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Biodegradation of eucalyptus urograndis wood by fungi
International Biodeterioration & Biodegradation 89 (2014) 95e102
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Biodegradation of eucalyptus urograndis wood by fungi Djanira Rodrigues Negrão a, *, Tadeu Antônio Fernandes da Silva Júnior b, José Raimundo de Souza Passos c, Cláudio Angeli Sansígolo d, Marli Teixeira de Almeida Minhoni b, Edson Luiz Furtado b a
Centro de Energia Nuclear na Agricultura (CENA), Universidade de São Paulo (USP), P.O. Box 96, CEP: 13400-970 Piracicaba, São Paulo, Brazil Departamento de Proteção Vegetal, Faculdade de Ciências Agronômicas (FCA), Universidade Estadual Paulista (UNESP), P.O. Box. 237, CEP: 18603-970 Botucatu, São Paulo, Brazil c Departamento de Bioestatística, Instituto de Biociências, UNESP, P.O. Box 510, CEP: 18618-970 Botucatu, São Paulo, Brazil d Departamento de Ciência Florestal, Faculdade de Ciências Agronômicas, UNESP, P.O. Box 237, Botucatu, São Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 7 January 2014 Accepted 8 January 2014 Available online 14 February 2014
We focused in selecting four fungi, naturally living in Eucalyptus sp. fields, for application in accelerating stump decay. The wood-rot fungi Pycnoporus sanguineus (Ps), Lentinus bertieri (Lb) and Xylaria sp. (Xa) were isolated from Eucalyptus sp. field and the fungus Lentinula edodes (Led) was obtained from a commercial strain. All fungi were studied according to their capacity to degrade eucalyptus urograndis wood. In order to evaluate mass losses of seven years old eucalyptus urograndis’ wood test blocks from heartwood were prepared added to glass flasks with red clay soil. The humidity of the soil was adjusted with 50 and 100% of its water retention capacity. Mass loss evaluations occurred at 30 until 120 days after eucalyptus wood degradation. Chemical analysis and soil pH were measured only in the last evaluation. Mycelial growth assays with potato-dextrose-agar, malt-agar and sawdust-dextrose-agar at three temperatures was carried out in order to get information about the best conditions of fungi growth. On the 120th day, Ps and Lb showed good capacity of wood degradation by leading to a high mass loss in soil with highest humidity. These fungi were the best consumers of lignin, hemicellulose, cellulose and extractives, caused acidification in the soil. Ps and Lb had faster mycelial growth in sawdust-dextroseagar, especially in high temperature, comparing to Xa and Led. Xa and Led are not good eucalyptus urograndis heartwood degraders, because they consume preferentially hemicellulose. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Eucalyptus stump White-rot fungi Mycelial growth Wood chemistry Soil pH
1. Introduction The Eucalyptus genus is the main forest specie cultivated in Brazil. In 2010, the planted area was around 4.9 million hectares (ABRAF, 2012). The most planted Eucalyptus tree in Brazil is the eucalyptus urograndis, generated from the crossing between Eucalyptus grandis Eucalyptus urophylla. Because of this, eucalyptus urograndis has no scientific name and is not considered specie by geneticists. The planting of Eucalyptus spp. can be done in new areas, replacing the pastures, using reformed areas and also by regrowth (ABRAF, 2012). The harvesting of planted forests is usually performed after the seventh year of planting with chainsaws or mechanical harvesters. The timber is removed leaving only leaves, bark, thinner branches and the stumps, which remain fixed by their
* Corresponding author. E-mail addresses: [email protected], [email protected] (D.R. Negrão). 0964-8305/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2014.01.004
roots. Seven years after the first planting, new seedling plants are planted in between the plants that were harvested. However, with successive plantings the stumps hinder management activities, such as transit machinery for planting and fertilization. After cutting the trees, mechanic stumps drawdown is performed to improve crop management. A complete withdrawal is required for the reuse of the area after the third crop rotation. This practice has a high cost, and usually, they are calculated by each hour worked. According to Pavan et al. (2010), for removing the stumps from 1 ha of a planted 21 year-old forest, is necessary 25 h costing approximately US$ 50.00/hr. The cost of stump removal does not vary by age, but by the density of the forest. For removing the roots, a widespread excavation is necessary, generally two or 3 m deep, to assess and remove the roots. Besides the high costs of Eucalyptus spp. production for companies and smaller producers, the environment also pays with grave disturbance. The stumps and roots removal not only causes severe disruption in the microbiota, but also modifies some
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physical and chemical characteristics of the soil for several years. After one year of removing roots, several changes occur in the soil, such as decrease in the concentration of nitrogen, carbon and sulfur, and increase in concentrations of phosphorus. It can take up to 10 years for nutrient levels to be normalized in soils after removal of these roots (Hope, 2007). In an attempt to minimize these problems, the biodegradation of roots has been studied. This may provide recycling of nutrients from the wood to the soil, promoting an increase of biodiversity and richness of microorganisms, also improving physical-chemical characteristics of the soil. Eucalyptus spp. reforestation areas with the system that maintain the soil undisturbed have high stable carbon content, with positive effects on the reduction of CO2 emissions. Regarding the organic matter present on the surface, the roots contribute for most stable forms of carbon in forest soils (Petersson and Melin, 2010; Kätterer et al., 2011). In nature, many fungi can degrade wood, but the best wooddegrading fungi are the basidiomycetes, such as white-rot fungi. These fungi are able to degrade efficiently wood components, especially lignin (Rayner and Boddy, 1988). The accelerated degradation of stumps and roots with white rot fungi has been studied as a way to prevent or reduce the disturbance of soil in planted forests. The degradation of Eucalyptus roots in reforestation fields is termed as “biological stump removal”, and consists of inoculating the stumps with wood degrading fungi. Some topics should be carefully considerate to achieve success in roots biodegradation, such as selection of the fungi according to their habitat, choosing the best season to inoculate (temperature, humidity) and confirm that the microorganism is not phytopathogenic (Alonso et al., 2007; Andrade et al., 2012). The great advantage of the “biological stump removal” application in Brazil is the high temperature in freshly cut areas of Eucalyptus fields, where the solar incidence is high during many months of the year. The temperature contributes to the activity of microorganisms that live in dead roots, reaching their climax in the range of 30e 40 C (Chen et al., 2000). The high frequency of rain during the summer also offers great conditions for fungi growth in Eucalyptus fields. The research of wood degradation by fungi is extremely important to forestry companies, because it enables proper management of the Eucalyptus roots and stumps in reforestation fields. Using properly, some fungi can be considered as a sustainable way to accelerate roots and stumps waste recycling. This study aims to select fungi with high wood degradation ability, in order to apply them in accelerating biodegradation of stumps and roots of Eucalyptus spp. 2. Materials and methods 2.1. Wood decay fungi During February 2009 till June 2011 we collected several fungi from Eucalyptus spp. fields located in southeast region of the State of São Paulo, Brazil. Pycnoporus sanguineus, Lentinus bertieri and Xylaria sp. were identified at Botanic Institute of São Paulo by Dr. Marina Capellari. The fungi P. sanguineus and L. bertieri were deposited in the Culture Collection of Algae, Cyanobacteria and Fungi of Botanic Institute e CCIBt, São Paulo-SP, Brazil, with respective numbers CCIBt 3817 and CCIBt 3818. Xylaria sp. and commercial isolate of Lentinula edodes (Led) are deposited in the Fungi Collection of the Mushrooms Module, Faculty of Agricultural Sciences, UNESP, Botucatu-SP, Brazil, with respective numbers BT23 and LED 98/47. The selection of these fungi between all those collected (around thirty isolates) was made by observations of mycelial growth in
different solid medium (described below) and Eucalyptus sawdust (data not shown). 2.2. Mycelial growth The mycelial growth of Ps, Lb, Xa and Led was evaluated in petri dishes with potato dextrose-agar, 2% malt extract agar (MA) and sawdust dextrose agar medium (SDA). The SDA medium was prepared according to Eira and Minhoni (1997), using Eucalyptus spp. sawdust, wheat and corn bran and calcium carbonate (41:1:1:1, v/ v) were mixed and autoclaved for 4 h. 80 g from this mix were boiled in 1 L of distilled water. After filtration of the boiled sawdust 12 g L1 agar were added to prepare the SDS solid media. All culture medium were autoclaved at 120 C for 20 min, and placed in disposable petri dishes (90 15 mm). The fungal growth was measured daily, until 7.8 cm diameter. The experimental design was completely randomized factorial with 4 3 3 (fungi culture medium temperatures), with five replicates. 2.3. Assay of accelerating wood degradation 2.3.1. Preparation of the experimental plots The experimental plots were based in the Standard ASTM D2017 (1997), using 600 mL glass bottles filled with 300 g of clay texture red soil, pH 4.7, water retention capacity of 29%. The soil was screened through a 5 mm mesh and dried in green house conditions (25 C, for 30 days). Soil humidity was obtained according to water field capacity. In the treatment with 50% (U50) 70 mL distilled water was used to get 50% of humidity and 140 mL to get 100% of soil humidity. After correction of the soil humidity, a Pinus taeda feeder (30 50 5 mm) was placed on the soil surface of each glass bottle and sterilized by autoclaving 121 C for 1 h during three consecutive days. Control treatments were divided in A, B and C. Control A was prepared with bottles with not sterilized dry soil. Controls B and C, with 50 and 100% of moisture, respectively, were sterilized. The experimental design was completely randomized in a 4 2 6 factorial (fungi humidity degradation evaluation time). The evaluation time of wood mass loss was at 30, 60, 75, 90, 105 and 120 days after inoculation, with eight replicates. 2.3.2. Eucalyptus wood samples In this study, the wood samples were obtained from 13 trees of the hybrid eucalyptus urograndis (named clone 105 urograndis) seven years old, donated by the Eucatex Company S/A. Each wood sample had 50 cm of length (cut from 10 cm above the ground), and stored under green house conditions (25 C, humidity bellow 20%) during 45 days. The bark and sapwood were removed, using only the heartwood to make the wood blocks, on dimensions of 25 25 10 mm (length, width, direction of the fibers). The inclusion criteria for the use of wood blocks were: no defects, as knots, resin or gums, and without visible evidence of fungus infection. From each tree sample were made approximately 200 wood blocks, totaling 2600 samples, which were mixed among the thirteen samples. Each wood block was sanded (emery 20 mm), labeled and dried in the oven (102 3 C) until reached constant weight (Gehaka BG 400). The initial mass (W1) of each sample was measured, stored in glass Petri dishes (120 by 20 mm) and sterilized by autoclaving at 120 C for 15 min. 2.3.3. Wood inoculation The parameters for accelerated degradation of wood by the fungi were based on Standard ASTM D-2017 (1997) and ASTM D1413 (1994). A 7 mm diameter mycelial disc of each fungus
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cultivated in PDA at 25 C during five days, were placed on the P. taeda wood chips under aseptic conditions. The flasks were placed in a climate-controlled room at 25 2 C, 70 10% of relative humidity, in the dark, during 25 days for complete Pinus wood colonization. After that, the wood blocks were placed under the mycelium, i.e. colonized P. taeda, and the time for all evaluations was calculated from this moment. Over the evaluation period, the mycelium developed under the wood blocks was manually removed under tap water; the wood of the controls treatments were also washed. All samples were placed in the oven (102 3 C) to reach constant mass (W2), and the mass loss (M) of each wood block was calculated using the following equation: M(%) ¼ [(Wi W2)]/W1] 100. 2.4. Chemical analysis Twenty-five additional bottles of each treatment were prepared for chemical analyses of wood components such as extractives, lignin, cellulose, hemicellulose and wood solubility. After 120 days of wood biodegradation, 25 wood blocks of each treatment were washed in tap water, cut manually in small pieces, mixed and dried at 45 C. The wood samples were analyzed in duplicate. Nonautoclaved wood (control A) and controls treatment B and C were also analyzed in duplicate and used as a reference. For chemical analysis was necessary to transform wood blocks in sawdust, using a macro Willey mill (10 mesh screen), and classified in 40e60 mesh (Fraction 40/60), which were used for all chemical analyzes. The preparation of wood for chemical analyzes was based on TAPPI Standard T264 cm-97 (TAPPI, 1997a). For extractive analyzes was used the Standard T204 cm-97 (TAPPI, 1997b), for Klason lignin was used the Standard TAPPI T222 om-83 (TAPPI, 1999b), for hemicellulose analysis the Standard TAPPI T249 cm-85 (TAPPI, 1999a) and for cellulose analysis was used the methodology according to Santos (2000) and Wright and Wallis (1998). The solubility of the wood in NaOH 1% was analyzed according to Standard T212om-02 (TAPPI, 2002). The experimental design was completely randomized in a 7 x 2 factorial (fungi and controls treatments soil humidity), in duplicate. The wood chemical analyses were performed in the Department of Forest Sciences, Laboratory of Wood Chemistry, FCA/ UNESP, Botucatu, São Paulo, Brazil. 2.5. Soil pH analyses Ten flasks were used to analyze the soil pH from the same glass bottles used in chemical analysis. Twenty five grams/flask of soil close to Pinus feeder were removed and mixed with 25 g from another flask, totalizing five samples from 10 flasks. Soil pH was analyzed in water, in duplicate. The experimental design was completely randomized in a 7 x 2 factorial (fungi and controls soil humidity), with five replicates. The analyses were carried out in the Department of Naturals Resources, Laboratory of Soil Science, FCA/ UNESP. 2.6. Statistical analysis A generalized linear model with gamma distribution error and log link function (Nelder and Wedderburn, 1972; Liang and Zeger, 1986) was fitted for the variables (covariates in the brackets) describes mycelial growth of wood degrading fungi (specie, temperature and time); assay of accelerating wood degradation (specie, humidity and time); chemical analysis of the eucalyptus urograndis wood (specie and humidity) and analysis of soil pH (specie and humidity). The adjustment of the models was measured by analysis of deviance (Nelder and Wedderburn, 1972).
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For calculating the correlation between wood mass losses and chemical components it was necessary to have the data on ordered pairs, or format data sets in the same dimension, considering only the last evaluation period (120 days). The average percentage difference was obtained for each combination, as follows: 1. between humidity and specie, for the wood degradation assay and, 2. between specie and soil humidity, for the average percentage differences of chemical compounds. 3. Results 3.1. Mycelial growth The substrate and the temperature influenced the mycelial growth of all fungi (Fig. 1). The fungus P. sanguineus grew faster in SDA medium, followed by MA, mainly at higher temperatures. L. bertieri did not suffer difference in its dynamic mycelial growth, in PDA and SDA medium at 27 and 31 C. Xylaria sp. had better mycelial growth in SDA medium, while its growth in PDA was slower; this fungus grew better at 27 and 31 C. L. edodes grew faster in SDA medium at 27 C. The same fungus had a long lag phase in the BDA e MA culture medium, mainly at 31 C. SDA medium was the best substrate to provide faster mycelial growth for all fungi evaluated. In vitro assays can give important insights for predicting the behavior of fungi in vivo (Gomes et al., 2007). This knowledge contributes to obtain the optimum conditions for fungi growth on a particular substrate under a range of temperature, besides incubation time. 3.2. Accelerated degradation of the eucalyptus wood Comparisons between biodegraded and non-biodegraded wood can be done according to the values observed in control A, which is the intact form of timber (Table 1). This ensures that the degradation of cellulose, hemicellulose, lignin and solubility levels presented were the result of biological activity (Paes et al., 2007). Large variation of wood mass loss was observed according to soil humidity. The lowest rates were observed in the U50 treatment, while in the U100 treatment we observed mass losses around of 50%. There was triple interaction between the fungi, humidity and time. Regardless to wood colonization, in 14 days P. sanguineus, L. bertieri and Xylaria sp. colonized completely P. taeda wood and L. edodes required 25 days to colonize the same wood specie. In U50 treatment, it is likely that the low amount of water damaged the mycelial growth of L. edodes, observed by its low strength on the Pinus and eucalyptus urograndis wood until the last assessment period. On the other hand, this fungus also had difficulties growing at U100 in both types of wood. Because of this, the assessments of mass loss in this treatment were made only at 75 and 120 days. Even under these conditions we observed reduction in mass loss of wood during the assays, exhibiting ability to adapt to adverse conditions. At 120 days, in the U50 treatment, the difference in mass loss between P. sanguineus and L. bertieri was of 22%. Among P. sanguineus and L. edodes was 11%. In the U100 treatment the difference in weight loss between P. sanguineus and L. bertieri had increased to 45%; with L. bertieri and L. edodes was 69%, and between L. bertieri and Xylaria sp. the difference was of 97%. Statistical differences between P. sanguineus and L. bertieri occurred at 60, 105 and 120 days. Eucalyptus urograndis wood mass losses caused by Xylaria sp. were not significant in either treatments, but there was a greater tendency of wood degradation in U100 treatment, being 38.4% higher than the U50 treatment, at 105 days. Low degradation ability
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Fig. 1. Mycelial growth of Pycnoporus sanguineus (1), Lentinus bertieri (2), Xylaria sp. (3) and Lentinula edodes (4) in PDA (A), SDA (B) and MA (C) solid media at 23 C (rhombus), 27 C (square) and 31 C (triangle).
can occur by its impartial colonization under and into the wood, which was observed in all eucalyptus urograndis wood. Unlike L. edodes, Xylaria colonized better P. taeda wood than eucalyptus urograndis wood. 3.3. Wood chemical analysis The consumption rates (%) of the chemical components of wood depended on the combination between species and moisture (Table 2). Through the chemical analysis we could identify which wood components were preferentially consumed by each fungus and thus, some physiological characteristics of each species could be observed.
All U50 treatments had higher extractives content from its degraded wood when compared to extractives content obtained from control A. In the U100 treatment an increase in the percentage of extractives content was observed in treatments with L. bertieri and L. edodes. The wood degraded by P. sanguineus had low extractives content. The largest consumers of lignin in U50 treatment were P. sanguineus and L. edodes. P. sanguineus and L. bertieri were the best lignin consumers at U100. In U50, cellulose content was low in the wood degraded by P. sanguineus and L. bertieri, differing between them in 8.6%, while in U100, this difference increased to 33%. Low cellulose consumption was observed in wood degraded by L. edodes and Xylaria sp.
Table 1 e Average difference of eucalyptus urograndis wood mass loss (interquartile in brackets), according species of fungi and soil humidity (H%) and incubation time (T), in days. H%
T
Species P. sanguineus
50
100
30 60 75 90 105 120 30 60 75 90 105 120
2.70 2.98 2.21 2.15 6.92 8.01 9.14 23.50 19.57 27.16 41.97 49.70
(2.76) (3.02) (2.21) (2.69) (6.91) (14.10) (5.16) (11.01) (15.29) (11.98) (30.11) (10.23)
L. bertieri AaA AadA ABbA AaA BCaA CbA Aab Bbb Bab Bab Cbb Ccb
2.91 2.82 2.23 3.36 7.48 6.72 9.34 16.32 18.58 21.67 28.95 28.17
(1.19) (0.76) (0.20) (3.39) (9.17) (10.0) (5.17) (7.64) (12.1) (9.94) (29.1) (6.59)
L. edodes ABaA ABabA AaA BaA CaA CabA Aab Bab BCab CDab DEab Eab
2.55 (0.61) 3.33 (0.70) 2.29(0.81) 4.12 (3.18) 5.81 (6.19) 7.38 (6.60) e e 9.31 (9.88) e e 10.29 (5.25)
Xylaria sp. ABaA ABbA AaA BaA CaA CaA e e Abb e e Abb
2.80 (0.51) 2.21 (0.28) 2.15 (0.15) 2.20 (1.90) 3.12 (3.12) 2.95 (1.35) 3.02 (0.72) 3.19 (1.80) 3.37 (1.41) 5.56(2.95) 5.07 (5.33) 3.77 (2.48)
Control Abbaa Aca Aaa BCaa BCba Cca Abb ACcb ADcb Bbb BEcb CDEda
2.71a 2.72 2.80 2.14 2.26 2.30 2.67b 2.89 2.78 3.05 3.22 2.50
(2.65) (2.54) (2.26) (2.11) (2.00) (0.19) (2.66) (2.03) (2.77) (2.96) (0) (0.12)
ABaa ABdca ABaba Aaa Bba Aca ADba ABca ACca ABcb CBda Deb
Average difference medians followed by the same lowercase letter line did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each soil humidity (H%) and incubation time (T). Average difference medians followed by the same uppercase letter column did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each soil humidity (H%) and species. Average difference medians followed by the same greek letters column did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each incubation time (T) and species. * Control B, subjected to 50% humidity; ** Control C, subjected to 100% humidity.
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Table 2 Average percentage of the chemical compounds of eucalyptus urograndis wood (percentage of compound losses in black italic) degraded by fungi, according to soil humidity (H %), at 120 days. H%
Treatment
Extractives
Lignin
Cellulose
Hemicellulose
Solubility
0
Control A
4.8
23.3
38.1
36.3
12.99
50
P. sanguineus L. bertieri Xylaria sp. L. edodes Control B P. sanguineus L. bertieri Xylaria sp. L. edodes Control C
5.4 6.3 5.2 6.5 5.2 4.2 5.8 4.8 6.4 4.8
100
12.5 31.2 8.3 35.4 8.3 12.5 20.8 e 33.3 e
21.1 21.7 23.9 21.5 22.6 12.2 16.4 22.8 20.7 23.7
9.4 6.9 2.6 7.7 3.0 52.4 29.6 2.1 11.1 1.7
34.6 34.9 37.0 36.6 38.0 20.0 26.0 35.7 35.8 38.5
9.2 8.4 2.9 3.9 0.2 47.5 31.7 3.3 6.0 1.0
32.0 31.2 34.0 30.6 33.5 18.0 24.2 32.8 29.9 32.6
11.8 14.0 6.3 15.7 7.7 50.4 33.3 9.6 17.6 10.2
18.2 17.4 16.1 17.2 15.4 19.5 21.0 15.8 18.5 15.9
40.1 33.9 23.9 32.4 18.7 50.5 61.7 21.6 42.4 22.4
Average calculated by two replicates, all analyses were made in duplicate.
Hemicellulose contents in controls B and C were high comparing to control A, and also compared to the others wood components analyzed. In U50 treatment, L. edodes and L. bertieri were the best degraders of hemicellulose. In U100 treatment, the biggest consumers of this component were P. sanguineus and L. bertieri, with the difference between them of 34%; between P. sanguineus and L. edodes the difference increased to 65%. Regarding wood solubilization, which indicates wood degradation, L. bertieri and L. edodes produced wood solubilization at similar rates in the U50. Xylaria sp. produced less wood solubilization and P. sanguineus caused the largest wood solubilization rate. In U100 treatment, L. bertieri caused increase in the solubility levels of wood at a higher rate than P. sanguineus. This may indicate that the ability of L. bertieri to degrade eucalyptus urograndis wood can become more severe over time. Data on correlation values between wood mass losses and chemical components that remaining after 120 days of biodegradation are showed in Fig. 2 and Table 4. The data for Control A was not used because there is no equivalent data in the wood degradation assay. There was high correlation (p < 0.001) between the mean difference percentage of wood mass loss and mean differences of wood components, except for extractives. This means, for example, when the average mass loss mass decrease (no degradation occurs) the average cellulose content increases. Therefore, the wood biodegradation influences directly cellulose, hemicellulose and lignin contents. Regarding the solubility, the opposite was observed. In addition, Fig. 2 shows a close profile between lignin, hemicellulose and cellulose at the maximum point and a positive correlation profile for solubility.
Soil pH There was interaction between species and humidity, and significant effect of humidity on the soil pH (Table 3). In U50 treatment neither fungus modified the soil pH, comparing to control. In this treatment, P. sanguineus, L. bertieri and L. edodes colonized the soil with apparently fragile mycelium; Xylaria sp. colonized briefly the soil close to the P. taeda wood, at least to the naked eye, because of its black color. In the U100 treatment, P. sanguineus, L. bertieri and L. edodes produced mycelium intensively branched into the soil. P. sanguineus and L. bertieri changed the pH of the soil causing its acidification. There was no statistic difference in the pH of soil colonized by L. edodes and the controls. 4. Discussion Mycelial growth of all fungi was bounded mainly to the temperature. P. sanguineus, L. bertieri and Xylaria sp. preferred high temperatures, growing faster at 27 and 31 C, while L. edodes grew faster only at 23 C. In natural environment, these fungi can be exposed to high temperature, very common in areas of Eucalyptus spp. reforestation. Detailed study regarding mycelial growth is important to manage the best season for inoculating these fungi in the fields, as Gomes et al. (2007) did with Armillaria sp., finding the optimum temperature for growing this fungus. According to Chen et al. (2000), an increase in temperature can be better for enzymatic activities and to metabolize nutrients obtained through wood decay. Besides temperature, the type of lignocellulosic substrate and other nutrients added can interfere with the fungal growth. When sugars are added to the culture medium, fungi usually use it as a primary source of energy to supply for its growth and vital
Table 3 Measurements of soil pH at 120 days from samples with 50 and 100% of humidity colonized and non colonized (Control) by fungi. Treatments
Humidity (%) 50
P. sanguineus L. bertieri Xylaria sp. L. edodes Control Fig. 2. Average percentage of the eucalyptus urograndis wood mass loss and chemical components after fungi degradation, at 120 days.
4.77 4.82 4.69 4.71 4.67
100 (0.18) (0.14) (0.01) (0.10) (0.06)
Aa Aa Aa Aa Aa
4.11 4.30 4.32 4.89 4.82
(0.03) (0.09) (0.07) (0.10) (0.02)
Ab Ab Bb Cb Ca
Average difference medians followed by the same lowercase (line) or uppercase (column) letter did not differ statistically among themselves by the LSMEANS Test (p > 0.05).
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Table 4 Means correlations values of the average percentage and p-values of eucalyptus urograndis wood mass losses and chemical components remaining after fungi degradation, at 120 days. Treatments
Correlation (r)
p-value(*)
Cellulose Extratives Hemicellulose Holocellulose Lignin Solubility
0.9904 0.2802 0.9820 0.9938 0.9895 þ0.8499
<0.0001 0.4329 <0.0001 <0.0001 <0.0001 0.0018
(*)
p-values related to the hypotheses:H0 : r ¼ 0vs. H0 : rs0
functions, and also to consume others complex substrates, like lignin (Kües and Liu, 2000). In this study, we observed fungi growth preference to SDA medium, made from Eucalyptus spp. Thus, one of the most important steps to understand what makes fungi able to colonize the wood is to characterize their nutritional needs (Rayner and Boddy, 1988). Although PDA and MA culture medium are commonly used in microbiology laboratories, they can disturb the metabolism of some fungi species, affecting the velocity of its growth and causing the suppression of the lignocellulosic system (Lonergan et al., 1993; Kachlishvili et al., 2005). With respect to wood degradation, we notice that the sterilization process by autoclaving caused mass loss in eucalyptus urograndis heartwood, as observed in controls B and C. The autoclaving process can cause extractives degradation and/or evaporation, as observed by Guilmo et al. (1993).) Wood mass loss after heating treatment is due primarily to the removal of wood extractives, such as separation of acetyl groups linked to xylan chains and dissolution of low molecular weight carbohydrates, and in some cases, even lignin. In this study, we found wood mass loss around 3%, similar to the 14 year-old Eucalyptus saligna wood treated by heating (Brito et al., 2008; Lourenço et al., 2011). In relation to the heartwood mass loss in the U50 treatment, after 120 days of degradation, the percentage difference in mass loss between P. sanguineus and L. bertieri was 22%, and between P. sanguineus and L. edodes only 11%. Similar results of low mass loss caused by rotting fungi in Eucalyptus wood were observed by Oliveira et al. (2005) and Aguiar et al. (2013) using Gloeophyllum trabeum. Under conditions similar to this study, the authors also observed mass loss in Eucalyptus and Pinus woods were below 10%. In other hand, when the E. grandis wood is submitted to termallymodified technique, the degradation ability of white and brown rot fungi can vary according to heat treatment. The brown rot fungus G. trabeum caused 53% of mass loss in Eucalyptus wood termally-modified at 200 C while the white rot fungi P. sanguineus caused 31% of mass loss (Calonego et al., 2013). P. sanguineus has great lignocellulolytic ability, but this can vary according to the substrate. By using this specie, Ferraz et al. (1998) observed mass loss of E. grandis wood around 23% after 120 days of degradation, whereas Silva et al. (2007) observed 9% of mass loss. However, other types of wood can also be susceptible to this fungus, such Fagus sylvatica and Pinus sylvestris woods (Poiting et al., 2003). In Brazil, some studies related to accelerated degradation of stumps and roots of Eucalyptus sp. were carried out by Alonso et al. (2007) and Andrade et al. (2012). Alonso et al. (2007) observed 26% mass loss in E. saligna wood caused by P. sanguineus. Andrade et al. (2012) observed 27 and 29% mass losses in E. grandis wood caused by P. sanguineus and L. edodes, respectively. However, these authors used different fungal growth conditions and also of nutrients and they did not study the effect of the humidity in wood decay. Under the conditions evaluated in this study P. sanguineus and L. bertieri caused mass loss near to 50% and 28%, respectively, at the higher
soil humidity. These rates are very relevant for the selection of these fungi to degrade the eucalyptus urograndis wood and much higher than previous studies. In the literature, there are few studies about L. bertieri and no study has been found about wood degradation ability by this fungus. This basidiomycete has great capacity of mycelial growth and can efficiently degrade the eucalyptus urograndis wood and it was often found in the fields of Eucalyptus spp. Besides, it has great potential for biotechnological purpose and deserves more attention. Xylaria sp. did not degrade efficiently any compounds from wood, but throughout the degradation time, this fungus generated several reproductive structures, in the form of long strands black with white edges. Based in these observations, we could suggest that the eucalyptus urograndis wood is not a natural substrate for Xylaria sp., at least in the non-degraded wood used in this study. According to Klein and Paschke (2004), the fungi sustain their mycelia only if they have nutrients available, otherwise sexual or asexual spores are formed quickly. After removing the mycelium of Xylaria sp. we observed darkened spots adhered to the surface of the eucalyptus urograndis wood, even after washing and brushing. Also, Xylaria polymorpha also can produce pigments in beech and sugar maple wood when it pH is around 5 and 4.5, respectively, similar pH of the soil used in the present study (Tudor et al., 2013). We also observed that this fungus has ability to degrade Pinus wood, due to the soft appearance after 120 days. However, Xylaria sp. can produce white areas in Fagus sp. (Poiting et al., 2003). Tropical fungi of the genus Xylaria may have an important ecological role as decomposers of plant debris and/or wood already in an advanced stage of degradation (Alexopoulos et al., 1996). We observed whitish areas only in samples colonized by P. sanguineus, L. bertieri and L. edodes. However, after breaking the eucalyptus urograndis wood, we observed some black depositions on the wood, mainly after 120 days of colonization by P. sanguineus and L. bertieri. This study revealed the influence and importance of water in the rates of biodegradation of eucalyptus urograndis wood that also influence on the soil pH after fungal growth. Optimum pH is very important to enable fungi growth and consequently cause decay (Tudor et al., 2013). The schedule of inoculation according to the best season in the Eucalyptus spp. stumps and roots is very important to get success in this method. Seasons of higher precipitation and temperature promote better fungi colonization on the substrate. Around 90% of all problems related to wood involve water and wood biodegradation does not occur if the amount of moisture is less than 30%. High amounts of moisture (about 60e70%) contribute to the diffusion of enzymes inside the pores of the wood, making the carbohydrates accessible to hyphae (Griffin, 1993; Hoadley, 2000; Thybring, 2013). The wood chemical analyses showed increased extractives levels in the U50 treatment. In the U100 treatment, the wood degraded by L. bertieri and L. edodes had increase in the extractives contents compared to control A. Lower amounts of extractives were observed in the wood degraded by P. sanguineus in relation to nonautoclaved wood. In this case, probably the most extractable components (sugars, polysaccharides and phenolic compounds) were thoroughly consumed, and less soluble compounds were removed by the extraction process. The extractives can give a special resistance in wood, and if P. sanguineus has the ability to consume them, it is the best candidate to be employed in stumps degradation (Kirker et al., 2013). High temperature can cause partial degradation of hemicelluloses, resulting in lower concentrations of polysaccharides. Lower hemicelluloses contents were observed in control B and C.
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Brito et al. (2008) observed 16% hemicellulose losses in 14 year-old E. saligna wood. The solubility of the wood can indicate the degree of degradation, caused by biotic or abiotic agents. If any wood component suffers degradation, as consequence there is increase in the percentage of soluble materials in alkaline treatments (TAPPI, 2002). An increase on the solubility was observed in all treatments, i.e. with fungi or without fungi. Also, the heat sterilization, although briefly, can cause the degradation of pentosans, becoming part of the sugars and volatile extractives, leaching easily (Campbell and Taylor, 1933). The rate of solubilization of wood can vary with the fungus and the wood species. According to Istek et al. (2005), the solubility of the timber at NaOH 1% of Abies bornmülleriana and Fagus orientalis increased by 11 and 9%, respectively, after biodegradation by Phaneochaette crysosporium. Searching for nutrients into the soil, some fungi can produce organic acids, like oxalic acid (Fransson et al., 2004). These acids, the most varied possible, are produced to use it in the dissolution of humic compounds and others nutrients, like minerals. As a result, the modification of the soil pH can cause a suppressive environment, that will help to protect themselves from others microorganisms (Griffin, 1993; Fransson et al., 2004; Kluczek-Turpeinen et al., 2007; Magan, 2008). 5. Conclusion The relationship observed in this study between wood biodegradation, including mass loss, wood components consumption, and modification of the soil pH by fungal activity contributes to ecological studies of wood decay fungi. The knowledge about the interactions of biological processes that occur during wood biodegradation is critical for the success of biological stump and roots degradation of Eucalyptus spp. In this study we observed great ability of P. sanguineus and L. bertieri to degrade eucalyptus urograndis heartwood. Mass loss in Eucalyptus spp. stumps can be achieved in seasons with high temperature, around 30 C, combined with high humidity, conditions found mainly during the spring and summer, in Brazil. Acknowledgements We would also to express our sincere appreciation to Eucatex Company S/A, specially to Mr. Eduardo Bernardo and Mr Hideyo Aoki (Horto Florestal de Avaré, São Paulo, Brazil) for providing us wood materials; Dr. Marina Capelari and Adriana de Mello Gugliotta (Instituto de Botânica de São Paulo, São Paulo, Brazil) for help us to identify the fungi species. The first author would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil for providing the scholarship. References Aguiar, A., Gavioli, D., Ferraz, A., 2013. Extracellular activities and wood component losses during Pinus taeda biodegradation by the brown-rot fungus Gloeophyllum trabeum. Int. Biodeterior. Biodegrad. 82, 187e191. ABRAF, 2012. Brazilian Association of Planted Forest Producers, Statistical Yearbook online at:. Alexopoulos, C.J., Mims, C.W., Blackwell, M., 1996. Introductory Mycology, fourth ed. John Wiley & Sons, New York. Alonso, S.K., Silva, A.G., Kasuya, M.C.M., Barros, N.F., Cavallazi, J.R.P., Bettucci, L., Lupo, S., Alfenas, A.C., 2007. Isolation and screening of wood white rot fungi from Eucalyptus spp. forests with potential for use in degradation of stumps and roots. Rev. Árvore 31, 145e155. Andrade, F.A., Calonego, F.W., Severo, E.T.D., Furtado, E.L., 2012. Selection of fungi for accelerated decay in stumps of Eucalyptus spp. Bioresour. Technol. 110, 456e461.
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Effects of temperature and moisture on carbon respired from decomposing wood roots. For. Ecol. Manag. 138, 51e64. Eira, A.F., Minhoni, M.T.A., 1997. Manual Theoretical and Practical Cultivation of Edible Mushrooms, second ed. Foundation for Research and Agricultural and Forestry. FEPAF, Botucatu, São Paulo, Brazil. Ferraz, A., Esposito, Bruns, R.E., Durán, N., 1998. The use of principal component analysis (PCA) for pattern recognition in Eucalyptus grandis wood biodegradation experiments. World J Microbiol Biotechnol. 14, 487e490. Fransson, A.M., Valeur, I., Wallander, H., 2004. The wood-decaying fungus Hygrophoropsis aurantiaca increases P availability in acid forest humus soil, while N addition hampers this effect. Soil. Biol. Biochem. 36, 1699e1705. Gomes, N.S.B., Auer, C.G., Grigoletti Júnior, A., 2007. In vitro growth of Armillaria sp. isolates obtained from Pinus elliottii var. elliottii under several temperatures. Summa Phytopathol. 33, 187e189. Griffin, D.H., 1993. Fungal Physiology, second ed. Wiley-Liss, New York. Guilmo, S.M.P., Auer, C.G., Barrichello, L.E.G., 1993. Effect of thermophilic fungi on wood of Eucalyptus saligna Sm. II. Aspergillus sp., Dactylomyces thermophilus Sopp., Penicillium bacillisporum Swift, Rhizomucor sp. and Sporotrichum sp. Bol. Pesqui. Florest. 26/27, 29e34. Hoadley, R.B., 2000. Understanding Wood: A Craftman’s Guide to Wood Technology. Newton, Tauton. Hope, G.D., 2007. Changes in soil properties, tree growth, and nutrition over a period of 10 years after stump removal and scarification on moderately coarse soils in interior British Columbia. For. Ecol. Manag. 242, 625e635. Istek, A., Sivrikaya, H., Eroglu, H., Gulsoy, S.K., 2005. Biodegradation of Abies bornmülleriana (Mattf.) and Fagus orientalis (L.) by the white rot fungus Phanerochaete chrysosporium. Int. Biodeterior. Biodegrad. 55, 63e67. Kachlishvili, E., Penninckx, M.J., Tsiklauri, N., Elisashvili, V., 2005. 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Biodegradation of eucalyptus urograndis wood by fungi Djanira Rodrigues Negrão a, *, Tadeu Antônio Fernandes da Silva Júnior b, José Raimundo de Souza Passos c, Cláudio Angeli Sansígolo d, Marli Teixeira de Almeida Minhoni b, Edson Luiz Furtado b a
Centro de Energia Nuclear na Agricultura (CENA), Universidade de São Paulo (USP), P.O. Box 96, CEP: 13400-970 Piracicaba, São Paulo, Brazil Departamento de Proteção Vegetal, Faculdade de Ciências Agronômicas (FCA), Universidade Estadual Paulista (UNESP), P.O. Box. 237, CEP: 18603-970 Botucatu, São Paulo, Brazil c Departamento de Bioestatística, Instituto de Biociências, UNESP, P.O. Box 510, CEP: 18618-970 Botucatu, São Paulo, Brazil d Departamento de Ciência Florestal, Faculdade de Ciências Agronômicas, UNESP, P.O. Box 237, Botucatu, São Paulo, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 7 January 2014 Accepted 8 January 2014 Available online 14 February 2014
We focused in selecting four fungi, naturally living in Eucalyptus sp. fields, for application in accelerating stump decay. The wood-rot fungi Pycnoporus sanguineus (Ps), Lentinus bertieri (Lb) and Xylaria sp. (Xa) were isolated from Eucalyptus sp. field and the fungus Lentinula edodes (Led) was obtained from a commercial strain. All fungi were studied according to their capacity to degrade eucalyptus urograndis wood. In order to evaluate mass losses of seven years old eucalyptus urograndis’ wood test blocks from heartwood were prepared added to glass flasks with red clay soil. The humidity of the soil was adjusted with 50 and 100% of its water retention capacity. Mass loss evaluations occurred at 30 until 120 days after eucalyptus wood degradation. Chemical analysis and soil pH were measured only in the last evaluation. Mycelial growth assays with potato-dextrose-agar, malt-agar and sawdust-dextrose-agar at three temperatures was carried out in order to get information about the best conditions of fungi growth. On the 120th day, Ps and Lb showed good capacity of wood degradation by leading to a high mass loss in soil with highest humidity. These fungi were the best consumers of lignin, hemicellulose, cellulose and extractives, caused acidification in the soil. Ps and Lb had faster mycelial growth in sawdust-dextroseagar, especially in high temperature, comparing to Xa and Led. Xa and Led are not good eucalyptus urograndis heartwood degraders, because they consume preferentially hemicellulose. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Eucalyptus stump White-rot fungi Mycelial growth Wood chemistry Soil pH
1. Introduction The Eucalyptus genus is the main forest specie cultivated in Brazil. In 2010, the planted area was around 4.9 million hectares (ABRAF, 2012). The most planted Eucalyptus tree in Brazil is the eucalyptus urograndis, generated from the crossing between Eucalyptus grandis Eucalyptus urophylla. Because of this, eucalyptus urograndis has no scientific name and is not considered specie by geneticists. The planting of Eucalyptus spp. can be done in new areas, replacing the pastures, using reformed areas and also by regrowth (ABRAF, 2012). The harvesting of planted forests is usually performed after the seventh year of planting with chainsaws or mechanical harvesters. The timber is removed leaving only leaves, bark, thinner branches and the stumps, which remain fixed by their
* Corresponding author. E-mail addresses: [email protected], [email protected] (D.R. Negrão). 0964-8305/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2014.01.004
roots. Seven years after the first planting, new seedling plants are planted in between the plants that were harvested. However, with successive plantings the stumps hinder management activities, such as transit machinery for planting and fertilization. After cutting the trees, mechanic stumps drawdown is performed to improve crop management. A complete withdrawal is required for the reuse of the area after the third crop rotation. This practice has a high cost, and usually, they are calculated by each hour worked. According to Pavan et al. (2010), for removing the stumps from 1 ha of a planted 21 year-old forest, is necessary 25 h costing approximately US$ 50.00/hr. The cost of stump removal does not vary by age, but by the density of the forest. For removing the roots, a widespread excavation is necessary, generally two or 3 m deep, to assess and remove the roots. Besides the high costs of Eucalyptus spp. production for companies and smaller producers, the environment also pays with grave disturbance. The stumps and roots removal not only causes severe disruption in the microbiota, but also modifies some
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physical and chemical characteristics of the soil for several years. After one year of removing roots, several changes occur in the soil, such as decrease in the concentration of nitrogen, carbon and sulfur, and increase in concentrations of phosphorus. It can take up to 10 years for nutrient levels to be normalized in soils after removal of these roots (Hope, 2007). In an attempt to minimize these problems, the biodegradation of roots has been studied. This may provide recycling of nutrients from the wood to the soil, promoting an increase of biodiversity and richness of microorganisms, also improving physical-chemical characteristics of the soil. Eucalyptus spp. reforestation areas with the system that maintain the soil undisturbed have high stable carbon content, with positive effects on the reduction of CO2 emissions. Regarding the organic matter present on the surface, the roots contribute for most stable forms of carbon in forest soils (Petersson and Melin, 2010; Kätterer et al., 2011). In nature, many fungi can degrade wood, but the best wooddegrading fungi are the basidiomycetes, such as white-rot fungi. These fungi are able to degrade efficiently wood components, especially lignin (Rayner and Boddy, 1988). The accelerated degradation of stumps and roots with white rot fungi has been studied as a way to prevent or reduce the disturbance of soil in planted forests. The degradation of Eucalyptus roots in reforestation fields is termed as “biological stump removal”, and consists of inoculating the stumps with wood degrading fungi. Some topics should be carefully considerate to achieve success in roots biodegradation, such as selection of the fungi according to their habitat, choosing the best season to inoculate (temperature, humidity) and confirm that the microorganism is not phytopathogenic (Alonso et al., 2007; Andrade et al., 2012). The great advantage of the “biological stump removal” application in Brazil is the high temperature in freshly cut areas of Eucalyptus fields, where the solar incidence is high during many months of the year. The temperature contributes to the activity of microorganisms that live in dead roots, reaching their climax in the range of 30e 40 C (Chen et al., 2000). The high frequency of rain during the summer also offers great conditions for fungi growth in Eucalyptus fields. The research of wood degradation by fungi is extremely important to forestry companies, because it enables proper management of the Eucalyptus roots and stumps in reforestation fields. Using properly, some fungi can be considered as a sustainable way to accelerate roots and stumps waste recycling. This study aims to select fungi with high wood degradation ability, in order to apply them in accelerating biodegradation of stumps and roots of Eucalyptus spp. 2. Materials and methods 2.1. Wood decay fungi During February 2009 till June 2011 we collected several fungi from Eucalyptus spp. fields located in southeast region of the State of São Paulo, Brazil. Pycnoporus sanguineus, Lentinus bertieri and Xylaria sp. were identified at Botanic Institute of São Paulo by Dr. Marina Capellari. The fungi P. sanguineus and L. bertieri were deposited in the Culture Collection of Algae, Cyanobacteria and Fungi of Botanic Institute e CCIBt, São Paulo-SP, Brazil, with respective numbers CCIBt 3817 and CCIBt 3818. Xylaria sp. and commercial isolate of Lentinula edodes (Led) are deposited in the Fungi Collection of the Mushrooms Module, Faculty of Agricultural Sciences, UNESP, Botucatu-SP, Brazil, with respective numbers BT23 and LED 98/47. The selection of these fungi between all those collected (around thirty isolates) was made by observations of mycelial growth in
different solid medium (described below) and Eucalyptus sawdust (data not shown). 2.2. Mycelial growth The mycelial growth of Ps, Lb, Xa and Led was evaluated in petri dishes with potato dextrose-agar, 2% malt extract agar (MA) and sawdust dextrose agar medium (SDA). The SDA medium was prepared according to Eira and Minhoni (1997), using Eucalyptus spp. sawdust, wheat and corn bran and calcium carbonate (41:1:1:1, v/ v) were mixed and autoclaved for 4 h. 80 g from this mix were boiled in 1 L of distilled water. After filtration of the boiled sawdust 12 g L1 agar were added to prepare the SDS solid media. All culture medium were autoclaved at 120 C for 20 min, and placed in disposable petri dishes (90 15 mm). The fungal growth was measured daily, until 7.8 cm diameter. The experimental design was completely randomized factorial with 4 3 3 (fungi culture medium temperatures), with five replicates. 2.3. Assay of accelerating wood degradation 2.3.1. Preparation of the experimental plots The experimental plots were based in the Standard ASTM D2017 (1997), using 600 mL glass bottles filled with 300 g of clay texture red soil, pH 4.7, water retention capacity of 29%. The soil was screened through a 5 mm mesh and dried in green house conditions (25 C, for 30 days). Soil humidity was obtained according to water field capacity. In the treatment with 50% (U50) 70 mL distilled water was used to get 50% of humidity and 140 mL to get 100% of soil humidity. After correction of the soil humidity, a Pinus taeda feeder (30 50 5 mm) was placed on the soil surface of each glass bottle and sterilized by autoclaving 121 C for 1 h during three consecutive days. Control treatments were divided in A, B and C. Control A was prepared with bottles with not sterilized dry soil. Controls B and C, with 50 and 100% of moisture, respectively, were sterilized. The experimental design was completely randomized in a 4 2 6 factorial (fungi humidity degradation evaluation time). The evaluation time of wood mass loss was at 30, 60, 75, 90, 105 and 120 days after inoculation, with eight replicates. 2.3.2. Eucalyptus wood samples In this study, the wood samples were obtained from 13 trees of the hybrid eucalyptus urograndis (named clone 105 urograndis) seven years old, donated by the Eucatex Company S/A. Each wood sample had 50 cm of length (cut from 10 cm above the ground), and stored under green house conditions (25 C, humidity bellow 20%) during 45 days. The bark and sapwood were removed, using only the heartwood to make the wood blocks, on dimensions of 25 25 10 mm (length, width, direction of the fibers). The inclusion criteria for the use of wood blocks were: no defects, as knots, resin or gums, and without visible evidence of fungus infection. From each tree sample were made approximately 200 wood blocks, totaling 2600 samples, which were mixed among the thirteen samples. Each wood block was sanded (emery 20 mm), labeled and dried in the oven (102 3 C) until reached constant weight (Gehaka BG 400). The initial mass (W1) of each sample was measured, stored in glass Petri dishes (120 by 20 mm) and sterilized by autoclaving at 120 C for 15 min. 2.3.3. Wood inoculation The parameters for accelerated degradation of wood by the fungi were based on Standard ASTM D-2017 (1997) and ASTM D1413 (1994). A 7 mm diameter mycelial disc of each fungus
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cultivated in PDA at 25 C during five days, were placed on the P. taeda wood chips under aseptic conditions. The flasks were placed in a climate-controlled room at 25 2 C, 70 10% of relative humidity, in the dark, during 25 days for complete Pinus wood colonization. After that, the wood blocks were placed under the mycelium, i.e. colonized P. taeda, and the time for all evaluations was calculated from this moment. Over the evaluation period, the mycelium developed under the wood blocks was manually removed under tap water; the wood of the controls treatments were also washed. All samples were placed in the oven (102 3 C) to reach constant mass (W2), and the mass loss (M) of each wood block was calculated using the following equation: M(%) ¼ [(Wi W2)]/W1] 100. 2.4. Chemical analysis Twenty-five additional bottles of each treatment were prepared for chemical analyses of wood components such as extractives, lignin, cellulose, hemicellulose and wood solubility. After 120 days of wood biodegradation, 25 wood blocks of each treatment were washed in tap water, cut manually in small pieces, mixed and dried at 45 C. The wood samples were analyzed in duplicate. Nonautoclaved wood (control A) and controls treatment B and C were also analyzed in duplicate and used as a reference. For chemical analysis was necessary to transform wood blocks in sawdust, using a macro Willey mill (10 mesh screen), and classified in 40e60 mesh (Fraction 40/60), which were used for all chemical analyzes. The preparation of wood for chemical analyzes was based on TAPPI Standard T264 cm-97 (TAPPI, 1997a). For extractive analyzes was used the Standard T204 cm-97 (TAPPI, 1997b), for Klason lignin was used the Standard TAPPI T222 om-83 (TAPPI, 1999b), for hemicellulose analysis the Standard TAPPI T249 cm-85 (TAPPI, 1999a) and for cellulose analysis was used the methodology according to Santos (2000) and Wright and Wallis (1998). The solubility of the wood in NaOH 1% was analyzed according to Standard T212om-02 (TAPPI, 2002). The experimental design was completely randomized in a 7 x 2 factorial (fungi and controls treatments soil humidity), in duplicate. The wood chemical analyses were performed in the Department of Forest Sciences, Laboratory of Wood Chemistry, FCA/ UNESP, Botucatu, São Paulo, Brazil. 2.5. Soil pH analyses Ten flasks were used to analyze the soil pH from the same glass bottles used in chemical analysis. Twenty five grams/flask of soil close to Pinus feeder were removed and mixed with 25 g from another flask, totalizing five samples from 10 flasks. Soil pH was analyzed in water, in duplicate. The experimental design was completely randomized in a 7 x 2 factorial (fungi and controls soil humidity), with five replicates. The analyses were carried out in the Department of Naturals Resources, Laboratory of Soil Science, FCA/ UNESP. 2.6. Statistical analysis A generalized linear model with gamma distribution error and log link function (Nelder and Wedderburn, 1972; Liang and Zeger, 1986) was fitted for the variables (covariates in the brackets) describes mycelial growth of wood degrading fungi (specie, temperature and time); assay of accelerating wood degradation (specie, humidity and time); chemical analysis of the eucalyptus urograndis wood (specie and humidity) and analysis of soil pH (specie and humidity). The adjustment of the models was measured by analysis of deviance (Nelder and Wedderburn, 1972).
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For calculating the correlation between wood mass losses and chemical components it was necessary to have the data on ordered pairs, or format data sets in the same dimension, considering only the last evaluation period (120 days). The average percentage difference was obtained for each combination, as follows: 1. between humidity and specie, for the wood degradation assay and, 2. between specie and soil humidity, for the average percentage differences of chemical compounds. 3. Results 3.1. Mycelial growth The substrate and the temperature influenced the mycelial growth of all fungi (Fig. 1). The fungus P. sanguineus grew faster in SDA medium, followed by MA, mainly at higher temperatures. L. bertieri did not suffer difference in its dynamic mycelial growth, in PDA and SDA medium at 27 and 31 C. Xylaria sp. had better mycelial growth in SDA medium, while its growth in PDA was slower; this fungus grew better at 27 and 31 C. L. edodes grew faster in SDA medium at 27 C. The same fungus had a long lag phase in the BDA e MA culture medium, mainly at 31 C. SDA medium was the best substrate to provide faster mycelial growth for all fungi evaluated. In vitro assays can give important insights for predicting the behavior of fungi in vivo (Gomes et al., 2007). This knowledge contributes to obtain the optimum conditions for fungi growth on a particular substrate under a range of temperature, besides incubation time. 3.2. Accelerated degradation of the eucalyptus wood Comparisons between biodegraded and non-biodegraded wood can be done according to the values observed in control A, which is the intact form of timber (Table 1). This ensures that the degradation of cellulose, hemicellulose, lignin and solubility levels presented were the result of biological activity (Paes et al., 2007). Large variation of wood mass loss was observed according to soil humidity. The lowest rates were observed in the U50 treatment, while in the U100 treatment we observed mass losses around of 50%. There was triple interaction between the fungi, humidity and time. Regardless to wood colonization, in 14 days P. sanguineus, L. bertieri and Xylaria sp. colonized completely P. taeda wood and L. edodes required 25 days to colonize the same wood specie. In U50 treatment, it is likely that the low amount of water damaged the mycelial growth of L. edodes, observed by its low strength on the Pinus and eucalyptus urograndis wood until the last assessment period. On the other hand, this fungus also had difficulties growing at U100 in both types of wood. Because of this, the assessments of mass loss in this treatment were made only at 75 and 120 days. Even under these conditions we observed reduction in mass loss of wood during the assays, exhibiting ability to adapt to adverse conditions. At 120 days, in the U50 treatment, the difference in mass loss between P. sanguineus and L. bertieri was of 22%. Among P. sanguineus and L. edodes was 11%. In the U100 treatment the difference in weight loss between P. sanguineus and L. bertieri had increased to 45%; with L. bertieri and L. edodes was 69%, and between L. bertieri and Xylaria sp. the difference was of 97%. Statistical differences between P. sanguineus and L. bertieri occurred at 60, 105 and 120 days. Eucalyptus urograndis wood mass losses caused by Xylaria sp. were not significant in either treatments, but there was a greater tendency of wood degradation in U100 treatment, being 38.4% higher than the U50 treatment, at 105 days. Low degradation ability
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Fig. 1. Mycelial growth of Pycnoporus sanguineus (1), Lentinus bertieri (2), Xylaria sp. (3) and Lentinula edodes (4) in PDA (A), SDA (B) and MA (C) solid media at 23 C (rhombus), 27 C (square) and 31 C (triangle).
can occur by its impartial colonization under and into the wood, which was observed in all eucalyptus urograndis wood. Unlike L. edodes, Xylaria colonized better P. taeda wood than eucalyptus urograndis wood. 3.3. Wood chemical analysis The consumption rates (%) of the chemical components of wood depended on the combination between species and moisture (Table 2). Through the chemical analysis we could identify which wood components were preferentially consumed by each fungus and thus, some physiological characteristics of each species could be observed.
All U50 treatments had higher extractives content from its degraded wood when compared to extractives content obtained from control A. In the U100 treatment an increase in the percentage of extractives content was observed in treatments with L. bertieri and L. edodes. The wood degraded by P. sanguineus had low extractives content. The largest consumers of lignin in U50 treatment were P. sanguineus and L. edodes. P. sanguineus and L. bertieri were the best lignin consumers at U100. In U50, cellulose content was low in the wood degraded by P. sanguineus and L. bertieri, differing between them in 8.6%, while in U100, this difference increased to 33%. Low cellulose consumption was observed in wood degraded by L. edodes and Xylaria sp.
Table 1 e Average difference of eucalyptus urograndis wood mass loss (interquartile in brackets), according species of fungi and soil humidity (H%) and incubation time (T), in days. H%
T
Species P. sanguineus
50
100
30 60 75 90 105 120 30 60 75 90 105 120
2.70 2.98 2.21 2.15 6.92 8.01 9.14 23.50 19.57 27.16 41.97 49.70
(2.76) (3.02) (2.21) (2.69) (6.91) (14.10) (5.16) (11.01) (15.29) (11.98) (30.11) (10.23)
L. bertieri AaA AadA ABbA AaA BCaA CbA Aab Bbb Bab Bab Cbb Ccb
2.91 2.82 2.23 3.36 7.48 6.72 9.34 16.32 18.58 21.67 28.95 28.17
(1.19) (0.76) (0.20) (3.39) (9.17) (10.0) (5.17) (7.64) (12.1) (9.94) (29.1) (6.59)
L. edodes ABaA ABabA AaA BaA CaA CabA Aab Bab BCab CDab DEab Eab
2.55 (0.61) 3.33 (0.70) 2.29(0.81) 4.12 (3.18) 5.81 (6.19) 7.38 (6.60) e e 9.31 (9.88) e e 10.29 (5.25)
Xylaria sp. ABaA ABbA AaA BaA CaA CaA e e Abb e e Abb
2.80 (0.51) 2.21 (0.28) 2.15 (0.15) 2.20 (1.90) 3.12 (3.12) 2.95 (1.35) 3.02 (0.72) 3.19 (1.80) 3.37 (1.41) 5.56(2.95) 5.07 (5.33) 3.77 (2.48)
Control Abbaa Aca Aaa BCaa BCba Cca Abb ACcb ADcb Bbb BEcb CDEda
2.71a 2.72 2.80 2.14 2.26 2.30 2.67b 2.89 2.78 3.05 3.22 2.50
(2.65) (2.54) (2.26) (2.11) (2.00) (0.19) (2.66) (2.03) (2.77) (2.96) (0) (0.12)
ABaa ABdca ABaba Aaa Bba Aca ADba ABca ACca ABcb CBda Deb
Average difference medians followed by the same lowercase letter line did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each soil humidity (H%) and incubation time (T). Average difference medians followed by the same uppercase letter column did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each soil humidity (H%) and species. Average difference medians followed by the same greek letters column did not differ statistically among themselves by the LSMEANS Test (p > 0.05) according each incubation time (T) and species. * Control B, subjected to 50% humidity; ** Control C, subjected to 100% humidity.
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Table 2 Average percentage of the chemical compounds of eucalyptus urograndis wood (percentage of compound losses in black italic) degraded by fungi, according to soil humidity (H %), at 120 days. H%
Treatment
Extractives
Lignin
Cellulose
Hemicellulose
Solubility
0
Control A
4.8
23.3
38.1
36.3
12.99
50
P. sanguineus L. bertieri Xylaria sp. L. edodes Control B P. sanguineus L. bertieri Xylaria sp. L. edodes Control C
5.4 6.3 5.2 6.5 5.2 4.2 5.8 4.8 6.4 4.8
100
12.5 31.2 8.3 35.4 8.3 12.5 20.8 e 33.3 e
21.1 21.7 23.9 21.5 22.6 12.2 16.4 22.8 20.7 23.7
9.4 6.9 2.6 7.7 3.0 52.4 29.6 2.1 11.1 1.7
34.6 34.9 37.0 36.6 38.0 20.0 26.0 35.7 35.8 38.5
9.2 8.4 2.9 3.9 0.2 47.5 31.7 3.3 6.0 1.0
32.0 31.2 34.0 30.6 33.5 18.0 24.2 32.8 29.9 32.6
11.8 14.0 6.3 15.7 7.7 50.4 33.3 9.6 17.6 10.2
18.2 17.4 16.1 17.2 15.4 19.5 21.0 15.8 18.5 15.9
40.1 33.9 23.9 32.4 18.7 50.5 61.7 21.6 42.4 22.4
Average calculated by two replicates, all analyses were made in duplicate.
Hemicellulose contents in controls B and C were high comparing to control A, and also compared to the others wood components analyzed. In U50 treatment, L. edodes and L. bertieri were the best degraders of hemicellulose. In U100 treatment, the biggest consumers of this component were P. sanguineus and L. bertieri, with the difference between them of 34%; between P. sanguineus and L. edodes the difference increased to 65%. Regarding wood solubilization, which indicates wood degradation, L. bertieri and L. edodes produced wood solubilization at similar rates in the U50. Xylaria sp. produced less wood solubilization and P. sanguineus caused the largest wood solubilization rate. In U100 treatment, L. bertieri caused increase in the solubility levels of wood at a higher rate than P. sanguineus. This may indicate that the ability of L. bertieri to degrade eucalyptus urograndis wood can become more severe over time. Data on correlation values between wood mass losses and chemical components that remaining after 120 days of biodegradation are showed in Fig. 2 and Table 4. The data for Control A was not used because there is no equivalent data in the wood degradation assay. There was high correlation (p < 0.001) between the mean difference percentage of wood mass loss and mean differences of wood components, except for extractives. This means, for example, when the average mass loss mass decrease (no degradation occurs) the average cellulose content increases. Therefore, the wood biodegradation influences directly cellulose, hemicellulose and lignin contents. Regarding the solubility, the opposite was observed. In addition, Fig. 2 shows a close profile between lignin, hemicellulose and cellulose at the maximum point and a positive correlation profile for solubility.
Soil pH There was interaction between species and humidity, and significant effect of humidity on the soil pH (Table 3). In U50 treatment neither fungus modified the soil pH, comparing to control. In this treatment, P. sanguineus, L. bertieri and L. edodes colonized the soil with apparently fragile mycelium; Xylaria sp. colonized briefly the soil close to the P. taeda wood, at least to the naked eye, because of its black color. In the U100 treatment, P. sanguineus, L. bertieri and L. edodes produced mycelium intensively branched into the soil. P. sanguineus and L. bertieri changed the pH of the soil causing its acidification. There was no statistic difference in the pH of soil colonized by L. edodes and the controls. 4. Discussion Mycelial growth of all fungi was bounded mainly to the temperature. P. sanguineus, L. bertieri and Xylaria sp. preferred high temperatures, growing faster at 27 and 31 C, while L. edodes grew faster only at 23 C. In natural environment, these fungi can be exposed to high temperature, very common in areas of Eucalyptus spp. reforestation. Detailed study regarding mycelial growth is important to manage the best season for inoculating these fungi in the fields, as Gomes et al. (2007) did with Armillaria sp., finding the optimum temperature for growing this fungus. According to Chen et al. (2000), an increase in temperature can be better for enzymatic activities and to metabolize nutrients obtained through wood decay. Besides temperature, the type of lignocellulosic substrate and other nutrients added can interfere with the fungal growth. When sugars are added to the culture medium, fungi usually use it as a primary source of energy to supply for its growth and vital
Table 3 Measurements of soil pH at 120 days from samples with 50 and 100% of humidity colonized and non colonized (Control) by fungi. Treatments
Humidity (%) 50
P. sanguineus L. bertieri Xylaria sp. L. edodes Control Fig. 2. Average percentage of the eucalyptus urograndis wood mass loss and chemical components after fungi degradation, at 120 days.
4.77 4.82 4.69 4.71 4.67
100 (0.18) (0.14) (0.01) (0.10) (0.06)
Aa Aa Aa Aa Aa
4.11 4.30 4.32 4.89 4.82
(0.03) (0.09) (0.07) (0.10) (0.02)
Ab Ab Bb Cb Ca
Average difference medians followed by the same lowercase (line) or uppercase (column) letter did not differ statistically among themselves by the LSMEANS Test (p > 0.05).
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Table 4 Means correlations values of the average percentage and p-values of eucalyptus urograndis wood mass losses and chemical components remaining after fungi degradation, at 120 days. Treatments
Correlation (r)
p-value(*)
Cellulose Extratives Hemicellulose Holocellulose Lignin Solubility
0.9904 0.2802 0.9820 0.9938 0.9895 þ0.8499
<0.0001 0.4329 <0.0001 <0.0001 <0.0001 0.0018
(*)
p-values related to the hypotheses:H0 : r ¼ 0vs. H0 : rs0
functions, and also to consume others complex substrates, like lignin (Kües and Liu, 2000). In this study, we observed fungi growth preference to SDA medium, made from Eucalyptus spp. Thus, one of the most important steps to understand what makes fungi able to colonize the wood is to characterize their nutritional needs (Rayner and Boddy, 1988). Although PDA and MA culture medium are commonly used in microbiology laboratories, they can disturb the metabolism of some fungi species, affecting the velocity of its growth and causing the suppression of the lignocellulosic system (Lonergan et al., 1993; Kachlishvili et al., 2005). With respect to wood degradation, we notice that the sterilization process by autoclaving caused mass loss in eucalyptus urograndis heartwood, as observed in controls B and C. The autoclaving process can cause extractives degradation and/or evaporation, as observed by Guilmo et al. (1993).) Wood mass loss after heating treatment is due primarily to the removal of wood extractives, such as separation of acetyl groups linked to xylan chains and dissolution of low molecular weight carbohydrates, and in some cases, even lignin. In this study, we found wood mass loss around 3%, similar to the 14 year-old Eucalyptus saligna wood treated by heating (Brito et al., 2008; Lourenço et al., 2011). In relation to the heartwood mass loss in the U50 treatment, after 120 days of degradation, the percentage difference in mass loss between P. sanguineus and L. bertieri was 22%, and between P. sanguineus and L. edodes only 11%. Similar results of low mass loss caused by rotting fungi in Eucalyptus wood were observed by Oliveira et al. (2005) and Aguiar et al. (2013) using Gloeophyllum trabeum. Under conditions similar to this study, the authors also observed mass loss in Eucalyptus and Pinus woods were below 10%. In other hand, when the E. grandis wood is submitted to termallymodified technique, the degradation ability of white and brown rot fungi can vary according to heat treatment. The brown rot fungus G. trabeum caused 53% of mass loss in Eucalyptus wood termally-modified at 200 C while the white rot fungi P. sanguineus caused 31% of mass loss (Calonego et al., 2013). P. sanguineus has great lignocellulolytic ability, but this can vary according to the substrate. By using this specie, Ferraz et al. (1998) observed mass loss of E. grandis wood around 23% after 120 days of degradation, whereas Silva et al. (2007) observed 9% of mass loss. However, other types of wood can also be susceptible to this fungus, such Fagus sylvatica and Pinus sylvestris woods (Poiting et al., 2003). In Brazil, some studies related to accelerated degradation of stumps and roots of Eucalyptus sp. were carried out by Alonso et al. (2007) and Andrade et al. (2012). Alonso et al. (2007) observed 26% mass loss in E. saligna wood caused by P. sanguineus. Andrade et al. (2012) observed 27 and 29% mass losses in E. grandis wood caused by P. sanguineus and L. edodes, respectively. However, these authors used different fungal growth conditions and also of nutrients and they did not study the effect of the humidity in wood decay. Under the conditions evaluated in this study P. sanguineus and L. bertieri caused mass loss near to 50% and 28%, respectively, at the higher
soil humidity. These rates are very relevant for the selection of these fungi to degrade the eucalyptus urograndis wood and much higher than previous studies. In the literature, there are few studies about L. bertieri and no study has been found about wood degradation ability by this fungus. This basidiomycete has great capacity of mycelial growth and can efficiently degrade the eucalyptus urograndis wood and it was often found in the fields of Eucalyptus spp. Besides, it has great potential for biotechnological purpose and deserves more attention. Xylaria sp. did not degrade efficiently any compounds from wood, but throughout the degradation time, this fungus generated several reproductive structures, in the form of long strands black with white edges. Based in these observations, we could suggest that the eucalyptus urograndis wood is not a natural substrate for Xylaria sp., at least in the non-degraded wood used in this study. According to Klein and Paschke (2004), the fungi sustain their mycelia only if they have nutrients available, otherwise sexual or asexual spores are formed quickly. After removing the mycelium of Xylaria sp. we observed darkened spots adhered to the surface of the eucalyptus urograndis wood, even after washing and brushing. Also, Xylaria polymorpha also can produce pigments in beech and sugar maple wood when it pH is around 5 and 4.5, respectively, similar pH of the soil used in the present study (Tudor et al., 2013). We also observed that this fungus has ability to degrade Pinus wood, due to the soft appearance after 120 days. However, Xylaria sp. can produce white areas in Fagus sp. (Poiting et al., 2003). Tropical fungi of the genus Xylaria may have an important ecological role as decomposers of plant debris and/or wood already in an advanced stage of degradation (Alexopoulos et al., 1996). We observed whitish areas only in samples colonized by P. sanguineus, L. bertieri and L. edodes. However, after breaking the eucalyptus urograndis wood, we observed some black depositions on the wood, mainly after 120 days of colonization by P. sanguineus and L. bertieri. This study revealed the influence and importance of water in the rates of biodegradation of eucalyptus urograndis wood that also influence on the soil pH after fungal growth. Optimum pH is very important to enable fungi growth and consequently cause decay (Tudor et al., 2013). The schedule of inoculation according to the best season in the Eucalyptus spp. stumps and roots is very important to get success in this method. Seasons of higher precipitation and temperature promote better fungi colonization on the substrate. Around 90% of all problems related to wood involve water and wood biodegradation does not occur if the amount of moisture is less than 30%. High amounts of moisture (about 60e70%) contribute to the diffusion of enzymes inside the pores of the wood, making the carbohydrates accessible to hyphae (Griffin, 1993; Hoadley, 2000; Thybring, 2013). The wood chemical analyses showed increased extractives levels in the U50 treatment. In the U100 treatment, the wood degraded by L. bertieri and L. edodes had increase in the extractives contents compared to control A. Lower amounts of extractives were observed in the wood degraded by P. sanguineus in relation to nonautoclaved wood. In this case, probably the most extractable components (sugars, polysaccharides and phenolic compounds) were thoroughly consumed, and less soluble compounds were removed by the extraction process. The extractives can give a special resistance in wood, and if P. sanguineus has the ability to consume them, it is the best candidate to be employed in stumps degradation (Kirker et al., 2013). High temperature can cause partial degradation of hemicelluloses, resulting in lower concentrations of polysaccharides. Lower hemicelluloses contents were observed in control B and C.
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Brito et al. (2008) observed 16% hemicellulose losses in 14 year-old E. saligna wood. The solubility of the wood can indicate the degree of degradation, caused by biotic or abiotic agents. If any wood component suffers degradation, as consequence there is increase in the percentage of soluble materials in alkaline treatments (TAPPI, 2002). An increase on the solubility was observed in all treatments, i.e. with fungi or without fungi. Also, the heat sterilization, although briefly, can cause the degradation of pentosans, becoming part of the sugars and volatile extractives, leaching easily (Campbell and Taylor, 1933). The rate of solubilization of wood can vary with the fungus and the wood species. According to Istek et al. (2005), the solubility of the timber at NaOH 1% of Abies bornmülleriana and Fagus orientalis increased by 11 and 9%, respectively, after biodegradation by Phaneochaette crysosporium. Searching for nutrients into the soil, some fungi can produce organic acids, like oxalic acid (Fransson et al., 2004). These acids, the most varied possible, are produced to use it in the dissolution of humic compounds and others nutrients, like minerals. As a result, the modification of the soil pH can cause a suppressive environment, that will help to protect themselves from others microorganisms (Griffin, 1993; Fransson et al., 2004; Kluczek-Turpeinen et al., 2007; Magan, 2008). 5. Conclusion The relationship observed in this study between wood biodegradation, including mass loss, wood components consumption, and modification of the soil pH by fungal activity contributes to ecological studies of wood decay fungi. The knowledge about the interactions of biological processes that occur during wood biodegradation is critical for the success of biological stump and roots degradation of Eucalyptus spp. In this study we observed great ability of P. sanguineus and L. bertieri to degrade eucalyptus urograndis heartwood. Mass loss in Eucalyptus spp. stumps can be achieved in seasons with high temperature, around 30 C, combined with high humidity, conditions found mainly during the spring and summer, in Brazil. Acknowledgements We would also to express our sincere appreciation to Eucatex Company S/A, specially to Mr. Eduardo Bernardo and Mr Hideyo Aoki (Horto Florestal de Avaré, São Paulo, Brazil) for providing us wood materials; Dr. Marina Capelari and Adriana de Mello Gugliotta (Instituto de Botânica de São Paulo, São Paulo, Brazil) for help us to identify the fungi species. The first author would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil for providing the scholarship. References Aguiar, A., Gavioli, D., Ferraz, A., 2013. Extracellular activities and wood component losses during Pinus taeda biodegradation by the brown-rot fungus Gloeophyllum trabeum. Int. Biodeterior. Biodegrad. 82, 187e191. ABRAF, 2012. Brazilian Association of Planted Forest Producers, Statistical Yearbook online at:
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