Allelopathic effect of (Eucalyptus camaldulensis) on peanut (Arachis hypogaea) crop and purple nutsedge (Cyperus rotundus) weed

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1 Scholarly Journal of Agricultural Science Vol. 5(6), pp July, 2015 Available online at ISSN Scholarly-Journals Full Length Research Paper Allelopathic effect of (Eucalyptus camaldulensis) on peanut (Arachis hypogaea) crop and purple nutsedge (Cyperus rotundus) weed Ghanuni, A.M.*, Elshebani, A., Moftah, M.A. and Lajili, A.N. *Department of Crop Science University of Tripoli, Tripoli, Libya, Accepted 27 July, 2015 The experiments were conducted in the laboratories of Crop Science Department and the Experimental Station of the Faculty of Agriculture-University of Tripoli during the growing seasons and The study included experiments testing the allelopathic effect of Eucalyptus camaldulensis leaves on peanuts (Arachis hypogaea) crop and purple nutsedge (Cyperus rotundus) weed. The rates 0.0, 152.9, 178.3, g/row (2 cm x 2 m) of powdered leaves of Eucalyptus were tested in the field experiment; and equivalent rates namely, 0, 30, 35, 40 g/pot were also examined using 10 cm pots. Extraction of Eucalyptus leaf components was carried out in aqueous medium using Soxhlet method, and the aqueous extract was subjected to partitioning into organic and aqueous fractions. The effect of the concentrations 0, 25, 50, 75 and 100% (0, 12, 24, 36 and 48 mg ground leaf /ml) of the aqueous extract on peanut seeds and purple nutsedge tubers was examined. The results of the treated soil in the pots exhibited a significant reduction in the germination and seedling growth of purple nutsedge and peanut plants. Percent reduction in the growth parameters ranged between 50 and 60% in the pots treated with 35 or 40 g/pot compared to the treatments 0 and 30 g/pot. The reduction in peanut seed germination was greater than 50% in the pots treated with 35 or 40 g. However, the difference between these two treatments was significant. Peanut seeds or purple nutsedge tubers were planted in rows in field plots experiment revealed significant differences in their germination. The results revealed significant reduction in the germination and seedling growth of both species treated with the higher rates and g/row) of the powdered Eucalyptus leaves added to the soil. Peanut seed and nutsedge tuber germination was significantly reduced in Petri dishes containing the concentrations 50% or more of the aqueous extract. The highest concentration (100 %) completely inhibited germination of the tubers, and reduced peanut seed germination to about 6%. Aqueous and organic fractions recovered from crude aqueous extract inhibited the germination of lettuce seeds. In general, the results obtained suggest that the Eucalyptus leaf powder can be tested further on row crops grown in fields infested with purple nutsedge, and provide opportunity to try this procedure in the area of organic agriculture and chemical weed control. Key words. Allelopathy, allelochemicals, aqueous extracts, purple nutsedge, peanuts. INTRODUCTION The phenomenon of allelopathy has been defined and thoroughly discussed by Rice (Rice, 1984). The publication of Rice encouraged many scientists to work on the effects of plant materials on the germination of seeds and the growth of different plant species. These *Corresponding author am_ghanuni@tripoliuniv.edu.ly. materials were obtained from different organs (Irons and Burnside, 1982, Lehle et al., 1983). Plant extracts were also examined (Abou Hussein et al., 1974, Alsaadawi and Rice. 1982, Awan et al., 2009). Powdered (El - Rokiek et al., 2010) or aqueous extracts (Al Hamdi et al., 2001, Burgos and Talbert, 2000, Iqbal et al., 2003, Lehle et al., 1983, Ohman and Kommedahl, 1964, Peterson and HHarrison, 1995, Scrivanti, 2010) of different plant organs exhibit allelopathic effects on the tested plant

2 Scholarly J. Agric. Sci. 190 seeds. Different phenolic compounds were extracted from plants, identified, and recognized as allelochemicals (Zhao-Hui Li et al., 2010). Recently, a review on allelopathy (Farooq et al., 2010) has indicated the possibility of utilization of plant residues and their extracts for weed control. Several studies focused on the allelopathic effects caused by forest trees (Blanco, 2007)). The forest trees, Eucalyptus species, were tested for their allelopathic effects on different plant species (del Moral and Muller, 1970), Sasikumar et al., 2001, Zhang and Fu, 2009, Kikuchi et al., 2009). Two studies were conducted using Petri dishes and pots to test the effect of different concentrations of sugar beet (Beta vulgaris) and eucalyptus aqueous extracts on the growth of Portulaca oleracea. The results indicated that the growth of seedlings was inhibited, and was more pronounced in the concentration 20 g/l (Khan et al., 2008). Eucalyptus camaldulensis contains volatile allelochemicals (Elaissi et al., 2011, Padhy et al., 2000), as well as other stable phenolic compounds, which exhibit allelopathic effects (Padhy et al., 2000). Eucalyptus camaldulensis is a forest tree originated in Australia (Zhang and Fu, 2009) and has been introduced to Libya about 100 years ago (Khan et al., 2008). Research in Libya on allelopathy is very limited, especially, on the allelopathic effects of Eucalyptus leaves. Nevertheless, some local studies using pots have indicated that several rates of dry ground leaves of Eucalyptus exhibited allelopathic effect on the rhizomes of bermuda grass (Cynodon dactylon) (Nasef et al., 2010) and the tubers of purple nutsedge (Cyperus rotundus) (Shammam and Ghanuni, 2011). This study was conducted to examine the allelopathic effects of Eucalyptus leaves on peanut, lettuce and purple nutsedge. Dry ground leaves, aqueous extracts obtained from the leaves and their organic fractions were used for the allelopathic assays. MATERIALS AND METHODS The experiments were carried out during the summer of 2011 and 2012 in the laboratories of Crop Science Department and the Experimental Station of the University of Tripoli (Tripoli Libya). Selected leaves (blade and petiole) of 7-10 cm long and 1-2 cm width were removed from the branches of Eucalyptus tree grown in the Experimental Station. The leaves were dried in the oven (70-80⁰) for hr and ground in a Wiley mill to pass a 40-mesh screen. Ground leaves were stored in polyethylene bags. Seeds of peanut and lettuce were obtained from local markets and the tubers were removed from purple nutsedge plants, which were collected from the station during the middle of March and April. The tubers of 0.5 cm diameter and cm long were selected, washed with tap water to remove soil particles, wiped and stored in the refrigerator. Data collected from each experiment were subjected to analysis of variance at 5% probability, and LSD was used to compare the significant differences between the means. Experiment I. Soil was collected from an area of the station fields, where no Eucalyptus trees are present, and has not been treated with any chemicals for the last three years. Plant materials and large soil particles were removed from the soil using a convenient sieve. Four hundred grams of dry soil was placed in polyethylene pots of 10 cm diameter and the ground leaves of Eucalyptus were sifted on the top and covered with a thin layer of soil. Four seeds of peanut or purple nutsedge tubers were planted in each pot. The pots were irrigated and placed in the open fields of the Experimental Station under normal condition. Completely randomized design with four replications was used in this experiment. The treatments were 0.0, 30, 35, and 40 g/pot of the ground leaves of Eucalyptus (Heisey and Heisey, 2003). The number of germinated seeds or tubers was recorded two weeks after planting date. Plant foliage length and their weight were measured 50 days after planting date. Experiments II. Field experiment was conducted in a selected area of the station, which has not been treated with chemicals for the last three years. The area was plowed to remove summer annuals and the perennial weed Bermuda grass (Cynodon dactylon).the area was divided into plots (2x2m) and two rows were designed in each plot. The dimensions of each row were 2m x 2cm separated by 80 cm distance. The plots were irrigated and irrigation continued whenever needed. The ground leaves of Eucalyptus mixed with soil were applied along the rows in each plot and ten seeds of peanut and ten tubers of the sedge were alternatively planted at 10 cm distance along each row. In this experiment 0.0, 152.9, 178.3, and gram of the ground leaves/row represented the treatments. The experiment was randomized complete block design with four replications and four treatments. After 100 days from the treatment application, the allelopathic effect was determined on the weed and the crop. Percent control of the weed was recorded (0, no effect and 100, complete kill) (Ghanuni et al., 1999). Peanut pods were removed from the crop plants collected from each row and the weight of the pods and seeds were determined Experiment III. Eucalyptus leaf components were extracted using Soxhlet method. Twelve grams of the ground Eucalyptus

3 Ghanuni et al. 191 Leaves were placed in the extraction tube, covered with glass fibers and the tube placed in the Soxhlet apparatus. Extraction was carried out for 8 hours using 250 ml water as a solvent (Ghanuni et al., 2003). The concentrations 0, 25, 50 75, and 100%, which represent treatments (Ghanuni et al., 1999) were prepared from the aqueous extract. Bioassay of different concentration of the aqueous extract was conducted to examine the allelopathic effect of this extract. Peanut seeds and purple nutsedge tubers were sterilized for 10 minutes in 10% solution of sodium hypochlorite (NaOCl) prepared from the commercial product Chlorox. After washing, three times in sterilized distilled water and drying, ten seeds or tubers were placed in sterilized glass Petri dishes. Ten ml of the treatment was placed in each dish and the dishes were covered and sealed with Parafilm strips to prevent evaporation and contamination. The experiment was completely randomized design with five replications and five treatments. Germination was measured one week after planting (treatment application) and the length of plumules and radicals were measured two weeks after treatment application. Experiment IV. In this experiment, fractionation of the aqueous extract into polar and nonpolar components was done using ether as an organic nonpolar solvent. Recovered aqueous fractions were stored in the freezer and the organic fractions in the refrigerator. In the first step aqueous extraction was carried out as in experiment III; one hundred ml from the recovered aqueous extract was placed in a 250 ml conical flask to adjust the ph to 7.7 using 0.1 M NaHCO3. The basic solution was then transferred to a separatory funnel containing 25 ml ether, and fractionation was carried out by shaking the contents several times. Ether layer was removed from the funnel and a second volume of ether (25 ml) was added to the funnel containing the aqueous fraction. Extractions were continued as before, and this step was repeated five times. Recovered ether fractions were combined (org1) and stored. The ph of the aqueous fraction was lowered to 2.0 by adding few drops of 6.0 M HCl, and repeated separation of the components was carried out five times, each time with 25 ml ether. Aqueous fraction was separated (aq 1) and stored. Twenty five ml of 0.1 M NaHCO3 was added to the combined ether fraction, and the extraction was continued as before. Ether fraction (org2) was stored, and the aqueous fractions were pooled and acidified to ph 2.0, and the components were fractionated five times using 25 ml ether. Ether fractions were combined (org3) and aqueous fraction (aq 2) were collected and saved. Lettuce seeds were used to examine the allelopathic effect of the aqueous and ether fractions. Five seeds of lettuce were placed in a Petri dish containing one filter paper and five ml of the fraction was placed in each dish. Five ml of distilled water was also included as a control treatment. The dishes were covered and sealed with Parafilm to prevent evaporation and contamination. The experimental units were placed in the lab (room temperature). Completely randomized design was used with four replicates and six treatments (org1, org2, org3, aq1, aq2, control). The number of seedlings was recorded 10 days after treatment application, and the length of the seedlings was measured two weeks after treatment application. RESULTS AND DISCUSSION Experiment I. Allelopathic effects of the ground leaves of Eucalyptus were recorded for peanut and purple nutsedge, and the results obtained were represented for each pot, which include percent germination and other growth parameters. The effects on the weed After 50 days from planting, no germination of some tubers was observed in the pots containing 35 or 40 g. Moreover, germinated tubers in these pots exhibited reduction in the number of leaves per tuber and plant height. Table 1 shows the effects of the treatments on germination, plant height of the shoots and roots, number of leaves per plant, and number of plants / pot. These allelopathic effects were more pronounced in the pots treated with the two highest rates, namely 35 and 40 g. Generally, these inhibitory effects were in the range 25-50% of the untreated soil, and more effective than the lowest rate (30 g/pot). Nevertheless, this lower rate significantly reduced the number of the weed plants produced from each tuber to about 30%, 3.3 plants per tuber in the control pots and 2.2 in the pots containing this treatment. However, this treatment had no effect on the other plant growth measurements. The trend of the allelopathic effects caused by the two rates 35 and 40g was also recorded for the number of leaves per plant, and number of plants per tuber. About 50% reduction in the number of plant leaves was recorded in the pots treated with 35g.The treatments 35 and 40g/pot were similar with respect to their effect on the number of leaves / plant or the number of plants /tuber. The effects on the crop Peanut seed germination was significantly reduced to more than 50% in the pots treated with 35 or 40g ground

4 Scholarly J. Agric. Sci. 192 Table 1. Effects of dry ground leaves of Eucalyptus camaldulensis on the growth* of purple nutsedge (Cyperus rotundus). Rate g/pot Germination Plant height Number of leaves per Number of plants per % shoots (cm) roots plant tuber LSD (5%) *Data were recorded 50 days after planting Table 2. Effects of dry ground leaves of Eucalyptus camaldulensis on the growth* of peanut (Arachis hypogaea). Rate g/pot Germination % Plant height Plant dry weight Number of leaves per shoots (cm) Roots g plant ` LSD (5%) Table 3. Effects of dry ground leaves of Eucalyptus camaldulensis on the growth* of purple nutsedge (Cyperus rotundus) planted in the field. Germination Number of plants Dry weight Number of leaves Length of Rate Per tuber of shoots per plant shoots roots g /row % g /row (cm) LSD (5%) leaves of Eucalyptus (table 2). The percent germination in these pots was 33.0 and 17.0%, respectively. Germination in the control pots was not different from that observed in the lowest rate. The percent was 91.7 in the control treatment and 74.9% in the pots containing 30g ground leaves. The effects of the two rates 35 and 40g on the growth of the crop plants were essentially similar to that recorded for the weed plants. In other words, the length of the shoots and roots, dry weight of the shoots, and the number of leaves per plant were significantly reduced in the pots containing 35 or 40g.The allelopathic effect was more than 50% of the untreated soil or the lowest rate. Experiment II. Field evaluations of the allelopathic effects of the dry ground leaves of Eucalyptus revealed almost similar results obtained from experiment III. These inhibitory allelopathic effects were observed on the weed or the crop. Effects on the weed Tuber germination was significantly reduced to 60% in the rows treated with g, and declined to 35% in the rows contained 203.8g.The number of plants per tuber, dry weight of the shoots per row and the number of leaves per plant were significantly reduced in the rows treated with and g/row. Dry weight of the shoots was reduced to more than 50% in the rows treated with the highest rate (203.8g) compared to the treatment 178.3g. The lowest rate g/row exhibited significant inhibition of the root length, but did not affect other growth parameters (table 3). The effect on the crop The lowest rate g/row did not affect the growth of peanut plants (table 4).However, increasing the rate to

5 Ghanuni et al. 193 Table 4. Effects of dry ground leaves of Eucalyptus camaldulensis on the growth* of peanut (Arachis hypogaea) planted in the field. Rate Germination Number per plants Number of pods Dry Weight ( g/row) (cm) Seed number g /row % Per row Per row SHoots pods seeds Per row LSD (5%) Table 5. Effects of leaf aqueous extract of Eucalyptus camaldulensis on the growth* of purple nutsedge (Cyperus rotundus) and peanut (Arachis hypogaea). Concentration Germination (%) Length of plumule (cm) Length of radicle (cm) % CY* * AR** CY AR CY AR LSD (5%) ** CY, Cyperus rotundus; AR, Arachis hypogaea or 203.8g/row caused significant inhibition of the plant growth. About 25% reductions in the germination and the number of plants/row were recorded in the plots treated with either rate. Moreover, the inhibitory effects of these two rates were also recorded on the other growth parameters. The range of inhibition was and in the plots treated with and 203.8g/row, respectively. The comparison between these two treatments indicated that the highest rate was more effective on the number of pods and the dry weight of the seeds than the rate g/row. Namely, these reductions were equivalent to 52 and 46% of the treatment 178.3g/row, respectively. Experiment III The data presented in table 5 shows that increased concentration of the aqueous extract from 25% to 50 or more caused significant growth inhibition of both peanut and purple nutsedge. The germination of the tubers gradually dropped from 81.3% in the treatment 25% (not significant compared to control) to zero in the highest concentration (100%). Almost similar trend was observed from these treatments in the dishes contained peanut seeds; however, the highest rate significantly reduced seed germination to 6.3%. The lowest concentration (25%) significantly reduced the length of the plumule to about 50% of untreated seeds or tubers. Moreover, this treatment significantly reduced the length of peanut radical, but did not affect the radical of the weed. The length of the plumule produced from peanut seeds and purple nutsedge tubers in the dishes treated with 50 % aqueous extract were significantly reduced to 75 and 50% of the control, respectively. In addition, high concentration (100%) caused reductions in the length of peanut plumules 10 times of that observed in the dished treated with 75% aqueous extract. Significant decrease in the length of the radicles of both plant species in the dishes treated with 75 or 100% extract. However, no differences were observed between these two treatments. Lettuce plant is very sensitive to allelopathic plant species. In this experiment (no data presented), germination of lettuce seeds was completely inhibited in the dishes containing the aqueous (aq1, aq2) and organic fractions (org1, org2, org3). The control treatment exhibited 100% germination and all the seedlings contained plumules and radicles with 2.1 and 3.9 cm long, respectively. Generally, the phenomenon of allelopathy encouraged the scientists; especially in the fields of weed science, environment, biochemistry, plant pathology and plant physiology. Some of the efforts focused on the allelopathic effects caused by Eucalyptus leaves and their soil residues (del Moral and Muller, 1970, 31), and leaf extracts (18, 21). According to the results obtained from this study, dry ground leaves of Eucalyptus plant and their different extracts generally, inhibited seed germination and the growth of peanut and lettuce plants and the tubers of purple nutsedge. A study (Nasef et al., 2010) showed that 50g/pot or more of dry ground leaves of Eucalyptus inhibited the growth of bermuda grass plants. Moreover,

6 Scholarly J. Agric. Sci. 194 another publication (Chandra and Kandasamy, 1997) indicated that aqueous extract from the leaves of this tree inhibit the growth of bermuda grass and purple nutsedge. In addition, radical growth of Lipidium sortive was inhibited by aqueous extract of the leaves of Eucalyptus (Moradshahi et al., 2003). The investigation conducted in this work and other findings in the literature reviewed suggest that the leaves of Eucalyptus tree contain allelochemicals released into the soil and exhibit allelopathic effects. In California (USA), the annual vegetation adjacent to the naturalized stands of E. camaldulensis often is inhibited severely (del Moral and Muller, 1970). This severe inhibition, as explained by the author, was mainly due to release of allelochemicals, which are adsorbed to the colloidal soil particles. These chemicals are toxic to germinating seeds and seedlings. Another study (Moradshahi et al., 2003) indicated that water soluble toxins found in the Eucalyptus litter inhibited herb growth in the laboratory, green house, and field experiments. A concentration as low as 50g/L of aqueous extracts from the leaves of this tree inhibited Hill reaction by 88% and the concentration 100g/L inhibited peroxidase activity by 95% relative to control. The results obtained from the field study (experiment II) indicate that dry ground leaves of Eucalyptus can be used to control purple nutsedge in the fields where crops are planted in rows. However, It is recommended that more experiments should be conducted to test these effects on different crop species. Moreover, purple nutsedge control should be considered when dealing with organic agriculture. REFERENCES Abou Hussein, M.R., Fadl, M.S. and Wally. Y.A. (1974). 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