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Article

Composition of the Scent in Some Ophrys Orchids Growing in Basilicata (Southern Italy): A Solid-Phase Microextraction Study Coupled with Gas Chromatography and Mass Spectrometry

by
Maurizio D’Auria
1,*,
Richard Lorenz
2,
Marisabel Mecca
1,
Rocco Racioppi
1 and
Vito Antonio Romano
1
1
Dipartimento di Scienze, Università della Basilicata, V.le dell’Ateneo Lucano 10, 85100 Potenza, Italy
2
AHO Baden-Württemberg, 64686 Lautertal, Germany
*
Author to whom correspondence should be addressed.
Compounds 2023, 3(4), 573-583; https://doi.org/10.3390/compounds3040041
Submission received: 6 October 2023 / Revised: 8 November 2023 / Accepted: 10 November 2023 / Published: 14 November 2023
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Abstract

:
Several methods have been used to determine the volatile organic compounds emitted by Ophrys orchids. The use of different methods results in incomparable data. Solid-phase microextraction (SPME) has not been used extensively on Ophrys orchids. The main components found in the SPME analysis of the scent in Ophrys orchids were as follows: O. apifera: benzyl benzoate and α-copaene; O. crabronifera subsp. biscutella: pentadecane, heptadecane, and nonadecane; O. bertolonii subsp. bertolonii: pentadecane and heptadecane; O. passionis subsp. garganica: i-propyl palmitate and heptadecane; O. holosericea subsp. apulica: α-copaene, pentadecane, and heptadecane; O. lacaitae: α-copaene, pentadecane, and heptadecane; O. bombyliflora: cyclosativene, pentadecane, and ethyl dodecanoate; O. insectifera: 8-heptadecene and pentadecane; O. lutea: heptadecane and docosane; O. tenthredinifera subsp. neglecta: α-copaene, caryophyllene, and i-propyl palmitate.

1. Introduction

Solid-phase microextraction (SPME) is a simple and cheap method to identify the composition of volatile organic compounds in natural matrices. SPME has proven to be an extremely powerful and versatile method. Considering only recently published articles, it is possible to find articles on natural matrices such as radish [1], Camellia oleifera [2], soy [3], and soybean flowers [4]. Furthermore, articles devoted to finding volatile organic compounds in microorganisms such as kombucha fermentation broth [5], Kosakonia cowani [6], and Bacillus aryabhattai AYG1023 [7] have been published. Finally, there have been several articles related to the chemical characterization of food and beverages, such as grapevine chips for wine production [8], virgin olive oil [9], oranges [10], coffee [11], rice milk [12], Jiang-flavor baijiu liquor [13], and meat [14].
Some years ago, a systematic study was initiated on the scent of spontaneous orchid species growing in Basilicata (Southern Italy). The aim of this study was to create a homogeneous picture of the composition of the aroma of these species using the same methodology for all of the species. In particular, we decided to use solid-phase microextraction (SPME) [15]. This study allowed us to identify the components of the scent of Platanthera bifolia subsp. osca [16], Platanthera chlorantha [17], Cephalanthera orchids [18], Orchis [19], Serapias [20], Himantoglossum [21]. Barlia robertiana [22], Dactylorhiza [23], Gymnadenia [24], Neotinea [25], and Anacamptis orchids [26].
Ophrys orchids are distributed between the two subgenera Fuciflorae and Ophrys, and they belong to different sections and subsections (O. apifera Huds. 1762, O. bertolonii subsp. bertolonii Moretti 1823, O. crabronifera subsp. biscutella (O. Danesch and E. Danesch) Kreutz and Kreutz 2013, O. bombyliflora Link (1779) 1800, O. holosericea subsp. apulica (O. Danesch and E. Danesch) Buttler 1986, O. insectifera L. 1753, O. lacaitae Lojac. 1909, O. lutea subsp. lutea Cav. 1793, O. passionis subsp. garganica E. Nelson ex H. Baumann and R. Lorenz 2005, O. tenthredinifera subsp. neglecta (Parl.) E. G. Camus, Bergon and A. Camus 1908) (Table 1 and Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5).
It is known that Ophrys flowers imitate the mating signals of some insect species and are pollinated by sexually excited males who mistake the flower for a female of the same species and pollinate it during a “pseudocopulation”.
Sexually deceptive orchids are unique in their exclusive and effective use of male insects, primarily aculeate Hymenoptera, but also other Hymenoptera and some Diptera [28].
In most of the European Ophrys species studied so far, male copulation attempts can only be elicited by a scent identical to the female sex pheromone of the pollinating species; visual cues appear to be less important [29].
The differences in odor between similar orchid species are often small. Small variations have been found between the bouquets of Ophrys fusca and O. bilunulata, as well as between the similar O. sphegodes and O. exaltata [29,30].
Sexually deceptive orchid species typically exploit one or a few specific species of pollinators and may have different pollinators in different regions. A single insect species can also pollinate more than one sexually deceptive orchid species in different regions [30].
All of the species examined in this work are sexually deceptive, with the sole exception of O. apifera, which is notoriously an autogamous (i.e., self-pollinating) species.
With this work, we wanted to use a rapid method (SPME) to test species belonging to different sections and subsections of the Ophrys genus in order to verify whether the scents they emit are very similar or different within the different groups that they belong to.
The scent of Ophrys orchids has been extensively studied. Most of the studies have been performed though the identification of the components of the extracts of labella. Thus, O. insectifera showed the presence of pentacosane, tetracosane, nonanoic acid, and nonanal as its main components in a study conducted in 1987 [31], as well as tricosene, pentacosane, 9-heptacosene, and 9-nonacosene in a study conducted in 2017 [32]. However, the absorption of the scent of O. insectifera subsp. insectifera on Porapak Q showed the main components to be pentadecane, heptadecane, and cyclosativene [33], then alkanes with a lower molecular weight than those determined in the other studies on orchids, and a terpene. In the scent of O. sphegodes, pentacosane and tricosane were found in labella extracts [34], while tricosane, pentacosane, and p-cresol were the main components of the scent obtained via steam distillation of the flowers [35]. The scent of O. lupercalis and O. iricolor was due to the presence of tricosane, pentacosane, and heptacosane [36]. Together with the same compounds, nonanal was found in O. lupercalis, O. bilunulata, and O. fabrella [37]. SPE collection of the scent of O. normanii showed the presence of octadecanal, tricosane, tricosene, and pentacosene [38]. SPE absorption of the scent of O. apifera showed the presence of butanol, butyl ether, and caryophyllene [39]. Pentacosene and tricosene were found in the labella extracts of O. holosericea [40], while nonanal was the main component of the scent of O. lutea [41]. These studies showed that the extraction of the labella led to the identification of high-molecular-weight alkanes and alkenes as the main components of the aroma of these orchids. However, when SPME was used to determine the composition of the scent of O. bertolonii subsp. Benacensis, 4-methyl tetradecane, nonanal, decanal, dodecanal, 3,5-octadiene-2-one, and caryophyllene were found as the main components of the aroma [42].
In this work, the scents emitted by ten species of spontaneous orchids growing in Basilicata (Italy), belonging to the Ophrys genus, were determined using solid-phase microextraction. This method allows us to define a homogeneous framework of the scent of several species in the genus.

2. Materials and Methods

2.1. Plant Material

The sample of O. apifera was collected at Piani del Mattino (Pz) on 8 June 2017. The sample of O. crabronifera subsp. biscutella was collected at Valico Faggeto in the municipality of Moliterno (Pz) on 11 March 2018. The sample of O. bertolonii subsp. bertolonii was collected at Monte Grosse (Pz) on 18 April 2018. The sample of O. bombyliflora was collected at Contrada Macchia Orsino in the municipality of Tolve on 9 April 2018. The sample of O. holosericea subsp. apulica was collected at Scalo di Grassano on 19 April 2018. The sample of O. insectifera was collected at Monte Zaccana in the municipality of Castelluccio Superiore on 2 May 2018. The sample of O. lacaitae was collected at Contrada l’Aia Antica in the municipality of Calvello on 6 June 2018. The sample of O. lutea subsp. lutea was collected at Scalo di Albano on 11 April 2018. The sample of O. passionis subsp. garganica was collected at Scalo di Campomaggiore on 16 April 2018. The sample of O. tenthredinifera subsp. neglecta was collected at Torrente Serrapotomo in the municipality of Laurenzana on 8 April 2018. The plants were collected by Vito Antonio Romano.
The plants were harvested taking all the clod of earth, taking care not to damage the root system. All of the plants had closed flowers to avoid using flowers that were already fertilized but not visible because they were at the beginning of fertilization. The plants were planted in special pots in the greenhouse of the University of Basilicata (Potenza 650 m. a.s.l.), in closed boxes, with transparent cloth to avoid fertilization (even if occasional). The correct classification of the species was carried out on the flowering plants. The plants were tested when the flowers were all open except for the last two.
The plants were tested whole, without being damaged, under a cylindrical glass bell (12 cm × 45 cm), in which only the inflorescence and the SPME probe were inserted [16,17,18,19,20,21,22,23,24,25,26].
To avoid contamination, the interior of the bell was isolated from the external environment with appropriate closing and sealing systems during the 24 h of the test (from eight in the morning to eight the following day).
In order to be sure that the internal environment of the bell was isolated from the external environment, various blank tests were carried out.
After the tests, the plants remained closed in the boxes to verify that at the end of flowering there were no fertile ovaries, and for this reason no herbarium samples were taken. The earthen breeds with the bulbs were brought back to the site.
In view of the fact that the investigated taxa are rare wild plants, in order to preserve the species, we chose to use a single plant for our analysis.

2.2. Analysis of Volatile Organic Compounds

The SPME analysis of ten different samples of Ophrys was performed. In this way, the identified plants were collected and placed in glass jars for 24 h, which also contained the fiber (DVB/CAR/PDMS) and SPME syringe. After this time, the fiber was desorbed in a gas chromatographic apparatus equipped with a quadrupole mass spectrometer detector. A 50/30 μm DVB/CAR/PDMS module with 1 cm fiber (57328-U, Supelco, Milan, Italy) was employed to determine the VOCs. The SPME fiber was maintained in the bell jar for 24 h. The analytes were desorbed in the splitless injector at 250 °C for 2 min. Analyses were accomplished with an HP 6890 Plus gas chromatograph equipped with a Phenomenex Zebron ZB-5 MS capillary column (30 m × 0.25 mm i.d. × 0.25 μm FT) (Agilent, Milan, Italy). An HP 5973 mass-selective detector in the range 0–800 m/z (Agilent) was utilized with helium at 0.8 mL/min as the carrier gas. The EI source was used at 70 eV. The analyses were performed using a splitless injector. The splitless injector was maintained at 250 °C, and the detector was maintained at 230 °C. The oven was held at 40 °C for 2 min and then gradually warmed at 8 °C/min up to 250 °C, where it was held for 10 min. Tentative identification of the aroma components was based on mass spectra and Wiley 11 and NIST 14 library comparisons. A single VOC peak was considered to have been identified when its experimental spectrum matched that present in the library with a score over 90%. All of the analyses were performed in triplicate.

3. Results and Discussion

The SPME-GC-MS analysis of Ophrys samples gave the results reported in Table 2. The main component of the scent of O. apifera was benzyl benzoate (22.52%), while other important components included a-copaene (9.11%), caryophyllene (8.07%), and cyclosativene (6.97%) (Table 2). The significant difference in the scent in comparison with that obtained using SPE absorption should be noted. In that case, butanol, butyl ether, and caryophyllene were the main components of the aroma [39,43]. The observed differences could be due to the different analyzed species, the different harvesting places (Basilicata and Catalonia), the different pollinator insects, and/or the different analytical procedures.
The analysis of the scent of O. crabronifera subsp. biscutella showed that the aroma is mainly due to the presence hydrocarbon compounds, as in several of the species reported above. However, the presence of pentadecene (6.43%), pentadecane (8.06%), heptadecane (8.37%), nonadecane (8.18%), and heinecosane (5.12%) was detected. Furthermore, i-propyl palmitate was detected at a relevant amount (5.06%) (Table 2). In other Ophrys species, the scent analysis performed through the labella extraction gave high-molecular-weight hydrocarbons (i.e., more than thirty carbon atoms), while, in our determination, the main component of the scent had seventeen carbon atoms. Unfortunately, other analyses of the same species are not available.
The scent of O. bertolonii subsp. bertolonii gave a similar result. The main component was pentadecane (28.62%), while other significant compounds included heptadecane (7.23%) and nonadecane (6.30%). Also, in this case, a significant difference was observed considering the results obtained in the SPME analysis of O. bertolonii subsp. benancensis [28]. The observed differences could be due to the different subspecies, the different harvesting places (Basilicata and Lecco), or the different pollinators.
When a sample of O. passionis subsp. garganica was analyzed, the main component of the scent was i-propyl palmitate (29.62%), while other components included caryophyllene (8.34%), pentadecane (8.69%), and heptadecane (9.22%). In this case, this is the first reported analysis of this species.
In the case of O. holosericea subsp. Apulica, the main components were a-copaene (11.30%) and pentadecane (11.48%), while other compounds found in the scent included caryophyllene (7.90%) and heptadecane (8.15%). The analysis of the labella extracts gave some alkenes as main components of the scent [40]. In this case, the difference in the analytical procedures was responsible for the observed differences.
The same trend was observed in the analysis of O. lacaitae: the main components were a-copaene (12.08%), pentadecane (13.40%), and heptadecane (14.43%), while caryophyllene was found in a relevant amount (6.68%).
The scent of O. bombyliflora showed the presence of cyclosativene (10.09%), pentadecane (10.33%), and ethyl dodecanoate (9.46%). The analysis of the scent of O. insectifera gave the following results: the main component was 8-heptadecene (18.88%), followed by pentadecane (13.73%) and heptadecane (7.98%). Previous results obtained from labella extracts showed the presence of high-molecular-weight compounds [41,42]. The compounds detected through absorption on Porapak Q were quite similar to the results presented here, with the exception of caryophyllene [33]. Finally, it is noteworthy that all of the high-molecular-weight hydrocarbons detected in the labella were solid and had a very low vapor pressure. As a result of these properties, it is very unlikely that they would be present in the scent.
Nonanal was the compound detected in a previous work in O. lutea [41]. SPME analysis showed the presence of heptadecane (39.37%), 8-heptadecene (7.25%), nonadecane (8.87%), and docosane (9.94%). These results are consistent with the trend of SPME analysis on Ophrys orchids, where, with some differences for different orchid species, the compounds that we detected were very similar. The differences from previous results can depend on the analytical procedure. Finally, the scent of O. tenthredinifera subsp. neglecta included a-copaene (11.61%), caryophyllene (11.73%), and i-propyl palmitate (14.73%), showing another case beyond O. apifera where hydrocarbons are not present in relevant amounts in the scent.

4. Conclusions

In this study, the composition of the aroma of some orchids belonging to the Ophrys genus was determined. The results were obtained using SPME coupled with GC-MS as an analysis technique. In the ten samples analyzed, 62 compounds were found; however, the compounds present in greater quantities were almost always the same, with variations (sometimes substantial) between species. It is important to note that there was never any correspondence between our analyses and those obtained through the chemical extraction of plant labels. In this case, high-molecular-weight hydrocarbons are always recovered, although they are unlikely to be constituents of any aroma, being solid compounds with a low vapor pressure.
This article is part of a complex study with the aim of analyzing all of the orchid species that can be recovered in Basilicata. This work will be completed in the near future.
Finally, the main problem connected with the use of SPME is the different absorption of the scent component on the fiber [45]. An important study should be performed in order to obtain calibration curves for the main components of the scent.

Author Contributions

Conceptualization, M.D. and V.A.R.; investigation, M.M. and R.R.; data curation, R.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Compounds 03 00041 g001
Figure 1. Ophrys apifera (left); Ophrys crabronifera subsp. biscutella (right) (photos of V. A. R.).
Figure 1. Ophrys apifera (left); Ophrys crabronifera subsp. biscutella (right) (photos of V. A. R.).
Compounds 03 00041 g001
Compounds 03 00041 g002
Figure 2. Ophrys bertolonii subsp. bertolonii (left); Ophrys passionis subsp. garganica (right) (photos of V. A. R.).
Figure 2. Ophrys bertolonii subsp. bertolonii (left); Ophrys passionis subsp. garganica (right) (photos of V. A. R.).
Compounds 03 00041 g002
Compounds 03 00041 g003
Figure 3. Ophrys holosericea subsp. apulica (left); Ophrys lacaitae (right) (photos of V. A. R.).
Figure 3. Ophrys holosericea subsp. apulica (left); Ophrys lacaitae (right) (photos of V. A. R.).
Compounds 03 00041 g003
Compounds 03 00041 g004
Figure 4. Ophrys bombyliflora (left); Ophrys insectifera (right) (photos of V. A. R.).
Figure 4. Ophrys bombyliflora (left); Ophrys insectifera (right) (photos of V. A. R.).
Compounds 03 00041 g004
Compounds 03 00041 g005
Figure 5. Ophrys lutea subsp. lutea (left); Ophrys tenthredinifera subsp. neglecta (right) (photos of V. A. R.).
Figure 5. Ophrys lutea subsp. lutea (left); Ophrys tenthredinifera subsp. neglecta (right) (photos of V. A. R.).
Compounds 03 00041 g005
Table 1. Taxonomic identification of the species utilized in this study. The nomenclature has been referred to [27].
Table 1. Taxonomic identification of the species utilized in this study. The nomenclature has been referred to [27].
SpeciesSubgenusSectionSubsection
O. apiferaFucifloraeApiferae
O. crabronifera subsp. biscutellaFucifloraeAraniferaeSphegodes
O. bertolonii subsp. bertoloniiFucifloraeAraniferaeBertoloniorum
O. passionis subsp. garganicaFucifloraeAraniferaeSphegodes
O. holosericea subsp. apulicaFucifloraeFuciflorae
O. lacaitaFucifloraeFuciflorae
O. bombylifloraOphrysBomyliflorae
O. insectiferaOphrysOphrys
O. lutea subsp. luteaOphrysPseudophrysFusci-luteae
O. tenthredinifera subsp. neglectaOphrysTenthrediniferae
Table 2. Volatile organic compounds detected by using SPME-GC-MS in Ophrys species.
Table 2. Volatile organic compounds detected by using SPME-GC-MS in Ophrys species.
Compoundr.t. [min.]KI aArea% ± 0.03
O. apiferaO. crabronifera subsp. biscutellaO. bertolonii subsp. bertoloniiO. passionis subsp. GarganicaO. holosericea subsp. apulicaO. lacaitaeO. bombylifloraO.
insectifera		O. lutea subsp. luteaO. tenthredinifera subsp. neglecta
Octanol10.821072 4.69
Undecane11.311100 1.05
Decanal13.1711950.700.74 0.48
Dodecane13.271200 0.58 0.45 0.53
Nonanoic acid14.3812722.00
Isobornyl acetate14.971285 1.650.96 1.27
2-Undecanone15.011291 5.18
Tridecane15.051300 3.374.48 3.630.563.081.901.562.32
Decanoic acid16.0213353,30
Cyclosativene16.3813446.97 5.156.79 10.090.961.09
a-Copaene16.5213539.113.81 11.3012.083.15 11.61
Tetradecane16.7414000.861.816.363.724.473.173.472.131.093.60
Dodecanal16.861407 3.49
Caryophyllene17.2814288.071.081.728.347.906.684.38 1.2311.73
Geranylacetone17.4614510.88 1.62
b-Farnesene17.621454 3.58
Alloaromadendrene17.7614560.86
Epi-b-santalene17.801460 0.74
2,6-Di-t-butyl-p-benzoquinone17.961458 1.971.691.770.80 1.06
1-Pentadecene17.991489 6.43
Pentadecane18.3015002.528.0628.628.6911.4813.4010.3313.735.535.04
b-Cadinene18.6515072.52
Methyl dodecanoate18.681509 1.00 1.09
d-Cadinene18.821524 1.276.94 1.04
Dodecanoic acid19.1115591.780.71 0.98
Cyclotridecane19.461565 0.64
Ethyl dodecanoate19.751579 1.404.161.835.31 9.465.180.994.16
Hexadecane19.801600 2.863.586.386.164.176.973.382.084.03
Tetradecanal20.0116111.632.034.10 3.722.93 1.02
i-Propyl dodecanoate20.0616180.86
Isolongifolen-5-one20.171622 3.81
Benzophenone20.2816251.97
Unidentified20.62 8.52
Methyl dihydrojasmonate20.661648 3.15 2.93
8-Heptadecene20.911664 3.122.20 4.613.91 18.887.25
Heptadecane21.2217004.788.377.239.228.1514.436.677.9839.374.40
Pristane21.3017091.141.89 5.492.391.862.121.90 1.57
2-(Phenylmethylene)-octanal21.8717281.34
Farnesal21.891738 0.54
Tetradecanoic acid21.9117611.891.60
Benzyl benzoate22.16176822.52
3,5-Di-t-butyl-4-hydroxybenzaldehyde22.241771 0.80
Ethyl tetradecanoate22.511774 0.90 1.08 1.531.490.591.47
Octadecane22.5718002.181.891.662.072.073.512.911.811.551.71
Phytane22.641814 0.99
Hexadecanal22.8218191.60 1.24 1.084.04 0.83
Farnesyl acetaldehyde23.181855 1.81 2.26 1.09
Nonadecane23.8519001.858.186.301.724.286.812.802.998.871.65
b-Springene24.161922 1.60
Methyl hexadecanoate24.201927 0.74
Hexadecanoic acid24.581935 1.95 0.83
a-Springene24.781940 5.45
Ethyl 11-hexadecenoate24.801974 2.58
Eicosane25.0620001.821.151.32 1.181.571.061.560.74
Geranyl-a-terpinene25.152005 5.66
i-Propyl palmitate25.4020130.965.062.2429.625.56 2.732.2614.73
Heinecosane26.252100 5.123.264.853.954.131.38 1.50
Ethyl oleate26.9821690.92 1.81
Docosane27.3822001.52 1.30 0.87 1.819.940.71
1-Heneicosyl formate28.102250 0.77
9-Tricosene28.142270 4.26
Tricosane28.4723001.042.97 1.44 2.450.621.74
a KI: Kovats index [44].
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MDPI and ACS Style

D’Auria, M.; Lorenz, R.; Mecca, M.; Racioppi, R.; Romano, V.A. Composition of the Scent in Some Ophrys Orchids Growing in Basilicata (Southern Italy): A Solid-Phase Microextraction Study Coupled with Gas Chromatography and Mass Spectrometry. Compounds 2023, 3, 573-583. https://doi.org/10.3390/compounds3040041

AMA Style

D’Auria M, Lorenz R, Mecca M, Racioppi R, Romano VA. Composition of the Scent in Some Ophrys Orchids Growing in Basilicata (Southern Italy): A Solid-Phase Microextraction Study Coupled with Gas Chromatography and Mass Spectrometry. Compounds. 2023; 3(4):573-583. https://doi.org/10.3390/compounds3040041

Chicago/Turabian Style

D’Auria, Maurizio, Richard Lorenz, Marisabel Mecca, Rocco Racioppi, and Vito Antonio Romano. 2023. "Composition of the Scent in Some Ophrys Orchids Growing in Basilicata (Southern Italy): A Solid-Phase Microextraction Study Coupled with Gas Chromatography and Mass Spectrometry" Compounds 3, no. 4: 573-583. https://doi.org/10.3390/compounds3040041

APA Style

D’Auria, M., Lorenz, R., Mecca, M., Racioppi, R., & Romano, V. A. (2023). Composition of the Scent in Some Ophrys Orchids Growing in Basilicata (Southern Italy): A Solid-Phase Microextraction Study Coupled with Gas Chromatography and Mass Spectrometry. Compounds, 3(4), 573-583. https://doi.org/10.3390/compounds3040041

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