​The common bacteriostatic and fungistatic active ingredient in thyme essential oil is thymol, chemically known as 5-methyl-2-isopropylphenol, which is a natural monoterpene. Thymol has the strongest inhibitory effect against molds, and 0.01–0.05 mg/mL of thymol is found to be effective against molds such as Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. In addition, thymol can effectively inhibit Gram-positive pathogens such as Bacillus subtilis and Staphylococcus aureus between 0.06 and 0.2 mg/mL, while the inhibitory concentration for lactic acid bacteria must reach 0.1–0.5 mg/mL (Marchese et al., 2016). Another highly effective bacteriostatic and fungistatic active substance in thyme essential oil is terpinene, which can exhibit an bacteriostatic and fungistatic effect equivalent to 70% of streptomycin at a concentration of 0.07 mg/mL against Phytophthora capsici (Yang et al., 2022). Thyme essential oil (Thymus kotschyanus), as a mixture of multiple bacteriostatic and fungistatic active substances, has a minimum inhibitory concentration of 0.0625 mg/mL against common molds such as A. niger, A. flavus, and Fusarium oxysporum in silage feed or raw materials (Ownagh et al., 2010), and the minimum inhibitory concentration for yeast is 0.5 mg/mL. The inhibitory concentration for some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus plantarum, and Lactococcus lactis must reach at least 3–10 mg/mL (Thymus vulgaris ct.linalol, Thymus serrulatus and Thymus schimperi Ronniger) (Gutierrez et al., 2008; Damtie and Mekonnen, 2020; de Oliveira Carvalho et al., 2020). Thus, whether the active ingredient is single or a mixture in thyme essential oil, it shows low inhibitory concentration against pathogens and high inhibitory concentration against lactic acid bacteria. This selective inhibition laid the foundation for its use in silage feed (Damtie and Mekonnen, 2020).

The main active ingredient in clove essential oil is eugenol, chemically known as 4-allyl-2-methoxyphenol, which belongs to the terpene class of compounds and has a broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria (Khalil et al., 2017). The minimum inhibitory concentration of eugenol for Gram-positive bacteria such as S. mutans and S. aureus is generally between 0.1 and 0.2 mg/mL. Its minimal inhibitory concentrations against Fusarium species commonly found in silage, such as Fusarium avenaceum, Fusarium graminearum and Aspergillus nidulans, ranged from 0.1 mg/mL to 0.14 mg/mL. The minimum inhibitory concentration against Saccharomyces cerevisiae is 0.25 mg/mL, whereas the minimum inhibitory concentration for lactic acid bacteria such as Lactobacillus casei must reach 1 mg/mL (Marchese et al., 2017). Low levels of eugenol (below the minimum inhibitory concentration) show strong inhibitory activity against the expression of the physiological functions of microorganisms and the production of toxins such as ochratoxin (Jiang et al., 2022). Clove essential oil also exhibits a remarkable bacteriostatic and fungistatic activity, with a minimum inhibitory concentration of 0.2–0.3 mg/mL for yeast and a minimum inhibitory concentration of 0.031, 0.031, and 0.25 mg/mL for fungi such as F. graminearum, Rhizopus stolonifer, and Penicillium crustosum, respectively, which is lower than its minimum inhibitory concentration for lactic acid bacteria such as Lactobacillus fermentum and Lactobacillus lactis (Syzygium aromaticum (L.) Merr. and L. M. Perry) (Sameza et al., 2016; Sharma et al., 2017; Chan et al., 2018). The low inhibitory concentration of clove essential oil against harmful microorganisms and high inhibitory concentration against lactic acid bacteria determines its use in later production.

The bacteriostatic and fungistatic activity of cinnamon essential oil is derived from cinnamaldehyde, which has the strongest bacteriostatic and fungistatic activity among other secondary metabolites. Cinnamaldehyde has shown antifungal activity against Candida (Choonharuangdej et al., 2021), A. flavus (Achar et al., 2020), F. graminearum (Gwiazdowska et al., 2022), Aspergillus ochraceus (Wang et al., 2018), and other fungi, as well as broad-spectrum antifungal activity against Escherichia coli and S. aureus (Zhang et al., 2016). Compared with other active components of plant essential oil, cinnamaldehyde has the strongest inhibitory activity on the synthesis of aflatoxin and ochratoxin. The minimal inhibitory concentration of cinnamaldehyde against F. oxysporum and F. gramineum is 0.8 mg/mL, and its minimal inhibitory concentration against spoilage yeast is 0.31–1.25 mg/mL (Muñoz‐González et al., 2022). However, its minimal inhibitory concentration against lactic acid bacteria varies, with a minimal inhibitory concentration of 5 mg/mL against Lactobacillus sakei and a minimum inhibitory concentration of 50 mg/mL against Lactobacillus brucei (Sameza et al., 2016; Sharma et al., 2017; Muñoz-González et al., 2022). Cinnamon essential oil also shows good application effects. Cinnamon essential oil inhibits E. coli, S. aureus, and Listeria at a concentration of 0.3–0.5 mg/mL (Cinnamomum zeylanicum) (Nematollahi et al., 2020), and its minimum inhibitory concentration against Penicillium citrinosus and Penicillium expandatum is between 0.4 and 0.5 mg/mL (Cinnamomum cassia) (Lucas-Gonzalez et al., 2023). However, the minimum inhibitory concentration against lactic acid bacteria is approximately 10 mg/mL (C. zeylanicum) (de Oliveira Carvalho et al., 2020).

​The main active ingredients in oregano essential oil are carvacrol and thymol. Thymol has been elaborated in the previous section, and carvacrol is also a class of phenolic compounds with strong bacteriostatic and fungistatic activity. The minimum inhibitory concentration of carvacrol against S. aureus is 0.3 mg/mL; the minimum inhibitory concentration of carvacrol against Streptococcus is 2.5 mg/mL, and its minimum inhibitory concentration against Gram-negative bacteria such as E. coli and Salmonella typhimurium is less than 0.25 mg/mL (Marinelli et al., 2018; Rathod et al., 2021). The minimum inhibitory concentration against a variety of yeasts is approximately 0.2 mg/mL. On the contrary, oregano essential oil containing a mixture of carvacrol and thymol shows considerable synergistic bacteriostatic and fungistatic effects. Its minimum inhibitory concentration against a variety of Fusarium species is less than 0.8 mg/mL, and its antifungal activity is mostly between 0.2 and 0.3 mg/mL, whereas its minimum inhibitory concentration against a variety of lactic acid bacteria is between 5.5 and 13 mg/mL (Origanum vulgare L.) (Gutierrez et al., 2008; Konuk and Ergüden, 2017; Sharma et al., 2017). The bacteriostatic and fungistatic activity of oregano essential oil against lactic acid bacteria is lower than that against pathogenic fungi. Furthermore, oregano essential oil can inhibit the biosynthesis of aflatoxin after it acts on aflatoxin (Origanum majorana L.) (Chaudhari et al., 2020).

Peppermint essential oil has been used as a preservative in the food industry for a long time, and its bioactive substances have good effects on inhibiting pathogenic bacteria and spoilage microorganisms, such as menthol, menthone, and neomenthol (Kazem et al., 2011). A number of reports have shown that peppermint essential oil inhibits the cell activity of S. aureus (Kang et al., 2019), Candida albicans, Pseudomonas aeruginosa (Mahboubi and Kazempour, 2014), Helicobacter pylori, and Salmonella enteritidis (Imai et al., 2001). The in vitro anti-aflatoxin concentration of menthol is 1.0 mg/mL, while menthol stereoisomers and menthone do not show evident antitoxin function, which is related to their structural types (Ownagh et al., 2010; Muñoz-González et al., 2022). Peppermint essential oil has weaker bacteriostatic and fungistatic activity than menthol. Peppermint essential oil has a minimum inhibitory concentration of 62 mg/mL against a variety of mold species and more than 150 mg/mL against lactic acid bacteria, but it has considerable bacteriostatic and fungistatic activity against S. cerevisiae, with a minimum inhibitory concentration of 1 mg/mL. The results show that antifungal sensitivity to mold and yeast is higher than sensitivity to lactic acid bacteria. (Mentha Piperita) (de Oliveira Carvalho et al., 2020).

Turmerone is a glycosides active substance derived from turmeric essential oil. It mainly includes ar-turmerone and β-turmerone. Given its special structure, it has strong antibacterial and antifungal activity. At a concentration of 0.1% ar-turmerone, this purified compound reduced the growth of Fusarium semitectum, Aspergillus ochraceous, and Calletotrichum musae by 70%, 55%, and 68%, respectively (Dhingra et al., 2007). At 1 mg/disk, ar-turmerone strongly inhibited the growthl of C perfringens and moderately inhibited the growth of E. coli without any adverse effects on the growth of four lactic acid bacteria(Lee, 2006). Given the synergistic effect of various bacteriostatic and fungistatic active substances in turmeric essential oil, the bacteriostatic and fungistatic activity of the mixture is remarkably enhanced. The minimum inhibitory concentration of turmeric essential oil to various yeasts is between 0.01 and 0.03 mg/mL (Curcuma longa L.) (Konuk and Ergüden, 2017), and the concentration of turmeric essential oil has a remarkable inhibitory effect on Fusarium verticillium at 0.072 mg/mL (C. longa) (Avanço et al., 2017). However, the minimum inhibitory concentration against Lactobacillus is as high as 11–14.5 mg/mL (C. longa) (Khorsandi et al., 2018).

All of the above mentioned essential oils show strong inhibition against yeast, fusarium, and other pathogenic bacteria but relatively weak inhibition against lactic acid bacteria. This unique bacteriostatic and fungistatic property of essential oil contributes to the inhibition of spoilage bacteria in silage and to the improvement of silage quality.

The synergistic bacteriostatic and fungistatic effects of plant oils have been gradually recognized with the exploration of their bacteriostatic and fungistatic properties. The bacteriostatic and fungistatic activity of plant essential oils results from interactions among different components, often by terpenoid compounds with the strongest internal bacteriostatic and fungistatic properties, as well as by a variety of compounds of different classes,For instance, there are interactions between compounds such as citral and thujone, camphor, ethyl acetate of borneol, and citronellal. Similarly, interactions between α-pinene and thujone, camphor, citral, citronellal, and geraniol occur. Moreover, the interaction of cineole oxide with compounds like thujone, hexanal, and hinokitiol also demonstrates a synergistic enhancement of cytotoxicity(Wright et al., 2007), and the fractional inhibitory concentration of each component against bacteria is markedly reduced. The mixed use of eugenol and linalool in basil essential oil shows higher bacteriostatic, fungistatic and antioxidant activity to some fungi and bacteria than each component itself (Juliani et al., 2009). The bacteriostatic and fungistatic active components of cinnamon essential oil, cinnamaldehyde and cinnamic acid, also show bacteriostatic and fungistatic properties when combined. The combination of components derived from different essential oils also shows synergistic effects, such as the chimerism of cinnamaldehyde and carotenoids to form a mixture of solid and liquid fats to cause defects in the structure of the lipid matrix and improve the bioavailability and retention ability of active ingredients (Procopio et al., 2022). The combination of cinnamon essential oil and clove essential oil also has synergistic effects, and the enhanced essential oil remarkably inhibits biofilm formation, destroying the cell wall structure and scavenging free radicals (Yen and Chang, 2008). The same study found that the combination of thyme and rosemary as well as the triple combination of thyme, rosemary, and cinnamon show synergistic effects on Bacillus cinesulata and Pseudomonas ectrosp (Nikkhah et al., 2017). The combination of oregano and thyme essential oils, bay and almond essential oils, and basil and thyme essential oils all showed synergistic bacteriostatic and fungistatic effects, with minimum inhibitory concentrations reduced by up to 128 times.

​The bacteriostatic and fungistatic effects of essential oils are directly related to their lipophilicity. The six-carbon aromatic phenol group of cinnamaldehyde allows it to cross the phospholipid bilayer of the bacterial cell wall and bind to the inner and outer membrane proteins, thereby preventing them from performing their normal function. Altered membrane permeability and loss of functional proteins that transport molecules and ions perturb microbial cells, which leads to cytoplasmic coagulation, enzymatic denaturation, and loss of proteins, as well as loss of metabolites and ions (Suxia Shen et al., 2015). Cell membrane damage by essential oil is usually manifested as the change in cell membrane surface structure (usually wrinkled or irregular), the increase of relative electrical conductivity, the decrease of membrane potential, the increase of extracellular nucleic acid and protein concentration, the change of membrane potential and conductivity, and the accumulation of intracellular active components of essential oil, which leads to the acidification of the cell membrane and the damage of the cell membrane caused by protein denaturation (Trinh et al., 2015).

Cellular ion leakage is also an important bacteriostatic and fungistatic mechanism of plant essential oils. The α- and β-unsaturated bonds of various cinnamaldehydes can be conjugated to the plasma membrane calcium ATPase on the fungal plasma membrane, which opens the ion pathway, induces Ca²⁺ efflux, and inhibits fungal activity (Hu et al., 2013). Under ergosterol inhibition, celery essential oil acts on the cell membrane of A. flavus to take part in the α-demethylation of lanosterol, and Ca²⁺ leakage is observed. Mg²⁺ influx is enhanced, leading to the depletion of nutrient uptake, inhibition of nucleic acid synthesis, and inhibition of ATPase-dependent respiratory activity, thereby leading to cell lysis (Das et al., 2019). P. capsici Leonian and A. flavus, which have no ergosterol production ability, and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid elution treatment were used to exclude the influence of exogenous Ca²⁺. The cells showed Ca²⁺ outflow and ergosterol reduction after treatment with cinnamon essential oil, fenestra essential, oil and peppermint essential oil (Dwivedy et al., 2017). Ca²⁺ is the most prevalent regulator in the whole living system, which is involved in the regulation of the proliferation, differentiation, and apoptosis of organisms. In fungi, Ca²⁺ regulates spore formation, spore germination, hyphal branching, apical growth, and structural differentiation (Shreaz et al., 2016). The disorder of Ca²⁺ may interfere with the amount of circulating calcium ions in the mitochondria and activate the mitochondrial permeability transition pore, thereby leading to the initiation of the fungal apoptosis program (Berridge et al., 2000).

Quorum sensing (QS) is a feedback intercellular communication system of bacteria based on the secretion and sensing of external signaling molecules. The formation of biofilms is highly correlated with density-dependent QS propagation, which affects the production of bacterial secondary metabolites and regulates the secretion of virulence factors. Bacteria in the form of biofilms are different from planktic bacteria because their association with abiotic surfaces generates a three-dimensional organizational structure that is protected from the threat of being killed by fungicides, disinfectants, and antibiotics. Bacterial cells produce extracellular polymeric substances, proteins, and extracellular DNA, which support structural stability and improve substrate exchange and nutrient cycling in biofilms (Tan et al., 2019). The inhibition of intercellular communication between bacteria and biofilm formation is considered as an bacteriostatic and fungistatic pathway of plant essential oils (Bo et al., 2023). After applying cinnamaldehyde to the biofilm, the hydrophobicity of the cell membrane decreases, resulting in the reduced adhesion of the hydrophobic surface to the biofilm surface. Aggregation also decreased, whereas self-aggregation formed the complete biological structure of several different biofilm strains, which played an important role in biofilm stability (Yu et al., 2020). Molecular docking analysis showed that cinnamaldehyde downregulated the expression of cellulose synthase (BcsA) and transcription activator protein (luxR) receptor genes, thereby inhibiting the synthesis of signaling molecules in QS (Liu et al., 2021). Diallyl disulfide in garlic essential oil inhibits the virulence factors of P. aeruginosa at MIC concentrations by affecting the transcription of key genes in three different QS systems (Li W.-R. et al., 2018). Carvacrol binds to homoserine lactone synthase (Expl) and transcriptional regulators (Khan et al., 2017), which in turn inhibits the production of QS signaling molecules and the expression of QS-controlled genes (Joshi et al., 2016). Eugenol inhibits protease, pyocyanin, pyranan biosynthesis, extracellular polysaccharide, and rhamnolipid and closely binds to the synthesis of carbonyl N-coa acylation regulatory protein (LasR) by P. aeruginosa, thereby leading to the inhibition of QS (Rathinam et al., 2017). Cyclic diguanosine monophosphate (C-di-GMP) is considered as a key cytoplasmic signal and second messenger that controls bacterial virulence, cell cycle reproduction, motility, and other behaviors, such as the biofilm life cycle in several bacteria. Cinnamaldehyde carbon can affect the level of nitric oxide by regulating C-di-GMP, thereby inducing biofilm dispersion (Topa et al., 2018), whereas the ginger essential oil test found that cinnamaldehyde carbon promoted the degradation of proteins with EAL (Glu-Ala-Leu) or HD-GUP (His-Asp-Gly-Tyr-Pro) domains. This domain is directly associated with C-di-GMP levels, thereby inhibiting biofilm formation (Kim and Park, 2013).

​The cell wall is an important structure in fungi, indispensable for maintaining shape integrity and fluidity, for interacting with its surroundings, and for regulating the fungal membrane. Chitin, as a scaffold for the cell wall, imparts integrity and strength to the cell wall, thereby offers protection against external stresses, mechanical damage, and immune responses from hosts (Baker et al., 2007). Glucans attached to chitin serve as attachment points for other structures, constituting amorphous outer and intermediate fillers of the fungal cell wall (Shenghui Shen et al., 2019). Glycosylated proteins anchor β (1,6)-glucan chains through glycosylphosphatidylinositol (GPI). Mannosyl proteins account for 40% of the cell wall structure. Damage to mannosyl proteins will lead to the decreased activity of proteins synthesizing the cell wall, thereby affecting the strength and integrity of the cell wall, which is of great importance to the dynamic system of fungal cell wall (Schmidt et al., 2005). The chitin–glucan complex or chitosan complex is the main component of fungal cell wall, accounting for 60% of the dry weight of the cell (Araújo et al., 2020).

The active substances in many plant essential oils are mixed inhibitors of 1, 3-β-glucan synthase (FKS-1) and chitin synthase in fungi. Diallyl disulfide downregulates the expression of the chitin synthase gene, and the treated cells have reduced chitin, damaged epidermis, and changed morphology and physiological activity (Shah et al., 2020). Previous studies found that cinnamaldehyde and eugenol could bind to the active sites of these two enzyme proteins through different amino acid residues (Ju et al., 2022). Artemisia monosperma Del., Callistemon viminals G. Don, Citrus aurantifolia Swingle, and Cupressus macrocarpa Hartw. ex Gordon essential oils also inhibited chitin production (Abdelgaleil and El-Sabrout, 2018). Some experiments have found that a sub-inhibitory concentration of cinnamaldehyde can directly or indirectly attack the target of azole, polyene, and echinocandin through the accumulation of reactive oxygen species to induce moderate downregulation of the transcription of ERG-2, ERG-3, ERG-4, and ERG-11, which is consistent with the change trend of ergosterol (Parks and Casey, 1995). The upregulation of sterol influx transporters (such as AUS-1 and TIR-3) and sterol metabolism regulators (such as SUT-1 and UPC-2) inhibits ergosterol in a dose-dependent manner and then affects cell membrane integrity (Li Q. Q. et al., 2018). Estragole and linalool were found to inhibit ergosterol in a similar way as azole antifungal agents, both of which showed an inhibitory effect on 14α-demethylase, a key enzyme in ergosterol synthesis (Khan et al., 2010).

​ATPases are involved in most biological and physiological activities in the cell through energy coupling. When plant oils disrupt the permeability and fluidity of plasma membranes, the membrane potential of organelles is reduced; proton pumping is disrupted, and the synthesis of H-ATPase, the key enzyme in ATP production, is inhibited. Some polyphenolic compounds block the activity of ATP synthase by binding to some chemical binding cavities of the enzyme, and the functional groups of inhibitors have established interactions with key amino acid residues of the enzyme through hydrogen bonding, hydrophobic interaction, etc. (Issa et al., 2019). After the inhibition of ATPase, related dependent enzymes are also affected. The decrease of protease and phospholipase activities can be attributed to the degradation of ATPase mediated by cinnamaldehyde (Pootong et al., 2017), and the expression level of fatty acid synthases such as acetyl-CoA carboxylase and fatty acid biosynthetic enzymes (fasI, fasH, and fasF) is inhibited. In addition, glycerophospho acyltransferase (plsX, plsY, plsC), cytidine diphosphate-diacylglycerol synthase A (cdsA), phosphatidylglycerol synthase A (pgsA), cardiolipin synthase (cls), and polypeptide resistance factor (mprF) glycerophospholipid biosynthesis pathways are inhibited; thus, the degree of damage of bacterial biofilm is aggravated (Pang et al., 2021). After cinnamaldehyde treatment, the ATP permeability of biofilms was increased; the ATP level in biofilms was remarkable decreased, and the intracellular ATP was rapidly consumed to maintain pH. After eugenol and citral treatment, Pseudomonas roqueforti was found to have an altered mitochondrial morphology and membrane potential, which further damaged the metabolic pathways of cellular energy and finally induced apoptosis (Ju et al., 2020).

The filamentous temperature-sensitive protein Z (FtsZ) is a GTPase with weak a sequence homology with tubulin, which plays an active role in guiding the binary division of bacteria. The FtsZ is assembled into a Z-ring structure at the future cell division site. The Z ring can promote cytodivision and recruit more than a dozen other division proteins into the Z ring. As the Z ring contraction leads to membrane closure, the cell divides to form two daughter cells (Osawa et al., 2008). The inhibition of the FtsZ by most plant oils is related to GTPase activity, FtsZ binding capacity, Z-ring assembly, and contraction. The majority of FtsZ inhibitory compounds in essential oils are phenylpropanoids and polyphenols. Cinnamaldehyde can bind to the T7 loop in the C-terminal region of the FtsZ monomer, which interferes with the formation of the Z ring and disrupts its morphology in vivo. The formation of the cell membrane is incomplete, and it is in a filamentous structure that is not completely divided (Domadia et al., 2007). By interacting with the GTPase binding cavity, curcumin enhanced the GTPase activity and interfered with the assembly and polymerization of the FtsZ, thereby shortening the steady-state duration of polymer assembly (Duggirala et al., 2014). Totarol is an bacteriostatic and fungistatic active ingredient derived from the plant Rhamnosus pinus. After its treatment, the cell GTPase activity and FtsZ polymerization are inhibited; the cells are filamentous, and the Z-ring assembly is misaligned (Jaiswal et al., 2007). Germarene and gemmarene D-4ol in pine needle essential oil can also bind to the hydrophobic cavity of the FtsZ (Anderson et al., 2012).

​Nucleic acid is the material in which genetic information is stored, copied, and transmitted. The expression of genetic material controls the synthesis and metabolism of cellular proteins. Therefore, nucleic acid and gene expression are also important antimicrobial pathways. Listeria monocytogenes were treated with lemongrass essential oil to observe their transcriptome response, and the virulence genes hly and inlj were downregulated in a dose-dependent manner, whereas the fatty acid biosynthesis gene accP was upregulated (Hadjilouka et al., 2017). Fennel essential oil downregulates genes related to ochratoxin A (Nowotarska et al., 2017) biosynthesis in A. niger, thereby reducing OTA levels, but no changes in fungal spore morphology were observed, so fennel essential oil may only inhibit OTA production by reducing the expression of virulence-related genes (El Khoury et al., 2016). The test of citrus essential oil on S. aureus also showed that the expression of cytotoxic genes (comC, comD, gtfB, gffC, and gbpB) was remarkably downregulated, and its main active components, namely, linalool and limonene, indirectly inhibited the production of glucans required for biofilm formation (Benzaid et al., 2021). Transcriptome analysis showed that the indirect exposure of essential oil could affect the expression of Penicillium rubens genes, and essential oil could inhibit the activity of P. rubens by affecting polysaccharide, carbohydrate, fatty acid, nucleotide, and nucleoside metabolism (Kisová et al., 2020).