Resistance in field populations of Tetranychus urticae to acaricides and characterization of the inheritance of abamectin resistance

Crop Protection 67 (2015) 77e83

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Resistance in field populations of Tetranychus urticae to acaricides and characterization of the inheritance of abamectin resistance Cecília B.S. Ferreira*, Fernanda H.N. Andrade, Agna R.S. Rodrigues, Herbert A.A. Siqueira*, Manoel G.C. Gondim Jr. Departamento de Agronomia, Entomologia, Universidade Federal Rural de Pernambuco, Recife, PE, 52171-900, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 May 2014 Received in revised form 26 September 2014 Accepted 29 September 2014 Available online

Tetranychus urticae Koch (Acari: Tetranychidae) is one of the most important mite pests in the world. This mite is mainly controlled with synthetic acaricides, and abamectin is one of the most widely used ones. However, control failures have occurred in the field that may be associated with the development of acaricide resistance. Studies were carried out to evaluate resistance to abamectin in four T. urticae populations using dipping test. Also, cross-resistance was assessed to nine acaricides and the genetic basis to the abamectin resistance was determined. Reciprocal crosses between resistant and susceptible ~o populations were carried out to test the inheritance of resistance. Populations from Bonito and Breja (state of Pernambuco, Brazil) were highly resistant to abamectin and to other acaricides tested, including METI group. Cross-resistance observed between abamectin and milbemectin was likely due to similar modes of action because milbemectin was not used in the field. The inheritance findings of this work ~o populations was autosomal, incomplete indicate that resistance to abamectin in Bonito and Breja recessive and polyfactorial. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Two-spotted spider mite Toxicity Cross-resistance Chemical control Resistance management

1. Introduction Brazil produces 5% of the world's fruit and is surpassed only by China and India (SEBRAE, 2009). The Northeast region of Brazil is the major area for tropical fruit production (Nascimento, 2001), especially the municipalities of Petrolina (PE) and Juazeiro (BA), ~o Francisco Valley (Agrianual, located in the Lower Basin of the Sa 2008). This region benefits from excellent insolation and heat, a semi-arid climate and soil favorable for irrigation, enabling yearround production and high production between October and April, when European, Asian and American markets are in the offseason (CODEVASF, 1989). However, despite being favorable for fruit production throughout the year, these environmental characteristics also favor the rapid development of arthropods. The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most important agricultural mite pests (Jepson et al., 1975; Bolland et al., 1998). It can develop on over 1100 host species of more than 70 different plant genera (Grbic et al.,

* Corresponding authors. Tel.: þ55 81 3320 6207, þ55 81 3320 6234. E-mail addresses: [email protected], cecilia.sanguinetti@hotmail. com (C.B.S. Ferreira), [email protected] (F.H.N. Andrade), agna. [email protected] (A.R.S. Rodrigues), [email protected] (H.A.A. Siqueira), [email protected] (M.G.C. Gondim). http://dx.doi.org/10.1016/j.cropro.2014.09.022 0261-2194/© 2014 Elsevier Ltd. All rights reserved.

2011). This mite can be particularly problematic in fruit, ornamental and vegetable plants (Moraes and Flechtmann, 2008). Abamectin is one of the acaricides most widely used to control this mite (van Leeuwen et al., 2009). This acaricide belongs to the class of macrocyclic lactones, which are derived from the soil microorganism Streptomyces avermitilis, and acts on gammaaminobutyric acid (GABA) and glutamate-gated chloride channels (Dekeyser, 2005; van Leeuwen et al., 2010). In Brazil, repeated use of abamectin in T. urticae populations has led to control failures in many areas (Sato et al., 2000, 2009; Nicastro et al., 2010). The high T. urticae reproductive potential, haplo-diploid sexual reproduction and short life cycle also facilitate the rapid development of resistance to many acaricides after limited numbers of applications (Nauen et al., 2000; van Leeuwen et al., 2010). Among all arthropods, T. urticae has the largest prevalence of pesticide resistance (van Leeuwen et al., 2010; Grbic et al., 2011). Arthropods develop pesticide resistance through physiological mechanisms such as reductions in cuticular penetration, alterations in the target site, and increased detoxification metabolism (Feyereisen, 1995; van Leeuwen et al., 2010). Enzymes, such as esterases, cytochrome P450-dependent monooxygenases and glutathione S-transferases, are also involved (Kasai, 2004; Sato et al., 2007). Biochemical studies suggest the involvement of two T. urticae resistance mechanisms to the acaricide abamectin and showed a metabolism

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increase by cytochrome P450-dependent monooxygenases and glutathione S-transferases (Stumpf and Nauen, 2002). Riga et al. (2014) reported that CYP392A16 (a cytochrome P450 associated with high levels of acaricide resistance in T. urticae) catalyzes the hydroxylation of abamectin. Recent studies have however indicated that the abamectin resistance is associated with a mutation on the target site (Kwon et al., 2010b; Dermauw et al., 2012). Other acaricides are being used in an attempt to reduce the incidence of resistance to abamectin, and their efficacy may limit in controlling T. urticae due to the possible cross-resistance (resistance to one product resulting in resistance to others with the same or similar modes of action) (Guedes and Omoto, 2000; Stavrinides and Hadjistylli, 2009). Information guiding producers to correct pesticide use is needed to delay acaricide resistance development and to efficiently control it. Knowledge of the resistance mechanisms, resistance dynamics and the existence of cross-resistance is important for proper resistance management. Failures in T. urticae control are common in production areas of fruit trees and ornamental flowers of the state of Pernambuco in northeastern Brazil. However, no survey of the resistance of this pest in these areas has been carried out. Additionally, due to the intensive use of abamectin in some areas, this study aimed to survey the resistance to abamectin and other acaricides. Understanding the cross-resistance spectrum and the genetic inheritance of resistance to abamectin in T. urticae populations from Pernambuco will be important to establish more efficient recommendations to control this pest in plantations of fruits and ornamental flowers of the state of Pernambuco, Brazil.

concentrations (variation from 0.0001 mg/L to 1000 mg/L). Cotyledon leaf discs of C. ensiformes, 5 cm in diameter, were dipped into each acaricide concentration for 5 s and then dried for 20 min. Later, each disc was placed on filter paper, polyethylene foam and a Petri dish in this order, each 9 cm in diameter. The dish was saturated with distilled water. A total of 10 T. urticae adult females were transferred to each leaf disc (representing a replicate). An exception occurred for the treatment with hexythiazox, in which nymphs were used because this product does not affect adults. Each treatment had two replicates and a total of 20 mites per treatment. The Petri dishes were kept in incubators at 25 ± 1  C, 85 ± 10% relative humidity (RH) and a 12 h photoperiod. Mortality was evaluated 48 h after treatment by counting the number of mites that were alive and dead. Mites that did not walk at least the distance of their body length were considered dead. 2.3.2. Doseeresponse curves Based on the preliminary tests, 7 to 8 concentrations (from concentrations that caused approximate mite mortality of 0%e 100%) were established for each acaricide. The control group was sprayed with distilled water. Acaricide application and the evaluation were similar to that in the preliminary test. Susceptibility bioassays included three replications with 10 mites each, totaling 30 mites for each treatment, including the control. The whole bioassay was repeated three times. Three replications for each concentration were used in the cross-resistance bioassays, and the whole bioassay repeated at least twice for each acaricide. 2.4. Genetic characterization of resistance

2. Materials and methods 2.1. Acquisition and maintenance of mite populations Tetranychus urticae populations were sampled at Petrolina ~o (Rosa (Carica papaya L. plantation; 9 230 3900 S; 40 300 3500 W), Breja sp. plantation; 9104400 S; 36 340 2600 W) and Bonito (Crysanthemum sp. plantation; 8 280 1300 S; 35 430 3500 W) in the state of Pernambuco. The mites were transported to the laboratory and maintained on jack bean plants (Canavalia ensiformis L.) under laboratory conditions (27 ± 1  C, 85 ± 10% relative humidity and a photoperiod of 12 h). A laboratory population originally from Piracicaba (22 420 3000 S; 47 370 5400 W), state of S~ ao Paulo, Brazil, was collected from a cotton crop (Gossypium hirsutum L.) in 2000. It has since been kept on C. ensiformes under laboratory conditions without undergoing selection pressure by acaricides. 2.2. Acaricides tested The commercial formulations Kraft 36 EC, CHEMINOVA BRASIL LTDA (abamectin), Milbeknock, Iharabras S.A. Chemical Industries ~o de Cultivo LTDA (milbemectin), Polo 500 WP, Syngenta Proteça (diafenthiuron), Ortus 50 SC, Arysta lifescience do Brasil Indústria ria (fenpyroximate), Torque 500 SC, BASF S.A. Química e Agropecua (fenbutatin oxide), Omite 720 EC, Chemtura Indústria Química do ~o Paulo/SP (spiBrasil LTDA (propargite), Envidor, BAYER S.A. Sa rodiclofen), Pirate, BASF S.A. (chlorfenapyr) and Talento, DUPONT DO BRASIL S.A e Barueri (hexythiazox) were used.

Toxicity tests for resistant T. urticae populations derived from ~o and Bonito were carried out for two generations after Breja sampling in a similar manner to the previously described tests. The same tests were carried out on a susceptible laboratory population from Petrolina. The autosomal inheritance and maternal effects were tested using reciprocal crosses between males (n ¼ 50) and virgin females (n ¼ 50) from resistant and susceptible populations to acquire progenies from two hybrid types: F1 (_ susceptible  \ resistant) and F10 (_ resistant  \ susceptible). Each F1 progeny (F1 and F10 ) was kept separately to obtain adults. Doseemortality curves were estimated as previously described using only females obtained from each F1 progeny. The F1 progeny and the resistant population were backcrossed to study the monogenic inheritance model of abamectin resistance. The first backcross (BC1) was carried out between males (n ¼ 50) of the resistant population and females of F1 progeny (n ¼ 50). The second backcross (BC10 ) was carried out with males (n ¼ 50) of F1 progeny and females (n ¼ 50) of the resistant population (resistant females (RR) crossed with resistant males (R) originated by F1 reciprocal crossing). In this case, the descendants were RR females. Progenies obtained from the backcrosses were kept separately to be used in the bioassays of doseemortality curve estimation. This model was also tested with abamectin concentrations from 0.015625 to 16 mg/L using the direct test for the monogenic inheritance of resistance model. 2.5. Statistical analyses

2.3. Toxicity of acaricides to T. urticae 2.3.1. Preliminary tests Preliminary tests were carried out following method n. 4 of a series of methods for susceptibility tests by the Insecticide Resistance Action Committee (IRAC, 2009). At least five concentrations of each acaricide were used with a 10-fold increase between

Probit analysis (Finney, 1971) was used to analyze the treatment mortality data after correcting for control mortality (Abbott, 1925). The software POLO-PC (LeOra Software, 1987) was used to estimate the concentrationeresponse curves. The resistance ratios (RR50) of the susceptible population were calculated for a 95% confidence interval (CI) using the method described by Robertson and Preisler

C.B.S. Ferreira et al. / Crop Protection 67 (2015) 77e83

(1992). Therefore, populations or progenies will have significant toxicity ratios to acaricides if the confidence interval does not include the value 1.0. For resistance ratio interpretations, the following ranges were considered: RR50 < 10 (low or no resistance), 10 < RR50 < 100 (moderate resistance), RR50 > 100 (high resistance). Parallelism and equality tests between the concentrationemortality curves were interpreted using the chi-square test at a 5% significance level. Autosomal inheritance and maternal effects on T. urticae were evaluated from the concentrationemortality curves estimated for the F1 progenies. The level of resistance dominance (D) was determined from the LC50 (Lethal Concentration 50%), estimated for the F1 progeny (F1 or F10 ) and for the susceptible and resistant populations. This method followed Stone (1968), which states that D ¼ 1 corresponds to complete dominance, 0 < D < 1 corresponds to incomplete dominance, 1 < D < 0 corresponds to incomplete recessiveness and D ¼ 1 corresponds to complete recessiveness. The standard error of the dominance degree was calculated using the equation proposed by Lehmann (1966) and interpreted according to Preisler et al. (1990). The direct test for monogenic inheritance was carried out with an adjustment for the mortality observed for the BC1 backcross at a given abamectin concentration and the expected mortality, as described by Georghiou (1969). Mortality rates of the resistant population and the F1 progeny were estimated from the doseemortality curves, following Georghiou (1969). The chi-square test was then carried out using the observed backcross mortality rates and the expected mortality rates using the equation described by Sokal and Rohlf (1981). Thus, the monogenic inheritance hypothesis is rejected when the probability is less than 5%, considering one degree of freedom.

3. Results 3.1. Toxicity of acaricides to T. urticae High variation in the response to abamectin was observed among the T. urticae populations sampled in the state of Pernambuco (Probit model fitted to mortality data e Chi-square not significant at P > 0.05) (Table 1). The Petrolina II population had the smallest LC50 and LC95 values for abamectin (0.0011 mg/L and 0.033 mg/L, respectively), indicating a high susceptibility compared to the other tested populations and to the manufacturer recommended dose. Therefore, this population was considered a reference for resistance to abamectin. The estimated LC50 values for the other populations varied from 0.0084 (Piracicaba) to 326 (Bonito) mg abamectin/L. These results lead to resistance ratios (RR50) to abamectin that varied from 8.0 (Piracicaba) to almost 300,000

79

(Bonito) compared to Petrolina II, indicating high resistance for some populations. The Petrolina II population was also considered a reference for susceptibility to diafenthiuron. Populations from Piracicaba, Bonito and Brej~ ao had resistance ratios of <10, >100 and >100, respectively, compared to Petrolina II (Table 2). However, the Piracicaba population was considered a reference for susceptibility to the other tested products. The highest resistance ratios were ~o populations. Milbealways detected for the Bonito and Breja mectin resulted in high resistance ratios of 650 and 700 compared ~o populations, respectively. The to Piracicaba in the Bonito and Breja ~o populations were respective RR50 values for the Bonito and Breja 200 and 150 (fenpyroximate), 3600 and 570 (chlorfenapyr), 390 and 400 (spirodiclofen), 350 and 2050 (fenbutatin oxide), 45 and 96 (propargite), 4 and 5 (hexythiazox), and 4 and 9 (spiromesifen) compared to the reference population (Piracicaba) (Table 2). These results suggest cross-resistance to some of these acaricides. 3.2. Genetic characterization of resistance Overall, the mortality data followed the Probit model (p > 0.05) ~o and Bonito, except for the F1 cross for the populations from Breja and the RC10 backcross of the Bonito population (Table 3). The ~o population had a significant resistance ratio to abamectin Breja compared to the Petrolina II population (RR50 > 60,000) (Table 3). The resistance ratio between both reciprocal crosses (LC50 values of 0.57 and 0.61 mg/L for the F10 and F1 progenies, respectively) was RR50 ¼ 0.99 (0.54e1.82), indicating not sex-linked. The dominance degrees D50 were 0.23 and 0.24, respectively, which indicates incomplete dominance for these progenies (Table 3). The observed mortality of the RC1 backcross at a single tested concentration was significantly different from the expected mortality. Moreover, a plateau was observed in the range of 30% mortality (Fig. 1). The Bonito population exhibited significant resistance to abamectin compared to the Petrolina II population (RR50 > 150,000) (Table 3). The resistance ratio between both reciprocal crosses (LC50 values of 0.43 and 1.03 mg/L for the F10 and F1 progenies, respectively) was RR50 ¼ 1.00 (0.64e1.55), indicating not sex-linked. The dominance levels (D50) for these progenies were, respectively, 0.24 and 0.09, indicating incomplete dominance. The mortality observed for RC1 in six out of 12 tested concentrations was significantly different from the expected mortality. Moreover, a late plateau, in the range of 90% mortality (Fig. 1), was observed. This result suggests a monofactorial inheritance of resistance. 4. Discussion The resistance of T. urticae to abamectin is alarming in populations of various crops in the state of Pernambuco. However, on

Table 1 Susceptibility of Tetranychus urticae populations from the State of Pernambuco to abamectin. Population

Crop

Na

Slope ± SEb

Petrolina II e PE Piracicaba e SP Petrolina I e PE Gravat a e PE Goi^ ania e PE Brej~ ao e PE Bonito e PE

Papaya Cotton Grape Rose Papaya Rose Chrysanthemum

613 714 676 787 584 610 693

1.10 1.84 2.18 1.03 1.38 1.17 1.62

± ± ± ± ± ± ±

0.09 0.12 0.20 0.08 0.12 0.10 0.12

LC50 (95% CI)c

LC95 (95% CI)c

c2 (DF)d

RR50 (95% CI)e

0.0011 0.0084 0.036 0.041 1.79 118 326

0.033 0.066 0.205 1.66 27.9 3,000 3,397

10.8 9.4 6.4 2.4 3.9 1.7 7.1

1.0 8.0 34.4 39.3 1,716 113,532 295,270

(0.0007e0.0016) (0.0062e0.0111) (0.026e0.046) (0.031e0.053) (1.38e2.23) (92e148) (259e400)

(0.016e0.113) (0.042e0.129) (0.147e0.343) (1.03e3.12) (19.6e45.1) (1,913e5,544) (2,283e6,032)

(6) (5) (5) (6) (5) (5) (6)

(0.7e1.4) (4.6e13.9)* (18.4e64.0)* (27.3e56.4)* (187e15,794)* (80,600e159,919)* (190,314 342,664)*

*Significant resistance ratio when the confidence limit does not included the value 1.0. a Number of mites tested. b Standard error. c Concentration in mg/L (confidence interval). d Chi-square test (P > 0.05) and degree of freedom. e Resistance ratio: ratio and 95% CI of the LC50 between resistance and susceptible populations, calculated by Robertson and Preisler (1992) method.

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Table 2 Cross-resistance of Tetranychus urticae populations from the State of Pernambuco to acaricides. Acaricide

Population

Na

Slope ± SEb

Diafenthiuron

Petrolina II Piracicaba Brej~ ao Bonito Piracicaba Petrolina II Bonito Brej~ ao Piracicaba Petrolina II Bonito Brej~ ao Piracicaba Petrolina II Brej~ ao Bonito Piracicaba Petrolina II Bonito Brej~ ao Piracicaba Petrolina II Bonito Brej~ ao Piracicaba Petrolina II Bonito Brej~ ao Piracicaba Petrolina II Brej~ ao Bonito Piracicaba Petrolina II Brej~ ao Bonito

426 484 340 401 484 455 373 380 315 469 378 387 424 481 477 524 401 547 538 414 465 538 477 459 472 397 395 391 416 404 440 415 418 487 467 470

1.60 1.66 1.20 1.33 1.39 1.29 1.61 2.07 1.38 1.22 3.36 2.87 1.92 1.91 2.18 1.25 0.82 0.41 1.26 1.76 0.60 0.58 0.72 0.79 2.58 2.58 3.09 1.93 1.22 1.20 0.98 1.14 0.97 1.05 1.10 1.13

Milbemectin

Fenpyroximate

Chlorfenapyr

Spirodiclofen

Fenbutatin oxide

Propargite

Hexythiazox

Spiromesifen

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.13 0.12 0.13 0.12 0.12 0.12 0.15 0.28 0.14 0.10 0.34 0.25 0.18 0.17 0.21 0.09 0.07 0.04 0.09 0.16 0.06 0.05 0.06 0.09 0.25 0.25 0.29 0.17 0.12 0.12 0.11 0.12 0.12 0.10 0.10 0.10

LC50 (95% CI)c

LC95 (95% CI)c

c2 DFd

RR50e

6.6 (5.3e8.1) 10.7 (8.2e14.4) 4,053 (2,992e5,366) 7,732 (5,202e11,840) 0.6 (0.4e0.7) 5.4 (4.1e7.0) 357 (280e443) 384 (270e492) 22 (16e29) 87 (68e111) 3,246 (2,572e4,202) 4,343 (3,383e5,359) 1.3 (1.0e1.6) 2.8 (2.3e3.4) 735 (602e877) 4,652 (3,690e5,886) 16.4 (10.6e25.1) 37.5 (18.0e71.5) 6,401 (5,073e8,031) 6,586 (5,215e8,073) 0.83 (0.51e1.34) 1.72 (1.08e2.72) 293 (196e442) 1,705 (1,076e2,462) 6.5 (5.5e7.6) 15 (12.7e17.8) 291 (251e337) 622 (506e753) 2,938 (2,284e3,805) 4,370 (3,371e5,854) 12,700 (9372e17,088) 13,814 (10,584e18,168) 373 (234e527) 487 (333e651) 1,388 (911e1,964) 3,201 (2,087e4,863)

70 105 93,708 133,440 8.3 101 3,726 2,386 341 1,929 10,014 16,234 9.3 20.1 4,157 94,598 1,590 370,870 127,750 56,390 436 1,093 52,892 197,990 28 66 990 4,410 64,871 100,510 605,400 381,630 18,404 17,752 42,781 90,424

4.03(6) 7.68(6) 3.25(5) 7.78(5) 2.71(6) 4.40(6) 3.29(5) 1.22(5) 0.82(7) 5.61(6) 7.64(5) 11.49(6) 4.09(5) 3.22(6) 1.43(6) 4.94(7) 3.94(5) 0.72(7) 5.55(7) 1.94(5) 2.99(6) 1.18(7) 2.39(6) 0.31(6) 2.98(6) 4.32(5) 2.60(5) 4.29(5) 3.95(5) 1.23(5) 1.76(5) 3.43(5) 1.98(5) 4.25(6) 6.29(6) 9.69(6)

1.0 1.6 619 1,180 1.0 9.9 650 700 1.0 4.0 200 150 1.0 2.2 570 3,600 1.0 2.3 390 400 1.0 2.1 350 2,048 1.0 2.3 45 96 1.0 1.5 4.3 4.7 1.0 1.0 3.7 8.6

(48e113) (64e216) (52,321e225,280) (60,818e553,080) (5.7e14.0) (66e182) (2,559e6,310) (1,754e3,851) (213e662) (1,167e3,844) (6,955e19,352) (11,836e27,581) (6.8e14.1) (14.9e30.1) (3,151e6,089) (59,899e172,110) (739e4,512) (90,451 2,778,500) (83,254e223,270) (40,713e88,181) (145e2,183) (388e4,574) (21,897e178,780) (86,155e736,320) (22e40) (51e93) (788e1,355) (3,222e6,764) (36,909e145,830) (53,172e256,230) (288,110e1896,100) (203,090e971,410) (9,565e52,320) (10,540e37,390) (22,616e115,770) (40,277e358,900)

(0.7e1.4) (1.2e2.2) (431e888) (854e1,632) (0.7e1.5) (6.8e14.5) (458e927) (471e1,039) (0.7e1.5) (2.7e5.9) (106e207) (142e276) (0.8e1.3) (1.6e2.8) (431e748) (2,648e4,900) (0.5e1.8) (1.0e5.2) (239e638) (249e656) (0.5e2.0) (1.1e4.0) (188e658) (1,092e3,839) (0.8e1.3) (1.8e2.9) (36e56) (74e124) (0.7e1.4) (1.0e2.2) (2.9e6.4) (3.2e6.8) (0.6e1.8) (0.6e1.8) (2.3e6.1) (5.3e13.8)

*Significant resistance ratio when the confidence limit does not included the value 1.0. a Number of mites tested. b Standard error. c Concentration in mg/L (confidence interval). d Chi-square test (P > 0.05) and degree of freedom. e Resistance ratio: ratio and 95% CI of the LC50 between resistance and susceptible populations, calculated by Robertson and Preisler (1992) method.

ornamental flower plantations, the problem is more severe; suggesting that resistance management practices need to be adopted in these crops. The resistance of T. urticae to abamectin is known worldwide (Campos et al., 1995, 1996; Stumpf and Nauen, 2002; Sato et al., 2005, 2009; Riga et al., 2014) and this mite is considered the most resistant to most pesticides (van Leeuwen et al., 2009). There are serious problems with T. urticae resistance to abamectin in crops of ornamental plants in Brazil. Nearly 75% of the populations have resistance frequency values greater than 30% (Sato et al., 2009). However, Brazilian populations have shown a complete loss of resistance to abamectin after six months without selection pressure (Sato et al., 2005). High resistance to milbemectin was observed. However, this compound is new and is still not widely applied in cultivation areas for T. urticae control. Thus, cross-resistance with abamectin is clear, which has been shown previously (Sato et al., 2005). Milbemycin and avermectin (group 6 echloride channel activators, IRAC) are characterized by the presence of a rigid complex of 16 macrocyclic lactones (Lasota and Dybas, 1991; Clark et al., 1995; Nicastro et al., 2010). The difference between milbemectin and avermectin is a disaccharide substitute of carbon-13 in avermectin (Shoop et al., 1995). Both compounds potentiate glutamate and gamma aminobutyric acid (GABA), which opens chloride channels, leading to arthropod paralysis and death (Shoop et al., 1995; Bloomquist, 2001). Abamectin should not be used by rotating it with

milbemectin to manage resistance in T. urticae. In this study, populations resistant to abamectin had a high resistance ratio to chlorfenapyr, which acts by changing the proton gradient of oxidative phosphorylase. However, this result was not previously found in studies of cross-resistance between these products (Sato et al., 2005). Thus, the resistance may have evolved in parallel because of the competing usage of the products in the area. The acaricide fenpyroximate is a Mitochondrial Electron Transport Inhibitor (METI). The cross-resistance between abamectin and fenpyroximate is controversial because it was found in some studies (Nauen et al., 2001; Sato et al., 2005) and not in others (Kim et al., 2004). In this study, populations resistant to abamectin were moderately resistant to fenpyroximate, which may be caused by the similar mode of detoxification to abamectin (Kim et al., 2004), suggesting cross resistance. Still, this result suggests the need for caution when recommending the use of fenpyroximate as Kim et al. (2004) also noted. Populations resistant to abamectin were highly resistant to spirodiclofen. However, there are no records in the literature of cross-resistance between abamectin and spirodiclofen for T. urticae populations (Nauen et al., 2000; Rauch and Nauen, 2002; van Pottelberge et al., 2009). Nevertheless, metabolism detoxification might be playing a role in the resistance of T. urticae for both acaricides, which still needs to be assessed. Populations resistant to abamectin were highly resistant to diafenthiuron, which was used in the cultivation areas where the populations

0.16 ± 0.08 0.31 ± 0.08

2.9(6) 14.6() 5.4(9) 6.3(6) 15.8 (6)* 7.9(6) 7.4(10) 21.0(10)* 0.46 ± 0.12

0.09 ± 0.03 0.24 ± 0.03

0.23 ± 0.05

(488e1,711) (1,539e8,319) (392e2,174) (90,735e259,972) (956e2,892) (399e1,229) (573e2,506) (25,414e106,298) 914 3,578 923 153,586 1,663 700 1,198 51,976 (0.39e0.92) (0.18e1.70) (1.09e4.48) (63e126) (0.55e2.12) (0.28e0.71) (0.58e2.37) (18.9e207) 0.57 0.57 2.22 95.13 1.03 0.43 1.25 54.45 0.15 0.09 0.07 0.43 0.16 0.16 0.08 0.08 ± ± ± ± ± ± ± ± 240 193 214 242 240 233 237 237 F10 BC1 BC10 Bonito F1 F10 BC1 BC10

1.15 0.71 0.67 2.77 1.49 1.40 0.75 0.66

0.24 ± 0.05

*Probit model did not fit. a Number of mites tested. b Standard error. c Lethal concentration that kills 50% and 95% of population, respectively (Confidence Interval). d Resistance ratio: ratio (95% CI) of the LC50 and LC95 between resistance and susceptible populations, calculated using the Robertson and Preisler (1992) method. e Dominance based on LC50 and LC95 ± Standard error. f Degree of Freedom.

(166e2,906) (4,963e151,546) (898e29,048) (6,400e44,303) (192e1,824) (92e915) (2191e35,462) (2,639,706e92,257,147) 694 27,426 5,108 16,838 592 290 8,814 15,605,503

0.28 ± 0.11

2.9(6) 4.7(5) 8.8(7) 1.0 (0.3e3.5) 26,421 (8757e79,713) 1,738 (430e7,021)

0.022 (0.011e0.071) 583 (349e1,435) 38.1 (12.8e320) 15.3 (6.3e66) 112 (21.6e2,815) 599 (181e3,609) 373 (257e779) 13.1 (5e141) 6.4 (3e28) 193 (78.7e699) 16,050 (2042e1,752,100) 0.0006 (0.0003e0.0009) 40 (20e61) 0.61 (0.32e1.08) 237 205 270 Petrolina Brej~ ao F1

1.06 ± 0.15 1.41 ± 0.23 0.91 ± 0.11

1.0 (0.5e1.9) 64,582 (31,987e130,389) 988 (525e1,859)

D50 ± SEe RR50d LC50(95% CI)c Slope ± SEb Na Population

Table 3 Response (mortality) of field-resistant Tetranychus urticae Koch, their hybrid F1, and backcross progenies to abamectin.

LC95 (95% CI)c

RR95d

D95 ± SEe

c2 DFf

C.B.S. Ferreira et al. / Crop Protection 67 (2015) 77e83

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were sampled. Sato et al. (2005) stated that there is likely no crossresistance between propargite and abamectin in T. urticae Brazilian populations. The high resistance to fenbutatin oxide (not registered for T. urticae in Brazil), diafenthiuron, and the moderate resistance to propargite suggest a development of cross-resistance among these three compounds. This cross-resistance may have been found because the mechanism of resistance to these acaricides may be common among them. No resistance to hexythiazox or spiromesifen was found. Hexythiazox is not reported as being used for T. urticae control in Brazil, and spiromesifen is not often used in crops in the state of Pernambuco. Spiromesifen and spirodiclofen both inhibit lipid synthesis (group 23, IRAC), but there is apparently no cross-resistance between these acaricides. In addition, no cases of cross-resistance have been reported in the literature among abamectin, hexythiazox and spiromesifen for T. urticae. Cross-resistance among different chemical groups and modes of action has been reported (van Leeuwen et al., 2009). In this study, it seems that there is crossresistance between products of the same (abamectin and milbemectin) and different chemical groups (diafenthiuron, chlorfenapyr and fenbutatin oxide). Specifically for chlorfenapyr, the effect on respiration (uncoupling oxidative phosphorylation) occurs at a different site than for diafenthiuron and fenbutatin oxide (ATP synthase inhibitors), indicating that cross-resistance could be associated with a metabolic mechanism of resistance. Riga et al. (2014) reported CYP392A16 (a cytochrome P450 associated with high levels of acaricide resistance in T. urticae) did not metabolize hexythiazox, clofentezine and bifenthrin, but was capable of metabolizing abamectin to a less toxic hydroxy-form. Cytoplasmic effect (maternal effect) was not observed (confidence intervals of the LC50 values of reciprocal crosses overlapped) ~o as well as in both reciprocal crosses between Petrolina II and Breja with Petrolina II and Bonito populations, suggesting that resistance is autosomally inherited. Resistance was incompletely dominant in both populations. However, He et al. (2009) reported an incomplete and recessive inheritance of resistance to abamectin for T. urticae (¼Tetranychus cinnabarinus (Boisduval)), and that resistance was likely controlled by one or more maternally inherited gene(s). Other studies recently showed that resistance in a strain of T. urticae was incompletely recessive, not maternally inherited, polyfactorial and dose dependent (Kwon et al., 2010a; Dermauw et al., 2012). Differences among these results may be associated with dominance plasticity (Bourguet et al., 1996), particularly in field populations or different genetic factors associated with each resistance in these populations. Regarding insects, resistance to abamectin was not sex-linked or caused by cytoplasmic factors in Leptinotarsa decemlineata (Say), but autosomal, incompletely recessive, and polyfactorial (Argentine and Clark, 1990). Also, Konno and Scott (1991) reported that resistance to abamectin in Musca domestica L. was recessive and not sex-linked, but the male DL50 was always lower than that of females because males were smaller. The T. urticae genome has gained and lost genes over the course of its evolution (Grbic et al., 2011). This may be an important factor for resistance. A large number of genes that encode proteins associated with resistance were found in the T. urticae genome, which may be important to understand resistance (Grbic et al., 2011). Molecular studies have shown that the enzymatic activities of monooxygenases that depend on cytochrome P450 (MFO) and glutathione S-transferase (GST) are high in populations resistant to abamectin compared to susceptible populations (Nauen et al., 2001; Stumpf and Nauen, 2002). The target-site resistance is also an important mechanism of resistance (Dermauw et al., 2012). Presence of mutations was not assessed in the study; however, the resistances found to most of the acaricides were high or very high, which suggest the involvement of target site alterations. Recent

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C.B.S. Ferreira et al. / Crop Protection 67 (2015) 77e83

~o and (B) e Bonito. Fig. 1. Inheritance of abamectin resistance in two fields populations of Tetranychus urticae: (A) e Breja

studies show that abamectin resistance is also associated with genetic mutations (Kwon et al., 2010b; Dermauw et al., 2012). Kwon et al. (2010b) reported that altered target-site resistance to abamectin in T. urticae is attributed to a G323D point mutation in the GluCl, which was tightly associated with a moderately resistant phenotype. Conversely, Dermauw et al. (2012) reported that a mutation on Tu-GluCl3 in T. urticae was associated with very high levels of abamectin resistance. However, such mutations still need to be assessed in these populations from Brazil. 5. Conclusion Based on these results, the failures of field control in the state of Pernambuco are due to the development of resistance to abamectin. There is also evidence that the efficiency of other compounds, especially milbemectin, has been compromised due to cross-resistance with abamectin. However, it is likely that other resistances have evolved in parallel with abamectin resistance, even toward acaricides that have been used less frequently. Understanding the genetic basis of this resistance breaks new ground to enable the development of new management strategies. The resistance found in ornamental plants indicates the need for special

care for asexually propagated plants that are commercialized in various regions; in this case, the introduction of resistant populations in different regions may occur. Acknowledgments We thank the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e gico, CNPq, Edital Universal/2009 Proc. 477973/2009-4) for Tecnolo financing the Project and for the scholarship granted to the student Cecília Batista Sanguinetti Ferreira and research fellowships for the co-authors. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265e267. rio da Agricultura Brasileira. FPN Editora, Sa ~o Paulo. Agrianual, 2008. Anua Argentine, J., Clark, J., 1990. Selection for abamectin resistance in Colorado potato beetle (Coleoptera: Chrysomelidae). Pestic. Sci. 28, 17e24. Bloomquist, J.R., 2001. GABA and glutamate receptors as biochemical sites for insecticide action. In: Ishaaya, I. (Ed.), Biochemical Sites of Insecticide Action and Resistance. Springer, New York, pp. 17e41.

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