INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT
Assessment of Insecticide Resistance of Lobesia botrana (Lepidoptera: Tortricidae) in Emilia-Romagna Region STEFANO CIVOLANI,1,2,3 MAURO BOSELLI,4 ALDA BUTTURINI,4 MILVIA CHICCA,1 ELISA ANNA FANO,1 AND STEFANO CASSANELLI5
J. Econ. Entomol. 107(3): 1245Ð1249 (2014); DOI: http://dx.doi.org/10.1603/EC13537
ABSTRACT The European grapevine moth, Lobesia botrana (Denis & Schiffermu¨ ller) (Lepidoptera: Tortricidae), is the key pest of vineyard, Vitis vinifera L. In Italy, failures in Þeld chemical pest control have been recently reported. The susceptibility to insecticides indoxacarb, methoxyfenozide, and emamectin benzoate was then evaluated in a L. botrana population collected from a vineyard in Emilia-Romagna (northeastern Italy) where pest management programs achieved unsatisfactory results. The Þeld trial showed that the indoxacarb efÞcacy toward L. botrana was very low in the two timings of application (7.9 and ⫺1.5%) in comparison with untreated control, while the efÞcacy of methoxyfenozide (76.1%) and emamectin benzoate (88.8%) was high. The decreased efÞcacy of indoxacarb was also supported by the results of the laboratory bioassay on neonate L. botrana larvae, in which the resistance ratio was 72-fold in comparison with that of the susceptible strain. KEY WORDS Lobesia botrana, insecticide resistance, indoxacarb, integrated pest management
The European grapevine moth, Lobesia botrana (Denis & Schiffermu¨ ller) (Lepidoptera: Tortricidae), is the major pest in European vineyards, Vitis vinifera L., wherein the number of generations completed per year is geographically different from two in European colder areas to four in warmer Mediterranean countries (Bovey 1966, Bournier 1977, Ioriatti et al. 2011). In northeastern Italy, L. botrana usually has two or three generations per year in both cooler and warmer grape growing areas (Pavan et al. 2006, 2010, 2013). Larvae feed on grape inßorescences and then on green and ripe berries, causing both direct damage and yield reduction. The damage favors secondary infections by fungi, especially the gray mold fungus (Botrytis cinerea Persoon: Fries), which develops rapidly on damaged grapes and rots entire clusters (Fermaud and Giboulot 1992). The control strategies allowed in integrated pest management include chemical insecticides and biological practices such as mating disruption and Bt toxin application (Ioriatti et al. 2008). Nevertheless, pest control is still mainly based on chemical insecticides (Ioriatti et al. 2008). Traditional organophosphate insecticides are gradually being replaced by more environmentally friendly compounds, such as new neurotoxic insecticides (indoxacarb, emamectin benzoate, methoxyfenozide, and tebufenozide; Boselli et al. 2000, 1 Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy. 2 InnovaRicerca s.r.l. Monestirolo, Ferrara 44123, Italy. 3 Corresponding author, e-mail:
[email protected]. 4 Servizio Fitosanitario Regione Emilia-Romagna, 40129 Bologna, Italy. 5 Department of Life Sciences, University of Modena and Reggio Emilia, 42100 Reggio Emilia, Italy.
Boselli and Scannavini 2001, Charmillot et al. 2004, Sa´enz-de-Cabezo´ n Irigaray et al. 2005, Pavan et al. 2005, Charmillot et al. 2006, Trona et al. 2007, Ioriatti et al. 2009). To be effective, these compounds must be applied at the most susceptible stage of the pest, which is identiÞed by population monitoring using pheromone traps, eggs scouting, and appropriate forecasting models (Ioriatti et al. 2008, 2011; Hardman 2012). Recently, some grape growers near Ravenna (EmiliaRomagna, northeastern Italy) mentioned unsuccessful chemical control of the pest, suggesting the possible selection of resistant populations to insecticides, mostly to indoxacarb. Although vineyard crop failures have been occasionally reported worldwide, there are currently no conÞrmed cases of L. botrana resistance in the literature and International Resistance Action Committee (IRAC) reports (http://www.irac-online. org/pests/lobesia-botrana/). The aim of this study was to assess the susceptibility of L. botrana to three insecticides (indoxacarb, methoxyfenozide, and emamectin benzoate) Þrst in a Þeld trial in a vineyard where crop failures have been recently observed and also in a laboratory diet bioassay. Materials and Methods Field Trial. EfÞcacy of indoxacarb, a voltage-dependent sodium channel blocker Insecticide Resistance Action Committee (IRAC) group 22 (Avaunt, 150 g liter⫺1 EC emulsiÞable concentrate formulation; DuPont Crop Protection, Cernusco sul Naviglio Milan, Italy); methoxyfenozide, a ecdysone receptor agonist IRAC group 18 (Intrepid, 240 g liter⫺1 SC suspension concentrate formulation; Dow Agro Science,
0022-0493/14/1245Ð1249$04.00/0 䉷 2014 Entomological Society of America
1246 Table 1.
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Vol. 107, no. 3
Field trial: insecticides tested and timings of application
N⬚
Insecticide (AI)
g AI ha⫺1
Timing
Application date
1 2 3 4 5
Untreated control Methoxyfenozide Indoxacarb Indoxacarb Emamectin benzoate
Ð 99.9 46.8 46.8 14.9
Ð Beginning of oviposition (T1) Beginning of oviposition (T1) Beginning of eggs hatching (T2) Beginning of eggs hatching (T2)
Ð 16 June 2011 16 June 2011 25 June 2011 25 June 2011
Bologna, Italy); and emamectin benzoate, a chloride channel activator IRAC group 6 (AfÞrm, 0.95% WG water granule formulation; Syngenta Crop Protection, Milan, Italy), in controlling L. botrana second-generation larvae was evaluated in the Þeld in summer 2011. According to the European and Mediterranean Plant Protection Organisation (EPPO) guideline PP/1/11(3), the efÞcacy trial was carried out in a commercial vineyard ÔTrebbiano RomagnoloÕ in Bagnacavallo (Ravenna, Emilia-Romagna, Italy). The control strategies against L. botrana were based on indoxacarb, and serious damages were observed in the last grape harvests. In the vineyard plots selected in 2011 for the Þeld trial, no treatment against L. botrana Þrst generation was performed. At the end of the Þrst generation an average of 3.9 nests per bunch was observed. Plots were arranged in a randomized block design, with four replicates of three vines each (14.4 m2). All treatments were applied at an equivalent water volume of 1,041 liters ha⫺1 using a spray mistblower (Stihl SR 420, Andreas Stihl S.P.A., Milan, Italy). The timing of Þrst application (T1), involving methoxyfenozide and indoxacarb, was at the beginning of oviposition (16 June), while the second application timing (T2), involving indoxacarb and emamectin benzoate, was at 12% eggs hatched (25 June; see Table 1 and Figs. 1 and 2). Both dates were established according to the MRVLobesia mathematical forecasting model (Baumga¨rtner and Baronio 1988; Buggiani et al. 1996; Butturini and Tiso 2002, 2007) that was run on the basis of temperature data of the area and also by scoring in situ the number of L. botrana male moths captured by a pheromone trap (Large Plastic Delta, Suterra Europe
Biocontrol S.L., Barcelona, Spain). On 25 July, 50 bunches chosen at random in the central part of each plot were examined in situ by checking both the number of bunches damaged and the number of damaged berries per plot. Laboratory Bioassay. Insects. A laboratory susceptible L. botrana (“S. Michele,” SM) strain was used as the reference population. L. botrana larvae were originally collected in vineyards in Trento (Italy), and the colony maintained in the laboratory on a semisynthetic diet for ⬎100 generations. The larval Þeld population (labeled as RA4) was collected from the Þeld trial vineyard during the summer of 2011, and only from untreated control plots. The larvae were reared on a semiartiÞcial diet (Rapagnani et al. 1990), at 23 ⫾ 1⬚C, 65% relative humidity (RH) and a photoperiod of 16:8 (L:D) h, until adult emergence. The adults were placed in plastic bottles for mating and egg laying. The colony was reared continuously without refreshing for 10 generations (12 mo) before laboratory bioassay. Dose–Response Bioassay. To assess the efÞcacy of commercial formulations of methoxyfenozide, indoxacarb, and emamectin benzoate, ⬍24-h-old (neonate) L. botrana larvae were tested by using 5Ð 8 different concentrations of the active ingredients (AIs) mixed to a semiartiÞcial diet (Rapagnani et al. 1990), according to IRAC guideline N⬚ 017. The control was exposed to semiartiÞcial diet with no active ingredients. Neonate larvae were individually transferred by a Þnehaired paintbrush to wells (2.5 ml) of a 128-well bioassay white plate (BioServ, Frenchtown, NJ), halfÞlled with semiartiÞcial diet. A batch of 32 larvae was
Fig. 1. MRV-Lobesia forecasting model: cumulative percentages of adults (Þrst generation), eggs, and larvae (second generation) predicted in the Bagnacavallo area (Ravenna, Italy; by Emilia-Romagna Plant Protection Service) from June to July 2011.
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CIVOLANI ET AL.: INSECTICIDE RESISTANCE OF Lobesia botrana
Fig. 2. Number of Þrst generation L. botrana male moths captured by a pheromone trap during the Þeld trial in 2011.
exposed to each active ingredient concentration for each replicate and the number of replicates ranged from 2 to 4; therefore, the total number of larvae exposed to each concentration ranged between 64 and 120. After exposure, larval mortality was recorded after 7 d, storing the plates at 23 ⫾ 1⬚C, 65% RH, and a photoperiod of 16:8 (L:D) h. Statistical Analysis. Results of the Þeld efÞcacy trials were subjected to an analysis of variance (ANOVA), and means were separated by FischerÕs least signiÞcant difference (LSD) test (P ⬍ 0.05; STATISTICA 6.0 for Windows; StafSoft Inc., Tulsa, OK). Estimates LC50 and LC90 for neonate larvae mortality and their 95% CI were obtained using the PoloPlus Version 2.0 (LeOra Software Company, Petaluma, CA) based on FinneyÕs (1971)probit analysis (Robertson et al. 2007). Significant differences were determined by nonoverlapping of the 95% CI, and the heterogeneity factor (2 by df) was calculated. Resistance factors were calculated by dividing the LC50 and LC90 values of the L. botrana RA4 by the LC50 and LC90 values of the laboratory susceptible strain L. botrana SM. Results Field Trial. Results of the Þeld efÞcacy evaluation are reported in Table 2. The untreated control shows an average 31.5 ⫾ 0.9 grapevine bunches (of the 50 sampled) damaged, with 222.7 ⫾ 10.2 berries damaged per plot. Concerning the efÞcacy of active ingredients, indoxacarb does not show signiÞcantly different values from the untreated control in both timings of Table 2.
Insecticide
Timing
1 2 3 4 5
Untreated control Methoxyfenozide Indoxacarb Indoxacarb Emamectin benzoate
Ð T1 T1 T2 T2
b
application (7.9 and ⫺1.5%). However, good results in terms of efÞcacy against grapevine bunch damages are obtained by emamectin benzoate and methoxyfenozide: their efÞcacy according to Abbott (Abbott 1925) is 88.8 and 76.1, respectively. In terms of number of berries damaged, indoxacarb maintains a low level of efÞcacy (32.0 and 20.8% in the two timings of application), signiÞcantly different from the control but much lower than the values of emamectin benzoate (93.4%) and methoxyfenozide (90.5%). Laboratory Bioassay. The responses of L. botrana neonate larvae exposed to the three insecticides are reported in Table 3. Concerning indoxacarb, the LC50 and LC90 values determined from the probit-log doseÐ response regression line for RA4 were signiÞcantly different in comparison with those of the susceptible laboratory strain SM, 2.186 mg (AI) liter⫺1 (1.07Ð 34.22; (minimumÐmaximum) and 0.177 (0.15Ð 0.20) respectively for LC50, and 24.390 (5.38 Ð104.5) and 0.336 (0.28 Ð 0.44) respectively for LC90. The resistance ratio (RR) of RA4 was ⬇12- and 72-fold respectively for LC50 and LC90 in comparison with the susceptible laboratory strain SM. When neonate larvae were exposed to methoxyfenozide, again RA4 showed signiÞcantly higher values of LC50 and LC90 than SM, 0.481 mg (AI) liter⫺1 (0.40 Ð 0.55) and 0.077 (0.05Ð 0.10), respectively, for LC50, and 1.012 (0.86 Ð1.26) and 0.365 (0.24 Ð 0.78), respectively, for LC90. The RR of RA4 was approximately sixfold for LC50 and threefold for LC90 in comparison to SM. Unlike results found for the previous two insecticides, the results of RA4 neonate larvae exposure to emamectin benzoate did not show signiÞcantly different LC50 and LC90 values in comparison to SM, namely 0.0011 mg (AI) liter⫺1 (0.0010 Ð 0.0012) and 0.0012 (0.0011Ð 0.0013), respectively, for RA4, and 0.0016 (0.0014 Ð 0.0022) and 0.0030 (0.0022Ð 0.0037), respectively, for SM. Discussion Rotation among different active ingredients with distinct modes of action has been suggested by IRAC (http://www.irac-online.org/content/uploads/ Principles-of-IRM1.pdf) to prevent the rapid development of insecticide resistance. The bioassays, although sufÞciently accurate, cannot completely mimic all factors acting in Þeld conditions (Kranthi 2005). Therefore, laboratory results should be compared with Þeld trials to get a more reliable evaluation of insecticide
Field trial results obtained by checking 50 grape bunches per plot on 25 July 2011
N
a
1247
Grape bunch damaged
Grape berries damaged
Mean ⫾ SE
P ⬍ 0.05a
Abbottb
Mean ⫾ SE
P ⬍ 0.05a
Abbottb
31.5 ⫾ 0.9 7.5 ⫾ 0.9 29.0 ⫾ 5.7 33.5 ⫾ 4.2 3.5 ⫾ 0.5
a b a a b
76.1 7.9 ⫺1.5 88.8
222.7 ⫾ 10.2 20.7 ⫾ 2.7 151.5 ⫾ 7.5 176.2 ⫾ 2.8 14.2 ⫾ 1.1
a d c b d
90.5 32.0 20.8 93.4
Different letters within columns indicate values that are signiÞcantly different using FischerÕs LSD test (P ⬍ 0.05). Values were corrected according to AbbottÕs formula.
2.68 1.32 0.40 0.0030 (0.0022Ð0.0037) 0.0012 (0.0012Ð0.0013)
0.077 (0.05Ð0.10) 0.481 (0.40Ð0.55)
0.0016 (0.0014Ð0.0022) 0.0011 (0.0010Ð0.0012)
1.89 ⫾ 0.20 3.97 ⫾ 0.48
4.59 ⫾ 0.31 27.94 ⫾ 3.72
0.68
2.77 0.365 (0.24Ð0.78) 1.012 (0.86Ð1.26)
0.177 (0.15Ð0.20) 2.186 (1.07Ð34.22)
6.24
1.47 2.81 72.58 Range (mg AI liter )
4.63 ⫾ 0.59 1.22 ⫾ 0.24
12.35
0.336 (0.28Ð0.44) 24.390 (5.38Ð104.5)
RRc LC90 (mg AI liter⫺1; 95% CI) RRc LC50 (mg AI liter⫺1; 95% CI) Slope⫾ SE
0.0001Ð0.005 0.0001Ð0.0033 Number of larvae tested. Number of concentrations tested. c Resistance ratio. d Heterogeneity factor ⫽ 2 by df. b
a
9.0 (133) 28.6 (252) 753 506
7 6
0.005Ð0.5 0.05Ð1.5 6 6 12.8 (86) 28.5 (242) 731 420
5 8 13.3 (45) 28.5 (242) 410 564
Indoxacarb SM RA4 Methoxyfenozide SM RA4 Emamectin benzoate SM RA4
N
0.03Ð0.3 0.09Ð2.0
⫺1 b
Concentration applied (mg AI liter⫺1)
Control mortality % Na Strain
Probit-log dose response regression line of L. botrana neonate larvae from RA4 and SM strains exposed with indoxacarb, methoxyfenozide, and emamectin benzoate Table 3.
2.71 0.80
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performance. For this reason, three new generation insecticides (indoxacarb, methoxyfenozide, and emamectin benzoate) were tested in our study by both a Þeld trial and a bioassay on a L. botrana population collected from a vineyard where failures in pest control were recently observed. According to IRAC, there are no previous literature reports concerning L. botrana resistance to the mentioned active ingredients (http:// www.irac-online.org/pests/lobesia-botrana/). In this study, Þeld trial results show that the indoxacarb efÞcacy toward L. botrana is very low in comparison with the untreated control, whereas the efÞcacy of methoxyfenozide and emamectin benzoate is still high. The low efÞcacy of indoxacarb is also supported by the results of the laboratory bioassay on neonate L. botrana larvae RA4 strain, in which the resistance factor is 72-fold higher than that of the susceptible strain SM. It is noteworthy that the high level of indoxacarb resistance in RA4 strain appears rather stable after 10 generations without refreshing, even in absence of selective pressure during the strain rearing. Concerning methoxyfenozide, the laboratory bioassay reveals a slight decrease of efÞcacy on RA4 in comparison with SM, which is still undetectable in the Þeld trials. On the contrary, emamectin benzoate shows a high efÞcacy both in the Þeld trial and in the laboratory bioassay, in this case no difference could be detected between RA4 and SM, unlike results found for indoxacarb and methoxyfenozide. A low or moderate (⬍10-fold) resistant ratio for methoxyfenozide has been described in multiresistant strains of Choristoneura rosaceana (Harris) and Cydia pomonella (L.) (Lepidoptera: Tortricidae) collected in Þeld, along with variable resistance values to indoxacarb (Ahmad et al. 2002, Dunley et al. 2006, MotaSanchez et al. 2008). Bioassays and biochemical studies carried out in C. rosaceana and in other Lepidoptera, such as Spodoptera exigua (Hu¨ bner) and Spodoptera littoralis (Boisduval), indicate that the oxidative metabolism might be involved in detoxiÞcation of both indoxacarb and methoxyfenozide (Smagghe et al. 2003, Ahmad and Hollingworth 2004, Mosallanejad and Smagghe 2009, Rodrõ´guez et al. 2011). As a whole, Þeld observations and biochemical Þndings suggest that the weak level of cross-resistance between methoxyfenozide and indoxacarb resistance could be because of the activity of nonspeciÞc monooxygenases, which are probably not responsible for emamectin benzoate detoxiÞcation. The results of our Þeld trial and bioassay apparently support a similar condition in the examined L. botrana strain, but further biochemical and molecular analyses are needed to conÞrm this hypothesis. Among the new generation insecticides, indoxacarb is the most widely used against L. botrana in Emilia-Romagna, and our study shows that the recurrent use of this insecticide is already selecting resistant Þeld populations. Although there are alternatives to insecticides for an effective resistance management of L. botrana (Ioriatti et al. 2011), further data in chemical ecology are required for improving the defense against this pest by promoting the development of new, more environmentally friendly techniques.
June 2014
CIVOLANI ET AL.: INSECTICIDE RESISTANCE OF Lobesia botrana Acknowledgments
This work was supported by Fondazione Cassa di Risparmio di Reggio EmiliaÐPietro Manadori (Reggio Emilia, Italy).
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