Mass Spectroscopic Analysis, MNDO Quantum Chemical Studies and Antifungal Activity of Essential and Recovered Oil Constituents of Lemon-Scented Gum against Three Common Molds

: The present study described the possibility of using wood-treated oil-fungicide of lemon-scented gum ( Corymbia citriodora ) from newly emerged leaves and unripened fruits against the infestation of Fusarium culmorum , Rhizoctonia solani and Penicillium chrysogenum . Air-dried wood samples of Melia azedarach were treated with the extracted oils from leaves and unripened fruits from C. citriodora . The main chemical constituents identified in the essential oil (EO) from leaves were citronellal (55.31%), citronellol (21.03%) and isopulegol (10.79%), while in unripened fruits were α -pinene (17.86%), eudesmol (13.9%), limonene (9.19%), γ -terpinen (8.21%), and guaiol (7.88%). For recovered oils (ROs), the major components from leaves were D -limonene (70.23%), γ terpinene (13.58%), β -pinene (2.40%) and isopregol (2.23%), while, 4-terpineol (21.35%), cis - β -terpineol, (19.33%), D -limonene (14.75%), and γ -terpinene (7.42%) represented the main components in fruits. EOs from leaves and fruits at the amounts of 100, 50 and 25 µL showed the highest inhibition percentage (IP) of 100% against F. culmorum and P. chrysogenum compared to control treatment, while at the amounts of 100, and 50 µL showed 100% IP of R. solani . Wood treated with ROs from leaves and fruits showed IPs of 96.66% and 93.33%, respectively, against the growth of R. solani . The mass spectra of the main components of C. citriodora leaves and fruits’ EOs have been recorded in electron ionization mode at 70 eV and fragmentation has been reported and discussed. On the other hand, different quantum parameters such as the heat of formation, ionization energy total energy, binding energy, electronic energy and dipole moment using the modified neglect of diatomic overlap (MNDO) semi-empirical method have been calculated.

A number of studies have demonstrated the antimicrobial properties of Eucalyptus essential oils (EOs) and their antimicrobial effects against a wide range of microorganisms have been studied. These species showed potential antifungal and antibacterial activities, especially E. citriodora (lemonscented gum) EO, which has been shown to have a wide spectrum of antifungal activity [10][11][12][13][14].
Several studies have reported the antifungal and antibacterial activities of the EOs from C. citriodora [10,12,14,17,29,30]. For example, strong antifungal activity against Macrophomina phaseolina, Colletotrichum lindemuthianum, Fusarium oxysporum f. sp. lycopersici, Helminthosporium oryzae, Alternaria triticina, Rhizoctonia solani, and Alternaria solani was found with the application of C. citriodora oil compared with Mancozeb [13]. Filter paper disks impregnated with C. citriodora leaf EO at 10 µL showed good activity against E. coli and S. aureus Elaissi [30]. With the presence of sabinene and terpinen-4-ol as main compounds in the essential oil of C. citriodora, the EO displayed potent antifungal activity against Trichophyton rubrum [31].
Recovery oil (RO) using n-hexane solvent from the distillate of Matricaria chamomilla fresh flowers after obtaining the EOs were reported to have potential antifungal activity against A. niger and A. terreus [6]. Therefore, the present study firstly aimed to extract the essential and recovery oils from C. citriodora leaves and unripened fruits; and secondly to explore their bioactivity as woodbiofungicides; and, finally, modified neglect of diatomic overlap (MNDO) quantum chemical studies have been reported.

Oil Extraction
C. citriodora plant materials (leaves and unripened fruits) were cut into small pieces then 100 g from each plant were hydrodistilled using the Clevenger apparatus for 3 h [32]. After collecting the essential oils, the recovered oils dissolved in water from the hydrodistillation were isolated using nhexane solvent. The n-hexane fraction or layer was separated using a funnel separator [6]. The essential and n-hexane oils were stored in glass tubes in the refrigerator at 4 °C until chemical and antifungal analyses.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of the Oils
Essential oils and n-hexane recovered oils were analyzed for their chemical composition using Focus GC-DSQ (Gas Chromatography-Dual Stage Quadrupole) Mass Spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m x 0.25 mm x 0.25 µm film thickness, Agilent, Palo Alto, CA, USA) apparatus at Atomic and Molecular Physics Unit, Experimental Nuclear Physics Department, Nuclear Research Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt. The column oven temperatures, injection properties, compound separation and identification can be found in previous works [3,6,33].

Antifungal Activity of Wood Treated with Oils
Three common molds namely Fusarium culmorum, Rhizoctonia solani and Penicillium chrysogenum with their accession numbers of MH352452, MH352450, and MH352451, respectively, were used for the bioassay [3,7,9]. Oils were applied at the amounts of 0, 25, 50, and 100 µL. Air-dried wood samples of Melia azedarach were prepared with the approximate dimension of 0.5 × 1 × 2 cm then autoclaved at 121 °C for 20 min and left to cool. Nine wood samples were treated with each concentration (three for each fungus) from each oil. Wood samples without oil treatments were used as a control. The antifungal effect of treated wood was measured following our previous works with minor modification [3,[34][35][36][37]

Statistical Analysis
Data of the antifungal activity were statistically analyzed with three factors (plant part, type of oil and the concentration) using analysis of variance, Statistical Analysis Software (SAS) system [38]. The differences among the mean of treatments were recorded using Fisher's Least Significant Difference LSD0.05.

Mass Spectrometric Investigations of the Main Components of Corymbia Citriodora Leaves and Fruits' Essential Iils
The 70 eV mass spectra of the major constituents of C. citriodora leaf EO are recorded and discussed as shown in Figure 1. The mass spectrum (MS) of the peak at retention time (RT) 13.63 min (Figure 1a) represent the citronellal component suggesting its molecular formula C10H18O ( Table 1). The molecular ion peak (MIP) was observed at m/z 154 with relative intensity (RI) = 5% and the peak at 69 (RI = 100%) representing the base peak (BP). Fragment ion (FI) of m/z 136 (10%), 95 (60%), 84 (20%) and 55 (60%) were also reported.
The MS of the peak at RT 15.77 min (Figure 1c) represent isopulegol component suggesting its molecular formula C10H18O ( Table 1). The MIP was observed at m/z 154 with RI = 5% and the peak at 67 (RI = 100%) representing the BP. Significant FI with m/z 121 (56%), 95 (57%), 84 (60%) and 55 (72%) were also observed.   The 70 eV mass spectra of the major constituents of C. citriodora fruits' EO are recorded and discussed as shown in Figure 3 and the chemical composition are shown in Table 2.
The MS of the peak at RT 3.47 min (Figure 3a) represent α-pinene component suggesting its molecular formula is C10H16 ( Table 2). The MIP was observed at m/z 136 with RI = 10% and the peak at 93 (RI = 100%) represent the BP. Fragment with m/z 77 (40%) was observed as other significant on.

Chemical Composition of Corymbia Citriodora Leaves and Fruits' Recovery Oils
The major components of the C. citriodora leaf recovery oils (RO, Table 3

Computation Method
The geometry of the studied molecules has been optimized based on semi-empirical calculations, using the molecular modeling program Hyperchem7.5 (W.Thiel 2003, HyperChemTM, Release 7.5 Pro 2002). Semi-empirical calculations were carried out using the routine MNDO and Polak-Ribiere conjugated gradient algorithm. For the optimized structure of the neutral and cation states, geometry optimization mode was carried out to give the molecular properties including heat of formations, total energy, binding energy, electronic energy and nuclear energy and dipole moment [40]. From the calculated data of the studied compounds (Table 5), values were obtained for heat of formation, ionization, total, binding, electronic energies and dipole moment. These thermochemical data are necessary in the description of the conformational properties of the studied molecules [41]. Table 5. Thermodynamic data of the studied molecules calculated within the modified neglect of diatomic overlap (MNDO) framework. From the calculated data of the studied molecule (Table 5), one can observe that the negative values of the heat of formations ∆F(M) and total energy for group 1 (citronellol , isopulegol, eudesmol guaiol, and isopregol) neutral molecules have negative values that mean these molecules are stable and the citronellol molecule is the most stable. This is due to the presence of the OH group in their structures, while group 2 α-pinene, limonene and γ-terpinene have the positive values of heat of formations. From these values the second group is relatively less stable than the first group which has OH group in their structures. This is confirmed by the values of dipole moment, hence the first group has approximately the same dipole moment (1.401, 1.374, 1.353, 1.447 and 1.227) in comparison with the second group (0.113, 0.122 and 0.028).

In Vitro Visual Observations of Dual Fungal Growth Against Oil-Treated Wood
To test the antifungal properties of essential (EO) and recovered (RO) oils from leaves and fruits of C. citriodora, oil-treated wood were bio-assayed against the growth of three fungi (F. culmorum, R. solani and P. chrysogenum) compared to control treatments in Figure 6. Nearly no growth of F. culmorum, R. solani and P. chrysogenum were found over wood treated with C. citriodora leaves and fruits oils after 14 days from incubation. On the other hand, the treated wood with EOs showed complete inhibition to the growth of F. culmorum and P. chrysogenum at all the oil amounts used (100, 50 and 25 µL), and also, at the amount of 100 and 50 µL of both oils, no growth of R. solani was observed. By visual observation and compared to control treatment, nearly no inhibition was found around the treated wood with ROs against the growth of P. chrysogenum but, also, no growth was observed over the treated wood samples. The extract from the unripened fruit prevents surrounding fungal growth in comparison to the extract from the leaf. Also, when little growth was observed it differed in appearance but it was still stopped by the ROs. Figure 6. In vitro antifungal bioassay of treated-wood with (1,2,3) C. citriodora leaf recovered oil; (4,5,6) C. citriodora fruit recovered oil; (7,8,9) C. citriodora leaf essential oil and (10,11,12) C. citriodora fruit essential oil. (A) Fusarium culmorum, (B) Penicillium chrysogenum, (C) Rhizoctonia solani.

Antifungal Activity of the Oils
Overall, leaves and fruits of C. citriodora showed the highest activity against F. culmorum and P. chrysogenum (Figure 7a). EOs were observed much higher activity against the studied fungi than ROs (Figure 7b). In addition, with increasing the oil amount, the activity was increased compared to the control (Figure 7c). The antifungal activity values of treated wood with C. citriodora leaf and fruits' EOs at the amounts of 100, 50 and 25 µL in Table 6 show that the highest inhibition percentage (IP) of 100% was observed against F. culmorum and P. chrysogenum compared to the control treatment. The treated wood with both EOs at the amounts of 100, and 50 µL showed 100% IP of R. solani.
The treated wood with ROs from leaves and fruits observed less activity against the growth of F. culmorum and P. chrysogenum, where IP reached 46.66% against F. culmorum on wood treated with C. citriodora leaf RO in the amount of 100 µL. Also, IP showed 60% against P. chrysogenum with wood treated at 100 µL of C. citriodora fruit RO. The ROs from leaves and fruits showed IPs of 96.66% and 93.33% against the growth of R. solani in the oil amount of 100 µL.

Discussion
In the present study, gas chromatography-mass spectrometry (GC-MS) with some calculations that were reported in the computation method was used for identification of the phytocompounds in EO and RO from C. citriodora leaves and unripe fruits [43][44][45]. For example, the fragmentation pathway of some identified main compounds such as D-limonene has been reported and discussed by Abd El-kareem et al. [42].
E. citriodora EO also inhibits the growth of phyto-and post-harvest pathogens [12,13,53], and its antifungal activity is attributed to citronellal, the major volatile constituent of this EO [12]. Significant inhibition of growth of Rhizoctonia solani was observed in Citronella (83.53%), and Lemon-tulsi (70.39%), Eucalyptus (68.63%), Pepper Mint (55.69%), and Patchauli (52.75%) which also effectively reduced the growth of the fungus [54]. E. citriodora and its major constituent citronellal was effective against rice pathogen R. solani and fully inhibited growth by the minimum concentrations [55]. Also, the synergism that occurred between citronellal and linalool showed strong antifungal activity [56]. Recently, the EO from C. citriodora leaves which contain α-citronellal (56.55%), α-citronellol (14.89%), and citronellol acetate (13.04%) was found to be highly toxic to the bacterial pathogen Ralstonia solanacearum phylotype II, the causal agent of brown rot disease [57]. The recovered compounds from hydrodistillation selected Lamiaceae species showed good antiradical and antioxidant activity [58]. Water-soluble oil was recovered by hexane extraction with 82.7%-83.3% dissolved in hot water and 90.0%-90.5% dissolved cold water from Tagetes minuta [59]. Recently, oil was recovered from hydrosol of Matricaria chamomilla flowers and showed good antifungal activity against A. niger [6]. Other study showed that linalyl acetate and limonene were recovered from from bergamot juice by supercritical and liquid CO2 [60].

Conclusions
In the present study, the treated Melia azedarach wood with C. citriodora leaf and fruits essential oils (25, 50 and 100 µL) showed the highest antifungal activity (100% inhibition) against F. culmorum and P. chrysogenum. Treated wood with both essential oils at 50 and 100 µL observed potent activity against the growth of R. solani with an inhibition percentage 100%. Recovered oils from leaves and fruits showed good activity against R. solani, where the inhibition percentage reached 96.66%, and 93.33%, respectively. Additionally, weak to moderate activity was observed against F. culmorum and P. chrysogenum as wood treated with recovered oils from leaves and fruits. Therefore, both oils could be used as natural antifungal agents for the treatment of several plant infection diseases and as wood bio-fungicide that can be used for packaging fruits or vegetables. Also, the mass spectra of the major components are recoded and discussed, where the main fragment ions were observed at m/z 67, 68, 69, 71, 93 and 161 for the main components of the studied samples. From MNDO calculations, citronellol molecule has the most negative values of heat of formations and it is the most stable molecule, while α -pinene is the least stable molecule.