Experimental and Applied Acarology 26: 243–251, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
Resistance to hexythiazox in Brevipalpus phoenicis (Acari: Tenuipalpidae) from Brazilian citrus FERNANDO JOLY CAMPOS and CELSO OMOTO* Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, 13418-900 Piracicaba, SP, Brazil; *Author for correspondence (e-mail:
[email protected]; phone: +55-19-3429-4199; fax: +55-19-3433-0562) Received 12 December 2001; accepted in revised form 31 May 2002
Key words: Acaricide resistance, Resistance management, Stability of resistance Abstract. The objective of this study was to collect baseline information for implementing an acaricide resistance management program of Brevipalpus phoenicis (Geijskes) to hexythiazox in Brazilian citrus groves. The egg susceptibility of B. phoenicis to hexythiazox was measured by a direct contact bioassay. The estimated LC 50 for the S strain was 0.89 mg hexythiazox L −1 of water (95% FL 0.75–1.03). After hexythiazox selection of a field-collected population associated with intense hexythiazox use, a resistance ratio greater than 10,000-fold was detected. Results from a survey revealed a great variability in the frequency of resistance in populations of B. phoenicis collected from citrus groves located in the State of São Paulo. No relationship was observed between the intensity of hexythiazox use and the frequency of resistance. Studies on dynamics of resistance showed that the resistance of B. phoenicis to hexythiazox is stable under laboratory conditions. Therefore, there is an urgent need to implement resistance management of B. phoenicis to hexythiazox in order to prolong its effective use in Brazilian citrus groves.
Introduction Brevipalpus phoenicis (Geijskes) is one of the most important citrus pests in Brazil. It can cause severe damage to the citrus industry because it vectors the citrus leprosis virus (Chiavegato et al. 1982; Oliveira 1986), which can cause premature leaf and fruit drop, or death of the tree (Knorr and Denmark 1970; Kitajima et al. 1972; Chagas and Rossetti 1980; Rodrigues et al. 1997). The acaricide hexythiazox has been used very often for controlling B. phoenicis since 1985, because of its high activity against eggs and other immature stages (Chiavegato et al. 1993) and the low toxicity to phytoseiid mites (Hoy and Ouyang 1986; Sato et al. 1995). Approximately US$ 80 million dollars are spent annually with acaricides in Brazilian citrus groves (Neves et al. 2001). Hexythiazox has been an important component in many integrated pest management (IPM) programs (Hoy and Ouyang 1986; Welty et al. 1989; Gravena 1994; Yamamoto et al. 1995), especially against tetranychid mites. Because of its primary mode of action (ovicide) and high persistence (Aveyard et al. 1986; Yamamoto et al. 1995), the evolution of hexythiazox resistance is a threat to this acaricide. High
244 level of resistance has already been detected in P. ulmi (> 2,500-fold) (Edge et al. 1987) and T. urticae (> 1,000-fold) (Gough 1990) in Australia, and in P. citri (> 24,000-fold) in Japan (Yamamoto et al. 1995). Approximately 15% of the acaricide-treated area for controlling B. phoenicis in Brazilian citrus groves has included hexythiazox alone or in combination with other acaricides. In recent years, inadequate control of B. phoenicis has been reported with hexythiazox (as well as with other acaricides) in some locations. Because of intense acaricide use we hypothesized that resistance could explain some of the field failures. We developed bioassay procedures to evaluate hexythiazox susceptibility in populations of B. phoenicis collected from Brazilian citrus groves with differing hexythiazox use. Laboratory selection was conducted to isolate hexythiazox-resistant mites to characterize the intensity of resistance. The dynamics of hexythiazox resistance in B. phoenicis were investigated under laboratory conditions to determine whether the resistance was stable or not.
Materials and methods Collection and maintenance of Brevipalpus phoenicis In this study, the population of B. phoenicis collected from an unsprayed citrus grove located in Piracicaba, São Paulo State, Brazil was designated as our susceptible reference strain (S). From January to June of 1999, 10 populations of B. phoenicis that received different regimes of hexythiazox use (Table 1) were collected from a commercial citrus grove of Fischer Agropecuária Co. in Barretos, São Paulo State, Brazil (populations are identified as Fischer-1 to Fischer-10). Approximately 50 mite-infested fruits were randomly collected per population. Maintenance of B. phoenicis populations in the laboratory was on citrus fruits (‘Valencia’ or ‘Pera Rio’ variety) collected from a citrus grove without pesticide applications. Fruits were washed with tap water, and after drying they were dipped in a heated wax, leaving an arena of approximately 10 cm 2 to confine the mites. Then, 50 to 60 field-collected mites were transferred per arena with the aid of a fine brush. Each mite population was kept on at least 30 fruits. The rearing room was set at 25±2 °C, 70±10% RH and photoperiod of 14:10 (L:D) h. Fruits were renewed every 30 to 40 days. Bioassay methods The egg susceptibility of B. phoenicis to hexythiazox was measured by a direct contact bioassay (Aveyard et al. 1986; Yamamoto et al. 1995). Twenty adult females were transferred from mite colonies onto a detached Ligustrum lucidum Aiton (Oleaceae) leaf placed with the lower surface up on a wet sponge in an open petri dish. The edge of each leaf was circled with wet cotton to maintain leaf moisture and to confine the mites. Females were left to oviposit for 2 days. Then, they
245 Table 1. Hexythiazox use from 1993 to 1998 in different population collected from a commercial citrus grove of Fischer Agropecuária Co., Barretos, São Paulo State, Brazil and the frequency of hexythiazoxresistant B. phoenicis (mean percent survivorship ± SEM at discriminating concentration of 18 mg a.i. L −1) in 1999. Population
Year 1993
Fischer-1 Fischer-2 Fischer-3 Fischer-4 Fischer-5 Fischer-6 Fischer-7 Fischer-8 Fischer-9 Fischer-10
1994
1995
1996
1997
X
M M
M M M
X M X
M
X X X X X X
M M
M
Resistance Frequency 1
220 163 222 178 252 239 393 273 282 341
29.89 (±3.67) e 60.47 (±7.11) de 46.82 (±7.31) bcd 43.60 (±7.20) cde 44.39 (±0.99) bcd 61.02 (±7.63) ab 75.15 (±4.62) ab 62.25 (±5.82) abc 79.42 (±5.57) bcd 93.60 (±2.12) a
1998
M X X
n
M
X = Use of hexythiazox alone M = Use of hexythiazox in mixture with another acaricide n = number of eggs tested 1Means followed by the same letter are not significantly different by the Tukey’s test (P < 0.05)
were removed and the number of eggs was recorded (20–40 eggs per leaf). Different concentrations of hexythiazox were prepared in distilled water from Savey® (50% wettable powder, DuPont do Brasil S.A.) and stirred with a magnetic stirring bar before each bioassay. Eggs on the leaf were treated with 2 ml of each concentration using a Potter spray tower (Burkard Manufacturing, Rickmansworth, Herts, England) producing an aqueous residue of approximately 1.6 mg cm −2. Control dishes were sprayed only with water. After treatment, bioassay dishes were moistened daily and held in an environmental chamber at 25 ± 1 °C and photoperiod of 14:10 (L:D) h. The number of unhatched eggs was assessed 11 days after treatment and percent mortality was calculated from the total number of eggs per leaf. Control mortality was less than 5%. The mortality was corrected by using the Abbott’s formula (Abbott 1925). Detection and characterization of hexythiazox resistance To isolate a hexythiazox-resistant strain (hexythiazox-R), a laboratory selection was conducted from the B. phoenicis population collected in a commercial citrus grove (Fischer-10), where the intensity of hexythiazox use was high and field failures in the mite control were reported frequently with the use of this acaricide. The same bioassay procedures described before were used to select for resistance. The mites that hatched from eggs treated at the concentration of 18 mg a.i. L −1 and developed to deutonymphs on the treated leaves were used for the next selection. Selected mites were transferred on citrus fruits for culturing as described before. Only 2 cycles of selection were performed to obtain the hexythiazox-R strain.
246 Hexythiazox concentrations ranging from 0.18 and 18 mg a.i. L −1 and from 10 to 10,000 mg a.i. L −1 were tested to characterize the responses to hexythiazox of the S and hexythiazox-R strains, respectively. Probit regressions for both strains were estimated (LEORA SOFTWARE 1987). The resistance ratio was determined by dividing the LC 50 of hexythiazox-R strain by the LC 50 of the S strain. Survey of susceptibility to hexythiazox The frequency of hexythiazox resistance in 10 populations of B. phoenicis was conducted with discriminating concentration bioassays of 18 mg a.i. L −1. For each population, bioassays were replicated 4 times. Each replicate consisted of 60 to 100 eggs. A simple linear correlation analysis was done to evaluate if there is a relationship between hexythiazox use and the frequency of resistance (␣ = 0.05). Proportional survivorship data (X) of each experimental unit were transformed using the square-root arcsine (X) and subjected to analysis of variance (PROC GLM; SAS Institute, 1989). Treatment means were separated by Tukey’s test at ␣ = 0.05. Dynamics of hexythiazox resistance Changes in the frequency of hexythiazox resistance were monitored monthly in 3 populations of B. phoenicis under laboratory conditions in the absence of selection pressure for a 6-month period. These populations were established with a mixture of S and hexythiazox-R strains at ratios of 80:20, 50:50 and 20:80 (populations are identified as 80S:20R, 50S:50R and 20S:80R, respectively). The S and hexythiazox-R strains served as controls. For each population, 12 fruits with a total of 100 mites each were established for this study. The rearing procedures were as described before. The frequency of resistance was estimated with the discriminating concentration bioassays of 18 mg a.i. L −1. For each population, bioassays were replicated 4 times. Each replicate consisted of 100 to 200 eggs. Proportional survivorship data (X) at the discriminating concentration of each experimental unit, collected during the 6-month period, were transformed using square-root arcsine (X) and subjected to a two-factor (population and time) analysis of variance with interaction (SAS Institute 1989). The significance level of the tests was ␣ = 0.05.
Results Detection and characterization of hexythiazox resistance The concentration-response data that represent the egg susceptibility to hexythiazox of S and hexythiazox-R strains of B. phoenicis are shown in Figure 1. The LC 50 for the S strain (n = 1,348) was 0.89 mg a.i. L −1 (95% FL, 0.75-1.03), slope
247
Figure 1. Concentration-response data of S and hexythiazox-R strains of Brevipalpus phoenicis to hexythiazox. Shaded area corresponds to a range of discriminating concentrations of hexythiazox for monitoring resistance.
(±SE) was 2.74 (± 0.15), and 2 was 4,18 (d.f. = 3; P > 0.05). It was not possible to estimate the LC 50 for the hexythiazox-R strain (n = 3,214) because at the highest concentration tested of 10,000 mg a.i. L −1, the mean egg mortality was only 43.2%. However, we conclude that the resistance ratio is greater than 10,000-fold. Based on these results, discriminating concentrations from 18 to 320 mg a.i. L −1 were defined for monitoring resistance (Figure 1). Survey of susceptibility to hexythiazox Results from a survey of susceptibility to hexythiazox in B. phoenicis populations with differing regimes of hexythiazox use showed a significant difference (F = 12.79; d.f. = 9, 39; P < 0.05) in the frequency of resistance (Table 1). The frequency of resistance ranged from 30% for Fischer-1 population to 94% for Fischer10, based on the percent survivorship at the discriminating concentration of 18 mg a.i. L −1. However, no relationship was observed between the number of hexythiazox sprays from 1993–98 and the frequency of resistance (r = 0.13; d.f. = 6; P > 0.05). Dynamics of hexythiazox resistance Hexythiazox resistance in B. phoenicis was very stable; that is, the frequency of resistance did not decline through time in the absence of selection pressure (Fig-
248
Figure 2. Changes in the frequency of hexythiazox resistance in populations of Brevipalpus phoenicis obtained from a mixture of S and hexythiazox-R strains at ratios of 80:20, 50:50 and 20:80 in the absence of selection pressure under laboratory conditions. Shown is the mean percent survivorship (± SEM) at the discriminating concentration of 18 mg a.i. L −1.
ure 2). The interaction of population and time was significant (F = 2,96; d.f. = 20, 90; P < 0.05), suggesting that populations differed in response to hexythiazox during the 6-month period. The populations 80S:20R and 50S:50R showed an increase in the frequency of resistance during the 6-month period. For example, the frequency of resistance reached up to 50% in the 80S:20R population after 2 months under absence of selection pressure. The frequency of resistance was fairly stable in the 20S:80R population.
Discussion A high intensity of hexythiazox resistance (> 10,000-fold) was detected in B. phoenicis populations from Brazilian citrus groves (Figure 1). B. phoenicis reproduces predominantly by thelytokous parthenogenesis and consists entirely of haploid females (Helle et al. 1980; Weeks et al. 2001), which could limit or inhibit genetic recombination; however, in this research we showed a high genetic variability in response to hexythiazox within B. phoenicis populations. Under thelytoky, females develop from unfertilized eggs (identical to maternal genome). Then, if there is genetic variability that confers resistance to a certain acaricide in a population of B. phoenicis, the selection pressure with this acaricide will rapidly increase the proportion of resistant genes. No relationship was observed between the hexythiazox use and the frequency of resistance (Table 1). This result does not mean that the intensity of hexythiazox use
249 did not affect the frequency of resistance, because we do not have the chemical use records before 1993 in the populations evaluated in this study. For example, in the population Fischer-9 that received only 1 spray of hexythiazox during 1993–98, the frequency of resistance was very high ( ⬇ 80%), much higher than populations that received more than 1 spray during the same period. Our laboratory study on dynamics of hexythiazox resistance in B. phoenicis showed that the resistance is stable due to possible co-adaptation process through time (Figure 2). Probably, because of the stability of resistance, the reversion to susceptibility has not been observed in the field either, even in situations where hexythiazox has not been used very often during 1993–98. In this case, the management of hexythiazox resistance becomes more difficult and challenging. A fairly stable hexythiazox resistance has also been reported in T. urticae in Australia (Herron et al. 1993) and in P. citri in Japan (Yamamoto et al. 1996) Hexythiazox was successfully used for many years and still is an important component in the citrus IPM programs. In the past few years, this product has provided inadequate control in some locations and recommended in combination with other acaricides (such as dicofol, propargite, cyhexatin etc.). This is an interesting approach because hexythiazox acts primarily on eggs and immature stages of B. phoenicis and the other acaricides could kill the adults. However, this strategy has been adopted mainly when the use of hexythiazox itself did not give good mite control; that is, the frequency of hexythiazox-resistant mites may be fairly high. Furthermore, when citrus growers use mixture of acaricides, hexythiazox has been used at half of the recommended rate for economical reasons. The recommended rate of hexythiazox is 15 mg a.i. L −1 (close to the lower limit of the discriminating concentration of 18 mg a.i. L −1). From the acaricide resistance management perspective, the mixture of products will be an interesting strategy when the frequency of resistance is still low for both compounds used in mixture, the persistence of both compounds is similar, each compound gives a high kill by itself, there is no linkage disequilibrium etc. (Mani 1985; Roush 1989; Tabashnik 1989). These conditions are rarely met with hexythiazox in B. phoenicis because the resistance has already evolved at high frequencies in some locations, the residual activity of hexythiazox is much longer than those of other acaricides, half-rate of hexythiazox is not enough to give a high kill, and the chance for selecting mites carrying resistance to both compounds (multiple-resistance) used in mixture is very high (Omoto 1998) due to its mode of reproduction predominantly by thelytoky and the kariotype of only two heterologous chromosomes of haploid females (Helle et al. 1980; Weeks et al. 2001). Because high variability in the frequencies of hexythiazox resistance was documented in B. phoenicis populations in this work, it is important for citrus growers to monitor the resistance before deciding whether to spray hexythiazox or not. If the frequency of resistance is high, hexythiazox should not be recommended (even in combination with other acaricides). On the other hand, if the frequency of resistance is still low, probably the best approach for using hexythiazox will be as a mixture with other acaricide at full rates each because of the stable nature of hexythiazox resistance in B. phoenicis. Synergism studies of hexythiazox and other
250 acaricides must be conducted to evaluate the possibility of reducing rates of the products used in mixtures. Adoption of rotation of acaricides with different mechanisms of action, resistance monitoring to all acaricides, strategies for preserving natural control agents, and other IPM recommendations will be essential for effective B. phoenicis control in Brazilian citrus groves.
Acknowledgements This work was supported by grants from FAPESP, FUNDECITRUS and PRONEX. A scholarship was given by CNPq to the senior author to obtain M. Sc. degree at University of São Paulo, Brazil. We thank Helton Carlos de Leão (Fischer Agropecuária S.A.) for help to collect mites from the field.
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