Essential Oils and Volatiles as Nematodicides against the Cyst Nematodes Globodera and Heterodera †

: The cyst nematodes of the genera Heterodera (HET) and Globodera (GLO) are among the most damaging obligate plant parasitic nematodes (PPNs) that parasitize cereals, rice, potatoes and soybean. In the absence of resistant crops, soil fumigation of pesticides provides a good strategy for population control. However, synthetic nematicides can cause negative environmental and public health impacts and are feared to lead to the development of resistance and immunity. The use of essential oils (EOs) could be a viable environmentally friendly alternative, which has been poorly explored on cyst nematodes but has shown very good results on other PPNs. The present work reviews the existing bibliography on the biological activity of EOs against GLO and HET. EOs from Allium sativum , Eucalyptus globulus , and Salvia ofﬁcinalis were the most active against GLO egg hatching. The EOs extracted from Hyssopus cuspidatus , Kaempferia galanga , Mentha canadensis , Ocimum basilicum , and Valeriana amurensis had the highest activity against HET J2 juveniles. Ethyl p -methoxycinnamate, a phenylpropanoid ester, was the EO volatile with the highest toxicity against HET, showing lower EC 50 values than the nematodicide fosthiazate. The study of EOs against cyst nematodes is still preliminary in comparison to other PPNs. Future works must expand this line of research and explore greener practices in cyst nematode pest management.


Introduction
The cyst nematodes (CNs) from the genera Heterodera (HET) and Globodera (GLO) are classified as the second most economically and scientifically important plant-parasitic nematode (PPN) group worldwide. These obligatory endoparasitic nematodes are highly distributed in global temperate regions and affect the productivity of essential food crops (e.g., potatoes, cereals, brassicas, tomatoes and sugar beet) [1]. Within this group, the soybean cyst nematode (SCN), Heterodera glycines; the potato cyst nematode (PCN), Globodera pallida and G. rostochiensis; and the cereal cyst nematode (CCN), Heterodera avenae and H. filipjevi, are considered the most damaging pests to food security with difficult to assess economical losses, and for which restricted quarantine regulations are imposed [2,3]. For example, in Europe, PCNs are responsible for potato yield losses estimated at EUR 220 million/year, while in East Africa (Kenya) potato losses are approximately USD 127 million/year, with only 9.9 t/ha of the potential yield of 40 t/ha [4].
The biology of cyst nematodes is quite exquisite. Similarly to the root-knot nematodes (RKNs), CNs are able to induce host-cell differentiation to establish a unique feeding structure for their development and reproduction (i.e., syncytium for CNs and giant cells for RKNs) [5,6]. Briefly, CN eggs are retained in the swollen females (cyst-like) and protected by a hardened cuticle. Upon stimulation by plant root exudates, eggs hatch and the infective J2 juveniles migrate towards the host root system, entering and moving intracellularly into the inner cortex, where a suitable cell to form the syncytium is selected [3]. An array of parasitism proteins (so called effectors) are secreted by the CNs to trigger root cell reprogramming as neighboring cells are incorporated into the syncytium, forming a multinucleate and highly metabolically active feeding structure [7]. The juveniles become sedentary, feeding on plant nutrients until reaching the adult stage. After mating, males leave the roots. On the contrary, females eventually die, and their cuticle undergoes tanning by polyphenol oxidases forming the cyst, which contains hundreds of embryonated eggs [3]. After the host plant dies, cysts are released from the roots into the soil, remaining dormant until the next susceptible host grows in its vicinity.
Control of CN populations is a challenging task due to the fact that they can persist in soil for long periods (up to 20 years), without a proper host plant, and withstanding extreme conditions of temperature and desiccation [8]. Pest management is normally performed by (a) improving cultural practices in order to increase plant tolerance and/or decrease CN hosts in the field; (b) through cultural control, by introducing crop rotation or cover crops with non-host plants and (c) chemical control using (hemi)synthetic chemical nematodicides. Pest management through cultural control can be an environmentally sustainable practice, but often provides no short-term farm income and may involve further expenses in additional equipment [9]. Chemical control is performed by the application of potent synthetic chemicals that kill or disrupt the feeding or reproductive behavior of nematodes and, although highly efficient, can show extremely negative environmental and public health impacts [10]. The use of essential oils (EOs), chemical mixtures of natural products, has begun to be regarded as a potential sustainable chemical control strategy against PPNs [10,11]. EOs are mostly composed of terpenoids (mainly monoand sesquiterpenes) and phenolic compounds, such as phenylpropanoids, that can often display additive, synergistic and antagonistic component interactions associated with their biological activities. Additionally, these mixtures have the advantage of not accumulating in the environment and having a broad range of activities, which diminishes the risk of developing resistant pathogenic strains [12]. The nematodicidal activities of EOs have been previously described for several PPNs [13] but biological assays against the cyst nematodes HET and GLO are still very few. In the present work, a bibliographic survey was performed on available publications reporting EOs tested against HET and GLO. Information was compiled on EO activity and chemical composition as well as the species and family of the plant source.

Nematodicidal Essential Oils
Research on nematodicidal EOs was performed with Web of Science ® and Google Scholar ® search engines, in all available databases, on published works reporting direct contact bioassays, using the topics "Heterodera" or "Globodera" and "essential oil". Information on the family and species of the plant source used for EO extraction and the respective EO half maximal effective concentration (EC 50 ) was collected when available. Only nine reports were found for the cyst nematodes.

Activity against Globodera
The activity of EOs extracted from plants against GLO was reported by three publications. Assays were performed on G. rostochiensis or on undefined GLO species [14][15][16]. EOs extracted from a total of 10 plant species were used in 24 bioassays. EOs used in the bioassays belonged mostly to plants from the Lamiaceae and Poaceae families ( Figure 1a). The species used were Allium sativum, Azadirachta indica, Cinnamomum camphora, Cymbopogon martinii, Eucalyptus globulus, Linum usitatissimum, Ocimum basilicium, Salvia officinalis, Tagetes erecta, and Thymus vulgaris. The highest hatching inhibition percentages were obtained for the EOs of A. sativum, E. globulus, and S. officinalis.
Tagetes erecta, and Thymus vulgaris. The highest hatching inhibition percentages were obtained for the EOs of A. sativum, E. globulus, and S. officinalis.

Activity against Heterodera
The activity of EOs against the genus Heterodera was reported by eight publications

Nematodicidal Volatiles from Essential Oils
In five publications, the main compounds of the most successful EOs against the genus HET were also tested, to pinpoint the compound(s) responsible for nematodicidal activity. The compounds with the highest activity were ethyl cinnamate, ethyl p-methoxy cinnamate, isovaleric acid, trans-cinnamaldehyde and α-terpineol. The EC50 values reported ranged from 0.08 to 0.21 mg/mL (Figure 2b). The most successful EO component was ethyl p-methoxy cinnamate, a phenylpropanoid ester that showed an EC50 value lower than that of the commercial nematodicide fosthiazate. Highly active compounds contained oxygen in their structure, a very electronegative element, and, with the exception of isovaleric acid and α-terpineol, were aromatic compounds (Figure 3).

Activity against Heterodera
The activity of EOs against the genus Heterodera was reported by eight publications

Nematodicidal Volatiles from Essential Oils
In five publications, the main compounds of the most successful EOs against the genus HET were also tested, to pinpoint the compound(s) responsible for nematodicidal activity. The compounds with the highest activity were ethyl cinnamate, ethyl p-methoxy cinnamate, isovaleric acid, trans-cinnamaldehyde and α-terpineol. The EC50 values reported ranged from 0.08 to 0.21 mg/mL (Figure 2b). The most successful EO component was ethyl p-methoxy cinnamate, a phenylpropanoid ester that showed an EC50 value lower than that of the commercial nematodicide fosthiazate. Highly active compounds contained oxygen in their structure, a very electronegative element, and, with the exception of isovaleric acid and α-terpineol, were aromatic compounds (Figure 3).

Nematodicidal Volatiles from Essential Oils
In five publications, the main compounds of the most successful EOs against the genus HET were also tested, to pinpoint the compound(s) responsible for nematodicidal activity. The compounds with the highest activity were ethyl cinnamate, ethyl p-methoxy cinnamate, isovaleric acid, trans-cinnamaldehyde and α-terpineol. The EC 50 values reported ranged from 0.08 to 0.21 mg/mL (Figure 2b). The most successful EO component was ethyl pmethoxy cinnamate, a phenylpropanoid ester that showed an EC 50 value lower than that of the commercial nematodicide fosthiazate. Highly active compounds contained oxygen in their structure, a very electronegative element, and, with the exception of isovaleric acid and α-terpineol, were aromatic compounds (Figure 3). Biol. Life Sci. Forum 2021, 3, 1 4 of 6 To assess the potential environmental and human health dangers for the use of EOs or EO components against CNs, some toxicological parameters are summarized for the most active compounds (Table 1). These EO compounds are reported to possess lower toxicities to mammals (higher LD50 values, in feeding tests with rats) and be lower potential environmental hazards than the commonly used nematodicide fosthiazate. Isovaleric acid and trans-cinnamaldehyde also appear to possess additional biological activities, which may be useful against multiple plant pests.  To assess the potential environmental and human health dangers for the use of EOs or EO components against CNs, some toxicological parameters are summarized for the most active compounds (Table 1). These EO compounds are reported to possess lower toxicities to mammals (higher LD 50 values, in feeding tests with rats) and be lower potential environmental hazards than the commonly used nematodicide fosthiazate. Isovaleric acid and trans-cinnamaldehyde also appear to possess additional biological activities, which may be useful against multiple plant pests.

Conclusions
In the present work, existing published information on the activity of EOs against the cyst nematodes HET and GLO was compiled and analyzed. Lamiaceae, Poaceae and Compositae plant families show potential as sources for EO-bearing plants with activity against cyst nematodes. Future projects with the aim of screening active EOs may benefit from exploring these families. Aromatic compounds with highly electronegative elements appear to be highly active and EOs rich in these components should be favored. Research on the mechanism of action of EOs on cyst nematodes is needed to ascertain the biological targets of the respective components, in order to devise sustainable and more ecofriendly pest management practices.