Department of Mycology & Plant Pathology, Quaid-e-Azam Campus, University of the Punjab, Lahore-54590, Pakistan, www.pu.edu.pk Email firstname.lastname@example.org
2Jodrell laboratories, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom. Email: email@example.com
3Natural Resources Institute, University of Greenwich at Medway, Central Avenue, Chatham Maritime, Kent ME44TB, United Kingdom. Email: firstname.lastname@example.org
In the search for natural alternatives for weed management, the allelopathic potential of three cultivars of sunflower (Helianthus annuus L.) was investigated through laboratory bioassays. The five most problematic weeds of wheat viz., Phalaris minor, Chenopodium album, Coronopis didymus, Rumex dentatus and Medicago polymorpha were selected for this study. Root, stem and leaf extracts from the sunflower varieties were applied in different concentrations of 10, 20, 30, 40 and 50% w/v against selected weeds. Results indicated the potential of sunflower allelochemicals as possible alternatives for achieving sustainable weed management. The crude aqueous extract of sunflower was fractionated by means of liquid-liquid extraction using dichloromethane and methanol. Possible causative allelopathic substances were isolated by means of high performance liquid chromatography (HPLC) and were assessed for their allelopathic activity individually and in combinations. The chemical characterization of effective allelochemicals by nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of sesquiterpene lactones in the dichloromethane fraction from crude leaf extract.
Helianthus annuus, allelochemicals, weeds, natural herbicides.
In agriculture, weeds are of concern because they compete with cultivated crop plants for growth factors. Modern agriculture relays on synthetic chemicals to get rid of these unwanted plants. Due to increased awareness about the risks involved in use of synthetic chemicals, much attention is being focused on the alternative methods of weed control. In past two decades, much work has been done on plant-derived compounds as environment friendly alternatives to synthetic herbicides for weed control. Allelochemicals are plant secondary metabolites that are submitted to biological and toxicological screens to identify their potential as natural pesticides. Contemporary research in allelopathy focuses on isolating, identifying and quantifying specific active allelochemicals. Once these substances are identified and characterized they can be used either as natural herbicides or as models for developing new and environment friendly herbicides.
Sunflower is well known for its allelopathic compounds. Several phenols and terpenes have been reported in various cultivars of sunflower (Spring et al. 1992; Macias et al. 2002). The present study was designed to evaluate the allelopathic potential of three sunflower cultivars against weeds known as most problematic in wheat including Chenopodium album L., Coronopis didymus (L.) Sm., Medicago polymorpha L., Rumex dentatus L. and Phalaris minor Retz.
H. annuus of varieties, Suncross42, Gulshan93 and Supper25 were grown and collected during the third plant developmental stage (plants 1m tall with flowers, 1 month before harvest).
For crude aqueous extract bioassays, fresh roots, stem and leaves were extracted in water (50gm/100ml) for 24 hours at room temperature. For fractionation guided bioassays, roots, stem and leaves were extracted in water (1:3) after 24 hrs soaking at room temperature. The crude aqueous extract was then partitioned by dichloromethane (DCM). The organic layer was removed by reduced pressure evaporation and residue was dissolved in water for assays or in 1-2 ml of methanol for HPLC analysis. The leaf DCM fraction was further fractionated through repeated HPLC and ten fractions were isolated and bioassayed with selected weeds.
Weed seeds were sown in 9cm diameter Petri dishes moistened with 5ml of various extracts and fractions, while control received distilled water in equal amount. Peteri dishes were placed in the dark at 20± 1°C for 10 days. Each treatment was replicated thrice. Dry weights were recorded at the end. Data was statistically analyzed using Duncan’s Multiple Range Test.
A Waters system consisting of a 600E pump, 717 autosampler and 996-photodiode-array detector with detection span from 200 to 700 nm was used for HPLC. To determine the chemical profiles of extracts, analytical HPLC was carried out on a Merck LiChrospher100 RP-18e column (250 mm x 4 mm i.d.) with a 5 μm particle size, and at flow rate of 1ml min-1. A linear solvent gradient of MeOH:H2O 25:75 to MeOH:H2O 100:0 over 40 min was used for separation. Positive ion-first-order MS were recorded using LC-MS (Thermo Finnigan LCQ) with an electrospray ionization (ESI) source. Atmospheric pressure chemical ionization mass spectra (negAPCI-MS) were acquired using a micromass LCT mass spectrometer calibrated with a PEG calibration solution (acetonitrile: water 50: 50). 1H NMR and 13C NMR spectra were recorded at 399.92 MHz and 100.6127 MHz, respectively, on a Bruker AM-400 NMR spectrometer at 30°C. Samples were dissolved in CDCl3 and TMS used as a primary reference.
Allelopathic effect of sunflower crude aqueous extract on dry weight of weeds
Various concentrations of sunflower root, stem and leaf extracts resulted in significant losses on dry weight bases of selected weeds. The effect was found directly proportional to the extract concentration.
Analysis of data obtained after checking root extract against weeds showed that a concentration of 10% extract supported the weeds dry weight, whereas 20% extract resulted in insignificant (P>0.05) losses in trait. Statistically significant reduction in dry weight was observed at 30% aqueous extract that increased with increase in concentration. 50% extract of Suncross42 incurred maximum losses of 86.6% in Rumex dentatus, which was closely followed by Chenopodium album (83.8%). Least effect was observed in Coronopis didyma that depicted 27.1% dry weight losses (Figure 1A).
In case of stem aqueous extracts, the maximum reduction by highest tested concentration was observed in Rumex dentatus (92.9%). This was followed by 84% losses in Chenopodium album and Phalaris minor. The smallest effect (55.1%) was observed in Medicago polymorpha. A concentration of 10% in the case of stem extracts resulted in insignificant (P>0.05) reduction in said trait. Significant decline was observed at concentrations 30% and higher (Figure 1B).
The leaf extract was found effective even at a low concentration of 10%. The maximum effect of Suncross42 was observed in case of Rumex dentatus (98.4%), which was closely followed by Chenopodium album (96.7%) and Phalaris minor (91.6%). Least effect was recorded in case of Medicago polymorpha that resulted in 43.8% losses in dry weight as compared to control (Figure 1C). Interaction analyzed among weed varieties and concentration means of sunflower varieties proved Suncross42 as most allelopathic variety that followed by Gulshan93 and Supper25.
Fractionation guided bioassays
On the bases of the above results, Suncross42 was selected for further analyses. Leaf DCM fraction of Suncross42 was found most efficient in reducing the dry weights of the weeds, this was followed by stem and root fractions. Largest reduction of 75.37% in dry weight was observed in Rumex dentatus that was closely followed by Chenopodium album (Figure 2). The DCM fraction of leaves was selected for further fractionation-guided bioassays. The leaf DCM fraction was sub-fractionated in to nine fractions using HPLC and the fractions were then checked against R. dentatus and C. album through aqueous extract bioassays. DCM combination was found most effective against selected weeds as compared to the tested fractions. Dry weight data analysis attested the equal efficiency of Fr1, Fr2, Fr5, Fr6, Fr7, Fr8, and Fr9 in both the tested weeds. The remaining two fractions i.e. Fr3 and Fr4 were not found significantly effective in reducing biomass of weeds (Figure 3).
Figure 1. Effect of crude water extract of roots (a) stem (b) and leaves (c) of sunflower varieties in different concentrations.
The possible effective compounds from above fractions were subjected to the structural elucidation. Their mass spectra were acquired and compounds from Fr2 and Fr3 were also analyzed through NMR. These spectra reveal the presence of sesquiterpene lactones.
Figure 2. Effect of DCM fraction of root, stem and leaves of Suncross42 on Dry weight of selected weeds.
Figure 3. Effect of sub-fractions of leaf DCM fraction of Suncross42 on Dry weight of R. dentatus and C. album.
Analysis of data acquired in crude aqueous extract bioassays showed strong allelopathic potential against selected weeds with slight variation. The reduction in dry biomass clearly indicates the phytotoxic effects of sunflower allelochemicals. Genetic variability was observed among the selected sunflower varieties in showing phytotoxic effect against weeds. This observation was found parallel to previously documented studies (Wu et al, 2000). Dry weight was least reduced in Phalaris minor. A larger effect was observed on broad-leaved weeds consistent with previous studies on rice hull (Kuk et al. 2001). In present study, among the broad-leaved weeds Rumex dentatus and Chenopodium album was affected the most in which dry weight reduction was recorded up to 98%. The fractionation guided bioassays also proved leaves as most effective source of allelochemicals to be used as natural herbicides. The MS and NMR spectra are in the process of interpretation for the structural elucidation of possible allelochemicals. Strong clues are there about the presence of sesquiterpene lactone in fractions analyzed. 1H NMR and 13C NMR data showed that analyzed compound contains a similar basic cyclohexanone ring of Annuionone, the apocarotenoids reported earlier by Macias et al (2004) with modified structures.
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