**1. Introduction**

Rotting of fruits and vegetables have been a frequent and serious problem for thousands of years. Even in developed countries, the citrus yield loss reaches up to 25%, with developing countries facing almost double this yield loss of citrus crop. This yield loss is causing serious annual economic losses and creating hurdles for the development of the citrus fruits-related industry [1]. Citrus fruits (Family Rutaceae, subfamily Aurantioideae) are highly susceptible to decay caused by more than 20 postharvest fungal infections leading, to approximately half the crop wasted due to fungal diseases [2]. Among various fungal strains, *Penicillium* fungi cause serious plant diseases and damage to the citrus crop during storage, accounting for about 10–30% [3]. The *P. digitatum* infects the citrus fruits, causes green mold disease, and generates huge (60–80%) yield losses under ambient conditions [4]. In order to control such fungal disease-mediated yield losses, prochloraz, imazalil, and thiabendazole are extensively used as potential antifungal agents [5]. The literature reported serious health issues

aroused by excessive use of certain routine and synthetic antifungal agents. Over the past few decades, the intentions have been to ensure the hygiene and safety levels of fruit products along with promoting maximum biodegradability. In order to achieve this ambition, researchers are still trying to screen natural products with strong antifungal potential from plants. Moreover, it has also been tried to explore new preservation technologies that can replace synthetic fungicides. Compared with synthetic fungicides, natural antifungal substances have attained much attention in recent years [6–9].

Various essential oils refined from *Melaleuca alternifolia* have strong inhibitory e ffects on multiple fungal strains, particularly *P. italicum* Wehmer and *P. digitatum* Sacc., and hence can be used as natural preservatives in agricultural and food processing [10]. Previously we have reported that cinnamaldehyde contained in cinnamon has a good antifungal e ffect against *P. italicum* and clarifies the method for dynamic detection of the antifungal e ffect of cinnamaldehyde [11]. In addition, GC-MS and GC-FID techniques successfully separated the essential oils components *viz*. limonene (87.02%), linalyl acetate (53.76%) and linalool (39.74%) from peels, leaves and flowers of *Citrus aurantium* var amara, neroli oil appeared as the best fungistatic agent, which reduced the *P. digitatum* sporulation to 25 % at a 50 mg/mL concentration of oil [12].

Carvacrol (5-Isopropyl-2-methylphenol) is the major volatile oil constituent from Labiaceae family plants, such as oregano, mint, and thyme [13]. Carvacrol has many medicinal and edible applications, including antifungal, antimicrobial, anti-inflammatory, insecticidal, antitumor, and other e ffects [14]. Carvacrol inhibited the growth of *P. digitatum* and *P. italicum* in in vitro and in vivo experiments of lemon [15]. Another study in Morocco, reported that *Thymus leptobotrys* and *T. riatarum* have carvacrol contents of 76.94% and 32.24%, respectively, and the former species has the highest bioactivity among the tested plant species in that study. In addition, the study reported a complete inhibition of germination of the spore of *P. digitatum* and *P. italicum* at 500 μL/L [16]. The results of the antimicrobial activity assay showed that carvacrol, cinnamaldehyde, and *trans*-cinnamaldehyde significantly reduced the growth of *P. digitatum*; particularly, the carviniol appeared as the most e ffective remedy to control *P. digitatum* [17].

Metabolomics is an important part of systems biology, which is quite similar to genomics and proteomics [18]. Metabolomics is distinguished from other related sciences in terms if its ability to find the relative relationship of metabolites with physiological and pathological changes through quantitative analyses of metabolites with low molecular weight (1000) [19]. Recently, the 1H-NMR based metabolomics approach was used to reveal the antimicrobial mechanism, which generally includes amino acid biosynthesis and energy-associated metabolism [20,21]. Although carvacrol already been proved to have significant antifungal properties, the underlying mechanism is not clear yet. In the current study, we applied the 1H-NMR-based metabolomics approach to evaluate the antifungal potential of carvacrol on *P. digitatum* and explored the underlying antifungal mechanism. Moreover, how carvacrol-induced oxidative stress disturbs the energy production and amino acid metabolism in *P. digitatum* shall also be studied through metabolic profiles.

#### **2. Materials and Methods**
