International Biodeterioration & Biodegradation 114 (2016) 87e93
Contents lists available at ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Fungicidal activity of essential oils from Brazilian Cerrado species against wood decay fungi Fernando C.M. de Medeiros a, Fernando N. Gouveia b, Humberto R. Bizzo c, udio H.S. Del Menezzi a, * Roberto F. Vieira d, Cla a
Departamento de Engenharia Florestal (UnB), Brasília, DF, Brazil rio de Produtos Florestais (SFB), Brasília, DF, Brazil Laborato Embrapa Agroindústria de Alimentos, Rio de Janeiro, RJ, Brazil d Embrapa Recursos Gen eticos e Biotecnologia, Brasília, DF, Brazil b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2015 Received in revised form 12 April 2016 Accepted 3 June 2016
This paper aimed to evaluate the fungicidal activity of essential oil from two Brazilian savannah species (Lippia origanoides Kunth and L. lacunosa Mart. & Schauer) and from clove [Syzygium aromaticum (L.) Merr. & L. M. Perry], tested against two wood decay fungi (Gloeophyllum trabeum and Trametes versicolor). Additionally, it was tested an adapted diffusion method along with digital images to assess the biological response. Four essential oil concentrations (100, 50, 25 and 12.5%) were tested. It was found that the essential oil from L. origanoides showed the highest fungicidal activity against G. trabeum and T. versicolor in any concentration tested. This activity is attributed to thymol, the major component of L. origanoides, and its several modes of action. L. lacunosa also showed fungicidal activity but dependent on the concentration used. The results showed that, in some cases, the essential oils presented higher inhibitions indexes than a commercial fungicide. The use of digital images has demonstrated the feasibility of making easier biological essays especially those in which there is growth without a specific geometric shape. Besides that, it gives a more sensibility and reliability to the process, since it is more accurate than human visual measurement. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Wood decay fungi Wood protection Essential oils/natural derived oils Digital image analysis Savanna vegetation Thymol
1. Introduction Although efficient, wood preservatives are toxic to man and to the environment (Ibach, 2005). New products to protect wood have been increasingly researched in order to reduce the damages and risks (Brand et al., 2006). One of the most promising alternatives is the employment of natural products from plants, such as essential oils (Singh and Singh, 2012). The development of a new preservative, however, requires essays not only in the laboratory, but also in the field to verify the efficiency and efficacy of the product. One of the principal barriers to the development of new preservatives, particularly the natural ones, is the discrepancy observed between results obtained among different tests (Singh and Singh, 2012). Currently, to attest the strength of a compound in wood protection three procedures have been adopted: (1) growth inhibition * Coresponding author. E-mail addresses:
[email protected] (F.C.M. Medeiros), fernando.gouveia@ florestal.gov.br (F.N. Gouveia),
[email protected] (H.R. Bizzo), roberto.
[email protected] (R.F. Vieira),
[email protected] (C.H.S. Del Menezzi). http://dx.doi.org/10.1016/j.ibiod.2016.06.003 0964-8305/© 2016 Elsevier Ltd. All rights reserved.
measurements on nutrient medium, (2) wood decay essays and (3) wood stakes field tests. Soil block tests are well established and usually brown and white-rot fungi are employed. Unlike other tests, there is not any standard procedure for the in vitro essays (growth inhibition measurements), which results in a wide range of different sort of experiments observed. When dealing specifically with new fungicides, there is basically two methods: (1) agar diffusion and (2) dilution (Pauli and Schilcher, 2010; Lang and Buchbauer, 2012; Saad et al., 2013). Within these two, there are many variations. In the diffusion method, a Petri dish fulfilled with solid nutrient medium is used, in which the essayed product is placed in the dish core. Therefore, the substance forms a gradient in the agar and the concentration in the medium is inversely proportional to the distance of the centre (White et al., 2001). The nutrient medium, however, must be previously inoculated with the microorganisms (Hammer and Carson, 2011; Pauli and Schilcher, 2010; White et al., 2001). The dilution method may be run in both liquid and solid medium (Saad et al., 2013). This technique requires the dilution of the target product
88
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93
in the nutrient medium and then the inoculation of the microorganism to be tested (White et al., 2001). Thereby, the minimum inhibitory concentration (MIC) is determined as the smallest concentration in which no growth of the microorganism is observed (Pauli and Schilcher, 2010). An important drawback of these methods is to measure precisely the degree of inhibition of the fungi growth. This way, in this paper, it is measured by using digital image processing procedure, which allowed calculating exactly the level of inhibition caused by the essential oil. Essential oils had proved a range of effects against insects (Isman et al., 2011), bacteria (Burt, 2004) and fungi (Singh and Singh, 2012; Boulogne et al., 2012; Graikou et al., 2012) that indicates a promising application in wood protection. The mechanism of action of the fungicidal activity of essential oils has been extensively discussed and several approaches have been presented. identified that a modification in the hydroxyl group (eOH) for less polar groups or no polar ones (acetyl or methyl) had a dramatic decrease in bioactivity of the compound making evident that the action is linked with the substance polarity (Cheng et al., 2008). On the other hand, the double bond position change turned the compound even more active. Isoeugenol has a more intense electron delocalization due to conjugated double bonds, which results, for example, in a higher antioxidant potential (Marteau et al., 2013). Phenolic compounds, such as thymol and eugenol, may inhibit wood decay fungi due to their ability to react with free radical and oxidants and also because of available hydroxyl groups (eOH) able to interact with metals (chelator effect) (Sova, 2012). These statements agree with the results of Cheng et al. (2008) who studied bioactivity of cinnamaldehyde and eugenol congeners. Their activity increased when there are conjugated bonds and acid or aldehyde groups since the electron delocalization effect is increased and thus the free radical scavenger potential. Hammel et al. (2002) suggest that reactive oxygen species are produced by reactions involving metals like Fe2þ demonstrating the role of some metals in the hole decay process. Therefore, as suggested by Schultz and Nicholas (2002), that antioxidant potential and metal chelators ability might have some influence in a wood preservative efficacy, because these properties affect many mechanisms developed by fungi to obtain food through wood degradation. In a recent paper, Teixeira et al. (2013) had evaluated the antioxidant activity of 17 commercial essential oils and found out that almost all oils containing phenylpropanoids or phenolic terpenes showed great antioxidant activity. Voda et al. (2003) evaluated the growth of Trametes versicolor and Coniophora puteana in nutrient medium filled with phenolic compounds and its derivates and it was noticed that thymol, trans-anethole, cumin aldehyde, carvacrol and methyl chavicol inhibited the development of wood decay fungi. In the same work, authors observed that less oxidized phenols had demonstrated higher activity reinforcing the theory in which antioxidant properties display a role in inhibition of wood decay fungi growth due to its higher free radical scavenging. Brazilian Cerrado, as the second largest Brazilian biome, with savanna-like vegetation, has a huge potential for producing the substances described above. Only a small fraction of the 12,000 known botanical species were chemically investigated, making Cerrado a very promising source for bioactive substances (Vieira et al., 2010). In spite of that several researches have been done to quantify and qualify essential oils of plants from Brazilian Cerrado, but few studies have focused on bioactivity of these compounds. In this context, the objective of this work was to assess the fungicide activity of the essential oil of two native species from the Brazilian Cerrado: Lippia origanoides Kunth and Lippia lacunosa Mart. & Schauer against wood decay fungi through an adapted diffusion method using digital images to assess the biological
response. Clove oil (Syzygium aromaticum (L.) Merr. & L. M. Perry) an active oil against decay fungi (Yen and Chanh, 2008; Ma-IN et al., 2014) was also employed, as a control, in order to verify and validate the methodology. 2. Materials and methods 2.1. Plant materials Aerial parts of Lippia origanoides were collected in May 2013 at the Embrapa Genetic Resources and Biotechnology campus. L. lacunosa material was collected at the Fazenda Agua Limpa, a protected area of the University of Brasília (UnB), in an area of Cerrado strictus sensu with a low human interference. Dried buds of Syzygium aromaticum were acquired in the local market. 2.2. Essential oil extraction and chemical analysis The essential oils were extracted by hydrodistillation of the dried material in a modified Clevenger-type apparatus for 3 h. The essential oils were collected, dried with anhydrous sodium sulfate and stored in the freezer for later analysis. The essential oils were analyzed in an Agilent 7890 A gas chromatograph fitted with a flame ionization detector (GC-FID) and equipped with a 5%-phenyl95%-methylpolysilixane (HP-5MS, 30 m 032 mm 0.25 mm) fused silica capillary column. The oven temperature was programmed from 60 C to 240 C at 3 C/min and the injector was kept at 250 C. Hydrogen was used as carrier gas at 1.5 mL/min. The oil was diluted at 1% in dichloromethane (v/v) and 1 mL of the solution was injected in split mode (1:20). The quantification of the constituents was done by normalized relative area of the peaks from a mean of three injections without using response factors. The mass spectra analyses were made in an Agilent 5973N gas chromatograph operating in the same conditions above. The carrier gas was helium (1.0 mL/min) and the mass detector was operated in electronic ionization mode (70 eV) at 3.15 scans/s with a mass range from 40 to 450 u. The linear retention indices (LRI) were obtained by the injection of a homologous serious of n-alkanes (C7eC28) in the same conditions and column as above. The indices were calculated by the Van Den Dool and Kratz (1963) equation. The identification of the compounds was made by comparison of their mass spectra with the NIST (NIST, 2013) database and the linear retention indices found in the literature (Adams, 2007). 2.3. Antifungal activity test and evaluation The antifungal activity of the essential oils was tested in Petri dishes (9 cm) filled with 15 mL of solid nutrient medium (30 g of malt extract and 18 g of agar in 1 L of deionized water). Both white rot fungus (Trametes versicolor) and a brown rot fungus (Gloeophyllum trabeum) were tested. In the centre of the dish, circular holes (5-mm) were made in the nutrient medium in which 20 mL of the three essential oils solutions were added (100, 50, 25, 12.5%, v/v in ethanol). Two 6-mm core of fungal mycelium cut from an actively growing colony were transferred onto the dish edges as shown in Fig. 1. Ethanol was used as a negative control while a commercial product whose active ingredient is tribromophenol (Jimo TBF Export 64 diluted in water, 4% v/v) as a positive control. The Petri dishes were incubated at 27 C and controlled humidity (70%) until the two mycelia from the negative control group reached each other. This took, in average, five days for the T. versicolor and 10 days for the G. trabeum. For each treatment 10 replicates were performed. The assessment of the essential oil concentration effect was established by the inhibition index according to eq. (1). This
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93
89
Fig. 1. Example of image processing: (a) actual color image, (b) gray scale image and (c) converted binary image.
equation was modified from literature to fit in this situation (Chang et al., 1999; Cheng et al., 2006):
Ið%Þ ¼
Aa 100 1 Ab
(1)
Where: I (%) ¼ percentage of growth inhibition index; Aa ¼ average area occupied by the culture in the presence of the tested product; Ab ¼ average area occupied by the culture in the presence of the solvent; The area occupied by the fungi culture was measured by digital photograph and image treatment.
different macro for each fungus was needed. The data was analyzed by analysis of variance (ANOVA) followed by Tukey test mean at a ¼ 0.05. 3. Results 3.1. Essential oils chemical composition analysis The essential oil yields (dry basis) were 2.92% (L. origanoides), 0.46% (L. lacunosa) and 11.2% (S. aromaticum). Ten compounds were identified in the oil of L. origanoides, 37 compounds in the oil of L. lacunosa and five compounds in clove oil (Table 1). The main compounds are thymol (L. origanoides), linalool (L. lacunosa) and eugenol (clove). 3.2. Bioactivity of the essential oils
The photos were taken with a digital camera Sony Cyber-shot DSC-WX7 (16.2 MP). The camera was adjusted without flash and zoom. The equipment was placed 12 cm vertically above the Petri dishes to be photographed. The images (RGB color, JPEG) were analyzed in the software ImageJ by the threshold process (Gonzalez and Woods, 2007; Marques Filho and Vieira Neto, 1999; Ferreira and Rasband, 2012). The color image was transformed in a gray scale image (8-bit) and then it was submitted to the threshold process resulting in a binary image in which the white area corresponds to the fungi occupied area. The process can be seen in Fig. 1. Images color and brightness were carefully manipulated in order to allow the image treatment. Too much light would reflect in the Petri dishes edges and would result in a wrong fungi area measurement. The image processing was used by macro programming. Because the fungi tested were from different colors, a
Fig. 2a and b shows the ordinary appearance of a Petri dish after the end of the experiment. Table 2 shows the growth inhibition index of the commercial fungicide for both fungi while Tables 3 and 4 show the growth inhibition indexes against G. trabeum and T. versicolor for the evaluated essential oils in the different concentrations tested. 4. Discussion 4.1. Bioactivity of the essential oils and possible mechanism of action The L. origanoides oil had a great antifungal activity as shown in Tables 3 and 4 These results are in agreement with some
90
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93
Table 1 Chemical constituents of L. origanoides, L. lacunosa and clove essential oils. Constituents
(E)-2-hexenol
a-pinene sabinene
b-pinene myrcene
a-terpinene p-cymene limonene 1,8-cineole (E)-b-ocimene g-terpinene linalool NId pinocarvone isopinocamphone terpinen-4-ol methyl salicylate myrtenol NI 2,6-dimethyl-3,5,7-octatriene-2-ol thymol methyl ether thymol myrtenyl acetate d-selinene eugenol a-copanene NI b-elemene gurjunene b-caryophyllene a-humulene g-muurolene germacrene D b-selinene d-selinene a-muurolene germacrene A g-cadinene d-cadinene eugenol acetate a-cadinene germacrene B germacrene- d-4-ol caryophyllene oxide NI epi-a-muurolol a-muurolol a-cadinol Total identified
LRIa
854 932 969 974 988 1014 1026 1024 1026 1044 1054 1095 e 1160 1173 1174 1189 1195 e 1207 1232 1289 1324 1335 1356 1374 e 1389 1409 1417 1452 1478 1484 1489 1492 1500 1508 1513 1522 1521 1537 1559 1574 1582 e 1640 1644 1652
LRIb
850 932 972 975 990 1016 1023 1027 1031 1045 1056 1101 1141 1159 1171 1179 1190 1193 1199 1207 1228 1289 1322 1333 1350 1372 1381 1389 1404 1413 1451 1471 1475 1479 1490 1495 1498 1508 1518 1511 1531 1549 1570 1575 1622 1635 1639 1647
Concentration (%)c L. origanoides
L. lacunosa
S. aromaticum
e e e e e 1.7 9.6 e 1.8 e 5.9 e 1.6 e e 0.9 e e e e 1.8 71.1 e e e 0.9 e e e 4.8 e e e e e e e e e e e e e e e e e e 98.5
0.3 2.2 0.3 2.5 0.3 e e 0.2 e 0.3 e 38.7 e 0.2 0.5 e e 0.5 0.2 0.4 e e 0.4 0.4 e 0.1 1.0 18.3 0.1 5.4 0.6 0.5 5.3 0.6 3.5 0.9 2.4 1.3 2.6 e 0.2 0.7 1.9 0.8 0.6 2.3 0.5 2.3 97.5
e e e e e e e e e e e e e e e e 1.3 e e e e e e e 81.6 e e e e 7.3 1.7 e e e e e e e e 8.1 e e e e e e e e 100
c The percentage composition was computed by the normalization method from GC peak calculated as mean values of three injections for each oil considering all response factors as 1 a Linear retention index from literature (Adams, 2007). b Linear retention index experimentally determined (van den Dool and Kratz, 1963). d NI ¼ compound not identified.
researchers that state this essential oil has biological action (Oliveira et al., 2007; Galvis et al., 2011; Veras et al., 2012). Nevertheless, this is the first time L. origanoides oil is tested against wood decay fungi. All works which describe its bioactivity agree that the observed effects come out because of the isomers thymol and carvacrol. The complete growth inhibition of G. trabeum in all concentrations tested proves the sensibility of this fungus against the oil. Although the growth inhibition of T. versicolor was lower, it was also very high especially considering that there was no significant difference between the three first concentrations (see Table 4). Even though L. origanoides oil had never been assessed against wood decay fungi, its main compound, thymol, has already been studied
to inhibit the growth of T. versicolor and Coniophora puteana and demonstrated a great capacity to avoid their growth (Voda et al., 2003). The comparison between the inhibitions indexes of the commercial fungicide and of the essential oils against G. trabeum (Tables 2 and 3) shows the superiority of the L. origanoides at any concentration, and of the S. aromaticum up to 50% concentration. Against T. versicolor (Tables 2 and 4) the three essential oils showed higher inhibitions indexes than that from commercial fungicide at least at 50% concentration. L. lacunosa oil was efficient in inhibiting the tested fungi development. The activity against T. versicolor was good, however, the inhibition index has decreased quickly, whilst, the inhibition of G. trabeum was reasonable. The L. lacunosa oil is rich in linalool
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93
91
Fig. 2. Fungi growth images at the end of the tests: (a) G. trabeum e (b) T. versicolor.
Table 2 Commercial fungicide inhibition index against G. trabeum and T. versicolor. Fungicide
Fungus
Jimo TBF Export 64 (4% v/v)
G. trabeum 96.81%
T. versicolor 59.99%
Table 3 Essential oil inhibition index tested against G. trabeum. Essential oil
L. origanoides L. lacunosa S. aromaticum
Concentration (%) 100
50
25
12.5
100.00aa 83.16a 100.00a
100.00a 32.71b 94.78a
100.00a 29.87bc 76.81b
100.00a 14.55c 26.04c
a At same line, values followed by the same letter indicate that there is no statistical difference according to the Tukey test at a ¼ 0.05.
Table 4 Essential oil inhibition index tested against T. versicolor. Essential oil
L. origanoides L. lacunosa S. aromaticum
Concentration (%) 100
50
25
12.5
100.00aa 100.00a 70.49a
98.85a 75.09b 67.06ab
94.50a 26.84c 54.58c
65.38b 1.19d 57.08bc
a At same line, values followed by the same letter indicate that there is no statistical difference according to the Tukey test at a ¼ 0.05.
(38.7%), a monoterpenic alcohol whose bioactivity is already known (Cheng et al., 2012). Cheng et al. (2006) demonstrated, however, that the activity of Cinnamomum osmophloeum essential oil rich in linalool is less intense against wood decay fungi than other chemotypes rich in eugenol or cinnamaldehyde. Oxygenated terpenes are more active to inhibit the growth of bacteria than hydrocarbon ones, meanwhile, phenols are stronger than alcohols, which in turn are more efficient than carbonyl compounds (Gallucci et al., 2009; Griffin et al., 1999). According to Gallucci et al. (2009) and Griffin et al. (1999), the main characteristic linked to bioactivity is the ability to make hydrogen bond. S. aromaticum essential oil, as expected, had a good activity, although not so strong as L. origanoides oil. There was no statistic difference between the higher concentrations against G. trabeum (100 and 50%), but the inhibition index has decreased quickly (Table 3). When analyzed beside T. versicolor, the oil demonstrated some activity.
There are plenty information about the biological activity of S. aromaticum oil against insects, bacteria and fungi (Ma-IN et al., 2014, Chaieb et al., 2007; Matan and Matan, 2007); Rana et al., 2011) showed that the antifungal activity is due to eugenol, the major component of the oil. In fact, Cheng et al. (2006) and Yen and Chanh (2008) demonstrated that eugenol is active against wood decay fungi especially against white rot fungi (Laetiporus sulphureus). In the same way, a lot of references state that among essential oils’ components, phenols, especially thymol, carvacrol and eugenol, have high antimicrobial action also against fungi (Saad et al., 2013; Burt, 2004; Lang and Buchbauer, 2012; Lima et al., 2013; Pauli and Schilcher, 2010; Scora and Scora, 1998). Nevertheless, the importance of antioxidant activity, as mentioned previously, it is not the main factor to decaying microorganism growth inhibition. As showed in this study, Voda et al. (2003) also demonstrated that thymol is more efficient than eugenol, even though the last one has higher antioxidant potential than the first one (Marteau et al., 2013), so it is not only one factor that will define the substance bioactivity. It is important to point out that oil containing thymol or its isomer carvacrol exhibit high antioxidant potential, but not as higher as oils containing eugenol (Teixeira et al., 2013). Saad et al. (2013) exposed that carvacrol action results from an ion gradient modification and assigned such effect due to the compound ability of bonding, as deprotonated, to cations and transfer them through the cell membrane. Lima et al. (2013) observed that the carvacrol toxic action against fungi do not evolve cations chelation, but cell permeability increase through cell membrane interactions. As carvacrol and thymol are very similar, it is believed that thymol behaves in a very close way and, as explained above, antimicrobial activity does not interfere in the other possible activities related to wood decay fungi: free radical scavenging and metal chelation. 4.2. Evaluation of the proposed methodology Although agar dilution method is the most used in scientific literature with essential oils, as mentioned, there are some situations where the diffusion methods may be an interesting alternative. Dilution technique has higher costs, demands laborious handling and the results are not absolute. Pauli and Schilcher (2010) explained that there are many variables in this kind of biologic essay (fungi mycelium size, pH of growth medium, incubation time and temperature) which may interfere in the results reproducibility. The important information understood through the aforementioned papers is that eugenol is a good product for further tests against wood decay fungi. However, it is possible to find it out
92
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93
through the diffusion method. In the present work, the growth inhibition index was comparable to a commercial product used against wood decay fungi indicating eugenol’s fungicidal activity. The disadvantage of using hydrophobic compounds does not forbid the achievement of valid results. In this study, thymol demonstrates high inhibition ability than eugenol even though the last is more soluble in water (2406 ppm) than the first one (846 ppm) (Griffin et al., 1999). On the other hand, diffusion method is much simpler, cheaper and demands less laboratory work to be performed, ideal characteristics for a preliminary assessment procedure. The possibility of having digital images and the employment of images processing techniques allowed the utilization of inoculation’s procedures more reproducible and less complex. There is no need to make solution with known quantities of fungi spores neither the risk of heterogeneous inoculation, because it can be used fungus mycelium, as in the dilution method. Through literature review there are not any papers, as far as we know, evaluating fungus area by digital image. All experiments studied needed a circular shape growth to enable area evaluating by physical measurement (Chang et al., 1999; Cheng et al., 2006). Thanks to the area calculation through image, fungi growth may have any shape and does not require anymore a circular shape. Although image measurements are commonly used in microbiological studies, it is employed in cell counting. Grishagin (2015) and Chiang et al. (2015) developed cell counting procedures and pointed out these techniques can achieve reliable results (acceptable errors) and can be 10 times faster compared to manual process. In the same way shown in these works, the image evaluation procedure adopted in this paper demonstrated to be easier and faster than physical measurements. In forest engineering, area measurements are important to establish leaf area. Patil and Bodhe (2011) described an image processing technique capable of calculated accurately leaf areas and salve time demonstrating again the reliability of image evaluation. Even though the benefits, the paper reported some errors because of plant disease or insect pests. The present work also had some of these troubles, as the two genres of fungus had not always the same colors. To overcome it, it was necessary to develop two different macros according to each fungus color. Santos et al. (2014), while measuring cacao genotypes leaf areas, also reported the necessity to adjust the method to the different genotypes. It was needed three different correction factors to accurately estimate the leaf area of seven cacao genotypes. It demonstrates the importance of assessing the algorithms for each fungus, bacteria or plant studied. The photograph action is another relevant issue. As explained in materials and methods section, image colors and brightness had to be carefully manipulated to enable good contrast among the fungus and environment. Chiang et al. (2015) worked with a black box completely closed, to avoid uncontrolled illumination, and a LED light to enable uniform illumination. In the present paper, light control was very important to avoid light reflection in the Petri dishes edges which would be miscounted as fungus surface and it was achieved by controlling lab’s illumination. Digital image evaluation approach can also be assimilated by agar dilution techniques since it is known the fungi growth is not always homogeneous and perfectly circular and the measurement by digital tools is more precise than physical scales. The employment of photographs and software to measure the inhibition index enable procedure automation by using the batch function in software ImageJ after the acquisition of the images. 5. Conclusion It was demonstrated that the essential oil from L. origanoides had the highest fungicidal activity against G. trabeum and
T. versicolor. Its ability was attributed to thymol high content, the major component, and its several modes of action. Considering the low toxicity of thymol to humans and the high oil yield (2.9%), both thymol and L. origanoides oil are potential raw materials to compose wood preservatives less aggressive to people and nature. The essential oil from L. lacunosa showed is also effective against T. versicolor. The results obtained showed that in some cases the essential oils tested in this work presented higher inhibitions index than a commercial fungicide. The next steps to ensure the essential oil of L. origanoides and L. lacunosa activity would be weight loss measurements on wood blocks (EN113) and, if verified its action, further investigation by longtime field essays. The employment of photographs to monitor the fungi growth has demonstrate the possibility to make easier biological essays, especially those in which there is growth without an specific geometric shape. Besides, it gives more sensibility and reliability to the process, since it is more accurate than human visual measurement. Even though its benefits, as far as we know, it is the first time a digital image technique is used to measure fungi growth. Therefore some tests must be performed to define its limits such as correlation with other techniques (physical measurement) and error evaluations. Acknowledgments ~o de Aperfeiçoamento de Authors are greatful to Coordenaça Pessoal de Nível Superior (CAPES), Conselho Nacional de Desen gico (CNPq), Fundaça ~o de Amparo a volvimento Científico e Tecnolo Pesquisa do Estado do Rio de Janeiro (FAPERJ), Universidade de Brasília (UnB), Serviço Florestal Brasileiro (SFB) and Empresa Braria (EMBRAPA) for financial support. sileira de Pesquisa Agropecua References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th. ed. Carol Stream: Allured Publishing Corporation. Boulogne, I., et al., 2012. Insecticidal and antifungal chemicals produced by plants: a review. Environ. Chem. Lett. 10, 325e347. http://dx.doi.org/10.1007/s10311012-0359-1. Brand, M.A., Anzaldo, J., Moreschi, J.C., 2006. Novos produtos para o tratamento ~o”. Floresta 26 (1), preservante da madeira. “Perspectivas da pesquisa e utilizaça 129e138. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods - a review. Int. J. Food Microbiol. 94, 223e253. http://dx.doi.org/ 10.1016/j.ijfoodmicro.2004.03.022. Chaieb, K., et al., 2007. The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother. Res. 21, 501e506. http://dx.doi.org/10.1002/ptr.2124. Chang, S.-T., et al., 1999. Antifungal compounds in the ethyl acetate soluble fraction of the extractives of Taiwania (Taiwania cryptomerioides Hayata) heartwood. Holzforschung 53 (5), 487e490. http://dx.doi.org/10.1515/HF.1999.080. Cheng, S.-S., et al., 2006. Chemical polymorphism and antifungal activity of essential oils from leaves of different provenances of indigenous cinnamon (Cinnamomum osmophloeum). Bioresour. Technol. 97, 306e312. http:// dx.doi.org/10.1016/j.biortech.2005.02.030. Cheng, S.-S., et al., 2008. Antifungal activity of cinnamaldehyde and eugenol congeners against wood- rot fungi. Bioresour. Tecnol. 99, 5145e5149. http:// dx.doi.org/10.1016/j.biortech.2007.09.013. Cheng, B.H., et al., 2012. Potential source of S-(þ)Linalool from Cinnamomum osmophloeum ct. linalool leaf: essential oil profile and enantiomeric purity. J. Agric. Food Chem. 60, 7623e7628. http://dx.doi.org/10.1021/jf302248w. Chiang, P.-J., et al., 2015. Automated counting of bacterial colonies by image analysis. J. Microbiol. Meth 108, 74e82. Dool, H.V.D., Kratz, P.D., 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatrography. J. Chromatrogr. 11, 463e471. Ferreira, T., Rasband, W., 2012. ImageJ User Guide. Disponivel em. Acesso em: setembro 2013. http://rsbweb.nih.gov/ij/docs/user-guide.pdf. Marques Filho, O., Vieira Neto, H., 1999. Processamento Digital de Imagens. Brasport, Rio de Janeiro. Gallucci, M.N., et al., 2009. Antimicrobial combined action of terpenes against the food-borne microorganisms Escherichia coli, Staphylococcus aureus and Bacillus cereus. Flavour Frag. J. 24, 348e354. http://dx.doi.org/10.1002/ffj.1948.
F.C.M. Medeiros et al. / International Biodeterioration & Biodegradation 114 (2016) 87e93 Galvis, L.B., et al., 2011. Antifungal, cytotoxic and chemical analysis of essential oils of Lippia origanoides H. B. K. in Colombia. Salud Uis Satander 43 (2), 141e148. Gonzalez, R.C., Woods, R.E., 2007. Digital Image Processing, 3. ed. PrenticeHall, Upper Saddle River, New Jersey. Graikou, K., et al., 2012. Chemical composition and biological activity of the essential oil from the wood of Pinus heldreichii Christ. var. leucodermis. Eur. J. Wood Prod. 70 (5), 615e620. http://dx.doi.org/10.1007/s00107-012-0596-9. Griffin, S.G., et al., 1999. The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour Frag. J. 14, 322e332. http:// dx.doi.org/10.1002/(SICI)1099-1026(199909/10)14:53.0.CO;2-4. Grishagin, I.V., 2015. Automatic cell counting with ImageJ. Anal. Biochem. 473, 63e65. Hammel, K.E., et al., 2002. Reactive oxygen species as agents of wood decay by fungi. Enzyme Microb. Tech. 30, 445e453. http://dx.doi.org/10.1016/S01410229(02)00011-X. Hammer, K.A., Carson, C.F., 2011. Antibacterial and antifungal activities of essential oils. In: Thormar, H. (Ed.), Lipids and Essential Oils as Antimicrobial Agents. Wiley, West Sussex, pp. 255e306. Cap. 11. Ibach, R.E., 2005. Biological properties. In: Rowell, R.M. (Ed.), Wood Chemistry and Wood Composites. CRC Press, Boca Raton, pp. 99e120. Cap. 5. Isman, M.B., Miresmailli, S., Machial, C., 2011. Comercial opportunities for pesticides based on plant essential oils in agriculture, industry and consumer products. Phytochem. Rev. 10, 197e204. http://dx.doi.org/10.1007/s11101-010-9170-4. Lang, G., Buchbauer, G., 2012. A review on recent research results (2008-2010) on essential oils as antimicrobials and antifungals. A review. Flavour Frag. J. 27, 13e39. http://dx.doi.org/10.1002/ffj.2082. Lima, I.O., et al., 2013. Antifungal activity and mode of action of carvacrol against Candida albicans strains. J. Essen. Oil Res. 25 (2), 138e142. http://dx.doi.org/ 10.1080/10412905.2012.754728. Ma-IN, K., Kittikun, A.H., Phongpaichit, S., 2014. Application of plant essential oils in prevention of fungal growth on Para rubber wood. Eur. J. Wood Prod. http:// dx.doi.org/10.1007/s00107-014-0790-z. Marteau, C., et al., 2013. Dual role of phenols as fragrances and antioxidants: mechanism, kinetics and drastic solvent effect. Flavour Frag. J. 28, 30e38. http:// dx.doi.org/10.1002/ffj.3123. Matan, N., Matan, N., 2007. Effect of cinnamon oil and clove oil against major fungi identified from surface of rubberwood (Hervea brasiliensis). In: The International Research Group on Wood Protection, vol. 38, pp. 1e5. Taipei. Proceedings… Stockholm: IRG/WP, 2007. IRG/WP 07-30446. NIST, 2013. National Institute of Standards and Technology - Spectral Database Fo Organic Compounds. NIST Webbook. Disponivel em. http://webbook.nist.gov/ chemistry. Accessed in November 2013. Oliveira, D.R., et al., 2007. Chemical and antimicrobial analyses of essential oil of Lippia origanoides H. B. K. Food Chem. 101, 236e240. http://dx.doi.org/10.1016/ j.foodchem.2006.01.022.
93
Patil, S.B., Bodhe, S.K., 2011. Betel leaf area measurement using image processing. Int. J. Comput. Sci. Eng. 3 (7), 2656e2660. Pauli, A., Schilcher, H., 2010. In vitro antimicrobial activities of essencial oils monographed in the European Pharmacopoeia 6th Edition. In: Baser, K.H.C., Buchbauer, G. (Eds.), Handbook of Essential Oils: Science, Technology and Applications. CRC Press, Boca Raton, pp. 353e548. Cap. 12. Rana, I.S., Rana, A.S., Rajak, R.C., 2011. Evaluation of antifungal activity in essential oil of the Syzygium aromaticum (L.) by extraction, purification and analysis of its main component eugenol. Braz. J. Microbiol. 42, 1269e1277. http://dx.doi.org/ 10.1590/S1517-83822011000400004. Saad, N.Y., Muller, C.D., Lobstein, A., 2013. Major bioactivities and mechanism of action of essential oils and their components. Flavour Frag. J. 28, 269e279. http://dx.doi.org/10.1002/ffj.3165. lise comparativa de me todos de determinaç~ Santos, S.N., et al., 2014. Ana ao de area tipos de cacau. Biosci. J. 30 (1), 411e419. foliar em geno Schultz, T.P., Nicholas, D.D., 2002. Development of environmentally-benign wood preservatives based on the combination of organic biocides with antioxidants and metal chelators. Phytochemistry 61, 555e560. http://dx.doi.org/10.1016/ S0031-9422(02)00267-4. Scora, K., Scora, R., 1998. Effect of volatiles on mycelium growth of Penillium digitatum, P. italicum and P. ulaiense. J. Basic Microbiol. 5 (6), 405e412. http:// dx.doi.org/10.1002/(SICI)1521-4028(199811)38:5/63.0.CO;2-2. Singh, T., Singh, A.P., 2012. A review on natural products as wood protectant. Wood Sci. Technol. 46 (5), 851e870. http://dx.doi.org/10.1007/s00226-011-0448-5. Sova, M., 2012. Antioxidant and antimicrobial activities of cinnamic acid derivatives. Mini-rev. Med. Chem. 12, 749e767. http://dx.doi.org/10.2174/ 138955712801264792. Teixeira, B., et al., 2013. Chemical composition and antibacterial and antioxidant properties of commercial essential oils. Ind. Crops Prod. 43, 587e595. http:// dx.doi.org/10.1016/j.indcrop.2012.07.069. Veras, H.N.H., et al., 2012. Synergistic antibiotic activity of volatile compounds from the essential oil of Lippia sidoides and thymol. Fitoterapia 83, 508e512. http:// dx.doi.org/10.1016/j.fitote.2011.12.024. Vieira, R.F., Bizzo, H.R., Deschamps, C., 2010. Genetic resources of aromatic plants from Brazil. Isr. J. Plant Sci. 58, 263e271. http://dx.doi.org/10.1560/IJPS.58.2.263. Voda, K., et al., 2003. Effects of the antifungal activity of oxygenatad aromatic essential oil compounds on the white-rot Trametes vesticolor and the brown-rot Coniophora puteana. Int. Biodeterior. Biodegrad. 51, 51e59. http://dx.doi.org/ 10.1016/S0964-8305(02)00075-6. White, D.G., et al., 2001. Antimicrobial resistance: standardisation and harmonisation of laboratory methodologies for the detection and quantification of antimicrobial resistance. Rev. Sci. Tech. Off. Int. Epiz. 20 (3), 849e858. Yen, T.B., Chanh, S.T., 2008. Synergistic effects of cinnamaldehyde in combination with eugenol against wood decay fungi. Bioresour. Technol. 99, 232e236. http://dx.doi.org/10.1016/j.biortech.2006.11.022.
