Production of wood decay enzymes, mass loss and lignin solubilization in wood by marine ascomycetes and their anamorphs

Abstract

2004). Production of wood decay enzymes, mass loss and lignin solubilization in wood by marine ascomycetes and their anamorphs. Fungal Diversity 15: 1-14. A study was carried out to establish the wood decay ability for a large number of diverse marine ascomycetes and their anamorphs. In vitro production of cellulase and xylanase was widespread among forty-seven fungi. Production of enzymes involved in lignin degradation was comparatively less common. Most isolates were capable of causing mass loss in a birch wood substrate although values were low (<5%) during a 24-week period. A few ascomycetes caused higher mass loss of up to 20.1%. In all cases wood decay was greater in exposed rather than submerged conditions. Ascocratera manglicola, Astrosphaeriella striatispora, Cryptovalsa halosarceicola, Linocarpon bipolaris and Rhizophila marina, were shown to solubilize significant amounts of lignin, with indices of lignin solubilization comparable to those of terrestrial white-rot basidiomycetes. Certain marine ascomycetes may therefore fulfill an equivalent ecological role.

Full text

Fungal Diversity
1
Production of wood decay enzymes, mass loss and lignin
solubilization in wood by marine ascomycetes and their
anamorphs
V.V.C. Bucher1, K.D. Hyde1, S.B. Pointing 1* and C.A. Reddy2
1Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong
Kong, PR China
2Department of Microbiology and Molecular Genetics, and NSF Center for Microbial Ecology,
Michigan State University, East Lansing, Michigan 48824-4320, USA
Bucher, V.V.C., Hyde, K.D., Pointing, S.B. and Reddy, C.A. (2004). Production of wood
decay enzymes, mass loss and lignin solubilization in wood by marine ascomycetes and their
anamorphs. Fungal Diversity 15: 1-14.
A study was carried out to establish the wood decay ability for a large number of diverse
marine ascomycetes and their anamorphs. In vitro production of cellulase and xylanase was
widespread among forty-seven fungi. Production of enzymes involved in lignin degradation
was comparatively less common. Most isolates were capable of causing mass loss in a birch
wood substrate although values were low (<5%) during a 24-week period. A few ascomycetes
caused higher mass loss of up to 20.1%. In all cases wood decay was greater in exposed rather
than submerged conditions. Ascocratera manglicola, Astrosphaeriella striatispora,
Cryptovalsa halosarceicola, Linocarpon bipolaris and Rhizophila marina, were shown to
solubilize significant amounts of lignin, with indices of lignin solubilization comparable to
those of terrestrial white-rot basidiomycetes. Certain marine ascomycetes may therefore fulfill
an equivalent ecological role.
Key words: marine fungi; wood decay; lignin
Introduction
Lignocellulosic substrates in the marine environment, particularly
mangrove wood, support a diverse mycota (Sarma and Hyde, 2001). Most
species described to date are ascomycetes, with fewer anamorphic states
known. Very few basidiomycetes are known, with many of the latter also
encountered infrequently (Jones and Alias, 1997; Sarma and Vittal, 2001).
Although marine borers are recognized as particularly aggressive wood
degraders in marine waters, they are not active in the upper intertidal zone and
so fungi are thought to be important wood degraders in this niche (Hyde,
*Corresponding author: S.B. pointing: e-mail: pointing@hku.hk

2
1991). Bacterial wood decay in marine environments is relatively slow and
superficial (Singh and Butcher, 1991).
The degradation mechanisms of wood by terrestrial fungi are well known
and it is assumed similar mechanisms exist in marine fungi (Pointing and Hyde
2000). Cellulose hydrolysis is achieved by endoglucanases and
cellobiohydrolases, collectively termed cellulases (Eaton and Hale, 1993).
Hydrolysis of hemicellulose, a mixed polymer, occurs via the action of
hydrolytic xylanases, mananases and possibly other hydrolases with broad
substrate specificity (Eaton and Hale, 1993). The mineralization of lignin
involves two peroxidases, lignin peroxidase and Mn-dependant peroxidase, and
a polyphenoloxidase, laccase, collectively known as lignin-modifying enzymes
(LME’s). These enzymes catalyze production of highly reactive radicals which
oxidize phenolic and non-phenolic lignin components (Pointing, 2001).
Three fungal wood decay types are recognized (Eaton and Hale, 1993).
Soft rot, a superficial decay where enzymatic decay of cellulose and
hemicellulose in surface layers of wood is accompanied by little or no lignin
degradation. This is characteristic of many ascomycetes and their anamorphs.
White rot, where rapid and extensive decay of all wood components due to
enzymatic degradation is observed, with characteristic wood bleaching as a
result of lignin removal. White-rot decay has been identified to date only
among basidiomycetes and a few higher ascomycete genera (Risna and
Suhirman, 2002; Urairuj et al., 2003). The role of white-rot fungi in
lignocellulose turnover is key since lignin is the most recalcitrant component of
wood. Brown rot, in which very rapid cellulose and hemicellulose decay is
attributed to non-enzymatic oxidation with little or no associated lignin
degradation. Certain marine fungi are known to cause soft rot (Leightley and
Eaton 1979a, Mouzouras 1986, 1989a) and white rot (Leightley and Eaton
1979b, Mouzouras 1986, 1989b) decay, although studies have generally
focused on taxa isolated from man-made wooden structures in temperate
locations. Brown rot fungi are not colonizers of very wet or waterlogged wood
and so are not found in marine environments.
The major gaps in our knowledge of marine fungal wood decay relate
firstly to the lack of data relating to enzyme production by species common to
natural substrates such as mangrove wood in the tropics. Secondly whether
they are significant wood degraders under conditions where they occur, such as
the intertidal region of mangroves. Thirdly it is not clear if marine fungi play a
significant role in lignin breakdown since basidiomycetes, conventionally
regarded as key lignin degraders in terrestrial environments, are rarely
encountered. The aim of this study is to improve our knowledge in these areas.
Firstly, by screening those fungi commonly encountered in tropical and sub-

Fungal Diversity
3
tropical marine habitats for in vitro production of wood decay enzymes.
Secondly, to establish the ability of such fungi to effect mass loss in wood
during simulated exposed (high tide) and submerged (low tide) conditions.
Thirdly, the ability of selected taxa to solubilize lignin from wood is assessed
in an effort to understand the extent to which fungal lignin degradation may be
achieved by marine ascomycetes and their anamorphs.
Materials and Methods
Organisms and culture conditions
Forty-five marine fungal taxa, with many commonly occurring in
mangroves (Sarma and Hyde, 2001), were obtained from The University of
Hong Kong Culture Collection or City University of Hong Kong Culture
Collection. Reference fungi were obtained from CABI Bioscience UK.
Cultures were maintained on potato-dextrose agar (Difco) supplemented with
1.5% (w/v) marine salts (Instant Ocean) at 25°C in darkness. Inoculum for
enzyme production assays and wood decay testing consisted of 5mm agar
plugs cut from the actively growing colony margin of 4-8 week cultures of
each fungus. Single agar plugs were used for agar based enzyme assays, whilst
five plugs were introduced to each conical flask for wood decay studies.
Enzyme production assays
The following growth conditions were used to test for lignocellulolytic
enzyme production (Pointing, 2000):
Cellulose-azure agar – 1% (w/v) cellulose-azure (Sigma), 0.1% (w/v)
mycological peptone (Oxoid), 0.01% (w/v) yeast extract (Difco), 1.5% (w/v)
marine salts (Instant Ocean), 1.6% (w/v) agar (Difco). Cellulolysis was
assessed by monitoring release of azure dye from cellulose-dye complex and
diffusion into clear agar not containing cellulose-azure. Ligninolytic
peroxidase production was indicated by subsequent decolorization of the azure
dye.
Xylan agar – 1% (w/v) birchwood xylan (Sigma), 0.1% (w/v)
mycological peptone, 0.01% (w/v) yeast extract, 1.5% (w/v) marine salts
(Instant Ocean), 1.6% (w/v) agar. Zones of xylanolysis were visualized after
flooding petri dishes with 0.25% (w/v) I2 and KI solution. Xylan is the major
component of hemicellulose and its hydrolysis in this study was used to
indicate hemicellulolytic ability.

4
Poly R agar – 0.2% (w/v) glucose (Sigma), 0.02% (w/v) Poly R 478
(Sigma), 0.1% (w/v) mycological peptone, 0.01% (w/v) yeast extract, 1.5%
(w/v) marine salts (Instant Ocean), 1.6% (w/v) agar. Production of lignin
modifying enzymes was recorded as clearance of the Poly R dye.
Syringaldazine agar - 0.2% (w/v) glucose, 0.1% (w/v) mycological
peptone, 0.01% (w/v) yeast extract, 1.5% (w/v) marine salts (Instant Ocean),
1.6% (w/v) agar. Formation of pink-purple zones around wells in the agar
flooded with 0.1% (w/v) syringaldazine (Sigma) indicated laccase production.
Each treatment was repeated in triplicate with mean values used to
determine enzyme production relative to control taxa. All incubations carried
out at 25oC in darkness.
Mass loss and lignin solubilization in wood
Untreated seasoned birch wood (Betula sp.) was chosen for its low
extractive content and low durability. It was not possible to obtain mangrove
wood of uniform quality for use in experiments and so the use of homogenous
wood samples from a relatively benign wood species was preferred in order to
limit bias due to substrate variability. Wood was cut into 1 × 1 × 2.5cm3 blocks
and leached in distilled water for 48 hours to remove water soluble
components. Initial dry mass was determined after drying at 60°C for 2 days.
Test blocks were then autoclaved and placed into 250ml Erlenmyer flasks that
had been previously colonized for 2 week, on either the surface of 50ml agar
(potato-dextrose agar, Difco) or submerged in 50ml liquid broth (potato-
dextrose broth, Difco) supplemented with 1.5% w/v marine salts (Instant
Ocean). Volume of liquid incubations was maintained by periodically adding
sterile distilled water to flasks. These treatments were used to simulate exposed
and submerged conditions respectively. High surface area to volume ratios of
culture medium ensured aerated conditions were maintained throughout the
experiment. Static incubation was chosen to minimize any bias due to physical
abrasion of wood blocks that can occur during shaking incubation. Incubation
was carried out at 25°C in darkness for 24 weeks. After incubation wood
blocks were removed and final dry mass calculated after first removing fungal
biomass from the surface of each wood block. Mass loss values were corrected
for that recorded in uninoculated controls. Results were expressed as the mean
of three replicates, plus/minus the standard deviation of the mean. Differences
among treatments were analyzed by one-way ANOVA.
For chemical analysis wood blocks were ground in a hammer mill and
the 250-500µm sized particle fraction prepared for analysis by ethanol-benzene
and hot water extraction according to TAPPI T12 os-75 (Anon, 1975).

Fungal Diversity
5
Determination of acid insoluble (Klason) lignin was carried out by measuring
residual mass after digestion of wood particles in 72% H2SO4 according to
TAPPI T222 os-98 (Anon, 1998), with uninoculated wood blocks serving as
controls. The index of lignin solubilization (ILS) (percent lignin loss/percent
mass loss) was calculated for each treatment. Results were expressed as the
mean of three replicates, with the standard deviation of the mean shown by
vertical error bars.
Results
Enzyme production assays (Table 1) revealed 89% of marine fungi were
cellulolytic (crystalline cellulose utilization) and 84% were xylanolytic. Few
isolates were cellulolytic only (13%) or xylanolytic only (9%). Some variation
within genera was observed, with two strains of Lulworthia sp. and two species
of Marinosphaera mangrovei variously able to produce xylanases and
cellulases in vitro respectively. There were no obvious differences in enzyme
production between ascomycetes or their anamorphs. The ability to produce
enzymes involved in lignin degradation in vitro varied greatly between taxa
used in this study. Of the anamorphs tested, 20% decolorized Poly R, whilst
none decolorized Azure B and 30% oxidized syringaldazine. Many of the
ascomycetes decolorized Poly R (77%), with 9% decolorizing Azure B and
17% oxidizing syringaldazine. The reactions of reference white-rot and soft-rot
taxa were typical and confirmed the suitability of the assay medium for each
test.
All marine fungi tested in this study caused mass loss in wood (Table 2),
although values were generally extremely low (<5%) after 24 weeks exposure.
Wood decay in exposed conditions was significantly higher (P <0.001) than in
submerged conditions for all groups, there was no significant difference
between ascomycetes or anamorphic species (P = 0.447). The anamorphs,
however, achieved significantly higher mass loss than ascomycetes in
submerged conditions (P <0.01). Six ascomycetes, Ascocratera manglicola,
Astrosphaeriella striatispora (HKUCC 7651), Cryptovalsa halosarceicola,
Lignincola laevis (HKUCC 6867), Linocarpon bipolaris and Rhizophila
marina, caused mass loss in excess of 10% during exposed incubation. Mass
loss values obtained for the three reference taxa were as expected under
exposed conditions, with extensive mass loss by basidiomycetes and lower
mass loss due to soft-rot of surface layers by Chaetomium globosum. During
submerged exposure wood decay by basiodiomycetes was reduced 8-fold, but
no significant change was observed for Chaetomium globosum.

6
Table 1. Production of wood decay enzymes in vitro by marine fungi.
HKUCC
Unless
stated*
Cellulase
Xylanase
Poly R
Azure B
Syringaldazine
TERRESTRIAL FUNGI
ASCOMYCETES
Chaetomium globosum 4097 + 0 0 0 0
BASIDIOMYCETES
Phanerochaete chrysosporium IMI 284010 + + + + +
Pycnoporus sanguineus IMI 307937 + + + + +
MARINE FUNGI
ANAMORPHS
Cytoplacosphaeria phragmaticola 6722 + + 0 0 +
Cytospora rhizophorae 6012 00000
Dactylaria sp. 6728 + + 0 0 +
Dendryphiella salina CY 2723 + + 0 0 0
Periconia prolifica 6724 + 0 + 0 0
Phoma sp. 6725 + 0 0 0 0
Phomopsis sp. 6155 + + 0 0 0
Trichocladium achrasporum 8266 0000+
Zalerion varium 5485 + + 0 0 0
ASCOMYCETES
Acrocordiopsis patilii 9145 + 0 0 0 +
Aigialus grandis 5796 0 + + 0 0
Aniptodera salsuginosa 6729 + + + 0 0
Ascocratera manglicola 9174 + + 0 0 0
Astrosphaeriella striatispora 5700 + + + 0 0
Astrosphaeriella striatispora 7651 + + + 0 +
Bathyascus grandisporus 6868 ++++0
Botryosphaeria sp. 8019 + + 0 0 0
Corollospora maritima CY 1520 + + 0 0 0
Cryptovalsa halosarceicola 9142 +++++
Dactylospora mangrovei 9141 + + + 0 NT
Eutypa sp. CY GJ94++000
Ascosalsum cincinnatula 6731 + + + 0 0
Helicascus nypae 5788 + + 0 0 0
Kallichroma tethys 6084 + + + 0 +
Hypocrea sp. 9144 + 0 0 0 0
Leptosphaeria sp. 6004 + + + 0 +
Lignicola laevis 6066 + + + 0 0

Fungal Diversity
7
Table 1 continued. Production of wood decay enzymes in vitro by marine fungi.
HKUCC
Unless
stated*
Cellulase
Xylanase
Poly R
Azure B
Syringaldazine
Lignincola laevis 6867 + + + 0 0
Lignincola laevis 6737 + + + 0 0
Linocarpon bipolaris 5790 + + + 0 0
Lulworthia grandispora CY 1303 + + + 0 0
Lulworthia sp.8054 + + + 0 0
Lulworthia sp. 8055 + 0 + 0 0
Marinosphaera mangrovei 8089 0 + + 0 0
Marinosphaera mangrovei 6914 + + + 0 0
Massarina achostrichi 6727 + + 0 0 0
Massarina thalassiae 9140 + + + 0 0
Massarina velatispora 5793 + + + 0 0
Neptunella longirostris 6712 + + + 0 0
Phragmitensis marina 6730 + + 0 0 0
Quintaria sp. 6726 + + + 0 +
Rhizophila marina 9143 ++++0
Salsuginea ramicola 6915 0 + + 0 NT
Savoryella lignicola 9176 + + 0 0 0
Verruculina enalia 6869 ++++0
IMI = CABI Biosiences UK; CY = City University of Hong Kong Culture Collection.
+ denotes a positive reaction, 0 denotes a negative reaction, NT = not tested due to pigment
production by the fungus.
The solubilization of lignin from wood blocks by those marine fungi causing
more than 10% mass loss was assessed (Fig. 1). With the exception of
Lignincola laevis (HKUCC 6867) all caused extensive lignin solubilization.
The ILS values obtained show Cryptovalsa halosarceicola and Linocarpon
bipolaris solubilized lignin from wood at a similar rate to other wood
components. The high ILS (1.9) produced by Rhizophila marina suggests this
fungus preferentially solubilized lignin over other wood components. Two
other isolates, Ascocratera manglicola and Astrosphaeriella striatispora
(HKUCC 7651), displayed an ILS value of 0.8 and 0.7 respectively. With the
exception of Ascocratera manglicola all marine fungi causing more than 10%
mass loss also produced LME’s in vitro. Lignincola laevis (HKUCC 6867),
however, was unable to solubilize lignin from wood in contrast to production
of LME’s in vitro. In the case of such ambiguities, data from direct

8
Table 2. Mass loss in wood under simulated exposed and submerged conditions by marine
fungi after 24 weeks incubation.
Fungus HKUCC
Unless stated
Percent wood mass loss
(± S.D. of the mean)
Exposed Submerged
TERRESTRIAL FUNGI
ASCOMYCETES
Chaetomium globosum 4097 6.0±0.8 7.2±1.3
BASIDIOMYCETES
Phanerochaete chrysosporium IMI 284010 56.4±4 7.0±9.8
Pycnoporus sanguineus IMI 307937 73.1±18.1 9.0±1.1
MARINE FUNGI
ANAMORPHS
Cytoplacosphaeria phragmaticola 6722 8.3 ±1.9 6.3 ±2.8
Cytospora rhizophorae 6012 2.3 ±0.4 1.5 ±0.2
Dactylaria sp. 6728 5.3 ±4 7.3 ±1.0
Dendryphiella salina CY 2723 6.7 ±0.7 4.1 ±2.0
Periconia prolifica 6724 2.4 ±0.2 3.3 ±1.6
Phoma sp. 6725 3.5 ±0.4 6.6 ±1.5
Phomopsis sp. 6155 7.7 ±0.9 4.0 ±2.6
Trichocladium achrasporum 8266 5.2 ±1.3 1.6 ±0.5
Zalerion varium 5485 6.4 ±0.7 3.9 ±0.1
ASCOMYCETES
Acrocordiopsis patilii 9145 ND 2.4 ±1.4
Aigialus grandis 5796 1.0 ±0.0 0.8 ±2.1
Aniptodera salsuginosa 6729 2.2 ±0.5 2.4 ±0.2
Ascocratera manglicola 9174 20.2 ±4.8 3.3 ±0.7
Astrosphaeriella striatispora 5700 9.6 ±1.0 4.7 ±3.9
Astrosphaeriella striatispora 7651 13 ±1.5 4.7 ±2.2
Bathyascus grandisporus 6868 3.3 ±1.4 1.6 ±0.7
Botryosphaeria sp. 8019 0.6 ±0.3 2.0 ±1.0
Corollospora maritima CY 1520 5.3 ±0.3 2.6 ±1.5
Cryptovalsa halosarceicola 9142 20 ±13 4.5 ±3.4
Dactylospora mangrovei 9141 2.4 ±0.5 0.9 ±0.2
Eutypa sp. CY GJ94 2.1 ±1.6 4.8 ±3.5
Ascosalsum cincinnatula 6731 3.1 ±0.9 1.0 ±0.4
Helicascus nypae 5788 1.1±0.6 0.6 ±0.1
Kallichroma tethys 6084 1.6 ±0.5 1.2 ±0.4
Hypocrea sp. 9144 3.2 ±0.5 4.0 ±1.8
Leptosphaeria sp.6004 4.3 ±0.6 2.3 ±0.6
Lignicola laevis 6066 2.5 ±0.4 2.7 ±0.8
Lignincola laevis 6867 11.1 ±2.6 4.5 ±0.6
Lignincola laevis 6737 1.3 ±0.2 1.0 ±0.4
Linocarpon bipolaris 5790 11.8 ±2.1 6.7 ±1.2

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Table 2 continued. Mass loss in wood under simulated exposed and submerged conditions by
marine fungi after 24 weeks incubation.
Fungus HKUCC
Unless stated
Percent wood mass loss
(± S.D. of the mean)
Exposed Submerge
d
Lulworthia grandispora CY 1303 5.5 ±0.5 2.9 ±1.6
Lulworthia sp. 8054 8.1 ±1.4 3.0 ±0.5
Lulworthia sp. 8055 3.5 ±0.0 2.5 ±0.1
Marinosphaera mangrovei 8089 6.4 ±1.1 2.6 ±0.5
Marinosphaera mangrovei 6914 3.8 ±0.6 4.9 ±3.1
Massarina achostrichi 6727 4.1 ±0.2 1.1 ±0.2
Massarina thalassiae 9140 0.4 ±0.4 0.1 ±0.9
Massarina velatispora 5793 4.8 ±0.5 1.8 ±2.0
Neptunella longirostris 6712 8.3 ±1.1 2.3 ±0.4
Phragmitensis marina 6730 1.2 ±0.3 4.0 ±2.1
Quintaria sp. 6726 6.8 ±0.6 1.6 ±0.9
Rhizophila marina 9143 12.9 ±4.8 2.6 ±0.5
Salsuginea ramicola 6915 ND ND
Savoryella lignicola 9176 8.1 ±0.6 5.4 ±1.8
Verruculina enalia 6869 6.4 ±0.5 4.0 ±1.6
IMI = CABI Biosiences UK; CY = City University of Hong Kong Culture Collection.
ND = not determined due to poor growth of fungus.
measurement of lignin solubilization or mineralization is regarded as more
indicative of fungal ability.
Discussion
The high incidence of cellulase and xylanase production indicates that a large
number of marine ascomycetes and anamorphic isolates are at least
physiologically capable of wood decay. This is unsurprising since cellulose and
hemicellulose serve as nutritional carbon sources in wood (Pointing and Hyde,
2000). There were no obvious differences in enzyme production between
ascomycetes and their anamorphs. Other studies have also qualitatively
demonstrated cellulolytic activity among a wide range of marine fungi
(Gessner, 1980; Leightley, 1980; McDonald and Speedie, 1982; Rohrmann and
Molitoris, 1992; Raghukumar et al., 1994; Pointing et al., 1998, 1999). The
ability of marine fungi to produce enzymes involved in lignin degradation in
vitro is less well studied, and results from this study varied greatly between
taxonomic groups. Anamorphic fungi were incapable of peroxidase or laccase

10
0
20
40
60
80
P. chr P. san A.man A.str C.hal L.bip L.lae R.mar
(%)
ILS
1.9R.mar
1.1L.bip
0.0L.lae
1.0C.hal
0.7A. str
0.8A.man
1.1P.san
1.0P.chr
Fig. 1. Mass loss and lignin solubilization in wood by selected marine ascomycetes after 24
weeks exposed incubation. Terrestrial controls: P.chr, Phanerochaete chrysosporium; P.san,
Pycnoporus sanguineus. Marine ascomycetes: A.man, Ascrocratera manglicola; A.str,
Astrosphaeriella striatispora (HKUCC 7651); C.hal, Cryptovalsa halosarceicola; L.lae,
Lignincola laevis HKUCC 6867); L.bip, Linocarpon bipolaris; R.mar, Rhizophila marina. ILS
= index of lignin solubilization. Shaded = mass loss; unshaded = lignin solubilization. Error
bars represent standard deviation of the mean.
type ligninolytic enzyme production, suggesting they are non-ligninolytic. The
reason for this difference between sexual and asexual states is unclear. In the
case of marine ascomycetes, laccase and/or Mn-dependant peroxidase are
probably the most commonly secreted enzymes involved in lignin breakdown,
since none of the isolates were capable of Azure B decolorization. Both these
enzymes are known to mediate lignin mineralization (Thurston, 1994; Orth and
Tien, 1995) and so indicates a possible physiological capability for lignin
degradation by these fungi. The inability of apparently ligninolytic marine
fungi to produce lignin peroxidase was also recorded by Raghukumar et al.
(1994). A demonstrated ability to mineralize 14C-labelled lignin is necessary to
confirm ligninolytic ability by marine fungi.

Fungal Diversity
11
That few fungi in this study were capable of effecting significant mass
loss in Birch wood during a 24-week incubation strongly indicates that many
fungi are unlikely to be important in terms of wood decay in the marine
environment. The use of birch wood rather than mangrove wood is highly
unlikely to result in underestimates of wood decay ability, since birch is known
to have an extremely low extractive content (responsible for decay resistance in
wood) and low durability. Those taxa capable of more aggressive wood decay
in this study are likely to play a key role, as mass loss data for marine
lignocellulosic materials are generally much higher, with values for mangrove
wood and marine grasses of up to 20 % and 51 % respectively under similar
conditions (Vrijmoed et al., 1999). The possibility of substrate specificity by
marine fungi was suggested by Hyde (1986), based on observed colonization
patterns of mangrove wood in the tropics. Furthermore, Mouzouras (1989b)
demonstrated that the basidiomycete Halocyphina villosa caused mass loss
only in certain mangrove wood species and not others, indicating specificity
also among naturally available wood substrates. The significantly higher mass
loss values obtained for wood incubated in simulated exposed conditions
strongly suggests that fungal decay is more significant in degradation of
substrates in the upper intertidal region. Decay probably occurs at much slower
rates when wood is submerged, although this must be offset against the
reduced role of fungi in wood decay where marine borers may be active
(Eltringham, 1971).
Five marine ascomycetes were demonstrated to solubilize significant
amounts of lignin from wood in this study. The ILS values obtained show
Cryptovalsa halosarceicola and Linocarpon bipolaris solubilized lignin from
wood at a similar rate to other wood components, a decay strategy only
previously associated with simultaneous white-rot basidiomycetes. The high
ILS produced by Rhizophila marina suggest this fungus is capable of
preferential white-rot, where lignin is solubilized more rapidly than other wood
components. Two other isolates, Ascocratera manglicola and Astrosphaeriella
striatispora (HKUCC 7651), displayed ILS values similar to terrestrial
ligninolytic ascomycetes (Worrall et al., 1997). It is interesting that the
strongly lignin-solubilizing isolates Astrosphaeriella striatispora and
Linocarpon bipolaris have only been previously recorded growing on Nypa
palm, a poorly lignified substrate, within intertidal mangroves (Yanna et al.,
2002). Only one other study reports lignin solubilization from wood by a
marine fungus, at relatively low levels (<12%) by a species of Zalerion
maritimum (anamorphic) after 6 months exposure (Henningsson, 1976).
The in vitro mineralization of radiolabelled synthetic lignin to CO2 has
been demonstrated for 12 marine strains including several ascomycetes and

12
anamorphic fungi, plus the basidiomycete Nia vibrissa, albeit at low levels
(<5% in 60 days) (Sutherland et al., 1982). More aggressive degradation of
synthetic lignin was recorded for a terrestrial basidiomycete Flavodon flavus
(24% in 24 days) isolated from decaying seagrass in the intertidal region
(Raghukumar et al., 1999). The enzymes lignin peroxidase, Mn dependant
peroxidase and laccase were responsible for lignin mineralization by this
isolate. Mineralization was optimal under non-saline conditions, but also
occurred significantly under conditions simulating full strength seawater
(approx. 20% in 24 day). It is not known if this fungus is capable of wood
decay and lignin solubilization in a wood substrate. The data from our study
shows a dramatic reduction in wood decay ability of terrestrial basidiomycetes
when submerged, and so it is likely that lignin mineralization rates would be
considerably reduced during high tides. Despite this, evidence suggests that
certain terrestrial basidiomycetes can tolerate saline growth conditions in vitro
(Castillo and Demoulin, 1997), and we have shown in this study that they are
capable of wood decay in submerged saline conditions in vitro at rates similar
to marine fungi. The relative lack of inhibition in wood decay during
submerged incubation for Chaetomium globosum is consistent with the
substrate recurrence of soft-rot fungi for wet or waterlogged wood (Zabel and
Morrel, 1992).
This study conclusively demonstrates the ability of certain marine
ascomycetes to solubilize lignin from wood with ILS values equivalent or
better than known terrestrial white-rot basidiomycetes, suggesting that marine
ascomycetes carry out a ‘white-rot like’ role in marine environments.
Terrestrial white-rot fungi have also been demonstrated to mineralize a range
of organic pollutants via their ligninolytic enzyme system (Reddy, 1995;
Pointing, 2001). It is therefore conceivable that marine fungi are also involved
in ameliorating pollution within estuarine environments such as mangroves,
which often receive a high pollution input. This places a high value on the
importance of fungi in coastal ecology.
Acknowledgements
This research was supported in part by The Hong Kong Research Grants Council, grant
number HKU 7235/99M awarded to SBP and CAR. The authors wish to thank L.L.P.
Vrijmoed for supplying selected fungal cultures. We are grateful to K.L.Y. Lau and H.Y.M.
Leung for technical assistance.
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(Received 15 October 2003; accepted 16 December 2003)