INSECTICIDE RESISTANCE IN MYZUS PERSICAE (SULZER) (HEMIPTERA: APHIDIDAE) IN SOUTHEASTERN AUSTRALIA

J. Aust. enl. SOC., 1993,,3 2 23-27

23

INSECTICIDE RESISTANCE IN MYZUS PERSICAE (SULZER) (HEMIPTERA: APHIDIDAE) IN SOUTHEASTERN AUSTRALIA G . A. HERRON, T. S. GIBSONand M. A. HORWOOD NS W Agriculture, Biological and Chemical Research Institute, PMB 10, Rydalmere, N . S . W . 2116.

Abstract Strains of Myzus persicae, collected from the Goulburn Valley, Victoria, and the Bathurst-Orange region, N.S.W., during 1988-89and 89-90 were screened for resistance to demeton-S-methyl, pirimicarb, methamidophos and mcthomyl. Bioassay was used to determine levels of resistance to each chemical. Electrophoresis was used to determine the frequency of individual aphids with enhanced levels of the resistance conferring E4 carboxylesterase, which was assumed to be the resistance mechanism. DemetonS-methyl resistance (RF range 14.8-40.0 x ) and pirimicarb resistance (RF range 7.5-20.2 X ) were detected in all strains tested by bioassay. Low level methamidophos resistance was detected (RF range 2.9-4.2 x ) in three of the seven strains tested. However, methomyl resistance was not detected in any strain. Electrophoretic examination of the resistant strains showed only one strain contained susceptible individuals. Most strains consisted predominantly of the highly resistant variant (R,) and some strains may contain the extremely resistant variant (RJ.

Introduction Myzus persicae (Sulzer) is a major pest of horticultural crops in Australia. Insecticides are widely used to control M. persicae. Resistance to organophosphorus insecticides was first documented in M . persicae during 1976, but no resistance to carbamate insecticides was found at that time (Hamilton and Attia 1978). Subsequent surveys did detect resistance to carbamate insecticides, including the highly selective aphicide pirimicarb, but control problems were not experienced (Attia et al. 1979). Recently, numerous control failures have been reported with the carbamate insecticides methomyl and pirimicarb. Only one biochemical resistance mechanism has been identified in studies of resistant strains of M. persicae from many countries (Devonshlre and Moores 1982). The E4 carboxylesterase enzyme detoxifies organophosphorus, carbamate and pyrethroid insecticides. The level of resistance conferred by the E4 carboxylesterase is dependent on gene amplification (Devonshire and Sawicki 1979). For simplicity, aphids are classified as moderately resistant (R,), very resistant (R,) and extremely resistant (R,) depending on the degree of amplification (Sawicki et al. 1978). Sawicki et al. (1978) showed that R, aphids are controlled by pirimicarb while a significant proportion of R, aphids will survive a pirimicarb treatment. lncreased levels of E4 carboxylesterase were detected in Australian multi-insecticide resistant strains of M . persicae (Hamilton et al. 1981), and the E4 carboxylesterase is probably responsible for M. persicae resistance in Australia. In this study bioassay techniques were used to determine whether populations surviving treatment in the field had higher levels of resistance to the carbamates methomyl and pirimicarb, and to the organophosphorus insecticides methamidophos and demeton-S-methyl than those previously recorded in Australia. We used electrophoresis for the first time in Australia as a diagnostic tool for resistance monitoring and relate the electrophoresis data to established bioassay methods and results. Materials and methods Sample collection Strains of M . persicae associated with control failures were collected fiom stone-fruit trees in the Goulburn Valley region of Victoria and the Bathurst-Orange region of N.S.W. during the seasons 1988-89 and 1989-90 (Table I). Strains Standen Farm (SF) and Spring Hill (SH) were from the Bathurst-Orange region, while strains Isrna Orchard (10), Anzolin Farm (AF) and Macheda Orchard (MO) were from the Goulburn Valley. Population samples were forwarded by overnight courier for processing the following day, divided into three parts and either bioassayed immediately in a discriminating dose assay, frozen for later electrophoresis, or used to start a parasite-free culture for subsequent log dose-probability assays. The response of the resistant strains was compared to baseline toxicity responses of a reference susceptible strain (S) that was collected at Rydalmere, N.S.W. and has been maintained under insecticide free conditions, in laboratory culture, For 15 years.

G. A. HERRON ETAL.

24

Table 1. The collection date and insecticide spray history of each M. persicae strain tested Strain Season Insecticide spray history Control failure collected reported with SF

1988-89

SH 10

1988-89 1988-89

AF

1988-89

MO

1988-89

SF

1989-90

MO

1989-90

pirimicarb 1987-88 methomyl 1988-89 methomyl 1988-89 used a range of chemicals-details unknown thiometon 1987-88 methomyl 1988-89 methamidophos 1988-89 pirimicarb 1988-89 used a range of chemicals-details unknown diazinon 1989-90 pirimicarb 1989-90 methamidophos 1989-90

methomyl methomyl not specific all chemicals

not specific both chemicals some control

Culturing method Strains were cultured by confining 100-200 apterous virginoparous adults in petri dish bases which were covered with a semi-permeable cellulose film. Each covered petri dish contained a seedling cabbage leaf with its stem inserted into moist foam. The petri dishes were maintained at 27 +2"C under constant illumination for 24 h. The juveniles produced were parasite free and were transferred to potted cabbage plants and reared in a mass culture laboratory. Aphids were reared under natural light at 25 +4"C until sufficient numbers were available for log dose-probability assays.

Bioassay The insecticides used were proprietary formulations of methomyl (225 g/L liquid concentrate), methamidophos (580 g/L emulsifiable concentrate), demeton-S-methyl(250 g/L emulsifiable concentrate), and pirimicarb (500 g/kg wettable powder). Discriminating dose tests were used to detect the presence of resistance. Aphids were sprayed directly with a 2 mL aliquot of an aqueous insecticide suspension through a Potter spray tower producing a spray deposit of 1.6 mg/cmz of aqueous insecticide solution. Aphids were sprayed in batches of 12-15 individuals on a 90 mm Whatman's filter paper disc. Two to five batches of aphids were sprayed with insecticide for each discriminating dose assay plus a control sprayed with water only. Sprayed aphids were transferred to petri dish bases with a fine sable hair brush and maintained at 25 + 1 "C. Knockdown, defined as the inability to walk in a coordinated manner, was assessed after 24 h and was corrected for control mortality (Abbott 1925). Aphids that were knocked down subsequently died. Discriminating doses were based on the KD,,, level of a standard susceptible reference strain, calculated using a computer program based on the probit method of Finney (1971). Discriminating doses were 3 g/L methomyl, 2 g/L methamidophos, 0.2 g/L pirimicarb, and 0.4 g/L demeton-S-methyl. Strains with survivors at the discriminating dose were tested further to confirm resistance and to determine resistance factors (RF). RFs were determined by spraying batches of 12-15 aphids from the cultured strain with five or more concentrations of insecticide. Each test was replicated four times and included a water only sprayed control. Test results were assessed as for the discriminating dose assays and RFs were based on KD,,s calculated using probit analysis.

Electrophoresis Esterases were separated by vertical polyacrylamide gel electrophoresis according to the method of Devonshire (1975). Separating gels contained 7.5% acrylamide, 100 mM Tris-HCI, pH 7.5, 0.32% (v/v) Triton X-100 and 0.012% (w/v) potassium ferricyanide. Each separating gel was overlaid with a stacking gel of 3.5% acrylamide, containing 80 mM Tris-HCI, pH 7.5, and 0.23% (w/v) Triton X-100. Single aphids were homogenised by hand in 2.5 mL Eppendorf centrifuge tubes using tapered disposable pestles. The homogenate was suspended in 25 pL of 10 mM sodium phosphate buffer, pH 7.0, containing 1 mM di-sodium EDTA, 0.5% (w/v) Triton X-100and 20% (w/v) sucrose. Samples were then centrifuged at room temperature for 1.5 min at 10,OOOg and 15 pL of the supernatant was loaded on to the sample wells in the gels. Electrophoresis was performed in a continuous system with a Pharmacia GE-2/4 apparatus with 40 mM sodium barbitone, pH 8.6, as the running buffer. Four gels (80 mm x 80 mm X 2.5 mm ) were run simultaneously at 150 V for 2.5 h at IOOC. Gels were incubated for 20 min in the dark at 35°C in a staining solution of 0.02% (w/v) I-napthylacetate and 5% (w/v) fast blue BB salt in 100 mM sodium phosphate buffer, pH 6.6. The staining solution was prepared fresh from a stock solution of 2% I-napthylacetate in AR grade acetone and IM sodium phosphate, pH 6.6. After incubation, I-napthylacetate esterases appeared on the gels as green/brown bands. The intensities of the E4 carboxylesterase bands on the polyacrylamide gel were subjectively assessed as described by Devonshire (1975) and were used to classify the aphids as susceptible (S) or resistant (R,, R2) (Fig. 1).

INSECTICIDE RESISTANCE IN MYZUS PERSICAE

25

FIG.1-E4 carboxylesterase bands showing susceptible (S), moderately resistant (R,) and highly resistant (R,) variants of M. persicae.

Results

Season 1988-89 All strains showed some resistance to demeton-S-methyl and pirimicarb (Table 2). Strains from the Goulburn Valley were similar in their resistance to pirimicarb (> 12 x ) and showed high levels of resistance to demeton-S-methyl(l9.3-24.4 x). All strains were susceptible to methomyl, while only strain AF showed methamidophos resistance. Fig. 1 shows the typical I-napthylacetate esterase .bands detected by electrophoresis in 1his study. Resistant variants could be distinguished as susceptible (S), R, or R, by the intensity of the E4 band (Devonshire 1975). Faint esterase bands, described by Devonshire (1975), which do not confer resistance, were also detected. Only one strain (SH) contained individuals classified as susceptible (Table 3). This strain from the Bathurst-Orange region had the lowest resistance factors found in this study (TabIe 2). All other strains consisted predominately of the highly resistant R, variant, with the two Goulburn Valley strains AF and MO consisting totally of the R, variant. Season 1989-90 Pirimicarb and demeton-S-methyl resistance levels in strain SF (17.4x and 26.6x ) were 1.8 x and 1.5 x those levels recorded for the 1988-89season. Similarly, resistance levels in strain M O (20.2x and 40.0x ) were both 1.6 x higher than the levels recorded for the previous season (Table 2). In contrast to the 1988-89season, methamidophos resistance was detected in strains SF and MO. Methomyl resistance was still not detected. Electrophoretic examination of the strains showed little change in the frequency of highly resistant, R, individuals in strain SF or MO from the previous seasons results (Table 3).

G. A. HERRON ET AL.

26

Table 2. Toxicity of insecticides to strains of M. persicae Season Region Strain Bioassay Results Methamidophos Demeton-S-methyl KD5O+ Slope R F + + KD50 Slope (95% F.L.) (95% F.L.) 1988-89 Bathurst/Orange SH

*

-

-

SF

* *

-

-

451 (405-502)

3.4

3.5

*

-

-

552 (496-615) 378 (333-428)

3.1

4.2

2.7

2.9

3.2

1.0

1988-89 Goulburn Valley I 0 AF MO 1989-90 Bathurst/Orange SF 1989-90 Goulburn Valley MO

Laboratory reference susceptible 130 (1 12-152)

-

RF

381 (323-450) 440 (380-509) 490 (416-577) 628 ( 5 5 1-716) 627 (526-748) 685' (590-797) 1027 (899-1 175)

3 .O

14.8

2.4

17.1

2.0

19.3

3.0

24.4

2. I

24.4

2.4

26.6

2.6

40.0

26 (22-29)

2.8

1 .o

Table 2 (continued). Season Region

Strain

Bioassay Results KD50 (95% F.L.)

1988-89 Bathurst/Orange SH SF 1988-89 Goulburn Valley I 0 AF MO

Pirimicarb Slope

RF

KD50 (95% F.L.)

3.3

7.5

*

3.1

9.4

*

2.1

12.8

*

2.9

12.5

3.8

12.4

* *

3.4

1.0

140

(124-158) 174 I 1 53- 198) ' 237 (191-295) 2331 (173-3 13) 231 1208-256)

Methomyl Slope

-

1989-90 Bathurst/Orange 1989-90 Goulburn Valley Laboratory reference susceptible 19 (16-22)

* +

+

I

+

2.5

127 (101- 159)

100% mortality for discriminating dose assay, therefore log dose-probability assay not performed. mg/L ai Resistance factor KD50 resistant strain KD50 susceptible strain Chi-squared goodness of fit test significant p