It is widely known worldwide that global food demand is increasing as the world´s population grows, which is projected to reach 8.6 billion people in 2030 and 9.8 billion in 2050 (United Nations, 2017). On the one hand, cereal grains, such as rice, wheat and maize, are major staple foods grown in most of the world, and supply a major proportion of people’s energy and nutritional needs. In particular, rice is staple food for people all around the world, particularly in Asia, Latin America and parts of Africa. In 2020, cereals were the main crops to be globally produced with slightly less than one third of total crops (FAO, 2022). The foods that are predominantly carbohydrate are important because they form the basis of most diets, especially for the poorest people in the developing world. In developing countries, these foods generally supply 70%, or more, of the population’s energy intake. On the other hand, tomato (Solanum lycopersicum L.) contributes considerably to human nutrition. It is a source of mainly carbs, vitamin C, fiber, vitamin k, among other components (United States Department of Agriculture (USDA), 2018). This crop is one of the most important horticultural crops in the world. According to FAOSTAT (2022), Spain ranked seventh as a world producer in 2021 with 4754.380 million metric tons, where tomato was grown on 56.11 ha. In Argentina, this crop is cultivated under greenhouse and field conditions. Argentina is another of the world’s largest producers, where tomato growing annually occupies around 15000 ha (Malbrán et al., 2020).

It is important to consider that hunger is still on the rise with almost 770 million people undernourished in 2021, which is an increase of 150 million since 2019. Agriculture and food production systems affect food availability and affordability, as well as diet quality and diversity (Food and Agriculture Organization of the United Nations (FAO), 2016). In line with all this, several scientists have been working hard in recent years to maximize food production and to reduce pre- and postharvest crop losses. The world’s production of primary crops has increased 52% between 2000 and 2020, which is mostly attributable to better crop management technology, among other factors (FAO, 2022).

Fungal pathogens are biotic adversities that cause major yield losses in agriculture, and reduce food quality and production worldwide (Almeida et al., 2019). Losses of between 35-55% caused by postharvest diseases of fruit and vegetables have been reported, with more significant values in developing countries (Verdeguer et al., 2020). Another important issue to highlight is that fungal proliferation in stored food and cereal not only causes significant harvest quality and yield losses (rancid odors and flavors), but some fungal species also produce secondary potentially toxins like aflatoxins and fumonosins, which pose human health problems (Soliman and Badeaa, 2002). Of the 17 Sustainable Development Goals (SDGs) agreed by world leaders in 2015, some particularly relevant ones are zero hunger and responsible consumption and production by considering not only higher production levels, but also reducing food losses throughout production and supply chains, including postharvest losses and better environmental care (Naciones Unidas, 2023; United Nations, 2023).

In recent years, society has tended to reduce chemical products like synthetic fungicides to control the phytopathogenic fungi of crops and postharvest diseases, which has led researchers to evaluate and develop new safer alternatives for the environment, and also for human and animal health. Aromatic plants and their essential oils (EOs) have been widely known since ancient times. They are used in foods to enhance flavor and organoleptic properties, and in the pharmaceutical industry for their beneficial effects on health. Nowadays however, some of the main interests of EOs are disease control and food preservation. Thus according to several reports, EOs have been proposed as an alternative to synthetic fungicides with promising results (Soliman and Badeaa, 2002; Santamarina et al., 2017; Verdeguer et al., 2020; Giménez-Santamarina et al., 2022).

The Mediterranean basin is especially rich in species of aromatic plants and is a center of their diversification. Species of the genus Thymus (Lamiaceae) are widely distributed in the Iberian Peninsula, and they form a taxonomically complex group of aromatic species. These plants are traditionally known for their medicinal and culinary purposes, and stand out for their antiseptic, antispasmodic and antitussive medicinal properties (Pina-Vaz et al., 2004; Martínez Marciales et al., 2023). Different Thymus species are used mainly as flavorings for foods, such as creams, sauces, vegetables, meat and fish. However, several studies have pointed out their potential as an antimicrobial agent and food preservative for cereals, grains, legumes, fruit and vegetables (Yakoubi et al., 2014; Pandey et al., 2017; Moukhles et al., 2022).

In the last few years, studies about the applications of Thymus EOs and their components have grown in number and these EOs been reported as effective against fungal phytopathogens (Santamarina et al., 2017; Costa Junior, 2018; Sapper et al., 2019; Aslam et al., 2022).

Additionally, it has been demonstrated that the biological activity of EOs depends on their chemical composition, which is influenced by factors like plant genotype, geographical origin, and environmental and agronomic conditions (Verdeguer et al., 2020). According to several authors, Thymus EOs present a widespread chemical polymorphism (Pina-Vaz et al., 2004; Rota et al., 2008). Thyme EOs are a complex mixture of different components that depend on plant species, of which the main ones are thymol, p-cymene, Ɣ-terpinene, carvacrol, (E)-β-cariophelene and linalool. They possess therapeutic and antimicrobial properties, namely antifungal, antioxidant, anti-inflammatory, etc (Vassiliou et al., 2023).

Currently, abundant information is available on the potential of T. vulgaris EOs against phytopathogenic and postharvest fungi, and also against: Alternaria citri, the causal agent of black rot in orange; Fusarium oxysporum f. sp. radicis-lycopersici, Phytophthora infestans and Rhizoctonia solani; Penicillium expansum and Penicillium crustosum, which are associated with the blue mold disease of grapes, among others (Ramezaniana et al., 2016; Ghuffar et al., 2021; Aksit et al., 2022).

Some studies have demonstrated that T. serpyllum EOs are able to inhibit the growth of Verticillium dahliae during in vitro tests (Arslan and Dervis, 2010). Sokolić-Mihalak et al. have revealed the inhibitory in vitro effect of T. serpyllum EOs against Aspergillus ochraceus, A. carbonarius and A. niger (Sokolić-Mihalak et al., 2012). Interestingly, others authors have proven the potential of the EOs of T. serpyllum for use in food preservation due to the dominant presence of thymol and carvacrol (Nedorostova et al., 2009; Galovičová et al., 2021).

Research into the T. piperella EO is very scarce and is related to the inhibition of food spoilage by fungi, with some inhibition on Aspergillus niger (Ruiz-Navajas et al., 2013).

The ability of EOs to protect foods against not only pathogenic and spoilage microorganisms, but also oxidation, has been reported by several researchers (Alves-Silva et al., 2013; Ruiz-Navajas et al., 2013). To achieve effective antimicrobial activity in direct food applications, high concentrations of EOs are generally needed, which might impact inappropriate flavors and odors in products (Seydim and Sarikus, 2006). To avoid this problem, EOs can be incorporated into bioactive film coatings, which would allow the compound to be fixed and retained on the product surface to, thus, increase its effectiveness. The major compounds in these coatings are biodegradable polymers and a relatively small amount of EOs can be used. Consequently, the application costs of EOs and/or other problems, such as the intense aroma and potential toxicity, could be minimized (Sánchez-González et al., 2010).

The objectives of this work were to: i) determine the chemical composition of the EOs of Thymus serpyllum and Thymus piperella chemotypes 1 and 2; ii) investigate the antifungal potential of EOs under “in vitro” conditions against Alternaria alternata, Bipolaris spicifera, Curvularia hawaiiensis, Fusarium oxysporum fsp. lycopersici, Penicillium italicum and Botryotinia fuckeliana; iii) evaluate a natural biofilm with the T. serpyllum EO, which we created as an antifungal product study, for rice grain (bomba Valencia Protected Designation of Origin of Valencian Rice), and for cherry tomato fruit conservation.

EOs are widely known for their several biological activities, such as bactericides, fungicides, insecticides, and also for their culinary and medicinal uses. Hence they have applications in food and pharmaceutical industries (Plaza et al., 2004; Jahani et al., 2020; Raveau et al., 2020). Of Lamiaceae family members, the genus Thymus comprises around 350 species and 36 subspecies with wild species and cultivated plants, and the Iberian Peninsula in general, and the Mediterranean Basin in particular, are centers of diversification. Most indigenous species grow all around the Mediterranean region (Zeljković and Maksimović, 2015; Vassiliou et al., 2023). The Thymus EOs are widely known for their high content of bioactive compounds, such as phenolic acids, flavonoids and terpenes, and for the diversity of chemotypes (Ruiz-Navajas et al., 2013; Yakoubi et al., 2014; Pandey et al., 2017; Moukhles et al., 2022). According to previous studies, their properties depend on their biologically composition (Vassiliou et al., 2023).

Thymol and carvacrol are two phenolic monoterpenes cited as the main components of the EOs of some Laminaceae and are produced by aromatic plants as a chemical defense mechanism upon exposure to biotic (herbivores, pathogens, pests) and abiotic factors as environmental stresses (Pulido et al., 2012; Naghdi Badi et al., 2017). Zeljović and Maksimović investigated 45 Thymus taxa from the Balkan Peninsula for their composition, where the aromatic thymol, carvacrol and p-cymene chemotypes were the most abundant (Zeljković and Maksimović, 2015).

The T. vulgaris EO is the most studied in the species belonging to the genus Thymus. These oils are rich in thymol and carvacrol, and are known to be effective against the fungi that infect humans, and also against phytopathogens (Affesa et al., 2022). According to several authors, high thymol and carvacrol concentrations are responsible for antimicrobial and antifungal properties (Yeddes et al., 2018; Kovačević et al., 2021).

Preliminary reports have demonstrated the antifungal activity of the EOs obtained from the plant species belonging to the genus Thymus against phytopathogens, such as P. expansum, Botrytis cinerea, Monilinia fructicola and Rhizopus oryzae, and cause postharvest fruit diseases of which thymol and carvacrol are their major components (Venturini et al., 2002; Plaza et al., 2004). Arora et al. reported that the T. vulgaris EOs showed 100% MGI against the fungi Colletotrichum capsici, Pythium aphanidermatum and Fusarium oxysporum, which cause significant pepper fruit Capsicum anuum losses (Arora et al., 2023).

The effect of the T. zygis EO has been demonstrated as a preservative in stored products by reducing postharvest fungi development and extending products’ commercial shelf lives (Sapper et al., 2019). Applying EOs to food as coatings helps to maintain the quality of products of natural origins, such as fruit and vegetables, by prolonging their shelf lives, and also with products that are environmentally friendly and safe for humans (Verdeguer et al., 2020).

Our study found that the major components of the (commercial) T. serpyllum EO were thymol (21.5%) and carvacrol (18.5%), followed by geraniol (12.4%) and p-cymene (12.3%), of 52 compounds. We demonstrated the fungistatic and fungicidal potentials of these EOs against A. alternata, B. spicifera, C. hawaiiensis, FOL, P. italicum and B. fuckeliana. Particularly, C. hawaiiensis was totally inhibited (100%), as was FOL (96%) at higher doses (300 µl/mL). Thus we highlight the effect of this EO against all the pathogens evaluated according to the obtained MGI values. Interestingly, several authors attribute their properties for both the pharmaceutical industry and food conservation purposes to thymol and carvacrol contents (Sokolić-Mihalak et al., 2012; Naghdi Badi et al., 2017).

Our results agree with preliminary reports, albeit with some variation in the reported percentages, that the major compounds of the T. serpyllum EO from plants that grow in the Slovak region were thymol (18.8%), carvacrol (17.4%), o-cymene (15.4%) and geraniol (10.7%) (Galovičová et al., 2021). According to their antimicrobial and antifungal studies, these authors suggested that the T. serpyllum EO could be used for storing root vegetables, and also as a Penicillium inhibitor in bread. Other studies have revealed that the dominant component of the T. serpyllum EO extracted from plants harvested in the flowering stage in Turkey were p-cymene, thymol and Ɣ-terpinene, which has a potential antifungal effect against Verticillium dahliae (Arslan and Dervis, 2010). Additionally, the Thymus serpyllum EO has a strong inhibitory effect against the growth and mycotoxin production of Aspergillus ochraceus, A. carbonarius and A. niger, which spoil food products and produce ochratoxin A (OTA), an important mycotoxin (Sokolić-Mihalak et al., 2012). These authors noted a significant inhibitory effect at lower doses (100 μL mL^-1) than those we used (150 and 300 μg mL^-1). However, we highlight in their tests that the EO was dissolved in 96% ethanol, which alone has a fungicidal effect.

Very few studies have been carried out with T. piperella. The few works that exist focused on studies related mainly to food conservation to reduce the oxidation and microbiological degradation caused by bacterial and fungi, which produce rancid odors and flavors, and come with widely known human and animal health concerns (Ruiz-Navajas et al., 2012; 2013). These authors reported the inhibition of fungal foodborne A. alternata, A. niger, A. flavus, P. chrysogenum, Mucor racemosus and M. circinelloides.

In our work, the T. piperella EOs were obtained from plants growing in two different locations in the Valencia region of Spain in the flowering stage. According to our results, we were able to determine two chemotypes. To Tp1, we assigned the chemotype carvacrol and chemotype thymol to Tp2. Our results agree with other authors, who have demonstrated that the major component of the T. piperella EO were monoterpenes, with phenolic compounds like thymol (Llorens-Molina et al., 2022). So previous studies about the chemical composition of 31 populations of T. piperella have demonstrated that an intraspecific variation of EOs compositions can determine three chemotypes according to major components: chemotype A p-cymene-carvacrol-Ɣ-terpinene; chemotype B: p-cymene-thymol, and chemotype C: p-cymene-carvacrol (Llorens-Molina et al., 2022). Several studies have proven the intraspecific variability of T. piperella populations, which determine different chemotypes (Boira and Blanquer, 1998; Llorens-Molina et al., 2022).

In our work, the major component of EOs were thymol and carvacrol, with a high potential against the five evaluated phytopathogens. All the studied fungi were more sensitive to Thymus serpyllum, whose main components are thymol and carvacrol. Therefore, we suggest that it may be related to the synergistic effects of its components. Both T. serpyllum and T. piperella had excellent results against Curvularia hawaiiensis, Bipolaris spicifera and Fusarium oxysporum. T. serpyllum grows in central and northern Europe, while T. piperella grows essentially in the Mediterranean basin. In addition, these EOs could be incorporated into bioactive films by retaining active compounds and to also increase their antifungal potential.

In this work, the EOs of T. serpyllum and the two of T. piperella (Tp1 and Tp2) reduced the fungal growth of all the evaluated phytopathogens. However, we emphasize the potential of the T. serpyllum EO because all the fungal pathogens were very sensitive, which markedly reduced growth, and even totally against C. hawaiiensis, but also with very good results against Bipolaris spicifera and Fusarium oxysporum. The natural biofilm created from Thymus serpyllum proved extremely effective in rice storage compared to the losses caused by the fungi Curvularia hawaiiensis and Bipolaris spicifera. It is also capable of controlling the Fusarium oxysporum fungus in cherry tomatoes during the postharvest and, thus, prolongs their shelf life in both warehouses and stores.

Considering our results and the fact that EOs have different advantages versus synthetic fungicides, for instance, they are not toxic to humans and animals, and they are biodegradable with short lives, we suggest that these natural products formulated as biofilms could be safely applied for food conversation purposes to replace synthetic products.

A natural film was obtained at a low Thymus serpyllum concentration that is not harmful for human health or the environment.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

JR: Conceptualization, Data curation, Investigation, Methodology, Resources, Writing – review & editing. FS-F: Data curation, Formal analysis, Investigation, Writing – review & editing. JL-M: Data curation, Formal analysis, Investigation, Methodology, Resources, Writing – review & editing. SL: Writing – review & editing. MPS: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing.