Eucalyptus camaldulensis , Citrus aurantium , and Citrus sinensis Essential Oils as Antifungal Activity against Aspergillus ﬂavus , Aspergillus niger , Aspergillus terreus , and Fusarium culmorum

: Several molds are able to colonize wood and many building products or solid wood causing losses for their valuable uses. Essential oils (EOs) from aromatic plants can be used as an ecofriendly biofungicide against the growth of several molds. EOs from Eucalyptus camaldulensis , Citrus aurantium , and C. sinensis have a broad-spectrum antimicrobial activity. EOs from of E. camaldulensis air-dried aerial parts, C. aurantium leaf and C. sinensis peel, and their combinations (1:1 v / v ) were evaluated for their antifungal activity against the growth of four common mold fungi ( Aspergillus ﬂavus , A. niger , A. terreus , and Fusarium culmorum ). The chemical compositions of the EOs were analyzed with GC / MS. The main compounds in EO from E. camaldulensis were spathulenol (20.84%), eucalyptol (12.01%), and sabinene (9.73%); in C. aurantium were linalyl acetate (42.29%), and linalool (29.76%); and in C. sinensis were D -limonene (73.4%) and γ -terpinene (22.6%). At 50 µ L / mL, C. sinensis EO showed the highest fungal mycilial growth inhibition (FMGI) percentage (86.66%) against A. ﬂavus . C. sinensis , E. camaldulensis , and E. camaldulensis / C. sinensis showed FMGI values of 96%, 91.66%, and 75.66% respectively, against A. niger . EOs from C. aurantium and C. sinensis showed potent activity against A. terreus (100% FMGI), while C. aurantium / E. camaldulensis and E. camaldulensis / C. sinensis showed FMGI values of 74.33% and 70.66%, respectively. Potent activity against F. culmorum with 100% was observed as the application of E. camaldulensis and C. sinensis EOs at 50 µ L / mL, while E. camaldulensis / C. sinensis (50 µ L / mL) showed FMGI value of 65.66%. The results suggest using the EOs and their combinations from E. camaldulensis , C. aurantium , and C. sinensis as a biofungicide against molds. The potent properties of EOs o ﬀ er the possibility of using them as eco-friendly, safe, and cost-e ﬀ ective antimicrobials for molds that could cause discoloration of the wood packaging or food spoilage.


Hydrodistillation Method for Isolation of Essential Oils
Eucalyptus camaldulensis air-dried aerial parts, Citrus aurantium green old leaves, and C. sinensis fresh peels were collected during January 2019, from Alexandria, Egypt. The raw materials were transferred to small pieces then approximate 100 g from each of them were inserted in a flask (2 L capacity) that contained 1500 mL of distilled water (DW). The flask with its contents was heated under refluxing in terms to hydrodistillate the material and extract the essential oil (EO) using a Clevenger apparatus for 3 h [6]. The collected EOs were stored in brown glass bottles in a refrigerator at 4 • C. Oils were prepared in equal ratio (1:1 v/v) [49] as presented in Table 1.

GC-MS Analysis of Essential Oils and Their Combinations
The chemical constituents of the EOs from E. camaldulensis aerial parts, C. aurantium leaves, and C. sinensis peels were performed using GC-TSQ Quantum mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). The conditions of the separation and identification of the EOs can be found in the previous works [12,[50][51][52][53].
The EOs and their combinations were dissolved in a dimethyl sulfoxide (DMSO 10%), Tween 40, and DW mixture in the ration of 1:0.5:1. The dissolved EO and prepared at the concentration of 50, 25, and 12.5 µL/mL were added to warm PDA medium (40 to 45 • C), before immediately pouring into 9 cm Petri dishes. The standard antibiotic Sertaconazol (3 g/L) was used as a control, the dilution mixture were used as positive and negative controls, respectively. From a 7-day-old colony, the fungus with discs of 9 mm diameter was transferred to the center of the treated PDA plates and controls. All the plates were incubated at 26 ± 1 • C for 14 days. All the tested concentrations as well as positive and negative controls were measured in triplicate.
After the fungal growth reached the edges in the negative control plates, the percentage of fungal mycelial growth inhibition (FMGI) was calculated using the following equation [56]; FMGI (%) = [(DC − DT)/DC] × 100, where DC and DT represent the average diameters of the fungal colony of control and treatment, respectively. The minimum inhibitory concentrations (MICs) of the EOs were prepared at concentrations of 4-50 µL/mL and were assessed using the broth dilution method according to CLSI [55].

Statistical Analysis
FMGI (%) values of the fungi diameter growth were statistically analyzed based on two factors (EO type or EO mixture and EO concentration) using analysis of variance in SAS system [57]. The differences between the mean of each treatment were recorded using LSD 0.05 and compared with positive control (Sertaconazol 3 g/L) and negative control (DMSO 10%).

Fungal Inhibition by Visual Observation
The visual observations of the fungal inhibition growth are shown in Figures 2-5 for Aspergillus flavus, A. niger, A. terreus, and Fusarium culmorum, respectively, as affected by the tested essential oils (EOs) from C. aurantium leaves, E. camaldulensis aerial parts, and C. sinensis peels, as well as their equal combinations. With increasing the concentration of EOs or their combinations, the fungal mycelial growth inhibition (FMGI) increased. No FMGI was shown in negative control (DMSO 10%) plates, while the positive control (Sertaconazol 3 g/L) of all the studied fungi showed good FMGI.

Fungal Inhibition by Visual Observation
The visual observations of the fungal inhibition growth are shown in Figures 2-5 for Aspergillus flavus, A. niger, A. terreus, and Fusarium culmorum, respectively, as affected by the tested essential oils (EOs) from C. aurantium leaves, E. camaldulensis aerial parts, and C. sinensis peels, as well as their equal combinations. With increasing the concentration of EOs or their combinations, the fungal mycelial growth inhibition (FMGI) increased. No FMGI was shown in negative control (DMSO 10%) plates, while the positive control (Sertaconazol 3 g/L) of all the studied fungi showed good FMGI.

Antifungal Activity of Essential Oils and Their Combinations In Vitro
The antifungal activity of the EOs and their combinations are presented in Table 5. C. sinensis peel EO showed the highest FMGI percentage of 86.66% against the growth of A. flavus, followed by E. camaldulensis EO (74.33%) at the concentration of 50 µL/mL. The bioactivity of EOs was decreased for all the combination treatments, but it was reached 64.66% as the EO combination of E. camaldulensis/ C. sinensis. However, these values are lower than of FMGI value from the positive treatment (88.66%). All the EO combinations showed limit impact on the growth of A. flavus.
It is worth noting that the potent toxicity was observed against the growth of A. terreus with 100% FMGI percentage with the application of EOs from C. aurantium and C. sinensis. These values were higher from those obtained from the positive control (91%), while the EOs from E. camaldulensis, C. aurantium/E. camaldulensis, and E. camaldulensis/C. sinensis observed good FMGI values of 79, 74.33, and 70.66%, respectively, against the growth of A. terreus. E. camaldulensis and C. sinensis EOs showed potent activity with 100% FMGI against the growth of F. culmorum. EOs from C. aurantium (50 µL/mL), C. sinensis (25 µL/mL), and E. camaldulensis/C. sinensis (50 µL/mL) were shown FMGI values of 65.66, 66.33, and 65.66%, respectively, which lower than the value from positive control (89.66%). Table 6 presents the minimum inhibitory concentrations for the EOs ranged between 8 and 40 µL/mL, 6 and 8 µL/mL, 6 and 12 µL/mL, and 6 and 40 µL/mL against the growth of Aspergillus flavus, A. niger, A. terreus, and Fusarium culmorum, respectively, while it was 8 µL/mL, 6 µL/mL, 8 µL/mL, and 6 µL/mL as measured for Sertaconazol for the same order of fungi.
Limonene, the main compound in peels of Citrus species, with percentage of 96.62% and other compounds of β-pinene, β-myrcene, α-pinene, and citral (Z and E) were identified in Citrus sinensis var. Valencia peel EO with good antifungal activity against of A. flavus [59]. The chemical composition peel EOs of C. sinensis from Uganda and Rwanda had limonene ranged from 87.9 to 92.5%, with small amounts of myrcene, α-pinene, and linalool [58]. Other study showed that the C. sinensis peel EO had limonene, β-myrcene, decanal, β-pinene, and linalool, as major compounds with good antioxidant activity [70]. Limonene in the present study reached 73.4% in C. sinensis, while in previous investigation it was 77.49% the peel oil of sweet orange followed by myrcene 6.27% [71,72]. Limonene (80.9%) and β-myrcene (4.19%) were the main constituents in fresh peel EO of C. sinensis [73]. Limonene fount in percentage of 87.9% and 92.5% from C. sinensis peels of Uganda and Rwanda, respectively [58]. C. sinensis peel EO with its main compound of limonene (98.54%) was observed potential of inhibition of mycelial growth (63.46%) of Sclerotinia sclerotiorum at the oil dose 300 µL [74]. D-limonene is highly useful in agriculture as antibacterial agent against economic phyto-pathogenic bacteria Ralstonia solanacearum isolated from potato as well as for insect repellent [12,75,76]. Moreover, in the pure form, monoterpene was reported to exhibit promising antifungal activity against Aspergillus niger, Fusarium oxysporum, Phytophthora digitatum, F. verticillioides, R. solani, and S. sclerotiorum [77,78].
It was reported that the combination of EOs could be probably resulted in a more effective [83][84][85]. E. globulus and Zingiber officinalis EOs in combination showed considerable activity against Giardia lamblia cysts [85]. C. maxima and C. sinensis EOs, with their main compounds dl-limonene, alone or in combination (1:1), showed potential fungitoxic spectrum against food-contaminating molds including A. flavus and completely inhibited aflatoxin B 1 [49]. In addition, EOs mixture of thyme and oregano exhibited potent antifungal activity [86]. The EO mixture of O. vulgare/Rosmarinus officinalis observed synergism effects against some microbes [87]. Checkerboard EOs of Lippia multiflora/Mentha piperita showed broad-spectrum synergism antibacterial activity [88]. Mixture EOs of S. aromaticum/R. officinalis showed synergism effects against Staphylococcus epidermidis, S aureus, B. subtilis, E. coli, Proteus vulgaris, P. aeruginosa, and Candida albicans and antagonism effects against A. niger [89].
For the generally accepted mechanisms of antimicrobial interaction that produce synergism, it was found that the combinations of EOs led to inhibition of the common biochemical pathway with inhibition of the protective enzymes, with subsequent use of cell wall-active agents to enhance the uptake of other antimicrobials [90][91][92][93][94][95].

Conclusions
This study revealed that the essential oils from Eucalyptus camaldulensis aerial parts, Citrus aurantium leaves, and C. sinensis peels singly and/or in combination showed qualitative differences in their chemical compositions. The results demonstrate that the essential oils possessed promising antifungal activity against Aspergillus flavus, A. niger, A. terreus, and Fusarium culmorum. Therefore, these essential oils could be considered for use as ecofriendly biofungicides to deter the growth of molds in food packaging or wood containers; however, for food preservatives, the toxicity test should be run before use is approved.