NewDe Lima Zealand et al.—Cold Journal ofdisinfestation Crop and Horticultural of citrus Science, 2007, Vol. 35: 39–50 39 0014–0671/07/3501–0039 © The Royal Society of 2007

Cold disinfestation of citrus (Citrus spp.) for Mediterranean ( capitata) and Queensland fruit fly ( tryoni) (Diptera: )

C. P. F. De Lima1 INTRODUCTION 2 A. J. Jessup Australian producers can supply fresh high quality L. Cruickshank2 citrus (Citrus spp.) “counter season” to meet the C. J. Walsh2 demands of markets in the Northern Hemisphere. However, quarantine restrictions are placed on E. R. Mansfield1 1 citrus because of the presence of Queensland Department of Agriculture fruit fly (QFF; (Froggatt)) and South Perth, WA 6151, Mediterranean fruit fly (MFF; email: [email protected] (Wiedemann)) on the Australian mainland. QFF is 2New South Wales Agriculture endemic to the entire east-coast region of Australia Gosford, NSW 2250, Australia extending inland as far as 600 km or more (May 1963) whereas MFF occurs in several pockets around major townships in Western Australia (White & Elson- Abstract Cold treatment of citrus (Citrus spp.) Harris 1992). Disinfestation methods require either fruits to achieve quarantine levels of disinfestation fumigation using methyl bromide, cold treatment, of fruit is a viable alternative to methyl bromide or irradiation (APHIS 2006). Methyl bromide is fumigation for export purposes. A series of trials, phytotoxic to citrus and shortens shelf life. Its use using standard bioassay procedures followed is also becoming restricted in Australia because it by large-scale export tests, were conducted on is ozone depleting and subject to being phased out five citrus cultivars at 2°C and 3°C against the under the Montreal Protocol. Irradiation does not Mediterranean fruit fly (MFF) (Ceratitis capitata) damage citrus and is straightforward to use. However, and the Queensland fruit fly (QFF) (Bactrocera issues of live sterilised in produce have not tryoni). The exposure periods required to achieve been resolved. The effect of cold storage on quality complete mortality in >30 000 insects varied with can be managed and is approved for disinfestation of fruit fly species and cultivar. For MFF, control at 2°C both MFF and QFF for export to Japan. The approved was obtained in 16 days in lemons (Citrus limon) and treatment for both species is 16 days at 1°C for oranges 18 days in oranges (Citrus sinensis) and mandarins (Citrus sinensis), mandarins (Citrus reticulata), (Citrus reticulata); whereas at 3°C, 18 days were and tangelos (Citrus paridisi × Citrus reticulata) required in lemons and 20 days in oranges and (Hill et al. 1988; De Lima 1993, 1997; Heather et mandarins. For QFF, control was obtained in 14 days al. 1996), and 14 days for lemons (Citrus limon) in lemons and 16 days in oranges and mandarins at (Jessup & De Lima 1989; De Lima 1991; Jessup et al. both 2°C and 3°C. The application of these results 1993). Advice from industry is that treatment at 1°C will provide more flexibility for the citrus industry generally requires that the thermostat be set at 0.4°C in satisfying quarantine treatments. for treatment in cold rooms and shipping containers and because probe records can vary by ±0.6°C the Keywords Mediterranean fruit fly; Ceratitis fruit can suffer damage at temperatures below 0°C. capitata; Queensland fruit fly; Bactrocera tryoni; Thus trials were conducted in five citrus cultivars at citrus; quarantine treatments; cold storage 2°C and 3°C to provide a temperature-time schedule of treatments giving exporters more choice in their selection of a suitable treatment. The Japanese Ministry of Agriculture, Forestry H05161; Online publication date 19 February 2007 and Fisheries has established a trial format Received 16 December 2005; accepted 7 January 2007 requiring quarantine levels of disinfestation to be 40 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35 demonstrated through a carefully conducted series under which the fruit fly colonies are maintained. of experiments. The first stage is a series of dose- To determine the course of development of the response tests (replicated 3 times) to determine the immature stages, test fruits were removed from cold LD50 and LD95 mortality values for each life-stage storage and held for 48 h at 26±1°C and 60–65% of QFF and MFF found in host fruit. The results RH to equilibrate to a temperature suitable for egg of these tests are used to select the life stage most and larval development. Two hundred fruit were tolerant to the treatment and to plan and conduct a selected without conscious bias and infested with series of large-scale trials under simulated export eggs. The methods of infestation for MFF and QFF conditions. The large-scale trials comprise three differs according to species of fruit fly and have been separate replicates of the selected treatment applied described previously (Friend 1957; Anderson 1963; to the most tolerant stage in each cultivar. To be Hill et al. 1988; Jessup et al. 1993). considered successful, each replicate trial should MFF does not readily oviposit citrus with demonstrate that no survivors are obtained when sufficient number of eggs to prepare satisfactory >10 000 individuals (total >30 000) are exposed disinfestation trials. Therefore, to obtain sufficient to the treatment. QFF trials were conducted at the insects of the different life stages it was necessary to Horticultural Research Laboratories in Gosford, artificially infest the citrus fruits. This was done by . MFF trials were conducted at the injecting each fruit to a depth of 10–15 mm (females Department of Agriculture, Entomology Laboratories oviposit 2–3 mm into fruit) with 0.5 ml of 6-h-old in South Perth, Western Australia. eggs in agar medium using a hypodermic syringe (c. 250 eggs from 2–3-week-old fertile females). The wound at the injection site was sealed with a MATERIALS AND METHODS drop of polyvinyl acetate adhesive. The eggs were obtained from a standard colony of 1–1.2 million Test fruit adult flies (four cages each containing 250 000–275 Five cultivars of citrus were tested—‘Valencia’ 000 adults, sex ratio 1:1) renewed with wild flies and ‘Navel’ oranges; ‘Ellendale’ and ‘Murcott’ every year, and maintained at 26±1°C and 60–65% mandarins; and ‘Lisbon’ lemons. For all QFF trials, RH in a dark:light cycle of 16:8 h. The colony is fruit was sourced from the Fruit Fly Exclusion Zone renewed by collecting infested fruit from backyard which is a fruit fly free citrus production area that properties in several towns over summer and autumn takes in the Murrumbidgee Irrigation Area of New each year. These insects are maintained in a separate South Wales, the Sunraysia district of Victoria, and building (to minimise cross-contamination) under the Riverland of South Australia. For MFF, the most- the above conditions until numbers are sufficient to tolerant-stage trial fruits were sourced from -free replace the existing colony. This process takes c. 6 areas within the Gingin, Chittering, Barekup, and months every year. Harvey Districts in Western Australia, and for large- QFF readily infests citrus, but ‘Valencia’, scale trials from the fruit fly free area of the Renmark ‘Navel’, and ‘Lisbon’ fruit require to be punctured District of South Australia. The fruit did not receive to increase numbers of eggs laid per fruit (Rigney direct applications and were free from 1976). The fruit was punctured to a depth of 3–4 mm pesticide residues. They were of good quality in (approximate depth of female oviposition) with a terms of sugar:citric acid ratio and brix values and hand-held wooden block studded with 28 fine pins, were suitable for the survival of immature stages and placed punctured-side down on top of cages of test insects at the time of infestation. The sugar: (1630 × 510 × 240 mm) holding 15 000–20 000 citric acid ratio and brix values are summarised sexually-mature flies (2–4 weeks old). Flies infested as: ‘Valencia’ (13.2, 9.4), ‘Navel’ (15.5, 10.9), the fruit through the gauze roof of the cage into the ‘Ellendale’ (15.7, 11.8), ‘Murcott’ (20.8, 11.6), and punctures made in the fruit. ‘Ellendale’ and ‘Murcott’ ‘Lisbon’ (1.2, 6.7). The fruits were obtained when fruit do not require to be punctured because their required and in season, and stored at 4–5°C and soft, thin skin allows females to oviposit quite easily. 90–95% relative humidity (RH). Fruit remained on the cages for 2–4 h. A standard QFF colony consists of 240 000–320 000 adults Development of immature stages (16 cages each containing 15 000–20,000 flies, sex of fruit flies in citrus fruit ratio 1:1) renewed with wild flies every year, and All experimental work in both laboratories was maintained at 26±1°C and 60–65% RH in a dark: conducted at 26±1°C and 60–65% RH, the conditions light cycle of 12:12 h. The colony renewal method De Lima et al.—Cold disinfestation of citrus 41 is similar to that described for MFF above although recorded by placing thermistor probes into the core replacement time is c. 2–3 months. of uninfested fruit placed in similar plastic labelled Infested fruits were stored at 26±1°C and 60–65% tote boxes in the cold rooms. RH for incubation. The course of egg and larval For MFF, the treatments consisted of 18 periods of development was determined by dissecting 10 fruit cold exposure and one untreated control. The 18 cold each day to obtain the required number of insects exposures were incremental doses of cold beginning and life stages. The fruit were dissected over a series with 48 h at the lowest dose and increasing by 24 h up of sieves (Endecotts Ltd, United Kingdom) ranging to 336 h (14 days). After this the dose was increased in aperture size from 2.0 mm to 125 microns. The by 48 h up to 576 h for both temperature trials, that is pulp was washed in a gentle stream of tap water to for 2.0 ± 0.5°C and 3.0 ± 0.5°C. Timing of exposure separate the eggs and larvae from the fruit medium. period began when the last probe in the fruit reached Thus, the number of days after infestation at which 2.0 ± 0.5°C or 3.0 ± 0.5°C. This was achieved each of the four target life-stages (eggs, 1st, within c. 6 h of starting each trial. The treatment 2nd, and 3rd ) predominated in the infesting conditions were held at 2.0 ± 0.5°C and 3.0 ± 0.5°C, population was determined. These data were used respectively, over the entire experimental period for to start the treatment of infested fruit at each life each cultivar. For QFF, the cold exposure periods stage separately. To determine the number of puparia were 1, 3, 5, 7, 9, 11, 13, and 15 days from the time obtained per fruit, a total of 50 fruit was set aside and the fruit core reached the target temperature. QFF incubated to pupation. This information enabled a was found to be very susceptible to cold at both 2°C decision to be made of the number of fruit required and 3°C with 99% mortality being achieved by 168 per replicate. h. The additional doses for both species were used to confirm 100% mortality and to establish at least two Most tolerant stage trials successive dose levels at 100% to enable confidence These trials were conducted from 1997 to 1999 for in selecting the treatment for the large-scale trials. both fruit fly species. For MFF, the experiments After exposure to the specified treatment, the box consisted of 19 treatments including the untreated containing the fruit was removed to the controlled control at both 2°C and 3°C. A total of 4560 fruits environment room containing the control fruit for of each and mandarin cultivar (‘Navel’, collection of surviving stages as puparia. The number ‘Valencia’, ‘Ellendale’, and ‘Murcott’) were used, of puparia emerging at each dose was compared with whereas 9120 ‘Lisbon’ lemons were required. More the number from the untreated controls to obtain the lemons were required because fewer MFF survived percentage responding to the treatment. The criterion in each fruit. A total of 27 360 fruit was treated for survival was the formation of an apparently at each temperature, making 54 720 fruit for both normal puparium (Baker 1939). temperatures. Thus, 20 (orange and mandarin) or refrigeration for the three cold rooms is supplied 40 (lemon) fruit were selected at random from the by three Lovelock Luke F26-120A air cooled belt- infested batch for each replicated treatment and driven condensing units with R22 refrigerant plus placed in large labelled plastic “tote” boxes. The six Muller MNDE17 induced draught evaporators boxes had a wire mesh bottom to permit emerging with refrigeration capacity of 7500 W at 1°C with larvae to drop into the sand contained in a second compressor speed 536 rpm. This capacity can be box fitted below. The box containing the infested increased to 9500 W at 1°C by raising the compressor fruit was covered with Terylene voile to allow air speed to 700 rpm. Defrost cycles are set at 12-h exchange and sealed in place with a plastic lid having intervals but defrosting may occur on demand from a large aperture in the centre. a gas pressure monitoring switch. Six fans, two for The fruit were held at 26±1°C and 60–65% RH each room (350 mm 4-blade propeller type) circulate for the period of time required for the fruit flies to air across the evaporator at an airflow averaging 800 develop to the stage required for the experiment. litres/s measured at various points in the room. The Each exposure therefore consisted of 3 replicates fans are switched off during the defrost cycle. (60 or 120 fruit) for each stage. At each treatment, Temperatures were recorded on a “Squirrel” (Grant four stages were tested, requiring 240 (orange and Instruments, Cambridge, England) data logger with mandarin) or 480 (lemon) infested fruits to be treated. an accuracy of ±0.05°C. Each cold room was supplied Each replicated trial was set up in one of the three with a logger to monitor a total of 16 thermistor purpose-built (34 m3) replicate cold rooms (thermostat probes, six to record air temperatures at various accuracy of ±0.1°C). Fruit pulp temperatures were positions in the cold room. One probe each was 42 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35 placed to record the inlet and outlet air temperatures containing c. 120 fruit (19 kg) stacked on pallets. of the cooler. The remaining four probes were placed There were six pallets in each replicate cold room. at various heights in other positions—top door-end, Each pallet carried 70 cartons arranged as 10 cartons middle, and left and right sides to calibrate the cold per layer and seven layers high to bring the treated room for uniformity of temperatures. The data from volume to 37.55%. Thus there were 7980 kg fruit in each probe site were recorded for the test report. each replicate cold trial of which 2400 fruit (oranges The remaining 10 thermistors were used to record and mandarins) or 4800 fruit (lemons) were infested fruit pulp temperatures by placing the probes in with 2nd instar MFF out of a total number of 50 400 the core of uninfested fruit at different positions fruit in 420 cartons. A similar procedure was adopted in the experimental stacks containing the infested for 1st instar QFF. The infested fruit were placed in fruit. Temperature recordings were automatically the centre of every carton throughout the stacks in logged at 60-min intervals throughout the trial. All the cold room so that a representative treatment could thermistor probes were calibrated in melting ice with be obtained in all cartons. a certified mercury glass thermometer, before and The cool-down period was accelerated by use of a after each trial to verify their accuracy. forced fan cooler placed in the cold room. When the temperature of the last probe was within the specified Large-scale trials treatment temperature (2.0 ± 0.5°C or 3.0 ± 0.5°C), For the MFF trials, from 1999 to 2001, a total of the forced cooling process was stopped and normal 126 400 fruits were infested for five cultivars at cold room operations were allowed to continue. The both temperatures, giving 63 200 at 2°C and 63 cool-down period ranged from 42 to 46 h. For MFF, 200 at 3°C. Thus for each temperature treatment, oranges and mandarins were exposed to 2°C for 18 10 600 fruit were infested for each trial of ‘Navel’, days and to 3°C for 20 days. Lemons were treated for ‘Valencia’, ‘Murcott’, and ‘Ellendale’ cultivars (42 16 days at 2°C and 18 days at 3°C. For QFF, oranges 400 fruits). For the ‘Lisbon’ lemon trials, 20 800 and mandarins were treated for 16 days, and lemons fruit were infested, because the number of puparia for 14 days at 2°C and 3°C since the most tolerant obtained was c. 50% less in lemons. In MFF, the 2nd stage trials showed no difference in susceptibility instar was shown to be the most tolerant stage to cold at both temperatures. After exposure to the cold treatment, whereas in QFF it was the 1st instar. treatment, the infested fruit were removed from the Fruits for the large-scale trials were infested in the cartons and taken to the controlled environment same way as described above for the most tolerant room and placed in containers over sand to collect stage trials. The trials required large quantities of puparia. infested fruit and these were placed in wire cages in single layers supported by mesh to promote uniform Data analysis exposure to the development conditions. The fruit MFF and QFF data obtained from a series of exposure infested for treatment and controls were held in wire periods (days) to 2°C and 3°C of the four stages— cages over sand in a controlled environment room eggs, 1st, 2nd, and 3rd instar were subjected to probit 26±1°C and 60–65% RH and covered with custom- analysis. The total number from all replicates was made Terylene voile covers. The infested fruits combined to obtain the number treated (puparia in in each treatment replicate were incubated to the control) and the number responding (from puparia required development stage for testing. Selection of obtained for each exposure period) and analysed using fruits for treatment and control was done at random. the Genstat package (GenStat Release 4.1 1999). For On the day of treatment the specified number of the analysis of MFF data the probit model uses a fruits for each stage for treatment and control were generalised linear procedure, assuming a binomial separated. The control fruits were placed in wire distribution for the number of responses and a probit cages for development to pupation. Extra fruit link function between the number of responses and were taken from each replicate trial for dissection the logarithm (log10) of the dose. Tests on data using to estimate the number of the target stage treated the logit link function and the complementary log- and other life stages present. log function did not significantly reduce the residual The infested fruits for cold treatment were taken and deviance and the probit link function was retained in placed in the centre of every export carton containing analysis. For the analysis of QFF data, independent uninfested filler fruit and packed following standard regression calculation was used for each stage in export practice. Each replicate trial was set-up in a each fruit since most of the regression lines were not separate cold room and consisted of 420 cartons each parallel. This method gave a better fit to the data. De Lima et al.—Cold disinfestation of citrus 43

Testing the data using Wadley’s analysis (Genstat ratio ranges from 3 to 11 times QFF at the LD50 Release 8.1 2005) did not give better insight into the estimate and 1.5 to 5 times at the LD95 estimate. data. The relative potency of cold to the fruit flies QFF exhibits very little change in response to cold was calculated at the LD50 and LD95 estimates and treatment at 2°C and 3°C. given as a ratio of MFF:QFF. Large-scale trials The selection of treatment periods was based on RESULTS obtaining consecutive 100% mortalities in the most tolerant stage trials (Tables 1 and 2). For MFF this Development of immature stages was 2°C for 18 days and 3°C for 20 days in oranges of MFF and QFF in citrus cultivars and mandarins, whereas for lemons the treatment The details of the life history of MFF and QFF were was 16 days at 2°C and 18 days at 3°C. In the control established before every series of trials. For MFF, fruit the number of puparia recovered from all 3 c. 50% of each stage was present on the following replicates exceeded 100 000 whereas no puparia days after infestation—eggs: 1–2; 1st instar: 3–5; were obtained from the cold treatment. In the QFF 2nd instar: 6–8; 3rd instar: 9–10. For QFF >50% trials, total insect mortality was achieved in 14 days of each stage was present on the following days for lemons and 16 days for oranges and mandarins after infestation—eggs: 1–2; 1st instar: 3–4; 2nd at both 2°C and 3°C. The number of puparia found instar: 5–6; 3rd instar: 7–9. The results were used to in the untreated control fruit ranged from >40 000 determine the stage of treatment as well as the date for lemons to >400 000 in ‘Ellendale’ mandarins. of treatment for each stage following infestation. The greater variation in the QFF infested fruit may be explained by the fact that infestation is by natural Most tolerant stage trials oviposition, with lemons clearly not favoured and Data of the percentage dying at each dose (exposure mandarins highly preferred by female QFF. period in days) for each test species, cultivar, and treatment temperature are given in Tables 1 and 2. The MFF data (Tables 3 and 4) show that the DISCUSSION 2nd instar is the most tolerant life stage at the LD50 estimate. However, the LD95 95% fiducial limits The results reported in this paper are based on a over-lap at 2°C for 1st and 2nd in lemons large body of work done over a period of 5 years (De and at 3°C in ‘Navel’ oranges, indicating that these Lima et al. 2002). The accumulated experience of lines are not parallel compared to the other stages. over 15 years of research (Hill et al. 1988; De Lima The QFF data (Tables 3 and 4) show that the 1st 1991, 1993, 1997; Jessup et al. 1993; Heather et al instar is, in general, the most tolerant life stage at the .1996; De Lima et al. 2002) was brought to bear on LD50 estimate for ‘Valencia’ and ‘Navel’ oranges and this project. The results obtained for MFF confirm ‘Lisbon’ lemons. However for ‘Ellendale’ mandarins and more clearly define (with the benefit of modern at 2°C the 2nd instar has a higher LD50 but the LD95 compressors, electronic thermostats, and data value for 1st instar is higher; for ‘Murcott’ mandarins loggers) treatment temperatures and times obtained the 3rd instar has a higher LD50 but the 2nd instar has over the past 90 years by other workers (Back & a higher LD95. At 3°C the 1st instar is more tolerant Pemberton 1916; Pettey & Griffiths 1931; Baker et for oranges, lemons, and ‘Murcott’ mandarins, but al. 1944; Richardson 1958). Powell (2003) assessed for ‘Ellendale’ mandarins it is the 2nd instar that the original data presented in this paper at 2°C for has the highest LD50 and LD95 estimates compared MFF (De Lima et al. 2002) and after comparing to the other stages. In summary, the 1st instar was data available from Back & Pemberton (1916), Nel considered the most tolerant stage in QFF since in (1936), Sproul (1976), Hill et al. (1988), Jessup et al. the majority of trials it is most tolerant at both the (1993), Santabella et al. (1999), and APHIS (2002) LD50 and LD95 estimates for both 2°C and 3°C. concluded that this data provided APHIS with >95% a comparison of the data across all fruit cultivars confidence in the treatment compared with other data for the two fruit fly species is given by the relative available in the APHIS treatment schedule, providing potency of cold treatment to MFF and QFF LD50 it with sufficient confidence to amend the schedule and LD95 points. This demonstrates that MFF is to 18 days at 2.2°C (36°F). Before this, APHIS had considerably more tolerant to cold treatment than established a panel of USDA scientists to review QFF at both temperatures. In general, MFF tolerance the cold treatment T107 because it was considered 44 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35

Table 1 Mortality of Mediterranean fruit fly Ceratitis( capitata) in citrus (Citrus spp.). Data are the average number of puparia obtained from three replicates at each stage in the untreated control and the average % mortality for each exposure period (days) at 2°C and 3°C. Trials continued for 24 days at both temperatures but no survivors were found at 20, 22, and 24 days.

2°C 3°C Fruit type Days treated Eggs 1st 2nd 3rd Eggs 1st 2nd 3rd Valencia Control 729 652 712 653 735 780 732 772 2 16.45 18.37 11.93 21.80 9.75 4.10 3.19 5.91 3 27.97 23.17 17.64 32.13 23.36 13.11 11.79 20.80 4 38.71 30.16 26.10 46.39 38.91 25.93 23.50 32.97 5 54.71 45.93 39.63 56.67 48.75 33.28 30.83 47.48 6 64.67 57.04 54.39 73.72 70.25 43.91 40.12 55.16 7 78.02 63.85 56.76 81.03 86.12 57.88 52.78 69.70 8 91.68 70.75 66.59 88.57 91.47 71.29 64.66 82.48 9 98.45 84.10 78.43 95.61 95.56 80.01 73.22 92.32 10 99.68 88.89 85.28 97.67 98.14 86.67 77.55 95.25 11 100.00 96.48 92.23 99.59 99.23 93.51 85.11 96.98 12 100.00 98.90 97.49 99.77 99.82 96.33 91.44 97.97 14 100.00 99.77 98.95 100.00 100.00 97.65 95.95 99.22 16 100.00 100.00 100.00 100.00 100.00 98.93 98.59 99.70 18 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Navel Control 1109 1073 1075 1071 1342 1363 1287 1293 2 10.90 11.87 13.82 16.61 13.31 5.50 4.84 8.76 3 20.79 20.94 20.13 25.02 22.27 12.61 12.02 23.56 4 36.86 36.86 35.69 38.60 40.69 23.78 18.67 37.35 5 53.02 44.85 44.22 50.11 52.09 36.32 34.11 50.10 6 69.18 56.20 56.23 66.45 67.97 48.59 44.57 63.61 7 83.57 64.64 65.55 75.88 79.49 54.93 53.51 71.44 8 93.57 72.87 73.57 83.45 88.51 63.85 63.58 78.45 9 99.61 81.11 80.02 93.81 94.76 76.19 73.89 89.18 10 99.97 87.56 88.40 97.54 97.99 80.81 81.22 93.84 11 100.00 95.61 95.04 99.22 99.90 90.61 87.21 98.45 12 100.00 98.86 98.53 99.79 100.00 96.16 96.11 99.20 14 100.00 99.91 99.58 99.97 100.00 98.04 98.06 99.74 16 100.00 100.00 100.00 100.00 100.00 99.22 99.35 99.95 18 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Lisbon Control 669 683 664 681 869 818 763 850 2 16.64 4.29 8.07 12.71 16.95 7.13 7.42 13.52 3 31.19 16.39 22.67 30.34 27.08 18.08 14.58 24.33 4 49.93 35.87 38.86 42.75 41.81 25.94 26.10 43.50 5 65.87 47.93 49.33 63.53 55.66 43.85 39.63 53.45 6 79.02 68.41 70.30 75.54 75.76 60.63 54.34 63.36 7 88.29 79.62 79.27 85.00 85.85 74.02 61.59 80.09 8 93.07 86.00 88.29 90.48 93.33 78.99 74.12 88.99 9 97.86 92.83 93.22 96.91 97.85 89.37 83.63 93.85 10 99.40 97.41 97.41 98.95 98.96 93.85 91.92 97.45 11 100.00 99.15 99.00 99.75 100.00 96.13 95.50 98.75 12 100.00 99.95 99.90 100.00 100.00 97.64 96.99 99.69 14 100.00 100.00 100.00 100.00 100.00 99.31 99.30 99.88 16 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 18 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Ellendale Control 792 842 773 793 818 806 761 756 2 14.78 6.44 9.52 14.53 11.85 9.71 6.92 11.85 3 26.48 16.04 19.87 26.44 22.44 19.18 18.65 24.76 4 43.58 28.84 31.92 40.34 36.09 28.69 28.42 36.26 5 57.43 44.51 44.38 57.14 49.86 40.18 38.31 49.74 6 70.95 56.00 55.28 66.57 64.68 48.37 46.76 60.18 7 82.27 70.82 67.16 77.68 81.63 58.99 56.83 74.76 8 94.61 80.59 77.31 88.51 86.31 69.28 66.68 83.30 9 99.37 87.79 85.81 94.95 94.22 81.89 80.39 89.56 10 99.83 93.64 93.39 97.52 99.19 89.62 87.78 94.10 11 100.00 96.04 96.46 98.57 99.84 92.93 91.07 96.96 12 100.00 98.44 98.19 99.37 100.00 96.86 95.75 98.77 14 100.00 99.66 99.71 99.92 100.00 99.34 98.82 99.87 16 100.00 100.00 100.00 100.00 100.00 99.75 99.65 100.00 18 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Murcott Control 715 788 782 764 662 685 723 655 2 10.11 2.10 1.77 5.12 11.42 4.04 5.81 10.12 De Lima et al.—Cold disinfestation of citrus 45

2°C 3°C Fruit type Days treated Eggs 1st 2nd 3rd Eggs 1st 2nd 3rd

Murcott cont. 3 29.39 14.58 14.02 24.55 23.39 15.96 15.39 24.82 4 54.35 31.77 26.73 44.39 42.91 29.93 24.98 36.72 5 63.67 43.32 41.17 53.84 60.71 40.63 36.96 52.34 6 73.31 59.90 54.87 70.14 76.46 45.84 44.75 66.94 7 86.26 66.93 69.73 76.57 88.08 59.76 60.05 81.18 8 96.09 81.70 81.23 88.22 93.36 70.32 68.20 87.89 9 98.74 87.47 81.74 93.57 95.57 83.16 78.85 91.05 10 99.81 95.95 92.27 97.90 98.24 89.05 86.73 95.02 11 100.00 97.72 94.64 99.07 99.95 93.63 92.76 95.78 12 100.00 99.35 97.21 99.81 100.00 95.86 95.39 98.58 14 100.00 99.95 99.12 99.95 100.00 98.15 98.02 99.59 16 100.00 100.00 100.00 100.00 100.00 99.08 98.85 99.64 18 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

Table 2 Mortality of Queensland fruit fly (Bactrocera tryoni) in citrus (Citrus spp.). Data are the average number of puparia obtained from three replicates at each stage in the untreated control and the average % mortality for each exposure period (days) at 2°C and 3°C.

2°C 3°C Fruit type Days treated Eggs 1st 2nd 3rd Eggs 1st 2nd 3rd Valencia Control 354 594 610 486 308 375 400 328 1 68.25 49.60 55.46 53.43 75.23 59.66 68.33 58.67 3 80.63 64.37 80.03 73.80 93.23 71.53 83.17 62.00 5 99.07 83.27 90.80 96.10 100.00 90.30 99.90 97.17 7 100.00 99.93 99.90 99.87 100.00 99.57 100.00 99.47 9 100.00 100.00 99.73 100.00 100.00 99.80 100.00 100.00 11 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 15 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Navel Control 1460 760 1248 822 778 782 642 601 1 75.43 51.25 61.92 67.40 72.50 54.93 56.86 64.43 3 89.13 69.80 80.90 73.17 80.70 61.63 64.00 73.83 5 99.83 86.97 91.20 97.80 86.40 74.83 89.97 92.87 7 100.00 99.43 99.93 100.00 90.70 92.50 95.77 99.70 9 100.00 100.00 100.00 100.00 100.00 99.80 99.73 100.00 11 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 15 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Lisbon Control 433 475 340 484 439 554 1116 703 1 84.42 52.15 79.24 72.30 76.94 68.35 79.00 81.76 3 99.53 80.33 98.33 91.30 93.50 80.80 92.30 94.03 5 100.00 90.40 99.87 99.30 95.33 98.67 100.00 99.53 7 100.00 100.00 100.00 100.00 100.00 98.67 100.00 100.00 9 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 11 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 15 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Ellendale Control 2790 2414 4542 3248 2206 2254 4084 2848 1 53.32 59.13 51.47 56.13 61.93 56.70 48.93 60.43 3 61.70 73.93 64.00 63.10 87.57 69.57 54.53 71.60 5 97.47 89.43 89.17 91.97 99.90 86.93 80.17 83.07 7 99.83 96.93 96.77 99.80 100.00 99.77 99.30 99.40 9 100.00 99.73 99.97 100.00 100.00 100.00 100.00 99.93 11 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 15 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 Murcott Control 1354 1808 2766 1976 2318 2042 2536 2298 1 69.77 53.65 57.73 49.83 46.72 43.03 51.63 62.77 3 83.73 80.87 69.90 86.33 55.80 54.33 66.80 78.60 5 99.67 97.07 99.03 97.60 87.27 74.97 92.03 94.57 7 100.00 100.00 100.00 100.00 99.90 98.27 99.23 99.80 9 100.00 100.00 100.00 100.00 100.00 99.77 100.00 99.93 11 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 13 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 15 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 46 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35

Table 3 Comparison of the number of days exposure at 2°C required to kill 50% (LD50) and 95% (LD95) with 95% fiducial limits (FL) of the four immature life stages of Mediterranean fruit fly Ceratitis( capitata) and Queensland fruit fly Bactrocera( tryoni) in five citrus Citrus( spp.) cultivars. The analysis is based on three replicate trials for each life stage.

Mediterranean fruit fly Queensland fruit fly Relative Citrus cultivar potency ratio and life stage LD50 (95% FL) LD95 (95% FL) LD50 (95% FL) LD95 (95% FL) mFF: QFF treated days days days days LD50 – LD95 (1) Valencia orange Eggs 4.202 9.822 0.729 3.897 5.76 – 2.52 (4.154, 4.250) (9.694, 9.955) (0.514, 0.938) (3.226, 4.929) 1st instar 5.521 12.690 1.327 6.679 4.16 – 1.90 (5.464, 5.578) (12.510, 12.880) (1.070, 1.579) (5.687, 8.119) 2nd instar 5.570 13.63 0.999 5.056 5.58 – 2.70 (5.512, 5.627) (13.44, 13.83) (0.769, 1.223) (4.257, 6.246) 3rd instar 4.339 9.834 1.089 4.875 3.98 – 2.02 (4.288, 4.389) (9.691, 9.983) (0.866, 1.305) (4.131, 5.973) (2) Navel orange Eggs 4.340 8.855 0.547 2.965 7.93 – 2.99 (4.303, 4.376) (8.760, 8.954) (0.340, 0.747) (2.408, 3.857) 1st instar 5.146 12.500 1.209 6.094 4.26 – 2.05 (5.099, 5.192) (12.340, 12.660) (0.962, 1.451) (5.171, 7.442) 2nd instar 5.168 12.630 0.852 4.854 6.07 – 2.60 (5.122, 5.214) (12.470, 12.800) (0.622, 1.078) (4.046, 6.074) 3rd instar 4.515 10.350 0.797 4.594 5.66 – 2.25 (4.473, 4.556) (10.230, 10.480) (0.571, 1.019) (3.817, 5.773) (3) Lisbon lemon Eggs 3.774 8.516 0.509 1.528 7.41 – 5.57 (3.726, 3.821) (8.391, 8.648) (0.175, 0.702) (1.249, 2.556) 1st instar 4.525 9.704 1.070 5.045 4.23 – 1.92 (4.476, 4.575) (9.572, 9.841) (0.843, 1.292) (4.269, 6.194) 2nd instar 4.636 9.741 0.532 1.922 8.71 – 5.07 (4.588, 4.684) (9.603, 9.886) (0.299, 0.713) (1.561, 2.703) 3rd instar 3.942 8.954 0.605 2.927 6.52 – 3.06 (3.894, 3.990) (8.824, 9.091) (0.398, 0.798) (2.394, 3.797) (4) Ellendale mandarin Eggs 4.083 9.086 1.202 5.478 3.40 – 1.66 (4.039, 4.127) (8.974, 9.202) (0.969, 1.430) (4.663, 6.665) 1st instar 4.758 11.320 0.970 5.980 4.91 – 1.89 (4.709, 4.807) (11.170, 11.480) (0.720, 1.215) (4.997, 7.458) 2nd instar 5.144 11.710 1.257 6.564 4.09 – 1.78 (5.093, 5.195) (11.560, 11.870) (1.001, 1.508) (5.569, 8.019) 3rd instar 4.249 9.993 1.148 5.950 3.70 – 1.68 (4.202, 4.296) (9.855, 10.140) (0.903, 1.389) (5.035, 7.295) (5) Murcott mandarin Eggs 3.929 8.385 0.684 3.560 5.74 – 2.36 (3.884, 3.973) (8.272, 8.502) (0.472, 0.887) (2.937, 4.531) 1st instar 4.640 10.910 1.023 4.289 4.54 – 2.54 (4.590, 4.689) (10.770, 11.070) (0.812, 1.226) (3.626, 5.286) 2nd instar 4.859 11.700 1.029 4.771 4.72 – 2.45 (4.807, 4.910) (11.540, 11.870) (0.806, 1.245) (4.030, 5.869) 3rd instar 4.161 9.652 1.055 3.880 3.94 – 2.49 (4.114, 4.208) (9.517, 9.792) (0.858, 1.243) (3.288, 4.785) De Lima et al.—Cold disinfestation of citrus 47

Table 4 Comparison of the number of days exposure at 3°C required to kill 50% (LD50) and 95% (LD95) with 95% fiducial limits (FL) of the four immature life stages of Mediterranean fruit fly Ceratitis( capitata) and Queensland fruit fly Bactrocera( tryoni) in five citrus Citrus( spp.) cultivars. The analysis is based on three replicate trials for each life stage.

Mediterranean fruit fly Queensland fruit fly Relative Citrus cultivar potency ratio and life stage LD50 (95% FL) LD95 (95% FL) LD50 (95% FL) LD95 (95% FL) mFF: QFF treated days days days days LD50 – LD95 (1) Valencia orange Eggs 4.369 9.281 0.556 2.593 7.86 – 3.58 (4.323, 4.415) (9.157, 9.410) (0.349, 0.746) (2.109, 3.414) 1st instar 5.789 12.950 0.987 5.665 5.87 – 2.29 (5.736, 5.842) (12.780, 13.130) (0.744, 1.226) (4.749, 7.031) 2nd instar 6.266 14.680 0.718 3.603 8.73 – 4.07 (6.207, 6.325) (14.480, 14.900) (0.507, 0.920) (2.983, 4.569) 3rd instar 4.921 10.950 1.084 5.534 4.54 – 1.98 (4.872, 4.970) (10.800, 11.100) (0.846, 1.316) (4.675, 6.800) (2) Navel orange Eggs 4.349 9.813 0.562 6.683 7.74 – 1.47 (4.313, 4.384) (9.717, 9.913) (0.319, 0.823) (5.333, 8.923) 1st instar 5.889 13.790 1.292 8.796 4.56 – 1.57 (5.847, 5.931) (13.650, 13.930) (0.997, 1.582) (7.350, 10.998) 2nd instar 6.106 13.980 1.126 6.690 5.42 – 2.09 (6.063, 6.149) (13.840, 14.120) (0.865, 1.381) (5.625, 8.278) 3rd instar 4.698 11.040 0.856 5.161 5.49 – 2.14 (4.659, 4.737) (10.920, 11.160) (0.620, 1.088) (4.296, 6.467) (3) Lisbon lemon Eggs 4.022 9.013 0.453 3.092 8.88 – 2.91 (3.980, 4.064) (8.901, 9.129) (0.250, 0.661) (2.473, 4.051) 1st instar 5.029 11.140 0.698 4.238 7.20 – 2.63 (4.981, 5.077) (11.000, 11.290) (0.476, 0.916) (3.493, 5.380) 2nd instar 5.369 12.140 0.473 2.554 11.35 – 4.75 (5.317, 5.421) (11.980, 12.300) (0.267, 0.670) (2.050, 3.381) 3rd instar 4.313 9.984 0.406 2.357 10.62 – 4.24 (4.268, 4.359) (9.854, 10.120) (0.202, 0.605) (1.863, 3.156) (4) Ellendale mandarin Eggs 4.468 9.901 0.827 3.355 5.40 – 2.95 (4.423, 4.513) (9.781, 10.030) (0.627, 1.013) (2.800, 4.243) 1st instar 5.364 13.110 1.088 6.076 4.93 – 2.16 (5.310, 5.427) (12.930, 13.290) (0.839, 1.332) (5.117, 7.495) 2nd instar 5.559 13.440 1.482 7.558 3.75 – 1.78 (5.504, 5.615) (13.250, 13.630) (1.209, 1.749) (6.451, 9.160) 3rd instar 4.606 11.050 0.993 6.417 4.64 – 1.72 (4.554, 4.656) (10.890, 11.210) (0.736, 1.246) (5.355, 8.019) (5) Murcott mandarin Eggs 4.117 8.760 1.469 6.736 2.80 – 1.30 (4.070, 4.164) (8.636, 8.889) (1.213, 1.720) (5.777, 8.108) 1st instar 5.543 12.740 1.675 8.084 3.31 – 1.58 (5.486, 5.600) (12.560, 12.930) (1.393, 1.950) (6.939, 9.719) 2nd instar 5.695 13.290 1.209 5.800 4.71 – 2.29 (5.638, 5.751) (13.100, 13.480) (0.968, 1.444) (4.929, 7.066) 3rd instar 4.472 10.430 0.850 4.668 5.26 – 2.23 (4.419, 4.525) (10.270, 10.600) (0.624, 1.072) (3.893, 5.837) 48 New Zealand Journal of Crop and Horticultural Science, 2007, Vol. 35

Table 5 Large-scale trials showing the total number treated of 2nd instar Mediterranean fruit fly (MFF; Ceratitis capitata) and 1st instar Queensland fruit fly (QFF;Bactrocera tryoni). Comparison of the number of days required to kill >10 000 individuals in three replicate trials in five citrus Citrus( spp.) cultivars at 2°C and 3°C.

Treatment: 2°C 3°C Fruit fly species Days No. of insects No. of Days No. of insects No. of and test fruit treated treated survivors treated treated survivors Mediterranean fruit fly Valencia 18 141 441 0 20 142 584 0 Navel 18 165 894 0 20 152 868 0 Lisbon 16 132 216 0 18 122 400 0 Ellendale 18 133 788 0 20 137 742 0 Murcott 18 108 732 0 20 105 678 0 Queensland fruit fly Valencia 16 74 088 0 16 124 961 0 Navel 16 111 636 0 16 251 231 0 Lisbon 14 45 878 0 14 41 754 0 Ellendale 16 466 071 0 16 350 649 0 Murcott 16 93 538 0 16 282 296 0 inadequate for treatment of Spanish Clementines days at all temperatures below 3°C and it is proposed (APHIS 2002). that APHIS treatment schedules for the QFF in citrus The QFF, Bactrocera tryoni, a species more be modified to accept treatment for 16 days for suited to tropical and subtropical conditions, has treatment temperatures below 3°C to benefit trade. been consistently shown to be more susceptible to cold treatment than the MFF which has been more adapted to cold climates. Data presented over ACKNOWLEDGMENTS the past 15 years (Hill et al. 1988; Jessup et al. The technical assistance of Ms H. M. Collie, Ms E. 1993; Heather et al. 1996; De Lima et al. 2002) have Hutchinson, Ms M. Taylor, Mr C. Brown, Mr R. Flores- consistently shown that all stages of the QFF are Vargas, Ms Z. Vogl, Ms N. Start, Mr D. Haldane, Mr killed in 12 days at 1°C. In the data reported in this G. Rogers, Mr D. J. Cruickshank, and Mr M. Coates paper there is no decrease in mortality with increase in the disinfestation trials is gratefully acknowledged. in temperature from 1°C to 2°C and 3°C. The greater Statistical advice was provided by Ms L. Spohr and Ms J. Speijers. The work was supported by the Citrus susceptibility of other Bactrocera species to cold (as Marketing Development Group; Horticulture Australia compared to MFF) has also been demonstrated by Limited; New South Wales Department of Agriculture; other researchers. For example Burditt & Balock Department of Agriculture, Western Australia; and the (1985) showed that all stages of the Oriental fruit Commonwealth Department of Agriculture Fisheries and fly Bactrocera dorsalis (Hendel) were killed in 12 Forestry Australia. days at 2.7°C and similarly all stages of Melon fruit fly Bactrocera cucurbitae (Coquillett) were killed in 10 days at 2.7°C. The Bactrocera species REFERENCES clearly are more susceptible to cold than the MFF Anderson DT 1963. The larval development of Dacus and the case is made for a shorter cold treatment tryoni (Frogg.) (Diptera: Tephritidae). I. Larval period for the QFF B. tryoni. In a review of the instars, imaginal discs, and haemocytes. Australian United States and Plant Health Inspection Journal of Zoology 11: 202–218. Service, Quarantine Treatment Manual of 1992, APHIS 2004. United States Animal and Plant Health Gould (1994) referred to the finding of Meats (1976) Inspection Service, Quarantine Treatment Manual. that QFF acclimates to cold temperatures. Current Risk mitigation for Mediterranean fruit flies with USDA-APHIS treatment schedules (APHIS 2006) special emphasis on risk reduction for commercial for QFF are as follows—<0°C: 13 days; 0.56°C: 14 imports of clementines (several species of Citrus days; 1.11°C: 18 days; 1.67°C: 20 days; 2.22°C: 22 reticulata) from Spain. Centre for Plant Health Science and Technology, Raleigh, NC. USDA- days. However, the data presented here demonstrate APHIS web site http//www.aphis.usda.Gov/oa/ that QFF are killed by continuous exposure for 16 clementine [accessed October 2004]. De Lima et al.—Cold disinfestation of citrus 49

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