A loop-mediated isothermal amplification (LAMP) assay for rapid identification of Ceratitis capitata and related species.

True fruit flies (Tephritidae) are among the most destructive agricultural pests in the world, attacking a wide range of fruits and vegetables. The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous species but, being widely established in the Mediterranean region, is not considered as a EU quarantine pest. Hence, it is important to discriminate Ceratitis capitata from non-EU tephritid species, present in imported fruit and vegetables, as non-EU species have a quarantine status. However, morphological identification of tephritid larvae, the most frequently intercepted stage in non-EU produce, is difficult and an easy-to-use molecular diagnostic tool would be helpful for rapid species identification. Therefore, a loop-mediated isothermal amplification (LAMP) method was developed for C. capitata and non-EU tephritids Ceratitis cosyra group1 and Ceratitis species from the FARQ complex, C. fasciventris, C. anonae, C. rosa and C. quilicii. LAMP assays were run with DNA from ILVO collected specimens and DNA samples collected during previous research surveys. LAMP primers were species-specific, with LAMP amplification occurring within 45 minutes for the targeted species. In addition, LAMP assays were successful for all C. capitata life stages or a limited amount of tissue. To conclude, the LAMP assays developed in this study were able to distinguish C. capitata from non-EU Tephritidae species and could be a useful tool for the rapid identification of C. capitata.

Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo.

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60-73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA.

High population densities of Macrolophus pygmaeus on tomato plants can cause economic fruit damage: interaction with Pepino mosaic virus?

BACKGROUND: The zoophytophagous predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a successful biocontrol agent against several pest species in protected tomato crops. This predator is considered to be harmless for the crop. However, in recent years, Heteroptera feeding punctures on tomato fruit in Belgian and Dutch greenhouses have been misinterpreted as Pepino mosaic virus (PepMV) symptoms. In this study, three hypotheses were tested: (1) M. pygmaeus causes fruit damage that increases with population density and surpasses economic thresholds; (2) the presence of prey or alternative prey reduces the damage; (3) an infection of the tomato plants by PepMV triggers or aggravates M. pygmaeus fruit damage. RESULTS: At increasing M. pygmaeus densities, the severity of fruit damage increased from a few dimples towards yellowish discoloration and deformed fruits. A correlation with an infection with PepMV was found. The severity of the symptoms was independent of the presence of prey. A minimum economic density threshold was estimated at 0.32 M. pygmaeus per leaf. CONCLUSION: M. pygmaeus can cause economic damage to tomato fruits at densities common in practice. An infection of the plants with PepMV enhances fruit symptoms significantly. Interacting plant defence responses are most likely the key to explaining this, although confirmation is required. © 2015 Society of Chemical Industry.

Efficacy of entomopathogenic nematodes against larvae of Tuta absoluta in the laboratory.

BACKGROUND: Previous studies have indicated the control potential of entomopathogenic nematodes (EPNs) against Tuta absoluta. Here, the potential of Steinernema feltiae, S. carpocapsae and Heterorhabditis bacteriophora is studied when applied against larvae of T. absoluta inside leaf mines in tomato leaf discs by means of an automated spray boom. RESULTS: The studied EPN species were effective against all four larval instars of T. absoluta but caused higher mortality in the later instars (e.g. fourth instar: 77.1-97.4% mortality) than in the first instars (36.8-60.0% mortality). Overall, S. feltiae and S. carpocapsae yielded better results than H. bacteriophora. Steinernema carpocapsae and H. bacteriophora performed better at 25 °C (causing 55.3 and 97.4% mortality respectively) than at 18 °C (causing 12.5 and 34.2% mortality respectively), whereas S. feltiae caused 100% mortality at both temperatures. Under optimal spraying conditions and with the use of Addit and Silwet L-77 adjuvants, a reduced dosage of 6.8 infective juveniles (IJs) cm(-2) yielded equally good control as a recommended dosage of 27.3 IJs cm(-2) . CONCLUSION: Under laboratory conditions, S. feltiae and S. carpocapsae showed good potential against the larvae of T. absoluta inside tomato leaf mines. Results need to be confirmed in greenhouse experiments. © 2015 Society of Chemical Industry.