Annals of Applied Biology ISSN 0003-4746
R E S E A R C H A RT I C L E
Plant extract contact toxicities to various developmental stages of Colorado potato beetles (Coleoptera: Chrysomelidae) A. Go¨kcxe1, M.E. Whalon2, H. C xam1, Y. Yanar1, _I. Demirtasx3 & N. Go¨ren4 1 2 3 4
Department Department Department Department
of of of of
Plant Protection, Gaziosmanpasxa University, Tokat, Turkey Entomology, Michigan State University, East Lansing, MI, USA Chemistry, Gaziosmanpasxa University, Tokat, Turkey Biology, Yıldız Teknik University, Istanbul, Turkey
Keywords Artemisia; Chenopodium; Hedera; Humulus; Leptinotarsa decemlineata; Lolium; plant extract; potato crops; Salvia; Sambucus; Verbascum; Xanthium. Correspondence A. Go¨kc xe, Department of Plant Protection, Agriculture Faculty, Gaziosmanpasxa University, Tokat 60250, Turkey. Email:
[email protected];
[email protected] Received: 28 February 2006; revised version accepted: 3 July 2006. doi:10.1111/j.1744-7348.2006.00081.x
Abstract The contact toxicities of methanol extracts from the nine plant species Hedera helix, Artemisia vulgaris, Xanthium strumarium, Humulus lupulus, Sambucus nigra, Chenopodium album, Salvia officinalis, Lolium temulentum and Verbascum songaricum were tested on the developmental stages of Colorado potato beetle (CPB) (Leptinotarsa decemlineata). About 2 mL of plant extract, 40% (w/w), was applied to the first instar to fourth instar larvae and adult beetles using a Potter spray tower. Most of the tested plant extracts caused relatively low mortality in all the beetle instars. Among the plant extracts, H. lupulus extract was the most toxic to all stages of the insect, except for the adult beetles. Larval mortality ranged from 40% in the fourth instars to 84% in the third instars. In a second series of experiments, dose–response bioassays using H. lupulus extract produced lethal concentration 50 (LC50) values ranging from 10%, 12%, 17% to 46% (w/w) active ingredient (plant material) for instars 1–4, respectively. This increasing mortality trend, however, did not extend to the adult stage where even the maximum dose of 40% plant material did not provide sufficient mortality to allow estimation of a LC50. These results demonstrated that the extract from H. lupulus has potential as an active ingredient in biological pesticides developed to manage larval instars of the CPB. The potential uses of this plant extract may be in conventional and organic pest management or as part of a mixture of plant extracts or conventional insecticides. Before extracts can be considered as biological control agents, their impact on natural enemies should be assessed.
Introduction The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is the most destructive pest of potatoes worldwide (Zehnder & Gelernter, 1989; Hare, 1990). CPB potato herbivory has been studied by many researchers (Ferro et al., 1983; Mailloux et al., 1991; Zehnder et al., 1995), and various forms of integrated pest management have been implemented, especially chemical suppression. Reliance on pesticide suppression, coupled with the beetles’ propensity to evolve resistance and cross-resistance, has greatly increased the economic importance of CPB worldwide
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(Stankovic et al., 2004). The US potato producers in the Upper Midwest have experienced intense economic and production challenges since the mid-1940s as a result of resistance development in CPB (Grafius, 1997). Introduction of neonicotinoid insecticides in the early 1990s averted impending resistance-mediated disaster for the potato industry (Grafius, 1997), and now, resistance and cross-resistance (neonicotinoids) are once again becoming prevalent in a number of locations throughout the Upper Midwest and US East coast (Nauen & Denholm, 2005). Given the resistance evolution, the search for promising CPB management tools continues. 197
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Various experiments using plant extracts in human and animal health protection, agriculture and household pest management have been particularly promising (PascualVillalabos & Robledo, 1999; Scott et al., 2004). The apparent societal hope for using plant extracts in place of more traditional pesticides has also increased the attention paid to natural products in the past decade (Duke et al., 2003). Plants belonging to Araliaceae, Asteraceae, Cannabaceae, Caprifoliaceae, Chenopodiaceae, Lamiaceae, Poaceae and Scrophulariaceae families are known to produce monoterpenes, sesquiterpene lactones and triterpenes; all of which may have commercial applications (Heywood et al., 1977; Tucker & Maciarello, 1994; Barney et al., 2005). These chemicals are known to affect insects in various ways. The chemicals may act as antifeedants or repellents as well as pesticides (Go¨kcxe et al., 2005; Go¨kcxe et al., 2006). Unfortunately, CPB resistance to relatively low-cost insecticides like organophosphates and carbamates has occurred across the world such that new, inexpensive yet effective pest control tools are not available, and this situation creates a somewhat urgent need for new pest management tools. ‘Plant-derived’ compounds also hold promise for use in organic production both in developed and developing countries. However, there are relatively few ‘organic’ insecticides that provide an efficient CPB suppression treatment in organic agriculture. Additionally, many organic farmers already use and make compost teas, and these methods often employ plant material for pest management purposes (Zinati, 2005). In a previous study, we evaluated extracts from 30 different plant species on third instar CPB larvae and reported nine natural product sources exhibiting high levels of contact activity (Go¨kcxe et al., 2006). This study reports the results of experiments to ascertain the contact toxicities of extracts from these nine promising plants on all stages of CPB, except the egg and pupae stages where impact is less probable.
Materials and methods Insects Colorado potato beetles were reared on potato plants (Solanum tuberosum L. cultivar Morfana) at Gaziosmanpasxa University Research Station in Tasxlıcxiftlik, Tokat, as described in Go¨kcxe et al. (2006). The larvae were hand collected from the field prior to the experiment and segregated using head capsule measurements of 0.6– 0.7 mm, 0.9–1.1 mm, 1.4–1.8 mm and 2.0–2.4 mm for first, second, third and fourth instars, respectively. Larvae with head capsule measurements not within these ranges were not used because of the ambiguity of instar 198
identification at these stages. The adults in the experiments were newly emerged 1- to 3-day olds. Plants and sample preparation The plant samples were prepared according to the procedure described by Go¨kcxe et al. (2006). Nine extracts were used in this study. The plants (Table 1) were all collected during spring and summer of 2002. Samples were dried at room temperature and were ground for 5 min in a mill (M 20 IKA Universal Mill, IKA Group, Wilmington, NC, USA). Fifty grams of dried plant samples were treated with 500 mL of methanol 99.9% (Sigma, St Louis, MO, USA) for 24 h, and the suspension was filtered through two layers of cheese cloth before excess methanol was evaporated in a rotary evaporator (RV 05 Basic 1B, IKA Group) at 32 ± 2C. The resulting residue was eluted with sufficient distilled water containing 10% acetone (w/w) to yield a 40% (w/w) plant suspension. For a subsequent dose–response bioassay using only Humulus lupulus L. extract, a stock suspension was prepared as described above, containing 50% (w/w) plant extract/water with 10% acetone and diluted in distilled water containing 10% acetone (w/w) to produce doses containing 2.5%, 5%, 10%, 20% and 40% (w/w) plant extract. Screening bioassay Effects of plant extracts on various life stages of Colorado potato beetle Extract contact effects were determined on first to fourth instar larvae as well as adult beetles. For each developmental stage and experimental treatment, 20 individuals were transferred to each of three Petri dishes. Each set of three dishes was sprayed with an experimental treatment, allowed to dry for 10 min at room temperature and maintained at 28 ± 2C and 16-h photophase. Mortality was assessed after 48 h. The experiment was repeated on three Table 1 Plant species extracts used against larvae and adult Colorado potato beetle Family Name
Scientific Name
Tissue Used
Araliaceae Asteraceae Asteraceae Cannabaceae Caprifoliaceae Chenopodiaceae Lamiaceae Poaceae Scrophulariaceae
Hedera helix L. Artemisia vulgaris L. Xanthium strumarium L. Humulus lupulus L. Sambucus nigra L. Chenopodium album L. Salvia officinalis L. Lolium temulentum L. Verbascum songaricum Schrenk
Leaves Leaves Fruit Inflorescences Fruit Leaves Leaves Leaves Leaves
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occasions, and for statistical analysis, the separate occasions were regarded as blocks. Treatments were applied using a Potter spray tower set at 0.7 kgf cm22 L21 with a 0.7-mm-diameter fine-droplet spray nozzle (Potter, 1952). Imidacloprid (ConfidorTM SL, Bayer CropScience AG, Rhein, Germany) was used as a positive control or standard according to the manufacturer’s recommended rate (1.5 lL mL21) in distilled water. Negative controls were treated with 2 mL of sterile distilled water containing 10% acetone. The plant extract suspensions were shaken for 1 min, and 2 mL of extract suspension was applied to each treatment. Toxicity of Humulus lupulus extract on various stages of Colorado potato beetle Additional toxicity studies using H. lupulus extract were carried out based on its promising effects to first to fourth larval stage and the adult stage as well. In each replicate, 20 larvae (first to fourth stage) or adults were placed on Whatman filter paper in a 90-mm Petri dish. The dorsal surface of each insect was sprayed under the Potter spray tower (described above) with 2 mL of H. lupulus suspension containing 2.5%, 5%, 10%, 20% or 40% (w/w) plant material. After treatment, insects were incubated and mortalities assessed after 48 h. Control insects (20 per replicate) were treated with 2 mL water containing 10% (w/w) acetone. Each bioassay was repeated three times in a trial, and three trials were completed for a total of nine replicates per dose. Data analysis Screening bioassay mortality results recorded at 48 h postapplication were corrected for mortality in the controls
using Abbott’s formula (Abbott, 1925), transformed to ensure normality and variance homogeneity using an arcsine transformation (Zar, 1999) and submitted to a randomised complete block analysis of variance, with significance being tested at the 5% level (P 0.05). Differences between treatments were additionally tested using the t-test (P 0.05), and all statistical analyses were carried out using MINITAB Release 14 (Pearson Education, Boston, MA, USA) (McKenzie & Goldman, 2005). Similarly, CPB mortality data obtained from dose–mortality bioassay using H. lupulus extract were corrected for control mortality using Abbott’s formula (Abbott, 1925), in POLO-PC (LeOra Software, 1994), to estimate lethal concentration 50 (LC50) and the regression line slopes. Homogeneity of regression lines between various developmental stages was tested using the maximum likelihood approximation test (P 0.05) (LeOra Software, 1994).
Results Effects of plant extracts on various life stages of Colorado potato beetle Evaluations of plant extract screening resulted in differences between effects of extracts at various life stages of the insect. First to third instars were very susceptible to H. lupulus extract while fourth instar and adults were less affected. Significant differences in mortalities were observed between treatments in first instar larvae (F = 45.13, d.f. = 10, 20, P 0.001). Relatively low ( 0.10) and L. temulentum (t = 21.58, d.f. = 20, P > 0.10) treatments were not significantly different from that of the control. Significant mortality was observed ranging from 9.6% (V. songaricum) to 40.0% (H. lupulus). H. lupulus extract again resulted in the highest level of mortality among the tested plant extracts. Imidacloprid was not as toxic to fourth stage larvae, yielding only 11.5% mortality, which was significantly lower than that seen in the treatment with H. lupulus extract (t = 3.24, d.f. = 20, P 0.05). Statistically significant differences were also seen between treatments in adults after 48 h (F = 5.67, d.f. = 10, 20, P 0.001). Among plant extracts, Artemisia vulgaris extract caused the highest adult mortality, and it was significantly more effective than any other plant extract, except C. album (t = 0.71, d.f. = 20, P > 0.40). Imidacloprid was also toxic to adult CPB, causing almost twice the highest observed plant extract mortality. Toxicity of Humulus lupulus crude extract on various stages of Colorado potato beetle Using multiple dose assays with H. lupulus extract, LC50 values varied with CPB developmental stage and LC50
values increased with increasing age of the insect stadium (Table 3). The first and second instar larvae yielded the lowest LC50 values, 9.98% and 11.8%, respectively, and these were significantly different from that of the other stages (Table 3). The LC50 of third instar larvae was significantly higher than that of the second instar larvae. The highest LC50 was observed for the fourth instar larvae, but this is an extrapolation as it exceeded the highest concentration tested in this study, and the LC50 was not estimable for adults (Table 3).
Discussion This study demonstrated that H. lupulus extract was the most promising plant extract among the nine extracts tested as it yielded larval and adult mortality in 48 h, and these results are in agreement with the contact and residual toxicity of plant extracts on third stage CPB (Go¨kcxe et al., 2006). Hop extracts contain many compounds, especially alpha and beta acids, prenylflavonoids, essential oils and proanthocyanidins (Stevens et al., 1997; Hoek et al., 2001; Taylor et al., 2003). Hampton et al. (2002) reported that natural and selected genotypes of native North American hops repelled chewing and sucking plant pests. The beta-acid fraction from H. lupulus demonstrated repellent and oviposition deterrent effects in Tetranychus urticae Koch (Jones et al., 1996). Jones et al. (2003) also reported tri-trophic interactions mediated by the beta acid from H. lupulus on the predatory mite Phytoseiulus persimilis Anthias-Henriot and its prey T. urticae, resulting in a significant reduction in the two-spotted spider mite population. Exposing the CPB developmental stages to selected plant extracts confirms and extends previous work that has shown that various extracts are toxic to this pest (Scott et al., 2003; Scott et al., 2004; Go¨kcxe et al., 2006). In our study, plant extracts elicited wide variability within plant extracts in toxicity to CPB larval stages and adults. Similar variability has been previously observed
Table 3 Dose–mortality responses of different instars of Colorado potato beetle treated with Humulus lupulus extract Tested stage of Leptinotarsa decemlineata
LC50 (%)
Fiducial Limits (%)
Slope SE
First stage larvae Second stage larvae Third stage larvae Fourth stage larvae Adult
9.98 11.80 17.19 46.39a >>40
8.49–11.64 8.99–15.29 16.17–19.45 39.74–56.95 >>40
1.29 1.48 2.69 2.07 1.21
0.12 0.13 0.24 0.24 0.34
Intercept SE 21.28 21.59 23.36 23.45 23.06
0.14 0.15 0.31 0.33 0.51
v2 0.46 5.08 2.05 1.84 0.53
SE, standard error. Five doses were used with three sets of 20 individuals per dose in each of three trials giving 45 observations for modelling (900 individual) per development stage. The v2 values less than 7.8 (d.f. = 3) are not significant (P > 0.05). a The calculated value is an extrapolation.
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following bioassays with other insects using other plant extracts (Pascual-Villalabos & Robledo, 1999). Interested readers should also see the discussion addressing plant extract effects on CPB in a previous publication (Go¨kcxe et al., 2006). Our work confirms previous observations (Scott et al., 2003) that the first three stages of CPB are more sensitive to mortality agents including plant extracts. Third instar larvae were previously reported to be the most susceptible stage in the life cycle of CPB to some insecticides, plant extracts and even biological control agents (Zehnder & Gelernter, 1989; Hilton et al., 1998; Scott et al., 2003). In addition to other putative tolerance mechanisms (ageing, metabolism, mobilisation of defence systems, etc.), lower tolerance of first to third instar larvae could also be related to their changing cuticular structure as the physical and chemical properties of cuticles excised from the various developmental stages are different (Hegazy et al., 1989). Therefore, the relatively thin cuticle of the first three instar compared with fourth instar may contribute to their sensitivity to plant extracts. The fourth stage was relatively less susceptible to plant extracts. This could be as result of development of (previous mentioned) mechanisms for tolerance and perhaps also to a rapid increase in cuticle thickness which occurs prior to onset of the pupal stage (Hegazy et al., 1989). Not surprisingly, CPB adults were less sensitive to plant extract than the larvae. Adult tolerance to plant extracts and insecticides has been previously reported by Scott et al. (2003), who found that adults were 10-fold less susceptible to Piper tuberculatum Jacq. extract than early instar larvae. Gouamene-Lamine et al. (2003) demonstrated that abamectin was less toxic to adult CPB than to the larvae. This sharp mortality decrease in the adult stage could be related to the elytra as they putatively reduce transport to the active sites internally. In addition, adults were reported to have nearly threefold more cytochrome P-450 than larvae such that they may be able to detoxify plant extract faster than larvae do (Gouamene-Lamine et al., 2003). Our dose–mortality study confirmed the earlier work showing differential response across developmental stages to some plant extracts. LC50 and fiducial limit values from the different stages of CPB showed that the adults were the least sensitive among the tested stages to H. lupulus toxicity, while the first, second and third instar larvae appeared to be the most susceptible to the plant extract. The fourth instar was intermediate in sensitivity. The existence of development stage specific susceptibility of CPB to toxicants was reported by Hilton et al. (1998), who reported that the LC50 values of azinphosmethyl varied threefold depending on the developmental stage of CPB. Gouamene-Lamine et al. (2003) tested aba-
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mectin on four larval instar stages of CPB and recorded 0.3, 0.14, 1.0 and 1.1 ng per beetle as LD50 values for the first, second, third and fourth instar larvae, respectively. This study demonstrated that H. lupulus extract may have potential as a natural plant product against CPB in pest management programmes. Perhaps, H. lupulus extracts could be used alone or in combination with conventional insecticides if synergist effects were found. Additionally, the incorporation of hop extracts as a component of CPB resistance management programmes may increase the useful lifetime of insecticides like the neonicotinoids if the mechanisms providing mortality for artificial and natural insecticides prove to be different. Also, mixtures of plant extract analogues may be more active than a single compound and may also delay the development of resistance in CPB. Purification, identification and synthesis of active compound(s) may allow this natural plant extract to compete successfully with conventional insecticides, especially in developing countries. However, the use of extract from this plant as an insecticide may cause some additional concerns such as mitigating beneficial insects and residues may have effects on humans. Therefore, more research and regulatory scrutiny must be expended on this extract before it can be used widely.
Acknowledgements This study was funded by The Scientific and Technical Research Council of Turkey (TUBITAK) grant number TOGTAG-2892 and Gaziosmanpasxa Universitesi Bilimsel Arasxtırma Projeleri Komisyonu (BAP) grant number 2001/43.
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