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Postharvest Biology and Technology ELSEVIER Postharvest Biology and Technology 8 (1996) 307-315 Cold disinfestation of Australian mandarins against Queensland fruit fly (Diptera: Tephritidae) Neil W. Heather a.*, Leonie Whitfort a, Richard L. McLauchlan b, Rosemary Kopittke a a Depanment of Prhary Industries, 80 Meiers Rd. Indooroopilly, Queensland 4068. Australia b Department of Primary Industries, 19 Hercules St, Hamilton, Queensland 4007, Australia Accepted 12 April 1996 Abstract Three cultivars of mandarins, ‘Imperial’, ‘Ellendale’ and ‘Murcott’ and one tangelo, ‘Min- neola’, were shown to be disinfested of Queensland fruit fly, Bactrocera tryoni (Froggatt) by cold treatment at 1°C for 16 days. From a total of 3885 treated fruit of all cultivars, estimated to contain 176,715 first instars - judged to be the most tolerant in-fruit stage of the pest - there were no survivors. This treatment efficacy meets all known international market requirements. Keywords: Mandarin; Bactrocera tryoni; Cold disinfestation; Quarantine treatment 1. Introduction Queensland fruit fly, Bactrocera tryoni (Froggatt) is indigenous to coastal eastern Australia (May, 1963) and consequently, host produce may require postharvest disinfes- tation treatment before it is acceptable to many markets. Consumer preference dictates that, wherever possible, a treatment should not leave chemical residues. Oranges are currently disinfested before export by cold storage at 1 f O.S’C for 16 days (Hill et al., 1988). Although this treatment is effective, it needed to be shown to be equally effective on Australian mandarins before those fruits could be considered for approval by some importing countries. Internationally, efficacies required of quarantine treatments differ according to the market. The USA is generally recognised as having the most stringent requirement against fruit fly, at 99.9968%; Japan requires 99.99%. Here we report the results of disinfestation trials on three cultivars of mandarins and one tangelo infested with B. tryoni, using cold treatment as the disinfestation method, * Corresponding author. Present address: Plant Protection Section, University of Queensland Gatton College, Lawes, Qld. 4343, Australia. 0925.5214/96/$1.5.00 Copyright 0 1996 Elsexier Science B.V. All rights reserved. PII SO925-5214(96)00022-l

Cold disinfestation of Australian mandarins against Queensland fruit fly (Diptera: Tephritidae)

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Page 1: Cold disinfestation of Australian mandarins against Queensland fruit fly (Diptera: Tephritidae)

Postharvest Biology and Technology

ELSEVIER Postharvest Biology and Technology 8 (1996) 307-315

Cold disinfestation of Australian mandarins against Queensland fruit fly (Diptera: Tephritidae)

Neil W. Heather a.*, Leonie Whitfort a, Richard L. McLauchlan b,

Rosemary Kopittke a

a Depanment of Prhary Industries, 80 Meiers Rd. Indooroopilly, Queensland 4068. Australia b Department of Primary Industries, 19 Hercules St, Hamilton, Queensland 4007, Australia

Accepted 12 April 1996

Abstract

Three cultivars of mandarins, ‘Imperial’, ‘Ellendale’ and ‘Murcott’ and one tangelo, ‘Min- neola’, were shown to be disinfested of Queensland fruit fly, Bactrocera tryoni (Froggatt) by cold treatment at 1°C for 16 days. From a total of 3885 treated fruit of all cultivars, estimated to contain 176,715 first instars - judged to be the most tolerant in-fruit stage of the pest - there were no survivors. This treatment efficacy meets all known international market requirements.

Keywords: Mandarin; Bactrocera tryoni; Cold disinfestation; Quarantine treatment

1. Introduction

Queensland fruit fly, Bactrocera tryoni (Froggatt) is indigenous to coastal eastern Australia (May, 1963) and consequently, host produce may require postharvest disinfes- tation treatment before it is acceptable to many markets. Consumer preference dictates that, wherever possible, a treatment should not leave chemical residues. Oranges are currently disinfested before export by cold storage at 1 f O.S’C for 16 days (Hill et al., 1988). Although this treatment is effective, it needed to be shown to be equally effective on Australian mandarins before those fruits could be considered for approval by some importing countries. Internationally, efficacies required of quarantine treatments differ according to the market. The USA is generally recognised as having the most stringent requirement against fruit fly, at 99.9968%; Japan requires 99.99%.

Here we report the results of disinfestation trials on three cultivars of mandarins and one tangelo infested with B. tryoni, using cold treatment as the disinfestation method,

* Corresponding author. Present address: Plant Protection Section, University of Queensland Gatton College, Lawes, Qld. 4343, Australia.

0925.5214/96/$1.5.00 Copyright 0 1996 Elsexier Science B.V. All rights reserved. PII SO925-5214(96)00022-l

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308 N.W Heather et al. /Postharvest Biology and Technology 8 (1996) 307-315

primarily for the Japanese market. Trials on the effects of the same treatment on fruit quality were done concurrently and have been reported separately (McLauchlan et al., 1993). Treated fruit were mostly injury free, although further research could be required on handling ‘Imperial’ fruit which occasionally exhibited minor skin blemish.

2. Materials and methods

The efficacy required by Japanese Plant Quarantine authorities needed to be demon- strated, this being no survivors from 3 trials each on >lO,OOO insects treated at the in-fruit stage most tolerant of the treatment (Jessup et al., 1993).

Test insects All testing was done using fruit flies from a colony of B. tryoni maintained at the

laboratories of the Department of Primary Industries, Indooroopilly, Queensland. The colony has been maintained continuously since 1971 with numerous additions of flies from field-infested fruit to sustain its genetic diversity. Adult flies were held in framed cages, 650 x 650 x 650 mm, in cohorts of approximately 25,000. Cage populations were only used for infestation between 3 and 7 weeks after eclosion, when fecundity was highest. The rearing method has been described in detail (Heather and Corcoran, 1985). All culturing was done at 26 f 1°C and incubation rooms for immature stages in fruit had humidity controlled at 75-80% RH.

Test fruit Testing was replicated over 4 cultivars viz. ‘Imperial’, ‘Ellendale’ and ‘Murcott’

mandarins and ‘Minneola’ tangelos. Their taxonomic status varies according to the variety (Bailey, 1976). ‘Imperial’ is a true mandarin or tangerine being a selection of Citrus reticuhtu, ‘Ellendale’ and ‘Murcott’ are tangors from mandarin and sweet orange crosses (C. reticulatu x Citrus sinensis), and ‘Minneola’ a tangelo is from a grapefruit and mandarin cross (Citrus purudisi x C. reticulutu).

Fruit used was from insecticide-residue-free commercial production, having been grown under integrated pest management programmes which minimised pesticide usage. This was to ensure that survival rates for eggs and larvae in untreated fruit were as consistent as possible. Fruit were pinholed before being placed in adult fly cages to optimise egg numbers by facilitating oviposition. After infestation, fruit were placed into incubation rooms at 26°C and 70% RI-l until the insects had developed to the stage to be tested.

Determination of life stages A destructive sampling method was used to determine development times of the fruit

fly immature stages in each cultivar at 26°C. Bulk lots of fruit were infested and samples of 2-4 fruit were removed from the incubation room at intervals until pupation had ceased. After removal from the incubation room each sample was quickly frozen at 18°C to kill the stages present. These fruit were subsequently thawed and dissected. For each time interval the numbers of each stage were recorded, based on larval characters (Anderson, 1963; Elson-Harris, 1988).

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Determination of the stage most tolerant of cold treatment Testing to determine which stage was most tolerant to cold was done at 1°C with time

as the variable since this is the disinfestation temperature in use for Australian oranges and it would be advantageous if mandarins could be treated jointly. Fruit for each stage to be tested were infested at differing times relative to development so that treatment of all stages could be started at the same time. When these stages were reached, they were checked by dissecting a small additional sample and infested fruit were then placed in the cold-room for treatment at 1 f 0.5”C. Samples of approximately 20 fruit were removed at 1, 2, 4, 6, 8 and 12 days and held over sand in incubation rooms at 26°C and 70% RH until all larvae had left. The sand was sieved three or more times over a 4-week period. All pupae were collected and the numbers compared with an untreated sample as the control. For each cultivar the resulting survivors from each life stage were recorded, enabling the LTsc (time of exposure in days that is lethal to 50% of insects) to be computed for each life stage, with the untreated fruit as a parallel sample from which numbers initially present in the treated fruit were estimated.

Large scale disinfestation tests The procedure used to naturally infest fruit to find the most tolerant stage was

also used for the large scale disinfestation tests on each fruit cultivar. Infested fruit were kept at 26°C in an incubation room for 3 days until eggs had developed to first instars (determined to be the most tolerant stage) before being subjected to cold treatment. Three replicated tests on a minimum of 10,000 individuals were done for each mandarin cultivar. The numbers of fruit flies developing in tangelo were variable and two replicates exceeded 10,000 but two others did not. However the total greatly exceeded 30,000.

Infested fruit were treated in cartons. After cold treatment was completed, fruit was held over sand at 26°C and 70% RI-I for pupation of any survivors. The sand was sieved twice or more for pupae, the last at 4 weeks after return to 26°C when fruit were examined to ensure no larvae remained. Control fruit which had been infested but not cold-treated were then held in separate incubation rooms from those containing the cold-treated fruit but at the same temperature and relative humidity. Pupation sand for the infested control fruit was sieved twice or more with the last 4 weeks after transfer to 26°C when fruit were checked to ensure that no larvae remained. Pupae were collected, counted and held in slightly moist sawdust for eclosion of adults.

The numbers of pupae from control fruit were used to estimate the numbers of larvae present in treated fruit at the same time of treatment as for the experiments to find the most tolerant stage. A minimum of one fruit in every six was segregated in an unbiased way as a control, so the number of insects estimated to be present in treated fruit was 5 (or fewer) times the number which emerged from control fruit. This method gave an estimated total after natural mortality.

Temperature recording Fruit core temperature was recorded every -15 min from sensors placed within fruit,

at 4 or more positions in the cold room. Air temperature in the cold rooms was recorded at the same time. All recording was done using electronic dataloggers with either

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thermocouple or resistance temperature detectors. Data were subsequently downloaded weekly and checked for inconsistencies.

Statistical analyses Insect survival data were analyzed using the Wadley’s Problem version of probit

analysis (Finney, 1971). Wadley’s method is appropriate for analyses where the number treated is not known but where information on the number treated is provided from a parallel sample of untreated fruit. The data for time of treatment were not log- transformed as this gave a better fit to the regression line. The Wadley’s Problem approach was judged to be superior to dissecting fruit and counting live and dead of the stage treated because it avoided operator error and took account of natural mortality over the whole immature life of the insects.

3. Results

Development times for insects in fruit Development times for stages of B. tryoni in mandarins and tangelos were consistent

across the fruit varieties when allowance was made for minor experimental variance (Table 1). Later stages developed more slowly in fruit than reported in carrot medium (Heather and Corcoran, 1985). Eggs hatched at almost the same time, but third instars were more than 2 days slower. Development times at treatment for each stage were selected for maximum numbers and to minimise overlap (Table 1). Third instars in ‘Ellendale’ mandarins were treated 0.5 days earlier because earliest pupation was recorded in this cultivar at 8 days.

Determination of the life stages most tolerant of cold treatment Probit analyses for these data are shown in Table 2. There was a general trend for

first instars to be the most tolerant stage, with all of the LTso values greater than those of any other stage, but there was overlapping of the 95% fiducial limits in each instance. In the cultivars ‘Imperial’ and ‘Minneola’, the LTso for eggs was lower than for larvae and the 95%-fiducial limits did not overlap. On this basis, the first instar was selected as the stage for large scale testing. No whole line comparisons were done because tests of parallelism proved negative (Fimrey, 1971).

Extrapolation of the data to probit 9 mortality was used to predict the treatment time to achieve the minimum efficacy required i.e. no survivors from 30,000 treated insects.

Table 1 Age from oviposition (days) at treatment of each immature stage of B. tryoni in mandarins and tangelos

Stage Age of stage from oviposition (days)

‘Imperial’ ‘Ellendale’ ‘Murcott’ ‘Minneola’

Egg 1.2 1.2 1.2 1.2 First instar 2.8-2.9 2.9 3.0 3.0 Second instar 5.8-5.9 5.8 5.8 5.9 Third instar 8.0 7.2 7.8 7.8-7.9

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Table 2 LT50 and LTgg.ggas (Probit 9) values (days) with 95%-fiducial limits from untransformed probit regression analyses (Wadley’s version) of immature stages of B. tryoni in naturally infested mandarins and tangelos exposed to a constant temperature of 1 f 0.5”C

Cultivar Stage LTso (95%-Fiducial Limits)

LT9939es

(95%-Fiducial Limits)

Imperial Egg

1st instar

2nd instar

3rd instar

Ellendale Egg

1 st instar

2nd instar

3rd instar

Murcott Egg

1st instar

2nd instar

3rd instar

Minneola Egg

1st instar

2nd instar

3rd instar

0.994 5.6 (O-l ,938) (3.72-52.98) 4.752 10.97

(2.909-5.585) (9.24-16.03) 1.763 11.45

(O-3.085) (9.17-16.94) 2.216 12.28

(O-4.153) (9.08-27.66)

0.247 (O-1.625) 4.167

(1.444-5.289) 2.364

(o-3.640) 2.190

(0.655-3.200)

8.59 (6.03-19.84) 12.94

(10.73-19.21) 12.53

(10.48-16.82) 13.13

(11.47-15.85)

2.486 (O-3.278) 3.735

(2.903-4.285) 2.312

(0.712-3.369) 2.582

(0.854-3.631)

6.01 (4.80-13.73) 10.85 (9.76-12.63) 14.04

(12.28-16.90) 12.71

(11.00-15.79)

0.008 (O-l ,407) 2.257

(O-4.869) 1.225

(o-2.030) 0.848

(O-l .902)

10.74 (8.24-17.07) 12.46 (8.72-85.39) 9.16 (7.72-l 1.79) 10.64 (8.78-14.19)

Probit 9 should over-estimate the Japanese requirement because to demonstrate probit 9 efficacy at P = 0.05 requires no survivors from 100,000 treated insects (Couey and Chew, 1986). Probit 9 predictions for first instars as the most tolerant stage required treatment times ranging from 10.97 to 12.94 days for B. tryoni, but as anticipated, the fiducial limits were large. The time of 16 days was used for large scale trials because it met the minimum efficacy, and because it is the approved treatment for Australian oranges for export to Japan based on the research of-Hill et al. (1988) for Ceratitis cupitutu.

This duration is acceptable because fruit damage studies showed that mandarins (except possibly ‘Imperial’) and tangelos would tolerate 16 days at 1°C (McLauchlan et al., 1993).

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312 N,W? Heather et al./Postharvest Biology and Technology 8 (1996) 307-??

Table 3 Large scale disinfestation trials: mortality of first instar B. tryoni in mandarins and tangelos cold-treated for 16 days at 1 f 0.W

Cultivar Replicate No. fruit Estimated no. insects treated

No. survivors

Imperial 1 220 24,010 2 220 25,478 3 209 17,251 Total 649 66,739

Ellendale 1 193 11,235 2 259 15,170 3 291 28,887 Total 749 55,292

Murcott 1 247 19,844 2 266 19,212 3 283 16,158 Total 796 55,214

Minneola 1 479 4,326 2 392 22,242 3 416 4,916 4 407 16,986 Total 1691 49,470

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0 0

Large scale disinfestation tests There were no survivors from 66,739 treated insects in ‘Imperial’ mandarins, 55,292

in’ Ellendale’ mandarins, 55,214 in ‘Murcott’ mandarins, or 49,470 ‘Minneola’ tangelos (Table 3). The large scale disinfestation tests thus confirmed the prediction that complete disinfestation of B. tryoni would occur by 16 days at 1°C. If the numbers for each variety are aggregated, the treatment could be judged to exceed probit 9 in efficacy (Couey and Chew, 1986).

4. Discussion

Published results of cold disinfestation studies on B. tryoni and other Tephritidae show no consistent patterns with respect to time-temperature efficacy for the life stages most tolerant of cold. Times for probit 9 (99.9968% mortality) ranged from a predicted 30 days at 1.7“C for Anastrepha suspensa (Loew) preconditioned at 10°C for 7 days (Benschoter, 1984), to 10 days at 2.8”C for Bactrocera dorsalis (Hendel) (Burditt and Balock, 1985). These were extremely wide differences for a fundamental physiological process in species of the same family and sometimes, the same genus (Hill et al., 1988 and Jessup et al., 1993 for B. tryoni).

In trials against both B. tryoni and C. capitata, Hill et al. (1988) concluded that larvae of B. tryoni were generally less cold-tolerant than those of C. capitata. However, the validity of this comparison is uncertain given substantial differences in experimental location and procedures between experiments for the two species. Jessup et al. (1993)

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predicted probit 9 mortality after 8.5 or 8.7 days at 1°C for first instars of B. tryoni

in two cultivars of lemons and 9.74 or 10.92 days for C. capituta, although in this species, second instars were the more tolerant at 11 .Ol and 12.33 days. Again, there were substantial differences in location and procedures between experiments on the two species including the method of infestation and almost certainly, differing cool-down times between the cold rooms which would have affected the total exposure to coId. The stages with greatest cold tolerance were always larvae although there was no consistency as to instar.

Hill et al. (1988) and Jessup et al. (1993) nominated different most tolerant stages. However given that infested fruit were exposed to cold in increments of 1 day, and that cool-down times probably differed, probit analyses would have over-estimated the precision of the LT values. Hill et al. (1988) appear to have identified their most tolerant stage by inspection and did not differentiate instars other than to observe that their ‘young larvae’ category contained mainly first and second instars and their ‘old larvae’ category, mainly third instars. At LT99.9a6s the data of Jessup et al. (1993) showed overlapping 95%-fiducial limits for all larval stages. Also, their data show symmetrical upper and lower fiducial limits. If the appropriate methods of Finney (1971) were used we would expect the limits to be asymmetrical, and probably wider.

Whilst comparisons at a specific LT would be valid for tests in the same laboratory, comparisons involving different laboratories, as in the comparisons between B. tryoni and C. cupitutu, are likely to be less reliable. Ideally, comparisons should be based on the whole response line but this is only valid if the lines are parallel (Finney, 1971). This might be expected on biological premises but the reality is frequently otherwise because of experimental variation. LT5e values enable a more robust comparison between stages or species than LTg~.~~hs values, where numbers responding are very few and fiducial limits are typically very wide. LT values at the lower end of the response line would have been proportionately more influenced by cool-down times, although there does not appear to be an obvious solution to this experimental problem. Across all of the previous studies in Australia, eggs were never more tolerant than larvae and in our trials, again, no instars were less tolerant than eggs. This could be due largely to the physical location of the eggs in fruit rather than for any major physiological reason. Where eggs were injected into fruit as in Mediterranean fruit fly in lemons (Jessup et al., 1993) the values were much closer to those for first instars.

We found that for B. tryoni, first instars were never numerically less tolerant than other stages but that their tolerance did not differ significantly from other instars. We infested fruit naturally (apart from pinholing), which gave uneven numbers and hence data more heterogeneous than the artificial infestation method used for Mediterranean fruit fly by Hill et al. (1988) and Jessup et al. (1993), which would have introduced approximately equal numbers of eggs to each fruit. However, the location of eggs and larvae in naturally infested fruit at treatment gives results which are likely to be of greater operational relevance. We also considered the method of Preisler and Robertson (1992) and others for use where numbers of test insects are unknown, but found the Wadley’s method to be superior. The highly important probability of cold acclimation of insects, identified by Benschoter (1984) for Anastrepha, remains to be investigated for B. tryoni.

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Regulatory authorities in USA (USDA, 1985) require treatment at l.ll”C for 12 days against C. capitata, for 20 days against Anastrepha ludens (Loew) and for 18 days against B. tryoni, which is not consistent with the observations of Hill et al. (1988) and Jessup et al. (1993). The importance of these differences lies with the Japanese requirement for the treatment to be based on the species which is most tolerant of the treatment. Concurrent trials in which the treatment was equally effective against C. capitata were done by Dr. F. de Lima, Department of Agriculture Western Australia (Biggs, 1993), but these are unlikely to resolve the issue of whether the species really differ, due to the closeness of response to cold of each and the confounding effects of differing experimental locations necessitated by Australian interstate quarantine requirements. The operational significance of any true differences between the species is likely to be negligible.

Our treatment for mandarins showed that 16 days at 1°C would achieve probit 9 efficacy, but it is highly probable that this could also be achieved at a time of 14 days or at a higher temperature for 16 days.

Acknowledgments

The technical assistance of Peter Leach, Elizabeth Pike and Aaron Nimmo is acknowledged. This research was supported by the Queensland Department of Primary Industries, the Horticultural Research and Development Corporation of Australia, grower groups and the Australian Joint Citrus Exporters.

References

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Bailey, L.H., 1976. Hortus Third - A concise dictionary of plants cultivated in the United States and Canada. Hortorium USA.

Benschoter, C.A., 1984. Low-temperature storage as a quarantine treatment for the Caribbean fruit fly (Diptera: Tephritidae) in Florida citrus. J. Econ. Entomol., 77: 1233-1235.

Biggs, T., 1993. Horticultural Research and Development Corporation Research Report for 1992/93. Rural Press, Melbourne, p. 23.

Burditt Jr, A.K. and Balock, J.W., 1985. Refrigeration as a quarantine treatment for fruits and vegetables infested with eggs and larvae of Dacus dorsalis and Dams cucurbitae (Diptera: Tephritidae). J. Econ. Entomol., 78: 885-887.

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

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