Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Insecticidal Activity of Selected Essential Oils against Drosophila suzukii (Diptera: Drosophilidae).

The spotted wing drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), is an invasive polyphagous pest of soft-skinned fruit that has started to threaten small fruit production in Europe since 2008. High reproductive capacity, short generation time, and difficulties in visualizing early damage contribute to its rapid spread. Currently, the control strategy against D. suzukii mostly relies on treatment with synthetic insecticides. Keeping in mind that this pest causes the greatest damage during the harvesting period, control using chemicals is not recommended due to the increased risk of high pesticide residue levels in the fruit. With the aim of reducing the use of insecticides, there is a need for developing an environmentally safer way of control. Alternative solutions could rely on the use of essential oils (EOs), which can be used in conventional and organic production systems. Four essential oils, geranium (Pelargonium graveolens), dill (Anethum graveolens), Scots pine (Pinus sylvestris), and bergamot (Citrus bergamia), were assessed for their insecticidal effects using four different tests (contact toxicity, fumigant-contact toxicity, repellent effect, and multiple-choice tests). The EOs applied were dissolved in acetone at three different concentrations. The most promising one was the geranium essential oil, which had the best effect in all conducted tests, even at the lowest applied concentration. Geranium oil caused very high mortality (95%), even at the lowest concentration applied, after 24 h in the fumigant-contact test, and it caused a mortality of over 85% in the contact test. It was also noticed that geranium demonstrated a deterrent effect by repelling females from laying eggs for four days after at the lowest applied concentration. Scots pine and dill EOs have moderate to strong effects in most tests. The mortality of 100% was achieved for the highest applied concentration in the fumigant-contact and contact tests. Bergamot EO did not have any significant insecticidal activity. Geranium, Scots pine, and dill have great potential to be used as an environmentally friendly way of controlling D. suzukii as they exhibit deterrent, repellent, and insecticidal effects.

Attraction of Egg Parasitoids Trissolcus mitsukurii and Trissolcus japonicus to the chemical cues of Halyomorpha halys and Nezara viridula.

Trissolcus mitsukurii and Trissolcus japonicus are two Asian egg parasitoids associated with different pentatomids such as Halyomorpha halys. Adventive populations of T. mitsukurii were found in Northern Italy, suggesting its employment as a biological control agent (BCA) against H. halys. Nevertheless, to reduce the latter's population, T. japonicus was released in Italy. Releasing an exotic parasitoid requires investigating the interaction between the BCA and the environment to avoid negative impacts on the entomofauna of the new habitat. Trissolcus mitsukurii is mainly associated with Nezara viridula in its native area. Therefore, we investigated and compared the ability of female T. mitsukurii and T. japonicus to distinguish between naturally released cues of H. halys and N. viridula. A single parasitoid was exposed to contact kairomones of both pests to evaluate its modifications in orthokinetic and locomotory behaviour. The behaviour of female T. mitsukurii was also tested on synthetic compounds simulating the cues of the two pentatomids. When naturally released cues were used, T. japonicus preferred the traces of H. halys, while T. mitsukurii preferred N. viridula's cues. Moreover, the attraction of T. mitsukurii to N. viridula's cues was confirmed with synthetic cues. Additional studies are needed to judge if this parasitoid can be used as a BCA.

Reduction of Post-Harvest Injuries Caused by Drosophila suzukii in Some Cultivars of Sweet Cherries Using a High Carbon Dioxide Level and Cold Storage.

Efficient strategies are required in sweet cherry fruits to control the spotted wing drosophila (SWD), Drosophila suzukii, due to its adverse economic effect on farmers. Cold storage (CS) and storage with elevated carbon dioxide (CO2) are environmentally safe approaches for the pest control of stored fresh fruit. These strategies are effective in controlling a wide variety of insect species, without allowing toxic compounds to accumulate. The purpose of this study was to assess the effectiveness of a post-harvest application of CO2 treatment at 50%, cold treatment at 4 °C (CT), and a combination of both (CO2-CT) in controlling the early stages of SWD within four cultivars of freshly harvested cherry fruit, namely "Burlat-Bigarreau", "Giorgia", "Ferrovia", and "Lapins". In addition, an evaluation of the quality attributes of the cherries (skin firmness, berry firmness, strong soluble material, and titratable acidity) was carried out at harvest and after 10 and 20 days of storage. All treatments significantly reduced the rate of emergence of SWD when compared to the control (untreated cherry at 24 °C), and 100% SWD mortality was obtained in Burlat-Bigarreau (CO2-CT). In addition, over the entire storage time, the quality parameters were preserved in the samples stored at 4 °C and in the samples with combined treatments in comparison with the control.

Influence of Distance from the Host on Parasitisation by Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae).

The olive fruit fly, Bactrocera oleae, is considered the main olive pest worldwide, and has been the target of biological control programmes through the release of the braconid parasitoid Psyttalia concolor. Laboratory tests were performed to evaluate the influence of distance from the host on parasitisation, placing larvae of the substitute host Ceratitis capitata at seven distances (0, 0.5, 1, 1.5, 2, 2.5, 3 mm) and four different time periods (7, 15, 30, 60 min). Moreover, field collected olives of Ogliarola Barese cultivar infested by B. oleae were exposed to P. concolor females to confirm its ability to parasitise B. oleae in small olives. Psyttalia concolor oviposition was inhibited at 2.5 and 3 mm due to the ovipositor length of the parasitoid females (2.7 mm). Hosts were easily parasitised at distances between 0 and 1.5 mm. The thin fruit pulp (up to 3.5 mm) of field collected olives allowed the parasitisation to occur also in mature fruits. At the best combination distance/time (0 mm, 30 min), tests performed with different larvae/parasitoid female ratio showed an increasing emergence of P. concolor (from 20% to 57%) with larvae/parasitoid ratio increasing from 0.11 to 0.74. The results of the present study might optimise the mass rearing of P. concolor, through a proper setting of its parameters, such as the host/parasitoid ratio, exposure distances, and interaction time.

Genetic variability in Italian populations of Drosophila suzukii.

BACKGROUND: Drosophila suzukii is a highly destructive pest species, causing substantial economic losses in soft fruit production. To better understand migration patterns, gene flow and adaptation in invaded regions, we studied the genetic structure of D. suzukii collected across Italy, where it was first observed in 2008. In particular, we analysed 15 previously characterised Simple Sequence Repeat (SSR) markers to estimate genetic differentiation across the genome of 278 flies collected from nine populations. RESULTS: The nine populations showed high allelic diversity, mainly due to very high heterozygosity. The high Polymorphism Information Content (PIC) index values (ranging from 0.68 to 0.84) indicated good discrimination power for the markers. Negative fixation index (F IS) values in seven of the populations indicated a low level of inbreeding, as suggested by the high number of alleles. STRUCTURE, Principal Coordinate and Neighbour Joining analysis also revealed that the Sicilian population was fairly divergent compared to other Italian populations. Moreover, migration was present across all populations, with the exception of the Sicilian one, confirming its isolation relative to the mainland. CONCLUSIONS: This is the first study characterising the genetic structure of the invasive species D. suzukii in Italy. Our analysis showed extensive genetic homogeneity among D. suzukii collected in Italy. The relatively isolated Sicilian population suggests a largely human-mediated migration pattern, while the warm climate in this region allows the production of soft fruit, and the associated D. suzukii reproductive season occurring much earlier than on the rest of the peninsula.