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Insecticidal Activity and Chemical Composition of Artemisia sieben Besser Essential Oil from Karaj, Iran
1. Asia-Pacific Entomol. 9 (1): 61 ~66 (2006) www.entomology.or.kr
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Insecticidal Activity and Chemical Composition of Artemisia sieberi Besser Essential Oil from Karaj, Iran Maryam Negahban', Saeid Moharramipour", Fatemeh Sefidkon2 IDepartment of Entomology, College of Agriculture, Tarbiat Modarres University, P. O. Box: 14115-336, Tehran, Iran 2Research Institute of Forests and Rangelands, P. O. Box: 13185-116, Tehran, Iran
Abstract Atremisia sieberi Besser is a widely distributed plant that grows in many areas of Iran and has strong insecticidal activity against stored product pests, so an experiment was conducted to investigate fumigant toxicity of the A. sieberi oil collected from Karaj region of Iran. The oil was applied against one to seven day old adults of three major stored product insects including: Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribollium castaneum (Herbst). The potency of fumigant toxicity of A. sieberi on C. maculatus was higher (LCso: 1.64 IJ.L per L) than S. oryzae (LCso: 4.41 IJ.L per L) and T. castaneum (LCso: 20.31 IJ.L per L). The relationships between the time exposure and oil concentration on mortality show that the mortality was increased as oil concentration and exposure time was increased. The concentration of 185 IJ.L per L and exposure time of 24h was enough to obtain 100% kill of the insects. It was also found that the regions where A. sieberi grows affect essential oil components of the plant and can play an important role in properties of fumigant toxicity. Key words Artemisia sieberi, fumigant tOXICIty, botanical insecticides, stored product insects, chemotype
Introduction Unbalanced and extensive uses of broad-spectrum pesticides have caused development of pesticide resistance, vast destruction of beneficial organisms, uncontrolled outbreak of secondary pests and undesirable environmental effect. Therefore, today there is a need to develop alternatives that is capable of reducing the large-scale utilization of synthetic *Corresponding author. E-mail: [email protected] Tel: +98-21-44196522; Fax: +98-21-44196524 (Received October 28, 2005; Accepted February 22, 2006)
pesticides for crop protection. Among methods used in integrated pest management, plants and their by products have played a significant role. Plants have always been rich source of chemicals and drugs for human (Amason et al., 1981). During the 20 th century a few of these natural compounds like nicotine, rotenone and pyrethrine have been used commercially as insecticide (Ware, 1798). A number of plant families are known to produce secondary metabolites having biological activity such as repellent, antifeedent and toxin (Philogene et al., 1981). During recent years, some plants have been receiving global attention and their secondary metabolites have been formulated as botanical pesticides in plant protection and biological control, since they do not leave toxic residues to the environment and have lower toxicity to mammalian and medicinal properties for human due to their natural origin (Duke, 1985). Artemisia species has been considerably exploited to contain toxic compounds. Many of these substances elaborated by the genus are toxic to pathogens or show other significant biological activity such as inhibition of the asexual reproduction of Aspergillus niger Tiegh and Penicillium italicum Wehmer (Tantaoui- Elaraki et al., 1993) and may be used in human diets or for animal fodder (Heywood and Humphries, 1977; Janssen et al., 1987). Moreover Artemisia like a large number of plants may be possessing insecticidal, repellent or antifeedent properties (Grainge and Ahmad, 1988; Amason et al., 1989; Jacobson, 1989; Shakarami et al. 2004a, b, c). Artemisia scoparia showed fumigant toxicity against stored product insects (Negahban et al. 2004, 2006; Negahban and Moharramipour, 2005). Extracts of Artemisia absinthium L. have been shown to possess a range of biological activities including insecticidal action of an alcoholic extract against the stored crop pest Sitophilus granarius L. (Ignatowicz and Wesolowska, 1994) and nematocidal action against Meoidogyne incognita (Kofoid & White) (Walker, 1978).
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J. Asia-Pacific Entomol. Vol. 9 (2006)
Essential oil from A. sieberi from dry lands near Qum, Iran, has been shown to possess fumigant toxicity against Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribollium castaneum (Herbst) (Negahban et al., 2006). Encouraged by these results, in the present study, fumigant toxicity of the essential oil of A. sieberi collected from the semi-arid lands of Karaj (in Tehran province) on three stored product insects and its chemical constituents was investigated and compared with those of results obtained previously from Qum collected plant.
a linear velocity of 30 cmls on DB- 5 column (30 m x 0.25 mm i.d, 0.25 urn film thickness). The oven was programmed to rise 60'C (3 min) isotherm, and then to 210 'C at a rate of 3'C Imin. Injector and detector temperatures were 300 and 270 'C, respectively. The GC mass analysis was carried out on a Varian 3400 equipped with a DB-5 column with the same characteristics as used in GC. The transfer line temperature was 260 'C. The ionization energy was 70 ev with a scan time of 1s and mass range of 40-300 amu. Unknown essential oil was identified by comparing its GC retention time to that of known compounds and by comparison of its mass spectra, either with known compounds or published spectra.
Materials and Methods Fumigant toxicity Insect rearing Callosobruchus maculatus, S. oryzae and T. castaneum were reared on bean grains, whole rice and wheat flour mixed with yeast (10:1, w/w) respectively. The cultures were maintained in the dark in growth chamber at 27 ± I 'C and 65 ± 5% R.H. All species had been kept in laboratory culture for over 3 years and were maintained as above conditions. All experiments were carried out under the same environmental conditions as the cultures.
Plant materials Aerial parts of A. sieberi (Asteracae) were collected at full flowering stage in December 2003 from Karaj region, Iran. The collected plant was dried naturally on laboratory benches at room temperature (23-2 7'C) for 5 days until it was crisp dry. The dried material was stored at -24 'C until needs and then hydrodistilled for recovery of its essential oil.
Extraction and analysis of the essential oil Essential oil was extracted from the plant samples using a Clevenger-Type apparatus where the plant material is subjected to hydrodistillation. Conditions of extraction were: 50 g of air-dried sample; 1:10 plant material/water volume ratio and 4 h distillation. The resulting oil was dried over anhydrous sodium sulfate (l0 min) and immediately placed into sealed glass tubes. Oil yield (2.9% w/w) was calculated on a dry weight basis. Extracted oil was stored in dark cold in a refrigerator at 4 'C. Gas chromatographic analysis was performed with a Shimadzu GC-9A with helium as a carrier gas with
To determine the fumigant toxicity of the A. sieberi oil, filter papers (2 em diameter) were impregnated with an appropriate concentrations (37 to 926 ul. per L) of the oil without using any solvent. Then the filter paper was attached to the under surface of the screw cap of a glass vial volume (27 mL). The cap was screwed tightly on the vial containing ten adults (1-7 days old with undefined sex) of each species of insect separately. Each concentration and control was replicated five times. Mortality was determined 3, 6, 9, 12 and 24 h after exposure. The mortality was calculated using the Abbott correction formula for natural mortality in untreated controls (Abbott, 1925). A bioassay was designed to determine median effective time to cause mortality of 50% of test insects (LTso values) at 37, 185 and 370 ul. per L air of the oil. The mortality was assessed by direct observation of the insects every hour for up to end point of mortality. Time-mortality data for each experiment were analysed by the method of finney (1971) with time as the explanatory variable to derive estimated hours for 50% mortality (LT 50). Estimates were compared usig overlap of the 95% fiducial limits. Non-overlap at the 95% fiducial limits is equivalent to a test for significant differences. Another experiment was designed to assess 50% (LCso) and 95% (LC9S) lethal doses. A series of dilutions were prepared to evaluate mortality of insects after an initial dose setting experiment. Control insects kept in the same condition without any essential oil. Each essential oil dose was replicated five times. Ten adult insects were put into each 280 mL glass bottle with screw lids. The number of dead and live insects in each bottle was counted 24 h after initial exposure to the essential oil. When no leg or antennal movements were observed, insects were
Insecticidal activity of Artemisia sieberi Essential Oil
the insecticidal activity of A. sieberi oil against C. maculatus, S. oryzae and T. castaneum adults was attributable to fumigant action. In all cases, a strong difference in mortality of the insects was observed as oil concentration and exposure time was increased. From the graph in Fig. 1, A. sieberi oil was relatively more toxic against C. maculatus than S. oryzae and T. castaneum. The lowest concentration (37 IJ.L per L) of the oil was able to induce 100% mortality of C. maculatus after 24h of exposure. Mortality of S. oryzae at the same concentration and exposure time was 92%, however complete mortality were achieved at 185 IJ.L per L after 24h of exposure. At 556 IJ.L per L the oil caused about 50 and 100% mortality against C. maculatus 3 and 9h after exposure, respectively, while at the same
considered dead. The treatment bottles were monitored at least 48 h after recording the data and no recoveries were taken into consideration. Then probit analysis (Finney, 1971) was employed in analyzing the dose-mortality response to estimate LC 50 and LC95 values using SAS program (SAS Institute, 1997).
Results Fumigant toxicity Experiments were conducted to determine whether
100
100
80
so
60
60
'10
'10
20
20
0 6
3
100
9
15
12
60 '10
18
21
24
6
12
9
18
15
21
24
80 60 '10
370~
444~
20 0
0 0
6
3
j
~
3
0
20
oa
185~
100
/;;
80
.'::
r:
0 0
e-,
63
9
15
12
.:
100 80 60 '10
21
18
0
24
3
6
9
12
15
18
21
24
100 80 60 '10
556~
741~
20
20
0
0 0
6
3
12
9
15
16
21
0
24
3
6
9
12
15
18
21
24
Exposure time (hours) 100
so 60 '10
926~
20 0 0
3
6
9
12
15
16
21
24
Exposure time (hours)
Fig. 1. Percentage mortality of Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum exposed to essential oil of Artemisia (Karaj, Iran) impregnated of filter paper discs.
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J. Asia-Pacific Entomol. Vol. 9 (2006)
concentration 100% mortality was achieved on S. oryzae and T castaneum after 24h. At highest concentration (926 !1L per L), the mortality of C. maculatus was obtained 100% 9h after treatment (Fig. 1). On the basis of the LT50s, C. maculatus was killed quicker than S. oryzae and T castaneum. For C. maculatus, LTso values were ranged from 3.09 h for the highest dose (37 !1L per L air) to 7.19 h for the lowest dose (37 !1L per L air) (Table 1). The estimate of LTsoS for S. oryzae was decreased from 13.09 h for 370 !1L per L to 8.83 h for 370 !1L per L air. With the T castaneum, LTso values were ranged from 11.96 h for the highest dose to 44.84 h for the lowest dose. On the basis of fiducial limits presented in Table 1, LT50 values at higher doses (185 and 370 !1L per L air) dose not seem to be
dose-dependent. The results of probit analysis showed that C. maculatus was comparatively more susceptible (LCso = 1.64 !1L per L) than S. oryzae (LCso= 4.41 !1L per L) and Ticastaneum (LCso= 20.31 !1L per L). LC9SS of A. sieberi oil were achieved at 8.84, 18.06 and 76.03 !1L per L on the following incest species respectively (Table 2).
Chemical constituents of A. sieberi The results of the chemical analysis are presented in Table 3. Twenty nine compounds in the oil were identified. The main constituents of the oils were Chrysanthenone (29.50%), Camphor (19.80%), 1,8Cineol (14.50%), Camphene (4.18%), Borneol (2.45%),
Table 1. LTses of Artemisia sieberi oil against Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum Insect species
C. maculatus
S. oryzae
T. castaneum
I
Concentration (ul, per Lair)
LTso (h)'
Slope±SE
Degree of freedom
Chi square (X 2)
37
7.19 (6.55-7.92)
2.86±0.33
8
7.61
185
4.06 (3.05-4.50)
3.48±0.45
4
4.56
370
3.09 (2.68-3.51)
2.57±0.29
5
4.81
37
13.09 (12.35-13.77)
4.73±0.47
11
2.96
185
10.25 (9.47-10.99)
3.35±0.37
11
4.23
370
8.83 (8.05-9.50)
3.61±0.44
9
3.79
37
44.84 (43.93-45.76)
9.32±0.78
18
0.86
185
13.02 (12.17-13.75)
4.83±0.61
9
4.19
370
11.96 (11.26-12.58)
5.38±0.64
8
2.54
95% lower and upper fiducial limits are shown in parenthesis.
Table 2. Fumigant toxicity of Artemisia sieberi oil (Karaj, Iran) against Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum
,
I
,
LC so (ul, per Lair)
LC95 (llL per Lair)
Slope±SEM
Degree of freedom
Chi square (X 2)
C. maculatus
1.64 (1.42-1.90)
8.84 (5.79-19.58)
2.25±0.36
5
7.11
S. oryzae
4.41 (3.97-4.92)
18.06 (13.91-26.3)
2.68±O.26
8
2.10
T. castaneum
20.31 (18.1-22.79)
76.03 (54.76-140.17)
2.87±0.45
5
5.93
Insect species
95% lower and upper fiducial limits are shown in parenthesis.
Insecticidal activity of Artemisia sieberi Essential Oil
Table 3. Chemical constituents of the essential oil from Artemisia sieberi (Karaj, Iran) Retention Index
% Composition
a-Thujene
921
0.17
a-Pinene
940
0.49
Camphene
948
4.18
Sabinene
966
0.39
13-Pinene
977
0.35
Myrcene
990
1.55
a-Phellndrene
1007
0.22
a-Terpinene
1013
0.15
p-Cymene
1021
3.03
1,8-Cineole
1028
14.50
(2)-13-0cimene
1037
0.03
(E)-13-0cimene
1048
1.06
cis-Sabinene hydrate
1068
0.87
a-Pinene oxide
1086
0.92
Nonanol
1103
2.08
f3-Thujene
1114
0.99
Chrysanthenone
1118
29.50
trans-pinocarveol
1133
2.54
Camphor
1140
19.80
Isoborneol
1156
0.58
Borneol
1159
2.45
Terpinene-4-oI
1172
0.94
p-Cymene-8-o1
1180
0.49
Myrtenol
1189
0.55
cis-Piperitol
1192
0.49
trans-piperitol
1203
1.32
Piperitone
1257
0.82
Cyciohexanol acetate
1310
2.63
13-Bourbonene
1390
0.29
Constituents
Other components
6.62
Cyclohexanol acetate (2.63%), Trans-pinocarveol (2.54%), P-cymene (3.03%), Nonanal (2.08%), Myrcene (1.55%), (E)-[3-0cymene (1.06%) and Transpiperitol (1.32%). Therefore, in this study Chrysanthenone, Camphor, 1,8-Cineol and Camphene were found to be the major constituents and accounted for 67.98% of the total oil.
65
Discussion Artemisia is a genus that grows in many areas of Iran. The essential oil of A. sieberi demonstrated fumigant toxicity against C. maculatus, S. oryzae and T. castaneum. The insecticidal activity varied
with insect species, concentrations of the oil and exposure time. The results showed high mortality rates in C. maculatus compared to S. oryzae and T. castaneum. Also, Papachristes and Stamopoulos (2002) have reported the higher susceptibility of the C. maculatus than T. castaneum. Moreover, our results indicated that the higher concentrations of the oil for a relatively short period are much more effective than lower concentrations for a long period. The Karaj A. sieberi oil showed potent toxicity giving 90-100% mortality within 24h exposure at 37 ul, per L of air for C. maculatus and S. oryzae. Lee et al. (2004) found that the essential oils from 42 species of Myrtacae caused 95% mortality at 45-50 ul, per L of air after 24h exposure against S. oryzae and LCsos were achieved 19 ul, per L to more than 100 ul, per L according to plant species. The A. sieberi from Karaj oil in the current study may be accounted more toxic than Myrtacae family since its LCso (4.41IlL per L) was at least four times more potent than results obtained by Lee et al (2004). The results of Qum region collected A. sieberi oil showed more toxic than Karaj oil against stored product insects and LCsos for C. maculatus, S. oryzae and T. castaneum were lower than results obtained from the present study (Negahban et al., 2006). At the concentration of 444 ul. per L the mortality obtained 100% after 9h for C. maculatus but 24h for S. oryzae and T. castaneum. However, the Karaj essential oil of A. sieberi might be extra fumigant toxicity compared with other related species. At the same concentrations, the slopes of the mortality curve from the Qum oil were very steep compared with the Karaj oil (Negahban et ai, 2006). For instance at 37 ul. per L the mortality was 100% for T. castaneum after 24h but Karaj oil did not show any mortality at the same condition. The chemical constituents of the Karaj and Qum collected A. sieberi oils, which were extracted with the same conditions, were compared. Camphor (54.67%), Camphene (11.73%) and 1,8-Cineol (9.90%) were found to be the major constituents in Qum oil (Negahban et al., 2006), whereas, Chrysanthenone (29.50%), Camphor (19.80%), 1,8-Cineol (14.50%) and Camphene (4.18%) were the major constituents in Karaj oil. It may be introduced the Qum and Karaj collected A. sieberi oils as new Camphor and Chrysanthenone chemotypes, respectively, however
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it need to be furthur investigated. Chemotypes are of especial interest in the world of essential oils. These are marked by differences in products of secondary metabolites such as essential oil composition that can occur even in morphologically stable species (Lahlou, 2004). It is probable however that many chemotypes of common aromatic plants have yet to be properly identified. The monoterpene Camphor might be a broad insecticidal activity against stored insects, since in a more detailed study Dunkel and Sears (1998) demonstrated potent toxic effects of Camphor from A. tridentata Nutt against T castaneum. Also, ObengOferi et al. (1997) found that 1,8-Cineol to be highly repellent and toxic against some stored product beetles. Therefore, higher toxicity of the Qum oil, as part, could be attributed to higher concentrations of the Champhor. These results showed that the chemical composition of the essential oil from A. sieberi could be changed according with geographical distribution and might be an effective factor on its insecticidal activity.
Literature Cited Abbott, W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Amason, J.T., T. Johns and R. Hebda. 1981. Use of plants for food and medicine by native peoples of Canada. Can. J. Bot. 59: 2189-2375. Amason, J.T., B.J.R. Philogene and P. Morand. 1989. Insecticides of plant origin. 224 pp. American Chemical Society, Washington, D.e. Duke. J.A. 1985. Handbook of medicinal herbs. 896 pp. CRC Press. Boca Roton. FL. Dunkel, F.V.and L.J. Sears. 1998. Fumigant properties of physical preparations from mountain big Sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects. J. Stored. Prod. Res. 34: 307321. Finney, DJ. 1971. Probit analysis, 3rd Edition. 333 pp. Cambridge University Press, London. Grainge, M. and S. Ahmed. 1988. Handbook of plants with pest-control properties. Wiley-Interscience, New York. Heywood, V.H. and G.J. Humphries. 1977. Anthemideae systematic review. vol. 2. pp. 851-898, in, The biology and chemistry of the Compositae Eds. V.H. Heywood, J.B. Harbone and B. Turner. Academic Press, London, New York. Ignatowicz, S. and B. Wesolowska. 1994. Insecticidal and deterrent properties of extracts from herbaceous plants. Ochrona Roslin. 38 (9): 14-15. Jacobson, M. 1989. Botanical pesticides: past, present, and future. pp. 1-10. in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogene and P. Morand. 224 pp. ACS Symposium Series No. 387. American Chemical Society, Washington DC. Janssen, A.M., Ll.C, Scheffer, A. Baerchein-Svendsen and A.B. Svendsen. 1987. Antimicrobial activities of essential oils. A 1976-1986 literature review on possible
applications. Pharrnaceut. Weekbl. 9: 193-197. Lahlou, M. 2004. Essential oils and fragrance compounds: bioactivity and mechanisms of action. Flavour and Fragrance J. 19: 159-165. Lee, B.B., P.C. Annis, F. Tumaalii and W.S. Choi. 2004. Fumigant toxicity of essential oils from the Myrtaceae family and 1,8-Cineole against 3 major stored-grain insects. J. Stored. Prod. Res. 40: 553-564. Negahban, M. and S. Moharramipour. 2005. Fumigant toxicity of essential oil from Artemisia scoparia Waldst & Kit. against Sitophilus oryzae (Coleo;tera: Curculionidae). p. 124, in Proceeding of the 2" Symposium of Medicinal Plants. Tehran, Iran. Negahban, M., S. Moharramipour and F. Sefidkon. 2006. Fumigant toxicity of essential oil from Artemisia sieberi Besser against three stored product insects. J. Stored. Prod. Res. (in press). Negahban, M., S. Moharramipour and M. Yousefelahi. 2004. Efficacy of essential oil from A. scoparia Waldst. & Kit. against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). P. 153, in Proceeding of the Fourth International Iran and Russia Conference Agriculture and Natural Resources, Shahrekord, Iran. Obeng-Ofori, D., C.H. Reichmuth, J. Bekele and A. Hassanali. 1997. Biological activity of 1,8-Cineol, a major component of essential oil of Ocimum kenyense (Ayobangira) against stored product beetles. J. Appl. Entomol. 121: 237-243. Papachristes, D.P. and D.C Stamopoulos. 2002. Toxicity of vapours of three essential oils to the immature stages of Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae). J. Stored. Prod. Res. 38: 365-373. Philogene, B.J.R. 1981. Secondary plant substance and insects: alkaloids phenolics. Ann. Entomol. Soc. Quessec. 26: 177 -187. SAS Institute. 1997. SAS users guide. 135 pp. SAS Institute, USA. Shakarami, J., K. Kamali and S. Moharramipour. 2004a. Application of Artemisia aucheri Boiss as a botanical insecticide. P. 31, in Proceeding of the 1st Iranian National Seminar on Development of Agrochemical Industries, Tehran, Iran. Shakarami, J., K. Kamali and S. Moharramipour. 2004b. Effect of essential oil of Artemisia aucheri Boiss on stored- product pest. P. 479, in Proceedings of the 3'd National Conference on the Development in the Application of Biological Products and Optimum Utilization of Chemical Fertilizers and Pesticides in Agriculture, Karaj, Iran. Shakarami, J., K. Kamali., S. Moharramipour. and P. Lachinani. 2004c. Insecticidal and deterrenccy effects of essential oils of Artemisia aucheri Boiss, Salvia braeteata L. and Nepeta cataria L. on Callosobruchus maculatus F. p. 490, in Proceedings of the 3'd National Conference on the Development in the Application of Biological Products and Optimum Utilization of Chemical Fertilizers and Pesticides in Agriculture, Tehran, Iran. Tantaoui-Elaraki, A., F. Ferhout and A. Errifi. 1993. Inhibition of the fungal asexual reproduction stages by three Moroccan essential oils. J. Essen. Oil. Res. 5: 535-545. Walker, J.T. 1995. Garden herbs as hosts for southern rootnot nematode (Meloidogyne incognita (Kofoid & White) Chitwood race 3). HortSci, 30: 292-293. Ware, G.W. 1978. Pesticides, theory and application. 308 pp. Freeman Co, New York.
PEST MANAGEMENT
Insecticidal Activity and Chemical Composition of Artemisia sieberi Besser Essential Oil from Karaj, Iran Maryam Negahban', Saeid Moharramipour", Fatemeh Sefidkon2 IDepartment of Entomology, College of Agriculture, Tarbiat Modarres University, P. O. Box: 14115-336, Tehran, Iran 2Research Institute of Forests and Rangelands, P. O. Box: 13185-116, Tehran, Iran
Abstract Atremisia sieberi Besser is a widely distributed plant that grows in many areas of Iran and has strong insecticidal activity against stored product pests, so an experiment was conducted to investigate fumigant toxicity of the A. sieberi oil collected from Karaj region of Iran. The oil was applied against one to seven day old adults of three major stored product insects including: Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribollium castaneum (Herbst). The potency of fumigant toxicity of A. sieberi on C. maculatus was higher (LCso: 1.64 IJ.L per L) than S. oryzae (LCso: 4.41 IJ.L per L) and T. castaneum (LCso: 20.31 IJ.L per L). The relationships between the time exposure and oil concentration on mortality show that the mortality was increased as oil concentration and exposure time was increased. The concentration of 185 IJ.L per L and exposure time of 24h was enough to obtain 100% kill of the insects. It was also found that the regions where A. sieberi grows affect essential oil components of the plant and can play an important role in properties of fumigant toxicity. Key words Artemisia sieberi, fumigant tOXICIty, botanical insecticides, stored product insects, chemotype
Introduction Unbalanced and extensive uses of broad-spectrum pesticides have caused development of pesticide resistance, vast destruction of beneficial organisms, uncontrolled outbreak of secondary pests and undesirable environmental effect. Therefore, today there is a need to develop alternatives that is capable of reducing the large-scale utilization of synthetic *Corresponding author. E-mail: [email protected] Tel: +98-21-44196522; Fax: +98-21-44196524 (Received October 28, 2005; Accepted February 22, 2006)
pesticides for crop protection. Among methods used in integrated pest management, plants and their by products have played a significant role. Plants have always been rich source of chemicals and drugs for human (Amason et al., 1981). During the 20 th century a few of these natural compounds like nicotine, rotenone and pyrethrine have been used commercially as insecticide (Ware, 1798). A number of plant families are known to produce secondary metabolites having biological activity such as repellent, antifeedent and toxin (Philogene et al., 1981). During recent years, some plants have been receiving global attention and their secondary metabolites have been formulated as botanical pesticides in plant protection and biological control, since they do not leave toxic residues to the environment and have lower toxicity to mammalian and medicinal properties for human due to their natural origin (Duke, 1985). Artemisia species has been considerably exploited to contain toxic compounds. Many of these substances elaborated by the genus are toxic to pathogens or show other significant biological activity such as inhibition of the asexual reproduction of Aspergillus niger Tiegh and Penicillium italicum Wehmer (Tantaoui- Elaraki et al., 1993) and may be used in human diets or for animal fodder (Heywood and Humphries, 1977; Janssen et al., 1987). Moreover Artemisia like a large number of plants may be possessing insecticidal, repellent or antifeedent properties (Grainge and Ahmad, 1988; Amason et al., 1989; Jacobson, 1989; Shakarami et al. 2004a, b, c). Artemisia scoparia showed fumigant toxicity against stored product insects (Negahban et al. 2004, 2006; Negahban and Moharramipour, 2005). Extracts of Artemisia absinthium L. have been shown to possess a range of biological activities including insecticidal action of an alcoholic extract against the stored crop pest Sitophilus granarius L. (Ignatowicz and Wesolowska, 1994) and nematocidal action against Meoidogyne incognita (Kofoid & White) (Walker, 1978).
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J. Asia-Pacific Entomol. Vol. 9 (2006)
Essential oil from A. sieberi from dry lands near Qum, Iran, has been shown to possess fumigant toxicity against Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.), and Tribollium castaneum (Herbst) (Negahban et al., 2006). Encouraged by these results, in the present study, fumigant toxicity of the essential oil of A. sieberi collected from the semi-arid lands of Karaj (in Tehran province) on three stored product insects and its chemical constituents was investigated and compared with those of results obtained previously from Qum collected plant.
a linear velocity of 30 cmls on DB- 5 column (30 m x 0.25 mm i.d, 0.25 urn film thickness). The oven was programmed to rise 60'C (3 min) isotherm, and then to 210 'C at a rate of 3'C Imin. Injector and detector temperatures were 300 and 270 'C, respectively. The GC mass analysis was carried out on a Varian 3400 equipped with a DB-5 column with the same characteristics as used in GC. The transfer line temperature was 260 'C. The ionization energy was 70 ev with a scan time of 1s and mass range of 40-300 amu. Unknown essential oil was identified by comparing its GC retention time to that of known compounds and by comparison of its mass spectra, either with known compounds or published spectra.
Materials and Methods Fumigant toxicity Insect rearing Callosobruchus maculatus, S. oryzae and T. castaneum were reared on bean grains, whole rice and wheat flour mixed with yeast (10:1, w/w) respectively. The cultures were maintained in the dark in growth chamber at 27 ± I 'C and 65 ± 5% R.H. All species had been kept in laboratory culture for over 3 years and were maintained as above conditions. All experiments were carried out under the same environmental conditions as the cultures.
Plant materials Aerial parts of A. sieberi (Asteracae) were collected at full flowering stage in December 2003 from Karaj region, Iran. The collected plant was dried naturally on laboratory benches at room temperature (23-2 7'C) for 5 days until it was crisp dry. The dried material was stored at -24 'C until needs and then hydrodistilled for recovery of its essential oil.
Extraction and analysis of the essential oil Essential oil was extracted from the plant samples using a Clevenger-Type apparatus where the plant material is subjected to hydrodistillation. Conditions of extraction were: 50 g of air-dried sample; 1:10 plant material/water volume ratio and 4 h distillation. The resulting oil was dried over anhydrous sodium sulfate (l0 min) and immediately placed into sealed glass tubes. Oil yield (2.9% w/w) was calculated on a dry weight basis. Extracted oil was stored in dark cold in a refrigerator at 4 'C. Gas chromatographic analysis was performed with a Shimadzu GC-9A with helium as a carrier gas with
To determine the fumigant toxicity of the A. sieberi oil, filter papers (2 em diameter) were impregnated with an appropriate concentrations (37 to 926 ul. per L) of the oil without using any solvent. Then the filter paper was attached to the under surface of the screw cap of a glass vial volume (27 mL). The cap was screwed tightly on the vial containing ten adults (1-7 days old with undefined sex) of each species of insect separately. Each concentration and control was replicated five times. Mortality was determined 3, 6, 9, 12 and 24 h after exposure. The mortality was calculated using the Abbott correction formula for natural mortality in untreated controls (Abbott, 1925). A bioassay was designed to determine median effective time to cause mortality of 50% of test insects (LTso values) at 37, 185 and 370 ul. per L air of the oil. The mortality was assessed by direct observation of the insects every hour for up to end point of mortality. Time-mortality data for each experiment were analysed by the method of finney (1971) with time as the explanatory variable to derive estimated hours for 50% mortality (LT 50). Estimates were compared usig overlap of the 95% fiducial limits. Non-overlap at the 95% fiducial limits is equivalent to a test for significant differences. Another experiment was designed to assess 50% (LCso) and 95% (LC9S) lethal doses. A series of dilutions were prepared to evaluate mortality of insects after an initial dose setting experiment. Control insects kept in the same condition without any essential oil. Each essential oil dose was replicated five times. Ten adult insects were put into each 280 mL glass bottle with screw lids. The number of dead and live insects in each bottle was counted 24 h after initial exposure to the essential oil. When no leg or antennal movements were observed, insects were
Insecticidal activity of Artemisia sieberi Essential Oil
the insecticidal activity of A. sieberi oil against C. maculatus, S. oryzae and T. castaneum adults was attributable to fumigant action. In all cases, a strong difference in mortality of the insects was observed as oil concentration and exposure time was increased. From the graph in Fig. 1, A. sieberi oil was relatively more toxic against C. maculatus than S. oryzae and T. castaneum. The lowest concentration (37 IJ.L per L) of the oil was able to induce 100% mortality of C. maculatus after 24h of exposure. Mortality of S. oryzae at the same concentration and exposure time was 92%, however complete mortality were achieved at 185 IJ.L per L after 24h of exposure. At 556 IJ.L per L the oil caused about 50 and 100% mortality against C. maculatus 3 and 9h after exposure, respectively, while at the same
considered dead. The treatment bottles were monitored at least 48 h after recording the data and no recoveries were taken into consideration. Then probit analysis (Finney, 1971) was employed in analyzing the dose-mortality response to estimate LC 50 and LC95 values using SAS program (SAS Institute, 1997).
Results Fumigant toxicity Experiments were conducted to determine whether
100
100
80
so
60
60
'10
'10
20
20
0 6
3
100
9
15
12
60 '10
18
21
24
6
12
9
18
15
21
24
80 60 '10
370~
444~
20 0
0 0
6
3
j
~
3
0
20
oa
185~
100
/;;
80
.'::
r:
0 0
e-,
63
9
15
12
.:
100 80 60 '10
21
18
0
24
3
6
9
12
15
18
21
24
100 80 60 '10
556~
741~
20
20
0
0 0
6
3
12
9
15
16
21
0
24
3
6
9
12
15
18
21
24
Exposure time (hours) 100
so 60 '10
926~
20 0 0
3
6
9
12
15
16
21
24
Exposure time (hours)
Fig. 1. Percentage mortality of Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum exposed to essential oil of Artemisia (Karaj, Iran) impregnated of filter paper discs.
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J. Asia-Pacific Entomol. Vol. 9 (2006)
concentration 100% mortality was achieved on S. oryzae and T castaneum after 24h. At highest concentration (926 !1L per L), the mortality of C. maculatus was obtained 100% 9h after treatment (Fig. 1). On the basis of the LT50s, C. maculatus was killed quicker than S. oryzae and T castaneum. For C. maculatus, LTso values were ranged from 3.09 h for the highest dose (37 !1L per L air) to 7.19 h for the lowest dose (37 !1L per L air) (Table 1). The estimate of LTsoS for S. oryzae was decreased from 13.09 h for 370 !1L per L to 8.83 h for 370 !1L per L air. With the T castaneum, LTso values were ranged from 11.96 h for the highest dose to 44.84 h for the lowest dose. On the basis of fiducial limits presented in Table 1, LT50 values at higher doses (185 and 370 !1L per L air) dose not seem to be
dose-dependent. The results of probit analysis showed that C. maculatus was comparatively more susceptible (LCso = 1.64 !1L per L) than S. oryzae (LCso= 4.41 !1L per L) and Ticastaneum (LCso= 20.31 !1L per L). LC9SS of A. sieberi oil were achieved at 8.84, 18.06 and 76.03 !1L per L on the following incest species respectively (Table 2).
Chemical constituents of A. sieberi The results of the chemical analysis are presented in Table 3. Twenty nine compounds in the oil were identified. The main constituents of the oils were Chrysanthenone (29.50%), Camphor (19.80%), 1,8Cineol (14.50%), Camphene (4.18%), Borneol (2.45%),
Table 1. LTses of Artemisia sieberi oil against Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum Insect species
C. maculatus
S. oryzae
T. castaneum
I
Concentration (ul, per Lair)
LTso (h)'
Slope±SE
Degree of freedom
Chi square (X 2)
37
7.19 (6.55-7.92)
2.86±0.33
8
7.61
185
4.06 (3.05-4.50)
3.48±0.45
4
4.56
370
3.09 (2.68-3.51)
2.57±0.29
5
4.81
37
13.09 (12.35-13.77)
4.73±0.47
11
2.96
185
10.25 (9.47-10.99)
3.35±0.37
11
4.23
370
8.83 (8.05-9.50)
3.61±0.44
9
3.79
37
44.84 (43.93-45.76)
9.32±0.78
18
0.86
185
13.02 (12.17-13.75)
4.83±0.61
9
4.19
370
11.96 (11.26-12.58)
5.38±0.64
8
2.54
95% lower and upper fiducial limits are shown in parenthesis.
Table 2. Fumigant toxicity of Artemisia sieberi oil (Karaj, Iran) against Callosobruchus maculatus, Sitophilus oryzae and Tribolium castaneum
,
I
,
LC so (ul, per Lair)
LC95 (llL per Lair)
Slope±SEM
Degree of freedom
Chi square (X 2)
C. maculatus
1.64 (1.42-1.90)
8.84 (5.79-19.58)
2.25±0.36
5
7.11
S. oryzae
4.41 (3.97-4.92)
18.06 (13.91-26.3)
2.68±O.26
8
2.10
T. castaneum
20.31 (18.1-22.79)
76.03 (54.76-140.17)
2.87±0.45
5
5.93
Insect species
95% lower and upper fiducial limits are shown in parenthesis.
Insecticidal activity of Artemisia sieberi Essential Oil
Table 3. Chemical constituents of the essential oil from Artemisia sieberi (Karaj, Iran) Retention Index
% Composition
a-Thujene
921
0.17
a-Pinene
940
0.49
Camphene
948
4.18
Sabinene
966
0.39
13-Pinene
977
0.35
Myrcene
990
1.55
a-Phellndrene
1007
0.22
a-Terpinene
1013
0.15
p-Cymene
1021
3.03
1,8-Cineole
1028
14.50
(2)-13-0cimene
1037
0.03
(E)-13-0cimene
1048
1.06
cis-Sabinene hydrate
1068
0.87
a-Pinene oxide
1086
0.92
Nonanol
1103
2.08
f3-Thujene
1114
0.99
Chrysanthenone
1118
29.50
trans-pinocarveol
1133
2.54
Camphor
1140
19.80
Isoborneol
1156
0.58
Borneol
1159
2.45
Terpinene-4-oI
1172
0.94
p-Cymene-8-o1
1180
0.49
Myrtenol
1189
0.55
cis-Piperitol
1192
0.49
trans-piperitol
1203
1.32
Piperitone
1257
0.82
Cyciohexanol acetate
1310
2.63
13-Bourbonene
1390
0.29
Constituents
Other components
6.62
Cyclohexanol acetate (2.63%), Trans-pinocarveol (2.54%), P-cymene (3.03%), Nonanal (2.08%), Myrcene (1.55%), (E)-[3-0cymene (1.06%) and Transpiperitol (1.32%). Therefore, in this study Chrysanthenone, Camphor, 1,8-Cineol and Camphene were found to be the major constituents and accounted for 67.98% of the total oil.
65
Discussion Artemisia is a genus that grows in many areas of Iran. The essential oil of A. sieberi demonstrated fumigant toxicity against C. maculatus, S. oryzae and T. castaneum. The insecticidal activity varied
with insect species, concentrations of the oil and exposure time. The results showed high mortality rates in C. maculatus compared to S. oryzae and T. castaneum. Also, Papachristes and Stamopoulos (2002) have reported the higher susceptibility of the C. maculatus than T. castaneum. Moreover, our results indicated that the higher concentrations of the oil for a relatively short period are much more effective than lower concentrations for a long period. The Karaj A. sieberi oil showed potent toxicity giving 90-100% mortality within 24h exposure at 37 ul, per L of air for C. maculatus and S. oryzae. Lee et al. (2004) found that the essential oils from 42 species of Myrtacae caused 95% mortality at 45-50 ul, per L of air after 24h exposure against S. oryzae and LCsos were achieved 19 ul, per L to more than 100 ul, per L according to plant species. The A. sieberi from Karaj oil in the current study may be accounted more toxic than Myrtacae family since its LCso (4.41IlL per L) was at least four times more potent than results obtained by Lee et al (2004). The results of Qum region collected A. sieberi oil showed more toxic than Karaj oil against stored product insects and LCsos for C. maculatus, S. oryzae and T. castaneum were lower than results obtained from the present study (Negahban et al., 2006). At the concentration of 444 ul. per L the mortality obtained 100% after 9h for C. maculatus but 24h for S. oryzae and T. castaneum. However, the Karaj essential oil of A. sieberi might be extra fumigant toxicity compared with other related species. At the same concentrations, the slopes of the mortality curve from the Qum oil were very steep compared with the Karaj oil (Negahban et ai, 2006). For instance at 37 ul. per L the mortality was 100% for T. castaneum after 24h but Karaj oil did not show any mortality at the same condition. The chemical constituents of the Karaj and Qum collected A. sieberi oils, which were extracted with the same conditions, were compared. Camphor (54.67%), Camphene (11.73%) and 1,8-Cineol (9.90%) were found to be the major constituents in Qum oil (Negahban et al., 2006), whereas, Chrysanthenone (29.50%), Camphor (19.80%), 1,8-Cineol (14.50%) and Camphene (4.18%) were the major constituents in Karaj oil. It may be introduced the Qum and Karaj collected A. sieberi oils as new Camphor and Chrysanthenone chemotypes, respectively, however
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J. Asia-Pacific Entomol. Vol. 9 (2006)
it need to be furthur investigated. Chemotypes are of especial interest in the world of essential oils. These are marked by differences in products of secondary metabolites such as essential oil composition that can occur even in morphologically stable species (Lahlou, 2004). It is probable however that many chemotypes of common aromatic plants have yet to be properly identified. The monoterpene Camphor might be a broad insecticidal activity against stored insects, since in a more detailed study Dunkel and Sears (1998) demonstrated potent toxic effects of Camphor from A. tridentata Nutt against T castaneum. Also, ObengOferi et al. (1997) found that 1,8-Cineol to be highly repellent and toxic against some stored product beetles. Therefore, higher toxicity of the Qum oil, as part, could be attributed to higher concentrations of the Champhor. These results showed that the chemical composition of the essential oil from A. sieberi could be changed according with geographical distribution and might be an effective factor on its insecticidal activity.
Literature Cited Abbott, W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Amason, J.T., T. Johns and R. Hebda. 1981. Use of plants for food and medicine by native peoples of Canada. Can. J. Bot. 59: 2189-2375. Amason, J.T., B.J.R. Philogene and P. Morand. 1989. Insecticides of plant origin. 224 pp. American Chemical Society, Washington, D.e. Duke. J.A. 1985. Handbook of medicinal herbs. 896 pp. CRC Press. Boca Roton. FL. Dunkel, F.V.and L.J. Sears. 1998. Fumigant properties of physical preparations from mountain big Sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects. J. Stored. Prod. Res. 34: 307321. Finney, DJ. 1971. Probit analysis, 3rd Edition. 333 pp. Cambridge University Press, London. Grainge, M. and S. Ahmed. 1988. Handbook of plants with pest-control properties. Wiley-Interscience, New York. Heywood, V.H. and G.J. Humphries. 1977. Anthemideae systematic review. vol. 2. pp. 851-898, in, The biology and chemistry of the Compositae Eds. V.H. Heywood, J.B. Harbone and B. Turner. Academic Press, London, New York. Ignatowicz, S. and B. Wesolowska. 1994. Insecticidal and deterrent properties of extracts from herbaceous plants. Ochrona Roslin. 38 (9): 14-15. Jacobson, M. 1989. Botanical pesticides: past, present, and future. pp. 1-10. in Insecticides of plant origin, Eds. J.T. Amason, BJ.R. Philogene and P. Morand. 224 pp. ACS Symposium Series No. 387. American Chemical Society, Washington DC. Janssen, A.M., Ll.C, Scheffer, A. Baerchein-Svendsen and A.B. Svendsen. 1987. Antimicrobial activities of essential oils. A 1976-1986 literature review on possible
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