Fungal Decay of Wood: Soft Rot—Brown Rot—White Rot

Abstract

Wood decay by fungi is typically classified into three types: soft rot, brown rot and white rot. Brown rot fungi are the most prevalent with regard to attack on coniferous, structural wood products in North America. The wood decayed by brown rot fungi is typically brown and crumbly and it is degraded via both non-enzymatic and enzymatic systems. A series of celluloytic enzymes are employed in the degradation process by brown rot fungi, but no lignin degrading enzymes are typically involved. White rot fungi are typically associated with hardwood decay and their wood decay patterns can take on different forms. White rotted wood normally has a bleached appearance and this may either occur uniformly, leaving the wood a spongy or stringy mass, or it may appear as a selective decay or a pocket rot. White rot fungi possess both cellulolytic and lignin degrading enzymes and these fungi therefore have the potential to degrade the entirety of the wood structure under the correct environmental conditions. Soft rot fungi typically attack higher moisture, and lower lignin content wood and can create unique cavities in the wood cell wall. Less is known about the soft rot degradative enzyme systems, but their degradative mechanisms are reviewed along with the degradative enzymatic and non-enzymatic systems known to exist in the brown rot and white rot fungi. As we learn more about the non-enzymatic systems involved in both brown and white rot degradative systems, it changes our perspective on the role of enzymes in the decay process. This in turn is affecting the way we think about controlling decay in wood preservation and wood protection schemes, as well as how we may apply fungal decay mechanisms in bioindustrial processe.

Full text

Chapter 2
Fungal
Decay
of Wood:
Soft
Rot—Brown Rot—White Rot
Barry
Goodell1
,
Yuhui
Qian
1
,
and Jody
Jellison
2
1Wood
Science
and
Technology
and
2
BiologicaI
Sciences,
1
Nutting
Hall/
AEWC,
and 2
Hitchner Hall,
Un ive rsi ty
of Maine, Orono,
ME 04469-5755
Wood
decay by
fungi
is
typically
classified
into
three
types:
soft rot, brown rot and white rot.
Brown
rot
fungi
are the most
prevalent
with
regard to attack on coniferous, structural wood
products in
North
America.
The wood decayed by brown rot
fungi
is
typically
brown and crumbly and it is degraded via
both non-enzymatic and enzymatic systems. A series of
celluloytic
enzymes are employed in the degradation process
by
brown rot
fungi,
but no
lignin
degrading enzymes are
typically
involved.
White rot
fungi
are
typically
associated
with
hardwood decay and their wood decay
patterns
can take
on
different forms. White rotted wood normally has a bleached
appearance and this may either occur
uniformly,
leaving the
wood
a spongy or stringy mass, or it may appear as a selective
decay or a pocket rot. White rot
fungi
possess both
cellulolytic
and
lignin
degrading enzymes and
these
fungi
therefore have
the potential to degrade the entirety of the wood structure
under the correct environmental conditions. Sof
t rot
fungi
typically
attack higher moisture, and lower
lignin
content
wood
and can
create
unique cavities in the wood
cell
wall.
Less
is known about the soft rot degradative enzyme systems,
but their degradative mechanisms are reviewed along
with
the
degradative enzymatic and non-enzymatic systems known to
exist
in the brown rot and white rot
fungi.
As we learn more
about the non-enzymatic systems
involved
in both brown and
©
2008
American
Chemical
Society 9
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
This PDF copy is for the author's own use for academic purposes, and it is not for
mass dissemination. It should be used only by academic students and colleagues.
Readers are encouraged to view the content in this Chapter as archival information recognizing that, as advances occur
in research, more recent/future reviews of the subject content will refine the information provided here.
Barry Goodell

10
white
rot degradative systems, it changes our perspective on
the role of enzymes in the decay process.
This
in turn is
affecting
the way we think about controlling decay in wood
preservation and wood protection schemes, as
well
as how we
may
apply fungal decay mechanisms in
bioindustrial
processes.
Introduction
Wood
decay
fimgi
have
long
caused problems for the durability of
structures, but
these
fungi
have also served an important
ecological
function as a
beneficial
agent
in breaking down waste materials in the environment. Since
wood
remains the most abundant natural resource used in construction (7),
methods to protect wood remain of paramount importance in the built
environment.
Although
insects, marine organisms, weathering and other
agents
of
deterioration cause losses in durability of wood and wood projets, more
destruction of wood is caused worldwide by decay
fungi
than by any of
these
other agents. Selected environments may favor other destructive organisms in
some locations however.
Typically
the term "decay" refers to the degradation of wood by fungal
action,
ultimately resulting in wood strength loss.
Wood
staining
fungi
and the
colonization
of wood by
mold
fungi
are not considered to be decay organisms
even though
these
fungi
can increase moisture uptake in wood and minor
strength losses in the wood are observed in some instances in
temperate
zones
because of the action of
these
fungi.
For example, it is
well
known
that
some
staining
fungi
can reduce the toughness of
stain-colonized
wood although other
mechanical
properties remain unaffected in this wood. Interestingly, significant
strength loss can occur in wood attacked by stain
fungi
in certain tropical
environments.
Wood
decay
fungi
may be
divided
into
three
categories.
Classically,
the
action
of white rot and brown rot
fungi
have been described because of the way
that
the wood
appears
after colonization by
these
fungi
and advanced
degradation of the wood has occurred. However, in the last 60 years, another
category of decay known as soft rot has been discovered and this fungal group
has also been observed to cause significant problems in wo od products.
Both
brown rot and white rot
fungi
are grouped in the Eumycota
(true
filamentous fungi) and more
specifically
in the sub-division o f Basidiomycetes
(Basidiomycotina)
fungi.
The soft rot
fungi
typically
have been found in the
Ascomycetes
(Ascomycotina) sub-division or they have been classed as
Fungi
Imperfecti
because their sexual
state
has not clearly been identified. In most
cases,
these
"imperfect"
fungi
key out to Ascomycetes once their sexual
state
is
revealed
or when molecular techniques are
utilized
for fungal analysis. The
Basidiomycete
decay
fungi
produce filamentous hyphae
that
are 20 to 30 times
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11
smaller
(1-5 πι) than the wood
cell
walls they invade.
Typically,
brown and
white
rot
fungi
attack the wood ray cells
initially
and most hyphae
will
pass
from
one
cell
to the next by growing through the pits
of
the wood
cells.
As th e growth
and invasion process continues beyond the
initial
stages,
the
fungi
extend
lengthwise through the lumens of the wood cells secreting non-enzymatic and
enzymatic
metabolites in an effort to degrade the wood
cell
wall
to obtain
nutrients
from
these
walls.
The soft rot
fungi
also
initially
invade the wood in a
similar
manner. For decay to occur, all decay
fungi
must have an
adequate
micro-environment
to foster the growth of the fungus. In addition this
micro-
environment must permit the secretion and
diffusion
of fungal metabolites
permitting
attack of the wood
cell
wall.
A favorable micro-environment
would
include
wood at the appropriate temperature, moisture content and pH.
Heartwood
containing natural fungitoxic agents, or wood treated
with
fungicidal
preservative chemicals, can act in several ways to prevent the growth or function
of
the decay
fungi.
Typically
wood must have a moisture content (MC) at or
above the fiber saturation point for wood degradation by
fungi
to progress.
Optimal
moisture contents
will
range
from
50% to beyond 150%
M C
depending
on
the specific gravity of the wood and the species of decay
fungi
colonizing
the
wood.
Many
soft rot
fiingi
are known to prefer high
MC
environments however,
and can attack wood at levels close to the wood-lumen saturati on
level.
Some
thermophilic
fungi
will
grow in, and degrade, wood
cell
walls at high
temperatures in excess of 80 °C; however, most decay
fungi
prefer temperatures
in
the range of 5-42 °C.
Optimal
temperature
ranges
will
vary considerably
dependent upon fungal species and isolate.
Above
and below the optimal range,
the
fungi
typically
may not be
killed,
but if th e temperatures are not extreme the
fungi
will
form
"resting" structures such as spores or resistant
mycelial
fragments. These resting structures can then germinate to
form
new fungal
hyphae when conditions are suitable for fungal growth and metabolism. Some
resting structures/spores are capable of
surviving
for years and can therefore be a
source of fungal
colonization
if
wood
is rewetted.
Detecting
the
Presence
of
Decay
Basidiomycetous
fungi
often produce large
fruiting
bodies, commonly
known
as conks, punks or bracket/shelf mushrooms in the later
stages
of wood
decay processes. In some
cases
however, at the macroscopic
level
the
fruiting
bodies can be indistinguishable
from
a
mycelial
mat growing on the wood
surface,
typically
on the underside.
Both
white and brown rot
fungi
can grow
through and decay wood even in the homokaiyotic (haploid)
state.
Because a
dikaryotic
state
from
the fusion of two homokaryon hyphae is required to
produce sexual
fruiting
bodies, and decay
fungi
in the homokaryon genetic
state
have been observed to produce active decay of wood, decay
fungi
do not
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12
necessarily
produce sexual
fruiting
bodies even in advanced decay
stages.
Production
of
fruiting
bodies can also be
dependent
upon environmental
conditions.
Even
among dikaryons, many decay
fungi
do not produce sexual
fruiting
bodies even in advanced decay
stages.
The purpose of the
fruiting
bodies
is
to produce sexual spores
which
can infect new wood and to provide a
mechanism
for sexual recombination. However, asexual spores and
mycelial
fragments have been found to be a primary source of fungal decay colonization
in
some
cases
(2).
Determining
if wood decay is occurring in its early
stages
can be
difficult
and has been examined
from
a number
of
perspectives. Isolation via culturing of
the
fungi
from
the wood can be used for determining if decay
fiingi
are
present,
but this method can pose problems in interpretation.
First,
only
living
fiingi
and
spores capable of germinating can be isolated in culture
from
wood undergoing
decay. If decay occurred in wood and caused a loss of strength, but the fungus
has become inactive over time, it may not be possible to culture the fungus
from
the wood. Conversely, r ec en t
colonization
of the wood may not have produced
significant
decay, yet the fungus can
still
be cultured
from
the wood. Therefore,
fungal
culturing and
allied
methods using immunological or molecular
techniques to
detect
the presence of decay
fungi
or fungal metabolites require
interpretation or they can be misleading.
Even
if
fungi
can be cultured
from
wood,
since many
fiingi
and bacteria often inhabit wood the
fungi
isolated must
be carefully sub-cultured, and techniques
which
favor the isolation of
basidiomycetes are often employed (5). Once pure, isolates of the decay fungus
must be keyed out, a time consuming and sometimes tedious process. For
positive
identification of some isolates, mating of the unknown
with
a known
candidate culture, maintained in a
collection,
must be performed. Methods
based on immunogenic response to components of the
fungi
or their metabolites
have been developed for detection of the presence of decay
fungi
in wood
(4-7).
The
specificity
of
these
assays
can be good, and a
field
test
detection system has
been developed (5-70); however,
like
microscopy a nd culturing analysis they
test
only
for the presence of the fungus, not for the presence of decay of the wood.
Newer
methods for detection of decay
fungi
have also been developed based on
DNA
analysis and
these
methods have the
ability
to
test
for either specific or
broad
classes of
fungi
(4). Polymerase
Chain
Reaction (PCR) methods exist
allowing
such
assays
to be used
reliably
in the laboratory (77).
With
regard to
the
assessment
of degraded wood or the presence of active or inactive decay
however, the
same
concerns exist
with
this method as
with
immunological
methods and
culturing
methods.
Other methods of decay detection include examining the decayed wood
microscopically.
This
is a good method for determining if decay
fiingi
are
present;
however, it
depends
upon the observation of key
features
indicative o f
decay.
Microscopic
analysis of wood necessarily requires
that
only
a
small
portion
of a sample be observed at any one time, and large samples
would
require extensive analysis in a number of locations to obtain an
overall
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13
assessment
of
the condition of sample as a
whole.
The presence of fungal hyphae
in
wood cells is not an accurate method of assessing the presence or absence of
decay because non-decay
fungi
may also be present in the wood. Some
basidiomycetous
fiingi
will
produce "clamp connections" as a method for
transferring genetic information in the
cell
division
process.
Clamp
connections
appear as
septate
nodes along the sides of the fungal hyphae but, as discussed
above, both brown and white rot
fiingi
can decay wood in the homokaryon
state
or
under other conditions where the
fungi
would
not have clamp connections.
Some
species of wood decay basidiomycetes however, do not produce clamp
connections even in the dikaryotic
state.
So, whereas the presence of clamp
connections in a wood-inhabiting fungus positively indicates the presence of a
decay fungus, the absence of
clamp
connections is not proof of a lack of decay.
Other methods of decay detection are continually being studied. These
methods range
from
chemical assays, electrical resistance analysis, mechanical
tests
such as "resistance
drilling",
to sonic and acoustical methods (4,12-14).
These methods all measure different wood properties
that
can be affected by the
action
of decay
fungi.
To
date,
however, no method has proven to be
fully
accurate in the detection of early
stage
decay in
field
samples where a
great
number of variables
typically
exist. The
difficulties
arise because the
variability
in
the wood
itself
is
typically
greater than any specific factor contributed by the
decay process, so a baseline control value is
critical
for accurate
assessment,
particularly
if early
stages
of decay are to be detected. Several of
these
systems
do work
well
for laboratory monitoring of decay, where careful controls and
baseline measurements can be taken.
Fungal
Decay
Mechanisms
The
decay processes employed by the brown-, white- and soft-rot
fungi
are
incompletely
understood and hence are
still
being examined by a number of
groups.
Although
enzymatic degradation has been studied for many years and a
number of
cellulolytic
and
lignolytic
enzymes have been isolated, research over
the last 15 years has also determined
that
non-enzymatic systems are
involved
in
decay processes. These processes
will
be detailed in the specific discussions of
the
fiingi
below.
Soft
Rot
Decay
Soft
rotted wood was
initially
described as a surface attack of
wood
caused
by
a variety of
Fungi
Imperfecti and Ascomycetes (15,16). The name soft rot
was suggested by Savory (17) to describe surface wood,
typically
in a
waterlogged condition,
that
was degraded by
these
fiingi.
The surface of this
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14
moist
wood
was softer than sound
wood.
Softening of
wood
is
typical
of all
types of decay, so the name may be considered a misnomer, but the term is
currently
accepted by experts in the
field.
Daniel
and
Nilsson
(18) broadened the
scope of
fiingi
included
as soft rot species to include al l Ascomycetes a nd
Fungi
Imperfecti
that
cause decay, but suggested
that
a more
definitive
classification
scheme should be explored before alternate names for
these
fungi
are developed.
Soft
rot
fiingi
typically
attack the outer surface of
wood
in relatively wet
environments (19). However they are
known
to cause extensive and
deep
degradation (extending several centimeters
deep
into the wood) of
utility
poles in
Scandanavia
and northern Europe
with
other reports of
deep
penetrating soft rot
from
other
areas
of the
world
appearing
periodically
(20).
Deeply
penetrating
soft
rot occurs when the
wood
is at a
high
moisture content, but not saturated. It
is
unknown whether this type of deep-penetrating degradation occurs more
extensively
in other
parts
of the
world,
or if it
simply
has been less frequently
reported because
of
the
limited
number of studies on this type of
fungal
attack.
As
soft rotted
wood
dries it develops surface checks across the grain as the
wood
shrinks. The
wood
becomes brown in
color.
Although
the decay may be
superficial,
the surface appearance may be
similar
to brown rot decay. In
advanced
stages
of decay the
wood
will
fail
in a brash or brittle manner when a
surface
sliver
is
lifted.
Typically
the
wood
is described as having a weathered
appearance
like
unpainted "barn board".
In
North
America,
decay by soft rot
fiingi
became more
widely
recognized
when
severe damage to
utility
poles treated using a new type of preservative
treatment
occurred. The process,
known
as the
Cellon
process (21,22), used
liquified
propane (LP) gas or other
volatile
solvents as the carrier for
pentacholophenol
which
was then pressure infused into the
wood.
The solvent
carrier
was
volatilized
from
the
wood
leaving
a clean, residue-free surface.
Unfortunately,
even though
bulk
chemical analysis indicated
that
adequate
retention of pentachlorophenol was present, the chemical often
only
passed into
the
cell
lumens without extensive penetration into the
wood
cell
walls,
or the
treatment
was
localized
because of the
volatilization
of the solvent producing a
variable
treatment
that
the
fiingi
could
"grow around". Washing the surface of
some poles
with
caustic to reduce pentachlorophenol
blooming
also resulted in
the surface leaching of pentachlorophenol resulting in even greater
treatment
variability.
Since soft rot
fiingi
have the capability to
penetrate
and extend
through the
wood
within
the S2 layer and also
typically
have greater resistance to
pentachlorophenol
treatments, they
could
invade and degrade the
wood
and were
less affected by the preservative
treatment
present in the
cell
lumens.
Over
800,000
utility
poles were treated using the
Cellon
process (23) starting in the
1960s,
and many were degraded in service by soft rot
fiingi
over a number of
years
following
installation.
Prior
to this experience
with
soft rot, this type of
fungus was
known
primarily
to attack
wood
in very wet locations such as the
wooden
slats commonly used in
cooling
towers in the previous century,
which
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15
are
still
widely
used. Soft rot in the US, although
still
less common than brown
rot, is now
better
understood to occur in wood
treated
with
a variety of different
preservative systems
Soft
rot fungi produce two different types of attack on the wood
cell
wall.
The first of
these
is known as cavity formation or Type I attack of the S2 layers
of
the wood
cell
wall
(24).
While
Type I cavity formation is always occurs in the
S2
layers of the wood
cell
wall,
these
cavities are also formed in some
cases
in
the SI layers, often in the
same
cell
(G.
Daniel,
personal communication, 2006).
The second type (Type II) is a general erosion of the wood
cell
wall
layers
starting from the S3-lumen interface and working outward. Type II soft rot is
similar
to white rot in this regard. Often, particularly in hardwoods, both Type I
and Type II attack can be produced by the
same
fungus in t he
same
sample.
Not
all soft rot fungi produce cavities in the S2 layer of the wood
cell
wall;
however, Type I cavity formation (24) has been studied more
because
of its
uniqueness.
Typically,
soft rot fungi initiate Type I attack by penetrating from
the lumen, using a fine microhyphae, and boring perpendicularly into the wood
cell
wall
(25,26).
The fungal micro-hyphae re-orient when they reach the S2
layer
to
align
the hyphae in the direction of the S2 cellulose
microfibrils.
Alternately,
the hyphae can branch, in a characteristic manner known as T-
branching,
to
allow
the hyphae to grow in opposite directions paralleling the
wood
cell
wall
S2
microfibrils.
The microfine hyphae
will
extend for a short
distance and then secretion of enzymes and other degradative metabolites for
attack of
cell
wall
material in the immediate environment around the fungal
hyphae is thought to occur. This secretion of metabolites results in the formation
of
cavities
within
the
cell
wall.
A complete complement of
cellulosic
enzymes,
including
endo-l,4-glucanase, exo-l,4-B-glucanase and 1,4-B-glucosidase have
been reported to be produced by soft rot fungi (27-29,26). Other
researchers
(18)
have hypothesized
that
a low molecular weight free radical generating system
also functions
with
phenol oxidase enzymes to oxidize
lignin,
and earlier work
(28) provides evidence
that
laccase is produced by
these
fiingi
to
degrade
the
wood
cell
wall
lignin.
The degradative action
typically
produces a diamond
shaped cavity
with
conically
pointed ends, presumably
because
of the way the
enzymes interact
with
the cellulose structure of the wood
cell
wall.
Following
production
of a cavity, the microhyphae extends further through the S2
cell
wall
and at this point it is known as a proboscis hyphae. The process of enzymatic
secretion and the cavity formation then
repeats
to produce a chain of diamond-
shaped cavities. It has been reported
that
soft rot cavity forming
fiingi
lose the
ability
to produce cavities when invading wood cells
that
have been
delignified,
suggesting
that
the orientation of
cellulose
microfibrils
or macrofibrils as
well
as
the encapsulating
lignin
matrix may play a role in cavity formation (18).
Multiple
hyphae often invade a wood
cell
wall
and can completely riddle the
wall
in advanced
stages
of degradation.
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16
Daniel
and
Nilsson
(18,26)
have reported on the chemical
nature
and the
amount of
lignin
in wood, and how
these
appear
to affect the type and severity of
attack by soft rot
fungi.
The
authors
were able to correlate high
lignin
levels in
wood
with
a reduced severity of attack. Hardwoods
posses
a combination of
syringyl-
and guiaicyl-type lignins and generally have lesser amounts of total
lignin
in the
cell
walls as compared to softwoods. Although bo th hardwoods and
softwoods are attacked by soft rot
fungi,
Type II erosion attack occurs in a more
limited
fashion in softwoods. The reduced capacity for attack of the S3 layer of
softwoods
appears
to be
because
of the highly
lignified
nature
of this layer in
softwoods and its
guiaicyl
lignin
composition. Although the macroscopic
appearance
of the wood may
appear
similar
to brown rot, the erosion
patterns
occurring
at the microscopic
level
appear
similar
to
those
produced by white rot
fungi.
Worrall
et al. (30) have shown however
that
white rot
fiingi
produce
erosion
troughs
with
rounded
ends
whereas the soft rot
fiingi
observed produce
angular erosion troughs.
Brown Rot
Decay
Brown
rot
fiingi
primarily metabolize the holocellulose component of w ood
and
cause
the wood to rapidly lose strength in comparison to the
rate
of wood
metabolism.
In later
stages
of decay, brown rotted wood becomes brown,
crumbly
and checked across the grain,
similar
to the
appearance
of soft rot decay
but
with
a coarser checking pattern. In early
stages
of decay however, the wood
may
appear
little changed other than being "water-stained" or wet. In
these
early
stages,
little
cell
wall
material is metabolized by the fungus; however, extensive
depolymerization
of the cellulose
within
the wood
cell
wall
occurs. In practical
application,
the lack of noticeable signs of decay in early decay
stages
presents
a
dangerous situation in load-bearing applications of the wood since the rapid
depolymerization
of cellulose in the wood cells results in early strength losses
during
the
initial
decay
stages.
Because the wood
does
not
visually
appear
to be
degraded at this
stage,
unexpected failures of wood structures can occur when
loads are applied. This points to the need for regular inspection and maintenance
of
wooden structures.
In
initial
stages
of
colonization,
brown rot
fiingi
selectively attack the simple
sugars
in the rays and the hemicellulose in the wood
cell
wall
(31). The fungi
ramify
through the wood, often using the pits to
pass
from one
cell
to another.
Once
established, pits may
still
be used to move through the wood; however, the
brown
rot hyphae also produce bore holes directly through the wood
cell
walls.
The hyphae
will
secrete
both enzymatic and non-enzymatic degradative
metabolites
within
a hyphal sheath (an extracellular glucan matrix). This hyphal
sheath surrounds the fungal hyphae tips, protecting the hyphae and connecting
the fungus to the wood
cell
wall
to provide a watery environment channeling the
diffusion
of the metabolites into the wood
cell
wall
(32,33).
The hyphal sheath
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

17
has been observed to
penetrate
within
degraded wood
cell
walls (34), permitting
passage
of degradative enzymes into the
wall
in advanced
stages
of degradation.
Enzymatic Brown Rot
Degradation
Mechanisms
Both
an enzymatic and non-enzymatic system are required for attack of the
cellulose
in the wood
cell
wall.
Prior
to the realization
that
a non-enzymatic
system was required,
cellulolytic
enzyme systems were studied extensively. For
this reason the enzymatic systems of the brown
rots
are
well
understood compared
to non-enzymatic systems. The proposed
"Goodell
non-enzymatic mechanism" is
diagramed
here
for
clarification
(Figure 1).
Brown
rot
fiingi
are known to produce
endoglucanases to cleave the
B-l,4-glucosidic
linkages, and B-glucosidases to
hydrolyse
cellobiose or other short oligosaccharides
(35,36).
Onl y
one brown rot
fungus, Coniophoraputeana, is known to also produce a cellobiohydrolase
(37,38).
Brown
rot fungi also produce a number of endo-xylanases and B-xylosidases
important in th e breakdown
of
hemicellulose
(39-41).
Non-Enzymatic
Brown Rot
Mechanisms
Cowling
(42) first recognized
that
all known enzymes were too large a
molecular
size to
allow
penetration into the pore structure of the intact wood
cell
wall
to produce the extent of
cellulose
depolymerization
that
had been observed
in
early
stages
of fungal attack.
Cowling
postulated at the time
that
an
undiscovered, very small
mass
"enzyme" may be
involved
(43)
because
at
that
time,
non-enzymatic catalytic systems had not yet been studied in
microbial
systems.
With
further study by
others
-see earlier brown rot review by
Goodell
(43) for reference citations- it became
apparent
that
a low molecular weight
system was necessary to
penetrate
the wood
cell
wall
permitting cellulose
depolymerization
in advance of
enzymatic
penetration
of
the wood
cell
wall.
Low
molecular weight catechols, quinones and hydroxybenzene derivatives
produced by brown rot
fiingi
were first proposed to act as low molecular weight
agents
in non-enzymatic degradation of the wood
cell
by
Goodell
et al. (44).
Prior
research by this group had shown
that
low molecular weight phenolate
compounds produced by the brown rot fungus Gloeophyllum
trabeum
(45,46)
could
catalyze the production of
hydroxyl
radicals in a mediated Fenton reaction
(47). Continuation
of
this research identified specific compounds produced by G.
trabeum
involved
in this mechanism (48). Other groups have confirmed this
mechanism and have identified other compounds produced by the
fiingi
that
also
function
to mediate the reaction, advancing research in this area and confirming
the
validity
of this mechanism
(49-52).
Research on an
alternate
non-enzymatic
mechanism
involving
a glycopeptide isolated from wood degrading
fiingi
has
also been explored for several years (29). This mechanism provides a potential
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Figure
1.
Simplified overview of the
Goodell
non-enzymatic brown-rot
decay
mechanism.
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

19
alternate pathway for free
radical
production required for non-enzymatic
degradation of
woo d
cell
wall
components. However, the glycopeptide(s) remain
only
partially
purified
and their chemical mechanism therefore remains unclear.
See
Goodell
et al. (43) for a more complete review of brown rot degradation
mechanisms.
White Rot
Decay
White
rot
fungi
attack
wood
and, in advanced
stages
of decay, cause the
wood
to appear bleached in appearance. In some common types of white rot
decay, the
wood
will
become soft and develop a "stringy" character where the
softened
wood
fibers can be easily separated
allowing
the
wood
to be peeled
apart.
In other types of white rot, pockets of softened, deteriorated zones of
wood
may appear.
The
unifying
feature of the white rot
fiingi
is
that
they produce a complete
enzymatic
system capable of directly or
indirectly
oxidizing
and
mineralizing
lignin
(53,54). The enzymes
involved
in this
ligninolytic
activity
include
lignin
peroxidase,
manganese peroxidase, and laccase (Figure 2).
Individual
white rot
fungal
species
will
possess one or more
of
these
enzymes.
Beyond
the
physical
appearance, two types of
white
rot are
known
based on
the manner in
which
the
wood
cell
wall
components are
oxidized.
"Simultaneous
white
rot" is the most common type of white rot of
wood
and
wood
products in
nature
and
typically,
cellulose,
hemicellulose and
lignin
are all degraded at some
relatively
uniform
rate
with
this type of white rot. "Selective white rot" occurs
when
hemicellulose and
lignin
are attacked preferentially to cellulose, in some
cases
and
with
some fungal species,
allowing
the cellulose to remain relatively
undegraded.
Fungi
that
produce this later type of degradation have been studied
for
several years in selective
delignification
systems for biotechnological
applications
(55-57). In
nature
there
is often an intergradation of
white
rot types
and
both simultaneous and selective white rot decays have been reported to be
produced
by the same fungus (58,59).
Historically,
white rotted
wood
has been used for a variety of applications
ranging
from
its use as insulated paneling in Russian refrigerator
trucks/vehicles,
and
white rotted
wood
and fiber has been used for cattle feed in isolated regions
of
the
world.
In the later application, the selective white rot
fiingi
free the
cellulose
from
the
lignin
fraction
of
the
wood
or fiber,
improving
the
digestibility
in
ruminant animals.
More
recently, but for several decades now,
uses
for white
rot
fiingi
in the
field
of
biotechnology
have been studied (60-64). The white rot
fungus Ceriporiopsis subvermispora and Phlebia subserialis have been used in
commercial
bio-pulping
applications to reduce the energy required in
refining
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

20
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

21
wood
chips in mechanical pulp production, and to improve certain strength and
brightness properties of paper produced from the inoculated chips. Other white
rot fungi such as Phanerochaete chrysosporium, Phanerochaete crassa, and
Pleurotus pulmonarius have also been studied as bio-bleaching
agents
for both
kraft pulp and sulphite pulp (65).
Cellulose
and hemicellulose degradation in the white rot fungi is known to
occur
via enzymatic processes and the white rot fungi produce both endo-
glucanases and exo-glucanases
which
can act synergistically on the crystalline
cellulose.
The non-enzymatic processes
which
are known to be
involved
(26,59)
particularly
for hemicellulose depolymerization and selective white rot attack are
not
well
understood. Enzymatic systems for break down of
holocellulose
include
endoglucanases,
β-glucosidases
and cellobiohydrolase enzymes
(66,67)
as
well
as xylosidase, xylanase, acetyl
xylan
esterase,
glucuronidase and
arabinofiiranosidase;
these
later enzymes being necessary for complete
depolymerization
and oxidation
of
hemicellulose
(68,69).
Because of the historic interest in white rot fungi for biotechnological
applications in the latter
half
of the 20th century, white rot enzyme biochemistry
for
lignin
oxidation was studied more intensely than the degradative mechanisms
in
the brown rot
fungi.
For this reason, more is known about white rot
lignolytic
enzyme systems; however, additional work remains to
fully
elucidate the
complete degradation schemes.
Work
revealing the non-enzymatic mechanisms
involved
in brown rot decay has to some extent stimulated research to explore
the role of non-enzymatic degradative systems in the white rot fungi
(70,71).
However,
non-enzymatic mediators
involved
in some white rot enzyme systems,
such
as laccase, have been known for more than a decade. Further research on
these
systems may lead to an improved understanding, not just of the basic
fungal
biochemistry, but on the improved use of white rot fungi in industrial
applications.
It is
well
known
that
non-enzymatic systems
penetrate
deeply into
the wood
cell
wall
in advance of, or preferentially to penetration of the enzymes
(26). Particularly i n selective white rot, low molecular weight
agents
have been
observed to
penetrate
completely through wood
cell
walls and the middle lamella
regions
(26,59).
Although metals and radical ions generated from enzymatic
action
have been identified using in vitro biochemical assays, and the penetration
of
these
low molecular weight components into the wood
cell
wall
has been
postulated, a conclusive study demonstrating the action of
specific
low molecular
weight compounds on the wood
cell
wall
is
lacking.
Lignin
degrading enzymes and the biochemical mechanisms employed by
these
enzymes to oxidize
lignin
have been described in a number of reviews
(72-74).
The discussion below
briefly
summarizes
these
mechanisms and also
introduces information on how some enzymatic systems employ the use of
naturally
produced
substrates,
or "mediators", where this has been reported in
the literature.
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

22
Lignin
Peroxidase
Lignin
peroxidase (LiP) has a relatively high redox potential (75) and is
known
to oxidize both phenolic and non-phenolic
lignin
groups; the latter
composing
approximately 90% of the
lignin
fraction of wood. The
substrate
typically
used for this enzyme in laboratory studies has been veratryl alcohol and
the mechanism for production of the veratryl alcohol radical in the presence of
hydrogen peroxide is shown in Figure 3. As
with
all enzymes,
lignin
peroxidase
is
incapable of penetrating the intact wood
cell
wall
because
of its size, and it is
capable only of attacking the exposed components of the wood
cell
wall
at the
lumen
surface. The veratryl alcohol radical however,
would
have the capacity to
penetrate
into the wood
cell
wall.
This radical has a relatively short
half-life
however (76); and if i t participates in
oxidizing
lignin
in the wood
cell
wall,
then
its short
half
life
would
help to explain the slow erosion of the wood
cell
which
occurs in simultaneous white rot. It
would
not however, explain the observed
degradation of the
cell
wall
in selective white rot decay
because
this type of
attack requires
cell
wall
penetration.
With
this enzyme's
ability
to oxidize non-
phenolic
lignin,
lignin
peroxidase was one of the early enzymes explored for use
in
bioindustrial applications, but it has proven to be less suited for
these
applications than desired and research on its application, for example in pulp
bleaching,
has declined.
Manganese
Peroxidase
and
Versatile
Peroxidase
The primary role of
manganese
peroxidase (MnP) in white rot wood
degrading systems is to oxidize
M n 2 +
to
Mn3 +
(73,77)
as overviewed in Figure 4.
It has been demonstrated
that
certain aliphatic organic acids such as oxalate,
malonate and lactate produced as de novo metabolites by white-rot fungi
(78,79)
function
as metal chelating
agents.
These organic acids increase the oxidation
rate
of
Mn2 +
(80) by binding
with
Mn3+,
resulting in a relatively stable complex,
and effectively
pulling
the oxidation
equilibrium
by the complexation of
M n 3 +
by
these
chelators. The more abundant
Mn
2 +
is then
oxidized
by MnP to
Mn
3 +
(59,72,81). The complexed
Mn3 + will
then diffuse into the wood
cell
wall
and
oxidize
phenolic
lignin
compounds.
The mechanism for this oxidation is not completely elucidated. It is
likely
that
it includes the redox of the
manganese
metal although other organic radicals
such
as phenoxy radical may also be
involved.
MnP has a lower redox potential
than LiP and can therefore only oxidize phenolic
lignin
structures under
physiological
conditions. Comparatively recently a new enzyme, Versatile
Peroxidase
(VP),
has been discovered
which
can be considered a hybrid between
MnP
and LiP
(82-85).
VP can directly oxidize non-phenolic
substrates
such as
veratryl
alcohol as
well
as phenolic
substrates.
It is currently unknown how
widespread VPs are in among the white rot fungi although they have been
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Ver at r yl
Alcoho l
o r
phenolic
compounds
Ot h e r
products
Figure
3.
Lignin
peroxidase activity involved in white-rot
decay.
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Other
products
Figure
4. Manganese peroxidase activity involved in
white-rot
decay.
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

25
discovered
in
fungi
such as Pleurotus eryngii (86) and Bjerkandera sp (87).
Timofeevski
et al. (88) reports
that
a mutant of
MnP
from
P. chrysosporium, in
which
only
one amino
acid
position had been changed, was able to
oxidize
both
Mn
2 +
and
typical
LiP
substrates
such as veratryl
alcohol.
This
points out the
close
similarity
between MnP and LiP enzymes.
More
information about the
action
and distribution
o f
this enzyme is expected in the future.
Laccase
Laccase
differs
from
LiP, M n P and VP in
that
it can use oxygen as an
oxidant
to degrade
lignin
and ultimately produce water
with
a four electron
reduction
of oxygen (Figure 5). Laccase is produced by many
microbial
organisms, both degradative and non-degradative and may play multiple
metabolic
roles. Laccase alone is unable to
oxidize
non-phenolic
lignin
compounds directly. However, it was discovered (89)
that
synthetic
substrates
known
as "mediator" compounds can be
oxidized
to their radical forms, and in
the presence of laccase
these
compounds then catalyze the oxidation of non-
phenolic
lignin.
Since laccase has
better
properties for industrial use, greater
exploration
of this enzyme for use in bioindustrial applications has been seen
(90)
.
Mediators such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate)
(ABTS)
and 1-hydroxy benzotriazole (HBT) have been used effectively in
trial
applications
but issues such as cost and
toxicity
remain.
More
importantly
from
the standpoint of understanding the fungal mechanisms, researchers in
Finland
(91) have demonstrated
that
several
fimgal
hydroxamate siderophores can be
successfully
used as natural mediators in laccase-aided
delignification
processes.
It was shown
that
iron-binding structures and free
hydroxyl
groups in
these
siderophores are the key
targets
for laccase. Iron chelating compounds
with
phenolate functional groups
similar
to other types of
microbial
siderophores
have also been isolated
from
brown rot
fungi
(44-48),
which
raises the intriguing
suggestion of phylogenetically-related mechanisms for
lignin
and cellulose
degradation by white rot and brown rot
fungi,
respectively.
Summary -
Conclusions
Soft
rot, brown rot and white rot
fungi
are the most destructive
microorganisms
of
wood
products.
Ecologically
they are
vitally
important, but it
is
their destructive
nature
in the built environment
that
has driven the interest in
understanding how
these
fiingi
function at the physical a nd
biochemical
level.
In
the first part of this chapter the progressive affects of the
three
types of
fungi
on
wood
are detailed, the similarities and differences in the modes of attack are
reviewed,
and the advantages and disadvantages of different methods for
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Figure
5. Laccase activity involved in white-rot
decay.
In Development of Commercial Wood Preservatives; Schultz, T., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

27
detecting decay are discussed. The biochemical mechanisms and spatial
relationships in the decay process are then discussed. In later chapters of this
book,
discussions of new methods to protect wood are presented, as are
discussions
of the fate of chemical protection methods using chemical
preservatives
that
have been withdrawn
from
use in several counties around the
world.
As older broad-spectrum chemical
treatments
are abandoned it
will
become increasingly important to understand the biochemical mechanisms and
microspatial
relationships between wood and the
fungi
involved
in the different
types of decay processes.
This
chapter reviews the known mechanisms and
spatial
relationships required for enzymes and non-enzymatic metabolites to
penetrate
and function as
cell
wall
depolymerization agents. New non-biocidal
processes for protecting wood
(92-94)
are already being developed based on
biochemical
studies of fungal decay functions
that
were uncovered
only
in the
last decade. Because information presented in this review chapter indicates the
mechanisms of the different decay organisms vary, combined specific
treatments
may
be needed to prevent attack by all types of decay
fungi.
Wood
modification
methods under development should also consider the variety of mechanism used
by
the decay
fungi
to
avoid
the failures of
past
treatments
that
were effective
against some, but not all
fungi.
Acknowledgements
The
authors
would
like
to thank our many collaborators worldwide who
offered
images for the
ACS
talk on this subject in
March
2005. These include:
Geoffrey
Daniel,
Jaime Rodriguez, Tor Schultz,
Darrel
Nicholas,
Jonathan
Schilling,
Holger
Militz,
Kurt
Messner and Tim
Filley.
Professor
Goodell
would
like
to thank his co-organizers for the
ACS
meeting
which
spawned this book.
Tor
Schultz as always has done an excellent j ob of
organizing
us
all.
In addition,
both Professors
Jellison
and
Goodell
would
like
to thank the many students,
current and
past,
including
Yuhui
Qian
an author on this paper, for their insight
into
decay biochemistry and the value of discussions
with
them in developing
new insights for research.
Funding
for the work research on this topic was by the
Wood
Utilization
Research
program and by the
CSREES
Mclntire-Stennis
program.
This
is paper
2976
of
the
Maine
Agricultural
and Forest Research Station.
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