Chemical Profiles, In Vitro Antioxidant and Antifungal Activity of Four Different Lavandula angustifolia L. EOs

Lavandula angustifolia L., known as lavender, is an economically important Lamiaceae due to the production of essential oils (EOs) for the food, cosmetic, pharmaceutical and medical industries. The purpose of this study was to determine the chemical composition of EOs isolated from four inflorescences of L. angustifolia L. collected in different geographical areas: central-southern Italy (LaCC, LaPE, LaPS) and southern France (LaPRV). The essential oils, obtained by steam distillation from plants at the full flowering stage, were analyzed using gas chromatography coupled with mass spectrometry (GC-MS). More than 70 components identified in each sample showed significant variability among the main constituents. The four EOs analyzed contained the following as main component: linalool (from 30.02% to 39.73%), borneol (13.65% in LaPE and 16.83% in La PS), linalyl acetate (24.34% in LaCC and 31.07% in LaPRV). The EOs were also evaluated for their in vitro antifungal activity against two white rot fungi (Phanerochaete chrysosporium and Trametes cingulata) as potential natural biodeteriogens in the artworks field, and against Sclerotium rolfsii, Botrytis cinerea and Fusarium verticilloides responsible for significant crop yield losses in tropical and subtropical areas. The results confirm a concentration-dependent toxicity pattern, where the fungal species show different sensitivity to the four EOs. The in vitro antioxidant activity by DPPH assay showed better scavenging activity on LaCC (IC50 26.26 mg/mL) and LaPRV (IC50 33.53 mg/mL), followed by LaPE (IC50 48.00 mg/mL) and LaPS (IC50 49.63 mg/mL). The potential application of EOs as a green method to control biodeterioration phenomena on a work of art on wood timber dated 1876 was evaluated.


Introduction
Essential oils (EOs) are accumulated in different organs and/or structures of aromatic plants, i.e., fruits, flowers, leaves, seeds, barks, and their components are being classified as secondary metabolites. They are relatively small chemical compounds, with ubiquitous distribution in the plant kingdom, though their role in the life of plants in most cases is not known [1]. Their wide range of activity is determined both by the plant genotype that determines its chemical composition and by other factors such as environmental condition, geographical location, time of harvest, stage of plant development or extraction methodology [2][3][4].
In recent years, the application of plant extracts and plant EOs has received increasing interest as a natural alternative to the use of commercial synthetic chemicals to control the main postharvest diseases of fruits and vegetables. As an example, different EOs such as oregano, winter savory, eucalyptus and peppermint, along with many of their principal components (limonene, carvacrol, etc.), have already demonstrated attractive and important antimicrobial, insecticidal, antioxidant and herbicidal activity for the agri-food industry [5]. Furthermore, EOs are biodegradable, have minimal effects on non-target The present study aimed to investigate the efficacy of EOs of L. angustifolia on different fungal species involved in both plant pathogen interaction and wood biodeterioration. Specifically, the following steps were performed: (i) comparison of the chemical composition of the EOs of four lavender flowers from different geographic regions; (ii) statistical analyses of data; (iii) in vitro antifungal activities against five phytopathogenic fungi; (iv) in vitro antioxidant activities of the EOs by DPPH free radical scavenging assay; (v) case study, as green biocidal (against biodeteriogen) on altered wooden artwork.
All extracts provided an essential oil characterized by a typical smell in a yield ranging between 3.1 and 5.9%, calculated according to the initial weight of 100 g each, respectively. The characterization of all EOs was evaluated using GC/MS and a set of standards: linalool, borneol, terpinen-4-ol, camphor and lavender oil (Sigma Aldrich, St. Louis, MO, USA). Table 1 summarizes the chemical composition of the EOs and their experimental retention indices compared with the retention indices reported in the literature [32], their percentage compositions and the abbreviations of the different classes of terpenes; the compounds are reported according to their elution on a Rtx ® -5 Restek capillary column. Approximately 70 components were identified for each sample, showing significant variability in some major constituents.

Explorative Data Analysis
In order to characterize the levels of diversity in the chemical composition of EOs, the classic Shannon entropy [33] has been considered with its relative version, the Pielou index [33]. Furthermore, to compare compositions between samples and to assess the lev-

Explorative Data Analysis
In order to characterize the levels of diversity in the chemical composition of EOs, the classic Shannon entropy [33] has been considered with its relative version, the Pielou index [33]. Furthermore, to compare compositions between samples and to assess the levels of dissimilarities between compositions of different types of lavender EOs, the percent model affinity (PMA) index has been calculated [34]. Mathematical details are presented in Section 4.5.
The results are shown in Table 3, where the reported mean and standard error of each indicator are calculated over the three sampling replicates. In addition, to better show which compounds characterize the levels of dissimilarities between types of lavender EOs, the cross plots between compositions are reported in Figure 2. The blue line is the bisector; it represents the situation in which a certain compound is present in the same percentages in both types of lavenders considered in each panel (Figure 2a-f). The points that are significantly distant from that bisector represent compounds observed in rather different percentages; they are highlighted by the specific names. Here, a certain point is considered distant from the bisector when its distance is larger than the standard deviation S d = 2.12. The dashed lines represent the tolerance interval, bisector ± S d . The points within this interval represent compounds with approximately the same percentage in both types of lavender EOs. Table 3. Similarity and diversity in the chemical compositions of lavender EOs from four different geographical provenances, assessed with the PMA similarity index (minimum similarity = 0; max similarity = 1) and the Pielou index (minimum diversity = 0; max diversity = 1), respectively. The respective standard errors over the three technical replicates are reported.

EOs
Similarity within this interval represent compounds with approximately the same percentage in both types of lavender EOs.  The results in Table 3 clearly show that two pairs of similarities are observed, LaPE with LaPS (PMA = 0.866) and LaCC with LaPRV (PMA = 0.719), while in terms of diversity, all the types of lavender EOs present the same level of entropy, suggesting a homogeneous structure in the chemical composition for all four types of EOs.

Antifungal In Vitro Test
The antifungal activity of the four lavender EOs against the growth of S. rolfsii, B. cinerea, F. verticillioides, P. chrysosporium and T. cingulata phytopathogenic fungi was carried out in vitro. LaCC-, LaPE-, LaPS-and LaPRV-characterized lavender EOs were used at different concentrations, ranging from 0 to 40 µL, as described in Section 4.6. Figure 3 reports the reduction in fungal growths, expressed as a reduction in mycelium radial growths on Petri dishes with respect to the negative control. The results in Table 3 clearly show that two pairs of similarities are observed, LaPE with LaPS (PMA = 0.866) and LaCC with LaPRV (PMA = 0.719), while in terms of diversity, all the types of lavender Eos present the same level of entropy, suggesting a homogeneous structure in the chemical composition for all four types of Eos.

Antifungal In Vitro Test
The antifungal activity of the four lavender EOs against the growth of S. rolfsii, B. cinerea, F. verticillioides, P. chrysosporium and T. cingulata phytopathogenic fungi was carried out in vitro. LaCC-, LaPE-, LaPS-and LaPRV-characterized lavender EOs were used at different concentrations, ranging from 0 to 40 µL, as described in Section 4.6.
The results show, first of all, a concentration-dependent trend in antifungal activity; then, each fungus shows a different profile of interaction and a different sensitivity among the tested EOs. Total inhibitory growth is generally reached using an amount ranging from 20 to 40 µL. F. verticillioides (Figure 3e) shows slightly different radial growth curves; in this fungus, EOs have a lower performance in terms of fungal growth inhibition. For example, on F. verticillioides, LaPE is less effective, with about 50% growth inhibition with 40 µL of the analyzed sample. On the contrary, LaPE applied on P. chrysosporium (Figure 3a) shows better inhibitory activity. In the same experimental conditions, it inhibits the growth of P. chrysosporium mycelium by 100% with only 20 µL of EO. Good inhibitory activity was found on S. rolfsii and B. cinerea using 30 µL of LaPS (Figure 3b,c), while the same amount of LaCC showed a better inhibition growth kinetic on T. cingulata (Figure 3d). LaPRV EO showed the best inhibition growth kinetics on F. verticillioides, although this fungus proved to be the least sensitive to treatment with EOs. The positive controls for antifungal activity were carried out using PDA plates added with Thiram (Tetrasar 50, powder, Isagro srl) at final concentrations in the ranges of 0 and 73 µg/mL (Figure 3f). B. cinerea and P. chrysosporium show linear and total mycelial growth inhibition at 73 µg/mL of Thiram. T. cingulata is 10 times more sensitive to Thiram, while F. verticillioides and S. rolfsii need a higher dose of fungicide to stop mycelial growth. In particular, the S. rolfsii toxicity curve flattens at higher doses of fungicide, indicating a lower sensitivity of the fungus to high doses of Thiram. Figure 3 reports the reduction in fungal growths, expressed as a reduction in mycelium radial growths on Petri dishes with respect to the negative control.

Antifungal In Vitro Test
The antifungal activity of the four lavender EOs against the growth of S. rolfsii, B. cinerea, F. verticillioides, P. chrysosporium and T. cingulata phytopathogenic fungi was carried out in vitro. LaCC-, LaPE-, LaPS-and LaPRV-characterized lavender EOs were used at different concentrations, ranging from 0 to 40 µL, as described in Section 4.6. Figure 3 reports the reduction in fungal growths, expressed as a reduction in mycelium radial growths on Petri dishes with respect to the negative control.
Molecules 2023, 28, 392 9 of 19 (e) (f) The results show, first of all, a concentration-dependent trend in antifungal activity; then, each fungus shows a different profile of interaction and a different sensitivity among the tested EOs. Total inhibitory growth is generally reached using an amount ranging from 20 to 40 µL. F. verticillioides (Figure 3e) shows slightly different radial growth curves; in this fungus, EOs have a lower performance in terms of fungal growth inhibition. For example, on F. verticillioides, LaPE is less effective, with about 50% growth inhibition with 40 µL of the analyzed sample. On the contrary, LaPE applied on P. chrysosporium ( Figure  3a) shows better inhibitory activity. In the same experimental conditions, it inhibits the growth of P. chrysosporium mycelium by 100% with only 20 µL of EO. Good inhibitory activity was found on S. rolfsii and B. cinerea using 30 µL of LaPS (Figure 3b,c), while the same amount of LaCC showed a better inhibition growth kinetic on T. cingulata ( Figure  3d). LaPRV EO showed the best inhibition growth kinetics on F. verticillioides, although this fungus proved to be the least sensitive to treatment with EOs. The positive controls for antifungal activity were carried out using PDA plates added with Thiram (Tetrasar 50, powder, Isagro srl) at final concentrations in the ranges of 0 and 73 µg/mL (Figure 3f). B. cinerea and P. chrysosporium show linear and total mycelial growth inhibition at 73 µg/mL of Thiram. T. cingulata is 10 times more sensitive to Thiram, while F. verticillioides and S. rolfsii need a higher dose of fungicide to stop mycelial growth. In particular, the S. rolfsii toxicity curve flattens at higher doses of fungicide, indicating a lower sensitivity of the fungus to high doses of Thiram.

Antioxidant Assay
The examined EOs of L. angustifolia L. showed different antioxidant activity, expressed in terms of IC 50 . The LaCC sample exhibited the highest antioxidant capability with an IC 50 value of 26.26 ± 0.21 mg/mL, followed by the LaPRV sample (IC 50 = 33.53 ± 0.23 mg/mL) ( Table 4). As shown in Figure 4, LaPE and LaPS EOs had comparable scavenging activity, with IC 50 values of 48.00 ± 0.35 and 49.63 ± 0.42 mg/mL, respectively. The scavenging activity ranged from 13.08% to 98.70% on LaPS and LaCC EOs, using concentrations ranging between 8.71 mg/mL and 261.21 mg/mL, respectively. The IC 50 value of standard ascorbic acid was 0.024 + 0.004 mg/mL.  The old wood appeared to be in an altered condition. A large portion of the scratched surface of the wood was affected by material gaps and traces of light white-gray residues of a nature yet to be ascertained ( Figure 5). The wood showed visible signs of degradation, with irregular loss of material and texture. White powdery residues were not visible on the opposite side of the wood, indicating that the wood may have been used less or that more care was taken when cleaning it after use.

Microbial Counts-Growth on Sample Surfaces Was Analyzed
The microbial growth that was detected revealed the sole presence of fungi and an absence of bacteria in almost all samples analyzed. Higher values for fungal counts were observed on the altered wooden timber, on the larger side surface with average values of < 10 CFU/cm 2 . The microbial diversity in the analyzed samples was very low and revealed the presence of one predominant fungal type; this may be due to the peculiar indoor environmental conditions, such as those present in the confined room of the Mostra Permanente (Museum). The inocula were smeared on the surface of Petri dishes containing malt agar 2% (Difco) added with streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL ampicillin (Fisher BioReagents, Monza, Italy). The plates were incubated at 26 °C for 4 days. The fungal isolate was identified as S. rolfsii and it was used together with four other plant pathogenic fungi to evaluate the antifungal activity of lavender EOs (see Section 2.3).  The old wood appeared to be in an altered condition. A large portion of the scratched surface of the wood was affected by material gaps and traces of light white-gray residues of a nature yet to be ascertained ( Figure 5). The wood showed visible signs of degradation, with irregular loss of material and texture. White powdery residues were not visible on the opposite side of the wood, indicating that the wood may have been used less or that more care was taken when cleaning it after use.  The old wood appeared to be in an altered condition. A large portion of the scratched surface of the wood was affected by material gaps and traces of light white-gray residues of a nature yet to be ascertained ( Figure 5). The wood showed visible signs of degradation, with irregular loss of material and texture. White powdery residues were not visible on the opposite side of the wood, indicating that the wood may have been used less or that more care was taken when cleaning it after use.

Microbial Counts-Growth on Sample Surfaces Was Analyzed
The microbial growth that was detected revealed the sole presence of fungi and an absence of bacteria in almost all samples analyzed. Higher values for fungal counts were observed on the altered wooden timber, on the larger side surface with average values of < 10 CFU/cm 2 . The microbial diversity in the analyzed samples was very low and revealed the presence of one predominant fungal type; this may be due to the peculiar indoor environmental conditions, such as those present in the confined room of the Mostra Permanente (Museum). The inocula were smeared on the surface of Petri dishes containing malt agar 2% (Difco) added with streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL ampicillin (Fisher BioReagents, Monza, Italy). The plates were incubated at 26 °C for 4 days. The fungal isolate was identified as S. rolfsii and it was used together with four other plant pathogenic fungi to evaluate the antifungal activity of lavender EOs (see Section 2.3).

Microbial Counts-Growth on Sample Surfaces Was Analyzed
The microbial growth that was detected revealed the sole presence of fungi and an absence of bacteria in almost all samples analyzed. Higher values for fungal counts were observed on the altered wooden timber, on the larger side surface with average values of <10 CFU/cm 2 . The microbial diversity in the analyzed samples was very low and revealed the presence of one predominant fungal type; this may be due to the peculiar indoor environmental conditions, such as those present in the confined room of the Mostra Permanente (Museum). The inocula were smeared on the surface of Petri dishes containing malt agar 2% (Difco) added with streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL ampicillin (Fisher BioReagents, Monza, Italy). The plates were incubated at 26 • C for 4 days. The fungal isolate was identified as S. rolfsii and it was used together with four other plant pathogenic fungi to evaluate the antifungal activity of lavender EOs (see Section 2.3).

Discussion
Fungal infections are not just a human health problem, they also affect the fields of agriculture and cultural heritage [36][37][38]. It is estimated that 20% and 40% of the total agricultural productivity loss is caused by animals, weeds and pathogens. These losses have implications for human health, the environment, and the economy [37]. To cite some data, in the 21st century, it is estimated that the loss of crops is due to 18% from animal parasites and 16% from microbial diseases (a large majority due to phytopathogenic fungi), for an average loss of 68% of the tonnage of agricultural production [39].
Microorganisms (lichens, algae, fungi and bacteria) are biodeteriogens and agents of colonization on artwork surface [29].
Plant extracts and EOs are ecological, protective, curative and antagonistic to many diseases. Therefore, plant extracts may have an important role in controlling soil-borne diseases, as they are a rich source of bioactive substances [40]. For example, thyme (Thymus vulgaris) EO is already known to be effective against fungi due to its high concentration of thymol and carvacrol [41].
Known in aromatherapy for its relaxing and sedative virtues, lavender EOs are evaluated in this study for their effectiveness against microorganisms, including fungi [42]. A comparison among the chemical components of four lavender EOs reveals linalool, linalyl acetate and borneol as the most representative compounds. However, differences occurred among samples, suggesting that a different chemical composition is subject to change under the influence of several aspects, such as climatic conditions and environmental factors. This resulted in variability in antifungal and antioxidant activity. The percent model affinity (PMA) index (Table 3) was used to identify similarities between the four lavender EOs. Two pairs of lavender EOs, LaPE/LaPS (PMA = 0.866) and LaCC/LaPRV (PMA = 0.719), were identified as having a high similarity level, while, in terms of "diversity" (Table 3), homogeneous structure in the chemical compositions was suggested for all the four types of the EOs. In the LaPE/LaPS pair, along with linalool, we observed a high level of borneol (13.65% and 16.83%), limonene (3.83% and 3.43%), camphor (5.68% and 3.87) and terpinen-4-ol (8.2% and 9.98%), while the concentration of linalyl acetate (1.8% and 2.41%) and lavandulyl acetate (0.49% and 0.98%) was very low (Table 1). In the LaCC/LaPRV pair, the representative components were linalyl acetate (24.34% and 31.07%) and lavandulyl acetate (6.51% and 3.2%), while borneol (1.05% and 4.43%) and limonene (0.25% and 0.75%) were in low concentrations.
A relatively small amount of linalyl acetate in comparison to the literature data has already been found in earlier studies [20]. The cross plots of Figure 2 reveals other components, particularly monoterpenoids (1,8 cineole, terpinen 4-ol and camphor), that contributed to the diversity among the lavender pairs. Most of these compounds belong to the alcohol group and, together with linalool, represent the monoterpenoid component that was the most abundant in all four lavenders ( Table 2). In our analysis, we also observed that the composition of the EO from LaCC was more complex than that of the other samples; E-β-ocimene (3.45%), terpinen-4-ol (5.42%), E-caryophyllene (2.78%), caryophyllene oxide (2.84%) and lavandulol (1.98%) were also detected. This feature could explain the better antioxidant activity observed with respect to the other EOs.
Several methods are used to evaluate the antioxidant activity of EOs obtained from different plants; however, differences in these methods may lead to different results that make comparisons difficult, and thus, investigations on the modification and improvement of these methods still continue to provide the most reliable technique [43]. We chose the DPPH method because it is a popular, quick, easy and convenient approach for the measurement of antioxidant properties involving the use of free radicals to assess the potential of substances to act as hydrogen donors or free radical scavengers [44,45]. Moreover, several parameters affect EO composition that may also result in different antioxidant activity values [46,47].
Furthermore, the results of the antioxidant activities of the four lavenders can be explained by considering the similarity between the same pairs (LaCC/LaPRV and LaPE/LaPS). Table 4 reports the IC 50 values (mg/mL) and scavenging activity range (%) of the four lavender EOs analyzed. The composition of the EOs may explain the different values of IC 50 in the antioxidant test. The higher antioxidant activity detected in LaCC EO (IC 50 26.26 mg/mL) could be related to the synergistic property associated with the minor components in the mixture, as reported in the literature [48][49][50]. LaCC EOs, along with linalool, linalyl acetate and lavandulyl acetate as the major components, contain several minor terpenoids, such as E-caryophyllene, which possess antioxidant, anti-inflammatory and analgesic properties [51]. Ruberto et al. [52] tested about 100 pure compounds present in EO for their antioxidant effectiveness. More recently, antioxidant activity has been shown in linalool, which is the dominant terpenoid in all four EOs tested [53].
Antifungal activity was performed in vitro on two white rot fungi (P. chrysosporium and T. cingulata) and three others responsible for rotting and diseases in various organs of the plant (S. rolfsii, B. cinerea and F. verticillioides) using four different L. angustifolia EOs. Results highlighted that LaPS shows efficacy against S. rolfsii, LaPE is more active against P. chrysosporium, LaCC inhibits T. cingulata and, finally, LaPRV is active against B. cinerea and F. verticillioides. In all cases, F. verticillioides appears to be more resistant to the toxic effect of the EOs used. These results are in accordance with [54,55], which indicate an ED 50 against B. cinerea, Fusarium spp. and Fusarium oxysporum of 223 µg/mL, 520 µg/mL and 372 µL/mL, respectively. Synergic effects and different cellular targets may be the key to interpretation. While major components have been extensively studied [56][57][58], others minority components play various roles. They may enhance or decrease the synergic effect by modifying the texture, color or density of the oil, but also EO cellular penetration or its lipophilic or hydrophilic nature, its membrane or wall fixation and its distribution within the cell, making the simultaneous inhibition of different cell targets possible [42,59]. In this regard, the lavenders used show a fairly wide range of monoterpenes and sesquiterpenes, between 5.78 and 13.11% and between 1.77 and 5.49%, respectively.
The comparison of data among the lavenders analyzed gives us the opportunity to select one or more populations with distinct EO chemical components with respect to the others. That is, choose a specific lavender carefully on the basis of its chemical composition to expect the results that it offers.
Preliminary results from our experiments could be useful from the perspective of controlling biodeteriogen activity (i.e., fungi) on altered wooden artworks. The indications will allow for the development of an organic green strategy for the recovery of altered works of art, as an alternative to the use of biocides and toxic compounds. The use of L. angustifolia EOs as a natural essence, for instance, inside a confined space such as a bag containing an old work of art to be recovered, could be a suitable technical solution [39].
However, not all that comes from nature is safe and free of hazard for human health. Since prehistorical times, the presence of toxic and therapeutic ingredients in natural plants and extracts (roots, leaves, fruits, fungi, etc.) has been well known and has been adopted or excluded in traditional and ethnic medicine around the world. Lavender derivatives, including EO, are not fully free of risk since they are included in the REACH list [60] among the substances that cause "serious eye irritation, are harmful to aquatic life with long-lasting effects and may cause an allergic skin reaction" [61].
The overall results suggest a careful use of each EO of lavender, taking into account its peculiarities, efficacy and limitations for utilization.

EOs Isolation
Flowers (100 g) of lavenders were hand-selected, cleaned and then separately subjected to hydrodistillation for 2 h according to the standard procedure described in the European Pharmacopoeia [62]. The EOs were dried over anhydrous sodium sulfate to remove traces of water and then stored in dark vials at 4 °C prior to gas chromatographymass spectrometry (GC-MS) analysis.

GC-FID Analysis and GC/MS Analysis
The characterization of the EO samples was determined using a gas chromatography system, GC 86.10 Expander (Dani), equipped with a FID detector, Rtx ® -5 Restek capillary column (30 m × 0.25 mm i.d., 0.25 um film thickness) (diphenyl-dimethyl polysiloxane), a split/splitless injector heated to 250 °C, and a flame ionization detector (FID) heated to 280 °C. The column temperature was maintained at 40 °C for 5 min, and then programmed to increase to 250 °C at a rate of 3 °C/min and held, using an isothermal process, for 10 min. The carrier gas was He (1.0 mL/min); 1 uL of each sample was dissolved in n-hexane (1:500) and injected. GC-MS analyses were performed on a Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) gas chromatography instrument equipped with a Rtx ® -5 Restek capillary column (30 m × 0.25 mm i.d., 0.25 um film thickness) and coupled with an ion-trap (IT) mass spectrometry (MS) detector Polaris Q (Thermo Fisher Scientific, Wal-

EOs Isolation
Flowers (100 g) of lavenders were hand-selected, cleaned and then separately subjected to hydrodistillation for 2 h according to the standard procedure described in the European Pharmacopoeia [62]. The EOs were dried over anhydrous sodium sulfate to remove traces of water and then stored in dark vials at 4 • C prior to gas chromatography-mass spectrometry (GC-MS) analysis.

GC-FID Analysis and GC/MS Analysis
The characterization of the EO samples was determined using a gas chromatography system, GC 86.10 Expander (Dani), equipped with a FID detector, Rtx ® -5 Restek capillary column (30 m × 0.25 mm i.d., 0.25 um film thickness) (diphenyl-dimethyl polysiloxane), a split/splitless injector heated to 250 • C, and a flame ionization detector (FID) heated to 280 • C. The column temperature was maintained at 40 • C for 5 min, and then programmed to increase to 250 • C at a rate of 3 • C/min and held, using an isothermal process, for 10 min. The carrier gas was He (1.0 mL/min); 1 uL of each sample was dissolved in n-hexane (1:500) and injected. GC-MS analyses were performed on a Trace GC Ultra The GC conditions were the same as those described above for the gas chromatography (GC-FID) analysis.

Identification of EO Components
The identification of the essential oil components was based on the comparison of their Kovats retention indices (Exp RI), determined in relation to the tR values of a homologous series of n-alkanes (C8-C20) injected under the same operating conditions as those in the literature [63,64]. The MS fragmentation pattern of each single compound with those from the NIST 02, Adams and Wiley 275 mass spectral libraries was compared [65,66]. The relative contents (%) of the sample components were computed as the average of the GC peak areas obtained in triplicate without any corrections [67]. All analytical standard components utilized (n-alcane C8-C20, linalool, borneol, terpinen-4-ol, camphor and lavender oil) were bought from Sigma Aldrich, St. Louis, MO, USA.

Statistical Analysis
The explorative data analysis was performed using the R software, available for free under the terms of the Free Software Foundation's GNU General Public License in source code form [68].
Concerning the results presented in Section 2.2, the levels of diversity in the chemical composition of the EOs were evaluated using the classic Shannon entropy: where p i is the proportion of the i-th of k compounds observed in the sample, and with its relative version, the Pielou index, Furthermore, the levels of dissimilarities between the compositions of the different types of lavender EOs were assessed using the percent model affinity (PMA) index, where A and B denote two generic samples.

Antifungal Activity Assay
The S. rolfsii and four other fungal strains (B. cinerea, F. verticillioides, P. chrysosporium and T. cingulata), previously identified and characterized (Figure 7) [69][70][71], were used in this study. Pure essential oils from the samples LaCC, LaPE, LaPS and LaPRV were dissolved in a final volume of 200 µL in ethanol and then added to 19 mL PDA (Oxoid Limited, Basingstoke, Hampshire, UK) plates to obtain the different final concentrations. Mycelial plugs (4 mm in diameter) from the edges of Petri dish cultures were incubated in the center of each PDA plate (90 mm diameter). Fungal cultures were incubated in the dark at 26 • C and 70% relative humidity (RH) for a variable number of days ranging from 3 to 12, depending on the fungal species analyzed (3 days for P. chrysosporium, 4 days for S. rolfsii and B. cinerea, and 12 days for T. cingulata and F. verticillioides). The tests were conducted in triplicate. The antifungal activity was determined by measuring the diameter (in mm) of the radial growth. The control growth was carried out on PDA plates prepared as described above, but without the EO samples. The positive controls for antifungal activity were carried out using PDA plates added with Thiram (Tetrasar 50, powder, Isagro Srl, Aprilia, Italy) at final concentrations in the range of 0-73 µg/mL. ter (in mm) of the radial growth. The control growth was carried out on PDA plates prepared as described above, but without the EO samples. The positive controls for antifungal activity were carried out using PDA plates added with Thiram (Tetrasar 50, powder, Isagro Srl, Aprilia, Italy) at final concentrations in the range of 0-73 µg/mL.

Antioxidant Activity
Antioxidant activity was determined by assessing the scavenging capacity of antioxidant compounds towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, using the standard method [72] adopted with suitable modifications [73]. In particular, for each EO, different aliquots were added to 1 mL of a freshly prepared DPPH methanolic solution (27 µg/mL) to obtain diverse EO concentrations (as reported in Figure 4). The samples were incubated in the dark at room temperature for 30 min. The absorbance (A) of each sample was measured using a UV-Vis spectrophotometer (Shimadzu UV-1601) at a wavelength of 517 nm. Measurements were also performed on control samples consisting of 1 mL of DPPH solution (27 µg/mL). Scavenging activity percentages-obtained by applying the formula [(Acontrol−Asample)/Acontrol] × 100-were correlated with EO concentrations. In this way, it was possible to calculate the IC50 value, which is a measure of antioxidant activity. Ascorbic acid was used as a positive control. Experiments were conducted in triplicate, and results were expressed as mean of the obtained IC50 values ± standard error (SE).

Antioxidant Activity
Antioxidant activity was determined by assessing the scavenging capacity of antioxidant compounds towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, using the standard method [72] adopted with suitable modifications [73]. In particular, for each EO, different aliquots were added to 1 mL of a freshly prepared DPPH methanolic solution (27 µg/mL) to obtain diverse EO concentrations (as reported in Figure 4). The samples were incubated in the dark at room temperature for 30 min. The absorbance (A) of each sample was measured using a UV-Vis spectrophotometer (Shimadzu UV-1601) at a wavelength of 517 nm. Measurements were also performed on control samples consisting of 1 mL of DPPH solution (27 µg/mL). Scavenging activity percentages-obtained by applying the formula [(A control − A sample )/A control ] × 100-were correlated with EO concentrations. In this way, it was possible to calculate the IC 50 value, which is a measure of antioxidant activity. Ascorbic acid was used as a positive control. Experiments were conducted in triplicate, and results were expressed as mean of the obtained IC 50 values ± standard error (SE).

Conclusions
This study showed significant variability in the EO composition of four L. angustifolia L. populations collected at full flowering from different geographical areas. We observed that two pairs of LaPE/LaPS and LaCC/LaPRV EOs, on the basis of both their chemical composition and percent model affinity (PMA) index, were identified.
The in vitro antifungal activity of lavender EOs against S. rolfsii, B. cinerea, F. verticillioides, P. chrysosporium and T. cingulata showed a wide range of variability responses. Furthermore, the in vitro antioxidant activity by DPPH assay showed variability related to the different compositions of EOs.
If confirmed by further studies, the antifungal activities of lavender EOs for artwork recovery could be useful in setting up an advanced green biological strategy as an alternative to synthetic biocides and toxic compounds.
Lavender oil, which is now used as a flavoring ingredient in food processing, could be used as an antioxidant to preserve foods and also protect artworks from biodeterioration. However, it is advisable to be very careful when handling lavender EOs because of their potential for harm from certain chemical components they contain.