Research Article Received: 21 February 2014
Revised: 11 August 2014
Accepted article published: 19 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3885
Cross-resistance and baseline susceptibility of Mediterranean strains of Bemisia tabaci to cyantraniliprole Carolina Grávalos,a Esther Fernández,a Ana Belando,a Inmaculada Moreno,a Caridad Rosb and Pablo Bielzaa,* Abstract BACKGROUND: The whitefly Bemisia tabaci Gennadius is a severe pest in many field and greenhouse crops worldwide and has developed resistance to insecticides from most chemical classes. The ease with which this pest develops resistance makes it essential to incorporate new compounds with different modes of action and no cross-resistance with those previously used into insecticide resistance management strategies. To that end, the systemic effect of the new diamide cyantraniliprole was tested with multiresistant, selected and field populations of Q-biotype B. tabaci from the Mediterranean area. RESULTS: Bioassays with multiresistant and laboratory-selected populations indicated no cross-resistance to cyantraniliprole in the B. tabaci strains exhibiting resistance to other insecticides. The LC50 values for nymphs from 14 field populations varied between 0.011 and 0.116 mg L−1 , a 10.5-fold natural variability. The LC50 values for adults from three strains ranged from 0.060 to 0.096 mg L−1 . CONCLUSION: These baseline data will be helpful for monitoring future potential shifts in susceptibility to cyantraniliprole in Mediterranean whitefly populations within an IRM programme. Cyantraniliprole may play an important role in mitigating insecticide resistance in B. tabaci because of its high efficacy and its lack of cross-resistance to other insecticides, even in multiresistant Q-biotype populations collected from a highly problematic insecticide resistance area. © 2014 Society of Chemical Industry Keywords: whitefly; anthranilic diamide; Q biotype; insecticide resistance; resistance management; Cyazypyr™
1
INTRODUCTION
The whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a key pest in many field and greenhouse crops worldwide, causing severe damage by direct feeding, by excretion of honeydew and by acting as a vector of viruses such as tomato yellow leaf curl virus (TYLCV). Currently this pest is considered to be a cryptic species complex1 with a number of species with differences in reproduction, host range, development rate, insecticide resistance and virus transmission. Use of insecticides has been the primary strategy for controlling B. tabaci, especially in virus-sensitive crops, where a great number of specific treatments against whiteflies are applied. Owing to this high insecticide pressure, this pest has developed moderate to extremely high resistance to insecticides from most chemical classes, including chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids, insect growth regulators and neonicotinoids.2 – 6 The ease with which this pest develops resistance renders the development and implementation of insecticide resistance management (IRM) strategies crucial. To that end, new compounds with different modes of action and no cross-resistance with those previously used must be incorporated into IRM strategies. However, prior to commercial use, baseline susceptibility data have to be developed as a reference to monitor potential resistance Pest Manag Sci (2014)
evolution. Similarly, studies must be done on whether existing resistance mechanisms confer cross-resistance to any new molecule. Cyantraniliprole is a novel anthranilic diamide insecticide with a new mode of action for sucking insects that belongs to group 28 of the classification of the Insecticide Resistance Action Committee (IRAC). It is a cross-spectrum second-generation anthranilic diamide insecticide discovered by the DuPont company. This compound has interesting physical properties (log P 1.9 at pH 4 and aqueous solubility 15 mg L−1 at 20 ∘ C) that contribute to its insecticidal spectrum, larger than for the classical diamides and including hemipteran pests such as Myzus persicae and hoppers. The trade name is Cyazypyr™. This new compound has both foliar and systemic activity (for direct application to the soil) and acts on a cross-spectrum of
∗
Correspondence to: Pablo Bielza, Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain. E-mail: [email protected]
a Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Cartagena, Spain b Biotecnología y Protección de Cultivos, IMIDA, La Alberca Murcia, Spain
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www.soci.org chewing and sucking pests by activating exclusively on their ryanodine receptors. This group of insecticides acts on selective ion channels, modulating the release of calcium. Cyantraniliprole molecules bind to the ryanodine receptors, causing uncontrolled release and depletion of internal calcium, preventing further muscle contraction. Anthranilic diamides, and specifically cyantraniliprole, show extremely low mammalian toxicity in all animal studies performed, conferring a favourable toxicological profile.7 – 9 This selectivity is due to the structural differences between insect and mammalian ryanodine receptors. Rapid degradation and low toxicity to non-target organisms confer a favourable environmental profile.7 – 9 High efficacy against B. tabaci and other pests has already been described both in laboratory and in field trials.10 – 13 Cyantraniliprole has a significant effect on reducing insect feeding,14,15 and has been shown to reduce virus transmission.16,17 Recently, high levels of resistance to other diamides (chlorantraniliprole and flubendiamide) have been reported for Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations from China, Brazil, the Philippines and Thailand.18 – 21 Diamide insecticides have been intensively used to control this pest in certain regions of those countries, which has led to the development of resistance only a few years after their introduction. Previous insecticide resistance issues to other compounds facilitated an excessive reliance on the then efficacious diamides. The situation of B. tabaci as regards resistance in certain areas mirrors that of P. xylostella, posing a serious threat through the misuse of diamides. Whitefly populations from south-eastern Spain (Almeria and Murcia) have been repeatedly reported as being resistant to many insecticides,22 and among them there are some of the most resistant strains ever reported.4 This makes this area a worst-case scenario in which to study cross-resistance. Moreover, owing to marked differences in insecticide susceptibilities among whitefly biotypes, and the fact that the Q biotype has been reported as the most resistant, it was important to test cyantraniliprole toxicity with field populations of this biotype, because only laboratory populations of Q-biotype whiteflies have as yet been tested against cyantraniliprole. Expression of resistance can be age specific in B. tabaci.6 Laboratory susceptible nymphs were 4–10 times more sensitive to imidacloprid than adults. For strains whose adults were significantly resistant, nymphs exhibited a susceptibility similar to adults from susceptible strains. Consequently, susceptibility and cross-resistance to a new compound ought to be studied for both nymph and adult stages. Knowledge of baseline susceptibility and cross-resistance to new insecticidal compounds is therefore essential for the rational development of antiresistance management strategies. The objectives of this research were to determine the adult and nymph susceptibilities of field populations of Q-biotype whiteflies from the Mediterranean basin (Spain, Italy and Greece), and to examine cross-resistance patterns to other insecticides used against this pest.
2
MATERIALS AND METHODS
2.1 Laboratory strains Five laboratory whitefly strains (Table 1) – a susceptible strain (LAB-S) and four multiresistant strains (MU-A, MU-MI, AL-MO and AL-PA) collected from tomato and sweet pepper crops in south-eastern Spain (Murcia and Almeria) in 2006,4 were used to test cross-resistance to cyantraniliprole with previously existing resistance mechanisms. In a previous study already published,4
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Table 1. Bemisia tabaci populations, origins and biotype information Strain
Country
Location
Original host
Year
LAB-S MU-A MU-MI AL-MO AL-PA MU-TF MU-TF9 AL-CA AL-RE AL-VI MU-AB MU-PE MU-CA AL-AL BA-CA IT-CA IT-RA GR-BER GR-PLA
Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Italy Italy Greece Greece
Murcia Murcia Murcia Almeria Almeria Murcia Murcia Almeria Almeria Almeria Murcia Murcia Murcia Almeria Barcelona Catania Ragusa Veria Platanos
Sweet pepper Tomato Sweet pepper Sweet pepper Sweet pepper Sweet pepper Aubergine Aubergine Aubergine Aubergine Cucumber Cucumber Sweet pepper Sweet pepper Cucumber Broccoli Aubergine Aubergine Cotton
2006 2006 2006 2006 2006 2008 2009 2009 2009 2009 2010 2010 2011 2011 2012 2009 2012 2010 2012
Biotype Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q
these strains were tested against some of the main insecticides used to control whiteflies, including methomyl (IRAC group 1A), imidacloprid (4A), pyriproxyfen (7C), buprofezin (16), pyridaben (21A), spiromesifen (23) and azaridachtin (UN) (Table 2). In the present work, these strains were also tested against alpha-cypermethrin (3A), thiamethoxam (4A) and pymetrozine (9B). In addition, seven resistant strains, selected in the laboratory for resistance to each insecticides, were used to test cross-resistance to cyantraniliprole. The resistant laboratory strains (R-ALFA, R-AZA, R-BU, R-PIME, R-PIB, R-SPI and R-PIX) were selected from the susceptible strain (LAB-S). Successive generations were exposed to increasing concentrations of each insecticide. These populations were selected at least 7 times. 2.2 Field strains Fourteen field populations of B. tabaci Q biotype from Spain (Almeria and Murcia, hot spots for resistance problems), Italy and Greece were collected from 2008 to 2012 (Table 1). Adults were collected directly from leaves of crop plants with a battery-operated entomological vacuum sampler. Insects were transferred to rearing cages with clean cotton, sweet pepper and aubergine plants. All populations were maintained at 27 ± 1 ∘ C, 60% RH and a 16:8 h light:dark photoperiod. Biotypes of all strains were characterised using PCR amplification of the mitochondrial cytochrome oxidase I gene, followed by restriction with the enzyme Tru9I.23 2.3 Insecticide Cyantraniliprole (Cyazypyr 20SC, Verimark™; DuPont Crop Protection, Newark, DE) was used in all bioassays carried out. Serial dilutions for the formulated compound were freshly made with deionised water for each bioassay. For selecting and testing the laboratory resistant strains, the following formulated insecticides were used: alpha-cypermethrin (Fastac; BASF, Seville, Spain), azadirachtin (Align; Sipcam Inagra,
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Table 2. Levels of resistance to cyantraniliprole and the main insecticides used in Spain for the control of Bemisia tabaci in five strains [one susceptible reference laboratory strain (LAB-S) and four multiresistant strains] of this insect pest LAB-S LC50 Insecticide
(mg L−1 )
MU-A LC50
(95% CL)
alphaCypermethrin
141.2 (40.3–255.6)
MU-MI
(mg L−1 )
LC50
(95% CL)
RR50
236.4 (90.6–780.3)
1.7
AL-MO
(mg L−1 )
LC50
(95% CL)
RR50
106.7 (48.5–294.7)
0.8
AL-PA
(mg L−1 )
(95% CL)
LC50 (mg L−1 ) RR50
63.94 (24.10–157.9)
(95% CL)
RR50
0.5
37.47 (10.11–120.6)
0.3 0.3
Azadirachtina
2.65 (0.53–6.55)
0.56 (0.13–1.34)
0.2
82.7 (36.5–166.7)
31
18.54 (11.49–26.37)
7
0.769 (0.003–3.011)
Buprofezina
8.73 (4.45–14.38)
96.2 (64.2–143.3)
11
10 159 (2158–1 389 658)
1164
390.4 (127.4–724.5)
45
518.1 (115.3–1723)
59
Imidacloprida
15.19 (1.29–81.08)
53.4 (16.0–110.5)
3.5
47.4 (6.7–206.6)
3.1
81.50 (36.01–168.2)
5
228.9 (82.43–1018)
15
19.9 (7.5–52.6)
1101 (558–3293)
55
1031 (406–1844)
52
590 (171–7212)
30
109.3 (36.9–467.8)
6
25
2181 (671–8595)
7
213.1 (17.42–1036)
0.7
157.6 (29.60–485.6)
0.5
Methomyla Pymetrozyne
317.1 (148.2–608.5) 7973 (5163–10529)
Pyridabena
0.34 (0.21–0.51)
Pyriproxyfena
0.32 (0.02–1.09)
20.86 (13.77–30.37) 25.92 (16.19–40.66)
0.9
2.95 (0.96–5.01)
9
0.76 (0.13–2.43)
2.2
3.08 (1.40–5.67)
9
1.2
402.4 (274.0–634.3)
19
322.2 (167.0–805.4)
15
243.4 (118.7–547.9)
12 1.3
Spiromesifena
1.08 (0.60–1.81)
1.21 (0.62–2.89)
1.1
3.38 (2.56–4.39)
3.1
7.72 (3.67–18.73)
7
1.39 (0.21–4.87)
Thiamethoxam
17.95 (8.12–34.38)
47.37 (3.00–116.4)
2.6
20.00 (8.82–38.66)
1.1
6.56 (0.55–19.56)
0.4
62.91 (30.20–155.0)
3.5
Cyantraniliprole
0.039 (0.034–0.045)
0.06 (0.038–0.089)
1.5
0.05 (0.039–0.057)
1.3
0.07 (0.041–0.099)
1.8
0.051 (0.038–0.065)
1.3
a
Data from Fernández et al.4
Valencia, Spain), buprofezin (Applaud; Syngenta Agro, Madrid, Spain), pymetrozine (Plenum; Syngenta Agro), pyridaben (Sanmite; BASF, Seville, Spain), pyriproxyfen (Juvinal 10EC; Kenogard, Barcelona, Spain), spiromesifen (Oberon SC; Bayer CropScience, Valencia, Spain) and thiamethoxam (Actara; Syngenta Agro). Serial dilutions of formulated compounds were prepared with deionised water containing 1 mL L−1 of Tween 20 (as non-ionic wetter) on the day of the bioassay. 2.4 Systemic bioassay A new systemic uptake bioassay was developed for testing cyantraniliprole susceptibility in nymphs and adults. For nymphs, cotton seedlings in the first true leaf stage (leaf size of about 2 cm diameter) were used (Fig. 1a). Plants were cut with a disinfected scalpel, leaving a stem length of 10 cm (Figs 1b and c). Then, the stem was introduced into a modified polystyrene plastic cage (Fig. 1.d) and maintained in a test-tube rack over a tray containing deionised water. Approximately 40 adults were released (unknown age) from the population tested into the plastic cage containing the cotton seedling for a 24 h oviposition period to allow synchronisation
(a)
(d)
of each developing stage (Fig. 1e). After oviposition, adults were removed and the total number of eggs on each leaf was counted. The desired serial dilutions of insecticide were prepared in deionised water using formulated insecticide. Deionised water was used as a control solution. The stem of the infested leaf was then placed in a glass scintillation vial containing 20 mL of the desired concentration of the insecticide (Fig. 1f ). Six replications (six seedlings) were used for each concentration (0.01, 0.05, 0.1 and 0.5 mg L−1 ). The bioassays were held at 27 ± 1 ∘ C and a 16:8 h light:dark photoperiod for the duration of the assay. Mortality was determined 14 days after oviposition by counting live nymphs and subtracting that number from the number of eggs counted on day 1 on each leaf. For adult bioassays, similar plants and methodology were used. Cotton seedlings were cut and maintained in laboratory conditions (27 ± 1 ∘ C and a 16:8 h light:dark photoperiod) in scintillation vials containing deionised water for 24 h. After this period, plants were transferred to new scintillation vials containing the desired concentration of the insecticide for a further 24 h. On the third day, plants were introduced into the modified polystyrene plastic cages. Ten healthy adults were then released into the cages.
(b)
(e)
(c)
(f)
Figure 1. Nymphal systemic uptake bioassay: (a) cotton seedlings; (b) seedling cut 10–cm long; (c) leaf size about 2 cm diameter; (d) modified polystyrene plastic cage for holding adults for oviposition; (e) plants with cages in a test-tube rack over a tray containing water; (f ) infested plants placed in glass scintillation vials containing 20 mL of the desired concentration of the insecticide.
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controlled environmental conditions (25 ± 2 ∘ C, 60% RH and a of 16:8 h light:dark photoperiod). Areas bounded by the clip cages were marked with a felt pen, and the cages and adults were removed. Eggs were counted and marked prior to treatment so that survival could be measured. Leaves with eggs were then dipped for 10 s in aqueous dilutions of formulated insecticide or the diluent only for untreated controls. Egg hatch was determined 10–12 days after treatment. Failure to hatch at this stage was considered as mortality. For all bioassays, each concentration had three replicates.
Figure 2. Adult systemic uptake bioassay detail.
Seedlings were maintained for 5 days in polypropylene conical centrifuge tubes (50 mL and self-standing base) with the desired concentration of the insecticide (Fig. 2). Mortality was determined 5 days after introducing adults into the cages by counting live and dead individuals on each plant. 2.5 Leaf-dip bioassay The susceptibility of whitefly populations to insecticides other than cyantraniliprole was tested using a leaf-dip bioassay. For nymphs, cotton plants with four true leaves were used. Approximately 40 adult whiteflies from the populations tested were caged on individual cotton leaves using small clip cages for a 48 h oviposition period to allow synchronisation of each developing stage. Plants were maintained at 25 ± 2 ∘ C, 60% RH and a 16:8 h light:dark photoperiod. After 15 days, when the majority of nymphs had reached second instar (N2), the leaves were dipped in serial dilutions of formulated material for 10 s. Control replicates were dipped in deionised water containing 1 mL L−1 of Tween 20. Each bioassay used three replicates at a minimum of six concentrations. Mortality was assessed by comparing the number of nymphs present on the day of treatment with the number remaining unenclosed or dead. This occurred when the last nymphal instar had been reached on control plants (15 days later). For pyriproxyfen, egg-dip bioassays were conducted on whole cotton plants (20–25 cm tall). Forty B. tabaci adults were confined to leaves using clip cages and allowed to oviposit for 24 h under
2.6 Data analysis When necessary, bioassay data were corrected for control mortality.24 Data were analysed using the POLO-PC program for probit analysis.25 The lethal concentration (LC50 ) plus the 95% confidence limits were calculated to determine significant differences between populations. Resistance ratios (RR50 ) for each population were calculated relative to the LC50 level of the susceptible reference strain.
3
RESULTS AND DISCUSSION
3.1 Cross-resistance The multiresistant strains and the susceptible reference strain were bioassayed with alpha-cypermethrin, pymetrozyne, thiamethoxam and cyantraniliprole to determine their levels of resistance (Table 2). These strains have been previously reported to exhibit low to very high resistance to other compounds (Table 2).4 Conversely, in spite of the marked differences in resistance among the whitefly strains, the susceptibility to cyantraniliprole was consistent among them. The LC50 values for cyantraniliprole ranged from 0.04 to 0.07 mg L−1 , with no significant differences. Nevertheless, the bioassays for the other insecticides were carried out months to years before the cyantraniliprole bioassays, and arguably the resistance might have changed over that period. To overcome this issue, seven resistant strains were selected in the laboratory for each insecticide and tested for their susceptibility to cyantraniliprole and their respective insecticides (Table 3). The resistance level achieved was low for alpha-cypermethrin (fourfold) and spiromesifen (16-fold), moderate for azadirachtin (39-fold), high for buprofezin (95-fold), pyridaben (108-fold) and pyriproxifen (112-fold) and extremely high for pymetrozine (2485-fold). Responses to cyantraniliprole in these strains were
Table 3. Levels of resistance to the insecticide selected for and to cyantraniliprole in laboratory-selected resistant strains of Bemisia tabaci Insecticidea Selected population R-ALFA R-AZA R-BU R-PIME R-PIB R-SPI R-PIX
LC50 (mg L−1 ) (95% CL) 557.7 (261.8–807.7) 102.6 (62.1–197.6) 830.1 (371.6–2325) 72 499 (21 651–23 200 000) 36.55 (18.05–61.35) 17.13 (7.76–26.84) 2333 (1052–7917)
Cyantraniliprole RR50 b
LC50 (mg L−1 ) (95% CL)
4.0 39 95 2485 108 16 112
0.011 (0.006–0.017) 0.047 (0.03–0.069) 0.129 (0.079–0.231) 0.051 (0.035–0.061) 0.081 (0.058–0.11) 0.030 (0.008–0.043) 0.096 (0.085–0.109)
RR50 b 0.3 1.2 3.3 1.3 2.1 0.8 2.5
a Insecticide selected for: R-ALFA, alpha-cypermethrin; R-AZA, azadirachtin,; R-BU, buprofezin; R-PIME, pymetrozyne; R-PIB, pyridaben; R-SPI, spiromesifen; R-PIX, pyriproxyfen. b Resistant factor = LC of selected population/LC of susceptible reference population LAB-S (Table 2). 50 50
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Table 4. Susceptibility data to cyantraniliprole in field populations and a susceptible reference population (LAB-S) of B. tabaci Q-biotype from the Mediterranean basin Population
Slope (±SE)
GR-PLA IT-RA MU-CA AL-VI AL-RE LAB-S AL-CA MU-PE IT-CA AL-AL MU-AB MU-TF BA-CA MU-TF9 GR-BER Field composite
1.780(±0.173) 4.379(±0.345) 3.375(±0.346) 2.208(±0.110) 2.116(±0.108) 2.146(±0.093) 1.248(±0.090) 5.173(±0.440) 1.327(±0.169) 1.238(±0.099) 1.575(±0.097) 2.093(±0.192) 1.407(±0.131) 2.268(±0.245) 1.347(±0.064) 1.812(±0.038)
LC50 (mg L−1 ) (95% FL)
RF50
LC90 (mg L−1 ) (95% FL)
RF90
0.011 (0.003–0.018) 0.024 (0.018–0.033) 0.031 (0.023–0.038) 0.032 (0.022–0.042) 0.037 (0.030–0.046) 0.039 (0.034–0.045) 0.045 (0.024–0.071) 0.057 (0.035–0.072) 0.059 (0.029–0.173) 0.060 (0.026–0.117) 0.063 (0.044–0.085) 0.065 (0.043–0.083) 0.081 (0.052–0.133) 0.097 (0.060–0.135) 0.116 (0.086–0.159) 0.048 (0.034–0.063)
0.3 0.6 0.8 0.8 0.9 1 1.2 1.5 1.5 1.5 1.6 1.7 2.1 2.5 3.0 1.2
0.056 (0.033–0.078) 0.047 (0.035–0.078) 0.075 (0.063–0.093) 0.121 (0.088–0.192) 0.151 (0.117–0.212) 0.156 (0.125–0.209) 0.481 (0.272–1.316) 0.101 (0.079–0.237) 0.543 (0.18–94.402) 0.651 (0.271–5.552) 0.41 (0.277–0.734) 0.265 (0.195–0.464) 0.661 (0.328–2.563) 0.356 (0.228–1.114) 1.041 (0.625–2.268) 0.242 (0.167–0.435)
0.4 0.3 0.5 0.8 1.0 1 3.1 0.6 3.5 4.2 2.6 1.7 4.2 2.3 6.7 1.6
relatively stable, with LC50 values ranging from 0.011 to 0.129 mg L−1 , with a maximum RR50 of 3.3 compared with the susceptible population (LAB-S, LC50 = 0.039 mg L−1 ), which is akin to the natural variability found in field populations (see below). A lack of cross-resistance between two diamides (chlorantraniliprole and cyantraniliprole), imidacloprid and pyriproxifen has also been reported for B-biotype field populations and Q-biotype laboratory strains of B. tabaci from Arizona.10 The results obtained show the absence of cross-resistance between cyantraniliprole and the current insecticides used to control whiteflies, suggesting that the existing resistance mechanisms do not affect the performance of this diamide. 3.2 Field populations The results obtained from the bioassays with cyantraniliprole in 14 field populations from Spain, Italy and Greece are presented in Table 4. Susceptibility to cyantraniliprole varied among the populations collected across the Mediterranean area. The LC50 values for nymphs varied between 0.011 mg L−1 (GR-PLA) and 0.116 mg L−1 (GR-BER) (Table 4), a 10.5-fold variability between the least and most sensitive populations. Two populations (GR-PLA and IT-RA) were significantly more susceptible than the susceptible reference population (LAB-S). Three populations, BA-CA, MU-TF9 and GR-BER, were significantly more tolerant than five field populations and the susceptible population LAB-S, but were not different to the remaining six field populations tested in these studies (LC50 values from 0.045 to 0.065 mg L−1 ). These data were consistent enough to be pooled to yield a composite LC50 of 0.048 mg L−1 for Q-biotype B. tabaci. In order to calculate resistance factors of field populations, these baseline data may be used as a reference.
Resistance monitoring studies before commercial use are carried out to determine variation in natural tolerance to a new compound. Knowing the extent of this natural variation in susceptibility is very important in order to distinguish it from genuine resistance after subsequent commercial introduction. The present laboratory bioassays of field populations of Mediterranean Q-biotype B. tabaci showed a slight variation (10.5-fold) in susceptibility to cyantraniliprole and were consistent in their response. The previous history of insecticide exposure may influence the response of whiteflies to cyantraniliprole; however, none of the populations tested had been exposed to cyantraniliprole under field conditions, so observed differences in LC50 values were attributable to natural variation. Other diamides such as chlorantraniliprole and flubendiamide have been used in recent years, and this may have an impact on susceptibility variability. Regional differences in susceptibility to cyantraniliprole have been recorded in several studies.10,13 Variations in susceptibility to cyantraniliprole were documented for Bemisia biotypes B and Q from Arizona10 and Florida.13 Overall, the LC50 values reported for B biotypes range from 0.013 to 0.065 mg L−1 , and for Q biotypes from 0.011 to 0.191 mg L−1 (including the present data). Therefore, these data indicate that there are no differences in the susceptible response to cyantraniliprole between B. tabaci biotypes and/or populations from different parts of the world. Nevertheless, there seem to be some Q-biotype strains that are more tolerant, which highlights the importance of carefully monitoring the resistance evolution to cyantraniliprole in this whitefly biotype. 3.3 Adults versus nymphs Adults from three Q-biotype laboratory populations were tested against cyantraniliprole (Table 5). The LC50 values ranged from
Table 5. Susceptibility data of adults of Bemisia tabaci to cyantraniliprole Population
Slope (±SE)
LAB-S MU-MI AL-PA
2.445(±0.237) 1.055(±0.101) 1.630(±0.222)
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LC50 (mg L−1 ) (95% CL)
RF50
LC90 (mg L−1 ) (95% CL)
RF90
0.071 (0.060–0.083) 0.060 (0.043–0.080) 0.096 (0.058–0.138)
0.8 1.4
0.238 (0.187–0.333) 0.979 (0.59–2.053) 0.588 (0.364–1.449)
4.1 2.5
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www.soci.org 0.060 to 0.096 mg L−1 , with a 1.6-fold variability and no significant differences. These values are slightly higher than those reported for B-biotype adults from Florida, which ranged from 0.037 to 0.059 mg L−1 .13 The LC50 values for nymphs of the same populations (Table 2) ranged from 0.039 to 0.051 mg L−1 . These results support a slightly greater toxicity for nymphs than for adults, as suggested previously.13 This difference may be due to a longer exposure time for nymphs (14 days) than for adults (5 days).13 Nevertheless, the difference is very small to be of significant importance in the field. When expression of resistance has been reported as age specific in B. tabaci,6 the variation between nymphs and adults is 4–10-fold.
4
CONCLUSIONS
The present results document the present status of high susceptibility to cyantraniliprole in Mediterranean Q-biotype whitefly populations. All the LC50 values reported in this study are similar to those reported in previous studies of cyantraniliprole against B. tabaci.10,13 There was no correlation between the toxicities of cyantraniliprole and other insecticides usually applied to control whitefly populations in southern Europe. Moreover, some populations highly resistant to other insecticides did not vary significantly in their response to cyantraniliprole compared with the susceptible reference. The hypothesis that existing resistance mechanisms do not confer any protection to this novel insecticide is substantiated by these data. The present results are consistent with previous studies that suggested the absence of cross-resistance between imidacloprid, pyriproxifen and cyantraniliprole.10 Therefore, pre-existing cross-resistance conferred by genes selected by other insecticides seems unlikely, as the resistance mechanisms in these populations gave no tolerance to cyantraniliprole. Contained cropping systems such as greenhouses are initially most at risk of resistance to novel insecticides developing because insecticides are applied frequently on enclosed insect populations. This provides ideal conditions for resistance selection, and these highly selected populations may be transported to new areas. The intensive vegetable-growing regions of south-eastern Spain, such as Almeria, with high frequencies of insecticide applications and the constant presence of hosts, provide an environment where selection for resistance is of concern.26 As a consequence, severe resistance problems in whiteflies have been reported in this area.4,22 In this present scenario, Q-biotype whitefly populations showed a consistent susceptibility to cyantraniliprole. These baseline data will be helpful for monitoring any future shifts in susceptibility to cyantraniliprole in Mediterranean whitefly populations that may occur within an IRM programme. In summary, cyantraniliprole may play an important role in mitigating insecticide resistance in B. tabaci because of its high efficacy and its lack of cross-resistance to other insecticides, although a high level of vigilance is required, as the threat from resistance developing to such efficacious insecticides can be high.
ACKNOWLEDGEMENTS The authors acknowledge anonymous referees for reviews and comments on the manuscript. They are grateful to JM Alvarez, DuPont Crop Protection, for his helpful comments on the manuscript. This research has been supported by DuPont and the Spanish Ministry of Economy and Competitiveness (MINECO)
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(AGL2011-25164) and by the European FEDER funds. C. Grávalos was supported by a FPI grant (BEF-2009-025572) funded by the MINECO.
REFERENCES 1 De Barro PJ, The Bemisia tabaci species complex: questions to guide future research. J Integr Agric 11:187–196 (2012). 2 Horowitz AR, Kontsedalov S, Khasdan V and Ishaaya I, Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance. Arch Insect Biochem Physiol 58:216–225 (2005). 3 Palumbo JC, Horowitz AR and Prabhaker N, Insecticidal control and resistance management for Bemisia tabaci. Crop Prot 20:739–765 (2001). 4 Fernández E, Grávalos C, Haro PJ, Cifuentes D and Bielza P, Insecticide resistance status of Bemisia tabaci Q-biotype in south-eastern Spain. Pest Manag Sci 65:885–891 (2009). 5 Nauen R and Denholm I, Resistance of insect pests to neonicotinoid insecticides: current status and future prospects. Arch Insect Biochem Physiol 58:200–215 (2005). 6 Nauen R, Bielza P, Denholm I and Gorman K, Age-specific expression of resistance to neonicotinoid insecticides in the whitefly, Bemisia tabaci. Pest Manag Sci 64:1106–1110 (2008). 7 Lahm GP, Selby TP, Freudenberger JH, Stevenson TM, Myers BJ, Seburyamo G et al., Insecticidal anthranilic diamides: a new class of potent ryanodine receptor activators. Bioorg Med Chem Lett 15:4898–4906 (2005). 8 Cordova D, Benner EA, Sacher MD, Rauh JJ, Sopa JS, Lahm GP et al., Anthranilic diamides: a new class of insecticides with a novel mode of action, ryanodine receptor activation. Pestic Biochem Physiol 84:196–214 (2006). 9 Sattelle DB, Cordova D and Cheek TR, Insect ryanodine receptors: molecular targets for novel pest control chemicals. Invert Neurosci 8:107–119 (2008). 10 Li X, Degain BA, Harpold VS, Marçon PG, Nichols RL, Fournier AJ et al., Baseline susceptibilities of B- and Q-biotype Bemisia tabaci to anthranilic diamides in Arizona. Pest Manag Sci 68:83–91 (2012). 11 Foster SP, Denholm I, Rison JL, Portillo HE, Margaritopoulis J and Slater R, Susceptibility of standard clones and European field populations of the green peach aphid, Myzus persicae, and the cotton aphid, Aphis gossypii (Hemiptera: Aphididae), to the novel anthranilic diamide insecticide cyantraniliprole. Pest Manag Sci 68:629–633 (2012). 12 Fettig CJ, Hayes CJ, McKelvey SR and Mori SR, Laboratory assays of select candidate insecticides for control of Dendroctonus ponderosae. Pest Manag Sci 67:548–555 (2011). 13 Caballero R, Cyman S, Schuster DJ, Portillo HE and Slater R, Baseline susceptibility of Bemisia tabaci (Genn.) biotype B in southern Florida to cyantraniliprole. Crop Prot 44:104–108 (2013). 14 Cameron R, Lang EB, Annan IB, Portillo HE and Alvarez JM, Use of fluorescence, a novel technique to determine reduction in Bemisia tabaci (Hemiptera: Aleyrodidae) nymph feeding when exposed to Benevia and other insecticides. J Econ Entomol 106:597–603 (2013). 15 Cameron R, Lang EB and Alvarez JM, Use of honeydew production to determine reduction in feeding by Bemisia tabaci (Hemiptera: Aleyrodidae) adults when exposed to cyantraniliprole and imidacloprid treatments. J Econ Entomol 107:546–550 (2014). 16 Govindappa MR, Bhemanna M, Arunkumar H and Ghante VN, Bio-efficacy of newer insecticides against tomato leaf curl virus disease and its vector whitefly (Bemisia tabaci) in tomato. Int J Appl Biol Pharm Technol 4:226–231 (2013). 17 Jacobson AL and Kennedy GG, Effect of cyantraniliprole on feeding behavior and virus transmission of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) on Capsicum annuum. Crop Prot 54:251–258 (2013). 18 Wang X and Wu Y, High levels of resistance to chlorantraniliprole evolved in field populations of Plutella xylostella. J Econ Entomol 105:1019–1023 (2012). 19 Wang X, Khakame SK, Ye C, Yang Y and Wu Y, Characterisation of field-evolved resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella, from China. Pest Manag Sci 69:661–665 (2013).
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Cross-resistance and susceptibility of B. tabaci to cyantraniliprole 20 Troczka B, Zimmer CT, Elias J, Schorn C, Bass C, Davies TGE et al., Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor. Insect Biochem Mol Biol 42:873–880 (2012). 21 Ribeiro LMS, Wanderley-Teixeira V, Ferreira HN, Teixeira AAC and Siqueira HAA, Fitness costs associated with field-evolved resistance to chlorantraniliprole in Plutella xylostella (Lepidoptera: Plutellidae). Bull Entomol Res 104:88–96 (2014). 22 Elbert A and Nauen R, Resistance of Bemisia tabaci (Homoptera: Aleyrodidae) to insecticides in southern Spain with special reference to neonicotinoids. Pest Manag Sci 56:60–64 (2000).
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www.soci.org 23 Bosco D, Loria A, Sartor C and Cenis JL, PCR-RFLP Identification of Bemisia tabaci biotypes in the Mediterranean Basin. Phytoparasitica 34:243–251 (2006). 24 Abbott WS, A method for computing the effectiveness of an insecticide. J Econ Entomol 18:265–267 (1925). 25 Russell RM, Robertson JL and Savin NE, POLO: a new computer program for probit analysis. Bull Entomol Soc Am 23:209–213 (1977). 26 Bielza P, Quinto V, Grávalos C, Fernández E and Abellán J, Impact of production system on development of insecticide resistance in Frankliniella occidentalis (Thysanoptera: Thripidae). J Econ Entomol 101:1685–1690 (2008).
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Revised: 11 August 2014
Accepted article published: 19 August 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3885
Cross-resistance and baseline susceptibility of Mediterranean strains of Bemisia tabaci to cyantraniliprole Carolina Grávalos,a Esther Fernández,a Ana Belando,a Inmaculada Moreno,a Caridad Rosb and Pablo Bielzaa,* Abstract BACKGROUND: The whitefly Bemisia tabaci Gennadius is a severe pest in many field and greenhouse crops worldwide and has developed resistance to insecticides from most chemical classes. The ease with which this pest develops resistance makes it essential to incorporate new compounds with different modes of action and no cross-resistance with those previously used into insecticide resistance management strategies. To that end, the systemic effect of the new diamide cyantraniliprole was tested with multiresistant, selected and field populations of Q-biotype B. tabaci from the Mediterranean area. RESULTS: Bioassays with multiresistant and laboratory-selected populations indicated no cross-resistance to cyantraniliprole in the B. tabaci strains exhibiting resistance to other insecticides. The LC50 values for nymphs from 14 field populations varied between 0.011 and 0.116 mg L−1 , a 10.5-fold natural variability. The LC50 values for adults from three strains ranged from 0.060 to 0.096 mg L−1 . CONCLUSION: These baseline data will be helpful for monitoring future potential shifts in susceptibility to cyantraniliprole in Mediterranean whitefly populations within an IRM programme. Cyantraniliprole may play an important role in mitigating insecticide resistance in B. tabaci because of its high efficacy and its lack of cross-resistance to other insecticides, even in multiresistant Q-biotype populations collected from a highly problematic insecticide resistance area. © 2014 Society of Chemical Industry Keywords: whitefly; anthranilic diamide; Q biotype; insecticide resistance; resistance management; Cyazypyr™
1
INTRODUCTION
The whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a key pest in many field and greenhouse crops worldwide, causing severe damage by direct feeding, by excretion of honeydew and by acting as a vector of viruses such as tomato yellow leaf curl virus (TYLCV). Currently this pest is considered to be a cryptic species complex1 with a number of species with differences in reproduction, host range, development rate, insecticide resistance and virus transmission. Use of insecticides has been the primary strategy for controlling B. tabaci, especially in virus-sensitive crops, where a great number of specific treatments against whiteflies are applied. Owing to this high insecticide pressure, this pest has developed moderate to extremely high resistance to insecticides from most chemical classes, including chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids, insect growth regulators and neonicotinoids.2 – 6 The ease with which this pest develops resistance renders the development and implementation of insecticide resistance management (IRM) strategies crucial. To that end, new compounds with different modes of action and no cross-resistance with those previously used must be incorporated into IRM strategies. However, prior to commercial use, baseline susceptibility data have to be developed as a reference to monitor potential resistance Pest Manag Sci (2014)
evolution. Similarly, studies must be done on whether existing resistance mechanisms confer cross-resistance to any new molecule. Cyantraniliprole is a novel anthranilic diamide insecticide with a new mode of action for sucking insects that belongs to group 28 of the classification of the Insecticide Resistance Action Committee (IRAC). It is a cross-spectrum second-generation anthranilic diamide insecticide discovered by the DuPont company. This compound has interesting physical properties (log P 1.9 at pH 4 and aqueous solubility 15 mg L−1 at 20 ∘ C) that contribute to its insecticidal spectrum, larger than for the classical diamides and including hemipteran pests such as Myzus persicae and hoppers. The trade name is Cyazypyr™. This new compound has both foliar and systemic activity (for direct application to the soil) and acts on a cross-spectrum of
∗
Correspondence to: Pablo Bielza, Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Spain. E-mail: [email protected]
a Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Cartagena, Spain b Biotecnología y Protección de Cultivos, IMIDA, La Alberca Murcia, Spain
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www.soci.org chewing and sucking pests by activating exclusively on their ryanodine receptors. This group of insecticides acts on selective ion channels, modulating the release of calcium. Cyantraniliprole molecules bind to the ryanodine receptors, causing uncontrolled release and depletion of internal calcium, preventing further muscle contraction. Anthranilic diamides, and specifically cyantraniliprole, show extremely low mammalian toxicity in all animal studies performed, conferring a favourable toxicological profile.7 – 9 This selectivity is due to the structural differences between insect and mammalian ryanodine receptors. Rapid degradation and low toxicity to non-target organisms confer a favourable environmental profile.7 – 9 High efficacy against B. tabaci and other pests has already been described both in laboratory and in field trials.10 – 13 Cyantraniliprole has a significant effect on reducing insect feeding,14,15 and has been shown to reduce virus transmission.16,17 Recently, high levels of resistance to other diamides (chlorantraniliprole and flubendiamide) have been reported for Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations from China, Brazil, the Philippines and Thailand.18 – 21 Diamide insecticides have been intensively used to control this pest in certain regions of those countries, which has led to the development of resistance only a few years after their introduction. Previous insecticide resistance issues to other compounds facilitated an excessive reliance on the then efficacious diamides. The situation of B. tabaci as regards resistance in certain areas mirrors that of P. xylostella, posing a serious threat through the misuse of diamides. Whitefly populations from south-eastern Spain (Almeria and Murcia) have been repeatedly reported as being resistant to many insecticides,22 and among them there are some of the most resistant strains ever reported.4 This makes this area a worst-case scenario in which to study cross-resistance. Moreover, owing to marked differences in insecticide susceptibilities among whitefly biotypes, and the fact that the Q biotype has been reported as the most resistant, it was important to test cyantraniliprole toxicity with field populations of this biotype, because only laboratory populations of Q-biotype whiteflies have as yet been tested against cyantraniliprole. Expression of resistance can be age specific in B. tabaci.6 Laboratory susceptible nymphs were 4–10 times more sensitive to imidacloprid than adults. For strains whose adults were significantly resistant, nymphs exhibited a susceptibility similar to adults from susceptible strains. Consequently, susceptibility and cross-resistance to a new compound ought to be studied for both nymph and adult stages. Knowledge of baseline susceptibility and cross-resistance to new insecticidal compounds is therefore essential for the rational development of antiresistance management strategies. The objectives of this research were to determine the adult and nymph susceptibilities of field populations of Q-biotype whiteflies from the Mediterranean basin (Spain, Italy and Greece), and to examine cross-resistance patterns to other insecticides used against this pest.
2
MATERIALS AND METHODS
2.1 Laboratory strains Five laboratory whitefly strains (Table 1) – a susceptible strain (LAB-S) and four multiresistant strains (MU-A, MU-MI, AL-MO and AL-PA) collected from tomato and sweet pepper crops in south-eastern Spain (Murcia and Almeria) in 2006,4 were used to test cross-resistance to cyantraniliprole with previously existing resistance mechanisms. In a previous study already published,4
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Table 1. Bemisia tabaci populations, origins and biotype information Strain
Country
Location
Original host
Year
LAB-S MU-A MU-MI AL-MO AL-PA MU-TF MU-TF9 AL-CA AL-RE AL-VI MU-AB MU-PE MU-CA AL-AL BA-CA IT-CA IT-RA GR-BER GR-PLA
Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Italy Italy Greece Greece
Murcia Murcia Murcia Almeria Almeria Murcia Murcia Almeria Almeria Almeria Murcia Murcia Murcia Almeria Barcelona Catania Ragusa Veria Platanos
Sweet pepper Tomato Sweet pepper Sweet pepper Sweet pepper Sweet pepper Aubergine Aubergine Aubergine Aubergine Cucumber Cucumber Sweet pepper Sweet pepper Cucumber Broccoli Aubergine Aubergine Cotton
2006 2006 2006 2006 2006 2008 2009 2009 2009 2009 2010 2010 2011 2011 2012 2009 2012 2010 2012
Biotype Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q
these strains were tested against some of the main insecticides used to control whiteflies, including methomyl (IRAC group 1A), imidacloprid (4A), pyriproxyfen (7C), buprofezin (16), pyridaben (21A), spiromesifen (23) and azaridachtin (UN) (Table 2). In the present work, these strains were also tested against alpha-cypermethrin (3A), thiamethoxam (4A) and pymetrozine (9B). In addition, seven resistant strains, selected in the laboratory for resistance to each insecticides, were used to test cross-resistance to cyantraniliprole. The resistant laboratory strains (R-ALFA, R-AZA, R-BU, R-PIME, R-PIB, R-SPI and R-PIX) were selected from the susceptible strain (LAB-S). Successive generations were exposed to increasing concentrations of each insecticide. These populations were selected at least 7 times. 2.2 Field strains Fourteen field populations of B. tabaci Q biotype from Spain (Almeria and Murcia, hot spots for resistance problems), Italy and Greece were collected from 2008 to 2012 (Table 1). Adults were collected directly from leaves of crop plants with a battery-operated entomological vacuum sampler. Insects were transferred to rearing cages with clean cotton, sweet pepper and aubergine plants. All populations were maintained at 27 ± 1 ∘ C, 60% RH and a 16:8 h light:dark photoperiod. Biotypes of all strains were characterised using PCR amplification of the mitochondrial cytochrome oxidase I gene, followed by restriction with the enzyme Tru9I.23 2.3 Insecticide Cyantraniliprole (Cyazypyr 20SC, Verimark™; DuPont Crop Protection, Newark, DE) was used in all bioassays carried out. Serial dilutions for the formulated compound were freshly made with deionised water for each bioassay. For selecting and testing the laboratory resistant strains, the following formulated insecticides were used: alpha-cypermethrin (Fastac; BASF, Seville, Spain), azadirachtin (Align; Sipcam Inagra,
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Cross-resistance and susceptibility of B. tabaci to cyantraniliprole
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Table 2. Levels of resistance to cyantraniliprole and the main insecticides used in Spain for the control of Bemisia tabaci in five strains [one susceptible reference laboratory strain (LAB-S) and four multiresistant strains] of this insect pest LAB-S LC50 Insecticide
(mg L−1 )
MU-A LC50
(95% CL)
alphaCypermethrin
141.2 (40.3–255.6)
MU-MI
(mg L−1 )
LC50
(95% CL)
RR50
236.4 (90.6–780.3)
1.7
AL-MO
(mg L−1 )
LC50
(95% CL)
RR50
106.7 (48.5–294.7)
0.8
AL-PA
(mg L−1 )
(95% CL)
LC50 (mg L−1 ) RR50
63.94 (24.10–157.9)
(95% CL)
RR50
0.5
37.47 (10.11–120.6)
0.3 0.3
Azadirachtina
2.65 (0.53–6.55)
0.56 (0.13–1.34)
0.2
82.7 (36.5–166.7)
31
18.54 (11.49–26.37)
7
0.769 (0.003–3.011)
Buprofezina
8.73 (4.45–14.38)
96.2 (64.2–143.3)
11
10 159 (2158–1 389 658)
1164
390.4 (127.4–724.5)
45
518.1 (115.3–1723)
59
Imidacloprida
15.19 (1.29–81.08)
53.4 (16.0–110.5)
3.5
47.4 (6.7–206.6)
3.1
81.50 (36.01–168.2)
5
228.9 (82.43–1018)
15
19.9 (7.5–52.6)
1101 (558–3293)
55
1031 (406–1844)
52
590 (171–7212)
30
109.3 (36.9–467.8)
6
25
2181 (671–8595)
7
213.1 (17.42–1036)
0.7
157.6 (29.60–485.6)
0.5
Methomyla Pymetrozyne
317.1 (148.2–608.5) 7973 (5163–10529)
Pyridabena
0.34 (0.21–0.51)
Pyriproxyfena
0.32 (0.02–1.09)
20.86 (13.77–30.37) 25.92 (16.19–40.66)
0.9
2.95 (0.96–5.01)
9
0.76 (0.13–2.43)
2.2
3.08 (1.40–5.67)
9
1.2
402.4 (274.0–634.3)
19
322.2 (167.0–805.4)
15
243.4 (118.7–547.9)
12 1.3
Spiromesifena
1.08 (0.60–1.81)
1.21 (0.62–2.89)
1.1
3.38 (2.56–4.39)
3.1
7.72 (3.67–18.73)
7
1.39 (0.21–4.87)
Thiamethoxam
17.95 (8.12–34.38)
47.37 (3.00–116.4)
2.6
20.00 (8.82–38.66)
1.1
6.56 (0.55–19.56)
0.4
62.91 (30.20–155.0)
3.5
Cyantraniliprole
0.039 (0.034–0.045)
0.06 (0.038–0.089)
1.5
0.05 (0.039–0.057)
1.3
0.07 (0.041–0.099)
1.8
0.051 (0.038–0.065)
1.3
a
Data from Fernández et al.4
Valencia, Spain), buprofezin (Applaud; Syngenta Agro, Madrid, Spain), pymetrozine (Plenum; Syngenta Agro), pyridaben (Sanmite; BASF, Seville, Spain), pyriproxyfen (Juvinal 10EC; Kenogard, Barcelona, Spain), spiromesifen (Oberon SC; Bayer CropScience, Valencia, Spain) and thiamethoxam (Actara; Syngenta Agro). Serial dilutions of formulated compounds were prepared with deionised water containing 1 mL L−1 of Tween 20 (as non-ionic wetter) on the day of the bioassay. 2.4 Systemic bioassay A new systemic uptake bioassay was developed for testing cyantraniliprole susceptibility in nymphs and adults. For nymphs, cotton seedlings in the first true leaf stage (leaf size of about 2 cm diameter) were used (Fig. 1a). Plants were cut with a disinfected scalpel, leaving a stem length of 10 cm (Figs 1b and c). Then, the stem was introduced into a modified polystyrene plastic cage (Fig. 1.d) and maintained in a test-tube rack over a tray containing deionised water. Approximately 40 adults were released (unknown age) from the population tested into the plastic cage containing the cotton seedling for a 24 h oviposition period to allow synchronisation
(a)
(d)
of each developing stage (Fig. 1e). After oviposition, adults were removed and the total number of eggs on each leaf was counted. The desired serial dilutions of insecticide were prepared in deionised water using formulated insecticide. Deionised water was used as a control solution. The stem of the infested leaf was then placed in a glass scintillation vial containing 20 mL of the desired concentration of the insecticide (Fig. 1f ). Six replications (six seedlings) were used for each concentration (0.01, 0.05, 0.1 and 0.5 mg L−1 ). The bioassays were held at 27 ± 1 ∘ C and a 16:8 h light:dark photoperiod for the duration of the assay. Mortality was determined 14 days after oviposition by counting live nymphs and subtracting that number from the number of eggs counted on day 1 on each leaf. For adult bioassays, similar plants and methodology were used. Cotton seedlings were cut and maintained in laboratory conditions (27 ± 1 ∘ C and a 16:8 h light:dark photoperiod) in scintillation vials containing deionised water for 24 h. After this period, plants were transferred to new scintillation vials containing the desired concentration of the insecticide for a further 24 h. On the third day, plants were introduced into the modified polystyrene plastic cages. Ten healthy adults were then released into the cages.
(b)
(e)
(c)
(f)
Figure 1. Nymphal systemic uptake bioassay: (a) cotton seedlings; (b) seedling cut 10–cm long; (c) leaf size about 2 cm diameter; (d) modified polystyrene plastic cage for holding adults for oviposition; (e) plants with cages in a test-tube rack over a tray containing water; (f ) infested plants placed in glass scintillation vials containing 20 mL of the desired concentration of the insecticide.
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controlled environmental conditions (25 ± 2 ∘ C, 60% RH and a of 16:8 h light:dark photoperiod). Areas bounded by the clip cages were marked with a felt pen, and the cages and adults were removed. Eggs were counted and marked prior to treatment so that survival could be measured. Leaves with eggs were then dipped for 10 s in aqueous dilutions of formulated insecticide or the diluent only for untreated controls. Egg hatch was determined 10–12 days after treatment. Failure to hatch at this stage was considered as mortality. For all bioassays, each concentration had three replicates.
Figure 2. Adult systemic uptake bioassay detail.
Seedlings were maintained for 5 days in polypropylene conical centrifuge tubes (50 mL and self-standing base) with the desired concentration of the insecticide (Fig. 2). Mortality was determined 5 days after introducing adults into the cages by counting live and dead individuals on each plant. 2.5 Leaf-dip bioassay The susceptibility of whitefly populations to insecticides other than cyantraniliprole was tested using a leaf-dip bioassay. For nymphs, cotton plants with four true leaves were used. Approximately 40 adult whiteflies from the populations tested were caged on individual cotton leaves using small clip cages for a 48 h oviposition period to allow synchronisation of each developing stage. Plants were maintained at 25 ± 2 ∘ C, 60% RH and a 16:8 h light:dark photoperiod. After 15 days, when the majority of nymphs had reached second instar (N2), the leaves were dipped in serial dilutions of formulated material for 10 s. Control replicates were dipped in deionised water containing 1 mL L−1 of Tween 20. Each bioassay used three replicates at a minimum of six concentrations. Mortality was assessed by comparing the number of nymphs present on the day of treatment with the number remaining unenclosed or dead. This occurred when the last nymphal instar had been reached on control plants (15 days later). For pyriproxyfen, egg-dip bioassays were conducted on whole cotton plants (20–25 cm tall). Forty B. tabaci adults were confined to leaves using clip cages and allowed to oviposit for 24 h under
2.6 Data analysis When necessary, bioassay data were corrected for control mortality.24 Data were analysed using the POLO-PC program for probit analysis.25 The lethal concentration (LC50 ) plus the 95% confidence limits were calculated to determine significant differences between populations. Resistance ratios (RR50 ) for each population were calculated relative to the LC50 level of the susceptible reference strain.
3
RESULTS AND DISCUSSION
3.1 Cross-resistance The multiresistant strains and the susceptible reference strain were bioassayed with alpha-cypermethrin, pymetrozyne, thiamethoxam and cyantraniliprole to determine their levels of resistance (Table 2). These strains have been previously reported to exhibit low to very high resistance to other compounds (Table 2).4 Conversely, in spite of the marked differences in resistance among the whitefly strains, the susceptibility to cyantraniliprole was consistent among them. The LC50 values for cyantraniliprole ranged from 0.04 to 0.07 mg L−1 , with no significant differences. Nevertheless, the bioassays for the other insecticides were carried out months to years before the cyantraniliprole bioassays, and arguably the resistance might have changed over that period. To overcome this issue, seven resistant strains were selected in the laboratory for each insecticide and tested for their susceptibility to cyantraniliprole and their respective insecticides (Table 3). The resistance level achieved was low for alpha-cypermethrin (fourfold) and spiromesifen (16-fold), moderate for azadirachtin (39-fold), high for buprofezin (95-fold), pyridaben (108-fold) and pyriproxifen (112-fold) and extremely high for pymetrozine (2485-fold). Responses to cyantraniliprole in these strains were
Table 3. Levels of resistance to the insecticide selected for and to cyantraniliprole in laboratory-selected resistant strains of Bemisia tabaci Insecticidea Selected population R-ALFA R-AZA R-BU R-PIME R-PIB R-SPI R-PIX
LC50 (mg L−1 ) (95% CL) 557.7 (261.8–807.7) 102.6 (62.1–197.6) 830.1 (371.6–2325) 72 499 (21 651–23 200 000) 36.55 (18.05–61.35) 17.13 (7.76–26.84) 2333 (1052–7917)
Cyantraniliprole RR50 b
LC50 (mg L−1 ) (95% CL)
4.0 39 95 2485 108 16 112
0.011 (0.006–0.017) 0.047 (0.03–0.069) 0.129 (0.079–0.231) 0.051 (0.035–0.061) 0.081 (0.058–0.11) 0.030 (0.008–0.043) 0.096 (0.085–0.109)
RR50 b 0.3 1.2 3.3 1.3 2.1 0.8 2.5
a Insecticide selected for: R-ALFA, alpha-cypermethrin; R-AZA, azadirachtin,; R-BU, buprofezin; R-PIME, pymetrozyne; R-PIB, pyridaben; R-SPI, spiromesifen; R-PIX, pyriproxyfen. b Resistant factor = LC of selected population/LC of susceptible reference population LAB-S (Table 2). 50 50
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Table 4. Susceptibility data to cyantraniliprole in field populations and a susceptible reference population (LAB-S) of B. tabaci Q-biotype from the Mediterranean basin Population
Slope (±SE)
GR-PLA IT-RA MU-CA AL-VI AL-RE LAB-S AL-CA MU-PE IT-CA AL-AL MU-AB MU-TF BA-CA MU-TF9 GR-BER Field composite
1.780(±0.173) 4.379(±0.345) 3.375(±0.346) 2.208(±0.110) 2.116(±0.108) 2.146(±0.093) 1.248(±0.090) 5.173(±0.440) 1.327(±0.169) 1.238(±0.099) 1.575(±0.097) 2.093(±0.192) 1.407(±0.131) 2.268(±0.245) 1.347(±0.064) 1.812(±0.038)
LC50 (mg L−1 ) (95% FL)
RF50
LC90 (mg L−1 ) (95% FL)
RF90
0.011 (0.003–0.018) 0.024 (0.018–0.033) 0.031 (0.023–0.038) 0.032 (0.022–0.042) 0.037 (0.030–0.046) 0.039 (0.034–0.045) 0.045 (0.024–0.071) 0.057 (0.035–0.072) 0.059 (0.029–0.173) 0.060 (0.026–0.117) 0.063 (0.044–0.085) 0.065 (0.043–0.083) 0.081 (0.052–0.133) 0.097 (0.060–0.135) 0.116 (0.086–0.159) 0.048 (0.034–0.063)
0.3 0.6 0.8 0.8 0.9 1 1.2 1.5 1.5 1.5 1.6 1.7 2.1 2.5 3.0 1.2
0.056 (0.033–0.078) 0.047 (0.035–0.078) 0.075 (0.063–0.093) 0.121 (0.088–0.192) 0.151 (0.117–0.212) 0.156 (0.125–0.209) 0.481 (0.272–1.316) 0.101 (0.079–0.237) 0.543 (0.18–94.402) 0.651 (0.271–5.552) 0.41 (0.277–0.734) 0.265 (0.195–0.464) 0.661 (0.328–2.563) 0.356 (0.228–1.114) 1.041 (0.625–2.268) 0.242 (0.167–0.435)
0.4 0.3 0.5 0.8 1.0 1 3.1 0.6 3.5 4.2 2.6 1.7 4.2 2.3 6.7 1.6
relatively stable, with LC50 values ranging from 0.011 to 0.129 mg L−1 , with a maximum RR50 of 3.3 compared with the susceptible population (LAB-S, LC50 = 0.039 mg L−1 ), which is akin to the natural variability found in field populations (see below). A lack of cross-resistance between two diamides (chlorantraniliprole and cyantraniliprole), imidacloprid and pyriproxifen has also been reported for B-biotype field populations and Q-biotype laboratory strains of B. tabaci from Arizona.10 The results obtained show the absence of cross-resistance between cyantraniliprole and the current insecticides used to control whiteflies, suggesting that the existing resistance mechanisms do not affect the performance of this diamide. 3.2 Field populations The results obtained from the bioassays with cyantraniliprole in 14 field populations from Spain, Italy and Greece are presented in Table 4. Susceptibility to cyantraniliprole varied among the populations collected across the Mediterranean area. The LC50 values for nymphs varied between 0.011 mg L−1 (GR-PLA) and 0.116 mg L−1 (GR-BER) (Table 4), a 10.5-fold variability between the least and most sensitive populations. Two populations (GR-PLA and IT-RA) were significantly more susceptible than the susceptible reference population (LAB-S). Three populations, BA-CA, MU-TF9 and GR-BER, were significantly more tolerant than five field populations and the susceptible population LAB-S, but were not different to the remaining six field populations tested in these studies (LC50 values from 0.045 to 0.065 mg L−1 ). These data were consistent enough to be pooled to yield a composite LC50 of 0.048 mg L−1 for Q-biotype B. tabaci. In order to calculate resistance factors of field populations, these baseline data may be used as a reference.
Resistance monitoring studies before commercial use are carried out to determine variation in natural tolerance to a new compound. Knowing the extent of this natural variation in susceptibility is very important in order to distinguish it from genuine resistance after subsequent commercial introduction. The present laboratory bioassays of field populations of Mediterranean Q-biotype B. tabaci showed a slight variation (10.5-fold) in susceptibility to cyantraniliprole and were consistent in their response. The previous history of insecticide exposure may influence the response of whiteflies to cyantraniliprole; however, none of the populations tested had been exposed to cyantraniliprole under field conditions, so observed differences in LC50 values were attributable to natural variation. Other diamides such as chlorantraniliprole and flubendiamide have been used in recent years, and this may have an impact on susceptibility variability. Regional differences in susceptibility to cyantraniliprole have been recorded in several studies.10,13 Variations in susceptibility to cyantraniliprole were documented for Bemisia biotypes B and Q from Arizona10 and Florida.13 Overall, the LC50 values reported for B biotypes range from 0.013 to 0.065 mg L−1 , and for Q biotypes from 0.011 to 0.191 mg L−1 (including the present data). Therefore, these data indicate that there are no differences in the susceptible response to cyantraniliprole between B. tabaci biotypes and/or populations from different parts of the world. Nevertheless, there seem to be some Q-biotype strains that are more tolerant, which highlights the importance of carefully monitoring the resistance evolution to cyantraniliprole in this whitefly biotype. 3.3 Adults versus nymphs Adults from three Q-biotype laboratory populations were tested against cyantraniliprole (Table 5). The LC50 values ranged from
Table 5. Susceptibility data of adults of Bemisia tabaci to cyantraniliprole Population
Slope (±SE)
LAB-S MU-MI AL-PA
2.445(±0.237) 1.055(±0.101) 1.630(±0.222)
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LC50 (mg L−1 ) (95% CL)
RF50
LC90 (mg L−1 ) (95% CL)
RF90
0.071 (0.060–0.083) 0.060 (0.043–0.080) 0.096 (0.058–0.138)
0.8 1.4
0.238 (0.187–0.333) 0.979 (0.59–2.053) 0.588 (0.364–1.449)
4.1 2.5
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www.soci.org 0.060 to 0.096 mg L−1 , with a 1.6-fold variability and no significant differences. These values are slightly higher than those reported for B-biotype adults from Florida, which ranged from 0.037 to 0.059 mg L−1 .13 The LC50 values for nymphs of the same populations (Table 2) ranged from 0.039 to 0.051 mg L−1 . These results support a slightly greater toxicity for nymphs than for adults, as suggested previously.13 This difference may be due to a longer exposure time for nymphs (14 days) than for adults (5 days).13 Nevertheless, the difference is very small to be of significant importance in the field. When expression of resistance has been reported as age specific in B. tabaci,6 the variation between nymphs and adults is 4–10-fold.
4
CONCLUSIONS
The present results document the present status of high susceptibility to cyantraniliprole in Mediterranean Q-biotype whitefly populations. All the LC50 values reported in this study are similar to those reported in previous studies of cyantraniliprole against B. tabaci.10,13 There was no correlation between the toxicities of cyantraniliprole and other insecticides usually applied to control whitefly populations in southern Europe. Moreover, some populations highly resistant to other insecticides did not vary significantly in their response to cyantraniliprole compared with the susceptible reference. The hypothesis that existing resistance mechanisms do not confer any protection to this novel insecticide is substantiated by these data. The present results are consistent with previous studies that suggested the absence of cross-resistance between imidacloprid, pyriproxifen and cyantraniliprole.10 Therefore, pre-existing cross-resistance conferred by genes selected by other insecticides seems unlikely, as the resistance mechanisms in these populations gave no tolerance to cyantraniliprole. Contained cropping systems such as greenhouses are initially most at risk of resistance to novel insecticides developing because insecticides are applied frequently on enclosed insect populations. This provides ideal conditions for resistance selection, and these highly selected populations may be transported to new areas. The intensive vegetable-growing regions of south-eastern Spain, such as Almeria, with high frequencies of insecticide applications and the constant presence of hosts, provide an environment where selection for resistance is of concern.26 As a consequence, severe resistance problems in whiteflies have been reported in this area.4,22 In this present scenario, Q-biotype whitefly populations showed a consistent susceptibility to cyantraniliprole. These baseline data will be helpful for monitoring any future shifts in susceptibility to cyantraniliprole in Mediterranean whitefly populations that may occur within an IRM programme. In summary, cyantraniliprole may play an important role in mitigating insecticide resistance in B. tabaci because of its high efficacy and its lack of cross-resistance to other insecticides, although a high level of vigilance is required, as the threat from resistance developing to such efficacious insecticides can be high.
ACKNOWLEDGEMENTS The authors acknowledge anonymous referees for reviews and comments on the manuscript. They are grateful to JM Alvarez, DuPont Crop Protection, for his helpful comments on the manuscript. This research has been supported by DuPont and the Spanish Ministry of Economy and Competitiveness (MINECO)
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C Grávalos et al.
(AGL2011-25164) and by the European FEDER funds. C. Grávalos was supported by a FPI grant (BEF-2009-025572) funded by the MINECO.
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