*Review* **Synergistic Field Crop Pest Management Properties of Plant-Derived Essential Oils in Combination with Synthetic Pesticides and Bioactive Molecules: A Review**

**Mackingsley Kushan Dassanayake 1, Chien Hwa Chong 2,\*, Teng-Jin Khoo 1, Adam Figiel 3, Antoni Szumny <sup>4</sup> and Chee Ming Choo <sup>5</sup>**


**Abstract:** The management of insect pests and fungal diseases that cause damage to crops has become challenging due to the rise of pesticide and fungicide resistance. The recent developments in studies related to plant-derived essential oil products has led to the discovery of a range of phytochemicals with the potential to combat pesticide and fungicide resistance. This review paper summarizes and interprets the findings of experimental work based on plant-based essential oils in combination with existing pesticidal and fungicidal agents and novel bioactive natural and synthetic molecules against the insect pests and fungi responsible for the damage of crops. The insect mortality rate and fractional inhibitory concentration were used to evaluate the insecticidal and fungicidal activities of essential oil synergists against crop-associated pests. A number of studies have revealed that plant-derived essential oils are capable of enhancing the insect mortality rate and reducing the minimum inhibitory concentration of commercially available pesticides, fungicides and other bioactive molecules. Considering these facts, plant-derived essential oils represent a valuable and novel source of bioactive compounds with potent synergism to modulate crop-associated insect pests and phytopathogenic fungi.

**Keywords:** phytochemicals; synergism; essential oils; fractional inhibitory concentration; insect mortality rate; phytopathogenic fungi; insect pests; pesticide resistance; fungicide resistance

#### **1. Introduction**

The demand for the production of crops is rising due to the increasing global population, which may exceed 35% by 2050 [1]. This has led to a 15–20-fold use of pesticides in order to enhance the availability of crop yields across the globe [2]. Pesticides are chemical agents that are either synthetically made or naturally occurring, which can be classified as insecticides, fungicides, herbicides, nematicides, rodenticides, etc. Approximately, 2 million metric tons of pesticides are used in agriculture across the globe annually, where countries like China, the USA and Argentina are the major contributors towards pesticide use, and it has been estimated that annual pesticide usage will soon increase up to 3.5 million metric tons worldwide [3]. It has been reported that around 47.5% of herbicides, 29.5% of insecticides, 17.5% of fungicides and the remaining 5.5% of other pest management

**Citation:** Dassanayake, M.K.; Chong, C.H.; Khoo, T.-J.; Figiel, A.; Szumny, A.; Choo, C.M. Synergistic Field Crop Pest Management Properties of Plant-Derived Essential Oils in Combination with Synthetic Pesticides and Bioactive Molecules: A Review. *Foods* **2021**, *10*, 2016. https:// doi.org/10.3390/foods10092016

Academic Editors: Ian Southwell and Oscar Núñez

Received: 24 July 2021 Accepted: 23 August 2021 Published: 27 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

agents account for all pesticides used worldwide [4]. However, the overuse of synthetic pesticides has led to serious health and ecological hazards, such as the increased risk of cancers, as well as cardiovascular, neurological, endocrine-related health issues and the potential damage done to non-target animals and plants that exist within the parameters of the agent applied [5]. For example, workers who were handling pesticides that consist of hexachlorocyclohexane (HCH) have experienced neurological symptoms. It was reported in 1992 by the National Institute of Occupational Health (NIOH) that paddy field workmen who were spraying insecticides containing methomyl showed abnormalities in their ECG, serum LDH and cholinesterase levels [6]. Chlorpyrifos is one of the most widely used synthetic pesticides in the history of agricultural practices, and the application of this agent can contaminate the soil and groundwater and known to be highly toxic to aquatic life [7]. The environmental, ecotoxicological and health consequences of the widespread application of synthetically made chemical pesticides and fungicides, as well as the development of resistance to these agents, have resulted in a heightened concern and interest among researchers and consumers to focus more on natural and sustainable products with fewer synthetic pesticides, insecticides, fungicides and herbicides [5].The quality of nutrition, food security and sustainability have become very important agenda issues in Sustainable Development Goal 2 (SDG2) established by the United Nations in 2015, and according to current estimates of SDG2, about 8.9% or 690 million people of the world population are in hunger; thus, it may not be possible to achieve zero hunger by the year 2030 [8]. Hence, the United Nations World Food Program aims to alleviate worldwide starvation by the year 2050. There exists a potential to integrate essential oils (EOs) and bioactive compounds from plants, herbs, fruit waste and enzymes of ripening fruits into agricultural practices. Essential oils (EOs) and bioactive compounds from plants, herbs, fruit waste and enzymes of fruits or biomaterials are potential crop protection agents [9]. Essential oils are odoriferous volatile natural oils that can be characterized by their aromatic and lipophilic nature [10]. These EOs are promising sources of naturally occurring bioactive compounds that show pesticidal and fungicidal activities [11]. Plants produce both primary (e.g., sugars and acids) and secondary metabolites, where EOs are largely composed of bioactive secondary metabolites like monoterpenes, esters, sesquiterpenes, phenols, aldehydes, oxides and ketones that are synthesized both internally and externally by plants [12,13]. Essential oils are abundantly found in aromatic plants, where more than 3000 types of EOs have been identified and about 300 essential oil variants have been commercialized [10,11,14,15]. Families of plants that are frequently studied for their essential oils include *Lauraceae*, *Myrtaceae*, *Lamiaceae*, *Rutaceae*, *Apiaceae*, *Asteraceae*, *Poaceae*, *Cupressaceae*, *Piperaceae* and *Zingiberaceae* [16–18]. Nonetheless, the demand for novel pesticidal and fungicidal products from natural sources is increasing, and it has been estimated that around 40%–50% of the crop yields of maize, barley, wheat, rice, potatoes, sugar beets and soybeans harvested worldwide are dissipated each year, largely due to pesticide resistance in crop-consuming insects [2]. The registration process for a new fungicide or pesticide usually requires the registrant (e.g., manufacturer) to analyze and conduct different laboratory-based tests [19]. These tests will define the chemistry of the new fungicide of pesticide, as well as the potential hazards to humans, domestic animals, and the proximal environmental and the impact on non-target organisms. Data that include the identity, chemical and physical properties of the active ingredient present in the product, as well as analytical methods, the proposed label and uses, human and environmental toxicity, safety data sheets, efficacy associated with the intended use, container management, residues resulting from the pesticide product usage and the disposal of product waste, are needed to support the application of a pesticide or fungicide registration during its full life-cycle [19,20]. The generation and verification of such data for a single compound may take many years and can be expensive [21]. Hence, there is a growing interest and continuous demand to discover new insecticidal, fungicidal and herbicidal agents with novel mechanisms of action, accompanied by efforts to ensure safety and reduce production cost.

Currently, research has been implemented on various chemical properties and biological activities like antioxidant, anticancer, antimicrobial, antiviral and pesticidal effects of plant-derived essential oils [22]. The following review paper emphasizes the impact of potent plant-derived essential oils and their bioactive compounds that synergistically integrate with synthetic pesticides and other novel molecules for crop preservation.

#### **2. Historical Background and Development of Natural Products in Agriculture**

Bioactive compounds present in these natural products can be applied as pesticidal, insecticidal and fungicidal agents [23]. The origins of many synthetic pesticidal, insecticidal and antifungal agents can be traced back from a variety of natural products since the introduction and commercialization of penicillin [24–26]. The use of plant-based pesticidal agents has been reported since ancient times, where extracts of poisonous herbs were used to control crop-consuming insect pests about 4000 years ago [27]. Nicotine sulfate, extracted from the leaves of tobacco plants, was applied as a natural insecticide in the seventeenth century, and compounds like pyrethrum derived from chrysanthemums flowers and rotenone extracted from the roots of tropical vegetables were used as natural pesticides in the nineteenth century [28]. The use of naturally occurring substances as fungicidal agents has been reported since the seventeenth century, when sea salt and lime were used to treat wheat in order to prevent the growth of bunt caused by fungi [29]. Another important discovery was made by the French botanist Pierre-Marie-Alexis Millardet, who concluded that copper sulfate, which is a naturally occurring substance, was able to effectively control and reduce downy mildew of certain fruits like grapes [30]. Natural products and their bioactive derivatives constituted about 36% of ingredients present in commercially available pesticides from 1997 to 2010. For example, soil-borne bacteria and *Streptomyces avermitilis* and *Saccharopolyspora spinosa* were used to produce natural pesticides known as avermectin and spinosyn, which can effectively cause the paralysis of insect pests [31]. Avermectin is an award-winning natural pesticidal agent that was isolated from the actinomycete species of bacteria known as *S. avermitilis*. Glufosinate, also known as phosphinothricin, is a naturally occurring broad-spectrum herbicidal agent produced by the bacteria of *Streptomyces* spp. [23]. This bacterial-derived compound was commercialized as an herbicide by the German pharmaceutical company named Bayer under the trade name of Finale [32,33]. The herbicidal action of glufosinate works by inhibiting the enzyme glutamine synthetase, resulting in the buildup of ammonia in the thylakoid lumen of plants and leading to photophosphorylation decoupling. The British pharmaceutical company named Corteva Agriscience commercialized a fungicide known as fenpicoxamid that was derived from antimycin, which is naturally produced by *Streptomyces* spp. bacteria. Fenpicoxamid works by inhibiting cellular respiration in fungi. The annual gross of fenpicoxamid and glufosinate exceeded USD 1 billion after introducing them to the market. Other examples of herbicides include the *Streptomyces* spp. produced tentoxin and the fungal *Alternaria alternate* (Fries)-derived thaxtomin [23]. These herbicidal agents were able to disrupt energy metabolism cellulose biosynthesis. Cornexistin is a fungal metabolite derived from *Paecilomyces variotii,* which acts as a broad-spectrum herbicidal agent against maize via the inactivation of enzymes known as aminotransferases [23,34].

#### **3. Sources and Chemical Composition of Plant-Derived Essential Oils**

Several species of plants consist of volatile essential oils, in which different plant parts like leaves, barks, peels, flowers, seeds, buds and roots can be diverse sources of various essential oils [35]. Plant-based essential oils are complex mixtures of naturally occurring polar and nonpolar compounds [36]. These essential oils have been classified into four primary groups as terpenes, derivatives of benzene, hydrocarbons and other forms of miscellaneous aromatic compounds [37,38]. Terpenes like monoterpenes and monoterpenoids are the most abundant and major representative molecules that constitute about 90% of EOs [39]. Plant-derived EOs are largely composed of carbon hydrocarbons including the following: acyclic alcohols like geraniol, linalool and citronellol; cyclic al-

cohols like terpeniol, menthol and isopulegol; bicyclic alcohol compounds like verbenol and borneol; phenols that include carvacrol and thymol; ketones like menthone, carvone and thujone; aldehydes that include citral and citronellal;acids like chrysanthemic acid; and oxides like cineole [35]. Terpenes present in these EOs are further classified into the following groups according to their molecular weight: hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Aromatic compounds occur less frequently compared to terpenes and are natural derivatives of phenylpropane compounds like cinnamaldehyde, aldehyde, cinnamic alcohol, as well as phenols that include eugenol and chavicol, methoxy derivatives like elemicine, methyl eugenols, anethole, estragole and methylenedioxy compounds like myristicine, apiole and safrole [14]. Although EOs are present in a variety of plants, their extraction and productivity are relatively time consuming and expensive processes, since very small amounts of pure EOs can be harnessed from a large amount of raw plant material [35,40].

#### **4. Pesticidal and Fungicidal Action Mechanisms of Plant-Derived Essential Oils**

Plant-derived essential oils consist of intrinsic properties that can interfere with biochemical, physiological and metabolic functions of insects and fungi by altering the biological activities of target sites of these organisms [41,42]. Anti-insect pest and antifungal agents from botanical EOs can have either narrow-spectrum or broad-spectrum activity, in which narrow-spectrum agents will only affect a particular species of insects or fungi and broad-spectrum agents are effective against a wide range of fungi or insect pests [43]. Additionally, these botanical agents can be classified as fungistatic, which only slow down the growth and multiplication of fungi but do not actually kill them, or asfungicidals that directly promote the cellular destruction of fungal organisms [44]. In case of anti-insect pest plant-derived agents, these can be classified as insect repellents, which consist of chemical properties that can simply repel insects, or as insecticides, which are lethal to insects and cause mortality upon contact [45].

#### *4.1. Mode of Action of Insecticidal Essential Oils*

Several molecular studies have revealed the action mechanisms of plant-derived essential oils that show the efficiency of pesticidal and insect repellent activity. The EO metabolite-mediated inhibition of acetylcholinesterase (AChE) and octopamine pathways of insects [46–50] (Figure 1) has been well investigated and documented. Among these mechanisms, the inhibition of AChE is one of the most exploited, since AChE is an enzyme that plays a crucial role in and neuromuscular and neuronal communication in insects [51–53]. AChE inhibition can cause neurotransmitter toxic effect on insect pests by the membrane disruption of the postsynaptic junction that leads to the interference of nerve current [54–56]. Octopamine is an important hormone associated with the nervous system of insects [57]. This neurohormone is present as octopamine-1 and octopamine-2 and respectively functions as a neurotransmitter and as a neuromodulator in insects, in which the inhibition of octopamine will cause the impairment of physiological modulation associated with muscle juncture and homeostasis of insect bodily fluids, which can alter their octopamine-mediated nervous system [58–63]. Plant-derived EOs are also capable of inhibiting GABA receptors present in insects, which can suspend GABA from binding with GABArs (GABA receptors) in extrasynaptic synaptic membranes [64–66] (Figure 1). Furthermore, phytochemical metabolites from plant-derived EOs can inhibit or interfere the activities of enzymes associated with the metabolism of xenobiotics and respiration of insects like CarEs, chitin, cytochrome P450s, ATP-binding cassette transporters and GSTs [67–69].

**Figure 1.** Insecticidal action mechanisms of plant-derived essential oils.

#### *4.2. Mode of Action of Fungicidal Essential Oils*

Plant-derived essential oils have multiple mechanisms of action to inhibit the growth and activity of fungi. Target sites of these EO metabolites include the biosynthesis of cell wall, ATPases activity, efflux pumps, quorum sensing/biofilm formation and cell membrane structure and integrity in fungi [70–74] (Figure 2). Essential oils that disrupt cell wall biosynthesis work by inhibiting the formation of components like chitin and β-glucans, which are necessary for the synthesis of fungal cell walls [75]. Ergosterol is an essential compound associated with fungal cell membranes and their biosynthetic pathways. The inhibition of ergosterol by EOs will cause structural, metabolic and osmotic instability in fungal cells, leading to compromised multiplication and virulence [76–79]. Certain EOs can affect the ATPases activity of fungi by interfering with the function enzymes associated with fungal mitochondria. The inhibition of mitochondrial enzymes like malate dehydrogenase, succinate dehydrogenase and lactate dehydrogenase can alter the level of reactive oxygen species and ATP, which leads to the diminishing of mitochondrial content that is essential for fungal metabolic pathways [80]. Efflux pumps are proteinaceous transporters localized in the cell membranes of both prokaryotic and eukaryotic cells. In fungi, these are important structures that mediate nutrient uptake, medium acidification and antifungal resistance. These efflux pumps are target sites of certain metabolites associated with plant-derived essential oils in modifying or reversing antifungal resistance [80–82]. Plantderived EOs are also capable of attenuating quorum-sensing (QS) activity in fungi, in which certain phytochemical metabolites present in these essential oils can inhibit cell-to-cell communicating QS signaling molecules like N-acyl homoserine lactones (AHLs), tyrosol, α-(1,3)-glucan and tryptophol, and fungal pheromones like a-factor and α-factor [83–88].

**Figure 2.** Antifungal action mechanisms of plant-derived essential oils.

#### **5. Synergistic and Hybridized Insect Pest Management Products of Botanical Essential Oils**

#### *5.1. As Homosynergistic Agents*

Plant-derived bioactive metabolites present in EOs are capable of interacting synergistically to increase pesticidal action. A study revealed that essential oil phytochemical compounds thymol and 1,8-cineole (Figure 3) interacted synergistically with pulegone to induce larvicidal activity against *Plutella xylostella* (Linnaeus) (diamondback moth). 1,8-cineole and pulegone (Figure 3) combination indicated the highest synergistic activity with a larval mortality rate of 90% in the study. The investigation further elucidated that thymol and 1,8-cineole were able to affect the levels of enzymes like carboxylesterase esterase, glutathione transferases and acetylcholinesterase associated with *P. xylostella* [89]. Rosemary essential oil compounds camphor (Figure 3) and 1,8-cineole indicated synergistic insecticidal action against the moth species known as *Trichoplusia ni* (cabbage looper). The study revealed that the mixture of these compounds (103 μg of 1,8-cineole and 150 μg of camphor) indicated a larval mortality rate >80% in both contact and fumigant assays with a penetration rate >40% in 60 min of application [90]. A similar study conducted by Tak and Isman [91] revealed that 1,8-cineole and camphor isolated from the essential oil of *Rosmarinus officinalis* were synergistically active when combined against *Trichoplusiani* (Hübner) larvae. A compound combination ratio of 60:40 of 1,8-cineole and camphor indicated a larvae mortality rate of 93.3 ± 6.7 in the study [91]. Binary mixtures of essential oil compounds α-terpineol (Figure 3) and thymol were able to synergize the biopesticidal activity of 1,8-cineole and linalool (Figure 3) against swinhoe larvae *Chilopartellus* (Swinhoe) at a dose of 189.7 μg [92]. An investigation conducted by Hummelbrunner and Isman [93] revealed that complex mixtures of *trans*-anethole, citronellal (Figure 3),

α-terpineol and thymol were able to interact synergistically and mediate acute toxicity to *S. litura* Fab. (tobacco cutworms) when topically administered at a dose of 40.6 μg [93]. Liu et al. [94] indicated that essential oils extracted from *Cinnamomum camphora* (L.) Presl. seeds and *Artemisia princeps* Pamp leaves exhibited synergistic insecticidal and repellent activity against crop pests like *Sitophilusoryzae* L. (rice weevil) and *B. rugimanus* Bohem when combined at a concentration ratio of 1:1 [94]. A study showed that cinnamon oil was able to synergize the larvicidal activity of rotenone against *Spodoptera litura* (F.) at a mixture ratio of 1:35 and concentration of 506 mg/L within 72 h of exposure [95]. Essential oil compounds γ-terpinene and terpinen-4-ol (Figure 3) isolated from the extracts of *Majorana hortensis* Moench were able to synergistically mediate insecticidal activity against *Aphis fabae* (Scopoli) and *S. littoralis* [96]. Andrés et al. [97] showed that binary mixtures of essential oil compounds terpinolene and safrole (Figure 3) extracted from *Piper hispidinervum* were able to induce synergistic antifeedant effect on crop-related pests like *Leptinotarsa decemlineata* (Say), *S. littoralis*, *Rhopalosiphum padi*(Linnaeus) and *Myzuspersicae* (Sulzer) [97]. Furthermore, an investigation showed that a binary mixture composed of limonene and carvone (Figure 3) at a concentration ratio of 6:2 displayed synergistic pesticidal activity against *Tribolium castaneum* (Herbst) (red flour beetle) adults at 10.84 μg and larvae at 30.62 μg [98]. Examples of insecticidal homosynergistic plant-derived EOs and their compounds are summarized in Table 1.

**Figure 3.** Phytochemical compounds isolated from plant-derived essential oil synergists.

#### *5.2. As Enhancers of Commercial Insecticides*

Certain essential oils and their representative phytochemical constituents are capable of enhancing the insecticidal action of commercially available synthetic chemical pesticides.

A study conducted by El-Meniawi et al. [99] showed that EOs from *Simmodsiachinesis*, *Allium sativum*, Fam. and *Mentha piperita* Fam. were able to synergistically enhance the activity of cyhalothrin, diuron and malathion, respectively, at concentrations ranging from 0.1 to 100 μm against *Bemisia tabaci* (Gennadius) (silver leaf whitefly). Further investigations in this study showed that these combinative agents induced the inhibition of the entomic enzymes ATPase, chitinase and acetylcholinesterase [99]. An investigation revealed the pesticide susceptibility of *Myzus persicae* (Sulzer) (green peach aphid) to imidacloprid and spirotetramat after individually combining them with *Thymus vulgaris* and *Lavandula angustifolia*, thymol and linalool, respectively. Imidacloprid with *L. angustifolia* combinative treatment indicated the highest synergism ratio of 19.8 in the study [100]. A similar study showed that rapeseed oil and soya oil enhanced the pesticidal action of pirimicarb and imidacloprid against *Myzuspersicae* (Sulzer) [101]. The essential oil compound linalool isolated from *Ocimumbasilicum* (Linnaeus) enhanced the pesticidal effect of deltamethrin against *Spodoptera frugiperda* (J.E. Smith) (all armyworm). The study showed that the dose of deltamethrin can be reduced by more than 6-fold by the application of 480 μg/μL of *O*. *basilicum* essential oil [102]. Another study indicated that deltamethrin at 9.62 μL and linalool at 0.177 μL combination induced enhanced insecticidal activity against *S. frugiperda* larvae, resulting in 95.75% mortality in 24 hours. The same study showed that linaloolatat0.177 μL enhanced the pesticidal activity of Decis® (25CE) at 0.25 μL, resulting in 100% larval mortality [103]. A recent research study conducted by Ismail (2021) showed that garlic oil was able to synergize and enhance the insecticidal action of chlorpyrifos and cypermethrin up to 9-fold against the crop pest *S. littoralis*. The study further elucidated that these combinative agents induced the inhibition of enzyme pathways associated with oxidase, glutathione S-transferase and general esterase (*α*´-β-EST) of the tested insect pest [104]. Mantzoukas et al. [105] stated that the cannabidiol (Figure 3) present in the essential oil of the Cannabis plant synergized the commercially available biopesticides madex, azatin and helicovex against the four crop pests *S. zeamais*, *Rhyzopertha dominica* (Fabricius), *Prostephanus truncates* (Horn) and *Trogodermagranarium* (Everts) at doses ranging from 500 to 3000 ppm [105]. Examples of commercially available synthetic pesticides used in combination with plant-derived essential oils and their compounds are summarized in Table 1.

#### **6. Synergistic and Hybridized Fungicidal Activity of Botanical Essential Oils**

#### *6.1. As Homosynergistic Agents*

Bioactive phytochemical metabolites present in EOs have been found to interact synergistically to mediate antifungal activity. A study revealed that EOs isolated from thyme, clove and lemongrass demonstrated high antifungal activity, which completely inhibited the growth of mycelium of *Fusarium oxysporum* (Sacc.) and *Fusarium circinatum* (Nirenberg and O'Donnell) at a concentration of 1000 μL/L [106]. Another study indicated that the essential oil combination of thyme, cinnamon, lime and clove induced antifungal activity against the crop-degrading fungus *Colletotrichum gloeosporioides* (Penz)and reduced the damage of crops [107]. Nardoni et al. [108] stated that EOs extracted from *Thymus vulgaris*, *Origanum vulgare*, *O. basilicum*, *Foeniculu mvulgare*, *Illicium verum*, *Syzygium aromaticum*, *Origanum majorana*, *Rosmarinus officinalis*, *Citrus sinensis*, *Citrus bergamia*, *Cymbopogon citrates*, *Salvia sclarea*, *Citrus aurantium*, *Citrus paradise* and *Citrus limon* showed synergistic antifungal activity against *P*. *funiculosum* and *M*. *racemosus* with a FICI of <0.5 for both fungi [108]. An investigation conducted by Bedoya-Serna et al. [109] showed that a nanoemulsion composed of a mixture of oregano and sunflower essential oil was synergistically active against *Fusarium* sp., *Cladosporium* sp. and *Penicillium* sp., which suspended their fungal spore formation at a concentration 0.1 mL [109]. Essential oils extracted from *Thymus vulgaris* and *O. vulgare* interacted synergistically to mediate antifungal activity against *Fusarium* spp. with FICIs ranging from 0.375 to 0.5 when used in combination. Moreover, the study showed that the best synergistic activity of the essential oil combination was demonstrated against *F. moniliforme* with a FICI of 0.375 at an indicative MIC

and MFC of 0.156 μL/mL [110]. An investigation carried out by Yen and Chang [111] indicated that cinnamaldehyde and eugenol isolated from cinnamon essential oil were synergistically fungicidal against *L. sulphureus*. The study revealed that the MIC of the cinnamaldehyde and eugenol (Figure 3) combination was90% lower compared to their stand-alone treatments [111]. Hartati [112] stated that combining essential oils extracted from *Cymbopogon nardus* (citronella) and *Azadirachta indica* (neem) at a concentration ratio of 1:1 was synergistic and effective against the fungal pathogen of patchouli plants known as *Synchytriumpogostemonis* S.D.Patil and Mahab [112]. A study revealed that a combination of essential oils from *Syzygium aromaticum* (Linn.) (clove) and *Cinnamonum zeylanicum* (cinnamon) mediated synergistic fungicidal activity against a crop disease causing *Aspergillus niger*, *Alternaria alternate* (Fries) Keissler, *Colletotrichum gloeosporioides* (Penzig), *Lasiodiplodia theobromae* (Patouillard) Griffon and Maublanc, *Plasmopara viticola* (Berkeley and Curtis) and *Rhizopus stolonifer* (Ehrenberg) Vuillemin. The best synergistic antifungal activity was observed for clove oil and cinnamon oil (9:1) with a FICI of 0.55 against *P. viticola* in the study [113]. A research conducted by Yu et al. [114] indicated that essential oil compounds terpinolene, terpinen-4-ol, δ-terpinene, α-pinene, 1,8-cineole, α-terpineol and α-terpinene (Figure 3) isolated from *Melaleuca alternifolia* (tea tree) interacted synergistically to mediate antifungal activity against *Botrytis cinerea* (Persoon). According to the results of the study, the highest antifungal synergism was observed for terpinen-4-ol and α-terpineol combination (1:1 ratio), which indicated a mycelial growth inhibition rate of 99.46% ± 0.76%, and scanning electron microscopic analysis revealed that these compounds made pronounced alterations in the cell wall ultrastructure, mycelial morphology and plasma membrane permeability [114]. Another investigation revealed that the essential oil compounds carvone, apiol and limonene (Figure 3) isolated from the seeds of the *Anathallis graveolens* (Pabst) F. Barros plant were synergistically active against *Aspergillus flavus,* which reduced ATPase and dehydrogenase synthesis, leading to fungal mitochondrial dysfunction and cell death induced by the accumulation ROS in *A. flavus* [115]. Moreover, Nakahara et al. [116] tested the combined activity of the EO compounds linalool and citronellal isolated from *C. nardus* against *Aspergillus* sp., *Eurotium* sp. and *Penicillium* sp., and found the combination to be synergistically fungicidal at a concentration of 112 mg/L [116]. Examples of fungicidal homosynergistic plant-derived EOs and their compounds are summarized in Table 1.

#### *6.2. As Enhancers of Commercial Antifungal Agents*

Plant-derived metabolites present in EOs are also capable of enhancing the antifungal action of existing synthetic chemical fungicidal agents. A study showed that the EO compound cinnamaldehyde potentiated the fungicidal action of fluconazole against *Aspergillus fumigatus* MTCC 2550 by reducing the MIC of the antifungal agent by up to 8-fold [117]. Gadban et al. [118] demonstrated that essential oil extracted from *Tagetesfilifolia* Lag. potentiated the fungicidal activity of difenoconazole, trifloxystrobin, cyproconazole and carbendazim up to 80% when used in combination against the phytopathogenic fungus *Colletotrichum truncatum* (Schweinitz) Andrus and W.D. Moore [118]. An investigation indicated that EO extracted from *Eupatorium adenophorum* leaves that consist of phytochemical compounds like 10Hα-9-oxo-agerophorone, 9-oxo-10, 11-dehydro-agerophorone and 10Hβ-9-oxo-agerophorone (Figure 3) was able to enhance the fungicidal action of mefenoxam and mancozebagainst *Pythium myriotylum* (Drechsler). According to the results of the study, the EO and mancozeb combination indicated the highest synergistic activity with a fungal mycelia growth rate of 100%, and light and transmission electron microscopic analysis revealed that the EO induced hyphae swelling, cell wall disruption, shortening of the cytoplasmic inclusion and degradation of plasma membrane and cytoplasmic organelles [119]. Camiletti et al. [120] tested and concluded the synergistic action of EO extracted from *Tagetes minuta* L., *Laurus nobilis* L. and *T. filifolia* with iprodione against a major crop-associated fungal pathogen known as *Sclerotium cepivorum* (Berkeley) Whetzel (withe rot). In the study, *T. minuta* in combination with iprodione showed the best synergistic activity, which induced 100% growth inhibition of the fungus. Furthermore, the

study elucidated that phytochemical compounds anethole, phenylpropanoids, sphatulenol and estragole (Figure 3) were abundantly present in the EOs of the tested plants [120]. An investigation revealed that EO extracted from *Pogestemon patchouli* mediated partial synergism with synthetic antifungal agents like ketoconazole and amphotericin B against *A. niger* and *A. flavus* with a FICI ranging from 0.52 to 1 [121]. Examples of commercially available synthetic fungicidal agents used in combination with plant-derived essential oils and their compounds are summarized in Table 1.

#### **7. Novel Developments in Synergistic Insecticidal and Fungicidal Plant-Derived Essential Oils**

Recent developments and novel strategies have been implemented to enhance pesticidal and fungicidal actions of plant-based essential oils. A study indicated that the essential oil compound carvacrol (Figure 3) was able to synergistically interact with the crystalline proteins produced by *Bacillus thuringiensis* MPU B9 and MPU B54 strains to mediate larvicidal activity against *Cydia pomonella* (Linnaeus)(codling moth) and *S. exigua* (beet armyworm moth). The best synergistic larvicidal action was observed at a 1:25000 (MPU B54 protein to carvacrol) concentration ratio, which induced a 96.7% (±3.33%) mortality rate [122]. A similar study elucidated that EOs from *A. indica* containing azadirachtin and *Sinapis alba* were synergistically active against crop pests, like *Spodopteraexigua* (Hübner), *C. pomonella* and *Dendrolimus pini* (Linnaeus), when used in combination with bacterial crystalline toxins of *B. thuringiensis* MPU B9 isolate. Hence, the results of the study indicated a 2-fold increase in larvicidal activity of the combined agents [123]. An investigation conducted by Radha et al. [124] stated that essential oils extracted from *Chenopodium ambrosoides* and *Thymus vulgaris* induced synergism with fungal secretions released by *Beauveria bassiana*(Balsamo) Vuillemin to mediate insecticidal and repellent action against *Callosobruchus maculates* (Fabricius) (*Cowpea bruchid*). According to the results of the study, the highest synergistic interaction was observed with *Chenopodium* oil, which induced a 76% mortality rate of *C. maculates* larvae in 168 h after treatment [124]. Yang et al. [125] tested the insecticidal efficiency of polyethylene glycol-coated garlic essential oil against adult *T. castaneum* and found that these nanoparticles are capable of inducing 100% mortality [125]. An investigation demonstrated that essential oil purified from *Pelargonium graveolens* induced 40% mortality of the *Agrotis ipsilon* (Hufnagel) (dark sword-grass) moth when encapsulated and deployed with solid lipid nanoparticles [126]. Research conducted by Pierattini et al. [127] demonstrated that diatomaceous earth molecules worked synergistically to potentiate the insecticidal activity of *O. basilicum* and *Foeniculum vulgare* against *Sitophilus granaries* (Linnaeus). The combinative treatment indicated a synergistic co-toxicity coefficient that ranges from 1.36 to 3.35 for *F. vulgare* and *O. basilicum* [127]. A novel study demonstrated that orange essential oil interacted synergistically with a baculovirus known as the nucleopolyhedrosis virus to induce enhanced larvicidal activity against *S. littoralis* (the cotton leaf wormmoth) [128]. Furthermore, a novel study conducted by Al-alawi. [129] demonstrated that pine essential oil synergistically interacted with secretions of *B. bassiana* BAU016 fungal isolate to induce enhanced larvicidal activity against *Tetranychus urticae* (Koch) (two-spotted spider mite) [129].

An investigation conducted by Nasseri et al. [130] showed that the EO of *Zataria multiflora* mediated synergistic fungicidal action against *Aspergillus ochraceus*, *A. niger*, *A. flavus*, *Alternaria solani*, *Rhizoctonia solani* and *Rhizopus stolonifer* (Ehrenberg) when loaded and used with solid lipid nanoparticles. The study demonstrated that these combinations inhibited 54%–79% of fungal growth [130]. Luque-Alcaraz et al. [131] tested the antifungal efficiency of chitosan and *Schinus molle* (pepper tree) essential oil conjunctive bio-nanocomposites against *Aspergillus parasiticus* and observed a 40%–50% reduction in fungal cell viability [131]. A study indicated that *M. piperita* EO coated with gold nanoparticles induced synergistically enhanced antifungal activity against *A. flavus* [132]. An investigation revealed that *Satureja khuzestanica* (Jamza) essential oil encapsulated with chitosan nanoparticles induced enhanced fungicidal action against *R. stolonifer* [133]. A research study conducted by Kalagatur et al. [134] elucidated that chitosan nanoparticles

mediated antifungal activity against the phytopathogenic fungus *Fusarium graminearum* (Schwabe) when incorporated with the EO of *Cymbopogon martini*, which indicated a MIC of 421.7 ± 27.14 and MFC of 618.3 ± 79.35 ppm. Scanning electron microscopic analysis in the study revealed detrimental changes in the fungal macroconidia and further elaborated antifungal action mechanisms like intracellular reactive oxygen species elevation, depletion of ergosterol content and lipid peroxidation. Moreover, the study revealed the abundance of geraniol (Figure 3) in the EO of *C. martini* [134]. Latha and Lal. [135] demonstrated that secretions produced by micro-algae were able to synergize and potentiate the antifungal action of thyme essential oil against the phytopathogenic fungus *Alternariabrassicae,* which causes a serious disease in pre-harvest and post-harvest broccoli crops [135]. A novel study showed that bioactive secretions of *Bacillus subtilis* B26 isolate synergistically enhanced the antifungal action of EOs obtained from myrtlewood, Leyland cypress needles, orange and lime when used in combination against phytopathogenicfungi *Ophiostoma perfectum*, *Trichoderma* spp. and *A. niger* [136]. Furthermore, a similar study elucidated that the essential oil extracted from *Zingiber officinale var. rubrum* induced enhanced fungicidal activity against an *A. niger* FNCC 6080 isolate when combined with the *Lactococcus lactis* produced bacteriocin lantibiotic known as nisin [137]. Examples of novel bioactive molecules used in combination with plant-derived essential oils and their compounds are summarized in Table 1.




**Table 1.** *Cont.*

N/S: Not specified.

#### **8. Concluding Remarks and Future Perspectives**

The issue of synthetic pesticide, insecticide and fungicide resistance is expanding rapidly across the globe. Hence, the prospects for the application of existing pesticides and fungicides in the future have become challenging and uncertain. Plant-derived essential oils and their phytoconstituents are remarkable sources of novel bioactive compounds with broad-spectrum insecticidal and antifungal properties. These compounds can exert homosynergistic action or synergistically interact with other pest management agents or bioactive molecules. This review summarizes and interprets the findings of experimental work based on plant-based essential oils in combination with existing pesticidal, insecticidal and fungicidal agents, as well as novel bioactive natural and synthetic molecules, against insect pests and fungi responsible for the spoilage of crops. These essential oil combinations have shown remarkable results as agents with different mechanisms for overcoming pesticidal, insecticidal and fungicidal resistance. For instance, several studies have elucidated that these synergistic combinative compounds can significantly reduce the insect mortality rate and MIC/MFC of fungi. The efforts in synergy research have led to the discovery and production of novel pest management agents. However, the underlying modes of actions associated with synergistic essential oil products have not yet been fully exploited. Hence, the broadening of molecular and biochemical studies based on combined synergists of essential oils are needed to establish a better understanding and further exploitation of their toxicological responses and bioactivity in order to determine their true potency and safety in agricultural application. At present, the availability of experimental data based on essential oil synergists is limited and, therefore, further studies are needed in order to broaden and elucidate their novel action mechanisms in modifying pesticidal, insecticidal and fungicidal resistance. Moreover, studies are needed on insecticidal and fungicidal activities of fruit waste and botanical enzymes, like bromelain combinative synergists, with plant-derived essential oils.

**Author Contributions:** C.H.C. contributed by giving the original idea, concept. M.K.D. collaborated in designing and writing, data interpretation and drafting of the article. T.-J.K., A.F., A.S., C.M.C. and C.H.C. contributed to editing the final version of the draft to be published. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors would like to extend their sincere gratitude to Faculty of Science and Engineering, University of Nottingham, Malaysia for funding this research through pump priming grant F0013.54.04.

**Institutional Review Board Statement:** The study did not require any consent for publication.

**Informed Consent Statement:** The study did not require the consent of the participants.

**Data Availability Statement:** The following review was based on data extracted from published research articles available in all relevant databases with no limitation up to 10 June 2021.

**Conflicts of Interest:** The authors have no competing interests to declare.

#### **Abbreviations**

*S. avermitilis*: *Streptomyces avermitilis*; *S. spinose*: *Saccharopolyspora spinosa* (Mertz and Yao); spp.: Species (multiple); *P. variotii*: *Paecilomyces variotii*; AChE: Acetylcholinesterase; GABA: Gamma aminobutyric acid; CarEs: carboxy-lesterase; ATP: Adenosine triphosphate; GST: glutathione S-transferase; β: Beta; α: Alpha; δ: Delta; QS: Quorum sensing; EO: Essential oil; ROS: Reactive oxygen species; ECG: Electrocardiogram; LDH: Lactate dehydrogenase; *P. xylostella*: *Plutella xylostella* (Linnaeus); *C. partellus*: *Chilo partellus* (Swinhoe); *T. ni*: *Trichoplusia ni* (Hübner); *S. litura*: *Spodoptera litura* (F.); *S. oryzae*: *Sitophilus oryzae (L.)*; *B. rugimanus*: *Bruchus rugimanus* Bohem; *A. fabae*: *Aphis fabae* (Scopoli); *S. littoralis*: *Spodoptera littoralis* (Boisduval); *L. decemlineata*: *Leptinotarsa decemlineata* (Say); *R. padi*: *Rhopalosiphum padi*(Linnaeus); M. *persicae*: *Myzus persicae* (Sulzer); *T. castaneum*: *Tribolium castaneum* (Herbst); *B. tabaci*: *Bemisia tabaci* (Gennadius); *L. angustifolia*: *Lavandula angustifolia* (Miller); *S. frugiperda*; *Spodoptera frugiperda* (J.E. Smith); *S. zeamais*: *R. dominica*: *Rhyzopertha dominica* (Fabricius); *P. truncatus*: *Prostephanus truncates* (Horn); *T. granarium*: *Trogoderma granarium* (Everts); *F. oxysporum*: *Fusarium oxysporum* (Sacc.): *F. circinatum*: *Fusarium circinatum* (Nirenberg and O'Donnell); *C. gloeosporioides*: *Colletotrichum gloeosporioides* (Penz); *P. funiculosum*: *Penicillium funiculosum*; *M. racemosus*: *Mucor racemosus* (Fresenius); sp.: Species (single); *L. sulphureus*: *Laetiporussulphureus* (Bull.) Murrill; *S. pogostemonis*: *Synchytriumpogostemonis* S.D. Patil and Mahab; *A. niger*: *Aspergillus niger*; *A. alternata*; *Alternaria alternate* (Fries) Keissler; *C. gloeosporioides*: *Colletotrichumgloeosporioides* (Penzig); *L. theobromae*: *Lasiodiplodia theobromae* (Patouillard) Griffon and Maublanc; *P. viticola*: *Plasmopara viticola* (Berkeley and Curtis); *R. stolonifer*: *Rhizopus stolonifer* (Ehrenberg) Vuillemin; *P. viticola*: *Plasmopara viticola* (Berkeley and Curtis); *A. flavus*: *Aspergillus flavus*; *A. fumigatus*; *Aspergillus flavus*; *C. truncatum*: *Colletotrichum truncatum* (Schweinitz) Andrus and W.D. Moore; *P. myriotylum*: *Pythium myriotylum* (Drechsler); *S. cepivorum*: *Sclerotium cepivorum* (Berkeley) Whetzel; *L. lactis*: *Lactococcus lactis*; *T. minuta*: *Tagetes minuta* (Linnaeus); *C. pomonella*: *Cydia pomonella* (Linnaeus); *B. thuringiensis*: *Bacillus thuringiensis*; *S. exigua*: *Spodoptera exigua* (Hübner); *D. pini*: *Dendrolimus pini* (Linnaeus); *B. bassiana*: *Beauveria bassiana* (Balsamo) Vuillemin; *T. castaneum*: *Tribolium castaneum* (Herbst); *A. ipsilon*: *Agrotis ipsilon* (Hufnagel); *F. vulgare*: *Foeniculum vulgare* (Miller); *O. basilicum*: *Ocimum basilicum* (Linnaeus); *T. urticae*: *Tetranychus urticae* (Koch); *A. ochraceus*: *Aspergillus ochraceus*; *A. brassicae*: *Alternaria brassicae*; *A. solani*: *Alternaria solani*; *A. ochraceus: Aspergillus ochraceus*; *R. solani*: *Rhizoctonia solani*; *R. stolonifer*: *Rhizopus stolonifer*(Ehrenberg) Vuillemin; *A. parasiticus*: *Aspergillus parasiticus*; *C. maculates*: *Callosobruchus maculates* (Fabricius) μg: Microgram; >: Greater than; mg/L: Milligram per liter; μm: Micrometer; μL: Microliter; ppm: Part per million; μL/L: Microliter per liter; FICI: Fractional inhibitory concentration index; <: Less than; mL: Milliliter; μL/mL: Microliter per milliliter; USD: United States Dollars; USA: United States of America; MIC: Minimum inhibitory concentration; MTCC: Microbial-Type Culture Collection and Gene Bank; MFC: Minimum fungicidal concentration; FNCC: Food and Nutrition Culture Collection.

#### **References**


## *Review Backhousia citriodora* **F. Muell. (Lemon Myrtle), an Unrivalled Source of Citral**

**Ian Southwell**

Plant Science, Southern Cross University, Lismore, NSW 2480, Australia; ian.southwell@scu.edu.au

**Abstract:** Lemon oils are amongst the highest volume and most frequently traded of the flavor and fragrance essential oils. Citronellal and citral are considered the key components responsible for the lemon note with citral (neral + geranial) preferred. Of the myriad of sources of citral, the Australian myrtaceous tree, Lemon Myrtle, *Backhousia citriodora* F. Muell. (Myrtaceae), is considered superior. This review examines the history, the natural occurrence, the cultivation, the taxonomy, the chemistry, the biological activity, the toxicology, the standardisation and the commercialisation of *Backhousia citriodora* especially in relation to its essential oil.

**Keywords:** *Backhousia citriodora*; lemon myrtle; lemon oils; citral; geranial; neral; iso-citrals; citronellal; flavor; fragrance; biological activity

#### **1. Introduction**

There are many natural sources of lemon oil or lemon scent. According to a recent ISO Strategic Business Plan [1], the top production of lemon oils comes from lemon (7500 tonne), *Litsea cubeba* (1700 tonne), citronella (1100 tonne) and *Eucalyptus* (now *Corymbia*) *citriodora* (1000 tonne). Lemon oil itself, cold pressed from the peel of *Citrus limon* L., Rutaceae, contains 2–3% of citral (geranial + neral) [2–4], the lemon flavor ingredient. Consequently, the oil, along with numerous other citrus species, is used more for its high limonene (60–80%) and minor component content as a fragrance, health care additive [5] or solvent rather than a citral lemon flavor. Citral- and citronellal-rich oils are the commercial lemon-scented oils. Significant sources [6] of these essential oils are listed in Table 1 [6–44].

The aim of this review is to examine investigations into Lemon Myrtle, *Backhousia citriodora* F. Muell. (Myrtaceae), a source of lemon-scented essential oil, that suggest that Lemon Myrtle is superior to other current commercial sources with respect to citral content, oil yield, organoleptic and medicinal properties.

The criteria used for selection of papers for review are so numerous that they are difficult to itemize. Little was covered prior to the classical The Essential Oil series [16,33] after which chemistry papers abounded, to be followed by more recent bioactivity, toxicology, standards and commercial communications as the industry expanded, all accessed from 'in-house libraries', electronic databases and published reference lists up until mid 2021.

#### **2. Taxonomy**

#### *2.1. Etymology*

In 1845, lemon-scented myrtle was named *Backhousia citriodora* F. Muell. by botanist Ferdinand von Mueller, the genus after the English botanist, James Backhouse and the species epithet from the distinctively strong lemon scent of the foliage [45]. The genus *Backhousia*, from the Myrtaceae family, is endemic to eastern Australia and is a close relative of the genus *Choricarpia*, with which it forms the *Backhousia* alliance [7]. The primary common name "Lemon-scented myrtle" was shortened to "lemon myrtle" for the native foods industry to market the leaf for culinary use. "Sweet Verbena Myrtle" and "Lemon Ironwood" are also common names. As *B. citriodora* has two chemoypes, distinction needs to be made between the citral chemotype and the L-citronellal chemotype [38,39].

**Citation:** Southwell, I. *Backhousia citriodora* F. Muell. (Lemon Myrtle), an Unrivalled Source of Citral. *Foods* **2021**, *10*, 1596. https://doi.org/ 10.3390/foods10071596

Academic Editors: Verica Dragovi´c-Uzelac and Ginés Benito Martínez-Hernández

Received: 31 May 2021 Accepted: 6 July 2021 Published: 9 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### *2.2. Habit*

Mature lemon myrtle trees reach 8 m (25 ft) in height, or higher (to 30 m) when crowned, but are often smaller. The leaves are evergreen, opposite, lanceolate, 4–15 cm in length and 1–5 cm (0.59–0.98 in) broad, glossy green, with an entire margin. The flowers are creamy white, 5–7 mm (0.20–0.28 in) in diameter, produced in clusters at the ends of the branches from summer through to autumn, and after petal fall, the calyx is persistent [45], as shown in Figure 1.

**Figure 1.** *Backhousia citriodora* in plantation and in flower, showing natural distribution.

#### *2.3. Distribution*

*B. citriodora* is endemic to only the east coast of Australia in Queensland from the Sunshine Coast regions of Eumundi, Maroochydore, Noosa and Woondum, to the ranges west of Miriam Vale and the Mackay, Whitsunday, Townsville and Herberton regions. Plantations have been established from north Queensland to northern New South Wales

for both the production of dried leaf and lemon essential oil [45]. The largest of these cover 200 and 70 acres, producing over 2400 tonne of fresh leaf on stem per annum [46].

#### *2.4. Chemotypes*

*Backhousia citriodora* has two essential oil chemotypes: the citral chemotype is more prevalent and is cultivated in Australia for flavoring and essential oil [45]. Citral as an isolate in steam-distilled lemon myrtle oil is typically 90–98%, and oil yield 1–3% from fresh leaf [10,20,45]. This is the highest-content natural source of citral (Table 1). The citronellal chemotype is uncommon and can be used as an insect repellent [5,44] as it has similarities to citronella (*Cymbopogon nardus*) and lemon-scented gum (*Corymbia citriodora*, formerly *Eucalyptus citriodora*). Although first reported by Penfold et al. in 1950 [38], it was only in 2001 that this chemotype was rediscovered and the oil fully characterized [39]. The unique characteristic of this chemotype is that the oil is a source of L-citronellal, whereas many sources contain either the racemic form or the D-isomer. This chemotype does not breed true as seed collected from a citronellal type tree has given progeny with a 1.05:1 ratio of the citronellal:citral chemotypes [39,45].

**Table 1.** Commercial and potentially commercial sources of citral (neral + geranial) and citronellal essential oil.


#### *2.5. Agronomy*

The silvicultural and agronomic aspects of this species, including plantation development, propagation, planting and tending, growth, pests, predators, diseases, harvesting and processing are very much dependent on the individual producer. One producer has detailed his approach [45]. The trees grow best near their natural habitat. There is an increasing demand for organic oil, hence using only organically approved pesticides, herbicides and fertilizers is recommended. The tree loves water but does not like wet feet. It is frost intolerant, with two or more nights below 0 degrees deadly to seedlings. They enjoy morning sun with growth aided by windbreaks. The species responds well to nitrogen

but excessive fertilizer leads to top-heavy plants, poor tree root structure and low leaf oil quality. Ornamental trees grow well in cooler climates as a shrub rather than a tree.

As *B. citriodora* is a tropical to subtropical rainforest tree, leaf production is reduced outside these natural environments. Irrigation is essential for the first years of establishment. Plantation rows need to allow for mechanized tending and harvesting with soft footprints to prevent root damage and spacing to allow for considerable foliage spread [45].

Suitable soils should be well drained and permeable, with a moderately acidic pH, with lime not recommended. Deep ripping the soil for plantation establishment allows for aeration and moisture retention. Mulching will retain moisture and reduce erosion. Planting on mounds is not recommended. Pests and diseases vary with location and climate but always need monitoring [45].

#### **3. Uses**

The organoleptic and bioactivity properties of citral have led to the essential oil of *B. citriodora* being used in a number of applications. Commercial production has two main applications [45]: fresh or dried herb sales and distillation for essential oil production. Chief secondary uses include use by florists and the plant nurseries, where flowers and leafy branches are very popular ornamentally and the tree itself is an asset to any garden.

Citral itself has a generally recognized as safe (GRAS) listing by the United States Food and Drug Administration (FDA), whereby when added to food, it is considered safe by experts [6]. Hence, lemon myrtle oil has been used for citral applications and added as a flavoring and scenting agent to foods, cosmetics, aromatherapy massage oils and various household products (such as detergents, soaps, air fresheners, and insect repellents) to give a lemon or verbena scent [6]. Citral is also an excellent starting material for the synthesis of vitamin A and the valuable fragrant ionones [6,10,20,45]. Additionally, citral has proven bioactivity for numerous potential applications [6,11,47,48] and *B. citriodora* oil or extract has been reported to possess antimicrobial [47–51], food pathogenic [52,53], postharvest pathogenic [54], skin infection [55,56] and anti-inflammatory and antioxidative [57,58] properties. Some of these will be detailed later in this review.

#### **4. Essential Oil**

The citral chemotype yields 1.1–3.2% (fresh weight of leaf) of oil with 80–98% citral [10,59]. For commercial equipment, consistent yields of 1.5% (*w*/*w*, containing some twig) were reported compared with a variable 0.4–3.2% for laboratory distillations [45].

A first report of the less common citronellal chemotype indicated yields of 0.5–0.9% (fresh weight) of oil with 62–80% citronellal [20,39]. Year-old trees from a progeny trial, however, yielded 1.8–3.2% (dry weight) with 85–89% citronellal [39]. Propagation of seed from a single citronellal-type mature tree gave mixed progeny with an approximate 1:1 ratio of the citral and citronellal chemotypes. In contrast, progeny from two citral chemotypes gave only 3/48 of the citronellal chemotype [39]. This rarer form of L or (-) citronellal provides a starting material for the stereospecific synthesis of terpenoids used in the perfume and flavor industry [20].

#### **5. Oil Chemistry**

The major components of the leaf essential oil of *B. citriodora* are shown in Table 2, Figures 2 and 3. Initially thought to be one compound, the major component was called citral because of its lemony aroma and flavor. This terpene aldehyde was found to be a mixture of the two geometric isomers neral 9 (IUPAC Name: (2E)-3,7-dimethylocta-2,6 dienal), and geranial 10 ((2Z)-3,7-dimethylocta-2,6-dienal) also known as citral a and citral b, respectively in the ratio of 1.2–1.5, as shown in Table 2 [45].

*B. citriodora* components were determined by gas chromatography using flame ionisation detection (GCFID) and gas chromatography–mass spectrometry (GC–MS). The most dominant of the minor components are the iso-citrals **5**, **6**, **8**. These isomers of citral seem to always co-exist with citral and are thought to be oxidative, thermal or acid/base rearrangement artefacts of citral sourced either naturally or synthetically [10,60–62]. A published patent reported the purification of citral by fractional distillation in a controlled acidic environment (pH 3–7). This procedure reduced the formation of iso-citrals [63].

**Table 2.** The percentage proportion ranges for key constituents in the essential oil of the citral chemotype of *Backhousia citriodora*.


<sup>a</sup> tr = traces < 0.01%. <sup>b</sup> Total citral is the addition of all five citral isomers. <sup>c</sup> On non-polar gas chromatography (GC) column stationary phases, nerol often co-elutes with neral.

Ά-Myrcene (**1**) 2,3-Dehydro-1,8-cineole (**2**) 6-Methyl-5-hepten-2-one (**3**) Citronellal (**4**)

**Figure 2.** Major constituents of *B. citriodora* essential oil.

**Figure 3.** Gas chromatographic trace of *B. citriodora* oil on a polar column.

With gas chromatography (GC), the preferred analytical method for determining essential oil quality, the choice of solvent for injection of aldehyde-rich oils such as *B. citriodora* is important. Alcoholic or ketonic solvents such as ethanol or acetone are unsuitable because of their tendency to form acetals and ketals if left in these solvents for a length of time [64]. This was also seen in the analysis of cinnamaldehyde from Cinnamomum species using methanol as a solvent [65].

#### **6. Bioactivity**

An increasing amount of data is now being published affirming the popularity of lemon myrtle as a complimentary medicine [48,66].

Many anecdotal reports of bioactivity are now being confirmed by in vitro and in vivo investigations. The Australian Therapeutic Goods Administration (TGA) is reported to have approved three *B. citriodora* essential oil medicines by 2006 [56] and by 2017 expressed an awareness of the increasing number of products containing citral [66].

Even when Rideal–Walker co-efficients were the chief measure of microbial activity, *Backhousia citriodora* essential oil scored well [45]. Lemon myrtle oil was shown to possess significant antimicrobial activity against the organisms *Staphylococcus aureus*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Candida albicans*, methicillin-resistant *S. aureus* (MRSA), *Aspergillus niger*, *Klebsiella pneumoniae* and *Propionibacterium acnes* comparable to its major component—citral [45,49–51]. For example, Minimum Inhibitory Concentrations (%*v*/*v*) against *Aspergillus niger* have been recorded as 0.1, i.e., lower than tea tree oil (0.4) and equivalent to citral (0.1) [49]. The antimicrobial activity of *B. citriodora* essential oils was found to be greater than that of citral alone and often superior to *Melaleuca alternifolia* essential oil. *B. citriodora* has significant antimicrobial activity that has potential as an antiseptic or surface disinfectant or for inclusion in foods as a natural antimicrobial agent [50].

The leaf paste has been confirmed for its antimicrobial and antifungal properties against many microbes including *Clostridium perfringens*, *Pseudomonas aeruginosa*, and a hospital isolate of methicillin-resistant *Staphylococcus aureus* (MRSA) [50,51]. Three others found the oil/extract to also be an effective antibacterial and antifungal agent against (a) food pathogenic bacteria and food spoilage yeasts [52], where damage of the yeast cell membrane through penetration caused swelling and lysis, leading to cell death; (b) against food-borne pathogens [53], where MIC values against *S. aureus* and *Escherichia coli* were 16- and 8-fold, respectively, better than tea tree oil; and (c) against the plant postharvest pathogen *Monilinia fructicola* [54], where in vitro inhibition of spore germination and mycelial growth was recorded.

Antiviral activity has been recorded in a clinical trial in treating *Molluscum contagiosum*, a skin virus causing pearly, flesh-coloured, dome-shaped papules with central umbilication frequently among children [55,56]. The trial showed that at the end of 21 days, there was a more than 90% reduction in lesions in 9/16 children treated with lemon myrtle oil.

Anti-inflammatory and antioxidative properties have also been investigated [57,58]. Lemon myrtle extract (LME) inhibited the production of inflammatory mediators such as nitric oxide (NO). Enzyme-linked immunosorbent assay and reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that pretreatment with LME suppressed the protein expression and mRNA levels of pro-inflammatory cytokines such as interleukin IL-6, and tumor necrosis factor (TNF)-α in a concentration-dependent manner, respectively. This activity suggested that lemon myrtle extract could be used as a potential therapeutic agent with potent anti-inflammatory effects that could be used to treat inflammatory bowel disease. Different drying and extraction techniques for optimizing the antioxidant activity of the leaf have also been investigated [67,68].

In another study, the efficacy of lemongrass (*Cymbopogon flexuosus*) essential oil and its bioactive part citral against dual-species biofilms formed by *Staphylococcus aureus* and *Candida* species was evaluated in vitro [69]. Biofilm staining and viability tests showed both lemongrass essential oil and citral were able to reduce biofilm biomass and cell viability of each species in the biofilm.

In addition, it has been suggested that lemon myrtle extract is suitable for use in ocular health nutritional products, not because of the presence of citral in the extract, but because the extract is a source of lutein and other antioxidants along with folate and the trace minerals, magnesium and calcium [57,70].

Studies with insects have shown that effective insect repellents based on natural active ingredients can deliver repellency on par with synthetic actives in the field. For example, Greive et al. [71] showed in preliminary studies that lemon myrtle oil has insect deterrent activity. Repellency of 82% was recorded against *Aedes aegypti* mosquitoes for 30 min in laboratory tests, with greater efficacy (97%) achieved when mixed (1:5) with *Melaleuca ericifolia* oil, a source of linalool.

#### **7. Toxicology**

Citral, the major component of *Backhousia citriodora* oil, has generally recognized as safe (GRAS) status and is listed by the United States Food and Drug Administration (FDA) and hence, when added to food, is considered safe by experts [6,47,48].

When a chemical or chemical category has been agreed by the Organisation for Economic Co-operation and Development (OECD) member countries, several documents are available to the public. The OECD-generated *profile* (called either the Screening Information Dataset (SIDS) Initial Assessment Profile (SIAP) or the Initial Targeted Assessment Profile (ITAP)) contains brief summaries of SIDS endpoints as well as the major conclusions of the hazard assessment. Hence, there is much information available at sites like: https: //hpvchemicals.oecd.org/UI/handler.axd?id=0ea83202-3f4f-4355-be4f-27ff02e19cb9 (accessed on 9 July 2021) [11,66,72–76] summarising the toxicology of citral. These reports draw the following conclusions:

(a) *"For human health, acute toxicity of citral was found to be low in rodents because the oral or dermal LD50 values were more than 1000 mg/kg. This chemical is irritating to skin and not irritating to eyes in rabbits, and sensitizing to skin in guinea pigs. In humans, this chemical was irritating and sensitizing to the skin at high concentrations but not by consumer products. Several repeated dose oral studies show no adverse effect of citral at less than 1000 mg/kg for 5 days to 13 weeks exposure and some histological changes in the nasal cavity or forestomach, the first exposure sites, probably due to irritation, at more than 1000 mg/kg. The NOAEL for repeat dose toxicity was 200 mg/kg/day"* and


A thorough investigation of lemon balm (*Melissa officinalis* L.) essential oil has been published [30] and its oral toxicity determined in mice. Although rich in citral, a high citronellal content makes this oil more like a typical *Leptospermum petersonii* oil [18–21]. In a similar manner, *Leptospermum petersonii* was evaluated by the Complementary Medicines Evaluation Committee as an oil with citral as major component to conclude that the oil "is suitable as an excipient ingredient up to 5% concentration in Listable topical medicines only" [76].

The antimicrobial and toxicological properties of *Backhousia citriodora* essential oil, have been investigated by Hayes and Markovic, 2002 [49]. In vitro cytotoxicity testing indicated that both lemon myrtle oil and citral had a very toxic effect against human cell lines: primary cell cultures of human skin fibroblasts. However, a product containing 1% lemon myrtle oil was found to be low in toxicity and could potentially be used in the formulation of topical antimicrobial products. These same authors performed in vitro percutaneous absorption investigations of the essential oil of lemon myrtle (*B. citriodora*) on freshly excised human full-thickness abdominal skin obtained from patients undergoing elective surgery [72]. Absorption of lemon myrtle oil in human skin discs was evaluated following topical application of neat lemon myrtle oil to the epidermal surface. Citral was the only component found to be absorbing into skin at all exposure periods. When a formulated product containing 1% lemon myrtle oil was applied, total absorption of citral was measured. The histopathological assessment indicated limited damage to epidermal cells. The combination of the above methodologies enabled the generation of data that could be used for a comprehensive evaluation of the toxicity effects of lemon myrtle oil for topical application.

In a review on the "Maternal reproductive toxicity of some essential oils and their constituents", a study on citral (6) affirms *B. citriodora* as the best source of citral and specifies its non-mutagenic and non-carcinogenic attributes [72–76] and reports on an inhibition of tissue morphogenesis and tumor production. The author then reviews a host of animal studies on the reproductive toxicity of citral for animals including reduced fertility in rats, dose-dependent malformations in chicken embryos, suppression of enzymes responsible for fetal development, teratogenesis in chicken embryos and restricted fetal cranial development. One suggested action mechanism indicates competition with estrogen for estrogen receptor sites. Consequently, the use of essential oils high in citral, such as *B. citriodora*, should be restricted during pregnancy because of a possible teratogenic hazard [6].

#### **8. Standards**

Only in recent years have standards been developed for the essential oil of *Backhousia citriodora*. There have, however, been a number of monographs, especially ISO Standards, elaborated for other citral-rich [4,12,17,22,31] and citronellal-rich [40,42,43] oils.

In 2001, Standards Australia's CH21 Essential Oil Committee elaborated a monograph entitled "Oil of *Backhousia citriodora*, citral type (lemon myrtle oil)", AS 4941-2001. This Standard [8] specified appearance, colour, aroma and physical constants, i.e., specific gravity, refractive index, optical rotation, solubility in alcohol and flash point. The chromatographic table, similar to Table 2 above, listed the major components giving typical

minimum and maximum percentages for each constituent. Additionally, supplied are typical chromatograms usually run on both a polar (similar to Figure 3 above) and non-polar stationary phase with significant peaks identified. Included in a 2011 amendment in this first Standard's trace were the regions where one would expect the alkanals n-octan-1-al, n-nonan-1-al, and n-decan-1-al, byproducts of the synthesis of citral to elute. Peaks in this region would indicate adulteration of the oil. This revised Standard was improved with a revision [8,77] of the geraniol percentage figures to 0.5–2.5%. This was achieved by examining the oil on gas chromatographic traces giving clear separation of geraniol and geranial which are difficult to resolve on many non-polar and intermediate-polarity stationary phases.

Approaches to the International Standard's Organisation's TC54 Essential Oil Committee in 2018 resulted in the adoption of a slightly modified version of this Australian Standard as an International Standard, which is expected to be published in 2022 [9].

#### **9. Commerce**

Although all parts of the tree, including the flowers, timber and, indeed the whole tree, can be used [45], it is the leaf that is most sought after and the main reason for plantation establishment. The leaf and terminal branches are steam distilled for a citral-rich oil used as a lemon flavor, fragrance and aromatherapy oil component. The leaf, processed as whole fresh leaf, whole dried leaf, or dried and milled herb, is also popular for lemon herbal tea and other culinary and lemon flavor uses. Lemon myrtle finds itself in teas, breads, biscuits, cakes, cheeses, chutneys, jams, pastas and vinegars, as a flavor; soaps, cosmetics and pot pourris as a fragrance; aromatherapy oils as a fragrant therapeutic; and as an air freshener, a disinfectant and in a range of body care products. Because of toxicity investigations on major component citral, topical use at less than 1% in a topical formulation is recommended [45,48].

There have been a host of industry production and use-related publications extolling the value and benefits of lemon myrtle and its essential oil [60,78–90].

Although past production figures have been difficult to acquire, several tonnes of oil and fresh or dried leaf are produced annually in Australia from millions of trees in several hundred of hectare of plantation. At the 2003 IFEAT International Conference in Sydney, an estimated current annual production of 5–8 tonne was reported [78].

The 2012 estimates of farmgate Australian production of lemon myrtle for 2011 were 575–1100 tonne leaf and 3–8 tonne oil, with a gross value of \$7–23 million with 90 per cent of oil exported, mainly to the United States and the European Union [79]. According to Biosecurity Australia [79,80], 57.4 tonne of organically certified lemon myrtle oil were exported from Australia to the European Union in 2011, virtually all to Germany. Most essential oil experts consider this a highly exaggerated figure but the importance of the species as an internationally and locally traded commodity cannot be understated. In a 2014 report summarising the industry [81] and relying on the 2011 figures [79], a leaf production figure of 838 tonne of leaf was recorded. A very recent (2020) market study [82] estimates the current state of the industry and projects growth forward to 2025. A current farm gate value of Aus \$12.2 m is larger than any of the other Australian native foods and botanicals and is predicted to double in the next five years. There are more than 50 enterprises producing leaf and/or oil with three of substantial size producing approximately 250 tonne of dry leaf and approximately 8 tonne of oil with farm gate values estimated at Aus \$37.50 and Aus\$ 350.00, respectively [82].

#### **10. Conclusions**

Lemon myrtle, *Backhousia citriodora,* citral type, is becoming established as an unrivalled source of citral lemon whether it be in leaf or oil form. With further development, this species may well become a superior source of citral. The oil yield is higher, the citral content better and the aroma cleaner, fresher and sweeter. In tree form, harvesting becomes more problematic as they do not recover and coppice from ground-level harvesting in the

same manner as tea tree (*Melaleuca alternifolia*) and blue mallee (*Eucalyptus polybractea*) will do. Leaf can be hand picked or tipped with a mechanical harvester.

The lesser known citronellal chemotype is unlikely to be developed commercially until further trials are performed despite the advantages of having an excellent source of the rarer L-enantiomer [39]. Because this chemotype does not breed true, plantation trials are still at early stages and genetic material for plantations is harder to source, immediate commercialisation is not envisaged.

The medicinal properties of the citral chemotype are being increasingly investigated as efficacy in many areas is being proven in both in vitro and in vivo research. Toxicity testing is proving that the product is generally safe when used in appropriate concentrations for most applications except for pregnant mums.

*Backhousia citriodora,* citral type, lemon myrtle oil, has attracted the world's attention in recent decades and is consequently assured a strong place in the flavor, fragrance and health care industries for decades to come. However, there is still much work to be performed, especially at the molecular level [91] and in detecting adulteration [92].

**Funding:** This research received no external funding.

**Acknowledgments:** The author is indebted to Gary Mazzorana for plantation information, to Murray Fagg and the Australia Plant Image Index (APII), Australian National Botanic Gardens, for the *Backhousia citriodora* flower photograph and to Gary Mazzorana and Joseph Brophy for comments on the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

