(2007) 465–470www.elsevier.com/locate/jmicmeth

Journal of Microbiological Methods 70

Activity of dehydroabietic acid derivatives against wood contaminant fungi

Sonia Savluchinske-Feio a,⁎, Lina Nunes c, Pablo Tavares Pereira b, Ana M. Silva a,José C. Roseiro b, Bárbara Gigante a, Maria João Marcelo Curto a

a Instituto Nacional de Engenharia e Tecnologia Industrial, Departamento de Tecnologia de Indústrias Químicas,Estrada ao Paço do Lumiar, 1699-038 Lisboa, Portugal

b Instituto Nacional de Engenharia e Tecnologia Industrial, Departamento de Biotecnologia, Estrada ao Paço do Lumiar, 1699-038 Lisboa, Portugalc Laboratório Nacional de Engenharia Civil, Av. do Brasil, 101, 1700-066 Lisboa, Portugal

Received 24 September 2006; received in revised form 11 May 2007; accepted 4 June 2007Available online 14 June 2007

Abstract

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and differentsubstituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. TheRelative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparisonwith those of control cultures; the results are expressed as EC50.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group ofcompounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means toinvestigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and insitu.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a goodlevel of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative toconventional fungicides.© 2007 Elsevier B.V. All rights reserved.

Keywords: Antifungal activity; Resin acid; Wood contaminant fungi

1. Introduction

Decay of wood by fungi results in devaluation from bothstructural and aesthetic standpoints and leads to economiclosses for wood producers. The fungi responsible for decay aredivided into white rots, brown rots, and soft rots (Zabel andMorrell, 1992). White rots break down lignin and cellulose, andcommonly cause rotted wood to feel moist, soft, spongy, orstringy and to appear white or yellow. Brown rots primarilydecay the cellulose and hemicellulose in wood, leaving behindthe brownish wood lignin. Wood affected by brown rot is

⁎ Corresponding author. Tel.: +351 217127250; fax: +351 217166966.E-mail address: sonia.feio@ineti.pt (S. Savluchinske-Feio).

0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mimet.2007.06.001

usually dry and fragile, readily crumbles into cubes because oflongitudinal and transverse cracks, and commonly forms a solidcolumn of rot in wood.

Soft rots are caused by both bacteria and fungi. They decaycellulose, hemicellulose, and lignin, but only in areas directlyadjacent to their growth. Soft rots grow more slowly than brownand white rots and usually do not cause extensive structuraldamage to wood.

Sapstain fungi are pigmented, they do not reduce the strengthof wood, but they do cause wood to stain detracting its appear-ance and posing a major economic problem for wood industry.

Mould grows on the surface of wood using sugars and othercarbohydrates. It gives the wood a ‘woolly’ or powderyappearance that does not affect the timber strength but discoloror stain infected timber.

Fig. 1. Dehydroabietic acid and derivatives tested.

Table 1Wood– decay fungi tested

Microorganisms

Wood surfacecontaminant fungi

Sapstain fungi Brown-rotfungi

White-rotfungus

Aspergillus niger L10 Aureobasidiumpullulans L3

Coniophoraputeana L54

Trametesversicolor L60

Rhizopus oryzae L122 Cladosporiumsphaerospermium L5

Gloeophyllumtrabeum L57

Penicilliumchrysogenum L34

Sclerophomapityophyla L43

Trichodermaharzianum L22

Trichodermakoningii L161

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The most common chemicals applied for wood decayproblems aiming at wood preservation have been creosote,pentachlorophenol, chromated copper arsenate (CCA), and morerecently, non-arsenic and non-chromium systems (Jorge et al.,2004). Although some of these formulationswere very efficient inextending the life of wood, the health hazard for operators and therisk of environmental impact in soil and landscape has to beseriously questioned.

Dehydroabietic acid, themain component of disproportionatedrosin, is a readily available hydrophenanthrene derivative whichbecame a useful starting material for the synthesis of industriallyand physiologically important products (Zinkel, 1975).

Several authors studied the antimicrobial activity of resinacids. Fukui et al. (1978) verified that pisiferic and dehydroa-bietic acids inhibited the growth of Gram-positive bacteria.Kobayashi et al. (1988) studied the antibacterial activity ofpisiferic acid derivatives against Gram-positive and Gram-negative bacteria, and concluded that the acid and hydroxylfunctions were the most important factors in the activity.

Franich et al. (1983) detected antifungal activity of oxidizedresin acid derivatives against Dothistroma pini, a leaf pathogenof young Pinus radiata.

In the course of photooxygenation studies on resin acidderivatives developed in our laboratory, new products wereobtained and several of them exhibited antimicrobial activity(Savluchinske Feio et al., 1997, 1999, 2002; Gigante et al.,2002).

In the search for alternatives, the use of natural plantproducts for the protection of wood is one of the most appealingfields of work with several reports being presented in this area(Bläske and Hertel, 2001; Cheng et al., 2004). The derivatiza-tion of natural products represents an additional approach forthe generation of compounds having desired biologicalactivities. In this work, dehydroabietic acid has been used as astarting material for the synthesis of biologically importantproducts (Savluchinske Feio et al., 2006).

The purpose of this study is the search of a benign alternativein wood protection. Ten dehydroabietic acid derivatives withdifferent A/B ring junction (natural steroidal or antipodal) withacid, ester, aldehyde or alcohol functionalizations at C-4 weretested against ten wood deteriorating fungi: moulds, sapstainand wood-rotting fungi.

2. Materials and methods

2.1. Test compounds

The compounds are C-4 functionalized derivatives ofdehydroabietic acid, trans-deisopropyldehydroabietic and cis-deisopropyldehydroabietic acids. Dehydroabietic acid, used asstarting material, was isolated from disproportionated rosin. Theisolation of dehydroabietic acid from disproportionated rosin(Halbrook and Lawrence, 1966) trans-deisopropyldehydroa-bietic, cis-deisopropyldehydroabietic acid and the C-4 deriva-tives (Fig. 1) were prepared by simple and sequential chemicalmodifications and their structures elucidated by physical andspectroscopic methods or by comparison with authenticsamples, as described earlier (Gigante et al., 2002).

2.2. Isolates

All the microorganisms were isolated from wood and belongto the Culture Collection of Timber Structures Division, LNEC,Portugal.

A list with the isolates with their specific characteristics isshown in Table 1.

Fungi were grown in malt extract agar, malt extract (Difco)20 g; dextrose (Difco) 20 g; bactopeptone (Difco) 1.0 g;bacteriological agar (Difco) 20 g, for 1000 mL of deionizedwater. The fungi were incubated at 25 °C during 4–7 days.

2.3. In vitro bioassay

The in vitro bioassay was performed according to Henrikset al. (1979). The test compounds dissolved in acetone weremixed with molten malt extract agar culture medium (culturemedium at 45 °C) in order to obtain 0.03%, 0.06%, 0.125%,0.25%, and 0.5% concentrations (test compound/culturemedium w/w) and distributed on Petri dishes containingsolidified malt extract agar. Control plates containing acetonewere also prepared. The plates were kept in a laminar flowchamber during 24 h in order to evaporate the solvent, andafterwards inoculated with agar plugs (7 mm diameter)containing active growing mycelia. The plates were incubatedat 25 °C during 4–7 days, after which the radial growth of the

467S. Savluchinske-Feio et al. / Journal of Microbiological Methods 70 (2007) 465–470

colonies was measured by comparison with those of controlcultures containing acetone.

The Relative Inhibition (RI) is calculated as follows:

RI ¼ ðDo� DÞ � 100Do

Do= Diameter of control coloniesD= Diameter of colonies treated with the test compounds

The results are expressed as EC50, the compound concen-tration required to inhibit the fungal growth at 50%. The testcompound concentrations are expressed on the abscissa with thegrowth inhibition (%) on a probability scale on the ordinate.

2.4. In situ bioassay

To evaluate a possible protection effect against fungal growthon green wood, a group of compounds was tested on Maritimepine (Pinus pinaster) sapwood. The efficacy against sapstain andmould fungi was performed according to previous methodology(Savluchinske Feio et al., 2004). The timber was stored in a deepfreezer (−18± °C) for 7 months. Blocks (50×50×8mm)were cutfrom green timber (mean initial moisture content expressed as apercentage of the oven dry weight of the timber was 137.0±32.8%) without visible staining.

Maritime pine wood blocks were immersed for 15 s in theselected chemicals: dehydroabietic acid (1) in the concentra-tions 1%, 2%, and 4% v/v in acetone, cis-deisopropyldehy-droabietanol (10) in the concentrations 1%, 2%, 4%, 6%, and8% v/v in acetone and resin (disproportionated rosin) in theconcentrations 1%, 2%, and 4% v/v in acetone. Untreated andacetone treated test specimens were used as negative controls.As positive controls, two commercial wood preservatives (Aand B) currently used in Portugal were also assayed in differentconcentrations in water. They were based, respectively, oncopper oxine (formulation A at 1%, 2%, and 4% v/v in water)and the other on a quaternary ammonium compound (formu-lation B at 2%, 4%, and 8% v/v in water).

Table 2Determination of EC50 for compounds 1–10 against wood-contaminant fungi

Fungi Test compounds (EC50)

1 2 3 4

A. niger 0.5 N0.5 0.03 0.1A. pullulans 0.5 N0.5 b0.03 N0.5C. puteana 0.5 N0.5 0.5 0.03C. sphaerospermium N0.5 N0.5 0.5 0.125T. versicolor 0.04 N0.5 b0.03 b0.03G. trabeum 0.25 N0.5 0.05 0.05P. chrysogenum N0.5 N0.5 N0.5 N0.5R. oryzae n. t. N0.5 b0.03 n. tS. pityophyla 0.5 N0.5 0.05 0.03T. harzianum 0.025 N0.5 b0.03 0.05T. koningii 0.05 N0.5 b0.03 b0.03

The effect of the test compounds, observed on the mycelial growth, is expressed as

After immersion, the blocks were left to stabilize for 1h which was needed to obtain full evaporation of the acetone.The treated and control blocks (6 replicates per variable understudy) were then inoculated, by spraying, with a 108 cells mL−1

fungal suspension (conidia) in physiological saline. The cellswere counted using a hematocytometer. The blocks were placedin plastic racks inside plastic bags at 22±2 °C and relativehumidity of 70±5% for 3 weeks.

Fungal growth was evaluated visually according to thefollowing scale 0=no growth; 1= traces of visible fungi;2=fungi established; 3=heavy sporulation; 4= full growth,underlying wood obscured. One way ANOVA and Tukey'sHonest Significant Difference (HSD) were used, whenappropriate, to compare the treatments tested. All differenceswere considered significant at pb0.05.

3. Results

3.1. In vitro bioassay

The antifungal activity of deisopropylated resin acidderivatives (5)–(7) and (8)–(10) was tested and comparedwith C-13 isopropylated analogues 1–4 against wood contam-inant fungi.

The results obtained with all the test compounds arepresented in Table 2. Among the compounds, only methyldehydroabietate (2) did not display antifungal activity againstthe microorganisms tested.

Dehydroabietic acid (1) showed good activity against Tra-metes versicolor (EC50=0.04) and both Trichoderma species,but presented weak antifungal activity towards all the otherfungi tested.

The absence of the isopropyl group in compounds (5) and(8) did not contribute to improve the activity (Table 2), pointingthat the stereochemistry of the molecule had no influence in theantifungal activity.

Compound (4), with an aldehyde function at C-4, revealed agood antifungal activity, but not as efficient as compound (3),with a hydroxyl function at C-4 that inhibited the growth of

5 6 7 8 9 10

n. t. N0.5 0.06 0.5 n. t. 0.05N0.5 N0.5 n. t. 0.5 N0.5 N0.5n. t. n. t. n. t. n. t. n. t. N0.5N0.5 0.5 b0.03 N0.5 N0.5 b0.030.05 n. t. b0.03 0.06 0.5 b0.030.5 0.5 b0.03 N0.5 N0.5 b0.03N0.5 0.5 b0.03 N0.5 N0.5 b0.03n. t. b0.03 0.03 n. t. n. t. b0.03N0.5 0.5 0.03 N0.5 N0.5 0.04n. t. n. t. 0.04. 0.05 n. t. 0.03n. t. N0.5 n. t. 0.05 n. t. 0.03

effective concentrations (EC50).

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several fungi, showing the importance of that group for theantifungal activity.

Cis-deisopropyldehydroabietanol (10) and trans-deisopro-pyldehydroabietanol (7) were the most active compounds(EC50 b0.03). However, Aureobasidium pullulans and Conio-phora puteana (EC50 N0.5) showed to be particularly resistantto the effect of these compounds. Penicillium chrysogenum issensitive to the action of the deisopropylated alcohol deriva-tives, but it is very resistant to the action of the othercompounds. The effect of cis-deisopropyldehydroabietanol(10) concentrations on fungal growth is presented in Fig. 2.Fig. 2a shows the effect of (10) in the inhibition growth ofwood surface contaminant fungi; P. chrysogenum was partic-ularly sensitive to that compound, and was inhibited at 100%even at the lowest concentration (0.03%). Fig. 2b shows thatamong sapstain fungi, Cladosporium sphaerospermium was themost sensitive (RI=100%) at concentrations comprised be-tween 0.06% and 0.5%. Gloeophyllum trabeum (Fig. 2c) wasinhibited at RI=100% at 0.06% concentration of (10) whereasC. puteana was inhibited at RI=80% at 0.5% concentration ofthe test compound. The white-rot fungus T. versicolor (Fig. 2d)was the most resistant among the fungi tested; RI=70% wasobtained at the highest concentration of cis-deisopropyldehy-droabietanol (10).

Fig. 2. (a) Antifungal activity of cis-deisopropyldehydroabietanol (10) agdeisopropyldehydroabietanol (10) against sapstain fungi. (c) Antifungal activity ofactivity of cis-deisopropyldehydroabietanol (10) against white-rot fungus. Concentr

3.2. In situ bioassays

The mean results and standard deviation of the absorption,colonization rating and final water content are presented inTable 3. The values of absorption obtained for acetone andacetone-diluted derivatives were extremely variable (68.5 to256.3 g−2) but no significant difference (ANOVA) was foundbetween the concentrations tested. In the case of the referenceproducts, used in water, no significant difference (ANOVA) wasdetected between treatments. The absorption can be considered,in the case of the present test, independent of the test product.

The average moisture content of the wood blocks at the endof the test varied between 46% and 63% being adequate for thedevelopment of the fungi involved.

As concerns the wood blocks, the values observed for thecontrol specimens (untreated and acetone treated) were asfollows: grade 4 was achieved in all test blocks. Theperformance of the reference products was also as expected,with control of sapstain and mould achieved for the two highestconcentrations in formulation A and the higher concentration informulation B.

Dehydroabietic acid did not show antifungal activity towardsthe fungi tested. The results obtained with the resin and cis-deisopropyldehydroabietanol were not conclusive for the 1%,

ainst wood surface contaminant fungi. (b) Antifungal activity of cis-cis-deisopropyldehydroabietanol (10) against brown-rot fungi. (d) Antifungalation is expressed in % (w/w).

Table 3Assays with wood

Test compounds Absorption (g.m−2) Moisture content (%) Colonization rating

Dehydroabietic acid 1% 136.24 (17.12) 45.99 (4.32) 4.00 (0.00) a2% 118.52 (9.15) 50.61 (1.43) 4.00 (0.00) a4% 103.68 (25.93) 53.47 (1.84) 4.00 (0.00) a

Cis-deisopropyldehydroabietanol 1% 120.72 (17.48) 51.37 (1.53) 3.67 (0.52) a2% 148.19 (60.43) 50.45 (3.05) 3.50 (0.49) a4% 111.09 (9.17) 50.09 (2.17) 3.33 (0.49) a6% 103.04 (19.32) 51.09 (2.17) 1.50 (0.44) bc8% 93.38 (23.07) 50.33 (3.29) 1.80 (0.43) b

Resin 1% 142.44 (21.96) 48.76 (2.29) 3.83 (0.41) a2% 125.38 (68.84) 51.80 (2.53) 3.83 (0.41) a4% 93.38 (23.07) 50.58 (2.04) 3.50 (0.55) a

Controls Absorption (g.m−2) Moisture content (%) Classification

Untreated acetone – 51.07 (2.72) 4.00 (0.00) a109.58 (27.03) 50.05 (3.34) 4.00 (0.00) a

Formulation A 1% 98.3 (23.31) 62.67 (0.56) 1.17 (0.75) bc2% 128.28 (12.98) 63.31 (0.80) 0.67 (0.52) c4% 126.99 (25.12) 63.49 (0.42) 0.83 (0.41) bc

Formulation B 2% 90.65 (25.63) 59.55 (6.96) 1.33 (0.52) bc4% 81.00 (24.22) 62.17 (2.00) 1.17 (0.41) bc8% 111.52 (13.04) 61.64 (1.05) 0.67 (0.82) c

Means and standard deviation of the absorption, final moisture, content and colonization rating. Means followed by the same letter are not significantly differentaccording to the Tukey's HSD test performed (pb0.05).

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2% and 4% concentrations as the increase of concentration ledto a little reduction in the fungal growth.

The results obtained with the two highest concentrations ofthe test compound (10) at 6% and 8% were not significantlydifferent from the reference products and a good level ofprotection of the wood against the organisms tested wasachieved.

4. Discussion

Natural occurring diterpenoids with a dehydroabietaneskeleton (ketones, alcohols, and phenols) are often found andisolated from plants and have been reported for their bioactivity(Ulubelen et al., 2000; Mensah et al., 2000).

The results obtained in the present work both in vitro and insitu show that the growth of several of the target fungi can bepartially or completely inhibited by some of the test compounds.

The compounds containing hydroxyl, ketone and aldehydefunctions inhibited several of the fungi tested. Henriks et al.(1979) observed that the oxidized resin acid 7-oxodehydroa-bietic displayed a strong antifungal activity towards Fomesannosus, a fungus responsible for one of the most importantdiseases of conifers (Annosus root disease) in the northtemperate zone of the world.

In the present work, the antifungal activity of dehydroabieticacid derivatives was increased by the presence of oxygenatedfunctions in the absence of the isopropyl group and showed tobe independent of the A/B ring configuration (cis/trans). Inprevious work (Gigante et al., 2002), we observed that thepresence of the isopropyl group decreased the antimicrobialactivity and that the stereochemistry of the molecule at the A/Bring junction did not display a significant role. From the results

obtained we concluded that the derivatization of the rosin acidderivatives could increase the bioactivity.

Generally, the resistance of fungi to the test compounds wasvariable between organisms of the same or different genus. Thereasons for differences in sensitivity could be associated withthe physiological way that different fungi may respond todifferent components (enzymes that inactivate the fungicide oralternative metabolic pathways in order to avoid the productreaction site). Such variation in antifungal activity may reflectdifferences in sites of action or the abilities of fungi to detoxifythe metabolites (Fiddaman and Rossal, 1994).

Also, differences in the physiology of the fungi could be anexplanation, as standard reproducible inocula is difficult, andfragments of mycelium as well as spores, origin colonies withina range of resistance (Gadd, 1989).

T. versicolor displayed a particular behavior in the presenceof cis-deisopropyldhydroabietanol (10). At the first threeconcentrations there is a reduction in the percentage of theinhibition growth, with an increase of this value in the lastconcentrations. That fungus is reported to do the biotransfor-mation of dehydroabietic acid (Van Beek et al., 2007) whichcould explain the results obtained in the present work.

Significant differences between the three higher concentra-tions of the test compounds were not observed (data not shown),which coincide with previous results with dehydroabieticderivatives (Savluchinske Feio et al., 2002). The hydrophobiccharacteristic of the compounds could contribute to a lessbioavailability of these poorly soluble compounds. Accordingto Sikkema et al. (1995), it is believed that only molecules ofcyclic hydrocarbons that are dissolved in the aqueous phase areavailable for intracellular metabolism. In order to decrease or toprevent mass transfer limitation, a co-solvent was used, that

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enables the substrate surface area to increase by dispersion ofthe particles. In the present work, high concentrations of the testcompounds reach a degree of saturation and may not bedissolved enough by the co-solvent (acetone). Therefore, it isless available to exert toxicity on the fungal cells. Anotherexplanation to the absence of significant differences betweenthe three higher concentrations of the test compounds could berelated, to the intern control exerted by the cell metabolism toavoid the excess of solute passage through the hyphae(Jennings, 1978).

The mode of action of this kind of compounds is poorlystudied. Studies by Transmission Electron Microscopy (Savlu-chinske Feio et al., 2002) on phytopathogenic fungi, Botryitiscinerea and Lophodermium seditiosum, treated with cis-7-oxodeisopropyldehydroabietate suggested an unspecific modeof action associated with a resistance mechanism based on theretention of that test compound by lipids in the young hyphae.

Nowadays, new wood preservatives are needed to replacetraditional compounds that have been banned or restricted dueto their general toxicity. Compounds derived of natural productsmay have less impact on the environment than synthetic agentsand would be more acceptable to consumers (Micales et al.,1994).

The antifungal activity of these dehydroabietic acid derivativessuggests their potential use as value-added wood preservativesbased on the selection of bioactive products aiming to obtain ahigher activity with less environmental impact.

Acknowledgements

Fundação para a Ciência e a Tecnologia, Projecto PRAXIS/PCNA/BIA/17.

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