Leaf extracts of Casearia sylvestris and Casearia decandra affect growth and production of ligninolytic enzymes in wood decay basidiomycetes 1

(Leaf extracts of Casearia sylvestris and Casearia decandra affect growth and production of ligninolytic enzymes in wood decay basidiomycetes). White-rot basidiomycetes are able to deteriorate wood products and be pathogenic to living trees, requiring, thus requiring control. The tropical flora is an important source of eco-friendly antifungal compounds; however, the knowledge on how leaf extracts affect the fungal physiology is limited. Therefore, in the present work we investigated the influence of ethanolic leaf extracts of Casearia sylvestris and C. decandra at 0.1 mg mL-1 on the production of ligninolytic enzymes by Trametes villosa, Ganoderma australe and Pycnoporus sanguineus. Overall, the extracts inhibited the mycelial growth and the production of biomass. Additionally, C. sylvestris extract reduced the production of manganese peroxidase and laccase; however, the exposure to C. decandra extract resulted in variable responses. Therefore, enzymes related to lignin degradation are potential targets to control wood decay fungi by plant bioactive compounds, as their ability to colonize the substrate may be impaired.


Introduction
White-rot wood basidiomycetes perform an essential role in nutrient cycling in ecosystems.In addition, these fungi have unique enzymatic system with applications in bioremediation, pulp and paper, textile, and food industries (Elisashvili et al. 2010).However, under certain conditions, many fungal species can deteriorate timber products such as railway sleepers, utility poles, and fences, causing significant economic losses.Furthermore, such microorganisms can act as phytopathogens attacking living trees in urban and forest areas (Harsh & Bisht 1997, Luley 2005).
In order to degrade lignin, wood decay fungi produce a complex set of extracellular ligninolytic enzymes including manganese peroxidase (EC 1.11.1.13)and laccase (EC 1.10.3.2).These enzymes are able to degrade oxidatively the lignin shield around the cellulose and hemicellulose components of the plant cell wall (Lundell et al. 2010, Levasseur et al. 2013).Thus, the action of ligninolytic enzymes provides access to cell wall polysaccharides that subsequently are converted to soluble sugars by hydrolytic enzymes as cellulases and hemicellulases.Therefore, it is hypothesized that compounds able to inhibit the ligninolytic system of fungi may affect negatively colonization and degradation of plant materials as the accessibility to carbon source is impaired.
Traditional methods to control wood decay and phytopathogenic fungi employ synthetic and inorganic compounds that can adversely affect the environment as well as the human health.The increasing concern on the use of chemicals have stimulated the development of alternative methods to control wood deteriorating microorganisms including the use of microbial antagonists and natural compounds as plant extracts and oils (Singh & Singh 2012, Tascioglu et al. 2013).
Tropical flora is rich in species that may serve as a source of new antifungal compounds, natural, and environmentally less harmful than chemical biocides.The genus Casearia (Salicaceae) has been used in traditional medicine and several pharmacological properties have been attributed to these plants, including antimicrobial activity (Santos et al. 2010, Ferreira et al. 2011).In previous study, we reported for the first time the antifungal activity of Casearia extracts on wood decay fungi.Gas Chromatography Mass Spectrometry (GC/MS) analysis identified a clerodane diterpenoid in leaf extract of C. sylvestris, and hydroquinone, β-sitosterol and cinnamic acid in leaf extract of C. decandra (Bento et al. 2014).Many publications have described the antimicrobial activity of extracts from plant species; however, there are few studies on their mode of action.The knowledge on how plant-derived compounds affect fungal physiology allows to determine vulnerable enzyme systems to be explored as targets and to predict the risk of resistance development.
Trametes villosa, Ganoderma australe and Pycnoporus sanguineus are among the most important saprophytic white-rot wood basidiomycetes, able to degrade completely all components of lignocellulose.However, these fungi are also capable of causing important economic losses in wood materials and are associated with wood decay in living trees in tropical and subtropical areas (Harsh & Bisht 1997, Luley 2005).Therefore, the present study was aimed at assessing the activity of ethanolic leaf extracts of C. sylvestris and C. decandra on growth and production of ligninolytic enzymes of the three white-rot wood fungi.

Materials and methods
Leaf extracts -The ethanolic extracts from leaves of Casearia sylvestris Swartz (RM17) and C. decandra Jacq (M742) were obtained from the plant extract bank located in the Center for Plant Physiology and Biochemistry Research of the Institute of Botany, Sao Paulo, Brazil.The leaf extracts were previously analyzed by GC/MS (Bento et al. 2014).Antifungal activity -Twenty milliliters of autoclaved Potato Dextrose Agar (PDA) at 45 °C were added in 9 cm diameter sterile plates containing 0.025 g mL -1 of sugarcane bagasse powder.After solidification, a 9 cm diameter cellophane membrane was aseptically added on the medium surface.A 2 mm diameter agar plug containing mycelia of each fungus was transferred to the cellophane membrane-covered PDA medium and the plates were kept at 28 ± 2 °C for 4 days.Thereafter, the cellophane membranes containing the fungal colony were aseptically removed and transferred to new plates containing 20 mL of PDA medium supplemented with 0.025 g mL -1 of sugarcane bagasse powder and ethanolic extract at a final concentration of 0.1 mg mL -1 .In the control plates, the ethanolic extract was replaced by the same volume of ethanol 99.8%.The plates with the fungal colonies were maintained at 28 ± 2 °C and every 4 days, during 16 days, sample plates were collected in order to determine mycelial growth, biomass production (fresh weight), and to perform enzyme extraction.The experiment was carried out in triplicate.

Microorganisms -The basidiomycetes
Enzyme extraction -The cellophane membrane containing the fungal colony was removed from the plate and the solid medium used for extracting the enzymes secreted to the medium according to Ballaminut et al. (2014), with modifications.The solid medium was homogenized in 50 mM sodium acetate buffer, pH 4.5, in the proportion 1:3 (solid medium: buffer; w/v).The homogenate was kept under constant agitation at 140 rpm for 1 h at 4 °C and subjected to vacuum filtration.The filtrate was collected and kept at -20 °C for one month prior to the enzyme assays.The protein concentration was determined by the Bradford method (Bradford 1976), using bovine serum albumin as standard.
Enzyme assays -Laccase activity was determined by monitoring the oxidation of 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonate) (ABTS) (ɛ = 36000 mol -1 cm -1 ) at 420 nm for 10 min at 25 °C.The reaction mixture contained 250 µL of 50 mM citrate-phosphate buffer, pH 4.0, 50 µL of ultrapure water, 100 µL of 5 mM ABTS, and 600 µL of protein extract.Total peroxidase activity was determined by using the same reagents employed for laccase activity, excepting that ultrapure water was replaced by 50 µL of 2 mM hydrogen peroxide.Total peroxidase activity was given by the difference between the value obtained in the reaction and the laccase activity value (Ballaminut et al. 2014).
The enzyme activities were expressed as U mg -1 protein, where one unit (U) corresponded to the amount of enzyme that oxidizes 1 µmol of substrate per min under the assay conditions.
Statistical analysis -Analysis of variance (ANOVA) using Tukey's test at P < 0.05 was used to examine significant differences between treatments.All results were expressed as mean ± standard deviation (SD).

Results and Discussion
Leaf extract of C. sylvestris reduced the mycelial growth of T. villosa by 48 % in the first 4 days of exposure; however, after 16 days of treatment the fungus was inhibited only by 15 % when compared to the control (figure 1a).G. australe (figure 1b) and P. sanguineus (figure 1c) were more susceptible to C. sylvestris extract as the mycelial growth was reduced by 76 and 72 %, respectively, after 16 days of exposure.Overall, the inhibitory effect of C. decandra extract was less evident as the mycelial growth of the fungi was not significantly reduced at the end of the experiment.Interestingly, despite having limited effect on the growth rate on plate, C. decandra extract altered the morphology of the colonies that produced sparse aerial mycelium (data not shown).Bento et al. (2014) reported microscopic changes in hyphal morphology of T. villosa and P. sanguineus exposed to extracts of Casearia spp., including hyphal branching and segmentation, and formation of defective clamp connections.Production of sparse aerial mycelium by the fungi exposed to Casearia extracts can result in lower production of biomass.In fact, all fungi presented reduced biomass after 16 days of treatment, approximately 72 % of inhibition for T. villosa (figure 2a) and 90 % of inhibition for G. australe (figure 2b) and P. sanguineus (figure 2c).Therefore, the production of biomass can be considered a better parameter than the mycelial growth to evaluate the antifungal activity of the extracts.
Reports on the antimicrobial activity of Casearia extracts are scarce and have been restricted mainly to human pathogens.Extracts from five species of Casearia inhibited the human pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans (Mosaddik et al. 2004).According to the authors, leaf extracts of Casearia sp. and C. grewiifolia presented the highest antimicrobial activity with minimal inhibitory concentration corresponding to >5 mg mL -1 , concentration 50-fold higher than that used in the present study.Bento et al. (2014) identified a clerodane diterpenoid in leaf extract of C. sylvestris.Most of the pharmacological properties described for Casearia species are attributed to clerodane diterpenoids found mainly in leaves (Ferreira et al. 2011).Oberlies et al. (2002) isolated from leaves and twigs of C. sylvestris three clerodane diterpenoids denominated casearvestrins with cytotoxic activity on tumor cell lines and antifungal activity on Aspergillus niger.Bento et al. (2014) also identified hydroquinone, β-sitosterol and cinnamic acid in leaf extract of C. decandra.Studies have shown the antimicrobial activity of these compounds (Amoroso et al. 2009, Sova 2012, Wong et al. 2012), including the development of a wood preservative formulation based on cinnamic acid able to protected wood against T. versicolor for three months (Kartal et al. 2006).
Despite the reduced development of the fungi exposed to C. sylvestris extract, it was observed increased levels of extracellular proteins (figure 3).Overall, C. decandra extract did not affect the production of extracellular proteins by T. villosa and P. sanguineus; however, increased production of extracellular proteins by G. australe was observed after 8 days of exposure to the leaf extract.Bento et al. (2014) reported that exposure of T. villosa and P. sanguineus to C. silvestris and C. decandra extracts increased the production of catalase and glutathione reductase, enzymes related to defense against oxidative stress triggered by biotic and abiotic factors.Studies on proteins secreted by fungi exposed to natural compounds are scarce, although it is important to comprehend the physiological effects of these substances on target organisms.
It is essential for white-rot wood decay basidiomycetes to produce extracellular ligninolytic enzymes for complete lignocellulose decomposition.The leaf extracts of C. sylvestris and C. decandra affected significantly the production of manganese peroxidase and laccase by the three basidiomycetes (figure 4).Overall, the extract of C. sylvestris inhibited completely the production of the enzymes by the fungal species.Conversely, the results with C. decandra were variable, as its leaf extract inhibited completely the three enzymes in G. australe, and manganese peroxidase in P. sanguineus; but, laccase was not significantly affected.C. decandra leaf extract inhibited manganese peroxidase and laccase in T. villosa; however, at the end of 16 days, the fungus recovered its normal production of manganese peroxidase, and the production of laccase overcame the control treatment by 50 %.
In normal conditions (control treatment), it was not detected production of total peroxidases by T. villosa and P. sanguineus; however, this enzyme activity was observed in G. australe after 12 and 16 days of growth.Furthermore, the three fungi produced manganese peroxidase and laccase; but, T. villosa produced higher levels of laccase than G. australe and P. sanguineus.Moreira-Neto et al. (2013) studying 12 basidiomycetes also observed higher ligninolytic potential of T. villosa compared with P. sanguineus.According to the authors, strains of T. villosa produced high levels of manganese peroxidase and laccase whereas P. sanguineus produced only low levels of laccase.
It was observed increased levels of total peroxidases in T. villosa after 16 days of exposure to C. decandra extract, whose activity had not been detected in the control treatment.The increased production of total peroxidases in T. villosa may function as a defense mechanism against chemical stress caused by the aromatic compounds present in the C. decandra extract.Bento et al. (2014) observed increased levels of glutathione reductase in T. villosa exposed to C. decandra extract, indicating an oxidative stress process.
According to Souza et al. (2004), induction of laccases by aromatic compounds constitutes a protective response to toxic compounds generated during lignin degradation.Elisashvili et al. (2010) also observed increased production of laccase by T. versicolor in presence of hydroquinone, a compound present in C. decandra extract.Furthermore, it was observed induction of laccase in G. lucidum exposed to pyrogallol and 2,6-dimethoxyphenol.Terrón et al. (2004) reported that aromatic compounds, particularly, p-coumaric acid and guaiacol, increased the production of laccase by Trametes sp.I-62.The authors also observed that even structurally closerelated compounds have different effects on the expression of three laccase isozyme genes.Whiterot wood basidiomycetes display a wide diversity of responses to exposure to aromatic compounds.Induction or repression of ligninolytic enzyme encoding genes depends on fungal physiological, genetic, or ecological peculiarities (Myasoedova et al. 2008).
Therefore, fungal inhibition by the C. sylvestris leaf extract was accompanied by reduced production of ligninolytic enzymes; consequently, the ability of the basidiomycetes to colonize lignocellulose-based substrates may be impaired.In contrast, the inhibition by the C. decandra extract was not correlated to reduced enzyme production.Thus, further studies are necessary as the mechanisms involved in the inhibition of the fungi by natural compounds are probably complex and multifactorial.The present work provides useful information for the development of eco-friendly formulations based on Casearia extracts to preserve wood materials and to protect urban and forest trees against these wood decay fungi.

Figure 2 .
Figure 2. Production of biomass by T. villosa (a), G. australe (b) and P. sanguineus (c) after 16 days of exposure to leaf extracts of C. sylvestris and C. decandra at 0.1 mg mL -1 .Values are means of three replicates (± SD).Bars with the same letters are not significantly different at P < 0.05, Tukey's test.

Figure 4 .
Figure 4. Total peroxidase, manganese peroxidase and laccase activities in T. villosa (a, b and c), G. australe (d, e and f) and P. sanguineus (g, h and i), respectively, exposed to leaf extracts of C. sylvestris and C. decandra.The time 0 indicates the moment when 4 day-old cultures were transferred to PDA medium supplemented with sugarcane bagasse powder at 0.025 g mL -1 and leaf extracts at 0.1 mg mL -1 .Values are means of three replicates (± SD).ns = non-significant difference from the control at P < 0.05, Tukey's test.(--) Control; (--) C. sylvestris; (-▲-) C. decandra.