Gamma radiation phytosanitary treatment against Trialeurodes vaporariorum (: Aleyrodidae) Guido A. Van Nieuwenhove1,2,*, Andrea V. F. Oviedo1,2, Yesica M. Dalto3, Juliana Perez3, Celina I. Horak3, Gerardo A. Gastaminza1, Eduardo Willink1, and Guy J. Hallman4

Abstract In Argentina the greenhouse ,Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), is one of the 2 main whitefly pest species af- fecting several fruit and vegetable crops. Both adults and immature stages (nymphs) are presently feeding on many commercial crops, and a single live can cause regulatory actions by importing countries. Irradiation is a promising postharvest phytosanitary treatment that is steadily increasing in use worldwide. Cobalt-60 gamma-ray target doses of 30, 60, 90 and 120 Gy were used to determine the effect on irradiated eggs, 2nd instars and late pupae. The late pupa was the stage most tolerant to irradiation. Based on probit and logit analysis of the data obtained using at least 30,000 late pupae, the estimated doses to provide 99.9968% prevention of adult emergence from irradiated pupae were 170 and 222 Gy. Moreover, when late pupae (2 replicates totaling 33,625) were irradiated with a target dose of 100 Gy (maximum absorbed dose of 108 Gy) only 1,146 (3.4%) emerged as normal-looking adults, and they did not lay any eggs. Egg laying and hatch in the non-irradiated controls was normal. Therefore, 108 Gy should guarantee a secure phytosanitary treatment against T. vaporariorum.

Key Words: ; irradiation; quarantine treatment; generic dose; phytosanitation

Resumen En Argentina, la mosca blanca de los invernaderos, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), es una de las dos princi- pales especies de moscas blancas plagas que afectan a numerosos cultivos de frutas y hortalizas. Tanto los adultos como las etapas inmaduras (ninfas) de T. vaporariorum están presentes alimentándose en numerosos cultivos comerciales y la sola presencia de un insecto vivo puede causar acciones reguladoras por parte de los países importadores. La irradiación es un tratamiento fitosanitario pos-cosecha prometedor a nivel mundial puesto que su uso está en continuo aumento. Actualmente, tratamientos fitosanitarios genéricos son propuestos y desarrollados utilizando miles de individuos para una amplia gama de plagas a fin de impedir su reproducción. Por lo tanto, el presente estudio se llevó a cabo utilizando, al menos, 30.000 pupas tardías de T. vaporariorum para proponer una dosis genérica de tratamiento cuarentenario seguro sobre los productos infestados por la mosca blanca de los invernaderos. Dosis de 30, 60, 90 y 120 Gy, mediante el empleo de una fuente de Cobalto 60, se utilizaron para irradiar huevos, ninfas de segundo estadio y pupas tardías a fin de determinar el efecto de la irradiación sobre los mismos. Las pupas tardías fueron el estadio más tolerante a la radiación gamma. Basándose en los análisis de probit y logit, respectivamente, las dosis estimadas para alcanzar 99.9968% de prevención en la emergencia de adultos de pupas irradiadas fueron 170 y 222 Gy. Además, cuando pupas en un estado de desarrollo avanzado (2 replicas totalizando 33,625) fueron irradiadas con una dosis objetivo de 100 Gy (máxi- ma dosis absorbida de 108 Gy), solo 1,146 (3.4%) emergieron como adultos aparentemente normales los cuales no depositaron huevos. La oviposición y eclosión en los controles no-irradiados fue normal. Por consiguiente, 108 Gy garantizaría un tratamiento fitosanitario seguro contra T. vaporariorum.

Palabras Clave: mosca blanca de los invernaderos; irradiación; tratamientos cuarentenarios; dosis genérica; sanidad vegetal

Whiteflies (Hemiptera: Aleyrodidae) are widely distributed pecially tomato, Solanum lycopersicum L., eggplant, S. melongena throughout the world attacking many commercial fruit and veg- L., potato, S. tuberosum L. and pepper, Capsicum annuum L.; and etable crops (Evans 2008). In Argentina Bemisia tabaci (Genna- common bean, (Phaseolus vulgaris L.; Fabales: Fabaceae); causing dius) and Trialeurodes vaporariorum Westwood are the main pest significant direct damage by sucking sap and indirectly by excret- species affecting fruit and vegetable crops (Viscarret et al. 2000). ing honeydew, which promotes the growth of fungi that reduce the Trialeurodes vaporariorum is the most prevalent and problematic photosynthetic capacity of plants. Females lay solitary or nested species in northwestern Argentina, feeding on important food crops eggs from which emerge the first instars (also called crawlers). This such as various cucurbits (Cucurbitales: Cucurbitaceae) especially 1st instar moves to a feeding site and affixes itself by its mouth- Cucurbita spp. and cucumber, Cucumis sativus L. (Cucurbitales: Cu- parts. It then goes through 3 more nymphal stages, N2, N3, and N4 curbitaceae); various solanaceous crops (Solanales: Solanaceae) es- (the last also known as the pupa), and from the latter stage a new

1Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Sección Zoología Agrícola, Tucumán, Argentina 2Facultad de Ciencias Naturales e IML-UNT, Miguel Lillo 205 (4000), S. M. de Tucumán, Tucumán, Argentina 3Comisión Nacional de Energía Atómica, Livestock and Technological Application Department, Centro Atómico Ezeiza, Buenos Aires, Argentina 4Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Vienna, Austria *Corresponding author; E-mail: [email protected] Copyright © International Atomic Energy Agency 2016. Published by the Florida Entomological Society. All rights reserved.

130 2016 — Florida Entomologist — Volume 99, Special Issue 2 Van Nieuwenhove et al.: Phytosanitary irradiation of the greenhouse whitefly on zucchini 131 adult emerges (CABI 2015). This cycle takes approximately 26 d in in wood-frame cages covered with organdy cloth. Then, 60 mated laboratory conditions of 25 ± 1 °C, 60 ± 10% RH and 12:12 h L:D, and females were placed inside of each cage for a period of 48 h to es- the life cycle is strongly influenced by temperature (Manzano & van tablish cohorts of uniform age. When the resulting eggs developed Lentern 2009). Reproduction in this species occurs through arrhe- to the respective stages and ages desired, all but 200 eggs, nymphs, notokous parthenogenesis where males develop from unfertilized or pupae were removed from each plant and taken to the irradia- eggs and females from fertilized eggs. tion facility. Irradiation treatment was conducted with a gamma Phytosanitary irradiation (PI) is a promising postharvest phyto- Co-60 irradiator with an activity of 583.8 Ci (21.6 PBq) and dose sanitary treatment that is steadily increasing in use in world trade rate of 40.7 Gy/min, at the National Atomic Energy Commission, (Hallman 2011). Moreover, PI is the ideal technology for developing Ezeiza Atomic Centre, Argentina. Each host plant with the stages of generic phytosanitary treatments because it is effective at doses whitefly were placed facing the Co-60 source. The absorbed dose that do not significantly lower the quality of most fresh commodi- was measured using Fricke dosimeters attached to the surfaces of ties. The generic phytosanitary treatment concept is that 1 treat- the host (ASTM 2004). ment dose can be used for a broad group of quarantine pests and/ After irradiation, the irradiated samples and controls were immedi- or commodities although not all pest species in a group have been ately returned to the lab and held under standard rearing conditions. tested for efficacy. A generic dose of 100 Gy is suggested for Ster- Then, each host plant was examined every 2 d under a stereomicro- norrhyncha, the suborder that includes the family Aleyrodidae scope (Leica MZ 95) to determine the fate of the eggs, nymphs, and pu- (Hallman 2012). pae, i.e., to determine if they had molted to the next stage or died. Live Unlike other disinfestation techniques, PI does not cause con- nymphs and pupae typically have a yellowish and white color, respec- siderable acute mortality, but renders pests incapable of complet- tively, whereas dead nymphs and pupae turn brown. Dead nymphs, ing development and/or reproducing; therefore, live but nonviable pupae, and unhatched eggs were counted, recorded, and removed at or reproductively sterile may occur with the exported com- each review event. Emerged adults were introduced inside cages each modity. with a zucchini summer squash plant to determine their reproductive Previous studies on irradiation of pupa and adult T. vaporariorum ability. found that 50–70 Gy seem sufficient to prevent reproduction (Genchev 1986, 1987; Calvitti et al. 1997; Moradi & Zarabi 2012). However, large- scale studies involving the treatment of many thousands of insects at Statistical Analysis 1 putative dose with no resulting reproduction are required to confirm that a proposed treatment dose suffices. Probit and logit analysis (PoloPlus, Petaluma, California) were used The objective of this study was to treat at least 30,000 late pupae to analyze emergence of normal-looking adults from irradiated late of T. vaporariorum at the minimum dose needed to prevent successful pupae using as dose the mean absorbed dose measured by dosimetry. reproduction in order to propose that dose as a phytosanitary treat- ment for commodities infested by the pest. The effects of irradiation on the egg and N2 stages were also evaluated. Results

Materials and Methods DOSIMETRY The results of routine dosimetry are presented in Table 1. Mea- sured doses were quite close to target doses, except at 30 and 60 Gy for Study Site and Insect Rearing the pupal tests where the measured doses were 40 and 28%, respec- The study was conducted at the Zoología Agrícola section of the tively, higher than the target doses. Nevertheless, the dose uniformity Estación Experimental Agroindustrial Obispo Colombres (EEAOC), lo- ratios (DUR) were relatively low throughout. cated in Las Talitas, Tucumán, Argentina. A native population of T. va- porariorum was collected from commercial greenhouses in 2010 from DEVELOPMENT OF IRRADIATED EGGS, 2ND INSTARS, AND PUPAE zucchini summer squash, Cucurbita maxima var. ‘Zapallito’, tomato, and cucumber plants located in Lules, Tucumán, Argentina. The col- At ≥ 60 Gy no 3–4 d-old eggs hatched, while 92.6 ± 2.1% of non- lected samples were transferred to laboratory conditions and released irradiated eggs hatched. At 30 Gy egg hatch was 80.5 ± 2.5%, while inside cages (100 × 60 × 50 cm) with zucchini plants. Trialeurodes va- 42.3 ± 4.1 and 1.25 ± 1.2% of eggs developed to 2nd and 3rd instars, porariorum was reared until suitable and sufficient eggs, nymphs, and respectively, 0.19 ± 0.12% pupated, and none emerged as adults. pupae were available for irradiation. None of the irradiated (≥ 30 Gy) 1–2 d-old 2nd instars developed to the emerged adult stage, while 65.9 ± 4.0 % of non-irradiated 2nd instars emerged as adults. Success in developing to 3rd instar and pupa Experimental Procedure was, of course, related to dose (Table 2). To determine the effect of irradiation on various developmental Because the absorbed doses for pupae were so different from tar- stages eggs (3–4 d old), second instars (1–2 d old) and late pupae (4–5 get doses for pupae at 30 and 60 Gy, the mean of the absorbed doses d old) were irradiated at the target doses of 0 (as a control), 30, 60, 90 rounded to the nearest gray (42, 77, 92, and 124 Gy for target doses 30, and 120 Gy with 3 replications for each stage and dose. 60, 90, and 120 Gy, respectively) were used to analyze and report data Zucchini summer squash was planted in 1,000 cm3 plastic pots for pupae. Adult emergence of 4–5 d-old irradiated pupae predictably filled with a sterilized mixture of 2/3 soil and 1/3 of peat as a grow- declined with increasing dose but was not eliminated at the highest ing medium. These seedlings were covered with organdy cloth dose used, 124 Gy. Percentage emergence (± SEM) for the non-irradiat- and kept in greenhouses free from insect contamination until they ed control, and pupae irradiated at 42, 77, 92, and 124 Gy was, respec- reached an adequate size. Zucchini summer squash plants with at tively, 75.5 ± 4.2, 22.0 ± 1.7, 5.7 ± 0.50, 3.9 ± 0.91, and 0.46 ± 0.56%. least 4 leaves (~25 d old) were placed for infestation individually By means of probit and logit analysis, respectively, we estimated the 132 2016 — Florida Entomologist — Volume 99, Special Issue 2

Table 1. Doses measureda at 4 target doses between 30–120 Gy for irradiation of eggs, 2nd nymphal instars and late pupae of Trialeurodes vaporariorum.

Stage Target dose (Gy) Minimum dose (± SEM, Gy) Maximum dose (± SEM, Gy) DURb Egg 30 29.51 ± 0.77 32.89 ± 0.88 1.10 Egg 60 58.87 ± 3.33 65.97 ± 2.02 1.11 Egg 90 87.78 ± 1.78 92.85 ± 1.08 1.08 Egg 120 118.63 ± 1.34 121.36 ± 2.13 1.02 Nymph 2 30 30.26 ± 0.94 33.84 ± 1.05 1.09 Nymph 2 60 61.88 ± 2.11 66.09 ± 1.97 1.06 Nymph 2 90 91.67 ± 1.08 92.63 ± 0.70 1.02 Nymph 2 120 118.84 ± 1.25 122.85 ± 1.54 1.03 Pupae 30 40.59 ± 0.37 42.92 ± 0.46 1.03 Pupae 60 75.04 ± 4.90 78.63 ± 4.73 1.03 Pupae 90 91.67 ± 1.62 92.84 ± 1.07 1.02 Pupae 120 121.91 ± 0.96 126.74 ± 0.78 1.02 Pupae 100 103.16 107.89 1.05 Pupae 100 98.60 103.12 1.05

aDoses are means for all tests except those at 100 Gy (confirmatory) where they are absolute. bDose uniformity ratio.

Table 2. Development of Trialeurodes vaporariorum following irradiation as 1–2 Discussion d old 2nd instars. Although the dosimetry system used was not accurate for the target Percentage of irradiated 2nd instars that reached subsequent stage (% ± SEM) doses 30 and 60 Gy in the pupal tests, it was quite accurate for doses closer to those that would be used for phytosanitary treatment of hosts of T. va- Dose (Gy) 3rd instar Pupa porariorum (~90–120 Gy). The target dose of 100 Gy applied to 33,250 late 0 86.8 ± 1.9 75.5 ± 3.2 pupae prevented oviposition, and thus, would serve as a phytosanitary 30 80.1 ± 3.8 18.6 ± 1.6 treatment dose for commodities at risk of carrying immature stages of the 60 62.3 ± 4.3 10.0 ± 2.0 pest. Because radiotolerance of an insect of any given species increases 90 71.7 ± 0.9 3.5 ± 0.86 with the stage of development, the most radiotolerant stage of concern 120 20.7 ± 2.5 2.5 ± 0.95 for phytosanitary treatment of most aleyrodids, including T. vaporariorum, would be the late pupa (Hallman et al. 2010). Because the highest dose measured near a target dose of 100 Gy was ~8% greater than the target dose, a minimum dose of 108 Gy could be proposed as the dose to provide doses to needed to provide 99.9968% prevention of adult emergence quarantine security against the pest for absolute security. from irradiated pupae to be 170 and 222 Gy (Table 3). Acknowledgments Reproduction of FEMALES Irradiated AS LATE pupaE A total of 33,625 T. vaporariorum late pupae (21,142 four to 5 d-old This work was part of the FAO/IAEA Coordinated Research Project pupae in 1 replicate and 12,483 in another) was irradiated with a tar- D62008 on Development of Generic Irradiation Doses for Quarantine get dose of 100 Gy (maximum absorbed dose of 108 Gy). Out of these Treatments. The research was funded through IAEA Research Contract irradiated late pupae only 1,146 (3.4%) emerged as normal-looking No. 15,641: Gamma Radiation Quarantine Treatments for Different adults, none of laid any egg. Egg laying and hatch in the non-irradiated Groups of , and Estación Experimental Agroindustrial Obis- controls was normal. po Colombres, Tucumán, Argentina.

Table 3. Results of probit and logit analyses of emergence of normal-appearing adult Trialeurodes vaporariorum from irradiated late pupae. A total of 33,625 T. vaporariorum late pupae (21,142 four to 5 d-old pupae in 1 replicate and 12,483 in another) was irradiated with a target dose of 100 Gy (maximum absorbed dose of 108 Gy).

Analysis

Parameter Probit Logit X2 4.8 6.2 Degrees of freedom 10 10 Probability (fits model?) 0.90 (yes) 0.80 (yes) Heterogeneity 0.48 0.62 Y intercept ± SEM −0.55 ± 0.14 −1.27 ± 0.25 Slope ± SEM 0.027 ± 0.002 0.052 ± 0.004 99% effective dose (95% fiducial limits), Gy 107 (100–117) 112 (104–123) 99.9968% effective dose (95% fiducial limits), Gy 170 (157–186) 222 (199–254) Van Nieuwenhove et al.: Phytosanitary irradiation of the greenhouse whitefly on zucchini 133 References Cited Genchev N. 1987: Radiation sterilization of the greenhouse whitefly (Trial- eurodes vaporariorum, Westwood) (Homoptera: Aleyrodidae): Sterilant effect of radiation on pupae and adults. Pochvoznanie, Agrokhimiya i Ras- ASTM [American Society for Testing and Materials International]. 2004. Stan- titelna Zashchita 22: 108-115. dard practice for using the Fricke reference standard dosimetry system, pp Hallman GJ. 2011. Phytosanitary applications of irradiation. Comprehensive Re- 261-268 In Annual book of ASTM Standards 2004, Vol. 2. ASTM Interna- views in Food Science and Food Safety 10: 143-151. tional, West Conshohocken, PA, USA. Hallman GJ. 2012. Generic phytosanitary irradiation treatments. Radiation CABI [Commonwealth Agricultural Bureau International]. 2015. Fallopia japon- Physics and Chemistry 81: 861-866. ica In Invasive Species Compendium. Wallingford, UK: CAB International. Hallman GJ, Levang-Brilz NM, Zettler JL, Winborne IC. 2010. Factors affecting www.cabi.org/isc. ionizing radiation phytosanitary treatments, and implications for research Calvitti M, Govoni C, Buttarazzi M, Cirio U. 1997. Induced sterility in greenhouse and generic treatments. Journal of Economic Entomology 103: 1950-1963. whitefly (Homoptera: Aleyrodidae) treated with gamma radiation. Journal Manzano MR, van Lenteren JC. 2009. Life history parameters of Trialeurodes va- of Economic Entomology 90: 1022-1027. porariorum (Westwood) (Hemiptera: Aleyrodidae) at different environmen- Evans GA. 2008. The (Hemiptera: Aleyrodidae) of the world and their tal conditions on two bean cultivars. Neotropical Entomology 38: 452-458. host plants and natural enemies. 591/1WF/World-Whitefly-Catalog. http:// Moradi M, Zarabi M. 2012. The best dose for sterilisation of greenhouse white- www.sel.barc.usda.gov fly, Trialeurodes vaporariorum (Westwood) (Hem.: Aleyrodidae) by gamma Genchev N. 1986: Radiation sterilization of the greenhouse whitefly (Trial- radiation. Journal of Life Sciences 6: 536-542. eurodes vaporariorum, Westwood) (Homoptera: Aleyrodidae): Lethal ra- Viscarret, MM, Botto EN, Polaszek A. 2000. Whiteflies (Hemiptera: Aleyrodidae) diation effects on pupae and adults. Pochvoznanie Agrokhimiya I Rastitelna of economic importance and their natural enemies (Hymenoptera: Aphelin- Zashchita 21: 125-133. idae, Signiphoridae) in Argentina. Revista Chilena de Entomología 26: 5-11.