Journal of Economic Entomology, 110(3), 2017, 954–960 doi: 10.1093/jee/tox068 Advance Access Publication Date: 21 April 2017 Research article Commodity Treatment and Quarantine Entomology Cold Disinfestation of “Hass” Avocado (Persia americana) of Three Species of Fruit Fly (Diptera: Tephritidae)— Ceratitis capitata, Ceratitis rosa, and Ceratitis cosyra A. B. Ware1,2,3 and C.L.N. du Toit1,2 Subject Editor: Lisa Neven Received 7 October 2016; Editorial decision 3 February 2017 Abstract The avocado industry is important in South Africa, but access to certain markets is impeded by the presence of phytosanitary pests. One of the ways of securing entry to these markets is to demonstrate that a mitigating treatment will result in there being a negligible chance of accidental importation. In cold treatment comparative studies at 0 C and 2 C of immature stages of Ceratitis capitata (Wiedemann), Ceratitis rosa Karsch, and Ceratitis cosyra (Walker) in “Hass” avocado, the third instar of C. cosyra was shown to be the most cold tolerant. This larval life stage was used in a large-scale trial to test treatment efficacy at 2 C, a temperature known to be the better for fruit quality. There were no survivors from the 49,795 individual fruit fly larvae subjected to the cold treatment at 2 C for 20 d. It is argued that, although this level of assessment falls short of the Probit 9 level normally required for fruit fly, they are rarely found in avocado fruit and that the level of disinfestation obtained is more than sufficient to achieve quarantine security. Key words: Mediterranean fruit fly, Ceratitis rosa, Ceratitis cosyra, disinfestation The South African avocado industry is located in Mpumalanga, Limpopo, and KwaZulu-Natal Provinces in the east of the country, with a small number of growers in the Eastern Cape. There are some 340 commercial growers on 15,500 ha producing 115,000 tons per annum and employing a labor force of 1,500 (information provided by the South African Avocado Growers’ Association). Approximately 45% of the crop is exported to Europe, but entry to many other markets is prohibited due to phytosanitary issues. International trade in plant products increases the risk of introduction of new pests and diseases. Consequently, government policies are developed to manage this risk. These policies normally dictate specific conditions for import of plant material that could harbor pests and diseases, which could become established in specific "pest-free" areas. The conditions imposed on product entry may range from the out-right ban to the imposition of pre- and postharvest mitigating measures. Examples of preharvest actions may include crop spraying, field monitoring, and inspections while postharvest activities may include one or more of the following: postharvest inspections, fumigation, irradiation, heat treatments, and cold treatments. These are all designed to negate the probability of accidental introduction (Heather and Hallman 2008). Not all pre- or postharvest mitigation treatments are suitable for all commodities. In the case of avocados (Persia americana Miller: Lauraceae) irradiation of 20 Kilorad often leaves brown lesions (Kamali et al. 1972) and the application of cold temperatures above 10 C may affect shelf life (Zauberman et al. 1977). However, a temperature of 2.0 C appears to be the treatment of choice in sterilizing the fruit of insect pests without undue damage to fruit quality or reducing the period it can be stored (Kok et al. 2010). In the United States, a 14-d treatment at 1.1 C or an 18-d treatment at 2.2 C of avocado is an acceptable mitigation intervention for the Mediterranean fruit fly [Ceratitis capitata (Wiedemann)] and Ceratitis rosa Karsch (U.S. Department of Agriculture–Animal and Plant Health Inspection Service [USDA-APHIS] treatment manual 2016). Unfortunately there are two other fruit fly species—Ceratitis cosyra (Walker) and oriental fruit fly [Bactrocera dorsalis (Hendel) ¼ Bactrocera invadens Drew, Tsuruta and White (Schutze et al. 2015)]—that could potentially oviposit in avocado fruit in South Africa (Brink et al 1997, Grové 2001, International Plant Protection Convention [IPPC] 2013) that are not included in the USDA-APHIS treatment manual. Research done in Kenya indicates that a cold treatment of 20 d at 2.0 C is an effective postharvest mitigation treatment for B. dorsalis (Ware et al. 2012). This article describes a cold tolerance comparative study of the three agriculturally important fruit fly species (C. capitata, C. rosa, and C. cosyra) indigenous to South Africa. C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. V All rights reserved. For Permissions, please email: journals.permissions@oup.com 954 Downloaded from https://academic.oup.com/jee/article/110/3/954/3746969 by guest on 13 June 2022 1 Department of Agriculture, University of Mpumalanga, Private Bag X 11283, Mbombela, South Africa (tonyware@mweb.co.za; Neethlingdt@gmail.com), 2Agri-Biotech Research Consultancies (ABRC), P.O. Box 7512, Mbombela, South Africa, and 3 Corresponding author, e-mail: tonyware@mweb.co.za Journal of Economic Entomology, 2017, Vol. 110, No. 3 955 Materials and Methods Test Insects and Test Fruit Fruit fly eggs were obtained from colonies kept at Citrus Research International (Pty) Ltd in Mbombela, Mpumalanga Province, South Africa. The flies were maintained in gauze cages. Ceratitis capitata and C. cosyra were kept in a constant temperature room (26.0 C) while C. rosa, because it is corpuscular insect, was kept in natural light at ambient temperature under a cover that had gauze sides. Ceratitis capitata oviposited through the gauze cages and the eggs were collected from a basin of water positioned under the cage. Ceratitis cosyra laid their eggs through the perforated lids of honey jars and the eggs were washed out using tap water. Ceratitis rosa oviposited into Granny Smith apples and that collection of the eggs required cutting the fruit and the washing out of the eggs with water. The collected eggs were placed onto agar-based larval food media where they developed through the larval stages. Immediately before pupation the larvae “jumped” from the media onto sand where they pupated. The colonies were supplemented annually with “wild” flies to maintain genetic variability. Hass avocados were sourced from H.L. Hall and Sons (Pty) Ltd near Mbombela and stored at 4.0 C until required. The day before infestation the required number of fruit was removed from cold storage and dipped in a quaternary ammonium compound fungicide (didecyl dimethyl ammonium chloride; Sporekill– Hygrotec (Pty) Ltd South Africa, Pretoria, South Africa) for 5 min and then allowed to air dry at ambient temperature. Previous research (Ware et al. 2012) has shown that maturity of the fruit is a factor in supporting the development of eggs and larvae. The softness (density) of the fruit was tested with a densimeter (Bareiss Analog Fruit Tester Type Shore A: Bareiss, Oberdischingen, Germany) and the fruit were infested once a Shore (unit of measurement equal to 0.0025 mm penetration) value of 70 or less was obtained. Cold Rooms The cold rooms, with dimensions 3.0 by 2.2 by 2.5 m (l by b by h), were constructed out of polystyrene sandwiched between aluminum sheets placed on a concrete floor. The chiller or heating coil units were placed near the ceiling. Defrosting (fans off) was done every six hours for 10 min. The temperature was controlled using Danfoss control units (Danfoss (Pty) Ltd, Rivonia, South Africa). A Squirrel meter and logger (Grant model 4F126-2040, Monitoring and control Laboratories (Pty) Ltd, Johannesburg, South Africa) connected to 16 two-wired type T thermocouples (Temperature Controls Pty Ltd, Randburg, South Africa) were used to measure fruit-core temperature. Before each treatment, the thermoprobes were calibrated by placing them on melting ice and on their reaching equilibrium recording the temperature. This was repeated 10 times, and the average calculated was used to adjust for individual thermoprobe variation. Cold room temperatures were recorded hourly. Two temperatures (0 C and 2 C) were investigated. General Procedure The inoculation procedure involved using an electric drill to make a single 6-mm-diameter hole to a depth through the skin and flesh to the pip of each avocado. Fruit fly eggs, collected within 12 h of oviposition, were placed in distilled water and diluted until there were 30–40 eggs per aliquot. This was then delivered into the hole which was then sealed with a plug of absorbent cotton wool. The fruit was then randomly placed into plastic boxes (0.48 by 0.28 by 0.30 m) together with an uninoculated fruit containing a thermoprobe. Most Cold Tolerant Life Stage Each of the three species investigated was processed simultaneously as shown in Fig. 1. The experiment was replicated three times at 2 C for 20 d for each species. Downloaded from https://academic.oup.com/jee/article/110/3/954/3746969 by guest on 13 June 2022 Fig. 1. Schematic representation of procedure followed to establish most tolerant life stage of fruit fly. 956 Journal of Economic Entomology, 2017, Vol. 110, No. 3 Table 1. Survival and mortality of three replicates of eggs, 3-d-old larvae, and 6-d-old larvae of C. capitata in “Hass” avocado when subjected to various periods of cold treatment of 0.0 C and 2.0 C C Exposure (d) 0 Three-day-old larvae (first and second instar) No. of fruit No. surviving Mortalitya (%) No. of fruit No. surviving 179 155 164 164 165 165 165 173 151 165 162 165 165 165 500.3 233.3 197.3 24.7 0.3 0 0 495.7 223.0 127.3 38.3 4.7 0 0 – 53.4 60.6 95.1 99.9 100 100 – 55.0 74.3 92.3 99.1 100 100 182 164 164 165 164 164 165 175 149 165 164 160 165 163 162 756.3 369.7 65.3 6.3 0.3 0 0 648.0 406.3 176.0 51.7 4.0 0.3 0 0 Mortalitya (%) – 51.3 91.4 99.2 99.7 100 100 – 37.3 72.4 92.0 99.4 99.6 100 100 Six-day-old larvae (third instar) No. of fruit No. surviving 180 165 165 164 165 164 165 174 161 165 161 164 165 165 165 165 392.0 364.3 180.7 12.7 0.7 0 0 361.7 330.3 174.0 34.3 10.3 3.0 0.3 0 0 Mortalitya (%) – 7.1 53.9 96.8 99.8 100 100 – 8.9 51.9 90.5 97.2 99.2 99.9 100 100 Average number of eggs inoculated (6 SD): Eggs 0.0 C—31.8 6 3.22; 2.0 C—35.1 6 3.7; 3-d-old larvae 0.0 C—36.8 6 4.4; 2.0 C—37.2 6 4.0; 6-d-old larvae 0.0 C—37.8 6 4.1; 2.0 C—38.4 6 4.0. a Corrected for natural mortality using Abbott’s formula (1925). Large-Scale Disinfestation Trials Based on the results of the above research that was designed to determine which life stage of the three species tested was the most cold tolerant, eggs were inoculated as described above into 2,500 fruit and allowed to develop into 6-d-old C. cosyra larvae. Approximately 80% of the fruit were placed into 2.0 C cold and the balance was used as the control. The cold chamber temperature was lowered over three days (an attempt to mimic commercial situation). Once 50% or more of the thermoprobes registered the target temperature the treatment was deemed to have begun. The first replicate was undertaken over 18 d and another three replicates were treated over 20 d. Statistical Analysis Statistical analysis was undertaken using regression analysis (Probit; Statgraphics Plus Version 5.1, Statpoint Technologies Inc., Warrington, VA) after Abbott’s (1925) transformation of the data to correct for natural mortality. Results Most Cold Tolerant Life Stage Data, after corrected for natural mortality using Abbott’s formula (1925), from the three life stages of the three fruit fly species tested at two different temperatures (Tables 1–3) were subjected to regression analysis (Table 4). Based on the lethal time taken for 99.9% (95% CL) of the insects to perish, it was determined that 6-d-old (third instar) C. cosyra larvae were the most cold tolerant at both the temperatures tested (10.6 d at 0.0 C and 13.6 d at 2.0 C). Large-Scale Disinfestation Trials There were two larvae that survived the 18-d treatment at 2.0 C in an estimated 35,490 individuals tested (Table 5). There were no survivors from an estimated 95,864 treated individuals for 20 d at the same temperature (Table 5; estimations based on the number of live insects per fruit in the control). The hourly cold room temperatures are shown in Fig. 2. Discussion Persea americana (cv. Hass) has a number of attributes that makes it resistant to colonization by fruit fly. The fruit is climacteric (does not ripen on the tree; De Villiers 2001). Preharvest fruit is hard and fruit flies find it an unsuitable host, but once harvested, it will soften and ripen after a few days. However, with the commercial export of “Hass” avocados from South Africa, the fruit is cooled to around 5 C and is shipped under controlled atmosphere and exported in a hard state. On removal from cold storage, the fruit physiology changes and it becomes soft and a suitable host (Ware et al. 2012). Part of the physiological change is that the hard green exocarp turns a black color on ripening (Newett et al. 2002). Colonization is dependent on the fruit flies ability to pierce the thick exocarp (Oi and Mau 1989, De Graaf 2009). Even once overcoming this physical barrier, tissue regeneration and the formation of calluses restricts survival (Kay and Schroeder 1963, Smith 1973, Ware et al. 2016) as does antibiosis (Mwatawala et al 2006). Avocado is not considered to be a host for C. capitata in field and laboratory studies (Du Toit and Tuffin 1980, De Lima 1995, Brink et al. 1997, Willink and Villagran 2007, De Graaf 2009). They are seldom trapped in avocado orchards (Grové et al. 1998), indicating a low pest pressure. It is suggested that C. capitata short aculei (Jones 1989) is unable to penetrate the exocarp (De Graaf 2009). De Graaf (2009) concluded that C. rosa and C. cosyra are able to develop in avocado, but development was restricted by fruit defenses. The latter species was shown to be the more cold tolerant, so the findings of previous research on the effect of cold treatment on C. rosa could not be used to determine its phytosanitary security. Downloaded from https://academic.oup.com/jee/article/110/3/954/3746969 by guest on 13 June 2022 2 0 3 5 7 9 11 13 0 3 5 7 9 11 13 15 17 Eggs Journal of Economic Entomology, 2017, Vol. 110, No. 3 957 Table 2. Survival and mortality of three replicates of eggs, 3-day-old larvae, and 6-d-old larvae of C. rosa in “Hass” avocado when subjected to various periods of cold treatment of 0.0 C and 2.0 C C 0 2 Exposure (d) Three-day-old larvae (first and second instar) Six-day-old larvae (third instar) No. of fruit No. surviving Mortalitya (%) No. of fruit No. surviving Mortalitya (%) No. of fruit No. surviving Mortalitya (%) 176 163 166 163 164 713.7 66.3 10.7 0 0 – 90.7 98.5 100 100 183 163 162 164 164 525.0 187.3 6.0 0 0 – 64.3 98.9 100 100 170 158 164 168 164 165 617.3 65.0 16.3 0.3 0 0 – 89.5 97.4 99.9 100 100 177 158 165 167 165 165 458.0 94.3 9.3 1.0 0 0 – 79.4 98.0 99.8 100 100 182 160 163 161 165 165 173 158 173 163 164 165 395.3 151.3 24.3 2.7 0 0 318.0 99.0 27.0 1.3 0 0 – 61.7 93.6 99.3 100 100 – 68.9 91.5 99.6 100 100 Average number of eggs inoculated (6 SD): Eggs 0.0 C—28.5 6 3.4; 2.0 C—29.7 6 3.5; 3-d-old larvae 0.0 C—27.8 6 4.2; 2.0 C—24.7 6 3.8; 6-d-old larvae 0.0 C—30.4 6 4.5; 2.0 C—24.7 6 3.8. a Corrected for natural mortality using Abbott’s formula (1925). Table 3. Survival and mortality of three replicates of eggs, 3-d-old larvae, and 6-d-old larvae of C. cosyra in “Hass” avocado when subjected to various periods of cold treatment of 0.0 C and 2.0 C C 0 2 Exposure (d) 0 3 5 7 9 11 13 15 0 3 5 7 9 11 13 15 17 Eggs Three-day-old larvae (first and second instar) a No. of fruit No. surviving Mortality (%) No. of fruit No. surviving 180 165 162 165 164 165 389.7 264.0 153.3 10.3 0 0 32.3 55.7 97.4 100 100 – 179 165 165 165 164 165 165 165 149 160 165 167 166 166 162 165 165 538.3 378.7 153.0 38.3 4.0 0.3 0 0 426.7 319.3 232.0 122.0 25.3 4.3 0.3 0 0 174 155 161 160 147 165 164 165 364.3 256.0 166.0 41.7 6.3 0.3 0 0 29.7 54.4 88.6 98.3 99.9 100 100 – Mortality (%) – 29.7 71.6 92.9 99.3 99.9 100 100 – 25.2 45.6 71.4 94.1 99.0 99.9 100 100 a Six-day-old larvae (third instar) No. of fruit No. surviving 180 165 159 164 164 164 165 165 173 161 164 166 188 165 164 166 164 363.7 222.7 113.0 32.3 6.3 0.7 0 0 342.3 268.7 175.0 95.7 45.0 6.3 1.7 0.3 0 Mortalitya (%) – 38.7 68.9 91.1 98.3 99.8 100 100 – 21.5 48.9 80.8 86.6 98.2 99.5 99.9 100 Average number of eggs inoculated (6 SD): Eggs 0.0 C—35.4 6 4.9; 2.0 C—37.2 6 5.8; 3-d-old larvae 0.0 C—31.0 6 4.6; 2.0 C—30.3 6 4.2; 6-d-old mature larvae 0.0 C—32.1 6 4.6; 2.0 C—31.1 6 4.6. a Corrected for natural mortality using Abbott’s formula (1925). A trait of fruit fly is that multiple insects may reside in an individual fruit—described as a contagious distribution (Follett and McQuate 2001). However, the fruit would receive a quarantine treatment that would result in a mortality of >99.9% and the rare survivors would have a random distribution within a shipment (Baker et al. 1990). It is argued that for a contagious distribution the Poisson distribution model may not be appropriate and that a negative distribution model is more applicable. However, for rare events such as fruit fly in avocado, the predictions are similar (Follett and McQuate 2001) and for either model the probability of finding one or more mating pairs is given by h i NFT 2 P¼ 1e 2 (Follett and McQuate 2001) where N is the number of fruit in a shipment, F is the field infestation rate per fruit, and T is the pest survival rate after a postharvest treatment (Liquido et al. 1995). NFT is the number of live insects after a postharvest treatment. De Graaf (2009) did not find any larvae in his field survey of 16,883 fruit. For the purposes of this study, it is assumed that 5 larvae were present in the 16,884 fruit surveyed. This assumption has been made because although fruit flies have a Downloaded from https://academic.oup.com/jee/article/110/3/954/3746969 by guest on 13 June 2022 0 3 5 7 9 11 0 3 5 7 9 11 Eggs 958 Journal of Economic Entomology, 2017, Vol. 110, No. 3 Table 4. Regression analysis of the survival of three life stages of C. capitata, C. rosa, and C. cosyra after various periods of cold treatment Species Egg C LT99.9 (d) (95% CL) Regression (y ¼ mortality, x ¼ days) P* 0.0 10.1 (9.9–10.4) 4.8 (4.7–4.9) 9.6 (9.4–9.9) 7.5 (7.3–7.7) 5.6 (5.4–5.8) 9.8 (9.6–10.0) 8.7 (8.5–8.9) 6.9 (6.7–7.1) 10.6 (10.3–10.9) 10.1 (9.9–10.3) 5.3 (5.1–5.4) 10.9 (10.7–11.3) 10.1 (9.9–10.4) 5.6 (5.4–5.8) 13.1 (12.8–13.4) 10.6 (10.4–10.9) 7.0 (6.8–7.2) 13.6 (13.3–14.0) y ¼ 0.4842x-1.809 y ¼ 1.1079x-2.2453 y ¼ 0.5748x-2.4287 y ¼ 0.7041-2.1785 y ¼ 1.0650-2.86 y ¼ 0.5426-2.2207 y ¼ 0.8107x-3.926 y ¼ 0.7539x-2.126 y ¼ 0.4711x-1.8833 y ¼ 0.4884x-1.6416 y ¼ 0.9689x-2.0086 y ¼ 0.4782x-2.164 y ¼ 0.4989x-1.9622 y ¼ 0.9432x-2.1910 y ¼ 0.3932x-2.0571 y ¼ 0.5570x-2.8238 y ¼ 0.7188x-1.9371 y ¼ 0.3682x-1.9388 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Three–day-old larvae (first and second instar) Six-day-old larvae (third instar) Egg 2.0 Three–day-old larvae (first and second instar) Six–day-old larvae (third instar) *P-value in the analysis of deviance <0.01 indicates there is a statistically significant relationship between the variables at a 99% confidence level. Table 5. The number of live 6-d-old (third instar) C. cosyra larvae in avocado that had undergone treatment at 2.0 C for 18 or 20 d Replicate 1 (18 d) 1 (20 d) 2 (20 d) 3 (20 d) Control Treated Live larvae No. of fruit Mean no. of flies/fruit No. of fruit Estimated no. of larvae treated No. of live larvae found 7,602 7,602 1,402 2,203 460 458 440 494 16.53 16.53 3.19 4.46 2,147 2,131 2,002 1,835 35,490 35,225 6,386 8,184 2 0 0 0 contagious distribution and egg packets laid by the fruit flies may contain 15 or more eggs, there is a large natural attrition rate due to encapsulation (Ware et al. 2016) and possible antibiosis. The five larvae represent a field infestation rate of one larva from 3,367 fruit. From the results of this study, the assumption is made that the 49,796 fruit sampled contained a living larva and indicates there is at most a 0.0000201 survival. It is estimated that there are 84,840 avocados in a container (based on 16 fruit/4-kg carton and 264 cartons per pallet with 20 pallets per 12.2-m-long container). Assuming a shipment of one container, the probability of one mating pair is 2.5 109. An alternative approach that measures risk is recommended and entails calculating the probability of the pest establishing after importation (Baker et al. 1990). For this study, it is assumed that a single C. cosyra survives in 49,796 treated individuals (see Table 5), resulting in a treatment survival rate that is 0.0000201. Assuming a natural infestation rate of 0.0000592 (one infested fruit from 16,883 [De Graaf 2009]) and the number of fruit per container is 84,480 (De Graaf 2009) then the probability of one mating pair per shipment is 2.5 10 9. There was no natural infestation in 16,883 fruit assessed by De Graaf (2009). Assuming for this study a single fruit was infested by five larvae then the probability of one mating pair being present in this consignment is 84.3%. This is derived from P¼ 1 x X k 1 21x expðkÞ x! x¼2 (Landolt et al. 1984) ¼ 1 þ expðkÞ – 2 exp ðk=2Þ (Baker et al. 1990) where k is the number of flies recovered. This translates to 1 larva in 3,376 fruit harvested. From the cold treatment results above, it was determined that there were no survivors in 49,795 individuals that were subjected to treatment. If it is assumed that a single fruit fly survived the treatment then to isolate this fly 168,107,920 fruit would have to be harvested. A 12.2-m shipping container accommodates 84,480 fruit (20 pallets made up of 264 cartons each containing 16 fruit [De Graaf 2009]); therefore, 1990 shipping containers would only contain the single fruit fly. If one assumed that a single shipment is 500,000 fruit then there would be a single fly in one of the 336 ships. As we require at least two flies if a colony is to be established, the number of ships required for that level of infestation would be double and then there is still only a 40% probability of there being a mating pair. These results can be viewed in another way: traditionally Probit 9 level of security (99.9968%) has been used for phytosanitary pest and disease threats. Baker (1939) determined this to be not more than 32 survivors out of a million—the true survival rate is less stringent at 136 survivors in 1,000,000 individuals (CL 95%) tested (Couey and Chew 1986). If there is only a single surviving fly from the 168,107,920 fruit harvested, then the Probit 9 level, assuming 32 flies in a million survive, indicate that the level of safety for C. cosyra is oversubscribed by >5,253 times. Downloaded from https://academic.oup.com/jee/article/110/3/954/3746969 by guest on 13 June 2022 C. capitata C. rosa C. cosyra C. capitata C. rosa C. cosyra C. capitata C. rosa C. cosyra C. capitata C. rosa C. cosyra C. capitata C. rosa C. cosyra C. capitata C. rosa C. cosyra Life stage Journal of Economic Entomology, 2017, Vol. 110, No. 3 959 These conclusions are conservative, as they do not include additional elements of a systems approach that may entail the monitoring of fruit fly and the subsequent application of bait and chemical sprays. The sanitation of the orchard through the removal and destruction of fallen fruit as well as packhouse inspection would further decrease the risk of fruit fly. It is concluded that the quarantine risk of any Hass avocado consignment having undergone a cold treatment of 2 C for 20 d poses a negligible risk of containing indigenous South African fruit fly species (C. ceratitis, C. rosa, and C. cosyra). Acknowledgments References Fig. 2. 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