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Bactrocera oleae (Rossi) (Diptera: Tephritidae) Response to Different Blends of Olive Fruit Fly-Associated Yeast Volatile Compounds as Attractants

1
Department of Applied Science, Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21000 Split, Croatia
2
Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Svetošimunska Cesta 25, 10000 Zagreb, Croatia
3
Department of Molecular Genetics and Physiology of Plants, Ruhr University Bochum, Universität 150, ND 3/30 P.O. Box 44, 44801 Bochum, Germany
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Gerardo Fernández Barbero
Agronomy 2022, 12(1), 72; https://doi.org/10.3390/agronomy12010072
Received: 24 November 2021 / Revised: 24 December 2021 / Accepted: 25 December 2021 / Published: 29 December 2021

Abstract

The olive fruit fly, Bactrocera oleae (Rossi) is economically the most important olive pest, causing yield losses in all olive growing areas where is detected. Considering that EU requires the reduction of pesticide use by up to 100% by 2050, more effective non-pesticide lures for B. oleae monitoring and/or controlling are needed. This research was aimed at investigating the attractiveness of different blends of olive fruit fly-associated yeast volatiles toward B. oleae. Three blends of olive fruit fly-associated yeast volatiles: isoamyl alcohol and 2-phenethyl alcohol; isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; and isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were selected and tested on yellow sticky traps for attraction of B. oleae in olive orchard. Results showed that traps containing all tested blends of olive fruit fly-associated yeast volatile compounds, in total, were significantly more attractive to B. oleae and were not significantly attractive to green lacewings, compared to the control. Among them, the most promising was the one containing the blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate because its attractiveness was constantly significant during investigation compared to the others. This blend in the future could lead us to the discovery of a new attractant for the monitoring and/or controlling of B. oleae.
Keywords: insect behavior; correlation with climatic parameters; isoamyl alcohol; isobutyl acetate; 2-phenethyl acetate; 2-phenethyl alcohol; volatility; yeast; green lacewings; Olea europea L. insect behavior; correlation with climatic parameters; isoamyl alcohol; isobutyl acetate; 2-phenethyl acetate; 2-phenethyl alcohol; volatility; yeast; green lacewings; Olea europea L.

1. Introduction

Olive fruit fly, Bactrocera oleae, (Rossi) (Diptera: Tephritidae) is the most harmful pest in all of the olive growing regions [1,2,3,4] and its presence has a negative effect on the entire olive production chain [5]. Bactrocera oleae is a monophagous that feeds exclusively on olive fruits of genus Olea, including Olea europaea L., O. verrucosa Willd., and O. chrysophylla Lam. [6]. Female flies lay their eggs in olive fruits whereas larvae feed on the pulp [3,6] causing fruit losses of up to 15% [7]. Collier and Van Steenwyk [8] point out that a total yield loss can occur in seasons with a high population of the olive fruit fly.
For years, the use of pesticides was the main method for B. oleae controlling [9,10]. It is an effective method that provides yields to olive growers, but also negatively affects the environment, beneficial organisms and olives [7], if used frequently. This is evident through the pesticide residues that have been detected in olive oils, olive fruits and olive growing areas [9,11,12,13] causing disruption of biodiversity [14] and insecticide resistance [9,15]. Therefore, the European Union is seeking to reduce pesticide use by 50% by 2030 and 100% by 2050 [2]. For this reason, implementation and improvement of non-pesticide control methods that would not have a negative effect on the environment or biodiversity, such as biotechnical, have an important role in olive protection.
Many researchers have reported that olive fruit fly is attracted to semiochemicals [16], host plant volatiles [17,18,19,20], microbial volatiles [21,22,23,24] and yeast volatiles [25]. Considering that, the essential part of biotechnical methods for olive fruit fly monitoring and/or controlling should be the implementation of their use as attractants. Semiochemicals, such as pheromones, are already in use in various pest control methods, such as “attract and kill”, mass trapping, sterile insect technique (SIT), mating disruption and monitoring [26], while plant, microbial or yeast volatile synthetic compounds, as new formulations of attractants, are not. Pheromones were usually used targeting specific sex [27], while yeast volatile compounds, as attractants, may be more successful because both sexes and all ages are targeted [25,28]. Recent studies have confirmed many yeast–insect interactions [29,30,31,32,33], while some of them point out that better understanding of yeast-tephritids interaction is needed and could lead to the development of new insect attractants [29,34]. Interactions between B. oleae and active or inactive formulation of yeasts associated with infested olives, olive fruit fly and other insects have been reported by Vitanović et al. [35]. During investigation authors also noticed that odors of some tested yeasts were highly attractive to green lacewings, catching large number of them. Green lacewings (Chrysopidae sp., Neuroptera) are one of the most important beneficial insects in olive orchards [36], as predators of olive pests such as Prays oleae [37], Euphyllura olivina [38,39] and Saissetia oleae [37,40]. Other insect predators as well as groups of parasitoids also play an important role in olive orchard as beneficial insects [41]. Therefore, many researchers are concerned because high number of beneficial insects is being caught on different types of traps in use [6,26,35,42,43].
According to Vitanović et al. [35], the active formulation of yeast strain Lachancea thermotolerans, isolated from B. oleae adult, was the most attractive to olive fruit flies as well as green lacewings in the olive orchard between 12 tested yeast strains. HE-SPME–GC/MS analysis showed that L. thermotolerans produced a unique and repeatable profile of volatiles, consisting of isoamyl alcohol, 2-phenethyl alcohol, isobutyl acetate and 2-phenethyl acetate, that were the most abundant compounds found [35]. These olive fruit fly (OFF)-associated yeast volatile compounds individually were attractive to olive fruit fly in both laboratory and in the field [35]. It was the first report that yeast volatiles, isolated from infested olive fruits or produced by yeasts associated with B. oleae or other insects, attracted olive fruit fly adults. In addition, the authors reported that some combinations of these volatiles, on yellow sticky (YS) traps, caught more olive fruit flies compared to control traps in olive orchards.
Therefore, the aim of this study was to investigate whether other blends of volatile compounds, produced by yeasts associated with olive fruit fly, be attractive to their adults and would those traps also be attractive to green lacewings. The influence of climatic parameters on the abundance of B. oleae and green lacewings caught on those traps was also studied. Furthermore, daily presence of OFF-associated yeast volatile compounds was tested during seven days in the olive canopy and analyzed by HS-SPME–GC/FID (headspace-solid phase microextraction–gas chromatography/flame ionization detection).
Results of this study can lead to discovery of new attractants that would be highly attractive to olive fruit fly and not attractive to green lacewings. This could be a useful tool for monitoring and/or controlling of B. oleae, as well as the other tephritids.

2. Materials and Methods

2.1. Synthetic Compounds

All chemical standards used for the bioassays were obtained from commercial sources. Isoamyl alcohol (≥99% purity) was purchased from Kemika d.d. (Zagreb, Croatia), while 2-phenethyl alcohol (≥99% purity) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Isobutyl acetate and 2-phenethyl acetate (98% purity) were purchased from Alfa Aesar (Kandel, Germany). Hexane (≥99% purity) was purchased from VWR International bvba (Leuven, Belgium) and was used as a control compound.

2.2. Field Bioassay

Yellow sticky (YS) traps (17 × 24 cm, Bio Plantella, UNICHEM d.o.o., Vrhnika, Slovenia) with dispensers were used in the olive orchard to test olive fruit fly response to three blends of OFF-associated yeast volatile compounds. 4 mL polypropylene vial (Cryotubes, BRAND GMBH + CO KG, Wertheim, Germany) with a 3 mm diameter hole onto vial cap served as a simple, slow-release chemical dispenser [25,44]. Dispensers were attached on YS traps with PVC cable ties (Brati Ritoša d.o.o., Pazin, Croatia) containing 0.1 g of cotton roll (Tosama d.o.o., Domžale, Slovenia) and 1 mL of pure chemical standard. Each chemical standard was added in a separated vial fixed with a cable tie in two or three blend combinations, as described below.
The trapping experiments consisted of four treatments: (1) 1:1 combination of two compounds (isoamyl alcohol and 2-phenethyl alcohol), (2) 1:1:1 combination of three compounds (isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate), (3) 1:1:1 combination of three compounds (isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate), and (4) control traps containing only hexane [35].
The YS traps with blends of OFF-associated yeast volatile compounds were placed in 0.5 ha olive orchard at Duilovo, split (coordinates: 43°30′19.4″ N 16°29′56.1″ E) from the beginning of July until the end of October 2018. During the investigation, climatic parameters (temperature, relative humidity and rainfall) were also observed. Climatic parameters data were taken from the Croatian Meteorological and Hydrological Service weather station (coordinates: 43.5167° N, 16.4167° E) (Table 1).
Traps were placed at 1.5–2 m height in the open shade of olive canopy in a random configuration between every third row in the olive orchard with the distance more than five trees apart. Every seven days, YS traps with dispensers were changed and the number of flies was recorded. The number of green lacewings (Neuroptera: Chrysopidae) was also recorded on the same YS traps. Each treatment was replicated 16 times at the site, in duplicate (N = 128). The YS traps were rotated forward sequentially by one location following each sampling date.
Number of flies per trap per day (FTD) of B. oleae was calculated, as follows. The number of B. oleae flies caught on each of tested YS traps (YS traps containing blend 1, YS traps containing blend 2, YS traps containing blend 3 and YS traps containing hexane) per day for each of three adult flights of B. oleae (1st generation, 2nd generation and 3rd generation) was calculated by adding the captures from each tested YS trap in each of the adult flight of B. oleae and dividing the count by the number of YS traps and exposed days [45]. It is an index of trapping success that makes no assumptions on trap efficiency caused by other factors affecting trapping success, except that all insects of the target species are equally attracted.

2.3. Measurement of Volatile Release

An additional study was conducted to analyze the volatile release rate over time from dispensers in three blends of OFF-associated yeast volatile compounds. Five dispensers containing volatile compounds (isoamyl alcohol, 2-phenethyl alcohol, 2-phenethyl acetate, isobutyl acetate and hexane) used in the previously conducted trial were attached to YS traps and hanged in the same olive orchard in split. The releasing devices were tested in October 2021 following the same protocol as described above. Each dispenser with a single OFF-associated yeast volatile compound, set in triplicate from each blend combination, was taken out every day from the olive canopy and the volatile release was analyzed during seven days using SPME–GC/FID. Each sampled dispenser containing OFF-associated yeast volatile compound was covered with Parafilm® until analysis.
Solid-phase microextraction (SPME) fiber (Divinylbenzene/Carboxen/Polydimethylsiloxane) was inserted through the parafilm for volatile collection. Prior to use, fiber was conditioned according to the manufacturer’s instructions. Sample extraction was performed in the headspace above the sample in 4 mL polypropylene vials. After equilibration, the vial content was absorbed onto SPME fiber, the fiber was retracted and directly inserted into the GC injection port for thermal desorption. All analyses were carried out using GC chromatograph (Nexis GC-2030, Shimadzu, Japan) coupled with a flame ionization detector. Compound analysis was performed using SH-Rtx-Wax capillary column (30 m × 0.25 mm × 0.25 µm) and the compounds were identified by comparing their retention times with those of standards analyzed under the same conditions. Helium (99.999% purity) was used as the carrier gas at a constant column flow of 1 mL/min. The oven temperature was held for 1 min from 40 °C to 225 °C using 30 °C/min. The total analysis time was 8.17 min. Injector temperature was kept at 200 °C.

2.4. Statistical Analysis

2.4.1. Analysis of Variance

One-way analysis of variance (ANOVA) was performed by SPSS software, version 25.0 (IBM Corporation, New York, NY, USA) to determine the difference between attractions of olive fruit flies and green lacewings for different blends of OFF-associated yeast volatile compounds. The data for captured insects was square root transformed before one-way ANOVA analysis. If there was a statistically significant effect between attraction of volatiles blends with the observed insects, least significant difference (LSD) test at the significance level of p ≤ 0.05 was performed.

2.4.2. Pearson Correlation Coefficient

In order to observe if there is a correlation between the abundance of olive fruit flies and green lacewings caught on YS traps containing different blends of OFF-associated yeast volatile compounds and climatic parameters (temperature, relative humidity and rainfall), Pearson correlation coefficient analysis was performed. The correlation was considered significant at the level of p ≤ 0.05. The analysis was performed using SPSS software, version 25.0 (IBM Corporation).

3. Results and Discussion

3.1. Bactrocera Oleae and Green Lacewings Attraction to Three Blends of OFF-Associated Yeast Volatile Compounds in Olive Orchard

During the investigation, from the beginning of July until the end of October 2018, 5961 olive fruit fly adults were caught on YS traps containing three blends of OFF-associated yeast volatile compounds (Figure 1), among which a significant difference was noted (p = 0.002).
These results support the hypothesis that B. oleae responds to volatile compounds produced by yeasts associated with olive fruit flies, in the field. Similar results were obtained by Vitanović et al. [25]. Their study was focused on olive fruit fly behavior when exposed to yeast volatile compounds associated with olives, olive fruit fly and other insects, both in the laboratory and in the field. The authors also tested olive fruit fly attraction to some blends of yeast volatile compounds in olive orchards. Those blends had different influence on olive fruit flies’ behavior, meaning that some of them were not attractive to flies [25]. Responses of other tephritid fruit flies to yeast volatile compounds have also been reported [29,46].
Yellow sticky traps containing a volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were the most effective in capturing B. oleae adults (2517), followed by YS traps that contained volatile blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (2118), and YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (1326) (Figure 1). These results were not expected because a recent study confirmed that B. oleae was more attracted to YS traps containing blends of three alcohols than YS traps that contained a binary volatile blend of alcohol and ester [25]. All tested OFF-associated yeast volatile blends in our research captured significantly greater numbers of olive fruit flies than the control traps (Figure 1). On the contrary, the results of Vitanović et al. [25] showed that one of the investigated blends of OFF-associated yeast volatiles (isoamyl alcohol, 2-phenethyl alcohol, 2-phenethyl acetate, isobutyl acetate and isobutanol) was not significantly different compared to control traps. In our study, YS traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (1258.50 ± 23.33; p = 0.001) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (1032.00 ± 35.36; p = 0.002) were significantly more attractive to olive fruit flies compared to YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (710 ± 98.99; p = 0.036) or YS traps containing hexane as the control (Figure 1). Moreover, YS traps that contained a binary blend of isoamyl alcohol and 2-phenethyl alcohol (710 ± 98.99; p = 0.036) caught a significantly greater number of flies than the control traps. Results also show that YS traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (1258.50 ± 23.33) did not significantly differ in B. oleae captures compared to YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (1032.00 ± 35.36; p = 0.059) (Figure 1). The activity of YS traps containing all tested blends compared to the control was expected because isoamyl alcohol and 2-phenethyl acetate were the most attractive volatile compounds to olive fruit flies in both laboratory and in the field, as well as was isobutyl acetate but only in the field [25]. Besides that, some researchers have confirmed isoamyl alcohol as an olive plant volatile compound, identifying it in leaves [47] or in fruits [1] and, as it is well known, host plants volatiles are attractive to B. oleae [1,17,18,19]. Davis et al. [44] state that isoamyl alcohol was also attractive to many of dipterans that were captured during his investigation. However, it is known that alcohols and esters are emitted by ripe or fermenting fruits [29,48] and our results may be related to that fact. Davis and Landolt [48] also claim that some of the fruit flies, such as Queensland fruit fly (Bactrocera tryoni, Froggatt) and Mexican fruit fly (Anastrepha ludens, Loew) respond to alcohols and esters as a food attractant. Nevertheless, all the above facts may be the reason why in our investigation on YS traps containing two blends of alcohols and esters together were significantly more attractive to olive fruit fly than YS traps containing only blends of alcohols.
Figure 1 also presents the results of green lacewings (Neuroptera: Chrysopidae) captured on the same YS traps containing different tested blends of OFF-associated yeast volatiles. During the investigation, 438 green lacewings were caught. The differences in attractiveness between YS traps that contained three blends of OFF-associated yeast volatile compounds to green lacewings were also tested. There was no significant difference in green lacewings attraction between YS traps that contained different tested blends. Furthermore, there was no significant difference in green lacewings catch between YS traps that contained tested blends compared to control YS traps (p = 0.961) (Figure 1). Mutualism between yeasts and green lacewings was confirmed long ago [49], but the relation between them is still not understood [50]. According to the previous discovery that some of the certain yeast strains associated with olives, olive fruit flies or other insects were attractive to green lacewings [35,51], we decided to investigate whether YS traps containing OFF-associated yeast volatile compounds be attractive to them, too. The results of our study are promising because olive fruit flies were highly attracted to YS traps containing different blends of OFF-associated yeast volatile compounds, while green lacewings were not. As is mentioned, many species of the green lacewing are beneficial insects, especially in olive orchards [36,37,38,40,52]. The implementation of new attractants that might be highly attractive to target pests and would not disturb their biodiversity, would be of great importance for olive protection.
Figure 2 shows the population dynamic (a) and FTD (b) of B. oleae for each of the tested YS traps containing blends of OFF-associated yeast volatile compounds during research, where adult flights of three generations of olive fruit fly are clearly seen.
In this part of the Mediterranean region, B. oleae usually develops three generations, but in years with favorable climatic conditions for its development, it can develop four generations [53,54]. During the investigation, for each olive fruit fly generation, significant differences in flies’ attraction to YS traps containing tested blends were found (Figure 2a). The most differences were noticed during the 2nd generation of B. oleae adult flight. Yellow sticky traps containing volatile blend of isoamyl alcohol, 2-phenethyl acetate, and isobutyl acetate (321 ± 35.36) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (314.5 ± 28.99) caught significantly more olive fruit flies compared to YS traps that contained a blend of isoamyl alcohol and 2-phenethyl alcohol (142.5 ± 95.46; p = 0.045 and p = 0.049, respectively) and control traps. These results may be related with temperatures because during the flight of 2nd generation, the values of the mean temperature were in the optimal range (24.2 °C–27.8 °C) for olive fruit fly reproduction, flying, and development [45] (Table 1). Results in Figure 2a show that between YS traps that contained a blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (321 ± 35.36) and YS traps that contained blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (314.5 ± 28.99), no difference in B. oleae attraction was found (p = 0.939), as well as between YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol and YS traps that contained hexane (142.50 ± 95.46 and 118.50 ± 19.09, respectively; p = 0.764). Observing population dynamics of the 1st and 3rd generation, YS traps containing isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were significantly more attractive to B. oleae compared to control traps (p = 0.034 and p = 0.028 respectively). The other two tested blends did not differ in olive fruit fly catch compared to control traps. Results in the same figure also show that during the two mentioned generations, YS traps containing different blends of OFF-associated yeast volatile compounds did not significantly differ in B. oleae captures.
Figure 2b shows the FDT during the investigation. The rate of population change was stable within each of the generations but varied greatly between them for all the tested traps. In general, FTD values for each of the tested YS traps decreased over generations, reaching their highest value in the 3rd generation, for each of the tested blends as well as the hexane (Figure 2b). Values of FTD for each of the tested YS traps decreased with increasing temperature and with decreasing relative humidity in the olive orchard (Figure 2b, Table 1).

3.2. Influence of Climatic Parameters on the Abundance of Bactrocera Oleae and Green Lacewings Caught on YS Traps Containing Different Blends of OFF-Associated Yeast Volatile Compounds

The attractiveness of YS traps containing tested blends to both, B. oleae and green lacewings, was influenced by climatic parameters (Table 2). Climatic parameters differed among different generations of olive fruit fly (Table 1). Bactrocera oleae is sensitive to high temperature, meaning that temperature above 31 °C causes mortality of all developmental pest stages [45]. Additionally, olive fruit fly activity decreases below 23 °C and completely stops at 17 °C. Observing the average values of all climatic parameters between three generations of B. oleae, it was noticed that the lowest and the highest values were recorded during the 1st or 3rd olive fruit fly generation (Table 1). Climatic conditions with the highest temperatures (up to 31.6 °C) and the lowest relative humidity values (up to 34%) were noticed during July and the beginning of August when the flight of 1st generation was recorded. Contrarily, during the flight of the 3rd generation in September and October, climatic conditions with the lowest temperatures (up to 14.5 °C) and the highest relative humidity values (up to 88%) were found. In both cases, the values of temperature and relative humidity were not favorable for B. oleae flying [55,56]. During the investigation, drought-marked climatic conditions were noticed (Table 1). The highest amount of rainfall (67.10 mm) was recorded at the end of October during flight of 3rd B. oleae generation.
The abundance of B. oleae caught on YS traps containing blend of isoamyl alcohol and 2-phenethyl alcohol (r = −0.739; p = 0.001) was significantly negatively correlated with temperature (Table 2). The abundance of B. oleae caught on the yellow sticky traps containing other tested blends was not correlated with temperature (Table 2). In addition to isoamyl alcohol, these blends also contained esters, of which blend 3 contained two esters, 2-phenethyl acetate, and isobutyl acetate. Yellow sticky traps that contained blend of alcohol and two esters were significantly more attractive to B. oleae compared to YS traps containing two alcohols during each of three B. oleae generations (Figure 2a,b).
For YS traps containing a blend of isoamyl alcohol and 2-phenethyl alcohol, besides the temperature, significant positive correlation with rainfall amount (r = 0.572; p = 0.041) was also observed (Table 2). In the same table, significant negative correlation between temperature and abundance of B. oleae caught on YS traps containing only hexane was also noticed (r = −0.632; p = 0.009). However, temperature as a climatic parameter was negatively correlated with the abundance of olive fruit fly in both cases, regardless of whether it was a control trap or YS trap containing isoamyl alcohol and 2-phenethyl alcohol. On the contrary, the abundance of green lacewings caught on YS traps that contained other tested blends was significantly positively correlated with temperature, as follows: blend of isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate (r = 0.729; p = 0.002) and blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate (r = 0.618; p = 0.014) (Table 2). In general, the abundance of green lacewings decreased with decreasing temperature, especially when the temperature dropped below 20 °C (Table 1, Supplementary Figure S1). The significant negative correlation was noticed between the abundance of green lacewings caught on YS traps containing isoamyl alcohol, 2-phenethyl alcohol, and 2-phenethyl acetate (r = −0.557, p = 0.048) and rainfall amounts (Table 2).
Relative humidity as climatic parameter did not show correlations with the abundance of B. oleae neither with green lacewings (Table 2).
Observing the population dynamic of both insects, we can conclude that the abundance of B. oleae increased with a temperature drop and relative humidity decreasing in the end of summer with the most significant peak in the beginning of October, while the abundance of green lacewings decreased, despite the YS traps difference (Figure 2a and Supplementary Figure S1). At the same time, YS traps containing blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were the most attractive to olive fruit fly among all tested traps with significance in relation to control traps (Figure 2a). These results are promising because October is olive harvest time, so the use of pesticides for B. oleae controlling is not allowed and at the same time the abundance of the olive fruit fly in the olive orchard is high.

3.3. Daily Volatile Release of Investigated Blends in the Field

Daily monitoring of the individual evaporation of each of the tested OFF-associated yeast volatile compounds was carried out in October 2021. As it is shown in Figure 3, a total of five volatile compounds (two alcohols, two esters and one alkane) were tested and identified using the HS-SPME–GC/FID method.
Samples were measured daily, for seven days, and analyzed using HS-SPME–GC/FID yielded peak area results, reflecting the amount of each sample present in the vial. Peak area of the measured samples was compared, and they reflected their proportion in the vials exposed in olive orchard. Hexane showed the largest decrease in peak area (89.29%), as did esters, especially isobutyl acetate (54.91%), while 2-phenethyl acetate decreased to a much lesser extent (25.81%). In contrast, by measuring alcohols in dispensers, isoamyl alcohol and 2-phenethyl alcohol showed an increase of 8.21 and 11.32%, respectively (Figure 3). During all tested days, as well as the last day of testing, the presence of all tested compounds was confirmed (Supplementary Figure S2) after exposure to outdoor field conditions.
Vitanović et al. [25] previously stated that these tested OFF-associated volatile compounds individually were attractive to B. oleae in the field, thus it was crucial to test their different blends in the olive orchard and to determine the release rate for each of them individually. Our results showed, by comparing the levels of OFF-associated yeast volatile components, that all of them were present in dispensers during their presence in the olive orchard (Figure 3). However, to our knowledge, the release of tested OFF-associated yeast volatile compounds has not been studied, both under controlled conditions and in the field. Important factors influencing the release of a chemical compound include temperature, airflow, wind, sunlight exposure, precipitation and relative humidity. In addition to these factors, the release rate is also affected by the characteristics of the compound itself and by the type of the used dispenser [57]. The vapor pressure of a compound directly affects its volatility, so a compound that has a higher vapor pressure, at a certain temperature evaporates faster than a compound that has a lower vapor pressure [28]. This explains the faster evaporation of isobutyl acetate release rate for OFF-associated yeast volatile compounds. In the case of dispensers placed in the field, variations in the release rate can lead to an increase in volatility of the chemical, changes in their composition, as well as degradation of the material of the dispenser in which it is applied [58].

4. Conclusions

The behavioral response of B. oleae to certain blends of OFF-associated yeast volatile compounds was confirmed. Yellow sticky traps containing all tested blends of OFF-associated yeast volatile compounds were significantly more attractive to olive fruit fly than the control traps, but traps that contained blends of alcohols and esters still stood out. These YS traps caught significantly more flies compared to YS traps containing blends of alcohols. Throughout the investigation, YS traps that contained blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate were constantly significant in B. oleae attraction compared to control traps, during the 1st, 2nd and 3rd generation. During the 2nd generation of B. oleae, YS traps containing isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate were also significantly more attractive to flies than the control traps but did not differ from YS traps containing other blend of alcohol and esters. Regarding climatic parameters, the abundance of olive fruit flies caught only on YS traps containing a blend of isoamyl alcohol and 2-phenethyl alcohol was significantly correlated with the temperature and rainfall.
None of the YS traps containing tested blends of OFF-associated yeast volatile compounds were attractive to green lacewings compared to control traps.
Furthermore, the presence of all tested OFF-associated yeast volatile compounds odor was confirmed during seven days in the olive canopy after exposure to outdoor field conditions. Esters, especially isobutyl acetate, as well as hexane, showed the largest decrease in abundance in dispensers exposed in the olive orchard.
Therefore, we can conclude that YS traps containing certain blends of OFF-associated yeast volatile compounds could be useful attractants for monitoring and/or controlling of B. oleae, especially blend of isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate, as the most attractive among them.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12010072/s1, Figure S1: Green lacewings population dynamic for three tested blends of OFF-associated yeast volatile compounds and control, in olive orchard during investigation. Figure S2: Enlarged view of HS-SPME-GC/FID chromatogram of hexane and four OFF-associated yeast volatile compounds (compared with matching retention time with standards): (1) hexane, RT = 0.8 min; (2) isobutyl acetate, RT = 2.45 min; (3) isoamyl alcohol, RT = 3.75 min; (4) 2-phenethyl alcohol, RT = 6.7 min; (5) 2-phenethyl acetate, RT = 7 min.

Author Contributions

Conceptualization and design of the experiments, E.V., M.J.Š., M.P. and M.V.B.; performed the laboratory and field experiments and analyzed the data, A.B., F.B., M.P., M.J.Š., M.V.B. and E.V.; statistical analyses, M.P., M.J.Š. and F.P.; writing—original draft preparation, A.B., F.B., M.P. and M.V.B.; writing-review and editing M.J.Š., and E.V. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the project KK.01.1.1.04.0002 “New methods in olive pests controlling using plant volatiles”, Split, Croatia, funded by European Union.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions generated for this study are included in the article/Supplementary Material; the data presented in this study are available on request from the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Total number of Bactrocera oleae and green lacewings captured on yellow sticky (YS) traps containing three blends of olive fruit fly-associated yeast volatile compounds in an olive orchard. Blends of volatile compounds were: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane. Bars labeled with different uppercase letter indicate significant differences between tested YS traps for olive fruit fly, while bars labeled with different lowercase letters indicate significant differences between tested YS traps for green lacewings; (LSD test at p ≤ 0.05).
Figure 1. Total number of Bactrocera oleae and green lacewings captured on yellow sticky (YS) traps containing three blends of olive fruit fly-associated yeast volatile compounds in an olive orchard. Blends of volatile compounds were: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane. Bars labeled with different uppercase letter indicate significant differences between tested YS traps for olive fruit fly, while bars labeled with different lowercase letters indicate significant differences between tested YS traps for green lacewings; (LSD test at p ≤ 0.05).
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Figure 2. (a) Bactrocera oleae population dynamics for three tested blends of olive fruit fly (OFF)-associated yeast volatile compounds and control, in olive orchard during investigation. Blends of OFF-associated yeast volatile compounds were attached on yellow sticky (YS) traps as follows: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane. Different uppercase letters indicate significant differences (LSD test at p ≤ 0.05) between tested traps separately for adult flight of each Bactrocera oleae generation (adult flight of 1st gen—first; 2nd gen—second; 3rd gen—third generation); (b) FTD (flies per trap per day) change between adult flights of three generations of Bactrocera oleae (1st–3rd) in relation to YS traps containing different blends of OFF-associated yeast volatile compounds and control, in olive orchard during investigation. Blends of OFF-associated yeast volatile compounds were hung up on yellow sticky traps as follows: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane.
Figure 2. (a) Bactrocera oleae population dynamics for three tested blends of olive fruit fly (OFF)-associated yeast volatile compounds and control, in olive orchard during investigation. Blends of OFF-associated yeast volatile compounds were attached on yellow sticky (YS) traps as follows: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane. Different uppercase letters indicate significant differences (LSD test at p ≤ 0.05) between tested traps separately for adult flight of each Bactrocera oleae generation (adult flight of 1st gen—first; 2nd gen—second; 3rd gen—third generation); (b) FTD (flies per trap per day) change between adult flights of three generations of Bactrocera oleae (1st–3rd) in relation to YS traps containing different blends of OFF-associated yeast volatile compounds and control, in olive orchard during investigation. Blends of OFF-associated yeast volatile compounds were hung up on yellow sticky traps as follows: (Blend 1) 1:1 isoamyl alcohol and 2-phenethyl alcohol; (Blend 2) 1:1:1 isoamyl alcohol, 2-phenethyl alcohol and 2-phenethyl acetate; (Blend 3) 1:1:1 isoamyl alcohol, 2-phenethyl acetate and isobutyl acetate; and (Control) control traps containing only hexane.
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Figure 3. Daily release of four olive fruit fly-associated yeast volatile compounds (isoamyl alcohol, 2-phenethyl alcohol, 2-phenethyl acetate, isobutyl acetate) and hexane as control during seven days.
Figure 3. Daily release of four olive fruit fly-associated yeast volatile compounds (isoamyl alcohol, 2-phenethyl alcohol, 2-phenethyl acetate, isobutyl acetate) and hexane as control during seven days.
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Table 1. Climatic parameters data during the investigation.
Table 1. Climatic parameters data during the investigation.
DateTemperature (°C)Relative Humidity (%)Rainfall (mm)
MeanMinMaxMeanMinMaxTotalMinMax
11 July 2018.26.325.527.648.831.070.00.80.80.8
18 July 2018.27.324.02949.936.070.07.11.57.1
25 July 2018.28.125.629.242.834.060.04.11.52.6
1 August 2018.29.226.631.647.040.057.00.20.20.2
8 August 2018.29.328.630.251.144.056.01.90.31.1
Adult flight of 1st gen28.0 47.9 14.1
15 August 2018.28.323.430.150.344.065.04.74.74.7
22 August 2018.29.32830.242.436.051.00.10.10.1
29 August 2018.25.722.228.051.543.064.04.34.34.3
5 September 2018.24.922.526.657.150.065.010.20.99.3
12 September 2018.25.523.926.548.140.061.04.44.44.4
Adult flight of 2nd gen26.7 49.9 23.7
19 September 2018.25.224.625.856.448.069.00.10.10.1
26 September 2018.21.214.525.157.632.068.06.26.26.2
3 October 2018.18.615.220.643.928.064.011.14.46.7
10 October 2018.20.319.021.262.333.082.076.60.167.1
18 October 2018.20.419.821.051.045.059.0
24 October 2018.17.914.820.258.542.088.0
Adult flight of 3rd gen20.6 54.9 94.0
Table 2. The correlation between climatic parameters and the abundance of Bactrocera oleae and green lacewings.
Table 2. The correlation between climatic parameters and the abundance of Bactrocera oleae and green lacewings.
Climatic ParameterTemperature (°C)Relative Humidity (%)Rainfall (mm)
Olive fruit flyBlend 1 r =−0.739r = 0.159r = 0.572
p = 0.001p = 0.557p = 0.041
Blend 2 r = −0.494r = 0.215r = 0.467
p = 0.052p = 0.423p = 0.108
Blend 3 r = −0.487r = 0.091r = 0.330
p = 0.056p = 0.737p = 0.270
Control r =−0.632r = 0.024r = 0.258
p = 0.009p = 0.929p = 0.394
Green lacewingsBlend 1 r = 0.434r = −0.068r = −0.429
p= 0.105p = 0.809p = 0.144
Blend 2 r = 0.729r = −0.297r =−0.557
p = 0.002p = 0.282p = 0.048
Blend 3 r = 0.618r = −0.178r = −0.454
p = 0.014p = 0.526p = 0.119
Control r = 0.664r = −0.214r = −0.485
p = 0.007p = 0.443p = 0.093
r = Pearson’s correlation coefficient; correlation was considered significant at level of p ≤ 0.05; absolute linear correlation coefficients ≥|0.50| are marked in bold.
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