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Micromorphological Studies on the Degradation of Commercial Woods by Brown Rot Fungus. Coniophora Putana.


In nature there is a dynamic equilibrium between the accumulation of woody biomass and it s decomposition [22] in forestry. Decay fungi play a major role in the processes of degrading in comparison with other microorganisms which keep balance between trees and degrading fungi. In fact, this phenomenon represents the state of their role in a co-evolutionary battle [22].

Different types of decay can originate from fungal species and condition within the wood. Within this spectrum three main types are commonly recognized: brown rots, white rots, and soft rots. The use of light microscopy techniques has provided a detailed picture of the decay patterns that occur among a wide range of host/fungus combinations. [1, 20,21,16,6].

Many aspects of the fungal activity on wood structure can be revealed by light microscopy, and this technique has provided information discussed in this research. Light microscopy with different staining techniques allows observation of early stages of decaying process.

The deterioration of wood caused by a combination of biological, chemical and physical process [23]. Along with a full understanding of the interactions between wood decay fungi and living trees requires anatomical, physiological and biochemical studies.

Softwoods are relatively homogenous in structure [13] and consists primarily of tracheids, uniseriate xylem rays, and in some genera, it has been composed by axial parenchyma and epithelial cells surrounding resin canals. By comparison, hardwoods are more heterogeneous, and its conducting functions are served by vessels, while fibres or fibre tracheids mainly supply mechanical strength and support.

Parenchyma is a more prominent feature in hardwood than in softwood. Furthermore, most genera have multiseriate xylem rays and varying amounts of axial parenchyma. Wood is an exceptionally difficult resource to decompose and fungal species differ in their degrading abilities [3]. Also, different compounds may be variously active or inactive under a given set of environmental conditions. In this context, the availability of oxygen, which may be greater in some parts of the wood than others, is particularly important [15,4,10,5].

Colonization of Wood by Hyphae:

The axial alignment of tracheids, vessels, fibers, radial arrangement of the xylem ray parenchyma allow to wide spread distribution of hyphae whitin the xylem [15,22,7,6]. Access to adjacent cells occurs via pit apertures or direct penetration may take place directly through the cell wall. Light microscopy reveal the more information from cell wall structure that influenced by hyphae.

In brown rot, cellulose and hemicelluloses are broken down in the wood substrate, but decomposition of lignin is limited [9]. compared with white rot fungi, where commercial application is of greater interest, little is known about the lignin decomposition capacity of brown rot fungi [13] except for a few reports of the presence of ligninolytic enzymes in brown rot fungi [19,20]. Brown rot is characterized by rapid degradation of the S2 layer, but the S3 layer and the lignin-rich middle lamellae appear to resist degradation [22].

Variations in the cell wall structure and/or distribution of cell wall constituents are reflected in the plasticity of wood degradation modes by wood decay fungi.

A typical anatomical feature of the wood of many trees is either the presence of apotracheal terminal parenchyma or strongly lignified late wood fibres at the border of growth rings. Numerous studies clearly indicate that parenchyma cells and late wood fibres of a range of broadleaved trees are resistant to degradation by brown rot and some weakly lignolytic white rot fungi

The aim of the present study was to test the hypothesis that decay fungi can be applied under controlled conditions as analytic tools for the exposure of the annual ring border in diffuse porous wood. For this purpose, wood samples of a range of diffuse porous trees were selected and artificially incubated with brown rot fungi.

Materials and Methods

The experiment was carried out in wood anatomy and preservation laboratory of natural resource of Tehran university during a period of October 2008- September 2010.

Wood Species and Wood Anatomy:

The fungal culture used for the incubation of the wood blocks was Coniophera puteana. cultures were obtained on to 90 mm plates containing 20 ml of malt extract agar (MEA) and incubated in the dark at 25[degrees]C for 2 weeks. Fungal contaminants were eliminated by sub-culturing on MEA plates. 60cc of MEA medium culture poured into every kolle flasks and tied with cotton and set in autoclave .Then they transferred to sterile culture cabin (lamin air flow). Every kolle was inoculated by small pieces of Coniophora puteana 14 days that cultured before in above plates and set in 25-30[degrees]c in dark for 14-21 days. During this time the mycelium covered all surface of medium culture.

The Vicinity of Fungi with Wood Samples:

Wood block (5*1*2 cm dimensions )of Parrotia persica, Juglans regia , Quercus persica and Buxus sempervirens dried at 100[degrees]C for 48 h, cooled in a desiccator, and then weighed.

All blocks were then re-wetted, so that their moisture content was in the range of 50-80% (by dry wt). Thereafter, wood blocks were autoclaved at 121[degrees]C for 30 min. After autoclaving, wood blocks were placed onto 14 day-old cultures on kolle dishes that were artificially incubated with brown rot fungi.

Study of Fungal Activities in Wood:

After 16 weeks, essential measurement was performed. The decrease in mass is evaluated according to following formula.

The dry mass of the test spices and the relative moisture ([F.sub.i]) were determined for the control of each series as follows:

[F.sub.i] = 1 - (mo - [m.sub.1]/[m.sub.0])

[F.sub.i] = initial moisture factor [m.sub.0] =conditional mass [m.sub.1] =initial oven dry mass

Having determined the mean [F.sub.I] for each series the oven dry mass ([m.sub.i]) of the equivalent set of the test spicemen was calculated using the following formula

[m.sub.1] = [m.sub.0] x [F.sub.i]

% Final loss of dry mass = ([m.sub.1]-[m.sub.2]/[m.sub.1])x100

Where [m.sub.2]=Final dry mass

In order to determine durability of species, we can use Findlay methods as follows
Very durable <5%
Durable 5%
Moderate durable 5-10%
Low durable 10-30%
No durable >30%

Systematic Position of Species:
Buxus sempervirens L. (from Buxaceae)
Juglans regia I.(from Juglandaceae)
Parrotia persica. D.C.(from Hamamelidaceae)
Quercus p ersica. J. et Sp. (from Fagaceae)
Anatomical characters are taken in Table 1
Table 1

Statically Analysis:

Analysis of variance (ANOVA methods) was employed to compare average dry mass. [S.sup.2] P and [S.sup.2]X were determined.

The following formula was used in order to determine the minimum amount of difference that makes the difference significant (LSD) [alpha] = 0.05

LSD = t(a.r (n - 1)[square root of 2/n[S.sup.2]P])

Study of Wood Decay under the Microscope:

The following process such as Soften and Cutting were done [14]. the cuts prepared by the microtome about with 12-16 [micor] thickness then staining process was done with fast green and safranin. After dehydration process and mounting on microscopic slide, species were studied under microscope.

Results and Discussion

Wood Anatomy of the Investigated Tree Spp:

In diffuse porous wood of Quercus sp , Parrotia persica, Juglans regia the vessels are solitary or distributed in radial clusters of 2-4 cells. Axial parenchyma cells consist of band of cell rows at the border of each annual ring (apotracheal terminal). Vessels and a parenchyma cells are embedded in a tissue consisting of thin-walled libriform wood fibres. (fig 2,3,4,7 and table 1).

In diffuse porous wood of Buxus sempervirens (fig 6), vessels are solitary and less distributed in radial clusters of 2-4 cells. The last few cell rows of libriform wood fibres at the border of the annual rings are more strongly lignified than the weakly lignified libriform wood fibres of the early wood. The apotracheal parenchyma is sparse and diffusely distributed. (table 1)

Dry Weight Loss of Incubated Wood Samples:

Table 2 shows the mean dry weight losses of wood samples incubated with Coniophora puteana 16 weeks .The lowest weight losses were recorded in Buxus sempervirens wood.

After 16 weeks, the highest weight losses by Coniophora puteana were recorded in Juglans regia (2.56 %), followed by Quercus persica ( 2.54 %).

At the macroscopic level, decay fungi showed differing abilities in exposing the border of annual rings. The quality of tree ring detection appears to be related to the selected host-fungus combination and the incubation period applied.

Generally, the application of decay fungi showed promising results with respect to the detection of annual rings in samples from which low weight losses were recorded.

After 16 weeks incubation, most samples showed extensive signs of decay although loss mass were not visible.

Studies carried out by Schwarze et al on some tree species show that parenchyma cells resists degradation by brown rot fungi, whereas the surrounding libriform wood fibres are strongly decayed [21]. Subsequently, the presence of a band of parenchyma cell rows at the border of the annual ring (apotraceal terminal) is strongly enhanced during early stages of brown rot ,this results accordance with Deflorio [5] work in wood decay fungi application in enhance annual ring detection.. Thus the non-uniformed degradation pattern in Buxus sempervernis results in a conspicuous exposure of tree rings.

The detection of tree rings in samples of studied wood was also improved with brown rot fungi. Although parenchyma cells are absent at the annual ring boundary of this species, Resistance of late wood fibres appears to be related to a higher degree of cell wall lignification, thus resulting in a greater resistance towards degradation.

This study has taken a step in the direction of proving whether biological methods could be used for macroscopically detecting annual rings and other characters.

Although the incubation of wood samples with decay fungi could lead to selective degradation of the wood cell wall, the wood samples sterilization after each incubation period prevents any further development of a decay process. Wood strength properties are also not critically affected.

Improving the macroscopic visibility of tree ring structures through controlled degradation could open up new perspectives for dendrochronology. Although the application of wood decay fungi for dendrochronological studies is still in an initial phase and the number of fungal species used so far reflect only a small spectrum of described species, screening the potential application of further decay fungi may prove to be very valuable for the future progress of dendrochronology.

At the initial stage of the decay the brown -rot fungi seem to operate by a mechansm which cause a extensive changes in the wood cell leading to a rapid decline in the strengh properties [16].

After 16 weeks of culture under certain condition Coniophora puteanea grew completely and became a dirty light brown mass there was a long delay period in the first stage of growth in comparison to coriolus versicolor that in which delay phase is short [12]. In all samples there was relative damage apparently. On the other hand, Mycelium coverage was about 100%. Besides, dry weight decreasing and changes in dimension were as follows:

(Table2). Plus, The mycelium of fungi was not seen in vessel in Buxus sempervirens, Perhaps it is due to little and small size of vessel in Buxus sempervirens and condensed fibres in annual ring. Although important differences were found in anatomical properties of commercial woods, no significant changes were found in relative weight loss in over 16 weeks for either fungal species. So, it was according to reserach of Humar et al in [11]. Also, results showed that high density woods had a little and small vessel had a high natural durability. If suitable substrate is present, the filamentous growth of Basidiomycetes forming mycelia cords (Baldrian, 2008) will represent a significant advantage for their growth. In wood, mycelia growth is allocated to lignocelluloses based resources and the creation of a network interconnecting the resource units [5].


In anatomical study, it was observed that the hyphae of Coniophera puteana occurs very sparsely in the wood, often restricted to the lumen of woody cells, and yet they cause a generalized decay in which the [S.sub.2] wall layer is almost completely degraded (fig1-8).

In studied species penetration of hyphe in vessel is detectable except for Buxus sempervirnes. It seems there was correlation with vessel dimension, frequency and density, While in Buxus sempervernis number of vessel and size is low. (Table 2).

However, all studied species were durable and resistant against Coniophera puteana. according to results of Schwarze in [22] on Brown rots that were examined microscopically in wood blocks such as Norway spruce, Betula, Oak, Robinia and Sycamore , most of the hyphae grew on the surface of the S3 layer and in the lumen.

In brown rot decomposition of cellulose. also, hemicelluloses take place at different stages. In fact, Cell wall degradation occurs not in the immediate vicinity of the hyphal sheath or out of the lumen (fig 1-8), but the ectoenzymes of the brown rot fungi must first diffuse very deeply into the cell wall through the S3 layer, to degrade the cellulose-rich S2 layer.

Thus, high content tends to delay the diffusion of the large molecules of the cellulose-degrading enzymes into the cell wall. primarily growth (delay phase) took a long time in Coniophera puteana because of these reasons in comparison with Coriolus versicola, [12,13] for examples.

When vessels are blocked by tyloses (Fig 2) or by extraneous deposits, penetration may occur through such prosenchymatous tissues since fibres are more important in preservative movement than wide rays in a way that they occur in oak and Beech wood. [12].








Mass loss caused by fungi is expressed in relative values (table 2). Besides data gained from the density and loss mass lead us to the same conclusions ;that density and durability are related to Mass losses, caused by wood decay fungi ,varied from 1.58% to 2.90%. Although differences are low, in general ,specimens with narrowest rings and low density were more decayed than specimen with average or the widest xylem growth rings. The highest mass losses were observed at specimens with narrow rings (table 1).

Wood is an important renewable and biodegradable natural resource with a multiple uses, also, wood is used extensively as a structural material for buildings,.. due to its high strength per unit weight, its versatility, and its variety. Hence, it is important for wood to be resistant to degrading. Therefore, the studied species are introduced as durable woods. According to results. Coniophora puteana effectively induce permeability changes in wood structures. It can be used to facilitate the identification of tree rings in diffuse porous , because the latewood of studied specimens limits the penetration of fungal mycelium.

Studying the microscopic characteristics of wood degradation and introduce the fungi involved in decay of living tree of this commercial forestry species will help to develop control measures against decay.


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[3.] Barron, G.L., 2003. Predatory fungi, wood decay and carbon cycle. Biodiversity 4: 3-9.

[4.] Blanchette, R.A., 1992. Anatomical responses of xylem to injury and invasion by fungi. In: Blanchette, R.A., Biggs, (eds), Defense Mechanisms of Woody Plants against Fungi. Springer, Berlin, pp: 72-92.

[5.] Boddy, L., J.C. Frankland, P. West, (eds) 2008. Ecology of Saprotrophic Basidiomycetes. Elsevier, Amsterdam G. Deflorio, et al., 2005. The application of wood fungi to enhance annual ring detection in three diffuse-porous hardwoods. Dendrochronologia., 22(2): 123-130.

[6.] Erwin, S.T., et al, 2008. Anatomical characterization of decayed wood in standing light red meranti and identification of the fungi isolated from the decayed area. J.Wood.Sci.54:233-241

[7.] Folmann, L.B., Klien LP. J. A. Gunnewiek, Boddy, de W. Boer, 2008. Impact of white -rot fungi on numbers and community composition of bacteria colonizing beech wood from forest soil, FEMS Microbiology Ecology, 63: 181-191.

[8.] European Committee for Standardization, 1994. Durability of Solid wood EN.350-1

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A.M. Olfat

Department of Wood and Paper Science and Industries, Faculty of Natural Resources University of Tehran, Iran.P.O.Box.31585-4314

Corresponding Author

A.M. Olfat, Department of Wood and Paper Science and Industries, Faculty of Natural Resources University of Tehran, Iran.

P.O. Box. 31585-4314; Tel-Fax: 0098-261-2249311.
Table 1: Anatomical characteristics of studied wood species.

Species Family Characteristics

 Buxus Buxaceae Wood diffuse -porous.
 sempervirens.L Growth ring boundaries distinct
 and demarcated by yellow- brown
 late -wood. Tylose in vessels is
 absent. Axial parenchyma
 apotracheal .Narrow growth ring
 but with dense fibre tissue. basic
 specific gravity 0.79-0.97 g.

Juglans regia.L Juglandaceae Wood diffuse-semi ring porous.
 Growth ring boundaries is
 distinct. Axial
 parenchyma-apotracheal with
 average growth ring. Tylose in
 vessels present. basic specific
 gravity 0.45-0.74
 g.[cm.sup.-3]Wood diffuse porous
 .Growth ring distinct, delineated
 by a dark line of denser
 summerwood. Pores extremely small.
 rather scanty.with narrow growth
 ring. Basic specific gravity 0.87
 g. [cm.sup.-3]

Parrotia persica Hmamelidaceae Quercus persica J. et sp
D .C FagaceaeWood ring porous .vessels
 in the early wood are distinctly
 larger than those in the late wood
 hence Growth ring boundaries
 distinct. Early wood is full of
 tylose with average growth ring.
 Basic specific gravity 0.75-0.85
 g. [cm.sup.-3]

Table 2: Changes in dry weight and
dimensions after 16 weeks incubation.

Spices [M.sub.1(gr)] [M.sub.2(gr)] [M.sub.2(gr)]

Juglans regia 6.9602 6.7831 0.1771
Parrotia persica 8.9331 8.684 0.2574
Buxus sempervirens 10.5347 10.3694 0.1653
Quercus persica. 8.208 8 0.208

Spices Loss mass (%) .sub.cm3]

Juglans regia 2.56 5.18*1.52*2.08
Parrotia persica 2.9 5.18*1.53*2.15
Buxus sempervirens 1.58 5.09*1.54*2.06
Quercus persica. 2.54 5.18*1.56*2.06

Where [m.sub.1] = initial oven dry mass and [m.sub.2] = Final dry mass
significant difference at p [less than or equal to] 0.05.
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Title Annotation:Original Article
Author:Olfat, A.M.
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Mar 1, 2011
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