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International Biodeterioration & Biodegradation 106 (2016) 88e96

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Antifungal activities of two essential oils used in the treatment ofthree commercial woods deteriorated by five common mold fungi

Mohamed Z.M. Salem a, *, Yassin E. Zidan b, Maisa M.A. Mansour b, Nesrin M.N. El Hadidi b,Wael A.A. Abo Elgat c

a Forestry and Wood Technology Department, Faculty of Agriculture (EL-Shatby), Alexandria University, Alexandria, Egyptb Conservation Department, Faculty of Archaeology, Cairo University, Giza 12613, Egyptc High Institute of Tourism, Hotel Management and Restoration, Alexandria, Egypt

a r t i c l e i n f o

Article history:Received 8 July 2015Received in revised form17 September 2015Accepted 13 October 2015Available online xxx

Keywords:Commercial woodsWood mold fungiBiodeteriorationAntifungal activityEssential oils

* Corresponding author.E-mail address: zidan_forest@yahoo.com (M.Z.M.

http://dx.doi.org/10.1016/j.ibiod.2015.10.0100964-8305/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

In the past ten years natural extracts have been used as important potential applications to prevent moldgrowth on in-service wood. The growth of fungal hyphae of five common mold fungi (Alternaria alter-nata, Fusarium subglutinans, Chaetomium globosum, Aspergillus niger, and Trichoderma viride) on woodsurface of Pinus sylvestris, Pinus rigida and Fagus sylvatica treated with the essential oil (EO) of P. rigida(wood) and Eucalyptus camaldulensis (leaves) was visually estimated. EOs were applied by vapor methodand the mold growth inhibition was measured. The chemical constituents of the EOs was analyzed byGC/MS, which referred to the presence of a-terpineol (34.49%), borneol (17.57%), and fenchyl alcohol(14.20%) as the major components in P. rigida wood oil, and eucalyptol (60.32%), a-pinene (13.65%), andg-terpinene (8.77%) in E. camaldulensis leaves. Complete inhibition against the growth of A. alternata,F. subglutinans, C. globosum, and A. niger except of T. viride by applying P. rigidawood EO at 5000 ppm andcomplete growth with all the studied fungi except of C. globosum at 156.25 ppm was found. Good in-hibitions against C. globosum at 5000 ppm and 156.25 ppm and no inhibition against A. niger and T. virideand little inhibition against F. subglutinans at high concentration was found by the application of EO fromE. camaldulensis leaves. These findings support the potential use of the EOs for wood protection againstmold infestation for surface-treatment or fumigation of wood products.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

For the building industry, the protection against mold growth onwood is a critical economic concern as well as choice of nontoxiccompounds which are applicable in interior wood protection.Wood deterioration is a complex biological process that can involvea wide variety of microorganisms, such as fungi and bacteria, and isinfluenced by the changing environmental conditions in which thewood is placed (Eaton and Hale, 1993; Zabel and Morrell, 1992).Fungi are important biodegrading organisms and are considered asserious degrading agents where the presence of vegetative cells orspores on the surface of wood or other materials like paper mayindicate a possible degradation in the future (Fabbri et al., 1997;Mesquita et al., 2009).

Salem).

Mold fungi growing onwood surfaces cause sapstain and simplesugars and starch present in ray cells and axial cell lumens areconsumed by molds (Kerner-Gang and Schneider, 1969; Mansourand Salem, 2015). Sapstain is a major problem for timber pro-ducers as well as pulp and paper manufacturers since fungalcolonization and disfigurement of freshly felled material prior todrying can result in significant economic losses.

Although mold fungi cause little or no significant damage to thestructural elements of the timber (Blanchette et al., 1992), they havea detrimental effect on the aesthetic value of the wood due to thecolonization by their pigmented mycelium. This is due to the pro-duction and deposition of granules of melanin in and around thefungal hyphae (Brisson et al., 1996). Ray parenchyma and cell lu-mens are colonized by mold fungi as well as scavenge of proteinsand triglycerides deep throughout the sapwood of the lumber(Breuil, 1998).

Molds like Alternaria and Trichoderma species, are verydestructive in museums, have a well-developed lignocellulolytic

M.Z.M. Salem et al. / International Biodeterioration & Biodegradation 106 (2016) 88e96 89

enzyme system (Garg et al., 1995).Alternaria fungi have black pigment (melanin) that causes

distinctly seen dark grey discoloration on wood joints (Domschet al., 2007). Alternaria alternata and other mold fungi isolatedfrom treated wood are recognized as soft-rot fungi and they havebeen found on painted or preservative-treated wood (Kim et al.,2007; Pournou and Bogomolova, 2009; Råberg et al., 2009).Fungal enzymatic activity onwood showed that A. alternatawas themost active tyrosinase producer and it was the hardiest to woodpreservatives fungal strain (Brid�ziuvien _e and Raudonien _e, 2013).A. alternata produces mycotoxins (tenuazonic acid) which causetoxic leucopenia in humans and is considered an important sourceof allergens as well (Breitenbach and Simon-Nobbe, 2002).A. alternata showed phenoloxidase activity that degrades lignin(Brid�ziuvien _e and Raudonien _e, 2013).

Trichoderma rarely have been reported to cause soft rot (Kimet al., 2007; Mansour and Salem, 2015). Trichoderma species arenot capable of invading living wood and are not an effective colo-nizer of living tissue (Lundborg and Unestam, 1980; Kubicek et al.,2008). They are associated with other fungi that are pathogenic tothe host tree (Jin et al., 1992), and were isolated with greater fre-quency from the roots of Douglas fir decayed by Phellinus weiriithan from sound, un-decayed regions (Goldfarb et al., 1989).Confocal micrograph showed Trichoderma viride hyphae in rayparenchyma cells of common radiata pine (Xiao et al., 1999).Greater persistence of green mold contamination in wood, woodenpallets and concrete were observed (Abosriwil and Clancy, 2002)and produce cellulase and hemicellulases (Gautam, 2010).

T. viride, Chaetomium globosum and Alternaria sp. are the fungiwith proven cellulolytic activity detected in the deterioratedwooden sculptures and art photographs temporarily stored in thequarantine room of the Cultural Center of Belgrade (Ljaljevi�c-Grbi�cet al., 2013). Aspergillus niger is a producer of many pectinases andhemicellulose degrading enzymes, like xylanases and arabinases(Delgado et al., 1992; van Peij et al., 1997; Parenicov�a et al., 2000).Also, xylan (major structural heteropolysaccharides of hardwood)degradation occurs in certain strains including A. niger and T. viride(Filho et al., 1996). A. niger and Fusarium oxysporum producescellulase and xylanase enzymes which are capable to biodegradethe forest waste from Pinus roxburghii, Cedrus deodara, Toona ciliataand Celtris australis (Kaushal et al., 2012) and hardwood has shownbetter degradation as compared to the softwood.

C. globosum is themost common species of Chaetomium found inbuildings (Andersen and Nissen, 2000) and produces the highlycytotoxic chaetomins and chaetoglobosins that inhibit cell divisionand glucose transport (Ueno, 1985). C. globosum was stated to be avery active organism in the decay of leather from book bindings(Strzelczyk et al., 1989) and was found in both wood-pulp paperand laid-paper (Mesquita et al., 2009).

Mycelial growth by most molds is hyaline and relatively insig-nificant in causing spoilage to Pinus sylvestris timber (Payne andBruce, 2001). Rapid air or kiln drying of wood or through the useof diffusible chemical preservatives can be used to control of sap-stain (Byrne, 1998). Mold fungi that grow on wood surface are ableto degrade lignin, but the phenoloxidases (peroxidase, tyrosinaseand laccase) that take part in lignin decomposition are not char-acteristic of every fungus (Brid�ziuvien _e and Raudonien _e, 2013).

Synthetic fungicides are commonly used to control and preventthe growth of mold and decay fungi on wood, but are not envi-ronmentally suitable for many indoor applications and the searchfor naturally, environmentally-friendly alternatives, which exhibitnegligible toxicity to human has become a necessity (Verma andDubey, 1999; Qi and Jellison, 2004; Li et al., 2013; Wang et al.,2005; Kiran and Raveesha, 2006). Natural extracts have thereforebecome potentially useful for protecting woods from mold fungi

(Philp et al., 1995; Jelokov�a and �Sindler, 1997; Qi and Jellison, 2004;Wang et al., 2005; Li et al., 2013; Mansour and Salem, 2015; Salemet al., 2015). Essential oils (EOs), a complex mixture of odorous andvolatile compounds, are known for their natural components, suchas monoterpenes, diterpenes and hydrocarbons with variousfunctional groups and, have been studied for their antibacterial andantifungal activities (Mazzanti et al., 1998; Hammer et al., 2002;Wang et al., 2005; Pawar and Thaker, 2006; Salem et al., 2014).

As part of the continuous research for using natural products asa bio-agents for protecting woods against mold fungi, our ongoingwork was to evaluate the antifungal effect of essential oils fromPinus rigida (wood) and Eucalyptus camaldulensis Dehnh. (leaves)against the growth of five commonmold fungi namely, A. alternata,Fusarium subglutinans, C. globosum, A. niger, and Trichoderma viride.The evaluation was done by application of these essential oils asbiocides for preservation of three commercial woods (P. sylvestris, P.rigida and Fagus sylvatica) against the biodeterioration caused bythe studied mold fungi.

2. Materials and methods

2.1. Chemicals

All of the chemicals used in the present study were of highanalytical grade from Fluka and SigmaeAldrich Co. (USA).

2.2. Preparation of wood samples

Sapwood samples of Scots pine (P. sylvestris L., Pinaceae), Pitchpine (Pinus rigida Mill., Pinaceae), and European beech (F. sylvaticaL.) (Fagaceae), were provided from a woodworking shop inAlexandria City, Egypt, August 2014. The wood samples(10 � 10 � 5 mm) were oven-dried at 105 �C for 24 h, thenautoclaved at 121 �C for 20 min.

2.3. Essential oil extraction

Fresh leaves of E. camaldulensis were cut to small pieces and theair-dried P. rigida ground wood was hydro-distillated for 3 h, in aClevenger apparatus (Salem et al., 2013). The essential oils (EOs)were dried over anhydrous Na2SO4, and measured with respect tothe mass weight (4.5 and 2.12 ml 100 g�1 sample weight forE. camaldulensis and P. rigida, respectively). The EOs were kept dryin sealed Eppendorf tubes and stored at 4 �C prior chemicalanalysis.

2.4. Preparation of essential oils

The extracted EOs were prepared at the concentrations of 5000,2500, 1250, 625, 312.5, and 156.25 ppm for antifungal activitybioassay. The respective amount of oil was diluted in 10% dimethylsulfoxide (DMSO): sterilized distilled water (SDW) (1:1 v/v) and0.5 ml of Tween 80 was added to emulsify carrier oils in water.

2.5. Vapor treatment of essential oils for mold inhibition

Wood samples were vapor treated with the EOs prepared at theconcentrations of 5000 ppm, 2500 ppm, 1250 ppm, 625 ppm,312.5 ppm, and 156.25 ppm using the evaporation method (Lopezet al., 2005; Nedorostova et al., 2009). Wood samples were put inpetri dishes contains 8 layers of filter papers (Wattman No. 1)overlaid by a mesh (polyethylene spacer). The dishes were auto-claved at 121 �C for 20 min and left to cool, then the oils with therespective concentration were impregnated over the filter papersand kept for 48 h to allow the EO evaporation which the fumigants

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(EO) subsequently absorbed by the wood samples.

2.6. Biodeterioration of wood by mold fungi in vitro

The biodeterioration of the studied commercial woods wasachieved by five common mold fungi namely; A. alternata, F.subglutinans, C. globosum, A. niger, and Trichoderma viride. 15-day-old potato dextrose agar culture (PDA) of each fungus was pre-pared. Three replicates from each wood were used for eachconcentration of the EOs. After treating wood samples with EOs,they were inoculated with a disc (5 mm diameter) of each fungusin a Petri dish containing PDA-culture and incubated for threedays for T. viride and one week for other fungi at 25 ± 1 �C.Mixture of 10% DMSO and SDW (1:1 v/v) was used as an alter-native in the control sample. The inhibition zones (IZs, mm) of theEOs around the treated woods against each fungus weremeasured and recorded using the recommendations of the pre-viously published works (Satish et al., 2007; Essa and Khallaf,2014; Mansour and Salem, 2015; Mansour et al., 2015). Themeasurements of IZs which means a clear zone (no growth offungus) around the treated wood samples were done by a rulerafter three days for T. viride and one week for other fungi, wherethe maximum growth (9 cm in diameter) was observed in thecontrol treatments.

2.7. Visual observation

After three days of the incubation period for T. viride and oneweek for other fungi, the fungal growth was visually evaluated bythe naked eye in accordance with GOST 9.048-75 (1975) standardand fungicidal activity of the EOs was estimated by fungal growthretardation, using the following visually determinedmarks (Humarand Pohleven, 2005):

0 mycelium growth more intense than control,1 normal growth, insignificant retardation (area of colony�90% of

area of controls),2 visible signs of retardation (colony <90% and �60% of controls),3 pronounced retardation (colony <60% and �25% of controls),4 very marked retardation (colony <25% of controls),5 no growth.

Additionally, the experiment was left in the laboratory condi-tions, and we checked it visually every week for four months. Allthe notes about the visual fungal growth were recorded.

2.8. GC/MS analysis of the essential oils

A Trace GC Ultra/Mass spectrophotometer ISQ (Thermo Scien-tific) instrument equipped with a FID and a DB-5 narrow borecolumn (length 10 m � 0.1 mm ID, 0.17 mm film thickness; Agilent,Palo Alto, CA, USA) was used. Helium was used as the carrier gas(flow rate of 1 ml min�1), and the oven temperature program was:45e165 �C (4 �C min�1) and 165e280 �C (15 �C min�1) with postrun (off) at 280 �C. Samples (1 ml) were injected at 250 �C, withsplit/split-less injector (50:1 split ratio) in the splitless mode flowwith 10mlmin�1. The GCeMSwas equippedwith a ZB-5MS Zebroncapillary column (length 30 m � 0.25 mm ID, 0.25 mm film thick-ness; Agilent). Helium (average velocity 39 cm s�1) was used as thecarrier gas and the oven temperature was held at 45 �C for 2 minthen increased from 45 to 165 �C (4 �C min�1), and 165e280 �C(15 �C min�1).

All mass spectra were recorded in the electron impact ioniza-tion (EI) at 70 electron volts. The mass spectrometer was scannedfrom m/z 50e500 at five scans per second. Peak area percent was

used for obtaining quantitative data with the GC with HP-ChemStation software (Agilent Technologies) without usingcorrection factors. Identification of the EO constituents was per-formed on the basis of MS library search (NIST and Wiley) (Davies,1990; Adams, 1995).

2.9. Statistical analysis

Results of application of the EOs from P. rigida (wood), andE. camaldulensis (leaves) on the three studied woods (P. sylvestris,P. rigida and F. sylvatica) against the growth of each fungus werestatistically analyzed using Analysis of variance (ANOVA) procedurein SAS version 8.2 (2001). Fisher's Least Significant Difference (LSD)at 5% level of probability was used to measure the differencesamong the means.

3. Results and discussion

3.1. Visual observation

Antifungal effects of essential oils (EOs) applied to the threewoods against five common mold fungi were assessed andcompared with the respective control (untreated) and the visualobservations of the growth are presented in Figs. 1 and 2.

Application of P. rigida wood EO (Fig. 1) to the three studiedwoods showed remarkable effects against the studied fungi.Complete inhibition against the growth of A. alternata,F. subglutinans, C. globosum, and A. niger except of T. viride wasfound by applying P. rigida wood EO (Fig. 1) at high concentra-tion (5000 ppm) and complete growth was found with all thestudied fungi except of C. globosum at low concentration(156.25 ppm).

The application of EO from E. camaldulensis leaves for the threewoods observed good inhibition against C. globosum (Fig. 2) at high(5000 ppm) and low (156.25 ppm) concentrations and no inhibi-tion against A. niger and T. viride and little inhibition againstF. subglutinans at high concentration.

Table 1 summarizes the visually fungicidal activity by fungalgrowth retardation which ranged from 1 to 5 marks (Humar andPohleven, 2005). EO from P. rigida wood applied at the concentra-tions of 5000 ppm and 156.25 ppm showed complete retardation ofthe studied fungi where the marks ranged from 4 to 5 and at lowconcentration (156.25 ppm) only against C. globosum and A. nigerwhere the mark reached 4. The growth of A. alternate andC. globosum was suspended at the high concentration ofE. camaldulensis leaves EO where the marks recorded were 4 and 5,respectively. Generally, EO of E. camaldulensis leaves were lessinhibitory than P. rigida EO.

Additionally, from the visual observations, wood of F. sylvatica(hardwood) is more susceptible to biodeterioration than P. sylvestrisand P. rigidawoods (softwoods). This behaviormay be due to higherlignification of secondary walls in the fibers of softwood than thatof hardwoods (Kaushal et al., 2012). Furthermore, the extractives insoftwood like resins and dihydric alcohols act as strong anti-microbial agents thus causing delay in the biodegradation process(Rowe, 1989). Moreover, hardwoods have higher hemicelluloses(glucan and xylan) and lower lignin content than softwood whichleads to better biodegradation of hardwood by fungi (Lee et al.,2008; Kaushal et al., 2012).

3.2. Antifungal activities of Fagus sylvatica wood treated with theessential oils

Results observed in Tables 2e4 show that there were highlysignificant effects of the studied EOs applied to the three woods

Fig. 1. Effect of P. rigida wood essential oil on the growing of five mold fungi on surface of three commercial woods (1-P. sylvestris, 2-F. sylvatica and 3-P. rigida); 0.5 ml (5000 ppm)and 0.015 ml (156.25 ppm) after three days for T. viride and one week for other fungi, where the maximum growth (9 cm in diameter) was observed in the control treatments.

M.Z.M. Salem et al. / International Biodeterioration & Biodegradation 106 (2016) 88e96 91

against the growth of A. alternata, F. subglutinans, C. globosum,A. niger, and T. viride. The results of antifungal activities of appli-cation of EOs to F. sylvatica wood are presented in Table 2.

The application of EO from P. rigida wood to F. sylvatica woodshowed good antifungal activity against the studied mold fungi atthe high concentration (5000 ppm) with IZ values of 18.00 mm,10.00 mm, 13.66 mm, 11.33 mm, and 10.00 mm against thegrowth of A. alternata, F. subglutinans, C. globosum, A. niger, andT. viride, respectively, and 13.66 mm against C. globosum at2500 ppm (Table 2). On the other hand, the application of EOfrom E. camaldulensis leaves had a little activity against thestudied mold fungi except against C. globosum (IZ, 11.66 mm), and

A. niger (IZ, 7.66 mm).

3.3. Antifungal activities of Pinus rigida wood treated with theessential oils

According to the results reported in Table 3, the EO from P. rigidawood increased the durability of P. rigida wood against the growthof the studied mold fungi at 5000 ppm with IZs values 6.66 mm,12.00 mm, 11.00 mm (12.66 mm at 2500 ppm), 6.00 mm, and10.33 mm against the growth of A. alternata, F. subglutinans,C. globosum, A. niger, and T. viride, respectively. EO fromE. camaldulensis leaves showed good activity against C. globosum

Fig. 2. Effect of E. camaldulensis leaves essential oil on the growing of five mold fungi on surface of three commercial woods (1-P. sylvestris, 2-F. sylvatica and 3-P. rigida); 0.5 ml(5000 ppm) and 0.015 ml (156.25 ppm) after three days for T. viride and one week for other fungi, where the maximum growth (9 cm in diameter) was observed in the controltreatments.

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and A. nigerwith IZs values 10.00mm, and 6.66mm, respectively, atthe concentration of 5000 ppm.

3.4. Antifungal activities of Pinus sylvestris wood treated with theessential oils

The EO from P. rigida wood increased the durability ofP. sylvestris wood against the growth of the studied mold fungi at5000 ppmwith IZs values 13.33mm, 6.33mm,11.66mm,11.66mm,and 15.66 mm against the growth of A. alternata, F. subglutinans,C. globosum, A. niger, and T. viride, respectively (Table 4).

Additionally, the EO from E. camaldulensis leaves showed good ac-tivity with IZs values of 11.00 mm, 9.00 mm, 10.00 mm, 9.00 mm,and 3.00 mm against the growth of A. alternata, F. subglutinans,C. globosum, A. niger, and T. viride, respectively, at the concentrationof 5000 ppm (Table 4).

3.5. Chemical composition of the essential oils from P. rigida (wood)and E. camaldulensis (leaves)

Data from Tables 2e4 revealed that the EOs from P. rigida (wood)and E. camaldulensis (leaves) showed good antifungal activity

Table 1Fungicidal activity was estimated by fungal growth retardation, using the following visually determined marks.

Treatments Fungal growth retardation marksa

Essential oil Concentration (ppm) A. alternata F. subglutinans C. globosum A. niger T. viride

P. rigida wood 5000 5 5 5 5 52500 4 4 5 5 51250 4 2 4 4 3625 4 1 4 4 1312.5 3 1 3 4 1156.25 3 1 4 4 2

E. camaldulensis leaves 5000 4 3 5 1 22500 3 1 4 4 21250 3 1 4 4 1625 2 1 4 3 1312.5 1 1 4 2 1156.25 1 0 4 1 1

a Measured according to Humar and Pohleven (2005).

Table 2Means of inhibition zones (mm) of essential oils at different concentrations applied to Fagus sylvatica wood against the growth of A. alternata, F. subglutinans, C. globosum,A. niger, and T. viride.

Treatments Inhibition zone (IZ, mm)

Essential oil (A) Concentration (ppm) (B) A. alternata F. subglutinans C. globosum A. niger T. viride

Air-dried of P. rigida ground wood Control (0) 0.000 0.000 0.000 0.000 0.0005000 18.000 10.000 13.333 11.333 10.0002500 6.000 4.333 13.666 8.333 4.0001250 3.000 1.333 4.333 9.333 0.000625 0.666 0.333 4.333 4.666 0.000312.5 1.666 0.666 4.333 2.666 0.000156.25 0.000 0.000 2.000 1.666 0.000

Fresh leaves of E. camaldulensis Control (0) 0.000 0.000 0.000 0.000 0.0005000 3.666 6.000 11.666 7.666 1.0002500 0.666 1.000 7.000 1.333 0.3331250 0.666 0.000 5.000 3.333 0.000625 0.333 0.000 2.333 2.333 0.000312.5 0.333 0.000 4.666 2.333 0.000156.25 0.000 0.000 2.666 1.333 0.000

L.S.D. at 0.05 A 0.593 0.457 0.774 1.473 0.516B 1.11 0.855 1.448 2.755 0.965A* B 1.778 1.02 2.654 ns 1.784

*Means with the same letter within the same column (capital letters) or row (small letters) are not significantly different according to LSD at 0.05 level of probability.

Table 3Means of inhibition zones (mm) of essential oils at different concentrations applied to Pinus rigidawood against the growth of A. alternata, F. subglutinans, C. globosum, A. niger,and T. viride.

Treatments Inhibition zone (IZ, mm)

Essential oil (A) Concentration (ppm) (B) A. alternata F. subglutinans C. globosum A. niger T. viride

Air-dried of P. rigida ground wood Control (0) 0.000 0.000 0.000 0.000 0.0005000 6.666 12.000 11.000 6.000 10.3332500 4.000 1.666 12.666 2.000 4.0001250 1.333 0.666 5.000 6.000 0.333625 2.000 0.000 3.000 1.666 0.000312.5 0.333 0.000 4.000 0.333 0.000156.25 0.000 0.000 2.000 0.000 0.000

Fresh leaves of E. camaldulensis Control (0) 0.000 0.000 0.000 0.000 0.0005000 1.666 1.666 10.000 6.666 1.3332500 2.000 0.666 4.333 2.000 0.3331250 1.666 0.000 3.000 2.000 0.000625 1.333 0.000 2.666 1.333 0.000312.5 1.333 0.000 4.000 0.666 0.000156.25 0.000 0.000 3.666 0.333 0.000

L.S.D. at 0.05 A 0.616 0.258 0.816 na 0.487B 1.154 0.483 1.526 1.815 0.912A* B 1.809 0.865 2.458 na 1.590

*Means with the same letter within the same column (capital letters) or row (small letters) are not significantly different according to LSD at 0.05 level of probability.Ns; not significant at 0.05 level of probability.

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Table 4Means of inhibition zones (mm) of essential oils at different concentrations applied to Pinus sylvestris wood against the growth of A. alternata, F. subglutinans, C. globosum,A. niger, and T. viride.

Treatments Inhibition zone (IZ, mm)

Essential oil (A) Concentration (ppm) (B) A. alternata F. subglutinans C. globosum A. niger T. viride

Air-dried of P. rigida ground wood Control (0) 0.000 0.000 0.000 0.000 0.0005000 13.333 6.333 11.666 11.666 15.6662500 3.666 5.000 11.333 9.000 7.6661250 2.666 0.000 2.000 9.000 2.000625 1.333 0.000 1.333 4.000 0.000312.5 0.000 0.000 2.000 4.000 0.000156.25 0.000 0.000 1.666 1.666 0.000

Fresh leaves of E. camaldulensis Control (0) 0.000 0.000 0.000 0.000 0.0005000 11.000 9.000 10.000 9.000 3.0002500 1.000 1.000 8.333 3.666 0.6661250 1.000 0.000 4.666 3.333 0.000625 1.333 0.000 3.000 2.666 0.000312.5 1.000 0.000 3.666 1.666 0.000156.25 0.000 0.000 2.333 0.666 0.000

L.S.D. at 0.05 A ns ns ns 1.112 0.609B 1.601 0.729 1.471 2.081 1.139A* B ns 0.970 1.725 ns 1.908

*Means with the same letter within the same column (capital letters) or row (small letters) are not significantly different according to LSD at 0.05 level of probability.Ns; not significant at 0.05 level of probability.

Table 5Chemical composition of the essential oil from Pinus rigida wood.

RT Compound name Area %

6.51 Tricyclene 0.256.90 a-Pinene 7.767.35 Camphene 2.157.79 Benzaldehyde 0.228.27 4(10)-Thujene 0.299.44 3-Carene 0.389.97 o-Cymene 0.5110.10 L-Limonene 3.3811.67 p-menth-1-ene-3b,7-diol 0.4812.29 p, a-Dimethylstyrene 1.2213.23 Fenchyl alcohol 14.2014.07 Pinocarveol 0.9314.30 (�)-Alcanfor 1.0514.74 Isoborneol 2.1114.90 pinene hydrochloride 1.0215.21 Borneol 17.5715.53 (�)-Terpinen-4-ol 1.1215.76 40-Methylacetophenone 0.2816.28 a-Terpineol 34.4916.37 Estragole 0.7416.72 L-Verbenone 1.1617.08 cis-carveol 1.0517.44 L-Carveol 0.2717.90 (S)-(þ)-Carvone 0.4319.41 (�)-Bornyl acetate 0.2523.29 Tetradecane 0.2123.44 Methyl eugenol 0.2525.33 Ethyl cinnamate 0.5326.26 exo-2-Hydroxycineole acetate 0.3926.44 Pentadecane 0.3729.39 Hexadecane 0.4432.16 Heptadecane 0.5132.30 2,6,10,14-Tetramethylpentadecane 0.2134.01 Octadecane 0.4735.24 Heneicosane 0.4336.21 Eicosane 0.3036.27 13a-methyl-13-vinyl-Podocarp-8(14)-ene 0.2138.07 Pimaric acid 0.35

Table 6Chemical composition of the essential oil from Eucalyptus camaldulensis leaves.

RT Compound name Area %

6.72 a-Phellandrene 1.396.88 a-Pinene 13.658.22 Sabinene 0.648.83 Myrcene 0.529.21 a-Phellandrene 0.549.92 p-Cymene 0.6210.05 1,3,8-p-Menthatriene 0.5110.41 Eucalyptol 60.3211.26 g-Terpinene 8.7712.24 a-Terpinene 1.0114.02 trans-Pinocarveol 0.7715.51 Terpinen-4-ol 4.2416 a-Terpineol 2.6817.36 Citronellol 0.2719.43 Carvacrol 0.224.48 Pyrogallol 0.52

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against the studied mold fungi. Table 5 shows the chemicalcomposition of the EO of P. rigida (wood) analyzed using GC/MS,where the major components are a-terpineol (34.49%), borneol(17.57%), fenchyl alcohol (14.20%), a-pinene (7.76%), L-limonene(3.38%), and isoborneol (2.11%). a-pinene caused more than 50%

inhibition of T. viride, cymene was generally most inhibitory,causing more than 86% reduction of all the tested fungi, andcamphene andmyrcene caused 74% inhibition of T. viride (De Groot,1972).

Table 6 presents the chemical constituents of the EO fromE. camaldulensis (leaves) by GC/MS. The major components wereeucalyptol (60.32%), a-pinene (13.65%), g-terpinene (8.77%), andterpinen-4-ol (4.24%), a-pinene was found in high ratio inE. camaldulensis (Salem et al., 2015). Eucalyptol has been reportedfor its strong antimicrobial properties against many pathogens(Rosato et al., 2007; Bakkali et al., 2008; Salem et al., 2015). a-pinene was found to be effective against the growth of Alternariasp., A. nidulans and A. niger (Gli�si�c et al., 2007). Essien and Akpan(2004) found that ethanolic and distilled H2O leaf extracts ofE. camaldulensis had marked fungicidal effect against clinical der-matophytic fungal isolates; Microsporium gypseum and Trichophy-ton mentagrophytes. The EO of E. camaldulensis showed moderateactivity at low doses against common phytopathogenic fungi,A. alternata, Colletotrichum corchori, Curvularia lunata, F. equiseti,Macrophomina phaseolina, Drecheslera oryzae and Botrydiplodiatheobromae (Begum et al., 1997; Barra et al., 2010). The induction ofmembrane damages of Saccharomyces cerevisiae genes involved inergosterol biosynthesis and sterol uptake, lipid metabolism, cell

M.Z.M. Salem et al. / International Biodeterioration & Biodegradation 106 (2016) 88e96 95

wall structure and function, detoxification and cellular transportare affected by the treatment with a-terpinene (Parveen et al.,2004).

From the previous results, the antifungal activity could berelated to the extractives found in wood itself as well as theapplied extracts for woods. For example, the resistance of beechand spruce wood against Trametes versicolor and Serpula lacry-mans was increased by tannins (Jelokov�a and �Sindler, 1997), butthe presence of sugar contents have a negative effect on the wooddurability of beech wood (Jelokov�a and �Sindler, 2001). Addi-tionally, P. rigida oil showed significant inhibition of mold growthand can serve as broad-spectrum biocides against the studiedmolds.

It is noteworthy to mention here that the experiment waschecked out from 2 to 4 months after wood samples had beencolonized with the 5 mold fungi. Visually, under laboratory condi-tions and after two months, some growths of microorganismsstarted to appear over and around the treated woods withE. camaldulensis leaves EO. These growths appeared over andaround the wood treated with P. rigida EO at the end of the thirdmonth from the inoculation with the studied fungi. At the end ofthe fourth month, huge growth or contaminations appearedaround and over the surfaces of wood. From this study, it is clearthat P. rigida oil provides significant inhibition of mold growth andcan play an important role in wood protection from molds. Theactivity could be related to the major components L-a-pinene, a-terpineol, borneol, and fenchyl alcohol presented in the oil. Also,some strong activity occurred by the application of E. camaldulensisleaves EO and could be related to the active components eucalyptoland a-pinene.

4. Conclusions

We herein report the antifungal activity of the three commercialwoods treated with two essential oils against the growth of fivecommon mold fungi. More interest has focused on the potentialapplications of the essential oils as wood protection agents toprevent mold and fungal growth on in-service wood.

Two sources of essential oils were used for the treatments,P. rigida wood and E. camaldulensis leaves. Visually, after twomonths, the growth of microorganisms started to appear over andaround the wood treated with E. camaldulensis leaves EO. Thesegrowths appeared over and around thewoods treated P. rigida EO atthe end of the third month from the inoculation with the studiedfungi. Complete inhibition against the growth of A. alternata,F. subglutinans, C. globosum, and A. niger except of T. viridewas foundby applying P. rigidawood essential oil at high concentrationwherethemarks ranged from 4 to 5, and complete growthwas foundwithall the studied fungi except of C. globosum at low concentration. Thegrowth of A. alternate and C. globosum was inhibited at the highconcentration of E. camaldulensis leaves essential oil where themarks recorded to be 4 and 5, respectively. The P. rigida wood oilvapor provides significant retaliation of mold growth on the surfaceof wood.

The chemical constituents of the essential oils analyzed by GC/MS were L-a-pinene, a-terpineol, borneol, and fenchyl alcohol asthe major components of P. rigida wood oil, and eucalyptol, a-pinene, g-terpinene, and terpinen-4-ol in the essential oil ofE. camaldulensis leaves.

The application by vapor essential oils, such as the oils fromP. rigida wood and E. camaldulensis leaves, can protect wood fromthe studied molds for a period of time under laboratory conditionsTherefore, further work is recommended to use these essential oilsas wood preservatives for outdoors, where the threat of raining, theanti-leaching properties have to be considered.

Conflicts of interest

The authors declare that there is no conflict of interest.

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