Introduction
Rapid population growth and increased urbanization are characteristics of modern society, making it imperative to address issues such as quality of life and food security (Ruel et al., 2017; Mouratidis, 2021). In this context, the nature of ecological interactions in agricultural and urban settings needs to be adapted to meet the demands of globalized society (Li et al., 2022). Plant natural products have a remarkable potential to develop sustainable food production systems as well as support human well-being. These products have been long used as antimicrobial agents and insect repellents (Mahady et al., 2008; da Silva and Ricci-Júnior, 2020). Among the many natural products, there has been an increased interest in using essential oils from plants as alternative options for pest management in both developed and developing countries, with the worldwide bioinsecticide market projected to grow from $2.2 billion in 2020 to $4.6 billion by 2025 (Mossa, 2016; Devrnja et al., 2022). Essential oils are volatile plant secondary metabolites that are composed of a complex blend of various bioactive compounds such as monoterpenes, sesquiterpenes and their oxygenated derivatives (Dhifi et al., 2016). Lamiaceae (mint family) species, frequently used in the food, cosmetics, and pharmaceutical industries, are some of the most economically important aromatic plants, producing a variety of volatile compounds that have been known to possess attractant, repellent, antifeedant, larvicidal, insecticidal, antimicrobial, and antioxidant activities among others (Nieto, 2017; Giatropoulos et al., 2018; Ebadollahi et al., 2020; Ramos da Silva et al., 2021; Nazar et al., 2022). For management of insect pests, plants from the Origanum, Thymus, Satureja, Lavandula, Rosmarinus, Melissa, Ocimum, Salvia among many other genera in the Lamiaceae family have been used because of their bioactive aromatic volatiles that exhibit activity against a variety of pests (Ebadollahi et al., 2020).
Two well-recognized plants from the Lamiaceae family, oregano (O. vulgare) and catnip (N. cataria) and, have been reported to exhibit a wide range of bioactivities, including antibacterial and insect repellent activities (Suschke et al., 2006; Peterson and Coats, 2011; Leyva-López et al., 2017; Sharififard et al., 2018; Gomes et al., 2021). Oregano essential oil has shown antibacterial activity against foodborne and other pathogenic bacteria such as Escherichia coli, Bacillus cereus, Salmonella enteritidis, Listeria monocytogenes, Vibrio vulnificus, Clostridium perfringens, and Staphylococcus aureus, leading to opportunities for its use as an antibacterial agent in the food and poultry industries (Kosakowska et al., 2021; Jin et al., 2022; Luo et al., 2022). In addition, use of oregano essential oil for managing pests of importance has also been studied. Oregano essential oil has been reported to have either lethal or sublethal activity against major pests of importance such as the maize weevil (Sitophilus zeamais) (Eesiah et al., 2022), stored product weevil (Sitophilus granarius) (Plata-Rueda et al., 2022), red flour beetle (Tribolium castaneum) (Licciardello et al., 2013), blowfly (Calliphora vomitoria) (Bedini et al., 2021), bed bugs (Cimex lectularius) (Sharififard et al., 2018), mosquitoes (Aedes albopictus) (Giatropoulos et al., 2022) and ticks (Ixodes ricinus) (Soutar et al., 2019).
Similarly, catnip essential oil has shown antibacterial activity against pathogens and pests of importance to human and animal health such as E. coli, S. aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Citrobacter freundii, Branhamella ovis, Serratia marcescens and Neisseria subflava among others (Ghosh et al., 2021) and insect repellent activity against important pests such as stable flies (Stomoxys calcitrans) and houseflies (Musa domestica) as well as American cockroaches (Periplaneta americana), red poultry mites (Dermanyssus gallinae), and disease vectoring mosquitoes (Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus) (Schultz et al., 2004; Zhu et al., 2009; Birkett et al., 2011; Zhu et al., 2014; Reichert et al., 2019). The antibacterial and insect repellent activities of oregano and catnip can be attributed to the major volatile terpene compounds such as carvacrol and nepetalactone respectively, found in the essential oil of the plants (Lambert et al., 2001; Peterson and Coats, 2011; Evergetis et al., 2018; Ghosh et al., 2021). Oregano and catnip essential oils have been registered with the Environmental Protection Agency (EPA) in the United States for use as a biochemical pesticide and as an insect repellent respectively (Environmental Protection Agency, 2010; Environmental Protection Agency, 2011). These two plants can be of strategic importance in mitigating damages related to plant pathogens and insect pests.
Xanthomonas and Pseudomonas are two genera of plant pathogenic bacteria that pose a serious threat to food security (Mansfield et al., 2012). X. perforans is known to cause bacterial spot, one of the most destructive diseases affecting economically valuable crops in the Solanaceae family, including tomatoes and peppers (Srivastava et al., 2021). P. syringae is one of the most common plant pathogens containing over 60 pathovars with distinct host plants, which collectively infect almost all commercially valuable crops (Xin et al., 2018). P. cichorii has a wide host range similar to P. syringae and it has been reported to cause bacterial spot on crops such as lettuce, escarole, and sweet basil plants in New Jersey (Xin et al., 2018; Patel et al., 2019; Patel et al., 2021). Bactericides, especially antibiotics and copper-based formulations, are widely used for controlling bacterial diseases of plants (Miller et al., 2022). However, efficacies of these controls have diminished substantially due to the ease at which resistance develops within bacterial pathogen populations (Sundin and Wang, 2018; Fan et al., 2022). Additional concerns regarding use of copper-based formulations include unfavorable effects on the environment such as decreased soil fertility, horizontal transfer of resistance genes to non-target microorganisms and phytotoxicity in plants, leading to a reduction in the quality and yield of the crop (Sundin et al., 2016; Lamichhane et al., 2018), highlighting the serious need for alternative methods of controlling bacterial plant diseases.
Similar to plant pathogens, arthropods such as common bed bugs (Cimex spp.), a well-known urban pest, are also of significant importance to food production and human well-being (Axtell, 1999; Zorrilla-Vaca et al., 2015). In addition to directly affecting humans, the common bed bug also poses a threat to the food industry, specifically the poultry industry (Kulash, 1947; Axtell, 1999). As a result of bed bug infestations in poultry farms, egg production yields have been reported to be decreased by up to 10% (Cater et al., 2011). Also, bed bugs can be transferred from infested poultry farms to domestic locations by poultry workers (Steelman et al., 2008). In addition to facing the significant cost of bed bug treatment and removal, those affected can also experience adverse effects on their mental and physical health as well as their overall quality of life (Scarpino and Althouse, 2019). Furthermore, increasing resistance has been reported in bed bug populations to various insecticides, including pyrethroids (Davies et al., 2012). Bed bugs in poultry breeding operations are incredibly difficult to control, especially in the presence of poultry due to limited availability of products safe for use in the presence of poultry (Tabler et al., 2015). Once established in a suitable environment, the common bed bug is challenging to control and nearly impossible to eliminate from poultry houses (Kulash, 1947; Tabler et al., 2015). Thus, additional strategies for control as well as more sustainable methods to overcome resistance in bed bugs are needed.
One of the challenges associated with the development of sustainable essential oil-based products is a steady supply of raw materials. Therefore, availability of genetic materials for specialty crops that produce high amounts of bioactive compounds is a vital aspect that needs to be addressed in the supply chain. As part of the effort to develop high essential oil yielding genetic materials, breeding programs have been focused on the Lamiaceae family to produce improved varieties with desirable phytochemical profiles that are adapted to different growing conditions. Among these new plants, a new patented cultivar of N. cataria, CR9, and a new patented cultivar of O. vulgare, Pierre, have been developed to produce high yield of essential oil and content of (Z,E)-nepetalactone and carvacrol respectively (Reichert et al., 2016; Reichert et al., 2021). These cultivars are some of the first for these species to be adapted to the North American and Mid-Atlantic regions and possess an upright growth habit allowing for a mechanical harvest and large-scale production (Reichert et al., 2016; Reichert et al., 2021).
While catnip and oregano essential oils have previously been tested against pathogens and pests of importance to agriculture and human health (Vasinauskiene et al., 2006; Peterson and Coats, 2011; Zomorodian et al., 2012; La Pergola et al., 2017), the study of antimicrobial and insect repellent effects of novel catnip cv. CR9 and oregano cv. Pierre remain underexplored. In addition, carvacrol-rich oregano essential oil has not been tested against P. cichorii and C. lectularius and catnip essential oil has not been tested against P. cichorii and X. perforans. Therefore, the aim of this study was to evaluate the chemical composition and potential of the essential oils of the new and unique N. cataria cv. CR9 and O. vulgare cv. Pierre as antibacterial agents against P. cichorii, P. syringae, and X. perforans; essential oil of O. vulgare cv. Pierre and mixtures of the essential oil of O. vulgare cv. Pierre and N. cataria cv. CR9 as insect repellents against C. lectularius.
Discussion
The aim of this study was to determine the antibacterial and insect repellent activities of catnip cv. CR9 and oregano cv. Pierre. The major component of the essential oil from novel catnip cultivar CR9 is (Z,E)-nepetalactone, a bicyclic oxygenated monoterpene that has shown to have remarkable insect repellent activity against malaria vectoring mosquitos, ticks, chicken mites, houseflies, and stable flies, among others (Gomes et al., 2020a). The chemical profile of catnip essential oil is consistent with the profiles reported in literature with (Z,E)-nepetalactone as the major constituent, followed by minor components such as β-caryophyllene and caryophyllene oxide, whose amounts vary (Reichert et al., 2016; Reichert et al., 2019). Nepetalactone and its various isomers have been reported to be the major constituent of N. cataria essential oil as well as the oil of other species of catnip in the Nepeta genus (Zenasni et al., 2008; Asgarpanah et al., 2014; Gomes et al., 2020a). Catnip genotypes possess distinct chemical profiles, with some catnip genotypes such as lemon catnip, lack the major compound nepetalactone (Gomes et al., 2020b). Therefore, having a genotype that produces high yield of nepetalactone is key for steady supply of effective products. Thus, catnip cv. CR9, a nepetalactone rich chemotype containing a single isomer of (Z,E)-nepetalactone as its major compound along with other desirable agronomic traits (Reichert et al., 2016), provides reliable genetic materials suitable for industrial applications.
Carvacrol was the most abundant compound in the essential oil of new oregano cv. Pierre, followed by p-cymene, a profile consistent with previously reported analyses (Reichert et al., 2021). Carvacrol is a commercially valuable molecule possessing antimicrobial, antioxidant and anti-inflammatory properties and has been approved by the United States (US) Food and Drug Administration (FDA) for use in food and as a chemical flavoring by the Council of Europe (Zotti et al., 2013). The high carvacrol yielding oregano cultivar Pierre, developed for the Mid-Atlantic region in Northern America (Reichert et al., 2021) can provide a reliable supply of essential oil with a consistent chemical profile to support the growing flavors and fragrance market which is projected to reach $5.4 billion in sales by 2025 in the United States (Statista, 2022). P. cichorii and P. syringae are bacterial plant pathogens that negatively impact economically valuable crops (Lamichhane et al., 2015). Both the catnip and oregano essential oils showed inhibitory activity against these two Pseudomonas species. The catnip essential oil showed significant antibacterial activity, however not as strong as the oregano oil, which was even superior to the antibiotic kanamycin in concentrations above 10%. Thus, its potential to serve as an antibacterial agent can be further evaluated against other plant pathogens of interest using the same or different methods. Essential oil and ethanolic extracts of novel catnip cv. CR9 have previously showed repellent activities against mosquitoes, ticks, and bed bugs (Reichert et al., 2019; Shi et al., 2021; González et al., 2022), but it is reported in this study for the first time that the essential oil from this genotype also displays antibacterial activity.
The antibacterial activity of essential oils from other N. cataria genotypes has previously been tested against food borne pathogens, respiratory tract pathogens and skin commensals, such as S. aureus, E. coli, P. aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis (Suschke et al., 2006; Suschke et al., 2007; Zenasni et al., 2008; Adiguzel et al., 2009; Zomorodian et al., 2012; Vukovic et al., 2016; Ashrafi et al., 2019). For Pseudomonas species, while the antibacterial effects of extracts and oils of N. cataria against P. syringae have been tested previously (Adiguzel et al., 2009), the inhibitory effects of catnip essential oil against P. cichorii are presented here for the first time. Essential oil of oregano cv. Pierre showed overall a stronger antibacterial activity than catnip cv. CR9 and, in higher concentrations, was even superior to the antibiotic kanamycin. Oregano essential oil has a well-established history of use as an antibacterial agent for applications in the food industry (Rodriguez-Garcia et al., 2016), and its activity against P. syringae and its various strains has been well documented (Vasinauskiene et al., 2006; Carezzano et al., 2017). However, similarly to the information previously discussed for catnip, to the best of our knowledge, there are no reports of the use of oregano essential oil against P. cichorii.
An interesting trend observed for both catnip and oregano was a reduction, with varying degrees of intensity, in the zones of inhibition of wells filled with the pure (100%) essential oils, in comparison to diluted essential oils, mainly at 20 and 40% concentrations, for both P. syringae and P. cichorii, but not for X. perforans, when the pure essential oils performed similarly or better than when diluted. One possible explanation is that the use of pure essential oil hinders its diffusion through the media due to having higher viscosity compared to the concentrations diluted with ethanol. More viscous essential oils have been reported to exhibit little antibacterial activity compared to less viscous oils in the agar well diffusion assay (Hood et al., 2003). The reason why X. perforans appears to be less affected by such phenomena is possibly related to the differences in media composition. The Pseudomonas species used in this study were cultured in King’s Medium B (KMB), while X. perforans was cultured in Yeast-Dextrose-Calcium Carbonate (YDC) medium. One of the main differences among these media that can affect the diffusion of viscous liquids is that KMB contains glycerol, which is a viscous liquid in itself, its presence in the medium may impede the diffusion of the essential oils. Additionally, previous studies have also reported different susceptibility among bacteria species to essential oil properties, some being inhibited more by vapor absorption (not directly dependent on media diffusion) and some being more susceptible to direct contact through the media (Inouye et al., 2001).
Despite the differences observed in the treatments with P. cichorii and P. syringae, when using the pure essential oil, X. perforans was also inhibited by catnip and, especially by oregano essential oils. Previously carvacrol, a major component of essential oil of oregano and in particular the major component of oregano cv. Pierre’s essential oil has been tested against copper resistant X. perforans in in vitro, greenhouse and field settings (Qiao et al., 2020). Carvacrol has shown to increase the sensitivity of the resistant X. perforans to copper-based treatments, promoted germination of tomato seeds, increased seedling vigor, reduced severity of bacterial spot and enhanced efficacy of copper against the resistant X. perforans strain (Qiao et al., 2020). As for catnip, previous studies of antimicrobial activity have been reported in other Xanthomonas species (Kokoskova and Pavela, 2006; Adiguzel et al., 2009), but this is the first report of inhibitory effects of catnip essential oil against X. perforans. The strong inhibitory effect displayed by the oregano essential oil suggests that it can be an excellent candidate for further studies to evaluate its potential in other in vitro tests as well as in agronomic evaluations of disease incidence and severity.
Different essential oils have been assessed for their antimicrobial activities, with mixed results (Ahluwalia et al., 2014; Mandal et al., 2021; Manjesh et al., 2022). These natural products are reported to have multiple modes of action that involve damage to the cell wall and membrane, changes to the fatty acid composition, leakage of ions and intracellular metabolites, disruption in glucose uptake and metabolic activity among others (Nazzaro et al., 2013; Angane et al., 2022; Chang et al., 2022). One of the many reported modes of action of oregano essential and oil and carvacrol, is their ability to permeabilize bacterial cell membranes (Hyldgaard et al., 2012). The efficacy of essential oils can be enhanced due to the synergy between their various components (Isman et al., 2011), making them ideal candidates for developing new antibacterial agents and crop protectants to combat resistant pathogens and pests.
In a study by Gavaric et al. (2015), both combinations of oregano and thyme essential oil as well as those of carvacrol and thymol, their major components, had an additive effect when tested against S. aureus, B. cereus, E. coli and Salmonella infantis. Carvacrol and thymol have also exhibited additive effects against S. aureus and P. aeruginosa (Lambert et al., 2001). A mixture of (Z,E)-nepetalactone and (E,Z)-nepetalactone has exhibited synergistic effects and antibacterial activity against five different Neisseria spp. (Ghosh et al., 2021). Literature regarding the potential synergistic, additive, or antagonistic interactions between the major and minor components of N. cataria essential oil against bacterial pathogens is scarce. Thus, additional studies need to be conducted to understand the type of interactions between the components and their effect on the overall activity of the oil.
In addition to carvacrol and nepetalactone, other compounds in the essential oils of catnip and oregano can influence their antimicrobial activity, either synergistically, additively, or antagonistically. Synergism between the major component of oregano, carvacrol and other components such as p-cymene, a weak antibacterial compound that is also a precursor of carvacrol, has been observed against B. cereus in vitro and in foods such as rice (Burt, 2004). The combination of carvacrol and cymene has shown to inhibit growth of V. cholerae in food, with cymene reportedly increasing the inhibitory activity of carvacrol (Rattanachaikunsopon and Phumkhachorn, 2010). The effectiveness of the combination of carvacrol and cymene may be due to the ability of cymene to expand the membrane, allowing an increase in the amount of carvacrol transported into the cell (Burt, 2004). In addition to p-cymene, γ-terpinene has also been reported to show varying levels of activity against several bacterial pathogens (Aligiannis et al., 2001; Cristani et al., 2007). Contrastingly, antagonistic effects of the combinations of carvacrol/thymol, carvacrol/eugenol and carvacrol/myrcene have also been reported in S. aureus, B. cereus and E. coli, with myrcene alone exhibiting no inhibitory activity against these pathogens (Gallucci et al., 2009).
Low molecular weight, hydrophobic terpenoids with weak antimicrobial activity of their own are reported to contribute to the overall antimicrobial activity of the essential oils due to their ability to disrupt the cell wall and enable the more active components to enter the cell (Álvarez-Martínez et al., 2020). The essential oil of oregano cv. Pierre has carvacrol as its major compound, followed by the terpenes p-cymene and γ-terpinene. It is possible that some of these compounds and other minor ones contribute to the observed antibacterial activity of the oregano oil. Future studies with the whole essential oil of oregano cv. Pierre as well as its individual constituents may help in further elucidating the potential synergism/antagonism between the components of the oil and their impact on the bioactivity of the oil. In contrast to the essential oils, the antibiotic kanamycin, belonging to the aminoglycosides class of compounds, leads to inhibition of protein synthesis by misreading the nucleotides and terminating the translation of mRNA (Dowling et al., 2017; Kapoor et al., 2017).
Essential oils can also affect the physiology and behavior of insects, with mono and sesquiterpenoid components of essential oils exhibiting sublethal effects on arthropods such as oviposition deterrence and repellency (Chang et al., 2022). In addition to demonstrating promising antibacterial effects, catnip cv. CR9 and oregano cv. Pierre essential oils also showed remarkable repellency against the common bed bug (C. lectularius). Catnip has been previously shown to repel bed bugs in a concentration-dependent manner (Shi et al., 2021), so in the present study only the novel oregano essential oil was studied to find the optimal concentration for repellency. Concentrations as low as 2.5% were found to be enough to cause complete repellency in petri dish assays, with results comparable to equivalent doses of DEET, insect repellent’s gold standard. Two isomers of nepetalactone [(4aS,7S,7aR) and (4aS,7S,7aS)] showed lower repellency against Anopheles gambiae compared to the catnip oil, indicating potential synergism between them, and when combined with (E)-(1R,9S)-caryophyllene, the resulting mixture was reported to have the same repellent activity as the original catnip oil (Birkett et al., 2011).
Previously, the essential oil from a different oregano genotype, mainly composed of terpineol and α-terpinene, has been tested against bed bugs in the petri dish repellency assay, repelling only 10% of the insects after 24 hours with a concentration of 2.5% (Sharififard et al., 2018). In the present study, the same essential oil concentration of oregano cv. Pierre showed 100% repellency. Such a contrasting result can be explained by the unique chemical composition of oregano cv. Pierre, which produces high levels of carvacrol and p-cymene, compounds widely regarded as strong repellents of different insects (Evergetis et al., 2018; Feng et al., 2021). The concentration of 1.25% of oregano cv. Pierre still showed high repellency after 24 hours of exposure in the petri dishes, although inferior to DEET and higher concentrations of the essential oil. In addition to repellency, oregano essential oil has been reported to display toxicity to bed bugs when applied topically in a study by Zha et al. (2018), however the chemical composition of the oregano oil used in the study is unknown. A study by Gaire et al. (2020a) demonstrated significant insecticidal activity of oregano essential oil against a deltamethrin resistant bed bug strain Knoxville. In another study, topically applied binary mixture of oregano essential oil and deltamethrin significantly increased mortality in resistant bed bugs (Gaire et al., 2021). The use of oregano essential oil as a repellent has been reported against other pests of importance to public health such as vectors of infectious diseases (mosquitoes and ticks), head lice and brown-banded cockroach (Alizadeh et al., 2021), however this is the first time that a carvacrol-rich oregano essential oil is reported to repel bed bugs.
After defining that the concentration of 2.5% was equivalent to higher concentrations of oregano essential oil, a subsequent study was conducted to assess the effectiveness of catnip and oregano mixtures along with carvacrol, the major component of oregano cv. Pierre’s essential oil as bed bug repellents at that concentration. Catnip, oregano, and their mixtures showed strong repellency against bed bugs after 1 hour of exposure, being equivalent to DEET. After 24 hours in the petri dishes, catnip essential at the 2.5% concentration shows a reduction in its bioactivity, being inferior to DEET and oregano essential oil. However, the different mixtures containing oregano remain equivalent to DEET at 24 hours, despite slight reductions in the average repellency, evidencing that combining catnip and oregano essential oils may help to increase the duration of repellency of products containing essential oil of catnip cv. CR9.
In this study, carvacrol showed repellency similar to that observed in the essential oil of oregano, demonstrating that it is the main contributor to the activity of the essential oil of oregano against C. lectularius. Additionally, carvacrol has demonstrated considerable topical and fumigant toxicity as well as significant neuroinhibitory effects in susceptible bed bugs (Gaire et al., 2019) and enhanced deltamethrin toxicity in resistant bed bugs (Gaire et al., 2021). Furthermore, carvacrol has shown to act synergistically when combined with other monoterpenoids such as thymol and eugenol in topical bioassays in bed bugs (Gaire et al., 2020b). Carvacrol exhibited acute topical toxicity against Musa domestica adults and showed significant synergy in the topical application assay when combined with p-cymene (Pavela, 2008). In another study by Pavela (2015), acute toxicity against the larvae of Culex quinquefasciatus was observed under the synergistic combinations of carvacrol and terpineol, carvacrol and carvone, and carvacrol and 4-allylanisole. In terms of repellency, carvacrol has been previously reported to have a repellent effect against bed bugs for up to 24 hours in the petri dish repellency assay (González-Morales et al., 2021).
Catnip cv. CR9’s essential oil has shown to be an effective repellent against ticks and mosquitoes, and it has also been tested previously in bed bugs (Shi et al., 2021). In addition, catnip essential oil and its main constituent nepetalactone have shown to repel other insects such as stable flies, house flies, ticks, chicken mites and many species of mosquitoes that vector diseases such as malaria (Gomes et al., 2020a). Catnip cv. CR9 offers a solution to challenges faced previously regarding catnip cultivation and essential oil production as it is a nepetalactone rich cultivar with high biomass yield and upright growth habit which is ideal for mechanical harvesting (Reichert et al., 2016; Gomes et al., 2020a). Catnip can be grown locally in rural communities as well to diversify crops and generate extra income, in addition to providing a source of insect repellents in areas where synthetic repellents may be hard to purchase (Gomes et al., 2020a). The oregano cv. Pierre can offer a high-quality source of oregano essential oil with a stable chemical profile for cultivation in the Mid-Atlantic Region of the United States (Reichert et al., 2021). With numerous applications in the food, beverage, personal care, cosmetics, and pharmaceutical industries, growing oregano can be profitable due to low cost of cultivation and increasing market demand for oregano oil (Dordas, 2009; Nazar et al., 2022). The strong repellency of both catnip and oregano essential oils against bed bugs makes them excellent candidates for plant-based insect repellents that can provide a more sustainable way to manage pests of human and agricultural importance.