Behavior of the Brown-rot Fungus Gloeophyllum trabeum on Thermally-modified Eucalyptus grandis Wood

In this study, we aimed evaluate the behavior of the brown-rot fungus Gloeophylum trabeum and white-rot fungus Pycnoporus sanguineus on thermally-modified Eucalyptus grandis wood. To this end, boards from five-year-eleven-month-old E. grandis trees, taken from the Duratex-SA company stock, were thermally-modified between 180 °C and 220 °C in the Laboratory of Wood Drying and Preservation at Universidade Estadual Paulista UNESP, Botucatu, Sao Paulo state Brazil. Samples of each treatment were tested according to the ASTM D-2017 (2008) technical norm. The accelerated decay caused by the brown-rot fungus G. trabeum was compared with the decay caused by the white-rot fungus P. sanguineus, studied by Calonego et al. (2010). The results showed that (1) brown-rot fungus caused greater decay than white-rot fungus; and (2) the increase in temperature from 180 to 220 °C caused reductions between 28.2% and 70.0% in the weight loss of E. grandis samples incubated with G. trabeum.


INTRODUCTION
The mechanism of wood degradation differs fundamentally between brown and white rot fungi. In general, the brown-rot fungi selectively removes cellulose and hemicelluloses compounds, whereas the white-rot fungi causes degradation of all cell wood components (Oliveira et al., 1986;Barreal, 1998). However, the white-rot fungus Pycnoporus sanguineus (L.) Murrill is a good producer of phenoloxidase, and preferentially degrades lignin (Esposito et al., 1993).
According to Oliveira et al. (1986), Barreal (1998) and Kleman-Leyer et al. (1992), the whiterot fungus attacks the surfaces of the microfibrils resulting in a progressive erosion of the polymers of wood. Yet, the brown-rot fungus completely cleaves the amorphous regions of the cellulose microfibrils, and subsequently, promotes significant loss in wood weight because of degradation in the crystalline region of the cellulose. At advanced stages of decay, the structural polysaccharides are quantitatively removed, and a modified lignin residue remains.
In general, the brown-rot fungi, including the species Gloeophyllum trabeum (Pers.) Murrill, produce extracellular hydrogen peroxide (H 2 O 2 ) and oxalic acid (H 2 C 2 O 2 ), which react with the iron ions present in lignocelluloses materials. The hydroxyl radicals produced by the Fenton's reaction were suggested to explain the cleavage of long chain cellulose molecules into small fragments. Thus, there is an increase of porosity of the cell wall allowing penetration of cellulolytic enzymes, which increase the decay on wood (Goodell et al., 1997;Xu & Goodell, 2001;Arantes & Milagres, 2009;Watanabe et al., 2010;Aguiar & Ferraz, 2011). Dutton et al. (1993) showed that the brown-rot fungi secrete large amounts of oxalate in culture medium reducing the pH of the substrate compared with the white-rot fungi, which do not reduce the pH of medium.
In evaluating the natural biological resistance of Aspidosperma desmanthum, Parinari excelsa, Mouriri callocarpa, Marmaroxylon racemosum, Peltogyne paniculata and Astronium sp. woods to the brown-rot fungus G. trabeum and white-rot fungus Pycnoporus sanguineus, it was founded that the decay caused by brown-rot fungus was greater. The woods studied presented weight loss between 1.97% and 12.2% when decayed to G. trabeum, and between 0.05% and 3.21% to P. sanguineus after accelerated decay test of 6 weeks (Alves et al., 2006).
The biological durability of wood can be increased by impregnation with chemical products, but in general, this technique is not positively regarded. Thus, increasing the biological durability of wood by thermal modification is considered to be more acceptable (Homan et al., 2000).
In general, thermal treatments expose the timber to temperatures approaching 200 °C for several hours, and change the chemical composition of wood. The equilibrium moisture content of wood and the availability of food (hemicelluloses) to fungi are reduced, new molecules that act as fungicides are produced, and there is a cross-linking between the lignin and the polymer from the thermal degradation of cellulose, making the recognition of the substrate by fungi difficult (Weiland & Guyonnet, 2003;Hakkou et al., 2006;Calonego et al., 2010Calonego et al., , 2012Severo et al., 2012).
In the accelerated decay test of Pinus pinaster untreated wood and wood that was thermallymodified at 230-260 °C, and then exposed to the brown-rot fungus Poria placenta (Fr.) Cooke for 16 weeks, a weight loss of 17.13% and 9.76%, respectively, was verified. The respective weight losses of untreated and treated Fagus sylvatica were approximately 22.92% and 5.94% (Weiland & Guyonnet, 2003).
In the accelerated decay test of untreated Eucalyptus grandis wood and wood that was thermally-modified at 180°, 200° and 220 °C, and then exposed to the white-rot fungus P. sanguineus for 12 weeks, a weight loss of 34.32%, 28.95%, 23.81% and 6.05%, respectively, was observed (Calonego et al., 2010).
However, there is little information about the effects of thermal treatment on the technological properties of Eucalyptus wood (Unsal & Ayrilmis, 2005).
Thus, the aim of this study was to evaluate the behavior of the brown-rot fungus G. trabeum and white-rot fungus P. sanguineus on thermallymodified E. grandis wood.

MATERIAL AND METHODS
In this study, we utilized wood from five-yeareleven-month-old Eucalyptus grandis trees from the Rio Claro Farm, managed by the Duratex-SA company, located in Lençóis Paulista, Sao Paulo state, Brazil. Six trees were randomly selected from inside the 2.2-ha stand. After felling, the trees were sectioned into 6.0-m logs. The first log from each tree with diameter between 20 and 22 cm was cut into flat saw boards. The boards that contained the pith were cut into 34-mm thick pieces for this study. Subsequently, all of the boards were dried from 75.7% to 10.0% moisture content in a dry kiln with capacity for approximately 2.5 m 3 of wood.

Thermal treatments of boards
The six dried boards were planed to 32-mm thick and cut into smaller pieces measuring 0.60 m in length. Regions with cracks and knots were discarded. One of these smaller pieces was kept in its original condition (untreated wood), and the other pieces were reserved for the thermal treatments (thermally-modified wood).
The material was placed in a thermal modification oven with programmable control. The treatment proceeded in steps from an initial temperature of 100 °C to 180 °C, and then to 200 °C and 220 °C over a period of 2.5 hours, according to the application of patent developed by Severo & Calonego (2009). Following the end of the thermal treatment, the oven was turned off and the wood pieces were kept inside. The pieces were allowed to cool naturally.

Accelerated laboratory tests of decay resistance of wood
The test to assess the attack of brown-rot fungus on the thermally-modified Eucalyptus grandis wood was conducted according to the standards presented in ASTM D-2017 (2008) norm. This procedure was performed simultaneously in the material inoculated with the white-rot fungus P. sanguineus studied by Calonego et al. (2010).
The samples were cut to create wood perfectly oriented in relation to the three anatomical planes (radial, tangential and longitudinal), and were approximately 40 mm from the pith of each piece of wood. The samples were sawn into test blocks measuring 25 by 25 by 9 mm in size, with the 9 mm dimension in the grain direction.
Although the ASTM D-2017 (2008) standard show that the necessary number of samples to characterize the decay resistance of wood is six, eighteen samples obtained from six boards were used to characterize each treatment (untreated wood and three other thermally-modified woods), totaling seventy-two samples by fungus tested.
In preparation for the test, the wood samples were dried in a drying oven at 103 ± 2 °C, until constant weight was reseached. As recommended by the ASTM D-1413 (2007) norm, the initial oven-dry weight (WI) of each test block was determined.
The soil-block test was prepared in 725 mL cylindrical culture bottles using 300 g of soil with a water holding capacity of 29%. After filling the bottles with distilled water, a feeder strip of Pinus sp. was added. Subsequently, the bottles were sterilized at 121 ±1 °C for 1 hour.
The culture bottles were then inoculated with the brown-rot fungus G. trabeum (collected from After sterilization, the test blocks were placed in the culture bottles with the cross-section face of the feeder strip facing down. The culture bottles were incubated in an incubation chamber in the dark to promote the growth of the fungus at 26.7 ± 1 °C and 70 ± 4% relative humidity for 12 weeks. At the end of the exposure period, the test blocks were removed from the culture bottles and any surface fungus growth was carefully brushed off. The blocks were then dried in drying oven at 103 ± 2 °C once again until constant weight was reseached. This weight was determined as the final oven-dry weight (WF) of each test block.
The percent weight losses in the individual test blocks from before and after exposure to the decay fungi were then calculated. The percent weight losses in the test blocks provide a measure of the relative decay susceptibility of the untreated and thermallymodified Eucalyptus grandis wood.

RESULTS AND DISCUSSION
The weight loss data of Eucalyptus grandis wood caused by the action of the brown-rot fungus G. trabeum was normally distributed and analysis of variance with a randomized block design was therefore used, taking into account the thermal treatments, as well as Tukey's test at 5% significance level for the comparison of the means.
The values for the amount of weight loss in untreated Eucalyptus grandis wood, found in Table 1, were 50.33% and 34.32% for the samples incubated with the brown-rot and white-rot fungi, respectively.
These results are similar to those cited by Andrade et al. (2012), who characterized the biological resistance of Eucalyptus grandis wood and concluded that material inoculated with the whiterot fungus P. sanguineus showed a weight loss of 29.0%, after 8 weeks in the accelerated test decay.
However, as shown in Table 1, the resistance of E. grandis wood to the brown-rot fungus G. trabeum was smaller than to the white-rot fungus studied by Calonego et al. (2010). According to Dutton et al. (1993), these variations were expected because the brown-rot fungi secrete large amounts of oxalate in culture medium, reducing the pH of the substrate during growth, and the amount of oxalate produced by white-rot fungi was not enough to reduce the pH of the medium during growth.
Several authors have reported that the brown-rot fungus G. trabeum produce extracellular hydrogen peroxide (H 2 O 2 ) and oxalic acid (H 2 C 2 O 2 ), which  Calonego et al. (2010); different letters -significant difference by Tukey's test at 95%probability between treatments; * -significant difference by "F" test at 95% probability between fungi; same letters and NS -non-significant difference. react with the iron ions present in lignocellulosic materials by the Fenton's reaction and cause the cleavage of long chain cellulose molecules into small fragments. Thus, there is an increase of porosity of the cell wall allowing penetration of cellulolytic enzymes which increase the decay on wood (Goodell et al., 1997;Xu & Goodell, 2001;Arantes & Milagres, 2009;Watanabe et al., 2010;Aguiar & Ferraz, 2011).
These explanations are in agreement with Alves et al. (2006), who evaluated the natural biological resistance of various tropical woods to the brown-rot fungus G. trabeum and white-rot fungus P. sanguineus and concluded that the decay caused by brown-rot fungus was greater.
However, the objective of this study was also to evaluate the resistance of thermally-modified E. grandis wood to brown-rot fungus in comparison with white-rot fungus.
The visual features of samples submitted to the action of the brown-rot and white-rot fungi are shown in Figure 1, and the values for the amount of weight loss and the average moisture content of E. grandis wood are presented in Table 1.
These evaluations indicated that although the thermal treatment reduced some of the mechanical properties of wood , there was a significant improvement in the decay resistance of Eucalyptus grandis wood by the increase in treatment temperatures. These results were expected because Homan et al. (2000), Millitz & Tjeerdsma (2001), Momohara et al. (2003), Weiland & Guyonnet (2003), Hakkou et al. (2006), and Calonego et al. (2010) found that thermal treatment at high temperatures increased the decay resistance of wood of other species and/or other fungi.
The decrease in weight loss caused by decay fungi (see Table 1) has already been explained by several authors, among them Weiland & Guyonnet (2003), Hakkou et al. (2006), Calonego et al. (2010) and Severo et al. (2012), because of changes in the chemical composition of wood, mainly the unavailability of food (hemicelluloses) to the fungi, the production of new molecules that act as fungicides, and the cross-linking between lignin and the polymer from the thermally degraded cellulose.
Verifying the effects of the thermal treatment on the decay caused by the white-rot fungus P. sanguineus and by the brown-rot fungus G. trabeum, it was possible to verify that the decay caused by brown-rot fungus was greater in all temperatures of thermal treatment.
These results can be explained by the ability of brown-rot fungi to secrete hydroxide complexes and oxalic acid able to cleave long chain cellulose Figure 1. Aspects of test blocks from untreated and thermally-modified Eucalyptus grandis wood after decay by brown-rot and white-rot fungi. molecules into small fragments, increasing the porosity of the cell wall, the penetration of cellulolytic enzymes, and the decay of wood (Goodell et al., 1997;Xu & Goodell, 2001;Arantes & Milagres, 2009;Watanabe et al., 2010;Aguiar & Ferraz, 2011).

CONCLUSIONS
This study shows that the decay caused by brown-rot fungus was greater than that caused by white-rot fungus. However, in verifying the effects of thermal modification on the decay resistance of E. grandis wood, it was possible to conclude that there was a decrease between 28.2 % and 70.0 % in the weight loss of wood exposed to the brown-rot fungus Gloeophylum trabeum.