Introduction

Bactrocera oleae (Diptera: Tephritidae) (Rossi), the olive fruit fly (OLF), can cause significant losses in olive production and is considered a key pest of wild and cultivated olive trees worldwide (Montiel Bueno and Jones 2002; Ordano et al. 2015; Daane and Johnson 2010). The trophic activity of the larvae and the oviposition punctures of the adults interfere with the biochemical composition of the olives, altering their quality and quantity (Corrado et al. 2023; Gómez-Caravaca et al. 2008; Malheiro et al. 2015; Pereira et al. 2004). The common control strategies concern the use of traps, biological control and chemicals (Daane and Johnson 2010; Lantero et al. 2023), even if it has changed over time (Nestel et al. 2016) due to the ongoing climate change (Caselli and Petacchi 2021). The rise in insect resistance and the risk to ecosystems resulted in a reduction of accepted synthetic molecules and stricter registration requirements (Siden-Kiamos et al. 2022). Recently, researchers have focused their efforts on the modification of pest microbiomes as a novel control strategy (Qadri et al. 2020). The change of microbial pest symbionts limits the adaptability of the hosts to the seasonal and nutritional trends due to the climatic circumstances (Ferguson et al. 2018; Higuita Palacio et al. 2021). Among the whole microbiome of the OLF, the obligate bacterial endosymbiont, described for the first time by Petri (1909), "Candidatus Erwinia dacicola" is the most important and abundant species (Ben-Yosef et al. 2010; Capuzzo et al. 2005; Estes et al. 2012, 2009; Savio et al. 2012). This symbiont is present in adults in the oesophageal bulb (OB), a cephalic organ connected to the pharynx, where it can rapidly multiply (Caleca et al. 2012; Sacchetti et al. 2008). The activity of "Ca. Erwinia dacicola" is fundamental in the development of the larval stages and high mortality has been registered with the administration of antibiotics or copper oxychloride (Belcari et al. 2005; Ben-Yosef et al. 2015; Bigiotti et al. 2019a, b; Estes et al. 2012; Sinno et al. 2020; Perin et al. 2023). On this biological basis, it is possible to hypothesize control strategies of the OLF by modifying the development and action of the endosymbionts.

Microorganisms, and in particular fungi, produce secondary metabolites (SMs) through highly specific and sometimes unusual pathways (Demain and Fang 2000; Herbert 1994; Vinale et al. 2021; Zeilinger et al. 2016). Fungal strains of the genus Trichoderma are well known producers of SMs with various biological activities like biocontrol against phytopathogenic fungi (Lorito et al. 2010; Nicoletti and Vinale 2018) and biostimulants that boost crop nutrition, growth and stress response (Hermosa et al. 2013; Marra et al. 2018; Mayo-Prieto et al. 2019; Sabzi-Nojadeh et al. 2024; Vinale et al. 2009; Vinale and Sivasithamparam 2020). 6-pentyl-α-pyrone (6PP) is a Trichoderma metabolite characterized by a strong and peculiar coconut flavor and strongly correlated to the biocontrol activities of the producing strains (Mutawila et al. 2016). This compound is involved also on antibiosis, microbial competition, induction of plant resistance, and plant promotion (Marra et al. 2019; Vinale et al. 2012; Ismaiel and Ali 2017). Harzianic acid (HA) is a tetramic acid derivatives with chelating properties on different metals (De Tommaso et al. 2020; Staropoli et al. 2023; Tommaso et al. 2021; Vinale et al. 2013), able to promote plant growth and to inhibit microbial development in a concentration-dependent manner (Vinale and Sivasithamparam 2020; Vinale et al. 2009; Sawa et al. 1994; Healy et al. 2015; De Filippis et al. 2021). Recent studies were conducted to evaluate the beneficial effects of fungal metabolites on B. oleae, such as viridiol from T. viride (Sinno et al. 2020) and natural compounds extracted from Metarhizium anisopliae (Juibari et al. 2023). In this framework, we aimed to evaluate the OLF adults' fitness and microbiome responses to applications of 6PP and HA, that are known for their antimicrobial activity (Ismaiel and Ali 2017; Sawa et al. 1994). Since the symbiosis helps the host to overcome difficulties and variations of the environment (Ben-Yosef et al. 2015; Siden-Kiamos et al. 2022), a study on seasonal variations of OLF-microbiome was carried out. This will contribute to a better understanding and correlate the SMs effects to the period of administration. Laboratory bioassays and subsequent real-time qPCRs were performed on wild adults for this purpose. Moreover, an additional study was also conducted on the olfactory response of wild adults to the volatile metabolite 6PP.

Materials and methods

Production and isolation of Trichoderma spp. secondary metabolites.

The beneficial fungi Trichoderma harzianum strain M10 and Trichoderma atroviride strain P1 were maintained on potato dextrose agar (PDA; Hi Media, Mumbai, India) at room temperature and sub-cultured bimonthly. 10 Plugs of M10 and P1 from actively growing margins of PDA cultures were inoculated in 5 L flasks containing 1 L of potato dextrose broth (PDB, Hi Media, Mumbai, India). The stationary cultures were incubated for 25 days at 25 °C, then filtered and exhaustively extracted with ethyl acetate. 6PP and HA were isolated as previously described (Vinale et al. 2012, 2009).

Chromatograms of T. harzianum M10 and T. atroviride P1 extracts used for harzianic acid (HA) and 6-penthyl-α-pyrone (6PP) isolation were obtained by LC–MS analysis (Fig. S3). In particular, the total crude extract and isolated metabolites (HA and 6PP) were analyzed using a quadrupole-time of flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) coupled to a liquid chromatograph (Agilent HP 1260 Infinity Series, Agilent Technologies). Separation was achieved with a Zorbax Extend C-18 column (4.6 × 50 mm, 3.5 µm, Agilent Technologies) at 37 °C. Elution gradient: 0.1% (v/v) formic acid in water (A), and 0.1% (v/v) formic acid in acetonitrile (B); from 5 to 100% B in 6 min, isocratic at 100% B for 2 min; from 100 to 5% B in 2 min (equilibration time: 2 min; flow rate: 0.4 mL/min; injection volume: 7 µL). MassHunter Data Acquisition Software, rev. B.05.01 (Agilent Technologies) was used to set spectrometric parameters (Mayo-Prieto et al. 2019). HA and 6PP isolated form liquid cultures showed the same chromatographic and spectroscopic data of the authentic standards present in our laboratories (Vinale et al. 2008, 2009).

Effects of 6PP and HA on fitness and microbiome responses of OLF adults

New-emerged adults collected from infested olives (cv. Leccino; experimental site UTM: 445,093,878 E; 4,518,644,785 N 33 T) sampled every two weeks were used in the tests. From the same place, a constant availability of fresh green olives was assured, protecting selected branches with different layers as previously described by Jesu et al. (2022). The collected fruits, pupae and adults were placed in a controlled chamber as reported by Jesu et al. (2022). The effects of SMs on the fitness of the adult OLFs exposed were evaluated through ingestion bioassays. The diets offered ad libitum consisted of 300 mg of sucrose solubilized in 200 µl of water (negative control, CNT). Positive controls were obtained using the same sucrose and water diet, in which 100 μg/ml of the antibiotic piperacillin (Sigma) (Piperacillin); 0.5%, 0.1% and 0.02% p/p copper oxychloride (Cu) dissolved (Caleca et al. 2010). The effects of pure harzianic acid (HA) and 6-pentyl-α-pyrone (6PP) are tested at the same concentrations chosen for Cu. The same concentrations of pure HA and 6PP were used as treatments. Five biological replications with 15 males and 15 females were conducted for each test. As water source, a 20 cm diameter cotton ball, soaked in water daily, was placed on the cover net of each cage. During the bioassays, diet consumption, mortality to the exposition, mean number of sterile stings and eggs laid per drupe/female, pre-pupal mortality and sex ratio of the emerged flies were computed as previously described (Jesu et al. 2022).

Following previously established protocols (Jesu et al. 2022), “Ca. Erwinia dacicola” was quantified considering only oesophageal bulbs (OBs) dissected with surgical micro scissors (Fine Science Tools, Foster City, CA, USA) and pins from the adults’ heads. Briefly, the 16 s regions of the “Ca. Erwinia dacicola” rDNA were cloned on Escherichia coli vectors using the TOPO® TA Cloning® Kit (Life Technologies-Thermo Fisher Scientific). The qPCRs were performed using a couple of primers specific for the Ca. E. dacicola 16S rRNA gene: EdEnRev (Estes et al. 2012) and EdF1 (Estes et al. 2009. These primers were previously used by Biogiotti et al. (2019b) in a similar work, for which with a good level of sensitivity was demonstrated even if some aspecific reaction may occur. All the analysis were performed on a StepOne™ Real-Time PCR System (Applied Biosystems) with a reaction mix consisted in 10 μL of SYBR® Green PCR 2X Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL of each primer (10 ng) and water up to 20 μL. The obtained CTs were then computed to get the actual amounts of the bacterium present in the hosts after the treatments.

Seasonal variability of “Ca. Erwinia dacicola” in field

For four years, during the period of activity of the fly, adult females were monthly collected as they emerged from the pupae collected from the experimental site (UTM: 445,093,878 E; 4,518,644,785 N 33 T), pooled in groups of 5 individuals and immediately stored at − 80 °C. A survey on the seasonal variations of the symbiont in natural conditions was completed on ten pools per month following the same methodology reported in Sect. 2.2.

Olfactory response of OLF to the volatile metabolite, 6-pentyl-α-pyrone.

The 6PP olfactometry assay was carried out in a Y-glass olfactometer (Fatouros et al. 2012; Ponzio et al. 2016). The lack of volatility of HA made it less indicated to be used in such assays. 12 fresh green olives were used as the positive control, corresponding to 90% of the attractiveness of OLF adults (n. 60 ♂ and 60 ♀) in pre-tests. In the other arm, the same number of olives were used in combination with 5 μl of subsequential dilutions (10–1, 10–2, and 10–3 M) of 6PP dropped on a 9 cm2 square of filter paper (Whatman®, No.4). All the trials were carried out on newly emerged adults (60 ♂ and 60 ♀ per test). The olfactometry room was air-conditioned (24 ± 2 °C; 60 ± 10% RH) and the tests were conducted at times when the biorhythm of the species is known to be positive (Bertolini et al. 2018). As a consequence of our results, a further trial was carried out using only 6PP 10–1 M without olives, to assess whether the insect response recorded in the trial was due to the combinations of the VOCs mixture present or only to the metabolite. One fly was placed in the Y-shaped tube and its choice was recorded after 5 min. The behavior was noted as indecision when the fly did not choose any arm. Between each fly’s run, the Y-glass tube was disassembled, cleaned, and reassembled with inverted positions of the arms, then left under a clean airflow for at least 5 min.

Statistical analysis

The biological and molecular data recorded were analyzed with the PRISM GraphPad 6.01 (GraphPad Software Inc., San Diego, CA, USA) and the SPSS (IBM Corp. Released 2021, version 28.0. Armonk, NY: IBM Corp). One-way ANOVA and post hoc Student–Newman–Keulsa,b tests were performed on the recorded data of mortality, sterile stings, eggs laid, F1 and on the “Ca. Erwinia dacicola” titer obtained after the laboratory trials and on its seasonal occurrence on the field collected adults (p value < 0.05). A χ2 test was applied for the olfactometry trials using the results of the controls as the expected ones (p value = 0.05).

Results

Effects of 6-pentyl-α-pyrone and harzianic acid on Bactrocera oleae fitness

The diet consumptions were similar among the treatments (Supplementary Fig. 1). 6PP at 0.02%, Cu at 0.5% and 0.1%, and the antibiotic piperacillin caused similar direct mortality to OLF (~ 50%, Fig. 1). The lowest mean mortality was recorded with HA at 0.5% (16.7%), which appeared to have beneficial effects on the treated population (Fig. 1). Tests conducted with HA at 0.1% and 0.02% showed similar effects of the control, while the effects of all the Cu and the 0.02% 6PP treatments did not differ from the positive control Piperacillin (Fig. 1). Females appeared to be more sensitive to the compound treatments (data not shown).

Fig. 1
figure 1

Effects of harzianic acid, 6-pentyl-α-pyrone and copper oxychloride on Bactrocera oleae expressed as mean mortality (%). The data were recorded after 14 days of continuous exposition ad libitum of the insects (ingestion bioassays). CNT () = negative control; Piperacillin (= positive control; Cu () = Copper oxychloride, positive control; HA (= Harzianic acid; 6PP () = 6-pentyl-α-pyrone. On the X axis are shown the concentrations used: 0.5% (p/p), 0.1% (p/p) and 0.02% (p/p). Piperacillin is reported for the only concentration used: 100 µg/ml. Each bar represents the mean ± standard deviations mean of five independent experiments. Different letters (a, b, c, d) mean significative differences (ANOVA and Student–Newman–Keuls tests, p value < 0.05)

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All the treatments altered the OLF’s reproductive activity, measured through the number of sterile stings and oviposition rates. Interestingly, all the treatments strongly increased the number of sterile stings (Supplementary Fig. 2) while reducing the number of laid eggs. The strongest effect was obtained with Cu at 0.5% (Fig. 2).

Fig. 2
figure 2

Effects of harzianic acid, 6-pentyl-α-pyrone and copper oxychloride on Bactrocera oleae expressed as number of laid eggs per female. CNT () = negative control; Piperacillin ( = positive control; Cu () = Copper oxychloride positive control; HA ()= Harzianic acid; 6PP () = 6-pentyl-α-pyrone. On the X axis are shown the concentrations used: 0.5% (p/p), 0.1% (p/p) and 0.02% (p/p). Piperacillin is reported for the only concentration used: 100 µg/ml. Each bar represents the mean ± standard deviations mean of five independent experiments. Different letters (a, b, c, d) mean significative differences (ANOVA and Student–Newman–Keuls tests, p value < 0.05)

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The effects of all SMs on the preimaginal development of the fly were evident (Fig. 3). The eggs laid after Cu at 0.5% and piperacillin administration did not hatch. The Cu-expositions strongly impacted the F1, where half of the observed individuals stopped their development as pupae (Fig. 3).

Fig. 3
figure 3

Number of living emerged F1 individuals of Bactrocera oleae per olive, after treatments with harzianic acid, 6-pentyl-α-pyrone and copper oxychloride. CNT () = negative control; piperacillin () = positive control; Cu () = copper oxychloride, positive control; HA () = harzianic acid; 6PP () = 6-pentyl-α-pyrone. On the X axis are shown the concentrations used: 0.5% (p/p), 0.1% (p/p) and 0.02% (p/p). Piperacillin is reported for the only concentration used: 100 µg/ml. Each bar represents the mean ± standard deviations mean of five independent experiments. Different letters (a, b, c, d) mean significative differences (ANOVA and Student–Newman–Keuls tests, p value < 0.05)

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The number of puparia (data not shown) and new adults (Fig. 3) were significantly higher for the control than for all the other treatments. The sex ratio estimated on the weighted averages of the exposed olives showed a majority of emerged females in most of the tests, particularly after the 6PP 0.1% application (3 ♀ per 1 ♂) (Supplementary Table 1).

Treatments with 6-pentyl-α-pyrone and harzianic acid affect the Bactrocera oleae symbiont “Candidatus Erwinia dacicola”

The analysis of the symbiont titer did not highlight significant differences (p > 0.05), even if there is a dose-dependent reduction trend, which is pointed up with the administration of HA, 6PP, and Cu. An exception is the administration of 0.1% Cu (Fig. 4), where there is an increase in the presence of “Ca. Erwinia dacicola”.

Fig. 4
figure 4

Amount of “Ca. Erwinia dacicola” in oesophageal bulbs of B. oleae after treatments with harzianic acid, 6-pentyl-α-pyrone and copper oxychloride, expressed in number of copies. The bacterium was detected by Real-Time qPCRs. CNT () = negative control; piperacillin () = positive control; Cu () = copper oxychloride positive control; HA () = harzianic acid; 6PP () = 6-pentyl-α-pyrone. On the X axis are shown the concentrations used: 0.5% (p/p), 0.1% (p/p) and 0.02% (p/p). Piperacillin is reported for the only concentration used: 100 µg/ml. Each bar represents the mean ± standard deviations mean of five independent experiments. No significative differences were recorded (ANOVA and Student–Newman–Keuls tests, p value > 0.05). Solid (HA), dashed (6PP) and dotted (Cu) lines represent the linear trend of the treatments

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Seasonal variability of “Ca. Erwinia dacicola” in field

The amount of “Ca. Erwinia dacicola” reached similar values during the same periods of the four years of the trial (Fig. 5). Each year, the highest peak of presence was recorded in August, the lowest in September and in December (Fig. 5). The overall recorded climatic conditions are similar considering the month and year of data collection. When differences between humidity and temperatures were recorded during the year, a subsequent and coherent difference in the symbiont titer was recorded (Supplementary Fig. 3).

Fig. 5
figure 5

Titer (number of copies) of “Ca. Erwinia dacicola” in oesophageal bulbs of newly emerged wild adults of B. oleae (10 pools of 5 females) from August (AUG) to December (DEC), in 2018, 2019, 2020 and 2021. Each bar represents the mean ± standard deviations of ten independent pools of five females each. Different letters (a, b, c, d) mean significative differences (ANOVA and Student–Newman–Keuls tests, p value < 0.05)

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Application of the volatile metabolite 6-pentyl-α-pyrone on Bactrocera oleae: olfactometry tests

The application of 6PP at 10–3 M to the olives did not alter the behavior of the OLFs, and the response was equivalent to the control carried out with only olives (Fig. 6). 6PP at 10–2 and 10–1 M cause a high number of cases of indecision: around 30% of the tested flies, while the remaining are approximately equally distributed between repellency and attractiveness. During the assays, the doubtful flies always showed a peculiar behavior: they remained stationary at the beginning of the tube and frantically cleaned their antennae, mouthparts and legs. Due to this behavior, additional olfactometry tests have been performed using 6PP 10–1 M alone. The data registered with this experiment confirmed this effect on flies (Fig. 6).

Fig. 6
figure 6

Distribution (%) of the choices of the Bactrocera oleae adults (60♀ + 60♂) in terms of attractiveness (black), repulsiveness (white) and indecision (gray) in two-way olfactometry tests with different concentrations of 6-pentyl-α-pyrone. Below the dashed line: AIR versus AIR = control with both the lateral chambers left empty; AIR versus OLIVES = control with 12 olives, attractive, in contraposition with clean air. Over the dashed line: AIR versus 6PP 10–3 + OLIVES = 150 μl of 6PP 10–3 M in combination with 12 fresh green olives and in contraposition to clean air; AIR versus 6PP−2 + OLIVES = 150 μl of 6PP 10–2 M in combination with 12 fresh green olives and in contraposition to clean air; AIR versus 6PP−1 + OLIVES = 150 μl of 6PP 10–1 M in combination with 12 fresh green olives and in contraposition to clean air. AIR versus 6PP−1 = 150 μl of 6PP−1 M were offered in contraposition to clean air. Each bar represents the percentage of choice. For each row, the sum of the white, black and gray bars is equal to 100%. The χ2 tests were carried out using the results recorded in the control tests as expected ones and are displayed separately: versus AIR versus OLVES control (external brackets); versus AIR versus AIR control (internal brackets). * = significance (p value < 0.05)

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Discussions

Insect pests are related to microbial consortia (mainly bacteria) that impact the physiology and all the possible multicomponent interactions (Gurung et al. 2019). The information on the composition of the microbiome is considered a crucial point to control specific pest (Gurung et al. 2019).

Insects and their symbionts coevolved for long time and have established dynamic relationships, offering a stable habitat, disposability of nutrients and effective defensive strategies (Moran and Telang 1998; Parker et al. 1997). Among the several known relationships between microorganisms and tephritids the relationship of OLF with “Ca. Erwinia dacicola” is characterized by the survival of the symbiont even during the destructive holometabolic process of the host (Degani and Gordani 2022; Estes et al. 2009). This characteristic makes the bacterium an interesting and persistent target for Integrated Pest Management (IPM) strategies. In this context, it could be useful to explore the use of this symbiont and its characteristics for managing tephritid pests more sustainably and effectively. The biosynthesis of 6PP by selected Trichoderma species is involved in the biocontrol activities of the producing strain against phytopathogenic fungi (Mutawila et al. 2016). This metabolite exhibits various actions in this way, including direct inhibition of spores germination, inhibition of mycelium pigmentation and suppression of hyphal growth (Degani and Gordani 2022; Parker et al. 1997). Noticeably, 6-PP is considered a food-grade volatile compound, which means it meets safety standards for use in food products. As a result, it could potentially be applied in crop protection with a large possibility of applications.

Harzianic acid (HA) is recognized for its involvement in both beneficial activities for the radical uptake of minerals (e.g., iron) and the promotion of growth in biological and ecological contexts. It serves as a key factor that influences the competitive interactions between microorganisms and can promote the growth of certain species while inhibiting the growth of others (Vinale et al. 2009).

The treatments with 6PP and HA influence the phenology of the OLF and similar results have been recorded with the application of essential oils (Rizzo et al. 2020), viridiol (Sinno et al. 2020) and smoke waters (Jesu et al. 2022). The mortality of treated OLF, similar to the control, and similar consumption among the treatments suggest that the tephritid does not detect the presence of the tested compounds in the diet. Further tests will be needed to assess whether the fly chooses diets supplemented with SMs even when it has the opportunity for different choices, to check the better possible formulation for the development of novel IPM technologies. The larger number of sterile punctures after the applications leads to a consequent better preservation of the fruits. The high mortality of the adults obtained with 6PP at 0.02% and Cu at 0.1%, comparable with that determined by the administration of the antibiotic piperacillin, confirmed a direct link between the survival of the fly and the presence of “Ca. Erwinia dacicola” described in the literature (Ben-Yosef et al. 2014, 2015; Capuzzo et al. 2005; Jurkevitch 2011). The antimicrobial activity of 6PP already reported (Ismaiel and Ali 2017) is confirmed in this study and explains the adults’ mortality and the difficulty in completing the larval development of B. oleae (Belcari et al. 2005; Caleca et al. 2010; Sacchetti et al. 2004; Tsolakis et al. 2011; Tzanakakis 1985). Ingestion of Cu at 0.5% reduced the adult emergences, while HA, with a dose-dependent effect, and 6PP determined a large number of sterile punctures. This effect has also been registered with Cu at 0.1% suggesting a direct effect on the microbiota of the host. The unexpected outlier in the molecular outcomes revealing an increase in bacterial titer with treatment with Cu 0.1% could be related to the non-high specificity of the premiers (Bigiotti et al. 2019b) or by an interference resulting from this Cu concentration. The dose-dependent bacteriostatic effect recorded for both HA and 6PP suggests that the use of fungal secondary metabolites acting on the pest symbionts is a promising control tool (Matloub et al. 2021; Pietri and Liang 2020). 6PP has been used also for olfactometer assays since this Trichoderma volatile metabolite in preliminary experiments revealed a good attitude to modify the fly behavior (unpublished data). The OLF responses at different concentrations of 6PP are consistent with what is already reported (Rizzo et al. 2020; Sinno et al. 2020; Jesu et al. 2022). The indecision is usually defined as the insect moving from one arm to the other and is related to the perception of a similar offer. This behavior for the OLF is usually shown when only air was injected into both arms of the olfactometer (Fig. 6, AIR-to-AIR control). The exposition to 6PP at 10–2 and 10–1 M induced the OLFs to continuously cleaning of the antennae, legs and mouth appendages, related to difficulties in the reception of odors or to a paroxysmal repellency. The analysis of the “Ca. Erwinia dacicola” titer after the administrations showed high variability within the replications but with an overall reduction trend. This bacterium is essential for insect development, in particular for its sustenance, both during the larval (Ben-Yosef et al. 2015) and at the adult stage (Ben-Yosef et al. 2010, 2014, 2015). A cyclical variability among the replicates correlated to the starting dates of the bioassays, has been demonstrated during the seasonal recording of the symbiont titer on adults. The higher titers were observed with higher temperatures and lower relative humidity corresponding to the natural conditions during which the OLF is known to slow down the number of generations related to a scarcity of nutrients and drupes to sting (Siden-Kiamos et al. 2022). The ability of the fly to adapt its reproduction rate when bad climatic conditions occur is already reported (Tzanakakis 2003). Moreover, the highlighted titer cyclical fluctuations can be related to several factors, following climatic and spatiotemporal patterns (Kounatidis et al. 2008) and other endogenous and exogenous variations (Ordano et al. 2015). The high variability among the replicates after the administration of SMs could be due to the seasonal variability of the titer in the OB of wild newly emerged adults we found during all the periods of activity of the fly. These fluctuations highlight how the starting conditions can modify the effectiveness of the treatments.

In conclusion, the data obtained suggest that B. oleae microbiota alteration using microbial metabolites is a promising tool for IPM and the application of Trichoderma-based products, using selected strains and metabolites (i.e., 6PP and HA), can affect the insect biology and improve olive production and quality (Dini et al. 2020; Lombardi et al. 2020).

Authors contributions

We want to thank the partners of the MIUR project LINFA (grant number Linfa 03PE_00026_1) as part of the Ph.D. doctoral scholarship of the Programma Operativo Nazionale Ricerca e Innovazione 2014-2020 (CCI 2014IT16M2OP005), Fondo Sociale Europeo, Azione I.1 “Dottorati Innovativi con caratterizzazione Industriale”, Codice Borsa: DOT178289 – Borsa 3. This study was carried out within the Agritech National Research Center and received funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4–D.D. 1032 17/06/2022, CN00000022). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them.