Introduction
The olive leaf gall midge, Dasineura oleae (Angelini, 1831) (Diptera: Cecidomyiidae), is an endemic monophagous species of Mediterranean countries hosted by olive trees (Olea europaea L.) (Dogănlar et al., Reference Dogănlar, Sertkaya and Skuhrava2011). In recent years, several outbreaks have been reported in the Mediterranean (Simoglou et al., Reference Simoglou, Karataraki, Roditakis and Roditakis2012; Caselli et al., Reference Caselli, Francini, Minnocci and Petacchi2021; Picchi et al., Reference Picchi, Tondini, Albertarelli, Monteforti and Petacchi2021). In particular, the Tuscany region (Italy) has registered a dramatic increase in infestations throughout its area, with large outbreaks recorded in Grosseto Province (i.e., Gavorrano and Capalbio) and Massa-Carrara Province since 2013 (Tondini and Petacchi, Reference Tondini and Petacchi2019). During 2020, the outbreaks extended farther north, reaching the olive areas around Sarzana on the border between Liguria and Tuscany.
As with other cecidomyiids, D. oleae is characterized by a very short adult life span of approximately 12–72 h under laboratory conditions (unpublished data). Adults therefore need to find young olive leaves on which to lay their eggs in a relatively short time. The capacity of the female midge to find a suitable host in the appropriate phenological stage for larval development is fundamental to guarantee the best chances of survival of the progeny (Hall et al., Reference Hall, Amarawardana, Cross, Francke, Boddum and Hillbur2012). In gall-forming insects, gall formation is generally caused by the trophic activity of the larva after hatching (Rohfritsch and Shorthouse, Reference Rohfritsch, Shorthouse, Kahl and Schell1982). When galls occur on leaves, the photosynthetic capacity of the plant is reduced, and the quantity and quality of the yield could be compromised (Martinez et al., Reference Martinez, Montenegro and Elgueta1992; DeClerck-Floate and Price, Reference DeClerck-Floate and Price1994; Gonzales et al., Reference Gonzales, Caballero and Medel2005; Huang et al., Reference Huang, Chou, Chang and Yang2014). Studies on the impact of D. oleae galls on olive leaf physiology have demonstrated that both net photosynthesis and stomatal conductance are negatively influenced by midge infestation (Caselli et al., Reference Caselli, Francini, Minnocci and Petacchi2021).
Several studies have demonstrated that plants under insect attack can trigger direct (e.g., necrosis and neoplasm formation) or indirect defense responses by changing volatile emissions, thus attracting natural enemies (Zong et al., Reference Zong, Zhang and Kampungu2012; Reymond, Reference Reymond2013). Therefore, some predators and parasitoids are attracted by plant-derived volatile organic compounds (VOCs). Other volatiles are released as herbivore-induced plant volatiles (HIPVs) due to the occurrence of the ‘crying for help’ phenomenon (Whitfield, Reference Whitfield2001; Gershenzon, Reference Gershenzon2007; Bruinsma and Dicke, Reference Bruinsma, Dicke and Schaller2008; Dicke, Reference Dicke2009; Dicke and Baldwin, Reference Dicke and Baldwin2010; Kaplan, Reference Kaplan2012; Heil, Reference Heil2014; Abraham et al., Reference Abraham, Giacomuzzi and Angeli2015). HIPVs are involved in multitrophic insect–plant interactions, and furthermore, the emission of HIPV signals can be detected by neighboring unaffected plants as a wake-up call (Giacomuzzi et al., Reference Giacomuzzi, Cappellin, Khomenko, Biasioli, Schütz, Tasin, Knight and Angeli2016). Consequently, neighboring plants are primed to increase their defense as a precautionary response in a phenomenon called ‘talking trees’ (Abraham et al., Reference Abraham, Giacomuzzi and Angeli2015; Arimura and Pearse, Reference Arimura and Pearse2017). From an agronomical point of view, HIPVs could be used very similarly to insect sexual pheromones for pest monitoring, mass trapping, and disruption of host-finding behavior (Regnault-Roger and Philogène, Reference Regnault-Roger and Philogène2008; Rodriguez-Saona and Stelinski, Reference Rodriguez-Saona, Stelinski, Peshin and Dhawan2009; Preti et al., Reference Preti, Knight, Mujica, Basoalto, Larsson Herrera, Tasin and Angeli2021a, Reference Preti, Favaro, Knight and Angeli2021b).
Studies on VOCs released by olive trees are currently scarce (Baratella, Reference Baratella2011; Malheiro et al., Reference Malheiro, Casal, Cunha, Baptista and Pereira2016) and none investigated the leaves volatile emissions following herbivore damage. The headspace collection of healthy olive leaf volatiles in Scarpati et al. (Reference Scarpati, Lo Scalzo and Vita1993) identified eight compounds. Among these compounds, toluene and α-pinene were the most abundant, having an important role in attracting and repelling Bactrocera oleae (Rossi), respectively, the major pest of O. europaea (Scarpati et al., Reference Scarpati, Lo Scalzo and Vita1993; Baratella, Reference Baratella2011). Other compounds such as styrene, xylene, octanal, nonanal, and (E,E)-α-farnesene were recorded as components of the VOC profile of O. europaea leaves (Baratella, Reference Baratella2011). In Malheiro et al. (Reference Malheiro, Casal, Cunha, Baptista and Pereira2016), toluene, (Z)-3-hexen-1-ol, and (Z)-3-hexenyl acetate were the main volatiles collected from the headspace of severed olive leaves. The main compounds identified after hydrodistillation of the leaves were (E,E)-α-farnesene, kongol, theaspiranes, and (E)-β-damascenone in cv Frantoio (Campeol et al., Reference Campeol, Flamini, Chericoni and Catalano2001), (E)-2-hexenal, (E,E)-α-farnesene, linalool, and caryophyllene in cv Olivastra Seggianese (Flamini et al., Reference Flamini, Cioni and Morelli2003), while the most abundant in cv Chemlali were (E)-2-hexenal, nonanal, (E)-β-damascenone, 3-ethenylpyridine, and caryophyllene (Abdeljelil Ben et al., Reference Abdeljelil Ben, Tekaya, Mechri, Flamini and Hammami2017). Studies on HIPVs are, to the best of our knowledge, completely absent concerning olive leaf pest infestations. Moreover, it is currently unknown how the VOC profile of O. europaea foliage changes after mechanical damage.
In this study, we characterized the headspace volatile compounds released by olive plants of cv Frantoio infested by D. oleae under both field and laboratory conditions, comparing these profiles to those of healthy and mechanically damaged plants. For the first time, we described induced volatiles released by infested O. europaea leaves. Collecting field data, we examined the synchronization between olive leaf gall midge attack and the phenology of O. europaea. Furthermore, we performed a morpho-anatomical analysis of the gall tissue to analyze leaf modifications due to D. oleae gall formation.
Materials and methods
Laboratory experiments
One-year-old rooted cuttings of O. europaea cv Frantoio were purchased from a plant nursery supplier (SPO, Società Pesciatina Olivicola, Pescia, Pistoia, Italy) in March 2020. Eighteen uniform plants were grown in 1.4-liter plastic pots containing sphagnum peat:pumice (1:1, v:v) and trained on a single shoot. Plants were drip-irrigated every day. The rooted cuttings were singly placed in cylindrical PVC cages (diameter: 25 cm, length: 80 cm) equipped with transparent chiffon fabric (mesh size: 0.05 mm) to allow aeration. On 10 April 2020, nine rooted cuttings were infested by placing five shoots with D. oleae galls taken from an infested orchard (length: 8 nodes) into each cage. Before the application, each infested shoot was carefully inspected to avoid any insect pests other than D. oleae. Adults of D. oleae emerged 24–48 h after the infested shoots were placed into the cages, mating occurred, and females laid eggs on the cutting leaflets. One month after the infestation (May 2020), leaf galls were visible. The other nine rooted cuttings were maintained not infested and treated as a control group. Infested and control plants were kept separated in two different climatic chambers under controlled conditions (24 ± 1°C, 16:8 (L:D) photoperiod) until the end of the experiment to avoid priming effects.
In order to distinguish pest-specific volatiles due to an active damage from the emissions of damaged tissues alone, we also sampled the volatiles emitted by mechanical damage, another 12 O. europaea cv Frantoio rooted cuttings (1 year old) were purchased from the nursery and kept under the same environmental conditions of the other laboratory plants. After 2 weeks of adjustment phase, six plants received damage, while the other six plants were considered as a control group. The mechanical damage was performed by using a metal punch (mod. 785, Zenith s.r.l., Manerbio, Italy) to cut 0.55 cm2 holes on the first ten fully expanded apical leaves (two holes per leaf) of the treatment group. The headspace volatiles were collected from mechanically damaged plants starting immediately after the damage.
Field experiments
Field investigations were done to characterize the volatiles released from D. oleae infested trees under field conditions. Three different olive orchards located in Castelnuovo Magra (La Spezia, Italy), an area in which a strong outbreak of D. oleae has been recorded, were chosen for these trials. For each orchard, six olive trees of cv Frantoio ranging from 30 to 50 years old were selected. Headspace volatiles were collected from one shoot per tree. The shoots analyzed were ca. 30 cm long with no fruits and were chosen at mid-canopy height by uniformity of leaf number and size through visual observation. Since the whole area surrounding the fields that were sampled registered outbreaks of D. oleae, it was not possible to find a field of trees that was not infested as a control group. Therefore, sampling fields were classified as highly infested (field H) when the rate of infested leaves was over 20% and gall density was over two galls per leaf or lightly infested (fields L1 and L2) when the infestation rate was below 20% and the gall density was below two galls per leaf, according to Tondini and Petacchi (Reference Tondini and Petacchi2019).
VOC collection
VOCs were collected using closed loop stripping analysis (CLSA) for 3 h (Kunert et al., Reference Kunert, David, Becher and Boland2009). A distal shoot portion of approximately 30 cm was selected and enclosed within a plastic bag (BVOC-bag, Cuki® oven bag, Cofresco, Volpiano, Italy). Six empty plastic bags were sampled as negative controls. Air samples were collected using an adsorbent trap (glass tube, 6.5 × 0.55 × 0.26 cm3, loaded with 1.5 mg activated charcoal; CLSA filter LR-type; Brechbühler AG, Schlieren, Switzerland). The trap was fitted to a 12-V graphite vacuum pump (Fürgut, Tannheim, Germany) using a short Teflon tube. The pump circulated air at a rate of ca. 1 liter min−1 within the VOC-bag. After collection, the volatiles were eluted from the CLSA filters with 100 μl of dichloromethane. The volatiles were analyzed by injecting 2 μl of each sample into a gas chromatography–mass spectrometer (GC/MS) (7890, Agilent Technologies, Santa Clara, USA) equipped with a mass selective detector-MS (5975C, Agilent Technologies). A GC/MS nonpolar HP-5 MS column (Agilent Technology, 30 m × 0.25 mm × 0.25 μm film thickness) was used for separation with a constant helium flow of 1.2 ml min−1 and at an average velocity of 39.723 cm s−1. The oven temperature program was started at 50°C for 1.8 min and heated to 250°C at a rate of 7.3°C min−1. The total run time was 34.19 min. The mass spectrometric detector was operated in scan mode (m/z 35–400 amu). Volatiles were initially identified by comparing their mass spectra with those found in two libraries: NIST 14 (National Institute of Standards and Technology, 2008) and Wiley 7 (John Wiley, NY, USA); a mixture of n-alkane standards (nC8–nC20, Sigma-Aldrich, St. Louis, Missouri, USA) was used to calculate linear retention indexes (LRIs) of the detected compounds (Van den Dool and Kratz, Reference Van den Dool and Kratz1963). The identification of VOCs was based on the comparison of their retention index r with the retention indexes reported in the literature (PubChem, Nist, and Pherobase) (Babushok et al., Reference Babushok, Linstrom and Zenkevich2011). The compounds were finally confirmed by comparing their retention times with those available from laboratory standards (Sigma-Aldrich, St. Louis, Missouri, USA). Compounds that were also detected in the negative control (VOC-bag) were considered contaminants. The amount of volatiles was reported as the 10−4 total ion current (TIC) mean ± standard deviation (SD).
Number of leaves, galls, and measurement of leaf area per sampled shoot
At the end of the collection of volatiles for both field and laboratory experiments, each shoot enclosed within the plastic bags was cut and defoliated to count the number of leaves and galls through visual observation and to measure the area of all the sampled leaves. For the area measurements, an LI-3000C Portable Leaf Area Meter (LI-COR, Lincoln, Nebraska, USA) was used.
Monitoring of the D. oleae flight pattern and phenology of O. europaea
To record the flight curve distribution of D. oleae, a field campaign was planned in an unirrigated olive orchard of ca. 1 ha, located at 60 m above sea level (a.s.l.) in Gavorrano (Grosseto, Italy) (42°54′28.30″N, 11°00′10.65″E), where pest infestations have been recorded in the last few years and in which weekly monitoring for the identification of the D. oleae developmental stage is ongoing. Standard transparent delta traps (Csalomon®, Budapest, Hungary) with a horizontal sticky base were used for these trials. Traps were nonattractive and odorless to avoid bias due to color and odor preference (Ranamukhaarachchi and Wickramarachchi, Reference Ranamukhaarachchi and Wickramarachchi2007). Twenty-five traps, placed 20 m apart from one another, were hung on trees at a height of 0.5 m above the ground, and D. oleae males and females captured per trap were recorded weekly during the entire month of April 2020.
O. europaea phenology has been determined by matching the growing degree days (GDDs) of olive trees during the sampling period and phenological observations in the field, with the BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale as the reference point for O. europaea (Cortés et al., Reference Cortés, Martínez-Calvo, Badenes, Bleiholder, Hack, Llácer and Meier2002). The GDDs were calculated using the formula by Orlandi et al. (Reference Orlandi, Ruga, Romano and Fornaciari2005) considering a threshold temperature of 10°C for O. europaea (Marchi et al., Reference Marchi, Giudotti, Ricciolini and Sebastiani2012). The Hydrological Service of Tuscany Region website was used to obtain daily maximum and minimum temperature data (Regione Toscana, 2020). The Braccagni (Grosseto, Italy) meteorological station was chosen because of its proximity to the sampling field.
Characterization of D. oleae attack by gall morphoanatomical observations
For the anatomical observations of the gall structures at the time of the experiment, ten leaves for both the control and infested treatments were chosen. A portion of 0.5 cm2 was cut from the central region of healthy leaves and from the gall of infested leaves and fixed in an FAA solution (45% ethyl alcohol, 5% glacial acetic acid, 10% formaldehyde 8:1:1, v/v/v). Later, leaf samples, once rinsed in water, were dehydrated in a graded ethanol series and embedded in Histoplast. The tissues were cross-sectioned (10 μm) with a Shandon microtome (Shandon Inc., Pittsburgh, Pennsylvania, USA), mounted on glass slides and stained with 0.01% toluidine blue (Sigma-Aldrich, St. Louis, Missouri, USA) for 12 min. Observations were performed under an optical microscope (Fluophot, Nikon, Shinjuku, Japan). Representative selected sections were photographed with a Leica EC3 digital camera equipped with a microscope.
Statistical analysis
The data were analyzed using R software (R Core Team, 2020). The leaf surface and the number of galls between groups in the laboratory and field trials were analyzed by analysis of variance (ANOVA). In the peak analysis, contaminants found in the solvent alone were excluded from the analysis. Peak area integrations (TIC values) were compared between treatments by ANOVA. The leaf surface and the number of galls were accounted for as controlling variables. Data were log-normalized to fit a normal distribution. In case of a statistically significant treatment or field effect, a Tukey post-hoc test was performed. Although the potted plants had no (control) or some (infested) galls, the field plants had different degrees of infestation. Thus, it was not possible to consider a control field, and the amount of galls was considered a controlling variable. In the laboratory trial, since no gall was present on the control plants, only the number of leaves and the leaf surfaces were controlled for. Principal component analysis (PCA) of the laboratory data was performed using the R package ‘factoextra’ (Kassambara and Mundt, Reference Kassambara and Mundt2020). The PCA calculated the combination of the VOC area data by extracting eigenvalues and eigenvectors of a correlation matrix and then highlighting principal components. A two-dimensional score plot was created to compare the volatile profiles from control and infested plants. All the data are reported as the mean ± SD. The figures were created by using the R packages ‘ggplot2’ and ‘cowplot’ (Wickham, Reference Wickham2016; Wilke, Reference Wilke2020).
Results
VOC characterization of laboratory experimental plants
Thirty-one compounds were detected in infested and uninfested olive-potted plants, and among them, a variety of green leaf volatiles, aromatics, and terpenes were found (table 1). Sixteen were identified using authentic standard compounds, and others were identified based on their mass spectra and available LRIs. For four compounds, it was not possible to assign any chemical structures due to the low similarity to any compounds in the databases. Among the collected VOCs, the main peaks were 2-ethylhexanol (584.3 ± 310.8), (Z)-3-hexenyl acetate (333.3 ± 263), and nonanal (145.4 ± 33.8) for the control group, while in the infested plants, (E,E)-α-farnesene (545.8 ± 256.1) and β-ocimene (393.9 ± 116.6) became the main volatiles, while 2-ethylhexanol remained one of the main volatiles (259.1 ± 256.9). Four VOCs were present only in the infested group: β-ocimene, cosmene, β-copaene, and unknown 1. Others increased their concentration in infested plants: (E)-4,8-dimethylnona-1,3,7-triene (DMNT), α-copaene, germacrene D, (Z,E)-α-farnesene, (E,E)-α-farnesene, and heptadecane. Within the same group, the leaf surface in the shoot influenced the amount of germacrene D (F 1,5 = 7.4, P < 0.05), (Z,E)-α-farnesene (F 1,4 = 20.68, P < 0.05), and heptadecane (F 1,5 = 15.47, P < 0.05). Three compounds were higher in the control group: (Z)-3-hexenyl acetate, 2-ethylhexanol, and methyl salicylate. Comparing laboratory and field experiments, three compounds were found only in the laboratory collection, regardless of the infestation status: 3-hexenyl butyrate, tetradecane, and β-oplopenone.
Table 1. Summary table of the volatile compounds identified by GC/MS analysis of the headspace of O. europaea cv Frantoio shoots and their amounts (10−4 TIC ± SD) under laboratory (n = 9 + 9) and field conditions (n = 6 + 5 + 6)
aLRI = linear retention index calculated in relation to n-alkanes.
bLRI = linear retention index already published in peer-reviewed journals and listed in Pherobase (1), NIST WebBook (2), or PubChem (3). The review of Babushok et al. (Reference Babushok, Linstrom and Zenkevich2011) has also been considered (4). When possible, the LRI was verified by a standard compound (†).
Sampling fields were classified as highly infested (H) or lightly infested (L1 and L2). The volatile compounds were collected by the CLSA technique for 3 h. The compounds were identified by mass spectrometry and confirmed by LRIs available from the literature or laboratory standards when available. The table also reports the P-value of the ANOVA test of the TIC amount. The number of samples where the compound was found is reported between brackets. Compounds in bold are present only in infested plants, while the gray background identifies the compounds varying between infestation conditions. Asterisks indicate statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001).
PCA allowed us to determine the clustering of the volatile profiles collected from infested and healthy-potted saplings. The score plot (fig. 1) reports the PCA results and shows the difference between infestation conditions. Although healthy plants are clustered together, infestation by D. oleae caused a remarkable deviation along the principal components. Since PC1 explains almost half of the total variability (45.4%) the power left to PC2 (12.9%) is only a few points away from PC3 (9.89%) and PC4 (7.12%), resulting in dimension 2 contributing a relatively low explanatory power.
Figure 1. PCA score plot of the volatile profiles from healthy (1–9) olive tree saplings and saplings infested with D. oleae (Diptera: Cecidomyiidae) (10–18). Clusters of volatiles attributed to infestation status are indicated by the circles.
VOC characterization of mechanically damaged plants
Five compounds were identified as released by mechanically damaged leaves: 3-hexen-1-ol, (E)-4-oxohen-2-enal, (Z)-3-hexenyl-acetate, 2-(2-butoxyethoxy)ethanol, and (E,E)-α-farnesene (table 2). Among these, 3-hexenyl-acetate was the most abundant (1603.3 ± 1023), while 2-(2-butoxyethoxy)ethanol had the lowest amount (44.4 ± 29.8).
Table 2. VOCs released by O. europaea cv Frantoio cuttings after mechanical damage and their amounts (10−4 TIC ± SD)
Volatiles were collected from six potted plants after 20 holes of 3 mm were made on ten leaves. Volatile emissions were compared with those of control plants collected on the same day. The volatiles were collected by the CLSA technique for 3 h. aLRI = Linear retention index calculated in relation to n-alkanes. bLRI = Linear retention index already published in peer-reviewed journals and listed in PubChem. When possible, the LRI was verified by a standard compound (†).
VOC characterization of infested field plants
A total of 37 compounds were collected from the field trees. Among them, caryophyllene and germacrene D were the most abundant, without differences between the olive orchards and the relative infestation level, while β-copaene and unknown 1 were remarkably more abundant in the more highly infested field (field H) (table 1). Some VOCs such as linalool, β-bourbonene, β-cubebene, β-elemene, aromadendrene, α-muurolene, β-copaen-4 α-ol, α-bisabolol, and unknown 4 (table 1) were found only in the field trials, regardless of the infestation level. Many VOCs were present only in highly infested plants: linalool, DMNT, cosmene, α-copaene, (E,E)-α-farnesene, unknown 1, and heptadecane, thus confirming what was observed under laboratory conditions. Additionally, (Z)-3-hexenyl acetate, higher in the control-potted plants, was more abundant in the low infested fields (fields L1 and L2). Benzaldehyde, which was not different between the potted plants, was instead not detected in field H. However, some compounds such as methyl salicylate showed a reversed pattern that increased in the highly infested field. The compounds β-bourbonene, β-cubebene, β-elemene, β-copaene, (Z,E)-α-farnesene, and δ-amorphene differed between the fields regardless of the infestation level (table 1).
The number of galls positively affected the amount of linalool (F 2,10 = 5.23, P < 0.05), β-copaen-4 α-ol (F 2,12 = 4.48, P < 0.05) and heptadecane (F 2,12 = 8.36, P < 0.05). The germacrene D amount was positively influenced by the leaf surface (F 2,12 = 9.44, P < 0.05).
Number of leaves, galls, and measurement of leaf area
Laboratory conditions
Leaf areas ranged between 179.59 ± 52.89 and 198.09 ± 58.13 cm2 per plant in the control and infested groups, respectively. Statistical analysis showed no significant differences between the two groups (F 1,16 = 0.499, P = 0.4363) (fig. 2a). The number of leaves per plant was not significantly different (F 1,16 = 0.674, P = 0.3296) between control (60.78 ± 10.83) and infested (71.56 ± 24.85) plants (fig. 2a). The number of galls per shoot in the infested plants was 134.89 ± 111.82, while the number of galls per leaf was 1.73 ± 1.06. In the cuttings used for the volatiles released by mechanical damage, the amount of leaves per shoot was 50.60 ± 16.77 and 50.33 ± 12.81 (F 1,10 = 0.582, P = 0.658) for control and damaged plants, respectively, while the leaf surface was 156.24 ± 52.89 and 163.16 ± 45.83 cm2 (F 1,10 = 0.637, P = 0.547), thus showing no significant difference between the groups.
Figure 2. (a) Sum of leaf area and number of leaves per shoot in O. europaea (cv Frantoio) rooted cuttings treated as control (n = 9) and infested by D. oleae (Diptera: Cecidomyiidae) (n = 9). (b) Sum of leaf area, number of leaves per shoot, and number of galls per shoot in three different olive orchards (cv Frantoio) located in Castelnuovo Magra (La Spezia, Italy) having different levels of D. oleae infestation: field H (n = 6) is highly infested, while fields L1 (n = 6) and L2 (n = 5) are infested at a lower level. Letters report statistical significance.
Field conditions
Measurements of the leaf area and the total number of leaves per shoot were performed for the three different olive orchards. The leaf area ranged between 227.81 ± 103.51 cm2 in field L1, 226.72 ± 53.31 cm2 in field L2, and 213.83 ± 100.44 cm2 in field H. Statistical analysis showed no significant difference between fields (F 2,15 = 0.049, P = 0.5873) (fig. 2b). The number of leaves per shoot was not significantly different (F 2,15 = 0.715, P = 0.5191 (fig. 2b). The number of galls per shoot was significantly higher in field H (594.33 ± 250.06) compared to that in fields L1 and L2 (44.16 ± 33 and 14.5 ± 7.12, respectively) (F 2,15 = 49.5, P = 0.0017) (fig. 2b). The number of galls per leaf was 6.68 ± 3.05 in field H, 0.49 ± 0.24 in field L1, and 0.22 ± 0.13 in field L2, confirming a significant difference between fields (F 2,15 = 24.9, P < 0.001).
Flight curve of D. oleae and O. europaea phenology
The flight curves of D. oleae males and females showed the same trend, with a greater presence of males than females during the whole period of sampling (fig. 3). On 8 April, a peak of adult flight activity was evident for both sexes, as the traps recorded a total of 2066 males and 1004 females. On this date, several swarms of D. oleae were noticed around young olive tree shoots and females during oviposition. The minimum presence of D. oleae was observed on 22 April, the last date of sampling, with a total catch of 512 males and 190 females.
Figure 3. Flight curves of D. oleae (Diptera: Cecidomyiidae) males and females (mean ± SD) and phenology of O. europaea expressed as the sum of GDDs and BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) stages (9 and 33). Data were collected from 1 April 2020 using a standard transparent delta trap (Csalomon®) in an olive orchard placed in Gavorrano (Grosseto, Italy). On 8 April, a peak of D. oleae adults, both males and females, was recorded. On the same date, researchers observed that the external leaflets of O. europaea started to open and their tips intercrossed (9th stage on the BBCH scale) at GDD values of 55.9. On 15 April, olive shoots reached 30% of the final length (33rd stage of the BBCH scale), at 75.7 GDD.
Through visual observation and referring to the BBCH scale for O. europaea, phenological stage number 9, corresponding to external leaflets opening with their extremity intercrossing, was reached on 8 April, at 55.9 GDDs. On 15 April, at 75.7 GDDs, phenological stage number 33 was identified (fig. 3).
Morphoanatomical observations
The healthy leaves of O. europaea cv Frantoio have a typical elongate-elliptical shape, with a smooth dark green adaxial surface and a lighter and less smooth abaxial surface (fig. 4a). A longitudinal healthy leaf section is presented in fig. 4b, in which palisade parenchyma I and II (PPI and PPII), spongy parenchyma (SP), and peltate trichomes (PT) are clearly recognizable. D. oleae galls are roundish, a few millimeters long (3–6 mm), and they are irregularly present just on the adaxial leaf surface (fig. 4a). The section in fig. 4c shows a belt of cell proliferation just near the larval chamber at the SP. The larval chamber is clearly recognized in fig. 4d. The thickness of this leaf section (2 mm) is threefold greater than the thickness of the healthy leaf (0.35 mm) (fig. 4b).
Figure 4. Adaxial and abaxial O. europaea (cv Frantoio) leaf surface in control and infested treatments (a) and cross sections of olive leaves stained with 0.01% toluidine blue (Sigma-Aldrich, St. Louis, Missouri, USA) and observed under an optical microscope (Fluophot, Nikon, Shinjuku, Japan) (b–d). (b) Section of a control leaf (10×): PPI and PPII, SP, and PT. (c) Leaves infested by D. oleae (Diptera: Cecidomyiidae): cell proliferation near the larval chamber (10×). The cut point for the cross section is represented in (a) with the letter ‘y’. (d) Leaves infested by D. oleae (4×) showing the larval chamber. The cut point for the cross section is represented in (a) with the letter ‘x’.
Discussion
Studies on olive leaf volatile profiles are currently scarce and based on hydrodistillate fractions, which are different from headspace emissions (Campeol et al., Reference Campeol, Flamini, Chericoni and Catalano2001; Flamini et al., Reference Flamini, Cioni and Morelli2003; Brahmi et al., Reference Brahmi, Flamini, Issaoui, Dhibi, Dabbou, Mastouri and Hammami2012; Malheiro et al., Reference Malheiro, Casal, Cunha, Baptista and Pereira2016). In the present study, headspace collections of olive volatiles were recorded under both laboratory and field conditions. Control-potted plants emitted a blend of 26 VOCs, including the aldehydes nonanal and benzaldehyde, the esters methyl salicylate, 3-hexenyl butyrate and hexyl acetate, the ketone sulcatone, and the sesquiterpenes caryophyllene, (E,E)-α-farnesene and germacrene D, previously identified by Malheiro et al. (Reference Malheiro, Ortiz, Casal, Baptista and Pereira2015, Reference Malheiro, Casal, Cunha, Baptista and Pereira2016). Other VOCs are reported here for the first time, such as myrcene, (Z)-3-hexenyl acetate, 2-ethylhexanol, DMNT, carveol, α-copaene, tetradecane, humulene, bicyclosesquiphellandrene, (E)-β-guaiene, (Z,E)-α-farnesene, δ-amorphene, liguloxide, β-oplopenone, heptadecane, γ-cadinene, and unknown 2.
This study, for the first time, provides evidence about HIPVs for olive trees due to insect attack to the leaves. Previous studies have only investigated fruit HIPV profile due to B. oleae larvae infestation (Alagna et al., Reference Alagna, Kallenbach, Pompa, De Marchis, Rao, Baldwin, Bonaventure and Baldoni2016) and the behavioral responses of its parasitoid Psyttalia concolor to olive drupe HIPVs (Giunti et al., Reference Giunti, Benelli, Flamini, Michaud and Canale2016a). Moreover, although gallers usually manage to suppress VOCs (Borges, Reference Borges2018), D. oleae induces a change of the volatile profiles of attacked plants in the amount of emissions and in the released compounds. A blend of 12 VOCs is distinctive of olive leaves infested by D. oleae: DMNT, β-ocimene, α-copaene, β-copaene, cosmene, germacrene D, (E)-β-guaiene, (Z,E)-α-farnesene, (E,E)-α-farnesene, unknown 1, unknown 3, and heptadecane. Some were found to be specific only to infested plants in laboratory trials (β-ocimene, cosmene, β-copaene, unknown 3, and unknown 4), while others showed increased emission in infested plants both in the laboratory and in field collection (DMNT, α-copaene, (E)-β-guaiene, (Z,E)-α-farnesene, (E,E)-α-farnesene, liguloxide, and heptadecane). This herbivore-induced emission observed in the study is in agreement with other studies that reported some of the same compounds in infested plants of other species (Suckling et al., Reference Suckling, Twidle, Gibb, Manning, Mitchell, Sullivan, Wee and El-Sayed2012). DMNT and α-copaene were released in Medicago truncatula Gaertn. following the feeding activity of Spodoptera exigua (Hübner) (Arimura et al., Reference Arimura, Garms, Maffei, Bossi, Schulze, Leitner, Mithöfer and Boland2008). In the study of Zeng et al. (Reference Zeng, Liao, Li, Zhou, Tang, Dong and Yang2017), α-farnesene and ocimene were released after tea leaf (Camellia sinensis (L.) Kuntze) stimulation by jasmonic acid combined with mechanical damage, simulating aphid damage. (E,E)-α-Farnesene and β-ocimene were also emitted by cotton plants treated with methyl jasmonate (Rodriguez-Saona et al., Reference Rodriguez-Saona, Crafts-Brandner, Paré and Henneberry2001). The sesquiterpene (E,E)-α-farnesene and the monoterpene β-ocimene are already known as constituents of olive oils, and when infested by B. oleae, olive fruits increase the emission of these two compounds (Giunti et al., Reference Giunti, Benelli, Conte, Mele, Caruso, Gucci, Flamini and Canale2016b). In the present study, (E,E)-α-farnesene and β-ocimene were the most abundant molecules in the infested potted plants, thus confirming a key role of these molecules in the olive tree–insect interactions. (E)-β-Ocimene is reported as an HIPV from B. oleae exploited by virgin males of the braconid parasitoid P. concolor to boost their mate searching activity (Giunti et al., Reference Giunti, Benelli, Palmeri and Canale2018).
The monoterpene cosmene, identified in the present study, was also found as an HIPV in apple leaves infested by Pandemis heparana Denis & Schiffermüller (Giacomuzzi et al., Reference Giacomuzzi, Cappellin, Khomenko, Biasioli, Schütz, Tasin, Knight and Angeli2016). Most of the other compounds are in agreement with other studies, as they were identified as VOCs of olive trees (Flamini et al., Reference Flamini, Cioni and Morelli2003; Baratella, Reference Baratella2011). The main VOCs collected from the field trials were caryophyllene and germacrene D, regardless of the infestation level between fields. Although caryophyllene is one of the main constituents of the olive leaf profile (Flamini et al., Reference Flamini, Cioni and Morelli2003), germacrene D might instead act as an HIPV since its emission increased in response to midge infestation in the laboratory collection. Moreover, statistical analysis revealed that germacrene D is also affected by the leaf surface of the sampled shoots both in laboratory and field trials. Two other VOCs, (Z,E)-α-farnesene and heptadecane, are commonly emitted in small amounts by apple leaves and olive leaves (Bengtsson et al., Reference Bengtsson, Bäckman, Liblikas, Ramirez, Borg-Karlson, Ansebo, Anderson, Löfqvist and Witzgall2001; Dursun et al., Reference Dursun, Güler, Özkan and Konuşkan2017). In the present study, they were directly related to the leaf surface of the shoots sampled in the laboratory trials, and their emission increased in infested plants.
A substantial number of VOCs are usually emitted due to oxidation processes after plant tissue injury. Considering the volatile emission due to mechanical damage allowed us to notice the difference between mechanical wounding and insect activity, thus identifying the set of specific herbivore-induced volatiles. The analysis revealed that (Z)-3-hexenyl acetate was the main released compound after mechanical wounding. Indeed, this ester is known to be released from damaged leaves (Oluwafemi et al., Reference Oluwafemi, Bruce, Pickett, Ton and Birkett2011), usually together with its original alcohol (Z)-3-hexen-1-ol (Kikuta et al., Reference Kikuta, Ueda, Nakayama, Katsuda, Ozawa, Takabayashi, Hatanak and Matsuda2011; Sufang et al., Reference Sufang, Jianing, Zhen and Le2013), which was also found in our collected VOCs. (Z)-3-Hexen-1-ol is found in a typical green leaf formed by lipoxygenase of linolenic acid of the cell membranes. Both 3-hexen-1-ol and (Z)-3-hexenyl acetate were indeed reported as the main VOCs collected by severed olive leaves in Malheiro et al. (Reference Malheiro, Casal, Cunha, Baptista and Pereira2016) confirming their emission after cell membrane breakup. Among the 12 compounds previously reported as HIPVs by D. oleae, (E,E)-α-farnesene was also released following mechanical damage in our study, but the 20-fold rise of emission in the infested plants and the consistency of the data under both laboratory and field conditions confirmed the role of this sesquiterpene as HIPV (Giacomuzzi et al., Reference Giacomuzzi, Cappellin, Khomenko, Biasioli, Schütz, Tasin, Knight and Angeli2016).
The laboratory trials allowed for the use of uninfested plants, thus revealing four molecules (β-ocimene, cosmene, β-copaene, and unknown 1) released only in the plants that were attacked and not detected in the control plants. However, although cosmene, β-copaene, and unknown 1 were also consistently higher in the plants of the highly infested field, β-ocimene was higher in the highly infested field and in one less-infested field. Some compounds were reduced in the infested plants in the laboratory trials. γ-Cadinene, although completely absent in infested potted plants, was present in the mature field plants but without a difference between infestation levels. (Z)-3-Hexenyl acetate and 2-ethylhexanol, which are more abundant in control-potted plant collections, were always present in field trials. However, the lack of uninfested field trees prevented comparison with the volatile profiles of controlled conditions and understanding whether the emission of these VOCs was actually reduced due to infestation. For this reason, the number of galls in the sampled branch was used to predict the amount of volatiles. Linalool, β-copaen-4 α-ol, and heptadecane were found to be dependent on the number of galls. Although linalool and heptadecane are known as common HIPVs (Moayeri et al., Reference Moayeri, Ashouri, Poll and Enkegaard2007; War et al., Reference War, Sharma, Paulraj, War and Ignacimuthu2011), β-copaen-4 α-ol has so far been reported only as a sesquiterpene characteristic of many plant species (Dória et al., Reference Dória, Silva, Carvalho, Alves and Cavalcanti2010; Magierowicz et al., Reference Magierowicz, Górska-Drabik and Sempruch2020), but not as an herbivory-induced compound. Moreover, β-copaen-4 α-ol was detected only in the field collection and not in the laboratory collection, thus suggesting an emission more related to mature plants or other parameters. Linalool, which was also not observed in the laboratory trial, is characteristic of highly infested plants in the field, together with DMNT, cosmene, α-copaene, (E,E)-α-farnesene, unknown 1, and heptadecane, already observed in potted plants as HIPVs. The reason linalool appeared only in the field collections might be dependent on the age or the phenological stage of the plant, since the plants used in the laboratory trials were young saplings. For instance, in Rao et al. (Reference Rao, Rajput, Patel and Purnanand2010) older leaves of palmarosa (Cymbopogon martinii (Roxburgh) Watson) contained more linalool than young leaves. Also in hop (Humulus lupulus L.), the linalool content increased with the age of the leaves (Matsui et al., Reference Matsui, Inui, Ishimaru, Hida and Oka2012). Hence, β-copaen-4 α-ol and linalool might act as HIPVs only in mature plants. A possible effect due to plant age might also explain the difference in the compounds β-bourbonene, β-cubebene, β-elemene, β-copaene, and δ-amorphene between field L1 and fields L2 and H. These sesquiterpenoids were not present in the young potted saplings, thus appearing only in field plants, regardless of the infestation level. Sesquiterpenoids are often involved in communication between species since they can travel long distances via advective transport in gas (Huang and Osbourn, Reference Huang and Osbourn2019). Other VOCs were detected only in the field collection, with no difference between fields (aromadendrene, α-muurolene, α-bisabolol, and unknown 4). Although certain studies pointed out differences in volatile emissions between young and old leaves (Takabayashi et al., Reference Takabayashi, Dicke, Takahashi, Posthumus and Van Beek1994; Rao et al., Reference Rao, Rajput, Patel and Purnanand2010; Matsui et al., Reference Matsui, Inui, Ishimaru, Hida and Oka2012), only a few studies are available about these differences when comparing young (few-week-old saplings) and mature trees (30-year-old olive trees). In the study of Street et al. (Reference Street, Owen, Duckham, Boissard and Hewitt1997), the emissions of Pinus pinea L. seemed to vary significantly from mature forest to young plantation, and age-related changes were found in the volatiles released by wounded phloem of Picea abies (L.) Karsten seedlings (Kännaste et al., Reference Kännaste, Zhao, Lindström, Stattin, Långström and Borg-Karlson2013). Whether similar changes can occur in broad-leaf species such as O. europaea has not been reported, but it might provide an explanation for the emissions observed in the present study. A genotype effect cannot be excluded, but it seems to be unlikely, since olive trees are reproduced mainly by cutting, and the cultivar chosen for the study was the same for all the trials.
The relationship between gall-inducing species and host plants is a complex association in which insects redirect plant physiology and, consequentially, the growth of the attacked organs for the purpose of gaining advantages (e.g., nourishment and shelter) (Shorthouse et al., Reference Shorthouse, Wool and Raman2005). For this reason, the synchronization of gall-inducing insects with their host is fundamental, particularly for short-lived insects, such as cecidomyiids (Yukawa, Reference Yukawa2000; Tondini and Petacchi, Reference Tondini and Petacchi2019). Furthermore, in a tritrophic interaction (host plant–pest–natural enemies) each component has its biological rhythm concerning the behavior and the physiology of all the members (Allemand et al., Reference Allemand, Pompanon, Fleury, Fouillet and Bouletreau1994; Fantinou et al., Reference Fantinou, Alexandri and Tsitsipis1998; Turlings et al., Reference Turlings, Lengwiler, Bernasconi and Wechsler1998; Zhang et al., Reference Zhang, Wei, Guo, Liu and Kang2010). The efficiency of the tritrophic system is maintained by synchronizing the rhythms across the trophic levels that is influenced by biotic and abiotic factors (Zhang et al., Reference Zhang, Wei, Guo, Liu and Kang2010). For instance, the emission of HIPVs by plants to recruit natural enemies of herbivore is a typical phenomenon involved in a synchronized tritrophic system (Zhang et al., Reference Zhang, Wei, Guo, Liu and Kang2010). However, studies about rhythm synchronization in tritrophic interactions are currently scarce. This paper gives the first demonstration of the synchronization between D. oleae adults (both females and males) and the phenology of O. europaea. A greater number of D. oleae adults during the 2020 sampling campaign was observed on 8 April. This is in accordance with our previous data that recorded the highest amount of D. oleae exactly in the same period also during the previous years, although in those cases only females were recorded (Tondini and Petacchi, Reference Tondini and Petacchi2019). In the same flying period, the phenology of O. europaea reached the 9th stage of the BBCH scale, in which the external leaflets opened and their tips intercrossed (Cortés et al., Reference Cortés, Martínez-Calvo, Badenes, Bleiholder, Hack, Llácer and Meier2002). The GDD on 8 April was 55.9. However, the GDD recorded in conjunction with the 9th stage of the BBCH scale usually ranged between 70 and –100 (Marchi et al., Reference Marchi, Giudotti, Ricciolini and Sebastiani2012). This phenomenon can be explained by the climatic conditions verified during the period between January and April 2020. The temperature during these months has never been too frigid, with a mean temperature of 9.9°C and a quite scarce rainfall, particularly in February (4.6 mm) (Regione Toscana, 2020). During March, rainfall was more abundant (57 mm) and concurrently with mild weather, abundant rainfall can be the cause of a vegetative restart of O. europaea, even if the GDD was not greater than 56 (Regione Toscana, 2020). During the third sampling date, on 15 April, researchers observed that the olive shoots reached 30% of the final length, classifying this phenological stage as the 33rd stage of the BBCH scale (Cortés et al., Reference Cortés, Martínez-Calvo, Badenes, Bleiholder, Hack, Llácer and Meier2002). During the whole period of sampling, D. oleae eggs were observed on olive leaflets, confirming the preference of this cecidomyiid for fresh leaves, unlike other gall midges colonizing hard leaves or other tissues, such as the yew gall midge, Taxomyia taxi (Inchbald) and the blueberry gall midge, Dasineura oxycoccana Johnson (Dernisky et al., Reference Dernisky, Evans, Liburd and Mackenzie2005; Miller and Raman, Reference Miller and Raman2019).
The hypothesis that the D. oleae sex ratio presents a bias in favor of females in the proportion of 2:1 by Hallett and Heal (Reference Hallett and Heal2001) is confuted in the current study, having observed exactly the opposite relationship. However, this phenomenon may result in a false estimation, since during direct sampling from the field several secondary factors are considered, such as the differential mortality of sexes due to food quality and/or overwintering diapause (Tabadkani et al., Reference Tabadkani, Ashouri, Rahimi-Alangi and Fathi-Moghaddam2012).
Leaf galls induced by cecidomyiid trophic activity generally show alterations in cells and tissues, with a consequent modification of the leaf blade shape (Albert et al., Reference Albert, Rana and Gandhi2013; de Alcantara Guimarães et al., Reference de Alcantara Guimarães, Brasil Bizarri, Silva Barbosa, Nakamura, Freiman de Souza Ramos and de Macêdo Vieira2013). A basic study on the leaf tissue modifications as a consequence of D. oleae feeding action has been recently reported by our group (Caselli et al., Reference Caselli, Francini, Minnocci and Petacchi2021). We observed that the first-stage larva forms an entrance hole on the adaxial olive leaf surface and then it develops in a gallery within the SP of the leaves. Spongy parenchymatic tissue is often modified after gall agent attack, similar to Clusia lanceolata Cambessèdes following Clusiamyia nitida Maia trophic action (de Alcantara Guimarães et al., Reference de Alcantara Guimarães, Brasil Bizarri, Silva Barbosa, Nakamura, Freiman de Souza Ramos and de Macêdo Vieira2013) and Copaifera langsdorffii Desfontaines following midrib gall development (Oliveira and Isaias, Reference Oliveira and Isaias2010). Additionally, the feeding activity of Dasineura mali on the upper apple leaves surface, causes characteristic distortions followed by a change in the foliage color (Lo et al., Reference Lo, Walker and Suckling2015). Near the larval chamber of D. oleae, the olive SP cells divide and become spherical with a small intercellular space, such as in Guarea macrophylla Vahl subsp. tuberculata Vellozo (Kraus et al., Reference Kraus, Sugiara and Cutrupi1996). Here, it is demonstrated that the formation of the larval chamber by D. oleae induces the development of new cell layers in the SP, as described for other cecidomyiids by Albert et al. (Reference Albert, Rana and Gandhi2013) and de Alcantara Guimarães et al. (Reference de Alcantara Guimarães, Brasil Bizarri, Silva Barbosa, Nakamura, Freiman de Souza Ramos and de Macêdo Vieira2013). We observed a spongy hyperplastic parenchyma, as described for Aspidosperma spruceanum Benth. ex Müll. Arg. by Formiga et al. (Reference Formiga, Gonçalves Soares and dos Santos Isaias2011) and consequentially, the galled leaf portion is thicker than the unaffected leaf portion. Further studies may focus on the metabolic compounds formed in the parenchyma tissues during the D. oleae gall development. Furthermore, parenchymal alteration may have a role in inducing a plant defense response (Sousa et al., Reference Sousa, Silva, Silva, Bento, Penãflor and Souza2020). Among plant defense strategies, the emission of HIPVs plays an important role in the recruitment of natural enemies that may control the attack of herbivore insect pests (Gebreziher, Reference Gebreziher2020).
Conclusions
The analysis of the volatiles emitted by plants infested by galls of D. oleae provided a clear set of 12 insect-induced compounds, and the data were consistent between the laboratory and the field trials. Following the insect attack, the emission amount of some VOCs increased significantly. Other HIPVs were not present in control plants and appeared only after the infestation. The sampling technique did not cause any mechanical damage that might interfere with the plant volatile emissions. Some volatiles seemed to be related to the age of the plant rather than to the infestation status.
This study confirms a close relationship between D. oleae and its exclusive host, O. europaea, highlighting a spring synchronization of midge flight activity and the vegetative restart of the olive tree. As a consequence of the larval feeding action, the olive leaves show modifications of the leaf shape blade due to the structural alteration of the spongy parenchymatic tissue. Further studies are needed to investigate the relationship between leaf structure modifications and VOCs emitted by O. europaea, shedding light on the ecological role of volatiles. Forthcoming behavioral tests might investigate whether the change in the volatile profile of the plant elicits a response in adults of D. oleae and its natural enemies.
Acknowledgements
The authors are grateful to Susanna Bartolini and Cristina Ghelardi for their laboratory assistance and technical help with the morphoanatomical observations and to Susanna Marchi for revisioning the section concerning Olea europaea physiology. The authors would like to thank the olive orchard owners Luisa Amadori, Roberto Cagnoli, and Massimo Lagomarsini for permission to perform monitoring and sampling. The authors would also like to thank the three anonymous reviewers for their helpful comments.
Author contributions
Conceptualization and methodology: AC, RF, RP and SA; literature search: AC and RF; writing – original draft preparation: AC and RF; writing – review and editing: AC, RF, RP and SA. All authors have read and agreed to the published version of the manuscript.
Financial support
This study has been realized with the funding provided by the scholarship granted by the Agrobiosciences PhD program at Scuola Superiore Sant'Anna of Pisa to Alice Caselli.
Conflict of interest
The authors declare none. The mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the author institutions.
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