VG323 Fruit fly disinfestation of cucurbits and capsicums with insecticides for New Zealand. Robert Corcoran QLD Department of Primary Industries

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1 VG323 Fruit fly disinfestation of cucurbits and capsicums with insecticides for New Zealand Robert Corcoran QLD Department of Primary Industries

2 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 Published and distributed by: Horticultural Research & Development Corporation Level 6 7 Merriwa Street Gordon NSW 2072 Telephone: (02) Fax: (02) hrdc@hrdc.gov.au Copyright 1997 **""- 6u HRD\C HORTICULTURAL RESEARCH & DEVELOPMENT CORPORATION Partnership in horticulture

3 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of detection" of 0.1 ppm, making this no longer practicable as a disinfestation treatment and approval for its use was withdrawn by the New Zealand Ministry of Agriculture and Fisheries (MAF).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 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

4 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 > 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.

5 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.

6 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.

7 3 INTRODUCTION 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 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

8 automatically switched off before dusk and on after dawn, subjecting the flies to natural conditions. Approximately 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. 4 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.

9 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, h; second instars, h; and third instars, 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 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.

10 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 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 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.

11 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 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 % or higher. Results for the four life stages range from probit 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

12 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, 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. 8 ACKNOWLEDGMENTS Funding for these trials was provided by Queensland exporters, Queensland Fruit and Vegetable Growers and the Horticultural Research and Development Corporation.

13 9 REFERENCES Anderson, D.T The larval development of Dacus tryoni (Frogg.) (DIPTERA: TRYPETIDAE) I. Larval instars, imaginal discs, and haemocytes. Aust. J. Zool., 11: Anon 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 Confidence limits and sample size in quarantine research. J. Econ. Entomol. 79: Cowley, J.M., R.T. Baker, K.G. Engleberger & T.G. Langi Methyl bromide fumigation of Tongan watermelons against Bactrocera xanthodes (Diptera: Tephritidae) and Analysis of Quarantine Security. J. Econ. Entomol. 84(6): Elson-Harris, M.M Morphology of the immature stages of Bactrocera tryoni (Froggatt)(Diptera: Tephritidae). J. Aust. Entomol. Soc. 27: Heather, N.W. & J.E. Bazeley 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 Dacus tryoni, pp 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 Post harvest quarantine disinfestation of zucchinis and rockmelons against Bactrocera cucumis (French) using insecticide dips of fenthion or dimethoate. Aust. J. Exp. Agric. 32: Lipton, W.J. & J.S. Tebbets Methyl bromide and ethylene dibromide as potential quarantine treatment of cantaloupe and 'Honey Dew' muskmelons against tephritid fruit flies. Acta Hortic. 157: Swaine, G., R.J. Corcoran & M.A. Davey 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 Dimethoate dipping of tomatoes against Queensland fruit fly Dacus tryoni (Froggatt). Aust. J. Exp. Agric. 24: Swaine, G., D.A. Ironside and R.J. Corcoran (eds.) Insect pests of fruit and vegetables (2nd edn). Queensland Department of Primary Industries, Brisbane.

14 Table 1. Weights of watermelons used in trials. Insect Treated Fruit (kg) Control Fruit (kg) stage treated* Range Mean Median Range Mean Median L L L L L L LI LI LI Egg Egg , Egg * Stages: Egg, first instar (LI), second instar (L2), third instar (L3).

15 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 24/3/94 5/7/ L3 21/7/ Totals mmmm L2 5/7/ L2 29/7/ L2 5/8/ wmmmmm Totals , ,886 3 Ll 13/7/ Ll 29/7/ Ll 5/8/ Totals , ,422 IHH 4 Egg Egg Egg 24/3/94 21/7/94 29/7/ Totals , ,347 lliilllll i * Stages: Egg, first instar (Ll), second instar, (L2) third instar (L3).

16 12 Table 3. Residues of dimethoate in watermelons ( kg) dipped in 400 mg/l solution for 1 minute DAY Recovery data* REP1 mg/kg Storage at 13 C REP 2 mg/kg Average mg/kg Storage at 20 C % 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

17 Table 4. Host susceptibility trials: Infestation levels in damaged (pinholed) and undamaged (entire) watermelons exposed to B. cucumis in laboratory trials. 13 Number of Fruit Total Number of Pupae Pupae per fruit Pinholed Entire Pinholed Entire Pinholed Entire Trial 1 (16/3/94) Trial 2 (17/6/94) Trial 3 (25/8/94)

18 14 Figure 1. Infesting fruit with Bactrocera cucumis. p m* llrrv m^ 9n&&S <**-izmi wa 33 Figure 3. Dipping fruit in insecticide.

19 I CD +- P co ^^^^^J^^^J^M^N^^^^J^^^^k^^^ KNXHH^MXHWHHHS.S.S.H.^TWn.S.TTT ^ttl>mh'oi y,yfyf' yjy kkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkj^kjr ' I I I I I I I I I I I I I I l l l l l l l l l l l l l l l ITTT..wmmm 00 r^ co U ii luuuuuu'l < o > _ k\^s.v\vsv\\\v' mimmiumuiiui LkkAkk\kk\kVkkk\kV^\\^VVA\\\k^k\\\WkkkkAkk^\Vkk\k\yvV CO CO co c2 Mmwmmi««mmi(^^ «<> o o 03 o CO o o CO 96BJS l 3B9 Ul 96B1U80J9CJ

20 16 Figure 4. Holding treated and untreated fruit for pupation.

21 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 > 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 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

22 3 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).

23 4 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 minutes to avoid premature collapse of fruit containing second and third instars. For the large scale tests using eggs, oviposition time was 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.

24 5 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 L h 95 HOh L h h 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).

25 6 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 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.

26 7 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, X 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. Stage Treated Mature eggs First instars Second instars Third instars Least significant difference (5%) = Least significant difference (1%) = Mean Mortality Transformed* Means a c a b Equivalent Means (%) *LSD testing is done on the arcsin transformed means; m eans followed by the same letter are not significantly different at the 5% level.

27 8 Large Scale Tests An estimated total of B. tryoni eggs, in three replicates of , , and 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 preexport inspections is less than 1 in 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 10 6 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 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 % 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 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.

28 9 Table 4. Mortality of eggs of Queensland fruit fly infesting capsicum fruit treated with a d for 1 minute Test Date Number of fruit Number of fruit Number of pupae from Estimated nu treated untreated untreated fruit eggs tre 8/3/ /3/ /3/ Totals

29 10 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.

30 11 REFERENCES Couey, H.M. & V. Chew Confidence limits and sample size in quarantine research. J. Econ. Entomol. 79: Heather, N.W. & R.J. Corcoran Dacus tryoni, pp In P. Singh & R.F. Moore [eds.], Handbook of insect rearing, vol. 2. Elsevier, Amsterdam. Heather, N.W. & J.E. Bazeley 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 Dimethoate and fenthion as packing line treatments for tomatoes against Dacus tryoni (Frogatt). Aust.J.Exp.Agric. 27: Preisler, H.K., and J.L. Robertson Estimation of treatment efficacy when numbers of test subjects are unknown. J. Econ. Entomol. 85:(4) Swaine, G., P.A. Hargreaves, D.E. Jackson, and R.J. Corcoran Dimethoate dipping of tomatoes against Queensland fruit fly Dacus tryoni (Froggatt). Aust.J.Exp.Agric. 24:

31 12 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.

32 Appendix I. Raw data used for the determination of the most tolerant stage of Bactrocera tryon Stage Treated Replicate a b c a b c a b c a b c L L2 M LI M M Eggs M a = b = c = number of fruit number of insects treated 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.

33 Appendix II DIMETHOATE RESIDUES IN CAPSICUMS Treatments: Analyses: Post harvest high volume (flood) with 400 mg/l dimethoate solution. Duplicate residue samples were taken and analysed at each sampling time. Lab. No. Application Days after Dimethoate Rate Treatment (mg/kg) (mg/l) 970/ / / / / / 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.

34 1 * «1 * - (/ > ^ t' Figure 1. Infesting fruit with Queensland fruit fly. Figure 2. Dipping fruit in insecticide.

35 Figure 3. Spraying fruit with insecticide. Figure 4. Holding treated and untreated fruit for pupation.