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Archives Of Phytopathology And Plant Protection
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Contact and residual toxicities of 30 plant extracts to Colorado potato beetle larvae
Ayhan Gökçe a; Mark E. Whalon b; Halt Çam a; Yusuf Yanar a; İbrahm Demrtaş c; Nezhun Gőren d a Department of Plant Protection, Gaziosmanpa a University, Tokat, Turkey b Department of Entomology, Michigan State University East Lansing, USA c Department of Chemistry Faculty of Arts and Sciences, Gaziosmanpa a University, Tokat, Turkey d Department of Biology Faculty of Arts and Sciences, Yildiz Teknik Univesity Davutpa a Kamp
s
, Istanbul, Turkey
First Published on: 15 June 2006 To cite this Article: Gökçe, Ayhan, Whalon, Mark E., Çam, Halt, Yanar, Yusuf, Demrtaş, İbrahm and Gőren, Nezhun (2006) 'Contact and residual toxicities of 30 plant extracts to Colorado potato beetle larvae', Archives Of Phytopathology And Plant Protection, 40:6, 441 - 450 To link to this article: DOI: 10.1080/03235400600628013 URL: http://dx.doi.org/10.1080/03235400600628013
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Archives of Phytopathology and Plant Protection December 2007; 40(6): 441 – 450
Contact and residual toxicities of 30 plant extracts to Colorado potato beetle larvae
_ C ¨ KC AYHAN GO ¸ E1, MARK E. WHALON2, HALIT ¸ AM1, YUSUF YANAR1, 3 4 } _ _ DEMIRTAS _ IBRAH IM ¸ , & NEZHUN GOREN 1
Department of Plant Protection, Gaziosmanpas¸a University, Tas¸liic¸iftlik Yerles¸kesi, Tokat, Turkey, Department of Entomology Michigan State University East Lansing, USA, 3Department of Chemistry Faculty of Arts and Sciences Gaziosmanpas¸a University, Tokat, Turkey, and 4Department of Biology Faculty of Arts and Sciences Yildiz Teknik Univesity Davutpas¸a Kamp} us} u, Istanbul, Turkey
2
(Received 31 December 2005)
Abstract Contact and residual toxicities of 30 plant extracts were investigated on third instar larvae of Colorado potato beetle, Leptinotarsa decemlineata. The plant samples were collected during the spring and summer of 2002 and were dried and ground. The plant samples were treated with methanol and the residue was eluted with distilled water containing 10% acetone, resulting in plant extracts. In contact bioassays, the beetle larvae were treated with 40% (w/w) plant extract using a Potter Spray Tower. The insects were incubated at 28+28C under a 16 h: 8 h photo regime and the mortality was recorded at 24 h intervals for 7 days. The plant extracts exhibited varying toxicity to the larvae ranging from 0 – 91% after 24 h incubation and Artemisia vulgaris, Hedera helix, Humulus lupulus, Lolium temulentum, Rubia tinctoria, Salvia officinalis, Sambucus nigra, Urtica dioica, Verbascum songaricum, and Xanthium strumarium extracts resulted in significantly higher mortality than the control. Generally, prolonged incubation time did not result in an increase in mortality. After 48 h of incubation, 10 plant extracts yielded a significant mortality and H. lupulus extract, the most toxic extract among those tested, caused 99% mortality which is similar the mortality caused by the chemical standard, imidacloprid. In residual assays, potato leaflets were treated with 20% (w/w) plant-extract concentrations using a Potter Spray Tower. Third instar larvae were added to a glass jar to which treated leaflets were transferred before incubation at the temperature and photo regime described above. Mortality was recorded at 24 h intervals for 7 days. Five plant extracts, H. lupulus, L. temulentum, Reseda lutea and Solanum nigrum, induced significantly higher mortalities compared with controls. Chenopodium album extract was the most toxic, killing 34.9% of exposed larvae, followed by L. temulentum, H. lupulus and S. nigrum after 72 h incubation. The results exhibited that certain plant extracts were toxic to the beetle larvae and may have potential for controlling this destructive pest under field conditions.
Keywords: Leptinotarsa decemlineata, Colorado potato beetle, plant extract, contact toxicity, residual toxicity
Introduction The Colorado potato beetle, Leptinotarsa decemlineata (Say), is a polyphagus member of the Chrysomelidae found on a wide range of host plants (Casagrande 1987). Adults and larvae Correspondence: Ayhan Go¨kc¸e, Department of Plant Protection Agriculture Faculty Gaziosmanpasa University Tokat 60250, Turkey. Fax: þ356 252 1488. E-mail:
[email protected] or
[email protected] ISSN 0323-5408 print/ISSN 1477-2906 online ª 2007 Taylor & Francis DOI: 10.1080/03235400600628013
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feed on the foliage of the host plants but larvae are the most damaging life stage and can cause economic injury if populations are moderate or above (Ferro et al. 1983; Mailloux et al. 1991). It feeds on all members of the Solanaceae family, such as potato, tomato, pepper, egg plant, and weeds including nightshades and buffalo bur (Hsiao 1978; Hare 1990). Colorado potato beetle is also a vector of bacterial ring rot (Christie et al. 1991) which can be of economic importance if climatic conditions favour the disease. Extensive use of pesticides has led to the evolution of resistance in L. decemlineata, phytotoxicity and residue problems (Mota-Sanchez et al. 2000; Stewart et al. 1997; Ioannidis et al. 1991). The occurrence of cross and multiple resistances have added complexity to management of this destructive pest. The evolution of resistance in arthropods and policy changes to ameliorate the negative environmental and ecological impacts of conventional insecticides has resulted in development of new pest control agents which have unique mode of actions. Phytochemical materials produced by plants can demonstrate a range of toxicity, antifeedant or repellent effects on insects (Dev & Koul 1997; Perry et al. 1998; Murray et al. 1999; Khambay & Jewess 2000). Some of these products have been registered for use as pest controls, e.g. pyrethrins, azadirachtin (Extoxnet 2005). Therefore, plant extracts may be good pest control candidates, particularly those from plants that have been used ingeniously for using pest control and other purposes in many area of the world (Duke 1990; Dev & Koul 1997). There have been growing efforts to discover new plant compounds that have insecticidal properties for developing plant derived insecticides (Murray et al. 1999; Scott et al. 2003). In previous studies, only a limited number of plant extracts have been tested on Colorado potato beetle larvae (Lopez-Olguin et al. 1999; Murray et al. 1999; Scott et al. 2003). However, exploration of plant extracts for possible use in control of this pest requires a larger screening of plant extracts. Thus, in the current work, we compared the contact and residual toxicities of 30 plant extracts on third instar Colorado potato beetle. The plant species (Table I) were chosen because they produce secondary compounds such as monoterpenes, sesquiterpenes and triterpenes (Heywood et al. 1977; Katsiotis et al. 1990; Latrasse et al. 1991; Baser et al. 1995; Baser et al. 1998) which show various activity on arthropods (Karadjova et al. 2000; Krupke et al. 2001; Jones et al. 2003; Go¨kc¸e et al. 2005). In Turkey, these plant species are associated with vegetable and orchard agro-ecosystems, but Colorado potato beetle larvae and adult have not been observed feeding on them (A. Go¨kc¸e personal observation). In addition, some of these plants are insect anti-feedants and repellents (C ¸ etinsoy et al. 1998; Johri et al. 2004; Pons 2004; Go¨kc¸e et al. 2005). Therefore, the toxicities of these plant species were evaluated on the important pest of potatoes. The objectives of the current study were: (i) to determine whether the selected plant extracts caused contact toxicity on Colorado potato beetle larvae, and (ii) to determine whether plant extracts demonstrated residual toxicities to Colorado potato beetle larvae.
Materials and methods Insects Colorado potato beetles were continuously reared on potato plants (Solanum tuborosum L. cultivar Morfana) at Gaziosmanpas¸a University Research Station in Tas¸lic¸iftlik, Tokat. The field was designated for organic potato production and there were no pesticide application for 3 years prior to the initiation of this project. The field was divided into three different plots separated by maize barriers. Planting occurred at 2-week intervals from April to June providing sufficient beetle stocks throughout the studies. Adult insects from a panmictic population collected throughout the region were released into successive plots when potato
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Table I. Plants used in contact and residual toxicity bioassay on Colorado potato beetle. Family name
Scientific name
Tissue used
Apiaceae Apiaceae Apocynaceae Araliaceae Asteraceae Asteraceae Asteraceae Asteraceae Asteraceae Cannabinaceae Caprifoliaceae Chenopodiaceae Cucurbitaceae Fabaceae Guttiferae Lamiaceae Lauraceae Poaceae Poaceae Poaceae Poaceae Ranunculaceae Resedaceae Rubiaceae Rubiaceae Scrophulariaceae Solanaceae Solanaceae Styracaceae Urticaceae
Bifora radians M. Bieb. Conium maculatum L. Nerium oleander L. Hedera helix L. Arctium lappa L. Artemisia vulgaris L. Chrysanthemum segetum L. Circium arvense (L.) Scop. Xanthium strumarium L. Humulus lupulus L. Sambucus nigra L. Chenopodium album L. Ecballium elaterium (L.) A.Rich. Glycyrrhiza glabra L. Hypericum perforatum L. Salvia officinalis L. Laurus nobilis L. Avena sterilis L. Cynodon dactylon L. Lolium temulentum L. Sorghum halepense (L.) Pers. Delphinium consolida L. Reseda lutea L. Galium aperina L. Rubia tinctoria L. Verbascum songaricum L. Datura stramonium L. Solanum nigrum L. Styrax officinalis L. Urtica dioica L.
Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Fruit Flower bud Fruit Leaves Fruit Fruit Leaves Leaves Leaves Leaves Leaves Leaves Fruit Leaves Fruit Leaves Fruit Leaves Fruit Fruit Fruit Leaves
plants were in the four or the five-leaflet stage. Third instar larvae were hand collected from the field prior to the experiments and segregated from other instars using a delimiter of 1.4 – 1.8 mm head capsule width measurement. Plants and sample preparation Thirty natural product sources were used per study. The plants (Table I) were all collected during spring and summer of 2002 in Tas¸lic¸iftlik, Tokat, a temperate region of Turkey, where the attitude is 600 m and the soil is sandy lime soil, except Styrax officinalis and H. helix obtained from Mersin. Samples were dried at room temperature for three weeks in the dark and subsequently were ground in a mill (M 20 IKA Universal Mill, IKA Group). Ground plants were stored in 2000 ml glass jars at 18+28C in the dark. Fifty grams of samples was placed into 1000 ml Erlenmeyer flasks with 500 ml of methanol (Sigma). Flask were covered with aluminum foil, placed on a horizontal shaker (HS 260 Basic, IKA Group) and shaken (120 oscillations min71 for 24 h) in the dark. The suspension was filtered through two layers of cheese cloth, transferred into a 250 ml evaporating flask and excess methanol evaporated in a rotary evaporator (RV 05 Basic 1B, IKA Group) at 32+28C. The resulting residue was weighed and eluted with sufficient distilled water containing 10% acetone (w/w) to yield a 40% (w/w) plant suspension.
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Bioassay Contact effects. Preliminary bioassays demonstrated that 2 ml of plant residue suspensions produced reproducible results; therefore, 2 ml of each plant extract was applied to 20 third instar larvae using the Potter Spray Tower set at 10 PSI and equipped with a nozzle of 0.7 mm internal diameter. Two ml of the distilled water containing 10% acetone was also applied to 20 larvae in each replicate as a negative control along with imidacloprid (ConfidorTM SL, Bayer) at manufacturer recommended rate, (1.5 ml ml71) in distilled water, as a positive control. After spraying, the larvae were transferred into 1000 ml glass jars and provided with fresh potato leaflets. The top of each jar was covered with cheese cloth and held at 28 + 28C and 16 h: 8 h light dark photo regime. Mortalities were recorded at 24 h intervals for 7 days. A randomized complete block design was used in this study. Each treatment was replicated three times within a trial and each trial was repeated three times. Residual effects. Stomach poison effects of the 30 plant extracts were assed by morbidity assays using third instar larvae fed on treated potato leaflets. Preliminary assays demonstrated that 20% (w/w) plant extracts in distilled water containing 10% acetone did not produce phytotoxicity and were easily and uniformly applied to leaflets. Leaflets were treated with 2 ml of each plant extract suspension in distilled water containing 10% acetone with the Potter Spray Tower set at 10 PSI with a 0.7 mm internal diameter nozzle. After treatment, the leaflets were dried at room temperature for about 5 min. The cut end of each leaflet petiole was covered with a 30655 mm piece of sterile cotton wool which was moistened with 2 ml of water containing 1% NPK (20-20-20) fertilizer. This treatment ensured that the excised leaflet remained green for at least 7 days. Treated leaflets were transferred into 1000 ml glass jars to which 20 third instar larvae were added before incubation as described above. Mortalities were assessed at 24-h intervals for 7 days. Control leaflets were treated with 2 ml of distilled water containing 10% acetone and the standard imidacloprid control was used as described above. A randomized block design was used in this study, as all the bioassays would not be conducted at a single occasion. Three blocks were assigned randomly over time and each block consisted of treatment of each plant extract, the standard and a control treatment. The whole experiment was carried out in consecutive 3 days and thus insects could be selected randomly from a single Colorado potato beetle population. Data analysis Data were corrected for mortality in the controls using Abbott’s formula (Abbott 1925) and then normalized using arcsine transformation (Zar 1999). Transformed data were analysed using ANOVA (/ ¼ 0.05) and Tukey’s mean separation (/ ¼ 0.05). Incubation time effects were assessed using a one-tailed paired-sample t-test (/ ¼ 0.05). All statistical analyses were carried out using MINITAB computer software Release 14 (McKenzie & Goldman 2005).
Results Contact effects The experimental protocol which involved limited handling, spraying and incubation did not appear deleterious to beetle larvae because few controls succumbed during any of the 8 d of the studies (Table II). The mortality induced by the crude plant extracts after 24 h of incubation varied from 0 – 91% and mortality from A. vulgaris, H. helix, H. lupulus,
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Table II. Mortality % (mean + SEM) caused by contact effect of plant extracts to third instar Colorado potato beetle larvae after 24 and 48 h incubation at 28 + 28C. Treatment Bifora radians Conium maculatum Nerium oleander Hedera helix Arctium lappa Artemisia vulgaris Chrysanthemum segetum Circium arvense Xanthium strumarium Humulus lupulus Sambucus nigra Chenopodium album Ecballium elaterium Glycyrrhiza glabra Hypericum perforatum Salvia officinalis Laurus nobilis Avena sterilis Cynodon dactylon Lolium temulentum Sorghum halepense Delphinium consolida Reseda lutea Galium aperina Rubia tinctoria Verbascum spp Datura stramonium Solanum nigrum Styrax officinalis Urtica dioica Imidacloprid Control
24 h 0.00 + 0.00 0.56 + 0.56 0.00 + 0.00 11.57 + 0.06 0.00 + 0.00 23.29 + 0.04 0.00 + 0.00 0.00 + 0.00 26.44 + 0.24 91.07 + 3.89 24.89 + 0.13 1.15 + 1.13 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00 19.84 + 0.13 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00 14.76 + 0.16 0.56 + 0.56 0.00 + 0.00 0.00 + 0.00 0.56 + 0.56 11.57 + 0.06 13.24 + 0.06 0.00 + 0.00 0.00 + 0.00 0.56 + 0.56 7.79 + 0.33 100 + 0.00 0.00 + 0.00
48 h e*A de A eA cA eA cA eA eA cA bA cA de A eA eA eA cA eA eA eA cA de A eA eA de A cA cA eA eA de A cd A aA eA
3.29 + 0.90 1.15 + 1.14 5.00 + 0.00 13.01 + 0.22 8.16 + 0.10 24.89 + 0.14 3.29 + 0.90 0.00 + 0.00 34.67 + 0.65 99.44 + 0.56 26.52 + 0.15 26.44 + 0.25 0.00 + 0.00 0.56 + 0.56 0.00 + 0.00 21.62 + 0.04 0.00 + 0.00 0.56 + 0.56 8.16 + 0.11 19.84 + 0.13 3.29 + 0.90 0.56 + 0.56 2.24 + 0.56 2.24 + 0.56 23.29 + 0.04 19.84 + 0.13 9.60 + 0.25 0.00 + 0.00 3.29 + 0.90 13.24 + 0.06 100 + 0.00 0.56 + 0.56
cd A dA cd B cA cd B bc A cd A dA bB aA bc A bc B dA dA dA bc A dA dA cd B bc B cd A dA dA dA bc A bc B cd B dA cd A cA aA dA
*Means in a column followed by a different lowercase letter are significantly different (p 5 0.05, ANOVA, Tukey test). Means in a row followed by a different uppercase letter are significantly different (p 5 0.05, Paired t-test).
L. temulentum, R. tinctoria, S. officinalis, S. nigra, U. dioica, V. songaricum and X. strumarium crude extracts were significantly higher than the control (F ¼ 50.08, d.f. ¼ 31, 64, p 5 0.05). Fifteen of the crude plant extracts did not cause beetle mortality from which Conium maculatum, C. album, S. officinalis, Galium aperina and Sorghum halepense were notable as they were the least toxic (Table II). After 24 h of incubation, the most toxic extract was from H. lupulus, which caused 91% mortality. Imidacloprid induced 100% mortality and it was significantly greater than all other treatments. For most crude plant extracts, increasing incubation time from 24 – 48 h did not cause a significant difference in mortality, but an increase was seen for Nerium oleander, Arctium lappa, X. strumarium, C. album, Cynadon dactylon, L. temulentum, V. songaricum and Datura stramonium extracts (Table II). The most dramatic increase in the 24 – 48 h was seen from C. album crude extract where toxicity increased from 1 – 26%. After 48 h of incubation mortality varied significantly between the tested extracts and 12 inflicted significant mortality (F ¼ 39.05, d.f. ¼ 31, 64, p 5 0.000). Humulus lupulus extract
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yielded 99% mortality after 48 h which is similar to mortality caused by imidacloprid. However, only 1% was recorded from C. maculatum, Glycyrrhiza glabra, Avena sterilis and Delphinium consolida extracts. Five plant extracts, Circium arvense, Ecballium elaterium, Hypericum perforatum, Laurus nobilis and S. nigrum, did not cause any mortality after 48 h and A. vulgaris, X. strumarium, S. nigra, C. album and R. tinctoria showed moderate mortality (Table II). Residual effects Twenty crude plant extracts caused some increase in mortality as compared to the controls values after 48 h incubation (Table III). Mortality varied from 0.6% (H. helix, A. lappa, A. vulgaris, H. perforatum, S. officinalis and U. dioica) to 20.9% (H. lupulus) and only H. lupulus, L. temulentum, R. lutea and S. nigrum were significantly different from the control (F ¼ 7.38, d.f. ¼ 31,64, p 5 0.000). Imidacloprid provided 71.9% mortality which was
Table III. Residual toxicities % (mean + SEM) of plant extracts to third instar Colorado potato beetle larvae after 48 and 72 h incubation at 28 + 28C. Treatment Bifora radians Conium maculatum Nerium oleander Hedera helix Arctium lappa Artemisia vulgaris Chrysanthemum segetum Circium arvense Xanthium strumarium Humulus lupulus Sambucus nigra Chenopodium album Ecballium elaterium Glycyrrhiza glabra Hypericum perforatum Salvia officinalis Laurus nobilis Avena sterilis Cynodon dactylon Lolium temulentum Sorghum halepense Delphinium consolida Reseda lutea Galium aperina Rubia tinctoria Verbascum spp Datura stramonium Solanum nigrum Styrax officinalis Urtica dioica Imidacloprid Control
48 h 1.15 + 1.13 0.00 + 0.00 0.00 + 0.00 0.56 + 0.56 0.56 + 0.56 0.56 + 0.56 0.56 + 0.56 2.24 + 0.56 0.00 + 0.00 20.91 + 0.61 3.29 + 0.90 5.64 + 1.46 0.00 + 0.00 3.29 + 0.90 0.56 + 0.56 1.75 + 1.73 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00 12.21 + 0.67 4.53 + 1.14 3.29 + 0.90 14.76 + 0.16 3.29 + 0.90 0.00 + 0.00 1.15 + 1.13 1.75 + 1.73 18.27 + 0.05 0.56 + 0.56 0.56 + 0.56 71.89 + 0.25 0.00 + 0.00
72 h bc* A cA cA bc A bc A bc A bc A bc A cA bA bc A bc A cA bc A bc A bc A cA cA cA bA bc A bc A bA bc A cA bc A bc A bA bc A bc A aA cA
1.75 + 1.72 5.00 + 0.00 3.29 + 0.90 2.24 + 0.56 6.11 + 2.24 4.25 + 1.31 4.53 + 1.13 5.18 + 1.76 2.24 + 0.56 22.46 + 0.77 12.56 + 0.50 34.85 + 0.27 0.00 + 0.00 6.49 + 0.11 2.24 + 0.56 18.12 + 0.18 0.00 + 0.00 2.24 + 0.56 4.53 + 1.13 23.96 + 0.92 6.49 + 0.11 9.60 + 0.25 22.46 + 0.77 6.49 + 0.11 0.56 + 0.56 6.87 + 1.73 2.37 + 2.33 26.15 + 0.50 2.24 + 0.56 3.29 + 0.90 83.64 + 0.23 6.49 + 0.11
cA bc B bc A bc A bc A bc A bc A bc A bc A bc A bc A bB cA bc A bc A bc A cA cA bc A bA bc A bc A bc A bc A cA bc A bc A bc A bc A bc A aA bc B
*Means in a column followed by a different lowercase letter are significantly different (p 5 0.05, ANOVA, Tukey test). Means in a row followed by a different uppercase letter are significantly different (p 5 0.05, Paired t-test).
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significantly higher than the crude plant extracts. Increasing incubation time from 48 – 72 h did not cause any significant increase in mortalities except from C. maculatum and C. album extracts (Table III). These data indicated that 48 h of incubation was usually sufficient to assess the residual toxicity potential. After 72 h, all plant extracts, except L. nobilis and E. elaterium, appeared to exhibit some lethality to the third instar larvae but overall significant variation occurred between plant extracts (F ¼ 6.28, d.f. ¼ 31,64, p 5 0.000). Rubia tinctoria killed 0.6% of larvae, whereas an intermediate level of toxicities was demonstrated by H. lupulus, L. temulentum, R. lutea and S. nigrum resulting in mortalities of 22.5, 24.0, 22.5 and 26.2% respectively. Chenopodium album was the most toxic extract, killing 34.9% of exposed larvae. Imidacloprid exhibited a toxicity (83.6%) which is nearly three-fold greater than that of the most toxic plant extract, C. album. Residual effects of crude plants extracts to third instar larvae were far less pronounced when compared with their contact toxicities. Leaflets treated with Bifora radians, A. lappa, X. strumarium, V. songaricum and C. maculatum extracts exhibited antifeedant effects as leaflets were only partly consumed by the larvae and this resulted in low mortalities but little foliage damage. Discussion In contact assays, H. lupulus crude extract caused 91.1% and 99.4% mortalities after 24 and 48 h respectively. The mortality recorded after 48 h was not significantly different from our chemical standard, imidacloprid, which has been used for controlling Colorado potato beetle in Turkey. These results demonstrated that H. lupulus crude extract could be as effective as currently used insecticides and after purification its effectiveness may further increase with potential in controlling the most destructive stage of Colorado potato beetle. In contact toxicities tests, a range of plant extract toxicities were observed with Colorado potato beetle larvae from 30 indigenous plants. Similar variations of plant extract toxicity have been reported by Muray et al. (1999) who investigated the effect of three plant extracts on fourth instar Colorado potato beetle and they observed significant variation in effectiveness of plant extracts and Hough-Goldstein (1990) who examined insecticidal activities of six plants on Colorado potato beetle larvae and adults and he reported that only tansy caused activity on this pest. The variation observed in toxicity of plant extracts between our results and previously reported results (Hough-Goldstein 1990; Mateeva-Radeva 1997; C ¸ etinsoy et al. 1998; Muray et al. 1999) could be due to a number of causes: chemical compositions of plants, the particular solvent and extraction process, the development stages of tested insects, photo and/or thermal sensitivity of compounds extracted from the plants. Mateeva-Radeva (1997) reported that U. dioica extracts caused around 60% mortality on third stage larvae; in our experiment, this extract produced only 13.2% mortality on the same stage. This difference between her results and our results may be a result of the chemical composition of U. dioica, resulting in a variation of insecticidal activity (Brader et al. 1992; Hampton et al. 2002). In this experiment, methanol, a polar solvent which dissolves many plant compounds, was used as an eluting solvent as has been done in many previous studies (Pascual-Villalobos & Robledo 1999; Wheeler & Isman 2001; Han et al. 2006). However Mateeva-Radeva (1997) and Hough-Goldstein (1990) used water as a solvent, and the use of different solvents for extraction of plant extracts may explain the difference between their results and our results. Using other solvents, e.g., hexane, acetone or chloroform, may result in different compounds’ yield and resultant morbidity. Our efforts were preliminary in nature and may lead to further extraction from these plant candidates. In our experiment, third instar Colorado potato beetle larvae were used and they
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appeared to be susceptible to an array of plant extracts. This developmental stage was chosen since previous studies (Hilton et al. 1998; Haffani et al. 2001; Martin et al. 2004) demonstrated that third instar larvae were moderately susceptible to certain insecticides and was good as a stage for exploration of plant extract toxicity. This stage is also the most destructive stage of the Colorado potato beetle (Perlak et al. 1993). Although we did not test the photo and/or thermal sensitivity of compounds in our experiments, it is unlikely that our method caused significant structural changes in the compounds, since temperatures used in the rotary evaporation step did not exceed 32+18C and most of plant compounds are stable at this temperature. There are some reports that certain plant extracts decrease in toxicity when exposed to full sunlight (Scott et al. 2003, 2004), but in our experiments the conditions of incubation were such that there was not any exposure to sunlight. Our results demonstrated relatively low residual activity of most of the crude plant extracts tested. Antifeedant effects may have masked morbidity potential, since some of larvae refused to feed on treated leaflets. Thus, low per oligosaccharide intake of candidate substances was certain. Similar results were reported by Trisyono and Whalon (1999) who reported that mortality of second instars of Colorado potato beetle larvae increased after 3 d of treatment. They speculated that this resulted from the antifeedant effects of azadirachtin. Moreover, the amount of compound intake depends on the amount of plant tissue consumed by the tested species. Given that we observed that potato leaflets treated with certain plant extracts, particularly B. radians, A. lappa, X. strumarium, V. songaricum and C. maculatum, were not readily consumed by the larvae, it is not surprising that the mortalities measured were moderate. The results suggest that the route of uptake may have affected toxicity. Generally our crude plant extracts demonstrated greater toxicities in the contact assays than in feeding assays. For example, H. lupulus was the most toxic plant extract in contact assays, yet it showed moderate toxicity in the feeding assays. Similar results were reported by Hilton et al. (1998) who showed that cypermethrin contact effect was greater than its residual effect. Thus insects in the contact assays may have died earlier than those in residual assays yielding the opportunity for prolonged exposure. Although the residual bioassay mortality data collected at 48 and 72 h were typically not significantly different, future assays may require longer incubation times to better demonstrate extract residual toxicity. Martin et al. (2004) waited 96 h before recording the numbers of Colorado potato beetle which had been killed by Photorhabdus luminescens and Haffani et al. (2001) employed a 6-d incubation period when examining the effectiveness of Bacillus thruringiensis. In this study, contact and residual toxicities of plant extracts to third instar Colorado potato beetle larvae were investigated. Some of the crude plant extracts were toxic to beetle larvae and may have the potential for controlling the pest under field conditions. However further investigations of plant extracts on the other development stages of Colorado potato beetle should be investigated to determine whether more of these plant-extract candidates could be useful under field conditions. Acknowledgements This study was funded by The Scientific and Technical Research Council of Turkey (TUBITAK) Grant number TOGTAG-2892 and Gaziosmanpas¸a Universitesi Bilimsel _ Aras¸tirma Projeleri Komisyonu (BAP) Grant number 2001/43. We would like to thank Izzet Kadio glu for identifying plant species used in this study. A Go¨kc¸e thanks all 2002, 2003 and 2004 students at Gaziosmanpas¸a University Agriculture Faculty Department of Plant Protection for their help.
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