, LUIS GONZLEZ and MANUEL J. REIGOSA Department of Plant Biology and Soil Science, University of Vigo, Lagoas-Marcosende Campus, Vigo, Spain
Water extracts that were obtained from the owers and phyllodes of Acacia melanoxylon were used to determine their allelopathic potential in relation to the germination and seedling growth of the native species, cocksfoot (Dactylis glomerata), perennial ryegrass (Lolium perenne), and common sorrel (Rumex acetosa), as well as a general biotest specie, lettuce (Lactuca sativa), in laboratory bioassays.The owers and phyllodes of A. melanoxylon were soaked separately in distilled water in a ratio of 1:1 (w/v) for 24 h in order to prepare the aqueous extracts. Distilled water was used as the control.The seeds of the target species were germinated in Petri dishes and counted daily for up to 7 days. The A. melanoxylon ower extract (100%, 75%, and 50%) decreased the seed germination of D. glomerata, R. acetosa, L. perenne, and L. sativa.The ower extract caused the most reduction in the germination index and germination speed in D. glomerata, L. perenne, and L. sativa. The mean LC50 value of the A. melanoxylon ower and phyllode extracts in relation to the germination inhibition of L. perenne was 43% and 41%, respectively, 40% and 38%, respectively, in R. acetosa, and 53% and 41%, respectively, in L. sativa. All four concentrations of the ower extract proved to be more phytotoxic than the phyllode extract, reducing the root length of all four species, while the phyllode extract decreased the root length of L. perenne and R. acetosa at the 100% concentration. The L. perenne and D. glomerata grass seeds were more sensitive regarding germination, as compared to L. sativa and R. acetosa.The ower aqueous extract of A. melanoxylon was more phytotoxic, as compared to the phyllode aqueous extract, even at the lowest concentration (25%). Keywords: Acacia melanoxylon, allelopathy, Dactylis glomerata, germination, Lolium perenne, Rumex acetosa.

In the 19th century, some Acacia species were introduced as an ornamental plant in southern Europe and have naturalized and become invasive in the Mediterranean and Atlantic regions, from Portugal to Italy (Sheppard et al. 2006). Acacia species affect crop growth by competing for various environmental resources as their litter interferes with the establishment and growth of the adjoining crop plants (Kohli et al. 2006), as well as releasing numerous chemical substances, including phenolic

Communicated by B.S. Ismail. *Correspondence to: M.I. Hussain, Department of Plant Biology and Soil Science, University of Vigo, Lagoas-Marcosende Campus, 36310 Vigo, Espaa. Email: mih76@uvigo.es Received 21 January 2010; accepted 29 October 2010

compounds, in the litter (Seigler 2003). Carballeira and Reigosa (1999) have demonstrated that the leachate from Acacia dealbata showed strong inhibitory effects on the germination and growth of Lactuca sativa during the owering of A. dealbata. Allelopathy was implicated by Duhan and Lakshminarayana (1995), Raqul-Hoque et al. (2003), El-Khawas and Shehata (2005), Al-Wakeel et al. (2007), and Lorenzo et al. (2008) when they observed that the water extracts of different Acacia species inhibited the germination, root and shoot length, and dry weight of different crops and weeds. However, allelopathic plants might have inhibitory, stimulatory, or no effect on the germination and growth of other plants (Reigosa et al. 1999). Blackwood (Acacia melanoxylon R. Br.) is an introduced species that has its origin in the temperate forests of the south-eastern Australian mainland and Tasmania. It doi:10.1111/j.1445-6664.2011.00401.x

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Allelopathic effect of Acacia melanoxylon is a versatile and highly adaptive tree specie that has spread all over the world (Knapic et al. 2006). It covers a considerable area in the coastal zone of the northwestern Iberian Peninsula, both in monocultures and in mixed stands, with Eucalyptus globulus, characterized by vigorous tree or root sprouts and seed germination that is stimulated by re. On invasion, it establishes quickly in the alien environment, thereby resulting in changes in the structure and dynamics of the native ecosystem. Although the species is found mostly in wastelands, it also grows well in cultivated elds, pastures, and along roadsides. This dominance might be related to chemical interference or allelopathy, which gives it an additional advantage over native plants.The forest plantations of A. melanoxylon started in the north-western Iberian Peninsula at the beginning of the 20th century (Areses 1953) and the plant is presently considered as invasive (Xunta de Galicia 2007). Its leaves have allelopathic effects on the germination and seedling growth of L. sativa (Souto et al. 2001).Allelochemicals are found in different parts of a plant body (roots, stems, owers, and leaves) and probably enter the soil from foliar leaching in rain water, exudation from roots into the soil water, or after leaf fall and the subsequent incorporation into the soil (Inderjit & Duke 2003). Different parts of the same plant also vary in their allelopathic effect on the germination and growth of crops. However, there is no information about the allelopathic effect of the water extract of the owers and phyllodes of A. melanoxylon on the native species that are present near the Acacia stands. Seed germination and seedling growth bioassays are widely used for the study of the depletion of germination and the stunting of growth of a susceptible plant due to the release of allelochemicals from a donor plant (Lottina-Hennsen et al. 2006). The aim of the present investigation was to assess the allelopathic potential of the ower and phyllode extracts of A. melanoxylon on the germination, emergence, root elongation, and seedling growth of three native species. This can provide basic information about the management of A. melanoxylon. MATERIALS AND METHODS Target species Two grasses, Dactylis glomerata L. cv. Amba (Poaceae) and Lolium perenne L. cv. Belida (Poaceae), and Rumex acetosa L. cv. Belleville (Polygonaceae) were used as the representative species because they naturally grow in the Galician forest ecosystem (north-western Spain) and under the canopy stands of A. melanoxylon. Lactuca sativa L. cv. Great Lakes California (Asteraceae) was selected as a general biotest specie because it is frequently used as a

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model specie in allelopathic bioassays (Macas et al. 2000).The use of L. sativa could demonstrate the possible mechanisms by which A. melanoxylon competes with other species, while the results concerning the three native species have more ecological signicance. The seeds of the test species were purchased commercially from Semillas Fito (Barcelona, Spain). Water extraction of Acacia melanoxylon The fresh shoots of A. melanoxylon were collected from a natural population in the surrounding area of Lagoas Marcosende Campus, University of Vigo, Spain, during the owering period. The owers and phyllodes (expanded petioles that form simple lamina; Atkin et al. 1998) were separated from the branches and each was soaked separately in distilled water in the ratio of 1:1 (w/v). Similar extraction techniques were used by Molina et al. (1991) in their studies of Eucalyptus spp. and Lorenzo et al. (2008) in their studies of the allelopathic effects of Acacia dealbata L.The extracts were prepared at room temperature and left in the laboratory for 24 h.The extracts were collected, ltered through lter paper, and described as 100%. Distilled water was added to the solutions to make different dilutions (75, 50, and 25%). Germination bioassays Glass Petri dishes (9 cm diameter) were used and contained blotting paper (3MM; Whatman, Maidstone, England).Twenty-ve seeds of each species were placed in the Petri dishes to which 3 mL of solution were added at the start, while the control received 3 mL of distilled water. An additional 1 mL of each solution was added every 48 h thereafter.Three replicates of each treatment were incubated in a germination chamber with the following germination conditions, each with a relative humidity of 80% (Hussain et al. 2008): 1 L. perenne: 25/15C day/night temperatures and 12/12 h light/darkness. 2 L. sativa: 18/8C day/night temperatures and 12/12 h light/darkness. 3 R. acetosa: 28/20C day/night temperatures and 9/15 h light/darkness. 4 D. glomerata: 25/20C day/night temperatures and 14/10 h light/darkness. The light was provided by cool, white uorescent tubes with an irradiance of 35 mmol m-2 s-1.The germination was assessed after every 24 h by counting the number of germinated seeds for up to 7 days. Germination was considered as the rupture of the seed coat and radicle emergence of 1 mm.

2011 The Authors Journal compilation 2011 Weed Science Society of Japan

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M.I. Hussain et al.


0.63 0.00* 0.00* 0.00* 0.00* 0.80 0.00* 0.00* 0.00* 0.00* 3.09 0.00 0.00 0.00 0.00 2.33 0.00 0.00 0.00 0.00 Table 1. Effects of the Acacia melanoxylon ower and phyllode water extracts on the germination of Dactylis glomerata at each exposure time during 1 week 0.63 0.88 2.02 0.57 0.88 3.09 0.00 2.67 3.33 1.33
* Signicant differences, compared to the control, at P < 0.05, according to the Dunnett test. The results represent the mean (SE) of three replicates.

Seedling growth bioassays The Petri dishes were placed in a cold chamber at 4C after 1 week in order to stop seedling growth and the radicle length was measured with a measuring tape (Reigosa & Pazos-Malvido 2007). Statistical analysis The germination rate index (GT) was determined, as described by Jderlund et al. (1996), and the speed of germination (S) was calculated, as proposed by Ahmed and Wardle (1994). The collected data were statistically analyzed by using a one-way ANOVA (Sokal & Rohlf 1995) and the Dunnett test was used to determine the differences between the treatment means at the 5% probability level. The mean LC50 value (the dose for 50% inhibition of seedling growth) was calculated by using a probit analysis, as described by Finney (1971). A logistic equation was tted to the germination data as a function of the logarithm of the concentrations of the A. melanoxylon ower and phyllode extracts by using SPSS for Windows (v. 15.0; SPSS, Chicago, IL, USA):

Day 7

Day 6

0.89 0.00* 0.00* 0.00* 0.00* 0.65 0.00* 0.00* 0.00* 0.00* 0.90 0.00* 0.00* 0.00* 0.00* 4.92 0.00 0.00 0.00 0.00 0.33 0.00 0.00 0.00 0.00 0.42 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.75 0.00 0.00 0.00 0.00 3.50 0.00 0.00 0.00 0.00

Day 5

Exposure time (days after sowing)

Day 4

Y = a + bX ,
where Y = the probit value, a = the intercept, b = the slope of the line, and X = the log10 concentration. The value of X was obtained in order to calculate the LC50 values of the concentrations of the A. melanoxylon ower and phyllode extracts. RESULTS Effect of the Acacia melanoxylon extracts on germination at each exposure time The laboratory test showed that the A. melanoxylon ower aqueous extract (100, 75, and 50%) signicantly inhibited the germination of D. glomerata, R. acetosa, L. perenne, and L. sativa (Tables 14). The A. melanoxylon phyllode extract (100% and 75%) completely inhibited the germination process of R. acetosa during the second, third, and sixth day, of L. perenne during the fourth, fth, and sixth day, and of L. sativa during the rst and second day. However, there was a tendency for a stimulation of the germination of R. acetosa and L. perenne by different concentrations of the phyllode extract. Effect of the Acacia melanoxylon extracts on the germination indices As shown in Table 5, the inhibitory effect of the water extracts on the GT and S depended on the extract 2011 The Authors Journal compilation 2011 Weed Science Society of Japan

Day 3

Day 2

Day 1

Flower aqueous extract Control 100% 75% 50% 25% Phyllode aqueous extract Control 100% 75% 50% 25%

Acacia melanoxylon

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00

0.42 0.00 0.00 0.00 0.00

0.33 0.00 0.00 0.00 0.00

4.92 2.00 4.00 3.33 3.67

0.90 1.15 1.15 0.41 0.88

4.75 4.00 4.00 2.00 4.33

0.65 2.00 1.15 2.00 1.20

3.50 3.00 2.67 2.22 2.13

0.89 0.57 0.66 0.83 0.76

0.80 0.00 1.76 2.02 0.66

2.33 1.67 3.33 2.00 1.67

Table 2. Effects of the Acacia melanoxylon ower and phyllode water extracts on the germination of Rumex acetosa at each exposure time during 1 week Exposure time (days after sowing) Day 1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.08 0.00 0.00 0.00 0.00 0.417 0.000* 0.000* 0.000* 0.000* 2.25 3.33 5.00 5.00 5.33 0.55 0.88 0.57* 0.15* 0.88* 2.17 2.00 3.67 1.33 2.67 0.405 1.000 0.330 0.330 0.330 1.25 1.67 2.33 2.00 1.67 0.37 0.66 0.33 1.15 0.88 0.27 1.67 2.33 1.00 1.67 0.19 0.88* 1.20* 1.00* 0.33* 0.00 0.00 0.00 0.00 0.00 2.08 0.00 0.00 0.00 0.00 0.417 0.000* 0.000* 0.000* 0.000* 2.25 1.67 1.33 1.33 2.67 0.55 0.33 0.33 0.34 1.66 2.17 1.67 4.33 2.33 3.33 0.405 0.330 0.880* 0.330 0.880 1.25 2.00 2.00 2.67 2.33 0.37 0.57 1.52 0.33 0.88 0.27 1.33 0.67 0.67 0.33 0.19 0.33 0.33 0.33 0.33 0.00 0.33 0.00 0.67 0.00 0.00 0.33 0.67 0.00 0.33 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 0.00 0.33 0.00 0.45 0.00 0.00 0.33 0.66 0.00 0.33

Acacia melanoxylon

Flower aqueous extract Control 100% 75% 50% 25% Phyllode aqueous extract Control 100% 75% 50% 25%

* Signicant differences, compared to the control, at P < 0.05, according to the Dunnett test. The results represent the mean (SE) of three replicates.

Table 3. Effects of the Acacia melanoxylon ower and phyllode water extracts on the germination of Lolium perenne at each exposure time during 1 week Exposure time (days after sowing) Day 1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.42 2.00 2.33 5.00 5.67 1.600 1.520 1.450 2.640 4.700 5.50 3.33 4.00 5.33 14.00 0.00 0.00 0.00 0.00 0.00 5.42 0.00 0.00 0.33 1.00 1.600 0.000* 0.000* 0.333* 0.570 5.50 0.00 0.00 0.00 0.67 1.04 0.00* 0.00* 0.00* 0.33* 1.04 3.33 1.73 3.38 3.51* Day 2 Day 3 Day 4 3.33 0.00 0.00 0.00 2.67 3.33 0.00 2.00 3.33 4.33 0.87 0.00* 0.00* 0.00* 1.45 0.87 0.00* 0.57 2.02 2.96 Day 5 4.58 0.00 0.00 0.67 3.33 4.58 0.00 0.33 0.60 3.67 1.010 0.000* 0.000* 0.666* 3.333 1.010 0.000* 0.333* 0.660* 0.330 Day 6 1.55 0.00 0.00 0.00 0.33 1.55 0.00 0.00 0.00 1.67 0.630 0.000* 0.000* 0.000* 0.333 0.630 0.000* 0.000* 0.000* 1.660 Day 7 0.50 0.00 0.00 0.00 0.00 0.50 0.00 0.00 0.00 0.00 0.337 0.000 0.000 0.000 0.000 0.337 0.000 0.000 0.000 0.000

Acacia melanoxylon

Allelopathic effect of Acacia melanoxylon

Flower aqueous extract Control 100% 75% 50% 25% Phyllode aqueous extract Control 100% 75% 50% 25%

2011 The Authors Journal compilation 2011 Weed Science Society of Japan

* Signicant differences, compared to the control, at P < 0.05, according to the Dunnett test. The results represent the mean (SE) of three replicates.

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Table 4. Effects of the Acacia melanoxylon ower and phyllode water extracts on the germination of Lactuca sativa at each exposure time during 1 week Exposure time (days after sowing) Day 1 7.25 0.00 0.00 0.00 0.00 7.25 0.00 1.67 3.67 5.00 1.58 0.00* 0.88* 1.85* 0.57 6.58 2.33 2.00 3.67 5.33 1.600 1.850* 1.150* 0.330* 2.900 2.67 1.00 2.00 1.00 1.67 0.730 1.040 1.150 1.000 1.200 1.00 1.67 0.33 1.33 0.33 0.570 1.660 0.330 0.880 0.330 0.75 1.67 0.26 0.33 2.00 0.37 1.66 0.06 0.33 2.00 0.82 0.00 0.00 0.00 0.33 0.423 0.000 0.000 0.000 0.330 1.58 0.00* 0.00* 0.00* 0.00* 6.58 0.00 0.00 0.00 0.67 1.600 0.000* 0.000* 0.000* 0.667* 2.67 0.00 0.00 0.33 1.33 0.730 0.000* 0.000* 0.330* 1.333 1.00 0.00 0.00 0.67 2.33 0.570 0.000 0.000 0.660 2.333 0.75 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.82 0.00 0.00 0.00 0.00 0.423 0.000 0.000 0.000 0.000 0.67 0.00 0.00 0.00 0.00 0.67 0.00 0.00 0.67 0.67 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 0.337 0.000 0.000 0.000 0.000 0.337 0.000 0.000 0.660 0.330

Acacia melanoxylon

M.I. Hussain et al.

Flower aqueous extract Control 100% 75% 50% 25% Phyllode aqueous extract Control 100% 75% 50% 25%

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Rumex acetosa S 1.24 0.00 0.00 0.00 0.00 1.24 0.70 1.16 1.22 0.93 0.200 0.110* 0.100 0.210 0.170 40.00 36.00 37.33 35.33 46.00 8.00 10.06 13.92 2.66 6.11 0.94 0.52 0.83 0.57 0.72 0.207 0.000* 0.000* 0.000* 0.000* 40.00 28.00 37.33 38.67 34.67 8.00 4.00 5.33 6.67 3.52 0.94 0.66 0.55 0.71 0.67 0.20 0.52 0.68 0.19 0.08 0.20 0.13 0.11 0.18 0.46 GT S 84.67 0.00 0.00 4.00 32.00 84.67 21.33 70.67 80.00 76.00 GT 4.05 0.00* 0.00* 4.00* 21.16* 4.05 19.36* 9.33* 10.06 4.61 1.79 0.00 0.00 0.08 0.47 1.79 0.61 1.61 2.17 1.67 Lolium perenne S 0.348 0.000* 0.000* 0.078* 0.251* 0.348 0.530 0.660 0.540 0.100 GT 78.67 8.740 0.00 0.000* 0.00 0.000* 4.00 4.000* 17.33 17.333* 78.67 8.740 22.67 9.610* 29.33 7.330* 28.00 6.110* 77.33 22.660 5.13 0.00 0.00 0.06 0.33 5.13 0.02 0.60 1.20 3.91 Lactuca sativa S 2.080 0.000* 0.000* 0.061* 0.338* 2.080 0.090* 0.150* 0.530* 0.820

* Signicant differences, compared to the control, at P < 0.05, according to the Dunnett test. The results represent the mean (SE) of three replicates.

Table 5. Effect of the different concentrations of the Acacia melanoxylon ower and phyllode aqueous extracts on the germination indices of Dactylis glomerata, Rumex acetosa, Lolium perenne, and Lactuca sativa

Acacia melanoxylon

Dactylis glomerata

GT

Flower aqueous extract Control 82.67 7.05 100% 0.00 0.00* 75% 0.00 0.00* 50% 0.00 0.00* 25% 0.00 0.00* Phyllode aqueous extract Control 82.67 7.05 100% 42.67 8.11* 75% 66.67 2.66* 50% 70.67 11.85 25% 70.67 11.85

* Signicant differences, compared to the control, at P < 0.05, according to the Dunnett test. The results represent the mean (SE) of three replicates. GT, germination rate index; S, speed of germination.

Allelopathic effect of Acacia melanoxylon concentration and the plant species. For D. glomerata, the GT and S were completely inhibited by the A. melanoxylon ower extract at all four concentrations. Both indices were less sensitive to the phyllode extract at the lowest concentration, which did not signicantly affect D. glomerata. For R. acetosa, the aqueous extracts of the A. melanoxylon owers and phyllodes did not signicantly inhibit the GT and S at any of the tested concentrations (Table 5). The ower extract (100, 75, 50, and 25%) signicantly suppressed the GT and S for L. perenne and L. sativa (Table 5), while the phyllode extract of A. melanoxylon signicantly decreased the GT for L. perenne at the 100% and 75% concentrations. For L. sativa, both the GT and S were signicantly decreased by the ower and phyllode extracts at the concentrations of 100, 75, and 50% (Table 5). Effect of the Acacia melanoxylon extracts on germination The effect of the A. melanoxylon ower and phyllode extracts on the germination of the native species and L. sativa after the probit analysis is presented in Tables 69. The total number of seeds, ungerminated seeds, expected response, and probability were determined against the four different concentrations (100, 75, 50, and 25%) of the A. melanoxylon ower and phyllode extracts.The data of the ungerminated seeds were tted to the probit model.The data regarding the ungerminated seeds were best tted to the probit model after log transformation of the data.The result of the c2-tests for goodness-of-t was d = 10 (at the 95% condence limit) for the ungerminated seeds. The regression equation was Y = -0.091 + 0.675X in relation to the D. glomerata germination data after exposure to the phyllode extract of A. melanoxylon. No regression equation was computed in relation to D. glomerata after exposure to the ower aqueous extract of A. melanoxylon because there was no seed germination. The concentration of 46% of the phyllodes of A. melanoxylon was a diagnosed concentration that inhibited 50% of the seed germination of D. glomerata (Table 6). For L. perenne, the regression equation was Y = 3.276 + 4.688X following exposure to the ower aqueous extract of A. melanoxylon.The concentration of 43% of the owers of A. melanoxylon inhibited 50% of the seed germination of L. perenne. The regression equation was Y = -0.016 + 1.177X following exposure to the A. melanoxylon phyllode extract.The concentration of 41% of the phyllodes of A. melanoxylon inhibited 50% of the seed germination of L. perenne (Table 7). The result of the c2-tests for goodness-of-t was d = 10 (at the 95% condence limit) for the ungerminated seeds of R. acetosa and the regression equation was Y = 0.447 + 0.204X following

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exposure to the ower aqueous extract of A. melanoxylon. The concentration of 40% of the A. melanoxylon owers inhibited 50% of the seed germination of R. acetosa.The regression equation was Y = 0.180 + 0.079X following exposure to the A. melanoxylon phyllode extract. The concentration of 38% of the phyllodes of A. melanoxylon inhibited 50% of the seed germination of R. acetosa (Table 8). The regression equation was Y = 1.673 + 1.855X following exposure to the ower aqueous extract of A. melanoxylon. The concentration of 53% of the owers of A. melanoxylon inhibited 50% of the seed germination of L. sativa.The regression equation was Y = 0.211 + 0.764X following exposure to the A. melanoxylon phyllode extract.The concentration of 41% of the phyllodes of A. melanoxylon inhibited 50% of the seed germination of L. sativa (Table 9). Effect of the Acacia melanoxylon extracts on root growth The effect of the A. melanoxylon ower and phyllode extracts on the root length of the plant species was concentration- and species-dependent. The root length of L. perenne and R. acetosa was signicantly reduced as a result of the A. melanoxylon ower extract at all concentrations (Fig. 1).The aqueous extract of the A. melanoxylon phyllodes signicantly reduced the root length of R. acetosa at all the tested concentrations, while the same extract signicantly decreased the root growth of L. perenne only at the 100% and 75% concentrations.The A. melanoxylon ower extract signicantly reduced the root length of D. glomerata and L. sativa at all concentrations. No signicant variation was detected due to the A. melanoxylon phyllode extract on the root length of D. glomerata and L. sativa at any of the tested concentrations (Fig. 2). DISCUSSION The allelopathic effects of exotic plants on the germination and seedling growth of native species have received more and more attention in recent decades (Tawaha & Turk 2003; Wakjira et al. 2005; Dorning & Cipollini 2006). The present study revealed that A. melanoxylon signicantly reduced the germination and seedling growth of the native species. Compared to the distilled water, the continuous application of the A. melanoxylon ower or phyllode extracts for up to 7 days signicantly reduced the seed germination of the native species.This nding is supported by the results of Souto et al. (2001), who identied several phenolic compounds in the A. melanoxylon phyllode extract.These compounds that are

2011 The Authors Journal compilation 2011 Weed Science Society of Japan

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M.I. Hussain et al.

Table 6. Probit analysis for the seed germination of Dactylis glomerata, exposed to four concentrations of Acacia melanoxylon phyllode aqueous extract Extract concentration (%) 100 75 50 25 Total no. of seeds 25 25 25 25 No. of ungerminated seeds 15 9 8 7 Expected response 11.59 10.75 9.60 7.76 Probability 0.464 0.430 0.384 0.309

Regression line parameters: Y = a + bX; Y = -0.091 + 0.675X; LC50 value = 3.48; diagnostic concentration = 46%.

Table 7. Probit analysis for the seed germination of Lolium perenne, exposed to four concentrations of Acacia melanoxylon aqueous extract Extract concentration (%) Flower 100 75 50 25 Phyllode 100 75 50 25 Total no. of seeds 25 25 25 25 25 25 25 25 No. of ungerminated seeds 25 25 24 17 20 7 6 5 Expected response 24.98 24.91 24.22 16.87 12.34 10.88 8.88 5.85 Probability

0.999 0.996 0.969 0.675 0.494 0.435 0.356 0.234

Regression line parameters (A. melanoxylon owers): Y = a + bX; Y = 3.276 + 4.688X; LC50 value = 2.33; diagnostic concentration = 43%. Regression line parameters (A. melanoxylon phyllodes): Y = a + bX; Y = 0.016 + 1.177X; LC50 value = 4.26; diagnostic concentration = 41%.

Table 8. Probit analysis for the seed germination of Rumex acetosa, exposed to four concentrations of Acacia melanoxylon aqueous extract Extract concentration (%) Flower 100 75 50 25 Phyllode 100 75 50 25 Total no. of seeds 25 25 25 25 25 25 25 25 No. of ungerminated seeds 18 16 16 15 16 16 14 11 Expected response 16.81 16.58 16.25 15.67 14.28 14.18 14.04 13.81 Probability

0.673 0.663 0.650 0.627 0.571 0.567 0.562 0.553

Regression line parameters (A. melanoxylon owers): Y = a + bX; Y = 0.447 + 0.204X; LC50 value = 2.23; diagnostic concentration = 40%. Regression line parameters (A. melanoxylon phyllodes): Y = a + bX; Y = 0.180 + 0.079X; LC50 value = 6.10; diagnostic concentration = 38%.

2011 The Authors Journal compilation 2011 Weed Science Society of Japan

Allelopathic effect of Acacia melanoxylon

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Table 9. Probit analysis for the seed germination of Lactuca sativa, exposed to four concentrations of Acacia melanoxylon aqueous extract Extract concentration (%) Flower 100 75 50 25 Phyllode 100 75 50 25 Total no. of seeds 25 25 25 25 25 25 25 25 No. of ungerminated seeds 25 25 21 16 19 18 8 6 Expected response 23.82 23.12 21.68 17.77 14.58 13.65 12.31 10.04 Probability

0.953 0.925 0.867 0.711 0.584 0.546 0.492 0.402

Regression line parameters (A. melanoxylon owers): Y = a + bX; Y = 1.673 + 1.855X; LC50 value = 1.8; diagnostic concentration = 53%. Regression line parameters (A. melanoxylon phyllodes): Y = 0.211 + 0.764X; LC50 value = 6.26; diagnostic concentration = 41%.

Fig. 1. Effect of the (a) ower and (b) phyllode aqueous extracts of Acacia melanoxylon on the root length of Rumex acetosa and Lolium perenne. Every column in each graph represents the mean value standard error from three replicates. *Signicant difference at the 0.05 probability level, compared to the control, according to the Dunnett test.

present in the phyllode extract might be responsible for the retardation of germination and other growth parameters of L. perenne, D. glomerata, and R. acetosa in the present study. Phenolics are widely recognized for their allelopathic potential in plants and can be found in a variety of plant tissues (Djurdjevic et al. 2004). Many other species of genus Acacia (Mimosaceae), like A. dealbata Link (Carballeira & Reigosa 1999; Lorenzo et al. 2008), Acacia confusa Merr (Chou et al. 1998), Acacia auriculiformis A. Cunn. ex Benth (Raqul-Hoque et al. 2003; Oyun 2006), and Acacia nilotica (El-Khawas & Shehata 2005; Al-Wakeel et al. 2007), are known to exhibit allelopathic activity.The effects of allelochemicals

have been studied mostly on seed germination and the suggested mechanisms for its inhibition are the disruption of mitochondrial respiration (Abrahim et al. 2000) through the inuence of allelochemicals on glycolysis, the Krebs cycle, electron transport and oxidative phosphorylation (Muscolo et al. 1999), and the mitochondrial membrane. For L. perenne, the LC50 values of the A. melanoxylon ower and phyllode extracts were 43 and 41%, respectively.The values for R. acetosa were 40 and 38%, respectively, while for L. sativa, they were 53 and 41%, respectively.The mean LC50 value of the A. melanoxylon phyllode extract in the germination inhibition of D.

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Fig. 2. Effect of the (a) ower and (b) phyllode aqueous extracts of Acacia melanoxylon on the root length of Dactylis glomerata and Lactuca sativa. Every column in each graph represents the mean value standard error from three replicates. *Signicant difference at the 0.05 probability level, compared to the control, according to the Dunnett test.

glomerata was 46%. Generally, the rate of seed germination decreased with an increasing concentration of the extracts, indicating that seed germination was quantitatively related to the extracts concentration.The increasing inhibitory rate with the increasing concentration was in accordance with previous reports (El-Darier &Youssef 2000; Singh et al. 2003; Batish et al. 2006) for other allelopathic species. Among the ower and phyllode extracts, the ower extract was observed to be the most inhibitory. The delayed seed germination with the ower extract of A. melanoxylon, compared with the phyllode extract, could be related to the more inhibitory effect of the allelochemicals that are present in the ower parts of the plant. These results are supported by the ndings of Kil andYun (1992), who reported that the effect of the aerial parts of Artemisia princeps on the germination and growth of different plant species was greater than the effect of the subaerial parts. The maximum decrease in the germination percentage of the seeds of the native species, when treated with the ower extract of A. melanoxylon, indicated the presence of water-soluble allelochemicals in maximum concentration. Dana and Domingo (2006) reported that the Acacia retinodes ower aqueous extract (100% concentration) decreased the germination of Carrichtera annua (33%) and L. sativa (86%); however, there was no effect on Conyza albida.The lower concentration of the phyllode extract actually promoted seed germination in some bioassay species, but the higher extract concentrations signicantly reduced the germination, which suggests that the stimulatory or inhibitory effect is a

function of the concentration (Saxena & Sharma 1996). Similarly, Reigosa et al. (1999) concluded that certain allelochemicals have a stimulatory effect or no action on various plant species at lower concentrations. The inhibition of seed germination was recorded in all the test species, although the extent of inhibition varied across the species and treatments.The aqueous extracts of the owers and phyllodes of A. melanoxylon suppressed the germination and seedling growth of L. perenne, D. glomerata, and R. acetosa and the inhibitory effect increased with increasing concentrations of the extracts. A marginal improvement in the germination at low concentrations of the phyllode extract could be the result of the detoxication of the allelochemical(s) through the conjugation, sequestration, or secretion of carbohydrates and the oxidation of phytotoxic compounds (Inderjit & Duke 2003). Al-Wakeel et al. (2007) reported that the lower doses of A. nilotica leaf residue (0.25 and 0.5%, w/w) stimulated the growth of the shoots and roots of pea, but the higher doses (0.75, 1.0, 1.5, and 2%, w/w) were inhibitory to seedling growth and the effect was concentration-dependent, although the ower extract of A. melanoxylon appeared to have a more negative effect on seed germination than the phyllode extract. This could be related to a greater concentration of diffusible active compounds in the owers than in the phyllodes or to a variation in the chemical composition between the tissues. As a result of the more dramatic effects of allelochemicals on seed germination than on the growth and

2011 The Authors Journal compilation 2011 Weed Science Society of Japan

Allelopathic effect of Acacia melanoxylon viability of adult plants (Weir et al. 2004), the seed germination bioassay commonly has been used to establish the allelopathic activity of plant extracts. However, in such bioassays, most researchers had used only one germination index or the total germination rate when Bewley and Black (1985) proposed the use of more than one index in order to reect the germination process more accurately. Thus, in the present study, two indices were used to assess the allelopathic effects on the germination of the test species. It is evident from the data that the GT, commonly used in allelopathic bioassays, was not sensitive enough at the lowest concentration of the phyllode extract to conclusively conrm allelopathic activity. It is because this index gives a global interpretation of germination (Jderlund et al. 1996) and does not take into account the speed of germination. In view of the failure of the GT to convincingly demonstrate allelopathic activity of the ower and phyllode extracts of A. melanoxylon, the S was used to monitor the germination behavior of the seeds of the target species because of its sensitivity (Chiapusio et al. 1997). The limitations of any index to adequately reect the effect of allelochemicals on germination behavior is evident from present studies and the use of multiple indices seems to be necessary in order to validate the allelochemical activity of plant extracts. The root length of all the target species was signicantly suppressed by the different concentrations of the A. melanoxylon ower extract, as compared to the phyllode extract. Jadhav and Gaynar (1992) concluded that the percentage of germination and plumule and radicle length of rice and cowpea decreased with an increasing concentration of A. auriculiformis A. Cunn. ex Benth leaf leachates. Similarly, Kamal et al. (1997) found that A. auriculiformis A. Cunn. ex Benth signicantly inhibited the germination and growth of rice and the radicle growth of cowpea. Carballeira and Reigosa (1999) have demonstrated that the leachate from A. dealbata showed strong inhibitory effects on the germination and radicle growth of L. sativa during the owering of A. dealbata. CONCLUSION The results that have been obtained in this study show that the aqueous extracts of the owers and phyllodes of A. melanoxylon possess allelochemicals that suppressed the germination and root growth of the native species, D. glomerata, L. perenne, and R. acetosa, and that the inhibition was concentration-dependent. These results were obtained under laboratory conditions.The target species are usually present in the Galician forest environment and in the adjacent elds. The evaluation of the allelochemicals and their isolation, identication, release, and

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movement under eld conditions are important future research guidelines.The effective natural products could be used as environmentally friendly herbicides to control weeds. ACKNOWLEDGMENTS Many thanks are due to Aldo Barreiro, Nuria Pedrol, Carlos Bolano, Alfredo Justo, Maite Ricart, and Paula Lorenzo for their assistance in the eld and laboratory work. REFERENCES
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2011 The Authors Journal compilation 2011 Weed Science Society of Japan