Profiling the Variability of Eucalyptus Essential Oils with Activity against the Phylum Nematoda †

The genus Eucalyptus (Myrtaceae) comprises more than 800 species, mostly native to Australia. Eucalyptus shoots essential oils (EOs) are well-known for their extremely high qualitative and quantitative variation in terpenes (mainly monoand sesquiterpenes). These EOs have a wide range of uses, from the taxonomic characterization of populations based on the chemical profiling of EO chemotypes, to industrial applications, including pharmaceutical, agrochemical, and in food and fragrances. In this study, we reviewed the available information concerning the chemical variability of EOs from Eucalyptus spp. assayed against nematodes. Among the most active EOs, those from E. globulus, E. staigeriana, and E. citriodora were more frequently used. EO chemical composition was mainly dominated by 1,8-cineole, limonene, p-cymene, citronellal and piperitone in varying proportions. Nematicidal activity of Eucalyptus EOs was reported against animal-parasitic nematodes, including gastrointestinal nematodes (e.g., Haemonchus contortus), plant-parasitic nematodes, such as root-knot nematodes (e.g., Meloidogyne incognita and M. chitwoodi) or the pinewood nematode Bursapelenchus xylophilus, and the free-living nematode Caenorhabditis elegans. Correlation between EO qualitative and quantitative composition with its respective activity may provide valuable information on the nematicidal specificity of EOs. This knowledge can be useful for devising environmentally safer pest management strategies in the conservation of ecosystems biodiversity.


Introduction
Essential oils (EOs) are volatile mixtures, exclusively obtained from plant material by hydro-, steam-or dry-distillation, or in the case of Citrus fruits, mechanically without heating [1]. These volatile mixtures are mainly composed of terpenes (mono-, sesqui-, and di-terpenes) and phenylpropanoids, and are usually dominated by one to three major components at relatively high amounts [2]. The genus Eucalyptus (Myrtaceae family) comprises more than 800 species, mostly native to Australia. Eucalyptus spp. have been extensively explored in the pharmaceutical and cosmetic industries due to their terpene-rich essential oils (EOs), namely high quantities of 1,8-cineole (also referred as Eucalyptol), an oxygenated monoterpenoid used extensively in flavorings, fragrances and cosmetics [3]. In traditional medicine, eucalypt leaves and 1,8-cineole are generally used as cough suppressants.
Eucalyptus spp. are well-known for the high foliar terpene qualitative and quantitative variations, at the taxa, population, and individual levels, and a large number of chemotypes have been identified [4][5][6][7][8][9]. Terpenes are a large class of secondary metabolites, with an important ecological role in mediating plant-plant and plant-animal interactions. In the Myrtaceae family, terpenes have been mostly implicated in defensive roles against herbivores and pathogens [7]. EO chemotypes are defined by qualitative and quantitative differences in the EO chemical composition among populations of the same species, due to genetic variations in the regulation of terpene biosynthesis [4,10,11]. The occurrence of EO chemotypes has been strongly associated with geographical variation and can reflect the different environmental conditions to which plants are exposed (e.g., altitude, solar exposition, or soil type) [1]. In addition, the frequent occurrence of EO chemotypes within Eucalyptus spp. could implicate differences in biological activities and should be carefully evaluated. Eucalyptus EOs have a wide range of biological activities including anti-microbial, fungicidal, insecticidal/insect repellent, herbicidal, acaricidal and nematicidal [12]. The phylum Nematoda (or Nemathelminthes) comprise roundworms and eelworms (parasites of plants). Nematodes are present in every ecosystem being found in freshwater, marine and terrestrial environments, and can be parasitic (parasites of plants, insects, humans and other animals) or non-parasitic (free-living). They are vermiform, unsegmented, bilaterally symmetrical pseudocelomates, having a pseudocoelom lined with mesoderm, in one side, and with endoderm on the other side. The nematode body is protected by the cuticle, a very complex and evolutionarily plastic structure that functions as protection and is involved in body movement and maintaining shape. Nematodes have digestive, reproductive, nervous and excretory systems, but lack circulatory or respiratory systems [13].

Chemical Variability of Eucalyptus Essential Oils
Chemical composition was reported for a total of 32 EOs tested against plant-parasitic (21), animal-parasitic (10) and free-living nematodes (1). E. citriodora, E. globulus and E. staigeriana were the most studied Eucalyptus species, with seven, three and five EOs tested, respectively. Cluster analysis was performed to determine the similarity between the composition of the tested EOs. Three main clusters were obtained (Figure 1a)

Chemical Composition of Active EOs
A total of 18 EOs were reported with a high activity against nematodes. Cluster analysis revealed three major clusters, with E. citriodora EOs included in one single cluster, E. globulus and one unidentified Eucalyptus sp. grouped together in another cluster, and E. staigeriana clustered together with E. dives and one unidentified Eucalyptus sp. (Figure 1b). No correlation could be established between EO clustering and activity against specific nematode groups. Despite the small number of reported nematicidal chemically characterized EOs some preliminary considerations could be made regarding activity against the groups of nematodes analyzed. Only one EO was tested against free-living nematodes, thus no substantial comparison can be made with the remaining nematode types. The oxygenated monoterpene 1,8-cineole could be found in EOs active against all nematode types, in amounts ≥20% (Table 1, Figure 2). The hydrocarbon monoterpene p-cymene was found in amounts ≥20% in EOs active against plant-parasitic nematodes while the hydrocarbon monoterpene limonene was only found in amounts ≥20% in EOs active against animal-parasitic nematodes.  The monoterpenic aldehyde citronellal was found in amounts ≥20% in EOs active against plant-and animal-parasitic nematodes while the ketone piperitone was only found in amounts ≥20% in EOs active against plant-parasitic nematodes (Table 1, Figure  2).

Conclusions
Chemical variability is an important trait of Eucalyptus EOs. In this study, EO composition from Eucalyptus spp. assayed against nematodes was reviewed. Cluster analysis grouped EOs from E. citriodora and E. globulus in two clusters based on the EO main components, namely citronellal and 1,8-cineole, respectively. E. staigeriana EO showed high variability in the EO main components, namely limonene, geranial, geraniol and methyl geranate. Concerning nematicidal EOs, p-cymene and 1,8-cineole were ubiquitously present, yet high proportions of limonene and piperitone were exclusive to animal-or plantparasitic nematodes, respectively. Citronellal showed high relative amounts for both animal-and plant-parasitic nematodes. Although supported by very few data, some chemical trends seem to indicate that specificity may occur for nematicidal EOs. A greater number of studies is necessary to understand how Eucalyptus EO chemical variability can influence nematicidal activity. This knowledge can be very valuable for the establishment of precision biocides with neutral environmental impacts.