Comparative transcriptome analysis of false codling moth, Thaumatotibia leucotreta in response to high and low-temperature treatments.

The false codling moth (FCM), Thaumatotibia leucotreta, is a major quarantine pest native to Africa. Physical postharvest phytosanitary measures such as cold and heat treatments are championed to control its spread to new regions. However, the molecular changes that T. leucotreta undergoes as it attempts to adjust to its surroundings during the treatments and withstand the extreme temperatures remain largely unknown. The current study employs RNA-seq using the next-generation Illumina HiSeq platform to produce transcriptome profiles for differential gene expression analysis of T. leucotreta larvae under thermal stress. The transcriptome assembly analysis revealed 226,067 transcripts, clustering into 127,018 unigenes. In comparison to the 25 °C treated group, 874, 91, 159, and 754 individual differentially expressed genes (DEGs) co-regulated at -10, 0, 40, and 50 °C, respectively were discovered. Annotation of the DEGs by gene ontology (GO) revealed several genes, previously implicated in low and high-temperature stresses, including heat shock proteins, cytochrome P450, cuticle proteins, odorant binding proteins, and immune system genes. Kyoto Encyclopedia of Genes and Genomics (KEGG) classification analysis revealed that substantive DEGs were those involved in metabolic pathways such as thiamine, purine, folate, and glycerolipid metabolism pathways. The RT-qPCR validation of several significantly up- and down-regulated DEGs showed congruence between RNA-seq and qPCR data. This baseline study lays a foundation for future research into the molecular mechanisms underlying T. leucotreta's cold/heat tolerance by providing a thorough differential gene expression analysis that has identified multiple genes that may be associated with the insect's ability to withstand cold and heat.

An 18-day, 3 °C cold treatment effectively kills Ceratitis capitata (Diptera: Tephritidae) in kiwifruit (Actinidia spp.).

A series of experiments were carried out to develop a phytosanitary disinfestation protocol to kill Ceratitis capitata (Weidemann) (Mediterranean fruit fly, Diptera: Tephritidae) in 'Hayward' kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson) and 'Zesy002' kiwifruit (Actinidia chinensis Planch.). Experiments on 4 immature life stages (eggs and 3 larval instars) with treatment durations of between 5 and 18 days showed that third instars were the most tolerant to temperatures around 3 °C, with the lethal time to 99.9968% (probit 9) mortality (LT99.9968) estimated to be 17.3 days (95% confidence interval (CI) 16.4-18.2). Larvae reared and treated in 'Zesy002' were significantly more susceptible to cold treatment than those reared in 'Hayward'. A large-scale trial testing a disinfestation protocol of 3 ±â€…0.5 °C for 18 days treated over 500,000 third-instar C. capitata with no survivors. These results demonstrate that a cold treatment of 3.5 °C or below for 18 days induces C. capitata mortality in kiwifruit at a rate that exceeds 99.9968% with a degree of confidence greater than 99%.

Ethyl Formate Fumigation for Control of the Scale Insect Asiacornococcus kaki, a Quarantine Pest on Sweet Persimmon, Diospyros kaki: Efficacy, Phytotoxicity and Safety.

Sweet persimmons are a valuable export commodity. However, the presence of live insects such as Asiacornococcus kaki limits their access to many export markets. Methyl bromide, traditionally used for pest control, is damaging to human health and the environment. Ethyl formate (EF) is a viable alternative; however, its effectiveness against A. kaki on sweet persimmon fruit is unknown. We evaluated the effectiveness of EF fumigation in controlling A. kaki present under the calyx of persimmon fruit. The hatching rate of eggs and the survival rates of nymphs and adults of A. kaki at low temperatures, its LCt50 and LCt99 after EF exposure, and phytotoxic damage caused by EF were evaluated in laboratory-scale and commercial-scale tests. The dose-response tests showed that the EF LCt99 at 5 °C was 9.69, 42.13, and 126.13 g h m-3 for adults, nymphs, and eggs, respectively. Commercial-scale tests demonstrated EF efficacy against all A. kaki stages without causing phytotoxic effects on persimmons, though the eggs of A. kaki were not completely controlled in linear low-density polyethylene (LLDPE)-packaged fruit. This study demonstrated that EF is a potential fumigant for quarantine pretreatment, especially before persimmon fruit is packed with LLDPE film, to control A. kaki infesting sweet persimmon fruit.

Comparison of Methyl Bromide and Ethyl Formate for Fumigation of Snail and Fly Pests of Imported Orchids.

Invasive snails and flies are major pests of imported orchids, controlled by methyl bromide (MB) fumigation in Korea. We compared the efficacy and phytotoxicity of ethyl formate (EF) and MB on four species of imported orchids using juvenile stages of Achatina fulica and third and fourth instars of Lycoriella mali. EF was as effective as MB. The LCt99 values of EF were 68.1 and 73.1 g h/m3 at 15 °C; and those of MB were 95.9 and 78.4 g h/m3 at 15 °C for A. fulica and L. mali, respectively. In the scale-up trials, EF treatment at 35 g/m3 for 4 h at 15 °C resulted in complete control of both pests. MB treatment based on the current treatment guidelines for imported orchids (48 g/m3, 2 h, at >15 °C) resulted in complete control of L. mali but not of A. fulica. Chlorophyll content and hue values of treated orchids were not affected by EF treatment but significantly changed by MB (p-value < 0.05). All four treated species of orchids died within 30 d of MB treatment, while only one species died from EF treatment. Our results suggest that EF is a potential alternative to MB in phytosanitary treatment of imported orchids.

Synergistic Effect of Cold Treatment Combined with Ethyl Formate Fumigation against Drosophila suzukii (Diptera: Drosophilidae).

Drosophila suzukii is a quarantine pest that is rapidly spreading in berries. This study evaluated the synergistic effect of combination treatment with ethyl formate (EF) and cold temperature for D. suzukii control on imported grapes. A higher insecticidal effect was observed at 1 °C than at 5 °C at all developmental stages, and the pupal stage showed the strongest tolerance to cold temperature. After EF fumigation alone, eggs showed the highest tolerance at 216.67 mg·h/L (LCT99 value), and adults showed the highest susceptibility at <27.24 mg·h/L. Among the combination treatment methods, cold temperature after fumigation resulted in the best synergistic effect. The effect of this combination was significant, with 23.3% higher mortality for eggs, 22.4% for larvae, and 23.4% for pupae than observed with EF fumigation alone. Furthermore, the period of complete D. suzukii control in the 12 L desiccator was shorter in the combination treatment group at the LCT80 value than at the LCT50 value of the egg stage. EF showed a very high sorption rate (24%) after 4 h of exposure at a grape loading ratio of 15% in a 0.65 m3 fumigation chamber. As the grape loading ratio for combination treatment decreased, D. suzukii mortality increased, but when EF was administered at the LCT80 value, there was little difference in the mortalities of the eggs and larvae but not the pupae. All D. suzukii developmental stages were completely controlled within 7 days after combination treatment, and phytotoxicity was not observed in grapes. These results suggest that the combination of cold-temperature treatment and EF fumigation could be used for D. suzukii control.

Hot Water Treatment for Post-Harvest Disinfestation of Bactrocera dorsalis (Diptera: Tephritidae) and Its Effect on cv. Tommy Atkins Mango.

Mango production and trade in sub-Saharan Africa is hampered by direct damage and the high quarantine status of B. dorsalis and the paucity of effective post-harvest phytosanitary treatments. The current study reports the development of a quarantine treatment protocol using hot water to disinfest B. dorsalis and assess its effect on cv. Tommy Atkins mango quality. We first determined the development of the eggs and all larval stages of B. dorsalis in cv. Tommy Atkins mango and used the information to establish a time-mortality relationship of the immature stages after subjecting infested fruits to a regimen of eight, time instances of hot water at 46.1 °C. Using probit analysis, we estimated the minimum time required to achieve 99.9968% mortality of each stage. Our results indicate that the egg was the least heat tolerant, followed by the first, second, and third instar. The time required to achieve 99.9968% control of the third instar in cv. Tommy Atkins mango (400-600 g) was determined to be 72.63 min (95% Cl: 70.32-74.95). In the confirmatory trials, the hot water treatment schedule of 46.1 °C/72.63 min was validated, and none of the 59,120 most heat-tolerant individuals treated survived. Further, there were no significant differences between hot water-treated and untreated mangoes recorded in weight loss, fruit firmness, pH, total soluble solids, moisture content, and titratable acidity eleven days post-treatment. These findings demonstrate an effectively optimum post-harvest disinfestation treatment against B. dorsalis in cv. Tommy Atkins mango that should be adopted commercially to facilitate access to profitable but strict export markets globally.

Preliminary study to evaluate ethanedinitrile (C2 N2 ) for quarantine treatment of four wood destroying pests.

BACKGROUND: Methyl bromide is an ozone depleting substance and is still being used for quarantine treatment of wood products and logs. Ethanedinitrile (C2 N2 ) is a new potential alternative fumigant and has been investigated as a timber fumigant because it has shown better penetration through timber and responds fast to insects and nematodes. This article reports on laboratory and commercial-scale trials with C2 N2 for important wood destroying pests namely Tomicus piniperda (Pine bark beetle) adults, Hyphantria cunea (Fall webworm) lavae, Reticulitermes speratus (Japanese Termite) adults and Cryphalus fulvus (Scolytidae) (Yellow minute bark beetle) which naturally infests pine logs. RESULTS: Tomicus pi:niperda adults were more tolerance than H. cunea lavae and R. speratus adults with lethal concentration × time affecting 99% mortality (LCt99 ) values of 27.84, 7.49 and 3.30 g h m-3 , respectively. Ethanedinitrile shows exceptionally high toxicity to adult R. speratus which was almost 26 times more toxic than methyl bromide. The complete control of larvae and adults stages of C. fulvus was achieved at concentration × time (Ct) products from 89.6 g h m-3 in a 6-8 L fumigation chamber. Temperature-depending trials on C. fulvus were conducted with C2 N2 doses of 30 at 21 ± 6 and 40 g m-3 at 9 ± 3 and 2 ± 2 °C for 24 h. The Ct products of 182.2, 227.8 and 268.6 g h m-3 were obtained and C. fulvus was completely controlled at all tested temperatures. CONCLUSION: Ethanedinitrile could offer quarantine treatment for wood products and logs for the control of wood destroying insect pests and termites.

Relative Tolerance of Three Morphotypes of the Anastrepha fraterculus Complex (Diptera: Tephritidae) to Cold Phytosanitary Treatment.

The Anastrepha fraterculus (Wiedemann) complex is currently comprised of at least eight morphotypes, including several that are likely to be described as new species. It is critical to evaluate whether the morphotypes differ in tolerance to phytosanitary treatments. Temperatures from 0 to 3°C are used as a phytosanitary treatment for some commodities exported from the region and at risk of infestation by the A. fraterculus complex. Description of A. fraterculus morphotypes as new species could result in the annulation of phytosanitary treatment schedules for the new species. This study compared the relative cold tolerance of five populations from three morphotypes of the A. fraterculus complex: Andean, Peruvian, and Brazilian-1. Both a laboratory and wild strain of the Brazilian-1 morphotype were studied. Differences in mortality of third instars of the five A. fraterculus populations reared on nectarines were observed only with short treatment durations at temperatures ranging from 1.38 ± 0.04°C to 1.51 ± 0.08°C (mean ± SEM). Estimated times to achieve the LT99.99682 (probit 9) showed that Brazilian-1 wild, Brazilian-1 laboratory, and Cusco population were the most cold tolerant, followed by Andean and Peruvian, the least cold tolerant morphotype (i.e., Brazilian-1 wild = Brazilian-1 laboratory = Cusco population > Andean > Peruvian). These findings suggest that the current cold treatment schedules of 15 d at ≤ 1.11°C and 17 d at ≤ 1.67°C can be applied as cold treatments to any potential new species that may arise from the A. fraterculus complex.

Effect of harvest seasonal and gamma irradiation on the physicochemical changes in pineapple fruit cv. Pattavia during stimulated sea shipment.

Gamma irradiation is used as a phytosanitary treatment for tropical fresh fruit from some producing countries. An experiment was carried out to study the effect of gamma irradiation and season of harvest on the quality 'Pattavia' pineapple fruit. Fruit harvested in the summer and the rainy cool (winter) seasons were exposed to gamma radiation at dose levels of 0 and 400-600 Gy from a 60 Cobalt source and the fruit stored at 13°C and 90% RH for up to 21 days. Gamma irradiation did not affect the ratio of TSS/TA, antioxidant content, or ascorbic acid concentration. However, gamma irradiation did delay color development and also induced internal browning over 50% of flesh discolored in fruit stored for 14 days, especially harvested winter fruit. Moreover, harvesting fruit in different seasons had a significant effect on fruit quality after harvest and during stimulated sea shipment storage. The result showed that gamma irradiation can be used as a phytosanitary treatment with minor changes in eating quality. However, the internal browning was greater if fruit were stored longer than 1 week at 13°C.

Phytosanitary Treatments Against Bactrocera dorsalis (Diptera: Tephritidae): Current Situation and Future Prospects.

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is arguably the most important tephritid attacking fruits after Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). In 2003 it was found in Africa and quickly spread to most of the sub-Saharan part of the continent, destroying fruits and creating regulatory barriers to their export. The insect is causing new nutritional and economic losses across Africa, as well as the losses it has caused for decades in infested areas of Asia, New Guinea, and Hawaii. This new panorama represents a challenge for fruit exportation from Africa. Phytosanitary treatments are required to export quarantined commodities out of infested areas to areas where the pest does not exist and could become established. This paper describes current phytosanitary treatments against B. dorsalis and their use throughout the world, the development of new treatments based on existing research, and recommendations for further research to provide phytosanitary solutions to the problem.

Efficacy of Nitric Oxide Fumigation for Controlling Codling Moth in Apples.

Nitric oxide (NO) fumigation under ultralow oxygen (ULO) conditions was studied for its efficacy in controlling codling moth and effects on postharvest quality of apples. NO fumigation was effective against eggs and larvae of different sizes on artificial diet in 48 h treatments. Small larvae were more susceptible to nitric oxide than other stages at 0.5% NO concentration. There were no significant differences among life stages at 1.0% to 2.0% NO concentrations. In 24 h treatments of eggs, 3.0% NO fumigation at 2 °C achieved 100% egg mortality. Two 24 h fumigation treatments of infested apples containing medium and large larvae with 3.0% and 5.0% NO resulted in 98% and 100% mortalities respectively. Sound apples were also fumigated with 5.0% NO for 24 h at 2 °C to determine effects on apple quality. The fumigation treatment was terminated by flushing with nitrogen and had no negative impact on postharvest quality of apples as measured by firmness and color at 2 and 4 weeks after fumigation. This study demonstrated that NO fumigation was effective against codling moth and safe to apple quality, and therefore has potential to become a practical alternative to methyl bromide fumigation for control of codling moth in apples.

Relative Tolerance of Six Bactrocera (Diptera: Tephritidae) Species to Phytosanitary Cold Treatment.

To compare relative cold treatment tolerance across the economically important tephritid fruit flies (Diptera: Tephritidae), Bactrocera carambolae Drew & Hancock, Bactrocera correcta (Bezzi), Bactrocera cucurbitae (Coquillett), four populations of Bactrocera dorsalis (Hendel), Bactrocera zonata (Saunders), and Bactrocera tryoni (Froggatt), eggs (in vitro), and larvae (in infested fruit or on carrot diet) were cold treated at 2.0 ± 0.2 °C for selected durations. The study was performed to assess whether a single (i.e., generic) cold treatment could be developed that would control the entire group of fruit flies that were tested. Probit regression models showed that the hierarchy of cold resistance was third-instar larvae reared on carrot diet > third-instar larvae reared on orange > eggs test in vitro. Differences in mortality responses of third-instar larvae reared in oranges across populations of B. dorsalis were observed only at subefficacious levels of control. The majority of Bactrocera species responded the same at the high levels of control demanded of phytosanitary treatments, which indicated that cold treatments would be similarly effective across the species and populations tested. B. cucurbitae was found to be the most cold tolerant of all the species tested.

Phytosanitation Methods Influence Posttreatment Colonization of Juglans nigra Logs by Pityophthorus juglandis (Coleoptera: Curculionidae: Scolytinae).

Several North American walnut species (Juglans spp.) are threatened by thousand cankers disease which is caused by the walnut twig beetle (Pityophthorus juglandis Blackman) and its associated fungal plant pathogen, Geosmithia morbida M. Kolarík, E. Freeland, C. Utley and N. Tisserat sp. nov. Spread of this disease may occur via movement of infested black walnut (Juglans nigra L.) wood. This study evaluated the ability of P. juglandis to colonize J. nigra wood previously treated with various phytosanitation methods. Steam-heated and methyl bromide-fumigated J. nigra logs, as well as kiln-dried natural wane J. nigra lumber (with and without bark) were subsequently exposed to P. juglandis colonization pressure in two exposure scenarios. Following a pheromone-mediated, high-pressure scenario in the canopy of infested trees, beetles readily colonized the bark of steam-heated and methyl bromide-fumigated logs, and were also recovered from kiln-dried lumber on which a thin strip of bark was retained. In the simulated lumberyard exposure experiment, during which samples were exposed to lower P. juglandis populations, beetles were again recovered from bark-on steam-heated logs, but were not recovered from kiln-dried bark-on lumber. These data suggest logs and bark-on lumber treated with phytosanitation methods should not be subsequently exposed to P. juglandis populations. Further beetle exclusion efforts for phytosanitized, bark-on walnut wood products transported out of quarantined areas may be necessary to ensure that these products do not serve as a pathway for the spread of P. juglandis and thousand cankers disease.