Phytochemical Compounds of Branches from P. halepensis Oily Liquid Extract and S. terebinthifolius Essential Oil and Their Potential Antifungal Activity

: In the present study, the antifungal activity of wood treated with Pinus halepensis branch n -hexane oily liquid extract (OLE) and Schinus terebinthifolius branch essential oil (EO) was evaluated against the growth of four phytopathogenic fungi— Bipolaris oryzae, Fusarium . wood of and each received 100 µL of the concentrated oils from P. halepensis and S. terebinthifolius . The main compounds identified in S. terebinthifolius branch EO were terpinen-4-ol (18.25%), cis - β terpineol (15.60%), γ -terpinene (12.46%), sabinene (9.83%), α -terpinene (8.56%), and 4-thujanol (6.71%), while the main compounds in P. halepensis branch HeO were 2-undecenal (22.25%), (8.43%), ( Z )-2-decenal (6.88%), nonanal (5.85%), (2 E )-2-decenal (4.65%), ( E , E )-2,4-decadienal (4.41%), arachidonic acid methyl ester (4.36%), and 2-(7-heptadecynyloxy)tetrahydro-2H-pyran (4.22%). P. halepensis OLE at a concentration of 3% showed the highest inhibition percentage of fungal growth (IPFG) of B. oryzae, followed by S. terebinthifolius EO at 3% and 2%, with IPFG values of 80%, 74.44%, and 71.66%, respectively. At a concentration of 3%, branch oils from S. terebinthifolius and P. halepensis were found to have the highest IPFG values with 45.55% and 40.55%, respectively, against F. oxysporum growth. Moderate to weak activity found against F. solani S. terebinthifolius P. halepensis to wood. EO and OLE-treated wood samples at 3% produced inhibitions of 54.44% and 41.11%, respectively, against R. solani .

flies [44][45][46][47]. Many researchers have reported that EO applications should be considered as resistance inducers and could affect pathogens through the production of phytoalexins and biochemical plant defense [48]. Indeed, even little amounts of EOs applied on plants in vivo have been shown to produce a greater reduction in disease severity [49]. There are many reasons that may contribute to the greater effect of EOs in vivo than in vitro, such as variation of the optimum temperature of fungal growth and the presence of microorganisms on the plant surface [50]. The present work aimed to evaluate the bioactivity of P. halepensis n-hexane oily liquid extract and S. terebinthifolius essential oil as wood-biofungicides.

Preparation of Essntial Oil and n-Hexane Oily Liquid Extract
Green branches (wood and bark) of Brazilian Peppertree, Schinus terebinthifolius Raddi (Anacardiaceae), were rapidly washed by tap water to remove the dust, then smoothly dried with paper towel and cut into small pieces. About 100 g of the cut branches underwent extraction of their essential oil (EO) using the hydrodistillation method of the Clevenger apparatus for 3 h [51]. Meanwhile, the green branches (wood and bark) of Aleppo pine, Pinus halepensis Miller (Pinaceae), after being cut into small pieces, underwent extraction via the soaking method, where about 100 g of the branch sample was soaked in n-hexane solvent (200 mL) for 6 h under shaking. The n-hexane oily liquid extract (OLE) was separated by filtration with Whatman No. 1 filter paper under suction pressure then the OLE concentrated by evaporating the n-hexane solvent under vacuum using a rotary evaporator at 45 °C. EO and OLE were stored in brown tubes until use.

The Antifungal Activity of Wood Treated with Essential Oil and Oily Lquid Extract
Wood samples of P. roxburghii Sarg were prepared at the approximate dimensions of 2 × 1 × 0.5 cm, autoclaved at 121 °C for 20 min, and then left to cool. The four fungi of Bipolaris oryzae, Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani were used for the bioassay. S. terebinthifolius (EO) and P. halepensis (OLE) were prepared at the concentrations of 1%, 2%, and 3%. For every wood sample, 100 µL of the concentrated EO or OLE was applied. The antifungal activity of the treated wood samples was compared with wood samples without oil treatments, which were used as a control. The fungal inhibition percentage was calculated with the formula of inhibition percentage of fungal growth (IPFG) (%)= DC-DT DC ×100, where DC is the diameter of fungal control and DT is the diameter of treatment. Four replicates were carried out for all of the treatments. The antifungal activities of the treated wood were measured following the method of our previous works [3,[52][53][54][55] with minor modification, namely, ensuring that each wood sample examined for its antifungal activity had received the tested amount of EO or OLE.

Gas Chromatography-Mass Spectrometry (GC/MS) Analysis of the EO and OLE
The chemical compositions of the EO and OLE were analyzed using the Focus GC-DSQ Mass Spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) apparatus at the Atomic and Molecular Physics Unit, Experimental Nuclear Physics Department, Nuclear Research Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt. The column oven temperature was initially held at 45 °C, and then increased by 5 °C/min to 200 °C, held for 5 min, then increased again to 300 °C by 5 °C/min. Other conditions and identification of the compounds using the WILEY 09 and NIST 14 mass spectral databases can be found in previous works [1][2][3][4][5]56,57].

Statistical Analysis
Data of the antifungal activity were statistically analyzed with two factors [(type of oil (EO and OLE) and the concentration)] using the SAS system [58]. The differences among the means of the treatments were recorded using Fisher's least significant difference (LSD0.05). Figure 1 shows the antifungal activity of the treated Pinus roxburghii wood with the n-hexane oily extract (OLE) from Pinus halepensis and the essential oil (EO) of S. terebinthifolius against the growth of Bipolaris oryzae. In the control treatment (wood without EO or OLE), the fungal growth almost reached full linear growth, but for the wood treated with both oils, the fungal mycelia were suspended and the inhibition was found at all of the concentrations used (1%, 2%, and 3%). Additionally, the fungal growth was found to grow on the side opposite to the treated wood with both EO and OLE.

Visual Observations of the Fungal Growth
Compared to the control condition, Fusarium oxysporum growth was inhibited and did not reach the treated wood with both EO and OLE, especially at the concentrations of 2% and 3% ( Figure 2). On the other hand, the treated wood with both EO and OLE showed weak activity against Fusarium solani ( Figure 3); however, P. halepensis OLE at 3% showed inhibition against its growth. Figure 4 shows that, in the control treatment, the fungal mycelia of Rhizoctonia solani covered and grew over the wood samples after the incubation period. Meanwhile, either the growth did not completely reach the direction of the treated wood, or no growth was found at all.
Furthermore, as shown in Figure 4, the fungal growth of R. solani was found in the opposite direction of the treated wood with the P. halepensis OLE and Schinus terebinthifolius EO.

B. oryzae with control wood
B. oryzae growth B. oryzae

Antifungal Activity of the Oils
Statistically, there was a significance effect of EO and OLE in terms of antifungal activity against B. oryzae, F. oxysporum, F. solani, and R. solani (Figure 5a), where P. halepensis OLE had the highest effect against the growth of F. oxysporum and F. solani. Meanwhile, S. terebinthifolius EO was found to have good activity against B. oryzae and R. solani. Furthermore, when increasing the EO or OLE concentration from 0% (control) to 3%, the inhibition percentage of fungal growth (IPFG) was increased ( Figure 5b). For the effect of EO/OLE-treated wood as affected by the interaction between the EO/OLE type and their concentrations (Table 1), the highest activity against B. oryzae growth was found for P. halepensis OLE applied at a concentration of 3%, followed by S. terebinthifolius EO at 3% and 2%, with IPFG values of 80%, 74.44%, and 71.66%, respectively. S. terebinthifolius EO and P. halepensis OLE at a concentration of 3% observed the highest IPFG, with values of 45.55% and 40.55%, respectively, against F. oxysporum growth.
Wood treated with P. halepensis OLE at the 3% and 2% concentration levels showed significant activity against the growth of F. solani, with IPFG values of 30.55% and 29.44%, respectively. Also, S. terebinthifolius EO (2%) and P. halepensis OLE (1%) showed IPFG values of 21.11% and 20%, respectively. The highest activity against the growth of R. solani was observed for wood treated with S. terebinthifolius EO and P. halepensis OLE at 3%, with IPFG values of 54.44% and 41.11%, respectively. However, S. terebinthifolius EO and P. halepensis OLE at 2% showed IPFG values of 35% and 30.55%, respectively, against R. solani.
Several cited chemical compositions of P. halepensis have been published regarding terpenes, fatty acids, aldehydes, and cyclic phenolic compounds [59][60][61]. The lipophilic extractives (resin acids, terpenes, fatty acids, glycerides, steryl esters, sterols) were extracted using n-hexane then the hydrophilic ones (stilbenes, flavanols, monosaccharides, sugar alcohols) with acetone/water from sapwood and heartwood of P. halepensis stem [62]. Additionally, lipophilic and hydrophilic extractives were analyzed with GC-MS that were more abundant in heartwood than in sapwood of P. halepensis [63]. Seed oil of P. halepensis extracted with hexane was rich in fatty acid composition with linoleic and oleic acids accounting for more than 76% [64].
In the present study, both oils were observed to have potential antimicrobial activity in terms of microbial inhibition when treating wood. The monoterpenic or sesquiterpenic hydrocarbons and their oxygenated derivatives are considered as potential antimicrobial agents by many studies. Thus, these results agree with other previous works [9,18,21,26,75,76].
Recently, schinol and a new biphenyl compound (4′-ethyl-4-methyl-2,2′,6,6′-tetrahydroxyl(1.1′biphenyl)-4,4′-dicarboxylate), isolated from hexane and dichloromethane fractions obtained from leaves and stems, showed marked antifungal activity against P. brasiliensis [34]. Santos et al. [77] found that EOs from S. terebinthifolius and S. molle could be a possible alternative to control diseases caused by phytopathogenic fungi that affect agricultural production. The EOs from S. terebinthifolius showed a pronounced fungicide effect against Botrytis ssp isolated from "gerberas" and roses [73]. The antifungal activity may be attributed to the presence of the said chemical components in the EO; for instance, O-cymene and limonene have been shown to have strong antifungal properties [78]. Nevertheless, the mechanism of antifungal activity of this EO is still unknown. However, a recent study on the induced damage to the cell membrane structure of yeast and isolated mitochondria suggests that phytoconstituents from the EO of S. terebinthifolius are likely to disrupt the permeability barrier of the cell membranes and thereby inhibit respiration [79,80]. Furthermore, it has been suggested that the differences in susceptibility of tested organisms to monoterpenes and the differences in the efficacy of different monoterpenes may possibly be explained by the variation in the rate of monoterpene penetration and characteristic membrane structure [80].
Several authors who have studied the antimicrobial activity of Pinaceae family [81][82][83] report that monoterpenes possess a high antifungal activity. The absence of monoterpenes in our EO can explain the absence of a very high activity of Aleppo pine EO, but aldehydes are often found as constituents of plant products [84] and may play an important role in the observed antimicrobial activity of plant aldehyde-containing material [85]. Their action, very likely due to an alteration in the function of membrane-associated proteins, seems to be exerted mainly at the cell surface. However, the capability to penetrate the outer layer of cells can help to explain the antimicrobial activity of some aldehydes, especially against Gram-negative bacteria [86,87]. On the other hand, the antifungal activity of our EO may due to the presence of n-caprylaldehyde (hexanal) which is commercially used to produce fruity flavors and to prevent fruit spoilage. Meanwhile, another study confirmed that the saturated aldehydes hexanal, nonanal, and (E)-2-octenal showed almost no antimicrobial activity against some clinical bacteria [88], while the same study proved that the complex of long chain aldehydes-which highly presented in our EO-has very good antimicrobial activity, as they stated that α,β-unsaturated aldehydes ((E)-2-eptenal, (E)-2-nonenal, (E)-2-decenal, and (E,E)-2,4-decadienal) were tested together (ratio = 1:1:1:1) against ATCC bacterial isolates and clinically isolated microbial strains, and a strong synergic effect was observed [88]. Moreover, the effectiveness of the aldehydes seems to depend not only on the presence of the α,β-double bond, but also on the chain length from the enal group and on the microorganism tested. Finally these oil α,βunsaturated aldehydes might be good alternatives to other highly toxic disinfectants, such as glutaraldehyde [89]. In another study [18], the P. halepensis EO was shown to possess antifungal activity against A. flavus, A. niger, F. oxysporum, and R. stolonifera, and thus can be used as a natural treatment for fungal infections, as well as a natural preservative in food.
The treated wood of Acacia saligna at the concentration of 5% with the methanolic extracts of Morus alba heartwood, Cupressus sempervirens wood, and Maclura pomifera bark showed no fungal growth of T. harzianum on the surface of the wood [52]. An inhibition zone was found against T. harzianum for A. saligna wood treated with the methanolic extract of C. sempervirens wood at concentrations of 5%, 10%, and 20% [52]. Wood samples of P. sylvestris, F. sylvatica, and P. rigida treated with the EOs or extracts from E. camaldulensis leaves or P. rigida wood had a potential effect against the growth of some phytopathogenic fungi [54,56].
Acer saccharum bark acetone extract, in combination with citric acid, applied to Leucaena leucocephala wood showed some activities against T. viride, F. subglutinans, and A. niger [4]. No growth of A. flavus and C. gloeosporioides was observed for sheets of Papyrus (Cyperus papyrus L.) strips pre-treated with P. rigida and E. camaldulensis or Salix babylonica extracts [90]. Melia azedarach wood samples treated with A. saligna flowers water extract showed activity and growth inhibition against F. culmorum, R. solani, and P. chrysogenum [8], while the same treated with the methanol extract of Musa paradisiaca peels showed potential antifungal activity against F. culmorum and R. solani [7]. Additionally, the same wood treated with oils extracted with n-hexane from E. camaldulensis and Vitex agnus-castus showed promising antifungal activities against F. culmorum, R. solani, and P. chrysogenum [3].
Among the chemical components of the oils, there are synergistic or antagonistic interaction effects. The mode of action of EOs has been investigated by many authors, who suggested that antimicrobial activity is produced by interactions provoked by terpenes in the enzymatic systems related with energy production and in the synthesis of structural components of the microbial cells [91]. Other reports have suggested that the components of the EOs cross the cell membrane, interacting with the enzymes and proteins of the membrane such as the H + -ATPase pumping membrane, thus producing a flux of protons toward the cell exterior, which induces changes in the cells and, ultimately, their death [92]. Furthermore, several works [93][94][95][96] have explained how the antimicrobial ability of terpenes stop the functions of cell membranes, not only the permeability, how they might penetrate the membrane to the cell interior organs and interact critically with intracellular sites. Moreover, Daferera et al. [97] reported that the antifungal activity of EOs may be due to formation of hydrogen bonds between the hydroxyl group of oil phenols and the active sites of the target enzymes.

Conclusions
The antifungal activity of the wood of Pinus roxburghii treated with P. halepensis branch n-hexane oily liquid extract and S. terebinthifolius branch essential oil was tested against B. oryzae, F. oxysporum, F. solani, and R. solani. Both the extracted essential oil and oily liquid extract inhibited F. oxysporum isolate growth with a percentage of ca. 40% at a concentration of 3%, and showed good bioactivity against the growth of B. oryzae, F. oxysporum, and R. solani. These activities could be related to monoterpenoids, the most abundant compounds identified in the extracts. Therefore, both natural extracted materials could be considered as a wood-biofungicide agent and for their possible use on a larger scale.

Conflicts of Interest:
The authors declare no conflicts of interest.