VG323 Fruit fly disinfestation of cucurbits and …...contested. The watermelon report will be sent to AQIS for submission to New Zealand, and until a response is received from NZ,

VG323 Fruit fly disinfestation of cucurbits and capsicums wi th insecticides for New Zealand

Robert Corcoran QLD Department of Primary Industries

VG323

This report is published by the Horticultural Research and Development Corporation to pass on information concerning horticultural research and development undertaken for the vegetable industry.

The research contained in this report was funded by the Horticultural Research and Development Corporation with the support of Harrowsmiths International and the Queensland Fruit & Vegetable Growers.

All expressions of opinion are not to be regarded as expressing the opinion of the Horticultural Research and Development Corporation or any authority of the Australian Government.

The Corporation and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests.

Cover price: $20.00 HRDC ISBN 1 86423 624 8

Published and distributed by: Horticultural Research & Development Corporation Level 6 7 Merriwa Street Gordon NSW 2072 Telephone: (02) 9418 2200 Fax: (02) 9418 1352 E-Mail: [email protected]

© Copyright 1997

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HRD\C

HORTICULTURAL RESEARCH & DEVELOPMENT CORPORATION

Partnership in horticulture

HRDC PROJECT NO: VG323

Fruit fly disinfestation of cucurbits and capsicums with insecticides for New Zealand.

1. SUMMARY

(a) Industry Summary

The objective of this project was to provide essential disinfestation treatment data to support an application to New Zealand to admit Australian capsicums, watermelons, honeydew melons and cucumbers. There has been substantial ongoing trade with New Zealand in these commodities using ethylene dibromide (EDB) fruit fly disinfestation treatments. On January 1994, the Maximum Residue Limit (MRL) for EDB was reduced to a "level of detection" of 0.1 ppm, making this no longer practicable as a disinfestation treatment and approval for its use was withdrawn by the New Zealand Ministry of Agriculture and Fisheries (MAF). This project aimed to provide an alternative treatment using insecticide until a residue-free treatment (for example heat or cold) could be developed.

Research commenced in October 1994 with capsicums. The method incorporated a packing-line spray system which delivers dimethoate at 400 mg/L through a series of fine sprays. This process was chosen after industry consultation and because a similar system has been used successfully for tomato exports to New Zealand for more than a decade. The research was completed and a submission was made to the New Zealand Ministry of Agriculture and Fisheries (MAF) in April 1994. In July we were advised by AQIS that New Zealand MAF had accepted and approved the research data concerning the dimethoate spray treatment for capsicums as a disinfestation method against Queensland fruit fly. In October New Zealand MAF reversed this decision following consultation with the New Zealand capsicum industry. Without approval from MAF this research cannot be used for exports. A strong rebuttal of the NZ industry criticisms has been made through AQIS.

Research was focused on watermelons in May 1994, as honeydew melons and cucumbers were already being exported to New Zealand under an interim arrangement. The process developed for watermelons was a one minute dip in dimethoate at 400 mg/L. This treatment is identical to the interim arrangements for most other cucurbits, which is based on previous research completed on rockmelons and zucchinis. As watermelons are larger than rockmelons, they were excluded from the interim arrangements because of the assumption that insecticide uptake, and hence efficacy on internally feeding insect stages, depends on the surface to volume ratio. Research was completed at the beginning of October with tests against all stages of Bactrocera cucumis (cucumber fly) meeting the current quarantine requirements. The submission is about to be made through AQIS to MAF. In November 1994 we received a copy of a New Zealand MAF draft standard on fruit fly disinfestation treatment efficacy, requiring different procedures than those used by us. The watermelon report has been re-written as far as possible in accordance with this

standard and the internationally recognised principle of equivalence will be claimed. This research also forms the basis on which the current interim arrangements for all cucurbits could be given full approval, because it meets the new New Zealand requirement to test all the immature stages and is otherwise equivalent in all respects.

(b) Technical Summary

The objective of this project was to provide essential disinfestation treatment data to support an application to New Zealand to admit Australian capsicums, watermelons honeydew melons and cucumbers. The research was conducted as two separate components: a packing-line spray system was developed for capsicums and a dipping system for watermelons.

Capsicums: Application of dimethoate at 400 mg/L through a packing-line spray system was shown to provide quarantine security against Queensland fruit fly, Bactrocera tryoni (Froggatt) in capsicums. There were no survivors in trials on fruit containing > 77000 eggs, shown to be the most tolerant stage to the treatment. The spray system achieved thorough wetting of each fruit for a minimum time of 1 minute.

Watermelons: A 1 minute dimethoate dip at 400 mg/L achieved a high level of quarantine security against cucumber fly, Bactrocera cucumis, watermelons. All juvenile stages of the insect were tested in large scale trials. There was little difference in tolerance to dimethoate between life stages and although rare survivors were recovered from experiments on each stage, mortality was always > 99.99% at 95% confidence which is believed to be the currently required post harvest treatment efficacy for critical quarantine pests for New Zealand.

Laboratory cage trials on watermelons as a host of B.cucumis showed that undamaged fruit were not infested. This is an indication of low and possibly non-host status. This indicates justification for a project to determine host status based on the MAP standard for host determination. Dimethoate residues were less than 0.2 mg/kg, compared with the Australian Maximum Residue Limit (MRL) of 2 mg/kg, at all times including the day of treatment

Since this research was done MAF has circulated a Standard Method for development of fruit fly disinfestation treatments, requiring different procedures to those used by us so it will be necessary to apply the principle of equivalence to the research methodology.

2. RECOMMENDATIONS

(a) Extension/adoption by industry

Quarantine disinfestation treatments such as those developed by this research cannot be adopted by industry without approval by the importing country, in this case New Zealand Ministry of Agriculture and Fisheries (MAF). Submissions are made to New Zealand MAF by the Australian Quarantine Inspection Service (AQIS). At present the capsicum submission has been rejected by New Zealand but this is being strongly contested. The watermelon report will be sent to AQIS for submission to New Zealand, and until a response is received from NZ, the industry will have to use the less preferred method of fumigation with methyl bromide.

(b) Directions for future research

Any future research on disinfestation of capsicums or cucurbits would be done using non residual methods such as heat or cold. The New Zealand draft standard on fruit fly disinfestation treatment efficacy uses methods which are much more suited to a non residual treatment and do not allow for the residual basis of insecticides. As such it is difficult to produce meaningful results with insecticide treatments. A project on host status could have a fair to excellent chance of success but would need to conform to the MAF standard method.

(c) Financial/Commercial benefits

Quarantine treatment research does not respond to normal cost-benefit analysis because it does not prevent damage or wastage nor does it increase productivity directly. Adoption of market access opportunities which flow from quarantine disinfestation research has advantages through increased market size, improved quality product for local and export markets and stabilisation of prices and incomes. It is therefore recommended that AQIS proceed with negotiations with New Zealand for the acceptance of the method as a way of ensuring market access for Australian capsicums, watermelons and other cucurbits to New Zealand.

2

DIMETHOATE DIPPING OF WATERMELON AGAINST

BACTROCERA CUCUMIS (French).

N.W. Heather, P.M. Peterson, D. Jackson, R. Kopittke Dept. of Primary Industries, Meiers Road, Indooroopilly

SUMMARY

A 1 minute dimethoate dip at 400 mg/L is proposed as a fruit fly quarantine disinfestation treatment for Australian watermelons exported to New Zealand. The treatment achieved a high level of quarantine security against cucumber fly, Bactrocera cucumis the only fruit fly which infests cucurbits in eastern Australia. All juvenile stages of the insect were tested in large scale trials because heterogeneity of response normally precludes meaningful results from small scale comparative trials to determine which stage is most tolerant of the treatment. There was little difference in tolerance to dimethoate between life stages and although survivors were recovered from experiments on each stage, in all cases mortality was > 99.99% at 95% confidence level.

Laboratory cage trials on watermelons as a host of B. cucumis showed that undamaged fruit were not infested. This is an indication of low and possibly non-host status. Dimethoate residues were less than the Australian Maximum Residue Limit (MRL) of 2 mg/kg at all times including the day of treatment.

Since this research was done the New Zealand Ministry of Agriculture and Fisheries has circulated a draft Standard Method for development of fruit fly disinfestation treatments, requiring different procedures to those used by us so it will be necessary to apply the principle of equivalence to the research methodology.

INTRODUCTION

3

Disinfestation of Queensland cucurbits against cucumber fly, Bactrocera cucumis (French), for the New Zealand market, was formerly done by fumigation with ethylene dibromide (EDB). Approval for EDB fumigation as a quarantine treatment for fruit fly host produce was withdrawn by the New Zealand Ministry of Agriculture and Fisheries (MAF) from 1 January 1994.

Dimethoate dipping at 400 mg/L is currently being used as an interim method for the export of most Queensland cucurbits to New Zealand, as it has been shown to be effective against B. cucumis in rockmelons and zucchinis (Heather et al. 1992). This treatment has proved very effective as a disinfestation treatment for tomatoes (Swaine et al. 1984, Heather & Bazeley 1989) exported from Australia to New Zealand, with an unblemished performance record over more than a decade. The treatment rate of 400 mg/L has a long history of use. It is believed to be the maximum which can be safely used across a range of fruit without exceeding Australian Maximum Residue Levels. As watermelons are larger than rockmelons and zucchinis they were not included in the interim quarantine arrangements for cucurbits exported to New Zealand because of the assumption that insecticide uptake, and hence efficacy on internally feeding insect stages, depends on the surface to volume ratio. Dipping in dimethoate is preferred by exporters because fumigation with methyl bromide (MB) is known to cause fruit injury eg. in cantaloupes and 'Honey Dew' melons (Lipton and Tebbets 1984), although Cowley et al. (1991) reported that watermelons from Tonga showed no deterioration.

During 1994, watermelons were exported to New Zealand following fumigation with MB, but this was suspended following detection of infestations of fruit fly in melons from Tonga (J. Snell AQIS pers.com.) presumably because the treatment had not been developed in Australia. Therefore an Australian developed treatment with a low risk of fruit damage is necessary to avoid trade interruptions of this nature.

MATERIALS AND METHODS

Test Insects

Fruit flies used to infest fruit in these trials were from a culture of B. cucumis maintained at the Department of Primary Industries Entomology Laboratory at Indooroopilly. Culture methods described by Heather and Corcoran (1985) for B. tryoni are identical except for the substitution of cooked pumpkin for dried carrot in the culture medium (Swaine et al. 1978). These cultures are supplemented with flies from field infested hosts annually to ensure that the gene pool is as relevant as practicable to field populations.

Adults were maintained in a controlled temperature (CT) room at 26+1 °C and 70+5% RH. Windows allowed natural light to enter, and overhead fluorescent light was

4

automatically switched off before dusk and on after dawn, subjecting the flies to natural conditions. Approximately 15 000 flies (sex ratio 1:1) were housed in wooden or aluminium framed cages 650 x 650 x 650 mm, with solid bases and mosquito netting on the sides and top. Cage populations were used only for the period of maximum fecundity, 2-6 weeks after eclosion.

Adults were fed on a diet of sugar cubes and enzymatic yeast hydrolysate. Water was available from a sponge wick in a plastic box.

Test Fruit

Watermelons (Gtrullus lanatus) were obtained from a Brisbane based export company. Fruit was taken from export stocks prior to fumigation and all fruit was thus of export size, quality and maturity. Fruit was individually weighed. The weight range and mean and median weight of treated and control fruit in each test was determined (Table 1).

Infestation

Watermelons were punctured 50 times with a 1 mm diameter steel pin, evenly distributed at the flower stalk end of the fruit. This procedure ensured an even distribution of eggs within the fruit and also tended to equalise numbers of eggs between fruits. However it did necessarily provide damage sites which were uncharacteristic of commercial fruit. The fruit was infested in cages containing B. cucumis of maximum fecundity ( 2 - 6 weeks after eclosion) for 30 to 60 minutes (Figure 1).

Determination of life stages

A destructive sampling method was used to determine development times for B. cucumis. Watermelons were infested and placed in CT rooms set at 26±1°C and 70+5% RH. Samples were removed from the rooms daily until pupation was complete. Larvae were removed by washing the fruit through a nest of fine sieves and then deep frozen to achieve a rapid kill. The sample was subsequently thawed and the proportion of each instar present at each sample time determined. Instars were identified using mouth hook and spiracle characters (Anderson 1962, Elson-Harris 1988).

Insecticides

A commercial emulsifiable concentrate formulation of dimethoate (400 g/L) was used. This was analysed for the actual concentration of active ingredient prior to the start of the trials, to avoid possible errors in dilution from decay of the active ingredient due to ageing of laboratory stock. This enabled the treatment strength to be made up with maximum accuracy.

5

Holding

Fruit were held at 26±1°C and 65+5% RH for development to the stages required for testing and after treatment until completion of each experiment. Holding times before treatment, selected on the basis of development studies (Figure 2), were first instars, 45-52 h; second instars, 91-98 h; and third instars, 143-172 h. Eggs were held for 19 h, which represented 80% development at 26°C. Treatment of eggs at an earlier age was unnecessary because the residual effect of the treatment would outlast development times for the stage.

Treatment

After infestation, one third of the fruit, chosen at random, were left untreated as controls. This is more than the usual proportion of one sixth. Treatment fruit were placed in a lidded wire cage and immersed for 1 minute in a large stainless steel tank containing a dimethoate solution of 400 mg/L (Figure 3). To ensure that treated fruit had the intended life stage present extra fruit were infested and sampled at the time of treatment to determine the proportion of the target stage present. Three trials were conducted on each of eggs, first, second and third instars.

Treated and untreated fruit were held separately over gauzed plastic boxes with sterilised sawdust as a pupation medium (Figure 4). Space available did not permit individual fruit to be held separately. All fruit were slit at the bottom to allow juice from the decomposing melon to drain through so the larvae would not drown, and at the top to ensure any survivors could escape the fruit and pupate. When pupation was complete, determined by a check of fruit for remaining larvae, pupae were sieved from the sawdust.

Residue Analysis

Watermelons were dipped in 400 mg/L dimethoate for 1 minute as for efficacy trials. After dipping they were allowed to dry for 10-20 minutes and packed into cardboard cartons. Day zero samples were let stand 1 hour after dipping. The remainder of the melons were placed in controlled temperature rooms at 13°C and 20°C to simulate transport and marketing conditions.

Two replicates, each of 4 melons were taken from produce stored at 13°C and 20°C at 0, 1, 2, 4 and 7 days after treatment. One quarter of each of the 4 melons was finely chopped in a Hobart Food Chopper and combined then a subsample extracted for residue analysis. Chopped samples were stored at -10°C.

A sample of untreated fruits was analysed and a residue of 0.03 mg/kg dimethoate was detected. Two sets of recovery data were calculated, one uncorrected and the other corrected for the residue level in the fruit used for the recovery analysis. Concentration of the dip solution was determined before and after treatment.

6

Host susceptibility

Further tests were conducted on export quality fruit to determine their susceptibility to B. cucumis. Fruit were placed in a cage of adults at maximum fecundity. Half of the fruit were pinholed while the other half were left entire. Equal numbers of pinholed and entire fruit were arranged alternately into the cage. After one hour they were removed from the adult cages and placed on gauzed boxes over sterilised sawdust in holding cages at 26±1°C and 65+5% RH. As in the efficacy trials, the melons were slit at the top and bottom, and pupae were sieved from the sawdust following pupation. This was replicated three times throughout the season.

RESULTS

Development times for B. cucumis in watermelons are given in Figure 2.

Three large scale disinfestation trials, each treating more than 10 000 insects were conducted on each life stage (Table 2). There were occasional survivors from each life stage tested. However for each the true mortality was greater than 99.99% at the 95% confidence level (Couey & Chew 1986). A total of 585 511 eggs and larvae were treated and 9 survived to pupae. Only five adults from treated fruit ecloded. The proportion of the target stage was determined for each trial (Table 2). In all cases it was greater than 50% which is normal because of overlap as development proceeds.

Results of residue analysis are given in Table 3. The measured dip strength was 375 mg/L before fruit were dipped and 379 mg/L after fruit were dipped. The variation from the nominal 400 mg/L is well within the range which might be expected using good handling and dilution practices (± 15%). Dilution error results from variation in concentrate formulation, analytical errors in measurement of the concentrate and dip solution and physical dilution errors.

Host susceptibility results (Table 4) show that while more than 100 larvae pupated from fruit that were pinpricked, none were recovered from entire fruit in any of the three replicates. Data were variable but it is not unusual to find variability of this magnitude to occur with fruit fly infestation.

7

DISCUSSION

The purpose of our research was to support a proposal to allow watermelons to be disinfested with dimethoate as a dip, for the New Zealand market, because the treatment is much simpler than fumigation and is less likely to cause fruit damage. It therefore forms the basis on which the current interim arrangement for all cucurbits could be given full approval. It meets a new Zealand requirement to test all the immature stages, the failure to do so having been given as a reason for only interim approval for smaller cucurbits. (These data were provided soon after the initial submission). In earlier experiments (Heather et al. 1992) only eggs and third instars were treated initially because the treatment has residual effectiveness which extends over the whole development time. Since the work was done MAP New Zealand has circulated a draft Standard for development of disinfestation treatments against fruit flies so it is necessary to apply the principle of equivalence to our method with respect to procedures used in its development.

Efficacy: Levels of security required for treatments against fruit flies vary from country to country. Cowley et al. (1991) used methyl bromide to disinfest watermelons for entry from Tonga to New Zealand and achieved probit 8 at the 95% confidence level. Current levels for treatments for Japan require zero survivors from 30 000 or more insects tested, which gives 95% confidence that the mortality is at least 99.99% (Couey & Chew 1986) and historically, treatments for Australian produce for New Zealand also have been required to achieve this minimum level of efficacy. Our data give 95% confidence that the mortality is 99.9935 % or higher. Results for the four life stages range from probit 8.83 - 8.98. They show that in a practical situation, that there would not be any significant difference in efficacy between the stages which might be present at treatment. An important factor is the residual effect of a dimethoate treatment evidenced by the residue analyses. These show that substantial levels of insecticide remain 7 days after treatment (although at all times these were < 10% of the permitted MRL). This means that if any early stage was aberrantly more tolerant, it would be killed as it developed through a subsequent more susceptible stage. We found no evidence that any stage was likely to present this problem. Also, our trials were designed to show that the established treatment of 400 mg/L dimethoate would achieve quarantine security. There was no intention to develop a treatment rate which would achieve a given security level.

We contend that testing of all stages or an early age within a stage is only appropriate and necessary for non-residual treatments such as heat, cold and fumigation. Earlier age eggs were not tested because the residual nature of the treatment ensured that in the unlikely event of younger eggs being less susceptible, they also would inevitably develop to the stage tested within 24 hours and it is evident from the residue data (Table 3) that there was no detectable reduction in residue levels in the first 24 hours after dipping. Also, it is highly improbable that younger eggs would be present in fruit to be treated commercially. Although Swain et al. (1991) anecdotally recorded that B. cucumis could enter packing sheds and infest fruit, observations and trapping over a number of seasons have not detected any occurrences of this nature (N.H. unpub.).

Residue Analysis: The Australian MRL for dimethoate in vegetables (except tomatoes and peppers) and fruits (except strawberries) is 2 mg/kg (Anon. 1988). Test fruit were

8

chosen from the smaller end of the weight range because, having the largest surface to volume ratio, they would be expected to have the highest residue levels. All test fruit in the residue trial were well below the MRL. The efficacy of the treatment at such low residue levels illustrates the adequacy of dimethoate dipping at 400 mg/L for 1 minute as a generic treatment against fruit flies.

Trials showed that commercial quality undamaged fruit were not infested by B. cucumis in laboratory cage trials with large initial populations > 15,000 6+9 at peak fecundity. Field cage trials are planned but were not complete at the time this report was prepared. The rarity of field infestation and the absence of infestation of undamaged fruit in laboratory cage trials is strong evidence of the low host status of watermelons and possibly, non-host status. This greatly enhances the end point security which would be conferred on treated melons.

ACKNOWLEDGMENTS

Funding for these trials was provided by Queensland exporters, Queensland Fruit and Vegetable Growers and the Horticultural Research and Development Corporation.

9

REFERENCES

Anderson, D.T. 1962. The larval development of Dacus tryoni (Frogg.) (DIPTERA: TRYPETIDAE) I. Larval instars, imaginal discs, and haemocytes. Aust. J. Zool., 11: 202-18.

Anon 1988. MRL Standard - Standard for maximum residue limits of pesticides, agricultural chemicals, feed additives, veterinary medicines and noxious substances in food. National Health and Medical Research Council, Canberra.

Couey, H.M. & Chew, V. 1986. Confidence limits and sample size in quarantine research. J. Econ. Entomol. 79: 887-890.

Cowley, J.M., R.T. Baker, K.G. Engleberger & T.G. Langi. 1991 Methyl bromide fumigation of Tongan watermelons against Bactrocera xanthodes (Diptera: Tephritidae) and Analysis of Quarantine Security. J. Econ. Entomol. 84(6): 1763-1767.

Elson-Harris, M.M. 1988. Morphology of the immature stages of Bactrocera tryoni (Froggatt)(Diptera: Tephritidae). J. Aust. Entomol. Soc. 27: 91-98.

Heather, N.W. & J.E. Bazeley. 1989. Dimethoate as a post-harvest quarantine disinfestation dip against Queensland fruit fly for 'Tristar' tomatoes. Queensland Department of Primary Industries Ref. note R8 Mar 89.

Heather, N.W. and R.J. Corcoran. 1985. Dacus tryoni, pp. 41-48. In: Pritam Singh & R.F. Moore [eds.], Handbook of insect rearing, Vol 2, Elsevier, Amsterdam.

Heather, N.W., D.E. Walpole, R.J. Corcoran, P.A. Hargreaves & R.A. Jordan. 1992. Post harvest quarantine disinfestation of zucchinis and rockmelons against Bactrocera cucumis (French) using insecticide dips of fenthion or dimethoate. Aust. J. Exp. Agric. 32: 241-244.

Lipton, W.J. & J.S. Tebbets. 1984. Methyl bromide and ethylene dibromide as potential quarantine treatment of cantaloupe and 'Honey Dew' muskmelons against tephritid fruit flies. Acta Hortic. 157: 161-169.

Swaine, G., R.J. Corcoran & M.A. Davey. 1978. Commodity treatments against infestations of the cucumber fly, Dacus (Austrodacus) cucumis French, in cucumbers. Queensl. J. Agric. Anim. Sci. 35: 5-9.

Swaine, G., P.A. Hargreaves, D.E. Jackson and R.J. Corcoran. 1984. Dimethoate dipping of tomatoes against Queensland fruit fly Dacus tryoni (Froggatt). Aust. J. Exp. Agric. 24: 227-229.

Swaine, G., D.A. Ironside and R.J. Corcoran (eds.). 1991. Insect pests of fruit and vegetables (2nd edn). Queensland Department of Primary Industries, Brisbane.

Table 1. Weights of watermelons used in trials.

Insect stage

treated*

Treated Fruit (kg) Control Fruit (kg) Insect stage

treated* Range Mean Median Range Mean Median

L3 2.3 - 3.9 2.90 2.80 2.3-3.9 2.90 2.80

L3 1.2-3.1 2.60 2.70 2.0-3.0 2.31 2.40

L3 1.7 - 4.0 2.39 2.20 1.6-2.5 2.14 2.20

L2 1.1-3.5 2.45 2.50 1.5-3.2 2.45 2.50

L2 1.8-3.2 2.36 2.40 1.8-2.6 21.8 2.10

L2 2.0-3.8 2.43 2.60 1.9-2.0 2.00 2.00

LI 1.6-4.0 2.51 2.50 1.7-3.0 2.36 2.30

LI 1.4 - 2.7 2.18 2.30 1.9-2.5 2.10 2.00

LI 2.0-3.4 2.42 2.30 1.7-1.8 1.78 1.80

Egg 2.3 - 3.9 2.90 2.80 2.3-3.9 2.90 2.80

Egg 1.8-3.0 2.32 2,10 2.0-2.6 2.32 2.35

Egg 1.2 - 3.0 2.13 2.20 1.6-2.3 1.92 2.00

* Stages: Egg, first instar (LI), second instar (L2), third instar (L3).

Table 2. Mortality of cucumber fly (JB. cucumis) infesting watermelon treated with a dimethoa

Stage* Test Date Number of fruit treated

Number of fruit

untreated

Number of pupae from

untreated fruit

Estimated number of

insects treated

Proportion of target

( « )

Numbe of pupa

L3

L3

L3

24/3/94

5/7/94

21/7/94

12

21

24

8

7

8

35 127

18 381

13 341

42 690

55 143

40 023

57

92

63

1

0

0

Totals 57 23 66 849 137 856 mmmm wmmMmm

1

L2

L2

L2

5/7/94

29/7/94

5/8/94

18

18

18

6

6

5

17 883

11 241

8 754

53 649

33 723

31 514

99

85

85

0

2

1

Totals 54 17 37,878 118,886 — 3

Ll

Ll

Ll

13/7/94

29/7/94

5/8/94

24

18

18

8

6

6

22 756

16 697

23 021

68 268

50 091

69 063

78

89

89

2

1

1

Totals 60 20 62,474 187,422 IHH 4

Egg

Egg

Egg

24/3/94

21/7/94

29/7/94

12

18

18

8

6

6

38 792

11297

19 756

48 188

33 891

59 268

100

100

100

0

0

1

Totals 48 20 69,845 141,347 l l i i l l l l l i * Stages: Egg, first instar (Ll), second instar, (L2) third instar (L3).

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Table 3. Residues of dimethoate in watermelons (1.8 - 2.6 kg) dipped in 400 mg/L solution for 1 minute

Storage at 13 °C

DAY REP1 mg/kg

REP 2 mg/kg

Average mg/kg

0 0.19 0.17 0.18

1 0.18 0.17 0.18

2 0.14 0.14 0.14

4 0.14 0.12 0.13

7 0.15 0.14 0.15

Storage at 20°C

0 0.19 0.17 0.18

1 0.18 0.20 0.19

2 0.14 0.20 0.16

4 0.14 0.10 0.12

7 0.12 0.16 0.14

Recovery data*

108.6% at 0.95 mg/kg level 125.4% at 0.18 mg/kg level

* Control Fruit had a residue of 0.03 mg/kg dimethoate (confirmed by GCMS)

Recovery Data corrected for control blank

105.2% at 0.95 mg/kg level 108.9% at 0.18 mg/kg level

13

Table 4. Host susceptibility trials: Infestation levels in damaged (pinholed) and undamaged (entire) watermelons exposed to B. cucumis in laboratory trials.

Number of Fruit Total Number of Pupae Pupae per fruit

Pinholed Entire Pinholed Entire Pinholed Entire

Trial 1 (16/3/94) 3 3 9 333 0 3 111 0

Trial 2 (17/6/94) 6 6 714 0 119 0

Trial 3 (25/8/94) 6 6 6 009 0 1001 0

14

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2

QUEENSLAND DEPARTMENT OF PRIMARY INDUSTRIES

TECHNICAL REPORT: DISINFESTATION OF CAPSICUMS

AGAINST FRUIT FLIES WITH DIMETHOATE

N. W. Heather, P.M. Peterson and R.A. Kopittke Department of Primary Industries, Meiers Road

Indooroopilly Qld 4068

SUMMARY

Application of dimethoate at 400 mg/L thorough a packing-line spray system was shown

to provide quarantine security against Queensland fruit fly, Bactrocera tryoni (Froggatt)

in capsicums. There were no survivors in trials on fruit containing > 77000 eggs, found

to be the most tolerant stage to the treatment. The spray system achieved thorough

wetting of each fruit for a minimum time of 1 minute.

INTRODUCTION

Disinfestation of capsicums against fruit fly was formerly done by fumigation with

ethylene dibromide (EDB), but approval for EDB fumigation as a quarantine treatment

of capsicums {Capsicum annum) and other fruit fly host produce exported to New

Zealand was withdrawn from 1 January 1994. Currently, there is no quarantine

treatment against fruit flies approved for disinfestation of capsicums for export to New

Zealand. Dimethoate dipping has proved very effective as a disinfestation treatment for

tomatoes exported from Australia to New Zealand, with an excellent performance record

over almost a decade. It was utilised because tomatoes suffer phytotoxic effects from

EDB fumigation. Because of the close botanical relationship between tomatoes and

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capsicums, dimethoate for capsicums can be expected to have similar advantages. For

tomatoes it is applied as a dip (Swaine et al. 1984, Heather & Bazeley, 1989) or as a

packing-line flood spray (Heather et al. 1987). For capsicums, only packing-line spray

application is advisable because the hollow centre of capsicums can take up insecticide

solution entering through holes left by larvae of Heliothis armiger.

Here, we report the conduct of experiments to show the efficacy achievable by a

disinfestation treatment with dimethoate applied by means of a packing-line spray

system against Queensland fruit fly Bactrocera tryoni (Froggatt).

MATERIALS AND METHODS

Procedure

The rates of development of juvenile stages of B. tryoni in capsicums were first

determined. Known stages were then dipped in a dimethoate solution to compare the

tolerances of each to the insecticide. This was followed by large scale trials to show the

efficacy of dimethoate at 400 mg/L which has become the standard concentration for

other produce.

Test Insects

Fruit flies used to infest fruit in these trials were from a culture of B. tryoni maintained

at the Department of Primary Industries Entomology Laboratory at Indooroopilly. This

culture is supplemented at intervals by wild flies infesting fruit in the Brisbane region

and, when available, other parts of Queensland. Culture methods have been described

by Heather and Corcoran (1985).

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Test fruit

Capsicums were grown by registered organic growers to ensure freedom from

insecticides which would inhibit fruit fly infestation, and were purchased from Allfresh

Pty Ltd., Brisbane Markets.

Insecticides

A commercial emulsifiable concentrate formulation of dimethoate (400g/L) was used.

This was analysed for the actual concentration of active ingredient immediately prior to

the start of the trials, to avoid errors in dilution from decay of the active ingredient due

to ageing. This enabled the treatment strength to be made up accurately.

Infestation

Capsicums were punctured 10 times with a steel pin, evenly distributed around the

crown at the stem end. This procedure ensured an even distribution of eggs within the

fruit, to reduce the possibility of localised fruit collapse, and also tended to equalise

numbers of eggs between fruits. The fruit was placed in cages containing B. tryoni at

maximum fecundity (3-6 weeks after eclosion) (Figure 1).

The time for oviposition differed according to the experiment. For the tests to compare

life stages, oviposition time was 10-15 minutes to avoid premature collapse of fruit

containing second and third instars. For the large scale tests using eggs, oviposition

time was 20-25 minutes.

After infestation, half of the fruit were left untreated as controls in the comparison tests,

while 1 in 6 fruits were left untreated in the large scale tests.

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Instar checks

To ensure that treated fruit had the intended life stage present extra fruit were infested

and sampled at the time of treatment to determine that the required life stage was

present.

Holding

Fruit were held at 26°C and 70% RH for development to the stages required for testing.

Holding times before treatment, selected on the basis of prior development studies

(Table 1), were first instars, 56h; second instars, 96 h; and third instars, 128 h. Eggs

were held for 32 h, which represented 80% development at 26°C.

Table 1. Development of Bactrocera tryoni in capsicums at 26°C and 70% RH.

Stage Rang ̂ Modal range

Eggs 0 - 40h LI 40 - 96h 50-•70h L2 70 - 140h 95 • • HOh L3 110 - 200h 125 - 140h Pupae 10 - 16d

Life Stage Comparison Tests

Dipping was used as the method of application for these tests because of the small

numbers of fruit involved in these trials. Fruit was infested on different days for each

life stage so that fruit containing either eggs or first, second or third instars, could be

dipped at the same time. Dipping was undertaken by wholly immersing a lidded wire

cage containing the fruit in a large stainless steel sink containing a dimethoate solution

of 4 mg/L, for 1 minute (Figure 2).

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The experiment was replicated six times. An arcsin transformation was applied to the

data. The data were analysed using a single factor analysis of variance. Treatment

means were compared using the protected least significant difference procedure.

Large scale tests

Dimethoate was applied at 400 rng/L by sprayers incorporated into a commercially

available module forming part of a tomato grading and packing conveyor system

(George and Courtier, Brisbane) (Figure 3). The module consisted of a variable speed

conveyor belt feeding fruit onto a system of brushes. Sprayers were top mounted above

the brushes. The conveyor system was adjusted to 0.5m/min for our trials.

Eight sprays were mounted, covering an area of 90 cm x 25 cm. The first test used 4

nozzles of with an aperture size of X4 (discharge of 4 gallons per hr at 250 k Pa) and

four nozzles with an aperture size of X2 (discharge of 2 gallons per hour at 250 k Pa).

The second and third tests used eight nozzles with an aperture size of X4 (Table 2).

Individual fruit were sprayed for an average of 67 seconds. The insecticide solution was

recirculated. Recovery of excess solution from fruit was via the conveyor brushes and a

drain tray with filtered return to the reservoir.

Three trials were conducted on an estimated total of 77 130 eggs in 730 capsicums.

Treated and untreated fruit (for both experiment types) were held separately over gauzed

plastic boxes with sawdust as a pupation medium (Figure 4). When pupation was

complete, pupae were sieved from the sawdust.

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Table 2. Packing-line spray application rates for dimethoate in large scale trials.

Run Nozzle size and number Dose

First large scale trial 4, X2 and 4, X4 7.25 L min'm"2 for 64 sec

Second large scale trial 8, X4 9.2 L min"1 m"2 for 65 sec

Third large scale trial 8, X4 9.2 L min"1 m"2 for 73 sec

RESULTS

Life Stage Comparison Tests

Stages differed significantly in response to the dimethoate dip (F=8.29; df=3,9; PO.01)

(Table 3). Mature eggs were significantly more tolerant than first instars (PO.01) and

third instars (PO.05). Significant differences also occurred in the larval stages; second

instars were significantly more tolerant than first instars (P<0.01) and third instars were

significantly more tolerant than first instars (PO.05). Although there was no significant

difference between eggs and second instars, the mean mortality for eggs was lower so

the large scale tests were performed on mature eggs. Raw data contained in earlier

advice to New Zealand are given in Appendix I.

Table 3. Determination of the most tolerant stage of Bactrocera tryoni in capsicums.

Mean Mortality

Stage Treated Transformed* Means

Equivalent Means (%)

Mature eggs First instars

Second instars Third instars

0.6798 a 1.1414 c 0.7632 a 0.9060 b

62.86 90.92 69.12 78.70

Least significant difference (5%) = 0.2137 Least significant difference (1%) = 0.3070

*LSD testing is done on the arcsin transformed means; m letter are not significantly different at the 5% level.

eans followed by the same

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Large Scale Tests

An estimated total of 77 130 B. tryoni eggs, in three replicates of 20 045, 15 195, and

41 890 was tested. No pupae were recovered from the treated fruit (Table 4).

DISCUSSION

Quarantine security is derived from a combination of the initial risk of infested fruit and

the efficacy of the disinfestation treatment component. Commercially produced

capsicums must have a very low incidence of fruit fly to meet consumer requirements.

It is virtually impossible to measure this incidence because it happens extremely rarely

and randomly. Anecdotal evidence suggests that the incidence of fruit rejected at pre-

export inspections is less than 1 in 104. Therefore it is likely that commercially

produced capsicums for export, infested with eggs or larvae, would be below the New

Zealand MPL of 5 insects in 1 x 106 fruit before a disinfestation treatment.

Required levels of security for treatments against fruit flies vary from country to country

and, within Australia, have varied from state to state. Current levels for treatments for

Japan require zero survivors from 30 000 insects tested, which gives 95% confidence

that the mortality is 99.99% or higher (Couey & Chew, 1986) and historically,

treatments for New Zealand also have been tested at this level. The results presented

here (Table 4) exceed that level of security with zero survivors from 77, 130 tested,

which gives 95% confidence that the mortality is 99.9961% or higher.

The treatment is thus at least as efficacious on capsicums as the dimethoate dip or

packing-line flood treatment approved for tomatoes by Australian and New Zealand

authorities, (zero survivors from 33 310 insects tested)(Heather et al. 1987). Our testing

used eggs as the stage most tolerant of the treatment although there was no significant

difference from second instars. When our raw data was supplied to New Zealand our

initial analyses showed no significant difference between any stage but subsequently,

analyses identified the differences shown in Table 3. These confirmed our decision and

New Zealand's agreement to use eggs as the stage for large scale testing.

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Table 4. Mortality of eggs of Queensland fruit fly infesting capsicum fruit treated with a d

for 1 minute

Test Date Number of fruit

treated

Number of fruit

untreated

Number of pupae from

untreated fruit

Estimated nu

eggs tre

8/3/94

10/3/94

16/3/94

170

360

200

34

72

40

4 009

3 039

8 378

20 04

15 19

41 89

Totals 730 146 15 426 77 13

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Since eggs are near the surface and could be expected to be exposed to a higher dose

for a longer time there is added assurance that eggs as the stage shown to be the most

tolerant are the most appropriate stage for testing a treatment.

Maximum wetting of fruit was considered desirable. In the large scale tests we changed

the size of nozzles and hence the application rate after the first replicate when extra

nozzles of size X4 became available. The treatment to be recommended from this work

would need to be at the higher rate of application if the efficacy level demonstrated is

required, although there were no survivors from the first replicate. In practice, once

fruit are thoroughly wetted any increase in the application rate should be irrelevant.

Time of wetting appears to have little influence beyond 1 minute (Swaine et al. 1984)

and it can be expected that, if residues on the fruit surface are not subsequently removed

maximum uptake of insecticide would occur from any fruit which had been thoroughly

wetted. Residue analyses at the application rate used in these trials were incomplete at

the time of compilation of this report. However analyses on fruit treated by a higher

volume of application (flood treatment at 16 L min"'m~2) showed that although the MRL

(lppm) was slightly exceeded in one sample on the day of application this had fallen to

safe levels by the third day (Appendix II). No problem is anticipated at the rate of

application used in our trials.

On the basis of our results we propose that a post harvest packing-line spray treatment

with 400 mg/L dimethoate which results in fruit being thoroughly wetted for 1 minute

be accepted as an appropriate fruit fly disinfestation treatment for capsicums to be

exported to New Zealand and interstate markets.

ACKNOWLEDGMENTS

This work was supported financially by Queensland Fruit and Vegetable Growers, the

Queensland Fruit Exporters Association, and the Horticulture Research and Development

Corporation.

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REFERENCES

Couey, H.M. & V. Chew. 1986 Confidence limits and sample size in quarantine

research. J. Econ. Entomol. 79: 887-890.

Heather, N.W. & R.J. Corcoran. 1985 Dacus tryoni, pp 41-48. In P. Singh & R.F.

Moore [eds.], Handbook of insect rearing, vol. 2. Elsevier, Amsterdam.

Heather, N.W. & J.E. Bazeley. 1989. Dimethoate as a post-harvest quarantine

disinfestation dip against Queensland fruit fly for 'Tristar' tomatoes. Queensland

Dept. Primary Industries Ref. note R8 Mar 89.

Heather, N.W., P.A. Hargreaves, R.J. Corcoran, and K.J. Melksham. 1987

Dimethoate and fenthion as packing line treatments for tomatoes against Dacus

tryoni (Frogatt). Aust.J.Exp.Agric. 27: 465-9.

Preisler, H.K., and J.L. Robertson. 1992 Estimation of treatment efficacy when

numbers of test subjects are unknown. J. Econ. Entomol. 85:(4) 1033-40.

Swaine, G., P.A. Hargreaves, D.E. Jackson, and R.J. Corcoran. 1984. Dimethoate

dipping of tomatoes against Queensland fruit fly Dacus tryoni (Froggatt).

Aust.J.Exp.Agric. 24: 447-9.

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The Authors: Dr Neil Heather, Senior Principal Scientist and Ms Pauline Peterson,

Entomologist, are members of the fruit fly disinfestation research group; Mrs Rosemary

Kopittke, Biometrician is a member of the Biometry Group. All are staff of the

Queensland Department of Primary Industries.

Appendix I. Raw data used for the determination of the most tolerant stage of Bactrocera tryon

Stage Treated Replicate

1 2 3 4

a b c a b c a b c a b c

L3 24 1224 87 31 60 90 25 1540 72 3 176 9

L2 M 30 307 88 12 1077 74 12 266 6

LI 20 850 93 M M - 10 428 9

Eggs M 27 165 84 19 428 64 11 551 6

a = number of fruit b = number of insects treated c = percent mortality based on pupal survival

M = missing value; caused in two cases by the collapse of the fruit, in 1 case by the failure of the ins control numbers and in the other case by survival of more insects in the treated fruit than in the control, le (1992) record this as a missing value.

Appendix II

DIMETHOATE RESIDUES IN CAPSICUMS

Treatments: Post harvest high volume (flood) with 400 mg/L dimethoate solution.

Analyses: Duplicate residue samples were taken and analysed at each sampling time.

Lab. No. Application Days after Dimethoate Rate

(mg/L) Treatment (mg/kg)

970/85 400 0 0.97 971/85 400 0 1.1 979/85 400 3 0.63 980/85 400 3 0.71 983/85 400 7 0.29 984/85 400 7 0.38

DIMETHOATE IN FORMULATION AND SPRAY SOLUTIONS

Sample

Formulation Spray soln. (pre-treatment*) Spray soln. (post-treatment*)

Nominal Cone.

400 g/L 400mg/L 400mg/L

Cone, by Analysis

413 g/L 387 mg/L 390 mg/L

* A sample of spray solution was taken before and after treatment of capsicums.

Residue analyses by P. Lynch and J. Cheyne Formulation and spray solution analyses by D. Jackson, Agricultural Chemistry Branch.

1 * «

* - 1 •

( /

> ^ t'

Figure 1. Infesting fruit with Queensland fruit fly.

Figure 2. Dipping fruit in insecticide.

Figure 3. Spraying fruit with insecticide.

Figure 4. Holding treated and untreated fruit for pupation.