IAWA Journal, Vol.

19 (2),1998: 169-180

ANATOMICAL STUDY OF THE DECAY CAUSED BY THE

WHITE-ROT FUNGUS TRAMETES TROGII (APHYLLOPHORALES)
IN WOOD OF SALIX AND POPULUS
by
Laura Leviu l & Maria Agueda Castro 2
1Laboratorio de Micologia Experimental & 2Laboratorio de Anatomia Vegetal,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pab. II, Pi so 4, 1428 Buenos Aires, Argentina

SUMMARY

Different stages of decay caused in vitro by Trametes trogii in Salix sp.

and Populus sp. wood are described. Anatomical features are reported
in three stages of this process. Decay progressed in a different pattern
in both species studied. In Populus sp. T. trogii caused a combination
of selective delignification and simultaneous decay within the same sub-
strate. In advanced stages wood blocks exhibited large empty holes and
a spongy structure. In Salix sp. a simultaneous white-rot decay took
place. Only vessels remained and the residual white-rotted wood devel-
oped a stringy appearance.
Key words: Wood decay, Salicaceae, white rot, Trametes trogii, LM,
SEM.

INTRODUCTION

White-rot fungi are the only type of wood-rotting fungi that can attack all wood com-
ponents and because of their ability to degrade lignin they are of interest to the pulp
and paper industry. Before white-rotters can be used industrially, it is necessary to
obtain knowledge of how they degrade different cells and about the degradation proc-
ess (Blanchette 1991).
White-rot basidiomycetes remove lignin from wood in several morphologically
distinct patterns. They can cause a simultaneous degradation of lignin along with cell
wall polysaccharides, leaving cells either riddled with holes and erosion troughs or
with extensively thinned secondary walls, or they can produce a selective type of
lignin degradation. During selective removal, lignin in the secondary wall and middle
lamellae may be almost entirely removed, leaving large quantities of cellulose in the
S2layer of the cell wall. However, the great variability observed makes it difficult to
place many species into anyone group (Blanchette 1994). In addition to species of
white-rot fungi that preferentially degrade lignin or those that simultaneously attack
all cell wall components, others produce both types of decay within the same substrate,
or selectively digest lignin during incipient stages of decay but subsequently remove
the residual cellulose in later stages of attack (Eriksson et at 1990). Several fungi can
cause a highly selective degradation of lignin in one type of wood and a simultaneous

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170 IAWA Journal, Vol. 19 (2),1998

degradation in another tree species (Ago sin et al. 1990). Environmental factors (e. g.,
temperature, moisture, oxygen and nitrogen concentration) also have significant ef-
fects on the amount oflignin degraded by a given isolate (Blanchette 1991).
Trametes trogii Berk. is a white-rot fungus, and attacks salicacean wood in Argen-
tina (Wright et al. 1973) and wood in poplar plantations in northern Italy (Anselmi
1990).
Salicacean woods are commonly used in the Argentinean pulp and paper industry.
Biopulping research with this fungus showed that it reduces energy input, and signifi-
cantly improves strength characteristics of finished paper (Planes et al. 1985). In vitro
production of cellulases, xylanases, pectinases and ligninases by this species has al-
ready been investigated (Levin & Forchiassin 1995, 1997; Vares & Hatakka 1997).
The present work was undertaken with the aim of obtaining a better knowledge of
the biology of this fungus, and insight into its decay capacity. This study reports the
main diagnostic features related to different stages of the decay, caused in vitro, by
Trametes trogii in willow and poplar woods. Anatomical characters visible with light
and scanning electron microscopy are described.

MATERIALS AND METHODS

Microorganism: Strain 463 (BAFC: Mycological Culture Collection of the Departa-

mento de Ciencias Biol6gicas, Facultad de Ciencias Exactas y Naturales, Universidad
de Buenos Aires) of Trametes trogii Berk. (Aphyllophorales, Basidiomycetes) was
used in these experiments. Stock cultures were maintained on malt agar slants at 4 0c.
In vitro weight losses of wood samples were determined according to the method-
ology used by Job and Wright (1986); 3 x 1 x 0.5 cm wood blocks of Populus sp. and
Salix sp. (Salicaceae) were used. Blocks were incubated 6 months at 28 ± 1°C. Every
two months, 10 blocks were withdrawn and cleaned of surface mycelia. Eight were
oven dried at 70°C until constant dry weight in order to verify weight losses, two
were reserved for anatomical sections. Uninoculated blocks served as controls.
For light microscopy (LM) studies, wood samples at different stages of decay were
selected, fixed in formaldehyde-acetic acid-alcohol, and embedded in paraffin wax.
Transverse and longitudinal (radial and tangential) sections 15-18 11m thick were cut
and double stained with safranin-fast green (Gram & Jorgensen 1953). They were
examined under a Zeiss microscope with and without polarized light. For scanning
electron microscopy (SEM) observations, sections were dehydrated in an acetone se-
ries, critical point dried, coated with gold-palladium, and observed in a Philips scan-
ning electron microscope.

RESULTS AND DISCUSSION

Weight loss by decay

Both types of wood lost similar percentages of weight during the different incuba-
tion periods (Fig. 1). The rate of degradation was relatively rapid for the first two
months, and slowed during months 2 to 4. Weight loss at 6 months was roughly 55%

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Levin & Castro - White rot in Salix and Populus l71

70,---------------------------------------~
--e-- Salix sp.
60 Populus sp.
'""'
E -0-
<l.l
U
~ 50
8-
'"'"
.9 40
:<:OIl
'v 30
~
/
<l.l

& 20
<l.l /
/
/

~ '/
/

10 ,-
"
0
0 60 120 180
Incubation period (days)

Fig. 1. Weight losses from poplar and willow wood decayed by Trametes trogii during diffe-
rent incubation periods.

for both wood species tested. Lopez and Deschamps (1978) and Anselmi and Nicolotti
(1988) reported similar results using other isolates of Trametes trogii to decay poplar
wood.

Morphology of decay
While no significant differences were found in the percentages of weight loss in
poplar or willow attacked by this fungus, a different pattern of structural and
ultrastructural changes could be seen during the decay.
In both types of wood studied, abundant clamped hyphae could be observed colo-
nizing the lumen of vessel elements, fibres and parenchyma cells and causing cell
wall thinning and erosion (Fig. 2A; 3A-F; 4C; 6A, C). Hyphae were numerous through-
out early and moderate stages of decay, but decreased in number with advancing
decay. Hyphal penetration was mainly through pits (Fig. 6D), whose inner apertures
were enlarged enzymatically during the decay, forming numerous and conspicuous
holes (Fig. 3E, F; 4C-E).

Decay in Populus sp.

The decay had a combined pattern of simultaneous and selective white rot. In some
areas a non-selective attack of all cell components occurred, and cell wall erosion
took place in the secondary wall adjacent to the hyphae (Fig. 3C-F). Decay pro-
gressed centrifugally from the lumen towards the middle lamellae. Once the erosion
reached the middle lamellae, this region was also degraded in a localized area. These
anatomical features could be observed under LM as disrupted or discontinuous walls
in transverse sections, in vessel elements, fibres and radial parenchyma; but the tissue

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172 IAWA Journal, Vol. 19 (2), 1998

could be generally observed lignified and stained red with safranin (Fig. 2A, B). With
SEM, holes with irregular rims, and axially elongated troughs, alongside the growing
hyphae, could be easily observed (Fig. 3F). Yet, in some areas a selective degradation
of lignin occurred, but it was not until advanced stages of degradation that the
delignification became clearly evident. The decay was not uniform, and practically
unaltered tissues coexisted with seriously damaged ones. In advanced stages of de-
cay, wood blocks presented a spongy structure. Large holes in the wood were evident,
where all cell types had already been disintegrated. In the remaining fibres and vessel
elements a selective removal of lignin could be observed. In transverse sections dou-
ble stained with safranin-fast green, their secondary wall stained green, indicating the
absence of lignin, whereas lignin present in the cell corners and in the compound
middle lamellae stained red (Fig. 2e, D). Delignification also affected the rigidity of
the vessel walls and some collapsed vessel elements could be seen under SEM (Fig.
(text continued on page 178)

Legends to Figures 2-6:

Fig. 2. White-rot decay caused by Trametes trogii in poplar wood; light micrographs. - A, B:
simultaneous decay: disrupted walls in fibres, vessel elements and radial parenchyma (arrow).
- C, D: selective decay in an advanced stage: delignified secondary wall (arrow) and lignified
middle lamella. - E, F: loss of cellulose birefringence, polarized illumination. - dl =de1igni-
fied; h =hyphae; H =hole; 1 =lignified. - Scale bars =50 f.lIIl.

Fig. 3. White-rot decay in poplar wood, early and moderate stages; SEM micrographs. In A-D
hyphae colonizing the lumen of parenchyma, vessel elements and fibres. - A, B: thick cell
walls; A: general aspect; B: fibres with clamped hyphae (arrow). - C-F: thinning and cell wall
erosion; C: general aspect; D: disrupted cell walls in fibres; E: holes in radial parenchyma,
superficial view; F: vessel elements: coalescence of holes and axially elongated troughs
alongside the growing hyphae. - Scale bars in A-C = 100 f.lIIl; in B = 10 11m; in D-F =50 f.lIIl.

Fig. 4. Advanced stage of decay in poplar; SEM micrographs. - A, B: vessel elements, fibres
and parenchyma cells partially destroyed, transverse sections, general aspects. - C-E:
longitudinal sections: eroded cell walls; C, D: in vessel elements; E: in fibres and radial
parenchyma. - Scale bars in A-C = 100 f.lIIl; in D = 10 f.lIIl; in E = 50 11m.

Fig. 5. White-rot decay caused by Trametes trogii in willow wood; light micrographs. - A-C:
early and moderate stages of decay; A: general aspect; B: cell wall cavities in latewood fibres
(arrow); C: lignified vessels and cell comers in earlywood (arrow). - D: advanced stage: lignified
vessels only remaining. - Scale bars in A, B, D = 100 11m; in C = 50 f.lIIl.

Fig. 6. Progressive decay in willow wood; SEM micrographs. - A: early stages, general aspect.
- B, C: moderate stages: note radial parenchyma and fibres partially destroyed. - D: hyphae
passing through the intervascular pit. - E, F: advanced stages. - E: stringy appearance in
longitudinal view. - F: general aspect, only vessels remaining. - Scale bars in A, B, E, F =
100 f.lIIl; in D = 10 flm; in C = 50 f.lIIl.

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Levin & Castro - White rot in Salix and Populus 173

.'1'14 ,r
. ', " '"~" .
.
.. ';,- ~
':' ~ , /' "
.-.~ ~ .
-'''.
,J~
"'.""r I.
, .:
.
4("'"
'.

{ A.\..( ' .
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-.r' -\oJ.". G
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Figure 2 - Legend on page 172.

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174 IAWA Journal, Vol. 19 (2),1998

.. -,.,;:' .: .
-'.
,
'-
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Figure 3 - Legend on page 172.

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Levin & Castro - White rot in Salix and Populus 175

Figure 4 - Legend on page 172.

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176 IAWA Journal, Vol. 19 (2), 1998

Figure 5 - Legend on page 172.

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Levin & Castro - White rot in Salix and Populus 177

Figure 6 - Legend on page 172.

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178 IAWA Journal, Vol. 19 (2),1998

4A, B). When lignin was depleted, the loss of birefringence was evident, indicating
generalized cellulose depolymerization along with the change in its anisotropic prop-
erties (Fig. 2E, F). As it was found in our study, there are many other examples where
one fungus produces both types of attack in the same substrate (Adaskaveg et al.
1990; Blanchette 1991, 1995). The reason for different forms of cell wall attack is
unclear, but is most likely related to differences in oxidative enzymes produced by
the fungus (Reid 1995) and differences in systems of cellulase and hemicellulase
repression (Blanchette 1995).

Decay in Salix sp.

In Salix sp., Trametes trogii caused only a simultaneous type of white rot. In early
and moderate stages of decay, the first signals of damage appeared in the thinner cell
walls of earlywood (Fig. S A). While fibres and ray parenchyma cells showed signs of
degradation, a remarkable resistance of vessel elements to the decay could be seen
(Fig. SC; 6A-C). In these decay stages, some cells had their secondary wall com-
pletely removed, and only the middle lamellae and cell comers remained (Fig. SC,
arrow). The lignin content is higher in the middle lamellae and cell comers and im-
parts greater decay resistance to these regions; the polysaccharides that are more abun-
dant in the secondary wall, contribute to its faster degradation. In fibres, the middle
lamellae are high in G lignin and the secondary walls contain a high proportion of
S lignin. Syringylpropyl (S) units of lignin are preferentially degraded by white-rot
fungi, whereas guaiacylpropyl (G) units are more resistant to degradation (Saka &
Goring 1988; Obst et al. 1994; Blanchette 1995).
In latewood fibres, round erosion troughs in the thick cell walls adjacent to the
hyphae were frequently detected (Fig. SB). The differences observed among earlywood
and latewood could be explained by latewood being less accessible to hyphae be-
cause of its thicker cell walls with fewer and smaller pits (Wilcox 1993).
In advanced stages of decay the residual white-rotted wood developed a stringy
appearance, fibres and parenchyma cells had been completely degraded, and only
vessels remained (Fig. 6E, F). Similar decay patterns were observed by Blanchette
et al. (1988), when studying white-rot decay in Acer and Tilia woods. The combined
anatomical and chemical composition of wood appears to govern microbial enzymes
responsible for the white stringy rot type of wood degradation resulting in an unusual
selective degradation of all cells except vessels. The high lignin content, large S3
layer, and increased concentration of guaiacyl versus syringyl lignin in the vessel
elements of some angiosperm wood, together confer a degree of resistance to enzymatic
attack by fungi that cause a white stringy rot (Blanchette et al. 1988). Phebia tremello-
sus has been shown to cause a preferential delignification of all woody cells including
vessels in Populus (Blanchette & Reid 1986). Likewise T. trogii decay on Populus
wood affected all xylary cells. Probably differences in chemical and ultrastructural
aspects exist in the vessel walls of the two wood species which modify the decay
pattern.

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Levin & Castro - White rot in Salix and Populus 179

CONCLUSIONS

The present study describes the main anatomical changes that could provide the basis
for diagnosing and evaluating wood decay by Trametes trogii. This white-rot fungus
caused similar weight losses in both woods tested. Although the woods have similar
xylotechnological properties, differences in the pattern of decay were evident. In
Populus sp. the decay had a combined pattern of simultaneous and selective white
rot. A non-selective attack of all cell components occurred in some areas and selec-
tive lignin degradation in others, but all woody cells were attacked. In advanced stages
of decay, wood blocks had a spongy structure and large holes in the wood were evi-
dent, where all cell types had already been disintegrated. In Salix sp. a simultaneous
white-rot decay took place, affecting fibres and parenchyma cells but not the vessel
elements. Only vessels remained in advanced stages of decay and the residual white-
rotted wood developed a stringy appearance.

ACKNOWLEDGEMENTS

We thank Dr. J.E. Wright, Dr. E. Ancibor and Dr. F. Forchiassin for their critical reading of the
manuscript, and Mr. D. Gimenez for his technical assistance during SEM observation. This work
was financially supported by CONICET, PID 3341/92 and University of Buenos Aires, Ex 134.

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