J Pest Sci (2014) 87:341–349 DOI 10.1007/s10340-013-0542-6 ORIGINAL PAPER Chemical analysis of essential oils of Eupatorium adenophorum and their antimicrobial, antioxidant and phytotoxic properties Vivek Ahluwalia • Ritu Sisodia • Suresh Walia Om P. Sati • Jitendra Kumar • Aditi Kundu • Received: 19 May 2013 / Accepted: 28 November 2013 / Published online: 8 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013 Abstract Essential oils from inflorescences and roots of Eupatorium adenophorum Spreng (Asteraceae) have been investigated for their antimicrobial, phytotoxic and antioxidant activities. Based on GC–MS, the oil from inflorescences is dominated by sesquiterpenes (55.9 %) with c-cadinene (18.4 %), c-muurolene (11.7 %), 3-acetoxyamorpha-4,7(11)-diene-8-one (7.4 %) and bornyl acetate (6.3 %) as the major constituents. The oil obtained from the roots contained both sesquiterpenes (34.3 %) and monoterpenes (32.5 %) in almost equal proportions with E,E-cosmene (19.9 %), c-muurolene (10.1 %), isothymol (7.5 %), b-cadinene (7.0 %) and a-phellandren-8-ol (5.9 %) as the major constituents. Both oils exhibited significant antifungal activity against five phytopathogenic fungi. The inflorescence oil showed higher antibacterial activity against Klebsiella pneumoniae, while the root oil was more effective against Staphylococcus aureus. The oils strongly inhibited or delayed germination and seedling growth of the weed Phalaris minor in a dose-dependent manner. As evidenced by a DPPH assay, the essential oils also exhibited significant free radical scavenging activity. Communicated by M. B. Isman. V. Ahluwalia  R. Sisodia  S. Walia (&)  J. Kumar  A. Kundu Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi, India e-mail: sureshwalia@gmail.com V. Ahluwalia (&)  O. P. Sati Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal), Uttarakhand, India e-mail: vivek.orgchem@gmail.com Keywords Eupatorium adenophorum  Essential oil  Antifungal activity  Antibacterial activity  Phytotoxicity  Antioxidant activity Introduction Insect pests, weeds and pathogens are our biggest competitors for food and non-food crops. They can reduce crop productivity by 25–50 % (Oerke 2006). To protect crops from pest infestation, enormous amounts of synthetic pesticides have been applied. Given the environmental risks associated with the use of synthetic pesticides, there is a growing interest in the development of crop protectants from natural sources (Seyran et al. 2010; Yangui et al. 2010). Insecticidal, nematicidal, fungicidal and other pest control properties of several phytochemicals are known (Isman 2004; Baldin et al. 2013). Among these, essential oils and their monoterpenoid and sesquiterpenoid constituents exhibit antioxidant, antimicrobial, insecticidal, antinemic and herbicidal properties (Isman 2000; Jiang et al. 2009; Akhtar et al. 2008; Douda et al. 2010; Nesci et al. 2011; Haouas et al. 2012). Isman (2000, 2006) has comprehensively investigated plant essential oils as natural sources of insecticidal substances. In addition, because of inhibitory effects of plant essential oils on certain plants, there is interest in their use as natural herbicides (Ghnaya et al. 2013). Eupatorium adenophorum, a native to Mexico, is now distributed worldwide (King and Robinson 1970). In India, it is widely distributed in the Himalayan region (Sharma and Chhetri 1977) and used as in folk medicine for its antimicrobial, antiseptic, blood coagulating, analgesic and antipyretic properties (Mandal et al. 1981; Ansari et al. 1983; Bhattarai and Shrestha 2009). Phytochemical 123 342 investigations of Eupatorium spp. have led to the isolation of several bioactive secondary metabolites (Okunade and Wiemer 1985; Clavin et al. 2000; Habtemariam 2001; Ruffinengo et al. 2005; Kundu et al. 2013). The essential oil extracted from aerial parts of the plant has been investigated for its chemical composition (Weyerstahl et al. 1997; Ding et al. 1999; Pala-Paul et al. 2002; Yong-ming et al. 2008; Padalia et al. 2009) as well as its insecticidal (Li et al. 2000; Cheng et al. 2007) and antibacterial activities (Kurade et al. 2010). No detailed information is, however, available on the antimicrobial, phytotoxic or antioxidant properties of the inflorescence and root essential oils of E. adenophorum, from the Northern Himalaya. Therefore, the present study aims at exploring the potential of these oils as a natural source of antimicrobial, phytotoxic and antioxidant agents. Materials and methods Plant material Inflorescences and roots of E. adenophorum were collected from Palampur, situated at an altitude of 1,220 m above mean sea level located in the mid-hills of the Northern Himalayas (Dhauladhar range) during May–June 2011. A voucher specimen (PLPEU 2011) of the plant was identified and deposited at CSK, HPKV, Palampur. Procurement of the chemicals The standard antioxidant butylated hydroxytoluene (BHT) and a,a-diphenyl-b-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich (India). Potato dextrose agar (PDA), nutrient broth and nutrient agar were procured from Hi-Media Pvt. Ltd. (India). Tween-20 and other chemicals/ solvents were obtained from Merck (India). Essential oil extraction The fresh inflorescences and finely chopped roots (500 g each) were subjected to hydrodistillation for 3 h using a Clevenger apparatus. To remove residual water, the extracted light pale yellow essential oils were dried over anhydrous sodium sulphate. The oils were stored in sealed vials at low temperature (\4 °C) until used for analysis. The yield of the inflorescence and root essential oil was 0.7 and 0.5 % v/w basis, respectively. GC–FID and GC–MS analysis Volatile oils from florescences and roots were analysed for their chemical compositions on an Agilent capillary GC– 123 J Pest Sci (2014) 87:341–349 FID (7890A) and GC–MS (5975) mass instrument equipped with a HP-5 column (30 m 9 0.25 mm, film thickness 0.25 lm). Helium was used as a carrier gas (1.0 mL min-1). The initial oven temperature was maintained at 60 °C and programmed to increase at 2 °C min-1 to 125 °C (held constant for 2 min), then to 160 °C at a rate of 2 °C min-1 (held constant for 5 min), and finally increased to 240 °C at a rate of 10 °C min-1. The injector temperature was maintained at 220 °C. The mass spectra were recorded with electron energy of 70 eV over a range of 50–650 amu and ion source temperature of 200 °C. In order to obtain the same elution order with GC–MS, simultaneous injection was done using the same column and appropriate operational conditions. The same column and analysis conditions were applied for both GC–FID and GC–MS. Identification of the essential oil constituents was carried out by comparison of their relative retention times with those of authentic samples or by comparing their relative retention index (RI) to series of n-alkanes, MS Library search (NIST & Wiley), and/or by comparison with the literature data (Adams 1995). In vitro antifungal activity A poisoned food technique was used to evaluate antifungal activity against five phytopathogenic fungi, Sclerotium rolfsii (ITCC 6263), Macrophomina phaseolina (ITCC 6267), Rhizoctonia solani (ITCC 4502), Pythium debaryanum (ITCC 95) and Fusarium oxysporum (ITCC 6246). Antifungal susceptibility testing methods The antifungal activity of the essential oils was evaluated using PDA medium (Ahluwalia et al. 2012) with slight modification. The stock solutions were prepared by dissolving essential oils in 2 ml of 0.1 % aqueous Tween 20. An appropriate quantity of essential oils in Tween 20 (0.1 % v/v) was added to molten PDA medium (65 mL) to obtain the desired concentrations of 0.25, 0.12 and 0.062 lL mL-1 of oil. A mycelial disc, 5 mm in diameter was cut from the 7-day-old culture and inoculated in the centre of each PDA plate and then incubated in the dark at 27 ± 1 °C for 7 days. PDA plates treated with Tween 20 (0.1 % v/v) alone was used as a negative control. In addition, PDA plates treated with hexaconazole, a standard reference fungicide was used as a positive control. The tests were repeated in triplicate, under aseptic conditions in a laminar flow chamber. The mycelial growth (cm) in both treated (T) and control (C) Petri dishes was measured diametrically in three different directions. From the mean growth of above readings, J Pest Sci (2014) 87:341–349 percentage inhibition of growth (% I) was calculated using Abbott’s formula (1925): I ð%Þ ¼ ½ðC  T Þ=C   100 IC ¼ f½I ð%Þ  CF=ð100  CFÞg  100 where IC is the corrected percent inhibition and CF is the correction factor obtained by equation CF = [(90 - C)/ C] 9 100, where 90 is the diameter of the Petri dish in mm and C is the diameter growth of the fungus in control plates. From the concentration (lL mL-1) the corresponding corrected percentage inhibition for each compound was calculated statistically by probit analysis with the help of Statistical Analysis System package (SAS package) software. EC50 values were calculated using the Basic LD50 programme version 1.1 (Trevors 1986). In vitro antibacterial activity Antibacterial activity was evaluated by the disc diffusion and agar dilution method (Aggarwal et al. 2011) against Gram-positive and Gram-negative bacteria. Two phytopathogenic bacterial cultures—Xanthomonas oryzae (ITCC B-47) and Erwinia chrysanthemi (ITCC B-40) were obtained from the Indian Type Culture Collection, Indian Agricultural Research Institute (IARI), New Delhi. Three human bacterial pathogens—Staphylococcus aureus (MTCC 3160), Pseudomonas aeruginosa (MTCC 2581) and Klebsiella pneumoniae (MTCC 7028) were procured from the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh. Disc diffusion assay 20 ml of nutrient agar medium was poured into the plates to a uniform depth and allowed to solidify. The standard inoculum suspension (106 c.f.u. ml-1) was streaked over the surface of the media using a sterile cotton swab to ensure the confluent growth of the organism. 10 lL of essential oil was diluted with two volumes of 5 % dimethyl sulphoxide, impregnated on filter paper discs, and used for the assays. On the surface of the plates, discs were placed with sterile forceps, pressed gently to ensure contact with the inoculated agar surface. Streptomycin (10 lg disc-1) was used as a positive control and hexane as a negative control. The plates were incubated in the dark at 37 °C (24 h) and the inhibition zones were calculated. All experiments were carried out in triplicate. 343 were used to inoculate 100 mL of nutrient broth so that an initial number of 2 9 106 c.f.u. mL-1 could be achieved. The dose range used in the test was selected based on preliminary screening of essential oils at higher concentrations. Essential oils were dissolved in 5 % DMSO and added to 20 mL inoculated media to get a final concentration of 0.40 lL mL-1. Twofold dilution of this essential oil concentration was achieved by transferring 10 mL of this 0.40 lL mL-1 concentration containing media to another 10 mL inoculated media to get a final concentration 0.20 lL mL-1 of the essential oil. All other dilutions were prepared in a similar fashion to obtain the minimal dilution to 0.025 lL mL-1. Each 10 mL inoculated media along with the test concentration was dispensed equally into 3 screw capped 10 mL glass culture tubes. The control tube contained the bacterium alone. 5 % DMSO was used as a negative control. The culture tubes were incubated at 37 °C for 24 h. After incubation, the growth of bacteria was determined by measuring optical density at 600 nm using a UV-Specord 200/1 (Analytik JENA InstrumentsÒ). The lowest concentration at which there was more than 90 % inhibition of bacteria relative to the negative control was taken as the minimal inhibitory concentration (MIC). Seed germination and seedling growth experiments Seeds of Phalaris minor (a weed) and Triticum aestivum (wheat) were used in herbicidal assays. The seeds were collected from parent plants growing in the harvest fields of the Indian Agricultural Research Institute, New Delhi. To avoid possible inhibition of germination due to fungal or bacterial toxins, seeds were surface sterilized with 15 % sodium hypochlorite solution for 20 min, then rinsed with abundant distilled water. Germination was carried out in Petri dishes where seeds were placed on double-layered Whatman No. 1 filter paper moistened with different concentrations (0, 0.125, 0.25, 0.5 and 1 lL mL-1) of essential oil in a 1 % solution of Tween 20 (Tworkoski 2002). Cultures were incubated under controlled conditions (25 °C, 70 % RH and 16:8 LD). The Petri dishes were sealed with adhesive tape to prevent the volatile oils from evaporating. The germinated seeds were counted and seedling lengths were measured after 7 days (ISTA 1996; Zahid et al. 2010; Amri et al. 2012). The assays were arranged in a completely randomized design with three replications (20 seeds each) including controls. Statistical analysis was done using the SAS package. Antioxidant activity Agar dilution method The test bacterial strains were grown at 37 °C in nutrient broth until the exponential growth phase. These cultures The antioxidant activity of essential oils was measured in terms of scavenging ability using a DPPH (1,1-diphenyl-2picrylhydrazyl radical) assay with slight modification 123 344 J Pest Sci (2014) 87:341–349 Table 1 Chemical constituents of the essential oils of E. adenophorum (inflorescence and root) Sr. No. RI a Constituent b RA (%) Inflorescences 1. 950 2. 3. 4. Table 1 continued Sr. No. RIa 47. 1,571 d-Gurjunene 48. 1,575 (?)-Spathulenol Constituent RAb (%) Inflorescences 1.93 1,005 a-Phellandrene 0.81 – 49. 1,580 Viridiflorol 1,010 1,018 3-Carene a-Terpinene 0.21 2.70 0.57 – 50. 1,581 Caryophyllene oxide 1.09 51. 1,640 s-Cadinol 0.48 5. 1,026 o-Cymene 2.23 52. 1,642 Cedrene-13-ol 6. 1,030 a-Limonene 4.78 53. 1,655 b-Eudesmol 7. 1,046 a-Ocimene 54. 1,682 a-Bisabolol – 8. 1,065 p-Mentha-3,8-diene – 0.36 9. 1,075 2-Allyl-p-cresol – 2.12 55. 56. 1,754 1,760 Elleryone Muurol-4-en-3,8-dione – 0.40 10. 1,080 a-Phellandrene-8-ol – 5.86 57. 1,790 11. 1,090 a-Terpinolene – 1.23 3-Acetoxyamorpha-4,7(11)diene-8-one 12. 1,096 Linalool – Monoterpenes hydrocarbons (%) 13. 1,134 E,E-Cosmene – 19.89 Oxygenated monoterpenes (%) 14. 1,158 (E)-2,3-epoxycarene – 0.43 15. 1,159 Cymen-4-ol – 0.18 16. 1,160 Isothymol – 7.51 a 17. 1,166 Borneol 0.52 2.86 b 18. 19. 1,186 1,218 Cis-a-terpineol Carveol 0.23 – – 0.35 20. 1,279 Thymol 1.00 2.27 21. 1,283 Bornyl acetate 6.30 2.88 0.39 0.16 1.34 – Camphene – – – 22. 1,299 m-Cymen-4-ol 1.38 23. 1,348 a-Cubebene 0.18 24. 1,380 Isolongifolene 25. 1,396 b-Vatirenene 2.45 – 26. 1,408 a-Gurjunene 1.53 – 27. 1,415 Caryophyllene 5.39 – 28. 1,424 s-Gurjunene 1.11 – 29. 1,430 b-Copaene 3.72 – 30. 1,431 Elixene 1.47 – 31. 1,432 b-Famesene – 0.41 32. 1,435 a-Bergamotene – 1.45 33. 1,440 Aromandrene – 34. 35. 1,439 1,450 a-Guaiene a-Terpinolene 36. 1,454 a-Himachalene – 37. 1,477 c-Muurolene 11.70 38. 1,479 c-Curcumene 5.66 39. 1,483 Germacrene-D 4.26 40. 1,492 b-Guaiene 41. 1,496 Zingiberene 0.21 – 42. 1,502 b-Himachalene 3.27 – 43. 1,509 c-Himachalene 44. 1,512 a-Chamigrene 45. 1,515 c-Cadinene 18.36 2.73 46. 1,531 b-Cadinene – 6.98 123 – 2.10 0.84 – – 0.53 Roots Roots 0.67 – 0.51 4.58 – – 1.67 10.11 2.59 – 2.42 – – 0.22 7.42 1.57 – 2.45 0.90 – 0.56 – 2.49 1.19 – – 16.85 32.52 7.21 19.46 Sesquiterpene hydrocarbons (%) 55.85 34.33 Oxygenated sesquiterpene (%) 13.41 8.26 Retention index on HP-5 MS column (Respect to n-alkanes) Relative area (Cavar et al. 2008). A stock solution of DPPH (10-4 M) was prepared in 30 % aqueous methanol. An aliquot (2 mL) of each sample (with different concentrations) was added to the DPPH solution (1 mL), shaken vigorously and allowed to stand at room temperature in the dark. After 30 min, the decrease in absorbance at 517 nm was measured against a blank (aqueous methanol solution) using a double-beam UV–Vis spectrophotometer (Perkin–Elmer Lambda EZ201). A mixture consisting of 1 ml of 30 % aqueous methanol and 3 ml of DPPH solution was used as the negative control. BHT was used as a positive control. The radical-scavenging activity of the samples was expressed as percentage inhibition of DPPH as per the following formula: I ð%Þ ¼ ½ðAB  AA Þ=AB   100 where AB and AA are the absorbance values of the control and the test sample, respectively. The oil concentration producing 50 % inhibition (IC50) was calculated from the graph of inhibition percentage plotted against oil concentration. 0.87 0.86 – Results Chemical composition of essential oils Hydrodistillation of E. adenophorum inflorescences and roots produced pale yellow oils with respective yields of J Pest Sci (2014) 87:341–349 345 Root Essential Oil 0.25 Pd Fo 0.125 Sr Rs 0.062 Mp Concentration µL mL Concentration µL mL -1 Inflorescence Essential Oil 0.25 Pd Fo 0.125 Sr Rs 0.062 Mp C C 0 20 40 60 80 100 0 20 Percentage Inhibition 40 60 80 100 Percentage Inhibition Fig. 1 Antifungal effect (percent inhibition) of E. adenophorum, inflorescence and root essential oils. Pd P. debaryanum, Fo Fusarium oxysporum, Sr Sclerotium rolfsii, Rs Rhizoctonia solanii, Mp Macrophomina phaseolina, C Negative control 0.7 and 0.5 % v/w. GC–MS analysis of the inflorescence essential oil led to identification of 34 monoterpenoid or sesquiterpeniod constituents representing 93.3 % of the oil (Table 1). Sesquiterpene hydrocarbons were most abundant (55.9 %) among which c-cadinene (18.4 %), c-muurolene (11.7 %), 3-acetoxyamorpha-4,7(11)-diene-8-one (7.4 %), c-curcumene (5.7 %) and caryophyllene (5.4 %) were identified as major constituents. Oxygenated sesquiterpenes represented only 13.4 % of total oil content. Monoterpenes were relatively less abundant (24.1 %), with 16.9 % monoterpene hydrocarbons and 7.2 % oxygenated monoterpenes. The major monoterpene hydrocarbons were identified as a-himachalene (3.3 %), limonene (2.9 %) and a-terpinene (2.7 %). Bornyl acetate (6.3 %) was the only predominant oxygenated monoterpene. Eupatorium adenophorum root essential oil was composed of 33 constituents representing 94.6 % of the total oil. The major constituents were sesquiterpene hydrocarbons (34.3 %) and monoterpene hydrocarbons (32.5 %). cMuurolene (10.1 %), b-cadinene (7.0 %) and aromandrene (4.6 %) were the main sesquiterpene hydrocarbons, whereas E,E-cosmene (19.9 %) and a-limonene (4.8 %) were the major monoterpene hydrocarbons. In addition to five oxygenated sesquiterpenes (8.3 %), isothymol (7.5 %) and a-phellandren-8-ol (5.9 %) were the two major oxygenated monoterpenes present in the oil (Table 1). Antifungal activity The volatile oils showed considerable increases in inhibition with concentration of essential oils (Fig. 1). At 0.25 lL mL-1, the inflorescence oil exhibited 70–90 % inhibition against all fungi tested. Maximum inhibition (90 %) was observed against S. rolfsii, while minimum inhibition (70 %) was observed against M. phaseolina. The essential oil showed maximum EC50 against M. phaseolina (EC50 = 0.076 lL mL-1) (Table 2). Table 2 Antifungal activity (EC50) of E. adenophorum essential oils and Hexaconazole Test fungi Inflorescence essential oil (lL mL-1) Root essential oil (lL mL-1) Hexaconazole (lg mL-1) Macrophomina phaseolina 0.076 0.153 4.45 Rhizoctonia solani 0.094 0.092 18.30 Sclerotium rolfsii 0.117 0.114 13.43 Fusarium oxysporum 0.120 0.103 22.01 Pythium debaryanum 0.083 0.093 25.92 Results shown are means of three experiments The root essential oil produced 82.8 % inhibition at 0.25 lL mL-1 against S. rolfsii. In terms of median concentration the root essential oil showed maximum antifungal activity against R. solani (EC50 = 0.092 lL mL-1) (Table 2). Antibacterial activity The assay for antibacterial activity revealed that among five bacterial species, K. pneumoniae and S. aureus with inhibition zones of 16 and 12 mm were most sensitive. The standard antibiotic streptomycin showed respective inhibition zones of 14 and 13 mm (Table 3). The smallest zones of inhibition was shown against E. chrysanthemi (9 mm) and X. oryzae (10 mm). The human bacterial pathogen K. pneumoniae showed the lowest MIC value (0.05 lL mL-1) while P. aeruginosa, X. oryzae and E. chrysanthemi exhibited the highest MIC values (0.20 lL mL-1) (Table 3). 123 346 J Pest Sci (2014) 87:341–349 Table 3 Antibacterial activity of E. adenophorum essential oils Bacteria Mean zone of inhibition (mm)a, b MIC (lL mL-1)c Inflorescence essential oil (10lL disc-1) Root essential oil (10lL disc-1) Streptomycin (10 lg disc-1) Inflorescence Root Klebsiella pneumoniae 16 ± 0.95 10 ± 1.50 14 ± 0.28 0.05 0.40 Staphylococcus aureus 12 ± 0.72 20 ± 0.92 13 ± 1.00 0.10 0.05 Pseudomonas aeruginosa Xanthomonas oryzae 09 ± 0.57 10 ± 0.65 13 ± 0.25 12 ± 1.05 11 ± 0.42 13 ± 0.33 0.20 0.20 0.10 0.10 Erwinia chrysanthemi 09 ± 0.57 11 ± 0.74 12 ± 0.52 0.20 0.10 a Results are mean ± standard deviation (SD) of three experiments b Disc diffusion assay c Agar dilution method Table 4 Effect of E. adenophorum essential oils on the germination of Phalaris minor and Triticum aestivum Concentration (lL mL-1) Inflorescence essential oil Phalaris minor Root essential oil Triticum aestivum Phalaris minor Triticum aestivum 0 96.67 ± 1.15 98.34 ± 0.57 96.67 ± 1.15 98.34 ± 0.57 0.125 63.34 ± 1.15 68.33 ± 1.15 76.56 ± 1.15 83.34 ± 1.15 0.25 31.66 ± 0.57 36.76 ± 1.73 41.33 ± 0.57 40.00 ± 1.00 0.5 1.0 13.43 ± 1.52 0±0 20.00 ± 1.00 0±0 20.00 ± 1.00 0±0 23.33 ± 0.57 0±0 Results shown are mean ± standard deviation (SD) of three experiments The root essential oil exhibited moderate to excellent antibacterial activity and the activity was bacteria specific. The Gram-positive bacterium S. aureus with an inhibition zone of 20 mm was most sensitive; the standard antibiotic streptomycin showed an inhibition zone of 13 mm. The volatile oil showed comparatively less inhibition against K. pneumoniae (10 mm) and E. chrysanthemi (11 mm). The lowest MIC value was 0.05 lL mL-1 against S. aureus and the highest MIC was 0.40 lL mL-1 against K. pneumoniae (Table 3). the essential oils showed more inhibitory activity towards P. minor than T. aestivum. Antioxidant activity The DPPH radical-scavenging activities of the essential oils were compared with the standard synthetic antioxidant BHT at six different concentrations. The IC50 (concentration to achieve 50 % inhibition) of the inflorescence and root oils were 2.21 and 1.86 lg mL-1, respectively, compared to the standard BHT (IC50 = 0.015 mg mL-1). Phytotoxicity Application of essential oils at 1.0 lL mL-1 completely inhibited germination and seedling growth of P. minor and T. aestivum (Tables 4, 5). At 0.5 lL mL-1, the germination of P. minor was only 13.4 % (inflorescence essential oil) and 20.0 % (root essential oil), whereas in T. aestivum the respective germination was 20.0 and 23.3 % (Table 4). At the lower concentration 0.125 lL mL-1, germination and seedling growth of T. aestivum and P. minor were not significantly reduced compared to control. The essential oils inhibited the germination and seedling growth in a dose-dependent manner and the effect was more pronounced in suppressing root growth (Table 5). In general, 123 Discussion GC–MS analyses of essential oils extracted from inflorescences and roots of E. adenophorum has led to the identification of 34 and 33 compounds in the respective oils (Table 1). While c-cadinene, c-muurolene, 3-acetoxyamorpha-4,7(11)-diene-8-one and bornyl acetate were found to be the major constituents in the inflorescence essential oil, E,E-cosmene, c-muurolene, b-cadinene and isothymol predominated in the root essential oil. An earlier study of an essential oil of E. adenophorum aerial parts from India revealed the presence of six major constituents, J Pest Sci (2014) 87:341–349 347 Table 5 Effect of E. adenophorum essential oils on the seedling growth of Phalaris minor and Triticum aestivum Concentration (lL mL-1) Inflorescence essential oil Phalaris minor Shoot length Root length Root essential oil Triticum aestivum Phalaris minor Shoot length Shoot length Root length Triticum aestivum Root length Shoot length Root length 0 7.05 ± 0.58 6.61 ± 0.34 9.11 ± 0.59 8.85 ± 0.65 7.05 ± 0.58 6.61 ± 0.34 9.11 ± 0.59 8.85 ± 0.65 0.125 5.23 ± 0.21 4.88 ± 0.59 8.18 ± 0.33 7.92 ± 1.08 5.00 ± 0.29 4.71 ± 0.50 8.06 ± 0.24 7.42 ± 0.29 0.25 3.97 ± 0.17 3.65 ± 0.25 5.23 ± 0.09 4.67 ± 0.10 3.80 ± 0.08 3.35 ± 0.31 4.95 ± 0.12 4.23 ± 0.05 0.5 1.40 ± 0.14 1.35 ± 0.21 2.85 ± 0.07 2.15 ± 0.21 1.10 ± 0.28 0.95 ± 0.07 2.65 ± 0.21 2.35 ± 0.07 1.0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 Results shown are mean ± standard deviation (SD) of three experiments namely 1-napthalenol, a-bisabolol, bornyl acetate, b-bisabolene, germacrene-D and a-phellandrene (Kurade et al. 2010). Nonetheless, there is great variability in the chemical composition of Eupatorium essential oils between different species. For example, essential oils from species of Eupatorium vary in their proportions of a-pinene (E. odoratum); cymene (E. capillifolium); b-caryophyllene oxide (E. cannabium); b-caryophyllene, humulene, c-muurolene (E. betonicaeforme); sabinene (E. macrophyllum); laevigatin and aristolone (E. laevigatum) (Bamba et al. 1993; Pino et al. 1998; Gurdip et al. 1999; Albuquerque et al. 2004; Maia et al. 2002). The essential oils were evaluated for their antifungal activity against five plant pathogenic fungi (Table 2). Both oils significantly inhibited growth of all five fungi, but were less effective than the commercial fungicide hexaconazole. Nevertheless, in view of their natural origin, their antifungal activity was quite noteworthy. As mentioned above, the inflorescence essential oil was characterized by relatively high contents of sesquiterpenes (c-cadinene and c-muurolene) and monoterpenes (bornyl acetate) that could be responsible for their antifungal activity. Similarly, in the essential oil of the roots, c-muurolene and b-cadinene were the major constituents. Cheng et al. (2005) had earlier reported antifungal activity of an essential oil of Japanese cedar heartwood and demonstrated that cadinene and muurolene were the major constituents (82.6 %) of oil, which have strong antifungal activity. In another study, Ambrosia trifida essential oil containing bornyl acetate as a major constituent showed high antifungal activity (Wang et al. 2006). Therefore, we infer that the antifungal activity of the essential oils may be attributed to the presence of these terpenoid constituents in the oils. However, synergy or antagonism among the various major and minor constituents are possibilities. Daferera et al. (2000) reported that the fungitoxic activity of essential oils might be a consequence of the formation of hydrogen bonds between the hydroxyl group in the oil constituents and active sites of target enzymes. The essential oils exhibited moderate to excellent antibacterial activity and the activity was bacteria specific (Table 3). The inflorescence oil was most active against the human pathogen K. pneumoniae while the root essential oil was most active against the Gram-positive bacteria S. aureus. The resistance of Gram-negative bacteria could be due to the complexity of their double-layered cell membrane in comparison to the single cell membrane of Gram-positive bacteria (Kalemba and Kunicka 2003). Other reports of the essential oil of E. adenophorum have shown moderate to significant antibacterial activity (200–12.5 mg mL-1) against Arthrobacter protophormiae, Escherichia coli, Micrococcus luteus, Rhodococcus rhodochrous and Staphylococcus aureus (Kurade et al. 2010). In addition, several studies indicate that the antimicrobial activity of essential oils is due to the ability of terpenes to affect permeability and other functions of cell membranes (Cristani et al. 2007). As far as we know, this is the first report of phytotoxicity of essential oils of E. adenophorum. The higher proportions of c-cadinene, c-muurolene, 3-acetoxyamorpha4,7(11)-diene-8-one, c-curcumene and bornyl acetate in the oils may be responsible for this phytotoxicity. An earlier study revealed that the radicle elongation of radish was significantly inhibited by Teucrium essential oil containing d- and c-cadinene as major constituents (De-Martino et al. 2010). Additionally, several terpenes like 1,8-cineole, citronellal, citronellol, linalool, a-pinene and limonene have been identified as potent inhibitors of seed germination and seedling growth (Romagni et al. 2000; Singh et al. 2006a, b). We suggest that the allelopathic activity of the E. adenophorum inflorescence and root essential oils may result from the combined additive or synergistic effect of their diverse constituents. Although the mode of inhibitory action of essential oils against germination still remains unclear, there are reports that volatile oils inhibit cell division and induce structural breaks and decomposition in roots (Singh et al. 2006b). 123 348 The antioxidant activity of essential oils is another biological property of great interest because they may preserve foods from the toxic effects of oxidants. Moreover, as scavengers of free radicals, essential oils may play a significant role in disease prevention (Aruoma 1998) through the antioxidant actions of their main constituents and/or other constituents in small quantities (Abdalla and Roozen 1999). The DPPH assay is often used as an indicator of free radical scavenging capacity which is an important mechanism of antioxidant activity. As evident from the IC50 values the inflorescence and root essential oils were approximately 100 times less effective than the positive control, BHT. The greater proportion of sesquiterpene hydrocarbons in the inflorescence essential oil might account for it’s low antioxidant activity compared to the root essential oil. Previous studies suggested that weak antioxidant activity of essential oils might be due to their sesquiterpenes hydrocarbons and oxygenated sesquiterpenes (Cavar et al. 2008). Further experiments are necessary to verify the relationship between chemical composition and antioxidant activity. Conclusion Chemical analyses of E. adenophorum essential oils revealed that the inflorescence essential oil was dominated by sesquiterpenoid constituents while the root essential oil was rich in monoterpenes. Both oils exhibited moderate but significant antifungal activity against plant pathogenic fungi and bacteria. The essential oils of inflorescences and roots showed considerable antifungal activity against M. phaseolina and R. solanii, whereas the inflorescence essential oil exhibited maximum inhibition and MIC against the human bacterial pathogen K. pneumoniae. Against S. aureus, the root essential oil was most effective. Both inflorescence and root essential oils displayed moderate to strong antioxidant activity. The phytotoxic effect of Eupatorium essential oils might be attributable to the presence of cadinenes and muurolene in the volatile oils. Interestingly, ours is the first report of E. adenophorum essential oils exhibiting phytotoxic and antioxidant activities. These essential oils have potential as antifungal and antibacterial agents as well as potential as a bio-herbicide. Further studies are required to determine activity under field conditions. Acknowledgments The authors are thankful to the Head, Department of Chemistry, HNB Garhwal University (A Central University), Srinagar, Uttarakhand, India and Head, Division of Agricultural Chemicals, IARI, New Delhi for providing the necessary facilities. The first author (VA) is thankful to National Innovative Agricultural Project (NAIP), Indian Council of Agricultural Research, New Delhi, India for financial support. 123 J Pest Sci (2014) 87:341–349 Conflict of interest interest. The authors declare that there is no conflict of References Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267 Abdalla AE, Roozen JP (1999) Effect of plant extracts on the oxidative stability of sunflower oil and emulsion. Food Chem 64:323–329 Adams RP (1995) Identification of essential oil components by gas chromatography-mass spectroscopy. 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