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Serpula lacrymans, the causative agent of dry rot timber decay in buildings, is a Basidiomycete fungus in the Boletales clade. It owes its destructiveness to a uniquely well-developed capacity to colonize by rapid mycelial spread from sites of initial spore infection, coupled with aggressive degradation of wood cellulose. Genomic methods have recently elucidated the evolution and enzymic repertoire of the fungus, suggesting that it has a distinctive mode of brown rot wood decay. Using novel methods to image nutrient translocation, its mycelium has been modeled as a highly responsive resource-supply network. Dry rot is preventable by keeping timber dry. However, in established outbreaks, further mycelial spread can be arrested by inhibitors of translocation.
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From: S. C.Watkinson and D. C. Eastwood, Serpula lacrymans, Wood and Buildings.
In Allen I. Laskin, Sima Sariaslani and Geoffrey M. Gadd, editors: Advances in
Applied Microbiology, Vol. 78, Burlington: Academic Press, 2012, pp. 121-149.
ISBN: 978-0-12-394805-2
© Copyright 2012 Elsevier Inc.
Academic Press
CHAPTER 5
Serpula lacrymans, Wood and
Buildings
S. C. Watkinson*
,1
and D. C. Eastwood
Contents I. Introduction 122
II. Evolutionary Origins 123
A. Ancestry and taxonomic affinities in S. lacrymans 123
B. Biogeography: The roles of vicariance, host
preference, long distance spread, and humans in
the present global and local distribution of
S. lacrymans 124
III. Genomic Analysis of the Wood Decomposing
Machinery in S. lacrymans 126
A. Evolution of nutritional strategies within
Agaricomycetes 126
B. Wood-decomposing enzymes 127
C. The role of secondary metabolism 130
D. Future directions 130
IV. Whole Organism Physiology and Adaptation
to Environment 132
A. Life history 132
B. The mycelium as a coordinated networked
organism 133
C. Carbon/nitrogen homeostasis in the mycelial
network: The role of nitrogen accumulation,
storage, and translocation 134
V. S. lacrymans in Buildings 139
A. History and background 139
B. Diagnosis 140
Advances in Applied Microbiology, Volume 78 #2012 Elsevier Inc.
ISSN 0065-2164, DOI: 10.1016/B978-0-12-394805-2.00005-1 All rights reserved.
*Department of Plant Sciences, University of Oxford, Oxford, United Kingdom
{
College of Science, University of Swansea, Swansea, United Kingdom
1
Corresponding author: e-mail address: sarah.watkinson@plants.ox.ac.uk
121
Author's personal copy
C. Control: Prevention 141
D. Control: Remediation 143
VI. Conclusion 144
Acknowledgments 145
References 145
Abstract Serpula lacrymans, the causative agent of dry rot timber decay in
buildings, is a Basidiomycete fungus in the Boletales clade. It owes
its destructiveness to a uniquely well-developed capacity to colo-
nize by rapid mycelial spread from sites of initial spore infection,
coupled with aggressive degradation of wood cellulose. Genomic
methods have recently elucidated the evolution and enzymic rep-
ertoire of the fungus, suggesting that it has a distinctive mode of
brown rot wood decay. Using novel methods to image nutrient
translocation, its mycelium has been modeled as a highly respon-
sive resource-supply network. Dry rot is preventable by keeping
timber dry. However, in established outbreaks, further mycelial
spread can be arrested by inhibitors of translocation.
I. INTRODUCTION
A problem in controlling building dry rot caused by Serpula lacrymans is
that we do not know what the determinants of its destructiveness are—
what particular characteristics make S. lacrymans more damaging to
buildings than its close relatives and other wood decay fungi which
inhabit forests. Several features of the fungus probably contribute. Its
ability to infect and colonize timber in buildings have long been linked
to a capacity to survive and flourish in a spatially discontinuous moisture
and nutrient supply. This adaptation was probably honed during its
evolution from ancestors that grew in temperate and boreal regions.
Phylogenomics has recently extended and confirmed our understanding
of the evolutionary origins of the fungus as an inhabitant of the forest
floor of pine forests, utilizing fallen dead wood as its carbon and energy
source. Its enzymatic repertoire, revealed by whole genome sequencing
and comparative genomics, suggests it has a unique capacity for selective
utilization of microcrystalline cellulose, the polymer that confers tensile
strength on wood. Moreover, the cellulose depolymerizing system may be
aided by a secondary metabolic pathway evolved within the Boletales
clade of Agaricomycetes, to which S. lacrymans belongs. Imaging methods
designed to capture amino acid movements throughout mycelial net-
works in realistic conditions reveal its capacity for scavenging and
importing scarce nitrogen to freshly colonized wood for protein synthesis
and for developing pathways of mass flow of solutions through mycelial
122 S. C. Watkinson and D. C. Eastwood
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cords. Based on biology, effective chemicals have been developed for
remediation and treatment of dry rot outbreaks and for wood preserva-
tion against dry rot, but in most cases, control is most economically
achieved by environmental management to avoid creating favorable
growth conditions for the fungus.
II. EVOLUTIONARY ORIGINS
A. Ancestry and taxonomic affinities in S. lacrymans
The Serpulaceae are a monophyletic group within the Agaricomycetes
group Boletales (Binder and Hibbett, 2006), which include saprotrophic
brown rot wood decay species and species symbiotic as ectomycorrhiza
on tree roots. Phylogenomic analysis estimates that the Boletales lineage
arose in the early Cretaceous period approximately 113 million years
ago (mya), when association with pinaceae and rosid tree species would
be expected. Transition between nutritional modes within the Boletales,
inferred from phylogenomics, has occurred both from brown rot wood
decay to ectomycorrhizal (i.e., Austropaxillus spp. in the Serpulaceae)
and from ectomycorrhizal (ECM) to brown rot (i.e., Hydnomerulius spp.).
Austropaxillus spp.have a southern hemisphere distribution forming
ECM with Nothofagus predominantly and Eucalyptus spp. The split
between Serpula and Austropaxillus occurred approximately 34.9 mya,
coinciding with the separation of South America and Australia from
Antarctica 31 mya, and suggests that the switch from Pinaceae to Nothofagus
association occurred before the breakup of Pangaea. Comparative genome
analysis of saprotrophic modes with the ECM fungus Laccaria bicolor (Agar-
icales; Martin et al.,2008) showed a common pattern of reduction in
lignocellulose-decomposing gene complement in brown rot and ECM
(Eastwood et al.,2011). The simplification of decay machinery associated
with the emergence of brown rot may also, by moderating the destructive-
ness of the plant cell wall degrading machinery, have enabled mutualistic
ectomycorrhizal associations between fungus and plant roots to evolve. In
combined culture, mycelium of S. lacrymans grows toward and ensheaths
living roots of Pinus sylvestris, suggesting an incipient or vestigial capacity
for a two-way nutrient interaction with living cells. Therefore, the transition
appears to be dynamic and may perhaps occur in either direction and in
more than one lineage (Hibbett et al.,2000).
Within Boletales, the Coniophoraceae, including Coniophora puteana,
known as the cellar fungus, are now seen to be more distant from Serpula
than was thought, forming a separate monophyletic clade from
Serpulaceae. Within the Serpulaceae, surprisingly, the symbiotic group
Austropaxillus, ectomycorrhizal on conifers, is nested between two
Serpula lacrymans, Wood and Buildings 123
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lineages leading to the brown rot decay species found in buildings:
S. lacrymans in Europe and Asia, and Serpula incrassata, the commonest
cause of dry rot in North American buildings. Intraspecific diversification
has occurred within the morphospecies S. lacrymans, leading to cryptic
speciation; the two resulting variants are S. lacrymans var. lacrymans and
S. lacrymans var. shastensis.
B. Biogeography: The roles of vicariance, host preference, long
distance spread, and humans in the present global and local
distribution of S. lacrymans
The geographical origins of extant building decay members of Serpula-
ceae have recently been clarified in a study (Skrede et al., 2011) which took
advantage of the extensive representation of this group in culture collec-
tions. Isolates originating from all over the world were used in the analy-
sis, and phylogenetic trees were constructed based on five highly
conserved genes to elucidate the historical biogeography of the group
through geological time. Serpulaceae originated in the past Cretaceous
period 94–74mya, in what is now North America, and probably grew in
boreal pine forest extending across the land mass known as Beringia,
which connected what are now Western Eurasia and Eastern North
America between 14 and 3.5mya. The fossil record confirms the existence
of pine wood at high latitudes and altitudes at this time. The breakup of
Beringia by the formation of the Bering Straits is believed to have sepa-
rated two populations of the most recent common ancestor and led to the
divergence of the ancestor of S. lacrymans var. lacrymans from S. lacrymans
var. shastensis. Subsequently, these two species extended their ranges
southward. S. lacrymans var. lacrymans is the dry rot fungus of buildings.
Extensive study of its population genetics shows that it expanded its
range in recent time (Kauserud et al., 2007), probably by the intervention
of humans. It is now found worldwide in the built environment from an
inferred origin in Asia. Its close relative S. lacrymans var. shastensis occurs
naturally in montane pine forests in Western North America but has not
as far as we know been reported from buildings (Skrede et al., 2011). In
North America, S. lacrymans now occurs mainly in the northern parts of
the USA and Canada, while the American dry rot fungus S. incrassata
(synonymous and homotypic with Meruliporia incrassata;(http://www.
mycobank.org/MycoTaxo.aspx?Link¼T&Rec¼288330) is commoner in
the southern states and the Pacific Northwest (Schmidt, 2006, 2007).
Serpula himantioides is the closest species to S. lacrymans occurring in
Europe and is found growing wild in woodlands. It is also occasionally
found decaying timber in buildings and can be mistaken for S. lacrymans
(Schmidt and Moreth, 1999; White et al., 2001).
124 S. C. Watkinson and D. C. Eastwood
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How did S. lacrymans var. lacrymans emerge from its forest origins to
become a destroyer of buildings? An extensive population genetics study
(Kauserud et al., 2004a,b, 2006a, 2007), based on isolates from buildings
throughout the global range of the subspecies, shows that isolates from
buildings throughout the world belong to five distinct genotypes. Each
population is characterized by a different set of alleles of mating type and
vegetative incompatibility genes, suggesting rapid expansions from sep-
arate founding events (Kauserud et al., 2007). Using highly variable
microsatellite sequences it has been shown that Japanese and European
populations of S. lacrymans var. lacrymans are different and separate, and
probably expanded rapidly from separate founder events representing
genetic bottlenecks (Engh et al., 2010). Analysis of the pattern of global
distribution of alleles at the vegetative incompatibility locus, vic, demon-
strated that all isolates could be grouped into only eight vegetatively
incompatible types (Kauserud et al., 2006b), each showing little variabil-
ity, consistent with recent worldwide dispersal of a few isolates. Genetic
recombination occurs throughout the S. lacrymans population of a
region, and there is no evidence of linkage disequilibrium that might
indicate clonal spread. Because there are so few incompatibility types, it
is likely that colonies formed by separate airborne spore infections can
fuse into large connected networks, which would result in rapid coloni-
zation of a building. Abundant meiospores are dispersed, produced
from fruiting bodies. These can be induced in culture, enabling the
preparation of a series of monokaryons, and paired mating experiments
(Schmidt and Moreth-Kebernik, 1991) that showed that mating
type alleles, like vic ones, are few in number compared with other
Agaricomycetes. Monokaryons of S. lacrymans grow slowly and show
little or no ability to grown on wood; this requires formation of a
dikaryotic (functionally diploid) mycelium by fusion of compatible
monokaryons. However, monokaryons are observed in culture to pro-
duce asexual arthrospores, not seen on the dikaryon. These may play a
part in dispersal and survival. They form when dikaryotic mycelium in
wood is slowly dried (Schmidt, 2007), perhaps because it then becomes
monokaryotic, and they can survive in wood for years. They may also
facilitate spread through wood and masonry in infected buildings via
water seepage.
In summary, S. lacrymans, a species of Serpulaceae within the Agar-
icomycete clade Boletales, emerged from forest ancestry into the built
environment in recent historic time, presumably as a result of acquiring
some new characteristic(s) that enhanced its fitness in the built environ-
ment. Evidently colonization of buildings requires more than mere arrival
of airborne spores, since many fungi occasionally colonize buildings, but
only S. lacrymans produces frequent invasions. Other wood decay Agar-
icomycetes are found causing decay of building timber. In Northern
Serpula lacrymans, Wood and Buildings 125
Author's personal copy
Germany, 37 species are listed as causes of building decay (Schmidt,
2007), but S. lacrymans, with 53 occurrences, is far commoner than the
next commonest, C. puteana with 12.
III. GENOMIC ANALYSIS OF THE WOOD DECOMPOSING
MACHINERY IN S. LACRYMANS
A. Evolution of nutritional strategies within Agaricomycetes
Recent whole genome sequencing of S. lacrymans and comparative geno-
mics of wood decay enzymes between fungi shows that S. lacrymans
possesses unique mechanisms for wood decay (Eastwood et al., 2011).
Fungal wood decay involves coordinated action of a battery of depoly-
merizing enzymes and nonenzymic depolymerizing agents that act in
concert on the polymer complex. Wood is composed of polysaccharide
polymers cellulose and hemicelluloses, and the phenylpropanoid polymer
lignin in which the celluloses are embedded as a result of secondary plant
cell wall development during the ontogeny of the wood cell. White rot decay
mineralizes all the components of the lignocellulose (including lignin, cellu-
lose, and hemicellulose) to carbon dioxide and water. Brown rot fungi such
as S. lacrymans, by contrast, achieve pervasive depolymerization and assimi-
lation of polysaccharide components, without the total decomposition of
lignin seen in white rot decay. The morphological relationship between the
hypha and the wood cell wall is different in brown and white rot wood
decay. Hyphae in brown rot initially grow in medullary ray cells from where
they grow through pits in walls between wood cells. Each hypha is
ensheathed in a mucilagenous coating through which enzymes, nonenzymic
agents, and the products of cellulose depolymerization presumably diffuse.
Hyphae are sparser than in white rot, and nonenzymic decay takes place at a
distance from the hyphal surface. Removal of cellulose, but not lignin, results
in shrinkage and darkening of the wood into the typical ‘‘cubical cracking’’
and ultimately leaves a powdery brown lignin residue.
White rot is the ancestral mode of wood decay. Phylogenomics indi-
cates that the orders of Agaricomycetes that harbor wood decay species—
Polyporales, Agaricales, and Boletales—diverged approximately 200–150
mya (Martin et al., 2011). Phylogenetic divergence between white rot fungi
in the Agaricomycetes (Eastwood et al., 2011) has resulted in some
lineage-specific adaptation in the cellular machinery of white rot decay,
but the underlying mechanisms are conserved. Enzymes encoded in the
genome include lignin oxidase enzymes (e.g., class II peroxidases and
laccases), and decay-related oxidoreductases (e.g., glucose-methanol-
choline oxidoreductases and glyoxal oxidases) employed in the decompo-
sition and metabolism of lignin. Wood polysaccharides are decomposed by
126 S. C. Watkinson and D. C. Eastwood
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a broad assemblage of wood-decomposing enzymes (glycoside hydrolases,
glycosyltransferases, carbohydrate esterases, polysaccharide lyases)
which convert cellulose, hemicellulose, and pectin to simple sugars.
These enzymes include families of endo- and exocellobiohydrolases,
b-glucosidases, cellobiose dehydrogenases, xylanases, mannanases, endo-
and exoglucanases, and pectinases. Genome analysis of sequenced white
rot fungi showed an expansion in numbers of genes associated with ligno-
cellulose decomposition compared with the predicted Agaricomycete
ancestor.
Brown rot fungi evolved from ancestors with the white rot mode of
decay. Brown rot fungi, including the genome sequenced S. lacrymans and
Postia (Poria) placenta, selectively decompose the cellulose and hemicellulose
parts of lignocellulose leaving lignin modified, but largely in situ.Brownrot
decay has independently evolved at least five times from white rot ancestry
(Hibbett and Donoghue, 2001); presumably there is a selective advantage for
the fungus in being able to extract utilizable carbohydrate from wood
without wasting energy on breaking down the nonutilizable lignin. Because
of their different evolutionary origins, it is likely that brown rot species in
different lineages may have differing decay mechanisms.
B. Wood-decomposing enzymes
Comparative analysis of their genomes showed that the plant cell wall
decomposing machinery of the brown rot species S. lacrymans (Boletales)
and P. placenta (Polyporales) have both shared (conserved) and divergent
features. Both have fewer lignocellulose-decay genes than white rots and
lack the lignin oxidation mechanisms characteristic of white rot decay.
While the overall complement of carbohydrate-active enzymes (CAZy)
(Cantarel et al., 2009) is reduced in the brown rots, gene duplications in
certain gene families, for example, glycoside hydrolase family GH5 (exo-
cellobiohydrolases) and GH28 (pectinases), represent a refinement in the
suite of enzymes associated with brown rot decay. The importance of
these enzymes was supported by transcriptomic and proteomic studies of
these fungi growing on wood (Fig. 5.1;Martinez et al., 2009; Vanden
Wymelenberg et al.,2010). Transcriptomic and proteomic analysis of
S. lacrymans wood cultures (Eastwood et al.,2011) showed CAZy accounted
for 50% proteins identified and 33.9% transcripts upregulated more than
20-fold when compared with glucose medium (Fig. 5.1). GH families 3, 5,
28, and 61 were prominent, while GH5 endoglucanase and GH74 endo-
glucanase/xyloglucanase were 100-fold higher on wood. The transcript
levels of putative hydrogen peroxide-generating oxidoreductase enzymes
were also increased on wood. Decay enzymes evident in the genome
of S. lacrymans differed in several respects from those of P. placenta.
While endocellobiohydrolase enzymes were not identified in P. placenta
Serpula lacrymans, Wood and Buildings 127
Author's personal copy
(Martinez et al.,2009), a single GH6 encoding gene was present in the
S. lacrymans genome, although it was not detected in either wood culture
transcriptomic or proteomic analysis, and so may not be expressed. More-
over, S. lacrymans differed from P. placenta in having a cellulose-binding
module in a gene derived from a cellobiose dehydrogenase that also
included an iron reductase (Fig. 5.2). The significance of this for S. lacrymans
cellulose decomposition is discussed below.
S. lacrymans also showed specific features in its machinery for nonen-
zymic attack on cellulose. Brown rot decay, occurring in the absence of
lignin oxidases, is theorized to involve initial breakages at amorphous
regions of crystalline cellulose mediated by free radicals generated
by Fenton’s reaction, the extracellular generation of highly reactive
hydroxyl radicals (
OH) from the oxidation of divalent iron:
Fe
2þ
þH
2
O
2
þH
þ
!Fe
3þ
þ
OHþH
2
O. Hydroxyl radical produced in this
reaction is generated by white rot fungi for breaking the aromatic phe-
nylpropanoid lignin polymer, and extensive evidence (Bagley and
Richter, 2002; Goodell, 2003) supports a similar mechanism for attack on
cellulose by brown rot fungi. The hydroxyl radical is the most powerful
biological oxidizing agent known and is also transient, with a half-life of
nanoseconds (Goodell et al., 1997), raising the question of how such a
reactive and transient molecule is targeted at the cellulose molecule.
Carbon metabolism
335267 1355
3
3.275
3.55
4.65
5.75
3.825
4.375
4.925
5.475
4.1
5.2
225
148
122
109
109
73
72
67
60
58
55
53
52
52
51
50
49
48
47
46
45
43
42
41
40
39
39
38
37
433209
453342
452187
445922
451883
447766
349170
372243
433208
453971
371352
480589
434626
417964
475099
362527
432855
454077
363565
477654
360767
357854
408489
453784
361086
465649
469302
439506
355924
Glycoside hydrolase, family 61
Gene ID Description Fold change
Average
MMN10d10log
Average wood
T10d10log
CBM, Glycoside hydrolase, family 5
Glycoside hydrolase, family 74
CBM9, CDH-like putative iron reductase
Glycoside hydrolase, family 43
Flavin-containing monooxygenase
Amidase signature enzyme
Glycoside hydrolase, family 10
Aminoacyl-tRNA synthetase
Glycoside hydrolase, family 5
Glycoside hydrolase, family 28
Oligopeptide transporter
Glycoside hydrolase, family 5
Bacterial alpha-L-rhamnosidase
Hypothetical protein
Predicted protein
Cytochrome P450
Sugar transporter
Glycoside hydrolase, family 3
Hypothetical protein
Lipase
Lipase
NAD-dependent epimerase
Glycoside hydorlase, family 61
Glycoside hydrolase, family 5
Glycoside hydrolase, family 61
Major facilitator superfamily
Glucose-methanol-choline oxidoreductase
General substrate transporter
Major facilitator superfamily
Oxidoreductase/monooxygenase activity
Transport
Lipid metabolism
Other functions
Predicted proteins with
orthologs in other fungi
Predicted proteins, new for
S. lacrymans
FIGURE 5.1 Functional characterization of S. lacrymans transcripts with significant
increased regulation (fourfold or greater; ANOVA P<0.01) when grown on wood com-
pared with glucose-based medium, identified by microarray analysis (n¼300 genes).
Gene list and relative expression level is provided for the 30 S. lacrymans genes with
greatest fold increase in transcript levels on solid wood substrate. Reproduced from
Eastwood et al. (2011).
128 S. C. Watkinson and D. C. Eastwood
Author's personal copy
Fenton’s reaction is dependent on hydrogen peroxide generation systems,
presumably similar to those of white rot, and a mechanism to reduce Fe
3þ
back to Fe
2þ
to continue the reaction. Low molecular weight compounds
(LMWC) have been implicated in the early decomposition of wood poly-
mers in both white and brown rot fungi since they penetrate the dense
lignocellulose polymer, in which micropore size excludes wood decay
enzymes. In brown rot, LMWC such as phenol derivatives and peptides
have been proposed to mediate the reduction of ferric iron. Phenolates
derived from the demethylation of lignin or secondary metabolites pro-
duced by brown rot fungi have been reported (Arantes et al., 2009, 2011;
Filley et al., 2002; Xu and Goodell, 2001). Oxalic acid production, wide-
spread in brown rot fungi, potentially regulates the generation of
hydroxyl radicals in Fenton’s reaction, via the effect of pH gradients
around hyphae and in surrounding wood, on iron chelation and the
Fe
2þ
/Fe
3þ
equilibrium (Arantes et al., 2009; Hyde and Wood, 1997).
There are several strands of evidence suggesting that the Boletales may
have a distinctive mode of nonenzymic breakage of cellulose chains. Iron
reducing capacity of LMWC was detected in wood decomposed by eight
brown rot species (Goodell et al., 2006), but not S. lacrymans or C. puteana
(Boletales) which showed lower levels of LMWC, comparable with white
rot fungi. A separate observation also suggesting that the Boletales may
have a unique cellulose decomposing system was the finding (Highley,
1988; Nilsson and Ginns, 1979) that the wood decaying members of this
family, but not other brown rots, also decompose cellulose in materials
where it is not associated with lignin, such as paper and cotton.
Iron reductase domain = cd00241; CBM — cellulose binding domain;
CDH —cellobiose dehydrogenase oxidoreductase domain; SIGN — signal
peptide cleava
g
e motif; LINK — linkin
g
sequence without specific function.
453175
453176
452187
417465
Serpula lacrymans var. lacrymans S7.9
Serpula lacrymans var. lacrymans S7.9
Serpula lacrymans var. lacrymans S7.9
Serpula lacrymans var. lacrymans S7.9
JUNK SIGN cd00241 LINK CDH
SIGN cd00241 LINK CDH
SIGN cd00241
SIGN cd00241 LINK CBM
Accession
number
Genome database Domain structure
FIGURE 5.2 S. lacrymans protein models (based on annotation of S. lacrymans mono-
karyon 7.9 genome) with a similar iron reductase domain (cd00241), including two with
putative cellobiose dehydrogenase genes (Genbank accessions: EGN95518 and
EGN94369) and one with putative carbohydrate-binding module (EGO21045). Eastwood
et al. (2011).
Serpula lacrymans, Wood and Buildings 129
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C. The role of secondary metabolism
S. lacrymans synthesizes a phenolate secondary metabolite derived from
the atromentin pathway of Boletales (Schneider et al., 2008) which has the
capacity to reduce Fe
3þ
. Genome analysis revealed S. lacrymans to be rich
in secondary metabolic enzymes, including a putative atromentin biosyn-
thesis pathway. Atromentin and its derivatives (e.g., variegatic and pulvi-
nic acids) are yellow, red, and orange pigments commonly associated with
members of the Boletales. In S. lacrymans, production of these pigments,
mainly variegatic acid, is induced during growth on carbon- and nitrogen-
rich media and accumulates in hyphae as well as being exuded into the
surrounding agar medium during growth in culture. Variegatic acid
isolated from S. lacrymans wood cultures demonstrated iron reduction
capacity comparable to the phenolate iron chelator 2,3-dihydroxybenzoic
acid (Eastwood et al.,2011).
An enzymic mechanism of iron reduction in S. lacrymans is also
inferred, separate from and additional to the nonenzymic system
described above. The genome was found to include an iron reductase
enzyme with a cellulose-binding domain, apparently derived from an
ancestral cellobiose dehydrogenase (Fig. 5.2). Its role in wood decay was
confirmed in the wood-induced transcriptome of S. lacrymans (Fig. 5.1),
where it was upregulated 122-fold. No homologue was found in the
genome of P. placenta (Polyporales), but it was present in the white rot
Phanerochaete chrysosporium (Polyporales; Martinez et al., 2004). Cellulose
binding of an iron reductase might play a role in targeting hydroxyl
radical generation to cellulose, by means of localizing the Fe
2þ
-dependent
Fenton reaction on the cellulose molecule. Figure 5.3 shows a proposed
scheme for nonenzymic cellulose chain breakage involving hydroxyl
radical attack on the lignocellulose complex and the putative role of the
CBM-iron reductase in docking the Fenton machinery close to the cellu-
lose molecule.
D. Future directions
Sequencing programs, particularly those supported through the USA
Department of Energy Joint Genome Institute, JGI, continue to increase
the number of Agaricomycotina genomes available for comparative
study. The current JGI Saprotrophic Agaricomycotina Project aims to
sequence a broad phylogenetic spread of fungi across the subphylum
including varying nutritional modes. In particular, sequences of fungi
from each of the independently evolved brown rot lineages will allow
further comparative analysis of how the mechanism evolved and help to
determine the level of convergence between species.
130 S. C. Watkinson and D. C. Eastwood
Author's personal copy
The recent genome sequencing of S. lacrymans var. shastensis, the
exclusively forest-inhabiting subspecies with vicariant North American
distribution, will provide an interesting comparison with the well studied
S. lacrymans var. lacrymans commonly associated with the built environ-
ment. Comparative genome analysis, combined with high-throughput
transcriptomic, proteomic, and metabolomic studies to provide insights
into niche adaptation, is expected to elucidate the evolutionary steps that
2H2+2O
2
H2O2+H
++Fe
2+
Fe3+
VA
VA
Fe3+
Fe3+
Fe2+
H2O
Oxidoreductases
Lignocellulose IR, VA,
and HQ
IR
CAZy
(GH3, 5, 28, 61)
Hemicellulose
decomposition
Hexoses and
pentoses
OH
OH
OH
OH
CBM1
FIGURE 5.3 Schematic overview of a proposed mechanism of brown rot wood decay by
S. lacrymans. Scavenging mycelium colonizes a new wood food source, inducing varie-
gatic acid (VA) production, and expression of oxidoreductase enzymes which drive
hydroxyl radical attack on the lignocellulose composite. Carbohydrate-active enzymes
(CAZy) thus gain access to the weakened composite structure and break down accessible
carbohydrates. The cellulose-binding iron reductase targets Fenton-generated hydroxyl
radical attack at cellulose chains, releasing chain ends for hydrolysis and assimilation. IR,
iron reductase; HQ, hydroxyquinones; CBM, cellulose-binding module (Eastwood et al.,
2011).
Serpula lacrymans, Wood and Buildings 131
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enabled the variant lacrymans to succeed in the built environment. Geno-
mic studies will be complemented by whole organism approaches,
including imaging of proteins suggested by genomic analysis to have
important physiological and developmental roles. Genetic transformation
of S. lacrymans isbeing developed using transformation technology. Hygro-
mycin B resistance and green fluorescence protein have been successfully
introduced into S. lacrymans monokaryon S7.9 by Agrobacterium-mediated
transformation of arthrospores (Eastwood, unpublished). Genes with
potential relevance to observed physiological activities of S. lacrymans
include those for fungal autophagy and TOR-regulated intracellular
nitrogen-sensing pathways (Pollack et al., 2009). Mycelial development
is sensitive to regulation by nitrogen limitation. In S. lacrymans, intracel-
lular amino acid level varies up to 40-fold in response to environmental
levels of nitrogen. We hypothesize that responses to nitrogen status,
including cord development, and the induction of an intracellular lyso-
somal-type proteinase activity (Watkinson et al., 2001) not found in other
fungi investigated, might be mediated through changes in intracellular
amino acid.
Brown rot wood decay fungi also have potential applications in indus-
try, being potentially useful for pretreatment of lignocellulosic materials,
including agricultural wastes, to separate and depolymerize wood cellu-
lose for conversions leading to ethanol. The phenolic lignin residues from
brown rot have potential value as feedstocks for the chemical industry.
IV. WHOLE ORGANISM PHYSIOLOGY AND ADAPTATION
TO ENVIRONMENT
A. Life history
The life history of a fungus comprises the adaptations of a species to
reach, exploit, and disperse from its food resources. Both S. lacrymans in
Europe and S. incrassata in North America are notorious for their ability to
spread through buildings by means of thick mycelial cords that develop
from a wood food base in damp timbers and grow at a rate of millimeters
per day across and through masonry and behind plaster until they reach
and colonize further sources of wood substrate. Eventually, after exten-
sive mycelial growth, the well-nourished and extensive mycelium initi-
ates sexual reproduction by forming massive sponge-like fruiting bodies
from which billions of rusty-red meiospores are released over periods of
weeks or months. Population genetic analyses show linkage equilibrium
throughout populations indicating that dispersal is by these spores
(Kauserud et al.,2004b). There is no evidence for clonal spread between
buildings. Thus the life history, the sequence of stages by which the fungus
132 S. C. Watkinson and D. C. Eastwood
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reaches and colonizes its food base, and exits to colonize new substrates, is
achieved by two main means: long distance travel by airborne spores and
colonization of the new habitat by mycelial growth. A damp masonry and
timber house presents the fungus with a similar environment to the forest
floor of the temperate forest where the species evolved.
B. The mycelium as a coordinated networked organism
S. lacrymans dry rot is pernicious because of its ability to spread through-
out a building. Its ancestors evolved to feed on scattered dead wood on
the forest floor like other cord-forming wood decay fungi (Tlalka et al.,
2008a). In seeking ways to control dry rot, it is helpful to consider how its
development and physiology are adapted to capture large pieces of wood
up to meters apart. Fungi share with us most of our cellular machinery
and biochemistry. Like us, they need carbohydrates for carbon skeletons
and energy, and amino acids to combine with carbon skeletons to synthe-
size proteins for growth and enzymes. The unique problem faced by a
fungus on the forest floor is that its carbon sources—pieces of wood —are
encountered in different places from its nitrogen sources. There is very
little nitrogen in wood and masonry, so it has to be scavenged opportu-
nistically and carbon and nitrogen metabolites brought together within
the mycelium for biosynthesis and growth. Many basidiomycete forest
floor dwellers like S. lacrymans have evolved a remarkable way of life that
involves developing not as a single multicellular body according to a
‘‘fate map’’ of tissue differentiation as in animals, but as a loose network
of separate hyphal filaments which communicate so that the whole dif-
fuse body reacts to its environment in a coordinated manner. Develop-
ment is environmentally cued. As wood resources are colonized,
transport pathways—mycelial cords—form between them linking them
into a resource-supply network (Bebber et al., 2007). Through the cords,
amino acids, sugars, mineral nutrients, and water are conducted respon-
sively according to spatial distribution of resources and nutrient require-
ments throughout the colony (Lindahl and Olsson, 2004). Mathematical
modeling of resource-supply networks showed that these fungal net-
works were found to be of the type that maximized connectivity and
robustness (Bebber et al., 2007), suggesting that cord-forming wood
decay fungi have been honed by natural selection both for exploiting
nutritionally heterogeneous and extensive habitats, and for robustness
to attack by grazing arthropods such as Collembola and other soil animals
that feed on fungal mycelium. Bebber et al. (2007) in theoretical analyses of
network architecture of different cord-forming species demonstrated a
trade-off in network architecture between connectivity and robustness to
attack (e.g., where mycophagic soil animals removed random links).
S. lacrymans, by growing in the built environment free from such attack,
Serpula lacrymans, Wood and Buildings 133
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may have been enabled to invest preferentially in connectivity and capac-
ity for spread, developing fewer, wider translocating cords than relatives
on the forest floor.
C. Carbon/nitrogen homeostasis in the mycelial network:
The role of nitrogen accumulation, storage, and translocation
Carbon/nitrogen homeostasis in the whole organism is achieved by long
distance translocation in response to variations in local nutrient status
across the mycelial network. S. lacrymans mycelium imports scavenged
nitrogen to colonized wood (Tlalka et al., 2008b). It has long been recog-
nized that S. lacrymans, like other fungi solely dependent on dead wood as
a sole nutrient source, shows physiological nitrogen conserving adapta-
tions, including mycelial autolysis and recycling of nitrogen under
N limitation, and accumulation under luxury conditions. The architecture
of mycelial networks also shows a range of morphological responses in
growing mycelium that can be interpreted as maximizing uptake from the
substratum and enabling translocation to equilibrate carbon and nitrogen
levels through the network. Nitrogen starvation induces development of
nutrient-translocating cords and suppresses diffuse hyphal branching.
Mycelium growing from wood in sand microcosms with manipulated
nitrogen supply (Tlalka et al., 2008a) demonstrates some of these puta-
tively adaptive developmental responses to the amount and spatial dis-
tribution of N. Abundant nitrogen in the sand produces a dense diffuse
growth that covers most of the sand area behind the advancing margin.
Without N additions, the thin mycelium is differentiated almost entirely
into mycelial cords.
Cues to switch between diffuse and corded development can come
from extracellular or intracellular nitrogen concentrations. Nitrogen star-
vation promotes development of long, radially directed cords that grow
rapidly away from the wood food base across the nutrient-free medium
fueled by nutrients translocated from the wood food base (Tlalka et al.,
2008b). Colonies fed with the amino acid a-aminoisobutyric acid (AIB, a
nonmetabolized amino acid that is actively accumulated into the free
amino acid and competitively inhibits uptake of utilizable amino acid;
Watkinson, 1984) do not display this ‘‘starved’’ morphology but instead
develop cords that are disoriented, while advance at the margins is
arrested (Tlalka et al., 2008b). The inhibition is transmitted between com-
patible colonies of S. lacrymans that have fused. Intracellular concentra-
tions of amino acids in fungi are labile (Klionsky et al., 1990), and
utilizable nitrogen in the form of amino acids is opportunistically accu-
mulated by S. lacrymans mycelium (Venables and Watkinson, 1989), with
up to 40-fold increases in intracellular amino acid levels in response to
high environmental concentrations. We hypothesize that AIB, which
134 S. C. Watkinson and D. C. Eastwood
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replaces utilizable amino acids in the intracellular pool, may subvert
intracellular nutrient-sensing to induce nitrogen-sufficient morphology
under N-limited (cord-inducing) conditions.
Sudden changes in nitrogen availability in different parts of an estab-
lished colony, induced by transferring a pregrown colony on to split
plates with different defined media, induced rapid responses in both
S. lacrymans (Tlalka et al., 2008a) and C. puteana, but not in the distantly
related Phanerochaete velutina. Extension growth ceased on the nitrogen-
rich medium. Instead, there was an increase in biomass relative to the
N-limited part of the colony, and pigments including variegatic acid and
pulvinic acid, produced by the secondary metabolic atromentin pathway
characteristic of Boletales (Schneider et al., 2008), were secreted into the
medium and accumulated in the mycelium.
A realistic experimental microcosm that mimics the S. lacrymans natu-
ral habitat is the compressed soil or sand plate, with wood blocks as
inocula and nutrient resources. Sand microcosms were used to test the
hypothesis that S. lacrymans mycelial networks selectively import myce-
lial nitrogen to a site of fresh wood colonization. Subinhibitory quantities
of
14
C-AIB were used to track amino acid translocation, since the labeled
AIB molecule is not broken down by metabolism and remains in the
intracellular free amino acid pool.
14
C-AIB was used to quantify amino
acid import to freshly colonized wood added to an established mycelial
network. Amino acid was preferentially translocated and accumulated
into the freshest wood resource (Fig. 5.4). Dynamic imaging methods
have been developed for mycelial translocation, at both cell and organism
scale (Fricker et al., 2008; Watkinson et al., 2005). Photon-counting scintil-
lation imaging (PCSI) enabled a dynamic record to be captured showing
real time redistribution of amino acid in wood sand microcosms over
periods of days or weeks. This confirmed the responsive redirection of
amino acid translocation into freshly colonized wood blocks placed in the
path of the advancing mycelium (Fig. 5.5), presumably to provide amino
nitrogen for biosynthesis using sugars obtained from the new cellulose
food source. Initially, a mycelium growing on sand from a wood block
was supplied with subinhibitory amounts of
14
C-AIB at the wood block as
a tracer and allowed to distribute the label evenly throughout the myce-
lium. A fresh block was placed in the path of the advancing mycelium and
imaged by PCSI as it overgrew and colonized the new food source.
Following contact, amino acid was withdrawn from distant parts of the
network and translocated into the mycelium over and around the new
resource, where it accumulated. Using a photon-counting camera, a video
record was captured and subsequently analyzed in MatLab. To quantify
the actual redistribution of amino acid, experimental microcosms with
more than one fresh wood block were fractionated at the end of the
imaging period and the
14
C-AIB quantified by scintillation counting.
Serpula lacrymans, Wood and Buildings 135
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The rate of amino acid translocation in cords was faster than diffusion,
confirming the existence of a mass flow pathway through cords.
The development, anatomy, and physiology of cords are thus shown
to be critical in colonization of separate woody resources by S. lacrymans,
but in spite of extensive study over more than a century, we know rather
little about their structure and function. Cords are initiated when wider,
aseptate hyphae appear in the mycelium and narrower, cytoplasm-filled
hyphae start to grow along them, both with and against the direction of
growth of the colony (Butler, 1958). The anatomy of mature functional
translocating cords is still poorly understood. The light microscopy of
early scientists, in particular, Falck (Falck, 1912), is still unsurpassed.
Wide, apparently apoplastic spaces seem to run longitudinally through
the center of the cord, apparently formed from ‘‘vessel’’ hyphae
which expand and lose septa, and these become ensheathed in exuded
Microcosm types
1A
M
M
I1
I1
I2
C
C
NW
NW
14C-AIB
14C-AIB
1B*
2B*
2C 52 1145 Not
colonized
542
222
55
52 81
97
652
706
314 1920
3717
2003
1296
2142 2291
(27)
1464
(17)
2923
(58)
4990
(34)
3431
(40)
5594 6057
(71)
170 243
711
41 5
16
16
50 N.A.
N.A.
N.A.
Inoculum 1 Inoculum 2 New
wood
(NW)
14C-AIB content
(pmol)
Cords Sand
myceliume
(M)
Tot a l e
(recovery
in %)
1C
2A
(I1) (I2) (C)
FIGURE 5.4 Experiment showing preferential allocation of
14
C-AIB to freshly colonized
wood via mycelial translocation, assayed by harvest and assay of
14
C by extraction and
scintillation counting of fractions. Results are shown for the
14
C-AIB content of inoculum
wood blocks, mycelial cords, and newly colonized fresh wood blocks as well as the
residue remaining in sand and in corded mycelium, estimated by scintillation counting of
14
C-AIB in ethanolic extracts. Wood blocks and cords were removed and extracted
separately. Total recovery and % recovered, and residual
14
C-AIB in uncorded mycelium
and in sand plus mycelium (Sand mycelium, M) were estimated
e
from counts extracted
from 10 randomized samples. Two microcosm arrangements were used, one with a single
inoculum block (microcosm type 1) and the other with two separate inoculum blocks to
create a more realistically complex network (microcosm type 2). Letters A, B, and C
denote replicate experiments, and the data for the colonies shown are asterisked. Images
show the colonies before application of
14
C-AIB and at 6weeks, immediately before
harvest. In one replicate of series two, the mycelium failed to capture the new wood
block, and maximum
14
C-AIB accumulated in the second block. From Tlalka et al. (2008b).
136 S. C. Watkinson and D. C. Eastwood
Author's personal copy
AB
E
DC
F
Cord 1
Cord length (x)
Time (t)
Cord 1
12 h
390 h
230 h100 h
I
NW
Cord 2
Cord 3
Cord 2 Cord 3
NW t1
t2
t3
t4
14C-AIB
FIGURE 5.5 Photon-counting scintillation imaging to show the dynamic pattern of
reallocation of
14
C-AIB within a mycelial network, induced by local colonization of fresh
wood. A 10-day-old mycelium grown from a colonized wood block over moist sand in a
22cm dish was loaded with 10ml of 0.9 mM AIB
14
C-AIB applied directly to the top of the
inoculum, and a sterile wood block (NW) was placed close to the mycelial margin (A).
Serpula lacrymans, Wood and Buildings 137
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extracellular matrix (Butler, 1958; Jennings and Watkinson, 1982). The
function of the loosely differentiated tissues is not understood. We do
not know how mass flow is driven, although osmotically generated
pressure gradients are inferred from water exudation at mycelial margins
and stimulation of mycelial extension over plaster by water additions at
the food base (Jennings, 1987). We have very little understanding of how
this mass flow translocation pathway is loaded and unloaded, but if the
vessel hyphae are truly nonliving, like xylem elements in the water
transport pathway of plants, there must be exchange interfaces between
them and the cytoplasm of living hyphae at both loading and unloading
ends. In future, cell imaging might elucidate activity at the loading inter-
face. Amino acids imported to sites of wood depolymerization by mass
flow are presumably unloaded for metabolism at the site of wood coloni-
zation. Gene expression of transport proteins including oligopeptides are
upregulated during S. lacrymans growth on wood (Fig. 5.1), and a case
might be made for live cell imaging using molecular probes to pinpoint
visually the expression of transport functions.
The ability of single hyphae that form the growing margin of the
mycelium to accumulate and translocate nutrients intracellularly has
been investigated using imaging and modeling approaches. Vacuoles of
filamentous fungi have been suggested to have role in translocation
of nutrients because of their elongated form and highly dynamic behavior
in which they show peristaltic movements associated with regular
blebbing off (Cole et al., 1997) and fusion. This occurs most actively near
the hyphal apex. Vacuoles maintain a high internal level of urea cycle
amino acids with high N/C ratio, especially arginine and ornithine
(Klionsky et al., 1990), thus their remarkably dynamic changes in
shape and frequent separations and fusions might facilitate diffusional
The whole plate, including the new wood block, was then covered with a scintillation
screen and imaged in a photon-counting camera during further growth of mycelium and
capture of the fresh wood resource (Fig. 5.3B–E). In all pictures, the photon signal was
integrated over 12h. Letters denote the inoculum loading site (I) and the new wood block
(NW); time following loading is given in pictures. The relative redistribution of the label
following localized resource capture is shown along the three chosen cords, indicated by
dotted white lines (D). Panel (F) shows changes in distribution of
14
C-AIB along cords 1, 2,
and 3 during the imaging period. Cord two developed to connect the original inoculum
with the fresh resource, which accumulated
14
C-AIB (marked with a star), while the other
two (cords 1 and 3) were not directly connected to it. In the xtdiagram, generated from
video images, the relative intensity of the signal elicited by the presence of
14
C-AIB is
shown as it changed along the length of each of the three selected cords simultaneously.
The cords are represented by the three parallel columns, and time is shown vertically
from top to bottom, representing a 390-h video recording period. In the columns
corresponding to the three selected cords, the xdimension is shown oriented outward
from the inoculum block toward the mycelial margin. From Tlalka et al. (2008b).
138 S. C. Watkinson and D. C. Eastwood
Author's personal copy
movement of nitrogen. Vacuolar activity can be imaged in real time by
confocal fluorescence microscopy, using a suitable vital dye. Oregon
Green was used as a marker to image vacuolar dynamics in a cord-
forming wood decay fungus, P. velutina, which has a similar growth
form to S. lacrymans. No suitable dye was found to image amino acid in
the vacuoles, so a modeling approach (Darrah et al., 2006) was adopted to
test the ability of vacuolar facilitated diffusion to sustain the inferred rate
of supply of amino acid from the mycelium to growing hyphal tip. Para-
meterizing the model with observed vacuolar amino acid concentrations,
nitrogen requirement for tip growth, and vacuolar rates of fusion indi-
cated that vacuolar amino translocation is adequate to supply the termi-
nal region of the hypha, but not longer, more branched regions of the
colony margin. Cords are required to operate long distance transport in
large natural mycelial systems.
V. S. LACRYMANS IN BUILDINGS
A. History and background
S. lacrymans has been intensively studied and documented in its building
habitat by both mycologists and architects. The result has been worldwide
sampling and specimen-based taxonomy and nomenclature of this spe-
cies. Mycologists are fortunate to have, in S. lacrymans var. lacrymans,
a uniquely tractable experimental subject: its growth in a building resem-
bles a Petri dish culture in being visible and often free of other fungi, and
unlike an agar culture, it shows growth in a realistic time and spatial scale.
Building conservators and architects have more extensive experience of
its growth characteristics in buildings than most mycologists. Their obser-
vations and reports are based on the need to spot attacks, identify the
organism responsible, and offer prognoses to clients; they offer scientifi-
cally valuable insights that may repay further investigation with the tools
of modern biology (Jennings, 1991; Singh, 1994).
Chemical aspects of the building environment suspected to affect dry
rot include the presence of calcium and iron in masonry, and the compo-
sition of indoor and outdoor air. The effect of atmospheric gas composi-
tion on the fungus has been relatively little-studied as yet. Carbon dioxide
sensing pathways have been identified in fungi (Bahn and Mu
¨hlschlegel,
2006) and might be involved in triggering sporophore initiation by sig-
naling differences between the inside and outside of structures in which
mycelium is growing and respiring.
Dependence on masonry for growth in buildings has been extensively
investigated in S. lacrymans, to test its importance for the fungus both as a
water-holding matrix and as a potential source of chemicals that might
Serpula lacrymans, Wood and Buildings 139
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influence growth either as nutrients or by interacting in other ways with
fungal metabolism. In wood block culture tests, added calcium has been
shown to reduce wood decay by S. lacrymans (Schilling, 2009), and it
seems likely that reports of increased dry rot growth in the presence of
plaster are most likely to be due to the moisture-retaining character of the
material, rather than a chemical enhancement of growth or decay.
Iron is needed for brown rot and absence of iron can limit wood decay.
Mycelium of brown rot fungi including S. himantioides, so probably also
S. lacrymans, has been shown to import iron into wood from iron-
amended gypsum plaster and thereby enhance the rate of wood decay
in pine wood blocks with very low iron content (Schilling and
Bissonnette, 2008). The experimental conditions critically affect the results
obtained in wood block tests of the effects of iron and calcium levels in
soil and plaster on S. lacrymans decay of adjacent wood.
B. Diagnosis
S. lacrymans and related fungi in buildings are identifiable by morpholog-
ical features (Huckfeldt and Schmidt, 2006; Schmidt, 2007) and by the
characteristics of decay. Visual methods have the advantage of simplicity
and speed. The parts of the building affected also give clues to the
probable identity of the fungus; moist masonry in contact with damp
softwood and conditions of poor ventilation are risk factors for dry rot.
Architects and historic building conservators have unique experience in
locating outbreaks of dry rot in buildings and predicting sites within
historic buildings that are vulnerable to dry rot. Experience shows that
S. lacrymans dry rot is likely to occur in poorly maintained, unoccupied
buildings where there has been persistent water ingress resulting in damp
masonry in contact with timber elements. Failed roofs and rainwater
goods that trickle water into walls, and damp infiltrating from walls in
contact with outdoor soil, indicate likely sites of fungal attack. Softwood is
more vulnerable to attack than seasoned hardwood, as expected from the
origin of the fungus in Montana and cool conifer forests and its preference
for conifer host wood. S. lacrymans mycelium established in damp timber
may extend through less damp areas where these are poorly ventilated,
but in our experience of attempting to reproduce realistic colonies of
S. lacrymans for trialling control methods under building conditions
(Dobson et al., 1993), extremely high humidity is required for the myce-
lium to extend over exposed surfaces. However, building structures may
contain pockets of unventilated air in cellars or behind paneling, within
which large masses of mycelium can develop.
Methods for diagnosis of S. lacrymans from building samples have
been put forward based on molecular methods used for species identifi-
cation in microbial ecology. At present molecular methods require the
140 S. C. Watkinson and D. C. Eastwood
Author's personal copy
participation of a molecular biology laboratory and so are not readily
available to the average householder or timber preservation firm; how-
ever, commercial kits are beginning to be developed (Jacobs et al., 2010).
Species-specific probes and primers (Horisawa et al., 2009; Schmidt, 2007)
have been shown to discriminate between S. lacrymans and other wood
decay fungi in buildings, using the ITS region of the genome, the DNA
sequence of which usually varies between species. As well as differing in
genotype, fungi including S. lacrymans each have characteristic metabolic
profiles including secondary metabolites some of which are volatile. Mass
spectrometric methods developed for bacterial identification have been
adapted to distinguish these (Schmidt and Kallow, 2005), and dogs have
been trained to sniff out S. lacrymans in buildings.
C. Control: Prevention
Because S. lacrymans has a requirement for damp wood and very high
relative humidity, it cannot grow in a well-constructed and maintained
building. In historic or unoccupied buildings, damp monitoring can warn
of a dry rot hazard. Physical conditions for fungal growth are well
described in the literature (Schmidt, 2006, 2007). The fungus is reported
to survive in infected wood that has dried out, particularly if drying is
slow, when it can form monokaryotic mycelium that develops arthros-
pores. Survival for a year or longer is reported, provided at temperatures
of 20
o
C or less, although it is reported to die below 6C. Optimum
temperature for growth is around 20–22C but 26–27C is tolerated.
Wood treated with preservatives in current use is protected from dry
rot attack. Wood preservatives against fungi are necessarily toxic because
fungi have similar metabolism to other organisms. We have investigated
potential uses of AIB as a low-toxicity water-based chemical that has a
specific effect in limiting mycelial extension in S. lacrymans and other
wood decay basidiomycetes (Dobson et al., 1993; Elliott and Watkinson,
1989). The preservative use of AIB has been investigated for protecting
wood in service against S. lacrymans attack. While widely used wood
preservatives, including Tanalith, a water-based copper-azole formula-
tion, are effective against S. lacrymans, there is currently a need to reduce
the biocide content of wood preservatives, without decreasing efficacy
(Leithoff, 2008). Being of very low toxicity, AIB was investigated as a
possible adjuvant that might enable toxic azoles to be used at lower
concentrations in an effective combined formulation. Response surface
models were developed, parameterized from agar plate tests of mycelial
growth inhibition by factorial combinations of AIB with either the azole
fungicide tebuconazole or 3-iodo-2-propynyl butyl carbamate (IPBC).
Based on the response-surface models, a narrower range of combinations
were selected for wood preservative testing using an accelerated
Serpula lacrymans, Wood and Buildings 141
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EN113 test (Fig. 5.6, Bota et al., 2010). Preservative decay tests such as
EN113 measure mass loss rather than strength loss. However, for
brown rot decay, a measure of the tensile strength loss, which occurs in
advance of substantial mass loss, may be preferred (Curling et al., 2002).
We attempted tensile strength measurements of wood veneer strips
exposed to challenge by agar cultures of S. lacrymans and C. puteana,to
try and obtain a dose/response relationship for preservative effects of
various formulations of AIB combined with toxic biocides including
tebuconazole and IPBC. However, biological sources of variability proved
too great. These included variation in time of colonization of veneers by
the mycelium, variation in annual ring structure of veneers, and
variability in water content of veneers due to import of water to the
wood by the fungus, with consequent inhibition of decay. As a result,
the accelerated EN113 test was preferred for these assays, although spec-
troscopic assay of the color of test veneers was promising in initial trials.
Mass loss test results showed adjuvant activity of AIB with IPBC but
not tebuconazole: the concentration of IPBC required for inhibition of
S. lacrymans was reduced by approximately 50% in the presence of 2g/l
AIB (Fig. 5.6).
Other protective applications of AIB might include impregnation of
building timber elements likely to be exposed to damp hazard with AIB,
possibly in a slow-release encapsulated form, so as to slow extension of
45
40
35
30
25
20
% Weight loss
Average % Weight loss
15
10
5
0
01025
IPBC concentration (g/l)
50 100
5
0 AIB 1 AIB 2 AIB 5 AIB
FIGURE 5.6 Wood preservative effect of combinations of IPBC at 0–100 g/l
combined with 0 or 1g/l AIB, measured as 5 weight loss of pine sapwood challenged with
S. lacrymans in an accelerated EN113 test. From Bota et al. (2010). Antifungal and wood
preservative efficacy of IPBC is enhanced by a-aminoisobutyric acid. Reproduced from
Bota et al. (2010).
142 S. C. Watkinson and D. C. Eastwood
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dry rot mycelium in the case of attack. Trials in brick towers with myce-
lium extending from an inserted precolonized wood block showed long-
term and complete arrest of mycelial extension (Dobson et al., 1993).
D. Control: Remediation
The dry rot problem is most serious in older and neglected buildings,
particularly larger buildings that have been allowed to deteriorate, have
been subject to fire damage and water soaking, and are shut up and
unoccupied for long periods. Where such buildings are of cultural and
historic importance, remedial treatment of dry rot may be necessary.
Ultimately, building repairs to keep all the timber components and adja-
cent masonry dry will be sufficient to control dry rot. Environmental
management can be assisted by electronic monitoring of moisture levels
(Phillipson et al., 2007) in vulnerable parts of the building. Complete
eradication of S. lacrymans from large old buildings by removal of all
affected and neighboring timber is normally neither realistic, nor is it
desirable where original components must be conserved for authenticity.
If the building is ventilated and the ambient temperature rises above the
low growth optimum of S. lacrymans,22
C, the mycelium of S. lacrymans
will eventually wither. An excellent critical review of the physiology and
control of wood decay fungi in buildings (Schmidt, 2007) provides evi-
dence that disproves the notion that dry wood can be attacked by the
fungus using imported water or water produced by metabolism. How-
ever, in massive leaky buildings, it may sometimes be desirable to adopt
‘‘first aid’’ procedures to limit mycelial spread of S. lacrymans, to prevent
decay spreading, particularly if the fungus threatens culturally valuable
wood or cellulosic materials. Having low-toxicity, and being water-
soluble, biodegradable, and with broad-spectrum fungal activity, AIB is
valuable in this case, as it immediately produces durable systemic arrest
of mycelial extension (Fig. 5.7). The advancing mycelial front receives AIB
in place of glutamate and other protein amino acids required for growth
(Fig. 5.8). A 10% (w/w) solution in water is applied to any point on the
actively growing mycelium, from where it permeates throughout
the connected mycelial network, reaching inaccessible parts that may be
growing concealed in plaster or masonry. We have used cellulosic mate-
rial such as jute sacking or absorbent paper into which the AIB solution is
infiltrated to ensure good contact between mycelium and solution and
enhance uptake. Treatment is not immediately lethal, but treated myce-
lium cannot grow to reach new wood food sources, so will eventually die.
The point of application is immaterial, because long distance transloca-
tion of AIB occurs throughout the network irrespective of the original
direction of mycelial growth. This is a well-proven and effective method
of stopping the spread of dry rot through damp buildings, but AIB is not
Serpula lacrymans, Wood and Buildings 143
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commercially available. Only being applicable within a niche market, its
projected returns have been considered insufficient to fund registration
trials. However, it is licensed by the UK HSE for use by qualified users on
a site-by-site basis.
VI. CONCLUSION
Knowledge of S. lacrymans has been vastly increased in recent years by the
advent of genomic and imaging technologies, and the recognition that its
biology is comparable to the ecologically important forest dwelling brown
rot basidiomycetes from which it evolved. Because of its economic impor-
tance and experimental tractability, it is an unsurpassed model for inves-
tigation of the physiology not only of cord-forming wood decay fungi, but
also of physiological machinery that fungi share with eukaryotes.
A
E
C
FD
B
H2O
AIB
10 mm 2 weeks
2 weeks
6 weeks
6 weeks
FIGURE 5.7 Rapid and widespread inhibition of mycelial extension by 0.1M AIB. Repli-
cate colonies of S. lacrymans were grown for 52days from paired adjacent precolonized
wood inocula over nutrient-free moist sand in 22cm square plastic dishes. At 10 days
after inoculation, filter paper disks were placed at the mycelial margin and infiltrated
with 300ml of either 0.1M AIB (A–C) or deionized water (D–F). The white dotted line
(A–C) indicates the position of the mycelial margin at time zero. AIB treatment induced
immediate global arrest of marginal extension and a change in the pattern of develop-
ment, with tightly reticulate mycelial cords in panels (A–C) compared with radial cords
in panels (D–F). Tlalka et al. (2008b).
144 S. C. Watkinson and D. C. Eastwood
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In particular, its highly developed sensing systems for intracellular nutrient
status, demonstrated in the experiments reviewed here, are almost unex-
plored. Comparative genomic approaches are elucidating the molecular
processes by which hyphae extract polysaccharide from wood and cellu-
losic materials, and these may have applications not only in controlling
wood decay but also in exploiting wood decay enzymes for the extraction
of wood polysaccharides for biofuel production and in better understand-
ing of the roles of brown rot wood decay in global carbon cycling.
ACKNOWLEDGMENTS
SCW is grateful to the curators of historic buildings affected by S. lacrymans for access and
collaboration: Scottish National Heritage, UK Ministry of Defence, UK former Property
Services Agency, University of Oxford, University College Oxford, HuttonþRostron Envi-
ronmental Investigations, and numerous private householders.
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Serpula lacrymans, Wood and Buildings 149
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... G. trabeum uses alcohol oxidase as an extracellular source of H 2 O 2 in the brown rot of wood, contributing to the Fenton reaction which, in turn, contributes to the decomposition of lignin. Coniophora puteana is a common brown-rot fungus, which is very common in European buildings (Watkinson and Eastwood 2012). It has been widely used as an important test fungus in decay tests, in which the tolerance of a fungus to several organic wood preservatives is determined. ...
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... Conversely, starving fungi of nitrogen offers a non-biocidal route to restricting decay. For example, a non-metabolized amino acid, a-aminoisobutyric acid (AIB) has been successfully used to control the dry rot fungus S. lacrymans [116]. AIB was also an effective adjuvant for the biocide iodopropynyl butyl carbamate (IPBC), reducing the concentration of IPBC required for inhibition of S. lacrymans by approximately 50% [116]. ...
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Serpula wood-decay fungi occupy a diverse range of natural and man-made ecological niches. Serpula himantioides is a forest-floor generalist with global coverage and strong antagonistic ability, while closely related species Serpula lacrymans contains specialist sister strains with widely differing ecologies. Serpula lacrymans var. shastensis is a forest-floor specialist in terms of resource preference and geographic coverage, while Serpula lacrymans var. lacrymans has successfully invaded the built environment and occupies a building-timber niche. To increase understanding of the cellular machinery required for niche adaptation, a detailed study of the P450 complement of these three strains was undertaken. Cytochrome P450 monooxygenases are present in all fungi and typically seen in high numbers in wood decay species, with putative roles in breakdown of plant extractives and lignocellulose metabolism. Investigating the genomes of these related yet ecologically diverse fungi revealed a high level of concordance in P450 complement, but with key differences in P450 family representation and expression during growth on wood, suggesting P450 proteins may play a role in niche adaptation. Gene expansion of certain key P450 families was noted, further supporting an important role for these proteins during wood decay. The generalist species S. himantioides was found to have the most P450 genes with the greatest family diversity and the highest number of P450 protein families expressed during wood decay.
... It is a member of the Basidiomycota (order Boletales). C. puteana is abundant on deciduous and coniferous wood, not only in nature, but also in buildings, cellars [7]. C. puteana was selected as a model species in the present study thanks its fast growth and production of a sufficient amount of hyphae. ...
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... Serpula lacrymans is a Basidiomycota of the order Boletales, which is the main agent of brown rot in buildings and historical monuments in Europe, Russia, North America, and Japan [1][2][3][4][5]. S. lacrymans is rarely detected alone in a building; other wood-degrading fungi accompany it which makes its detection difficult [6,7]. The observation of brown rot and the analysis of the mycelium and fruiting bodies can direct the identification of wood-damaging fungi to a presence of S. lacrymans [8]. ...
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... Hence, the fungal EPS matrix provides highly specialized microenvironments that allow certain decomposition reactions to take place only in the right time and in the right place, while preventing damaging reactions. In the brown rot wood decomposer Serpula lacrymans, for example, extracellular hydroxyl radicals are also generated through the Fenton reaction to attack wood cell walls during initial stages of decay (Watkinson and Eastwood, 2012). The β-glucans in the EPS matrix of this fungus have been shown to efficiently scavenge hydroxyl radicals, suggesting that the polysaccharides in fungal EPS matrices may protect fungal cells from oxidative damage (Zhu et al., 2017). ...
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"Wood and Tree Fungi" provides an up-to-date overview of the various wood and tree fungi that damage trees, lumber, and timber, with special focus given to identification, prevention, and remediation techniques. First, the fundamentals of cytology and morphology, growth and reproduction, formal genetics, and enzymatic wood decay are addressed. Causative factors are then analyzed, followed by descriptions of interactions like antagonism, succession, and mycorrhiza. The more practical section describes damages by viruses on trees and by bacteria on the xylem of trees and on structural timber, wood discoloration caused by molds and stain fungi, wood decays by rot fungi, and preservation methods. The habitats of wood fungi are described as well as tree care. Important tree pathogens and wood decay fungi are characterized for prevention and identification. The final section focuses on the positive effects of wood-inhabiting microorganisms, covering past and classical methods like myco-wood, mushroom cultivation, biological pulping, and "palo podrido", as well as recent biotechnological processes.
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