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Short Communication

Decomposing capacity of fungi commonly detected inPinus sylvestris needle litter

Johanna B. BOBERG*, Katarina IHRMARK, Bjorn D. LINDAHL

Uppsala BioCenter, Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, Box 7026,

SE-750 07 Uppsala, Sweden

a r t i c l e i n f o

Article history:

Received 25 May 2010

Revision received 20 August 2010

Accepted 3 September 2010

Available online 30 October 2010

Corresponding editor: Petr Baldrian

Keywords:

Cellulose

Decomposition

Dothideomycetes

Helotiales

Humus formation

Lignin

Litter fungi

Pinus sylvestris

* Corresponding author. Tel.: þ46 18 671806;E-mail address: Johanna.Boberg@mykopa

1754-5048/$ e see front matter ª 2010 Elsevdoi:10.1016/j.funeco.2010.09.002

a b s t r a c t

Amajor part of the fungal community in coniferous litter consists of fungi whose taxonomic

position and ecology are unknown. Here, nine isolates from within commonly occurring

phylogenetic groups were tested for their ability to decompose Pinus sylvestris needles. In a 1-

yr long incubation study, needle mass loss as well as changes in cellulose and lignin content

were determined and compared to those caused by two litter basidiomycetes (Marasmius

androsaceus and Mycena epipterygia) with recognized ability to decompose needles. A basid-

iomycetous Clavulina/Sistotrema strain appeared to be cellulolytic but not ligninolytic. Chalara

longipes and three other strains within Helotiales also decomposed cellulose but not lignin,

whereas Mollisia cinerea (also Helotiales) and two Dothideomycetes e Sydowia polyspora and

a Mytilinidion sp., seemed unable to cause significant mass loss of cellulose. Lophodermium

pinastri (Rhytismatales) readily decomposed cellulose, and also caused considerable loss of

lignin.

ª 2010 Elsevier Ltd and The British Mycological Society. All rights reserved.

Saprotrophic fungi are the main decomposers of coniferous

wood and litter (Rayner & Boddy 1988) and, thereby, play

important roles in nutrient and carbon circulation in the

boreal forest. The decomposing capacity of fungi associated

with broad leaf litter in temperate forests has been studied

extensively (reviewed by Osono 2007). Still, the ecophysio-

logical properties of many dominant litter fungi associated

with coniferous litter in the boreal zone remain uncertain.

Basidiomycetes are considered particularly important for the

decomposition of plant material, due to their production of

a wide range of lignocellulolytic enzymes (Osono & Takeda

fax: þ46 18 673599.t.slu.se (J.B. Boberg).ier Ltd and The British M

2002). However, recent studies using molecular methods

have indicated that a major part of the fungal community

present in decomposing litter consists of ascomycetes

(O’Brien et al. 2005; Lindahl et al. 2007). About 50e70 % of PCR

amplicons obtained from coniferous and deciduous litter were

attributed ascomycetous taxa, and the most abundant geno-

types were assigned either to the Leotiomycetes or to the

Dothideomycetes. Earlier observations have indicated that the

litter decomposing abilities of basidiomycetes to a large extent

exceed those of ascomycetes, and that most ascomycetes

have limited ability to decompose lignin, Xylariales

ycological Society. All rights reserved.

Decomposing capacity of fungi 111

(Sordariomycetes) being an exception (Osono & Takeda 2002).

In this study we investigated the capacity of some commonly

detected needle litter fungi to decompose different chemical

fractions of Pinus sylvestris needle litter.

The study included six Leotiomycete strains: the facultative

endophyte Lophodermium pinastri (Rhytismatales), Mollisia cin-

erea, Chalara longipes and three unidentified strains within the

Helotiales. In addition, two Dothideomycete strains e Sydowia

polyspora (Dothideales) and a Mytilinidion sp. (Pleosporales),

were included (Table 1). A strain within the Clavulina/Sistotrema

group (Cantharellales) was also tested together with the

recognized litter decomposing fungiMarasmius androsaceus and

Mycena epipterygia (Cox et al. 2001; Boberg et al. 2008, 2009).

M. androsaceus and M. epipterygia were isolated from sporo-

carps, and the ascomycetes together with the Clavulina/Sisto-

trema strain were isolated from Scots pine needles collected

from the forest floor at different localities in central Sweden

(Table 1). Stock cultures were maintained on Hagem agar

(Stenlid 1985), except for L. pinastri which was cultured on

vegetable juice agar (200ml Granini� vegetable juice, mainly

containing tomato, celery and carrot juice, 800 ml H2O, 20 g

agar). To confirm the identities ofM. androsaceus, M. epipterygia

and L. pinastri, and to identify the rest of the isolates, the ITS

region of the ribosomal DNA was sequenced following the

method described by Persson et al. (2009). The obtained

sequences were compared with identified reference sequences

at NCBI using the BLASTn algorithm. When no close matches

were found in the database, similar ITS sequences were

selected, aligned and analysed for similarity by neighbour

joining. All fungal strains are deposited at the Department of

Forest Mycology and Pathology, SLU, Uppsala, Sweden

(the isolate of L. pinastri is also deposited at CBS), and the

sequences are deposited at NCBI (Table 1).

Decomposing capacity was assayed in plastic Petri dishes

filled with 30 ml of 1.5 % water agar. Approximately 600 mg of

freeze dried, weighed and autoclaved (121 �C for 15 min)

brown, abscised P. sylvestris needles was added to the Petri

dishes. The dishes were inoculated with 4 mm diameter plugs

obtained from fungal stock cultures, sealed with Parafilm to

keep the systems axenic and restrict evaporation, and incu-

bated in darkness at 20 �C. Seven replicates for each strain, as

well as uninoculated control systems, were prepared. After

365 d the systems were harvested and the needles were dried

and weighed for estimation of mass loss. Lignin and cellulose

contents of the decomposed needles, pooled for each fungal

strain, were determined by proximate analysis using the acid

detergent fibre (ADF) sulphuric acid method (Rowland &

Roberts 1994). Briefly, a milled sample was boiled in acid

detergent to remove protein, lipids, pectin, starch, water

soluble carbohydrates and hemi-cellulose (ADF soluble frac-

tion), leavingafibre residue. Cellulosewas removedby treating

the fibre fraction with 72 % sulphuric acid, leaving ‘lignin’ and

ash. Thus, cellulose content is defined as the difference

between the weight before and after sulphuric acid treatment.

Lignin was determined by weight loss of the residue upon

ashing. Chitin, a constituent of fungal cell walls, will mainly

end up in the ADF soluble fraction (Fioretto et al. 2005).

All fungal strains were able to grow on the needles and

covered the Petri dishes at the end of the incubation

period. Needles colonized by M. androsaceus, M. epipterygia

and L. pinastri were clearly bleached, a feature associated

with lignin break down (Osono 2007), whereas needles

colonized by the other fungal strains appeared unaffected

or darker than control needles. M. cinerea, S. polyspora and

the Mytilinidion strain also produced dark coloured aerial

hyphae.

The five helotialean strains caused between 16 and 28 %

needle mass loss (Table 1). They lacked ligninolytic capacity,

but the decreases in the cellulose fraction (5e29 %) caused by

four of the five strains show that they exhibit cellulolytic

capacities, although to a lower extent than the basidiomycetes

(33e69 %). The order Helotiales encompasses a diverse group of

ecologically different fungi; plant endophytes and pathogens,

saprotrophs on wood and litter and mutualistic ericoid- or

ectomycorrhizal fungi (Vralstad et al. 2002; Allmer et al. 2006;

Korkama-Rajala et al. 2008). C. longipes caused the highest

mass loss of the helotialean strains. In earlier studies, decom-

position of spruce needles by C. longipes appeared limited

(Koukol et al. 2006b; Osono & Takeda 2006). Two helotialean

strains belonged to the subfamily Hyaloscyphoideae within the

familyHyaloscyphaceae (Cantrell &Hanlin 1997), and appeared

to be varieties of the same species. Although closely related,

they differed in cellulolytic capacity. Similar genotypes were

frequently detected in litter from Swedish pine forests

(Helotiales group A in Lindahl et al. 2007; J. Stenlid et al.

unpublished) and appeared to persist during later decomposi-

tion stages. The taxonomic placement of Helotiales 1 remains

uncertain. M. cinerea caused the lowest needle mass loss of the

helotialean strains and did not appear to alter the cellulose

fraction. The species M. cinerea includes a range of different

varieties, of which many are reported as endophytes, but are

also found frequently in decomposing litter (Kowalski & Rys

1996; Korkama-Rajala et al. 2008). M. cinerea f. minutella was

found in thewhole organic horizon in the study by Lindahl et al.

(2007) and in well decomposed litter by J. Stenlid et al.

(unpublished). It has been reported to decompose spruce nee-

dles (Koukol et al. 2006b) and compete well with other fungi in

a laboratory study (Koukol et al. 2006a).

TheDothideomycetes S. polyspora and theMytilinidion strain,

caused the lowest needle mass loss, 14 and 8 %, respectively

(Table 1). For the latter, needle mass loss equalled that of the

uninoculated control needles. These two strains caused no

decrease in the cellulose fraction, but instead a slight increase

with up to 4 % (Table 1). Thus, these fungi seem to lack explicit

cellulolytic capacity and mainly utilise soluble compounds in

needles. S. polyspora frequently associates with living, or

recently fallen, needles (Muller et al. 2001; Sinclair & Lyon 2005;

J. Stenlid et al. unpublished). In contrast to the results presented

here, S. polyspora has previously been shown to be capable of

spruce needle decomposition (Muller et al. 2001). Specieswithin

the genera Mytilinidion are typically associated with coniferous

substrata (e.g.Minter 2007). In the studies by Lindahl et al. (2007)

and J. Stenlid et al. (unpublished), Mytilinidion genotypes

(Dothideomycete group B) were mainly found in more decom-

posed needle litter. The apparent lack of litter decomposing

capacity might indicate that this group of fungi depends on the

activity of other decomposers, in order to persist during later

stages of decomposition.

TheClavulina/Sistotrema strain (Moncalvo et al. 2006) caused

33 % needle mass loss and appeared to be cellulolytic with

Table 1e Fungal taxon, isolate number, origin, GenBank accession number andmass loss of pine needles and changes of the cellulose, lignin and the ADF soluble fractioncaused by the respective fungal isolates after 365 d

Taxa Isolate Origin GenBankaccession number

Needle massloss (%)a

Ligninmg g�1needlesb

Lignin massloss (%)c

Cellulosemg g�1needlesb

Cellulosemass loss (%)c

ADF sol.mg g�1needlesb

ADF sol. massloss (%)c

Basidiomycetes

Marasmius androsaceus JB14 Uppsala GU234007 63.9� 1.8 53 76 84 69 223 47

Mycena epipterygia JB13 Uppsala GU234008 51.5� 2.0 83 63 184 33 217 49

Clavulina/Sistotrema BCE Jadraas GU453167 32.6� 0.8 245 �9 152 45 276 35

Ascomycetes

Lophodermium pinastri 96e78 Nacka AF473555 39.8� 1.8 168 25 184 33 250 41

Chalara longipes BDJ Jadraas GU453171 27.7� 0.6 241 �7 194 29 288 32

Helotiales 1 BCX Jadraas GU453165 21.2� 0.7 252 �12 243 11 289 32

Hyaloscyphaceae 1 BDK Jadraas GU453170 22.9� 0.3 256 �14 221 19 293 31

Hyaloscyphaceae 2 BDI Jadraas GU393951 18.0� 0.2 258 �15 259 5 300 29

Mollisia cinerea BCZ Jadraas GU453168 15.5� 0.7 255 �13 275 �0.3 318 25

Sydowia polyspora HomC Jadraas GU453166 13.5� 0.3 267 �18 281 �3 316 25

Mytilinidion BBC Uppsala GU453169 7.9� 0.1 257 �14 286 �4 378 10

Control (uninoculated ) e e e 7.6� 0.7 225 e 274 e 423 e

Original needles e e e e 177 e 273 e 547 e

a % of initial weight; data are mean values of 5e7 replicates� SEM.

b mg g�1 dw initial needles; data are from pooled replicates.

c % of uninoculated control weight; data are from pooled replicates.

112

J.B.Boberg

etal.

Decomposing capacity of fungi 113

limited ability to break down lignin (Table 1). Species within

this clade include both saprotrophic as well as ectomycor-

rhizal fungi (Di Marino et al. 2008). The strain tested here was

genetically similar (97 % ITS similarity) to a Cantharellales

taxon (Genbank accession AM999656.1) obtained from bryo-

phytes in a boreal forest in Norway and identical to a sequence

(C1z 3.5) from the moss dominated L horizon in the study by

Lindahl et al. (2007), suggesting thatmossesmay be its primary

substratum.

In systems with species not causing any lignin mass loss,

the ‘lignin’ fraction increased by 7e18 % compared to control

needles (Table 1). It appears as recalcitrant compounds, not

hydrolysable by sulphuric acid, formed due to the presence and

activities of fungi. Even in needles colonized by theMytilinidion

strain, where mass loss did not exceed the control, the acid

insoluble fraction increased during the incubation. A similar

net increase of the acid-unhydrolysable fraction has been

found for decomposing litter in earlier studies (Osono et al.

2006; Preston et al. 2009). �Snajdr et al. (2010) also found that

a significant part of a synthetic lignin analogue was trans-

formed into humic substances during litter decomposition. In

addition, autoclaving of the needles increased the acid insol-

uble fraction by 27 % (Table 1). The cellulose fraction remained

unaltered, suggesting that some compounds found in the ADF

soluble fraction of original needles were converted into more

recalcitrant compounds during autoclaving. As litter undergoes

decomposition, some of the organic material is gradually con-

verted to humus, which has a very long turnover time and

generally accumulates in soils. The exact process of humifica-

tion and the chemical nature of humus are to date not fully

understood (Kogel-Knabner 2002; Sutton & Sposito 2005). The

results presented here suggest that fungal colonisation of litter

could contribute to the formation of recalcitrant compounds,

even without significant decomposition.

L. pinastri caused 40 % needle mass loss and decomposed

both cellulose and lignin (Table 1). L. pinastri caused 25 % lignin

loss relative to autoclaved but uninoculated control needles,

but only a 5 % decrease relative to the original needles before

autoclaving (Table 1). It is therefore not fully certain whether

L. pinastri was able to break down the plant derived lignin or

merely recalcitrant compounds formed during the steriliza-

tion. Still, the lignin mass loss concurred with the observed

lignin break down caused by L. pinastri decomposing pine litter

in a study by Osono & Hirose (2010). L. pinastri is a well known

facultative endophyte, which colonises the needleswhen they

are still attached to the tree, but remains active as a sapro-

troph in litter on the ground. In spite of the extensive ability of

L. pinastri to decompose needles, the species has been found to

be restricted to relatively fresh litter components (Lindahl

et al. 2007; J. Stenlid et al. unpublished), and a low competi-

tive strengthmay limit the overall contribution of L. pinastri to

litter decomposition.

Species belonging to the genera Mycena and Marasmius are

very common in decomposer communities in forest litter

(Frankland 1998; O’Brien et al. 2005; Lindahl et al. 2007). The

isolates of M. androsaceus and M. epipterygia, as expected,

decomposed needles effectively with mass losses reaching 64

and 52 %, respectively (Table 1). Lignin decomposition was

also extensive with a reduction of 76 and 63 % for needles

inoculatedwithM. androsaceus andM. epipterygia, respectively.

These results confirm that these species are well adapted to

use needles as a resource and verify their importance in litter

decomposition. Interestingly, M. epipterygia decomposed

lignin at a higher rate than cellulose compared to M. andro-

saceus, which caused similar cellulose and lignin loss (Table 1).

Selective lignin decomposition has been observed for other

fungal species belonging to the Agaricales decomposing

different types of litter (Osono et al. 2003; Osono & Takeda

2006). Possibly this difference can be related to their ecolog-

ical strategies; M. androsaceus is mainly found in the newly

shed litter in the L horizon (Holmer & Stenlid 1991; Lindahl

et al. 2007), where cellulose is abundant. Mycena species, in

contrast, often colonize litter at slightly later stages of

decomposition (Frankland 1998; Lindahl et al. 2007) with lower

cellulose to lignin ratios.

In conclusion, four out of five tested helotialean strains

decomposed cellulose, whereas the Dothidiomycetes

seemed to lack cellulolytic capacity. Neither of these strains

appeared to have any lignin decomposing capacity.

However, the observed ‘ligninolytic’ capacity of L. pinastri

illustrates that other groups of ascomycetous fungi than the

Xylariales may exhibit significant decay capacity. The

detailed ecological strategies remain unclear for most of the

tested taxa. In the light of the intense focus on boreal litter

decomposition in relation to carbon cycling and global

climate change, it is striking that we know almost nothing

about a major part of the fungal community found in

decomposing needle litter.

Acknowledgements

Financial support from SLU is gratefully acknowledged. The

authors would also like to thank Ond�rej Koukol for identifying

Chalara longipes.

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