ORIGINAL RESEARCH ARTICLE
published: 20 May 2014
doi: 10.3389/ micb.2014.00230
Loss o di e si y in wood-inhabi ing ungal communi ies
a ec s decomposi ion ac i i y in No way sp uce wood
La a Valen ín1,2, Tiina Rajala1, Mikko Pel oniemi1, Jussi Heinonsalo3, Taina Pennanen1and
Raisa Mäkipää1*
1Van aa Resea ch Uni , Finnish Fo es Resea ch Ins i u e, Van aa, Finland
2Depa men o Chemical Enginee ing, Technical School o Enginee ing, Uni e si a Au ònoma de Ba celona, Ba celona, Spain
3Depa men o Food and En i onmen al Sciences, Uni e si y o Helsinki, Helsinki, Finland
Edi ed by:
John J. Kelly, Loyola Uni e si y
Chicago, USA
Re iewed by:
S e an Be ilsson, Uppsala
Uni e si y, Sweden
Pe a F ansson, Swedish Uni e si y
o Ag icul al Sciences, Sweden
*Co espondence:
Raisa Mäkipää, Van aa Resea ch
Uni , Finnish Fo es Resea ch
Ins i u e, PO Box 18,
Jokiniemenkuja 1, FI-01301 Van aa,
Finland
e-mail: [email p o ec ed]
Hund eds o wood-inhabi ing ungal species a e now h ea ened, p incipally due o a lack
o dead wood in in ensi ely managed o es s, bu he consequences o educed ungal
di e si y on ecosys em unc ioning a e no known. Se e al expe imen s ha e shown
ha p ima y p oduc i i y is nega i ely a ec ed by a loss o species, bu he e ec s
o mic obial di e si y on decomposi ion a e less s udied. We s udied he ela ionship
be ween ungal di e si y and he in i o decomposi ion a e o sligh ly, mode a ely
and hea ily decayed Picea abies wood wi h indigenous ungal communi ies ha we e
dilu ed o examine he in luence o di e si y. Respi a ion a e, wood-deg ading hyd oly ic
enzymes and ungal communi y s uc u e we e assessed du ing a 16-week incuba ion.
The numbe o obse ed OTUs in DGGE was used as a measu e o ungal di e si y.
Respi a ion a e inc eased be ween ea ly- and la e-decay s ages. Reduced ungal di e si y
was associa ed wi h lowe espi a ion a es du ing in e media e s ages o decay, bu no
e ec s we e de ec ed a la e s ages. The ac i i y o hyd oly ic enzymes a ied among
decay s ages and ungal dilu ions. Ou esul s sugges ha unc ioning o highly di e se
communi ies o he la e-decay s age we e mo e esis an o he loss o di e si y han less
di e se communi ies o ea ly decompose s. This indica es he accumula ion o unc ional
edundancy du ing he succession o he ungal communi y in decomposing subs a es.
Keywo ds: biodi e si y, woody deb is, espi a ion ac i i y, unc ional edundancy, enzymes
INTRODUCTION
The loss o species di e si y al e s ecosys em unc ion, s abili y
and he abili y o p o ide goods and se ices o socie y (Lo eau
e al., 2001; Hoope e al., 2005; We z e al., 2006; Ca dinale e al.,
2012). Se e al expe imen s ha e shown ha p ima y p oduc i i y
is nega i ely a ec ed by a loss o plan species (Lo eau e al., 2001;
Hoope e al., 2012) and ecosys em s abili y is educed by dec eas-
ing unc ional di e si y (Hoope e al., 2005). Biodi e si y a ec s
he a e o key ecosys em p ocesses such as decomposi ion and
nu ien cycling (Lo eau e al., 2001; Hä enschwile e al., 2005;
Gessne e al., 2010). In bo eal o es s, whe e ungal communi ies
a e majo decompose s (Rayne and Boddy, 1988; Lindahl and
Bobe g, 2008; S enlid e al., 2008), a high di e si y o soil ungi
can enhance he decomposi ion a e (Tiuno and Scheu, 2005),
especially unde en i onmen al luc ua ions such as a ia ions in
empe a u e egimes (Toljande e al., 2011). Hund eds o ungal
species in Fennoscandian o es s a e now h ea ened, p incipally
due o a lack o dead wood in a o es landscape ha is in en-
si ely managed (Sii onen, 2001).Theconsequenceso educed
ungal di e si y on decomposi ion a e unknown. Fu he mo e,
he s abili y and unc ion o o es ecosys ems migh be d ama i-
cally a ec ed i he ungal capaci y o esis habi a pe u ba ion is
con inuously exceeded, as is he case o many species in managed
o es s (S enlid e al., 2008).
Since ungal species p oduce complemen a y enzymes, he
p esence o many species can imp o e he communi ies’ e i-
ciency o deg ade a wide ange o li e cons i uen s and hus
enhance decomposi ion a e (Gessne e al., 2010). Expe imen s
wi h selec ed ungal species and hei combina ions e ealed a
s ong posi i e ela ionship be ween ungal di e si y and decom-
posi ion a e, which became asymp o ic a a ela i ely low le el
o di e si y (Se älä and McLean, 2004; Tiuno and Scheu, 2005).
In expe imen s in ol ing a ew species, acili a ion and esou ce
pa i ioning ha e been obse ed (Tiuno and Scheu, 2005). In
mo e di e se e es ial ungal communi ies, an agonis ic mech-
anisms migh p e ail (Boddy, 2000; Gessne e al., 2010)and
he coloniza ion sequence o wood-deg ading ungi can u he
a ec decomposi ion (Fukami e al., 2010). S udies o he decom-
posi ion a e in manipula ed ungal communi ies a e mos ly
pe o med wi h a ew cul u ed species, and he esul s migh be a
poo e lec ion o na u al communi ies, especially i ce ain com-
muni y membe s ha e a g ea e p opo ional in luence on he
decomposi ion a e han he o ali y o he ungal communi y
(Robinson e al., 2005).
Decaying No way sp uce logs ha bo di e se ungal commu-
ni ies (O askainen e al., 2010; Rajala e al., 2011; Kuba o á
e al., 2012), whe e he numbe o ac i e species is a highe
han ha conside ed in expe imen al s udies. Species numbe
www. on ie sin.o g May2014|Volume5|A icle230|1
Valen ín e al. Loss o di e si y a ec s decomposi ion
ends o inc ease wi h mass loss (and ela ed changes in he sub-
s a e quali y) and peaks in he mos decayed logs (Rajala e al.,
2012). Decomposi ion o cell wall polyme s in wood is a com-
plex p ocess ha is d i en by a succession o species wi h di e en
li e -deg ading enzymes (Bald ian, 2008; S enlid e al., 2008).
Du ing he decay p ocess o dead wood o e se e al decades,
he ungal decompose communi y changes om one domina ed
by ascomyce es o one o whi e- and b own- o basidiomyce es,
be o e slowly becoming one composed mainly by he myco -
hizal species ound in he unde lying soil (Rajala e al., 2010,
2012). Wood-deg ading ungi (whi e- o and b own- o ungi)
decompose polysaccha ides by p oducing hyd olases, bu only
whi e- o ungi e icien ly deg ade lignin ia oxida i e me alloen-
zymes (e.g., laccase and manganese pe oxidase) (Lundell e al.,
2010). Expe imen s wi h decomposing lea es and needles ha e
linked enzyme ac i i ies (EA) wi h ungal communi ies (Šnajd
e al., 2011; Ži èáko á e al., 2011), bu o ou knowledge, no s udy
has ye analyzed such a ela ionship wi h he ungal communi y
in decomposing wood. The e o e, we s udied wood subs a es in
di e en s ages o decay o examine he e ec s o manipula ed
ungal di e si y on enzyme p oduc ion and decomposi ion a e
du ing he en i e p ocess.
Theo e allobjec i eo hiss udywas oexamine he ela ion-
ship be ween ungal di e si y and decomposi ion a e by manip-
ula ing ungal communi ies ob ained om decaying No way
sp uce wood. We designed a mic ocosm expe imen o in es i-
ga e: (1) whe he a educed di e si y o wood-inhabi ing ungi
a ec s he decomposi ion o No way sp uce wood and, i so,
(2) whe he unc ional edundancy and s abili y o he decom-
posi ion p ocess a ies among decay s ages, and (3) whe he
changes in ungal di e si y and a e o decomposi ion a e ela ed
ochangesinhyd oly icenzymep oduc ion.We es ed he
hypo hesis ha CO2p oduc ion and enzyme ac i i y in he
dead wood a e a ec ed by he decay s age o he subs a e and
di e si y o he decomposing species. Fu he mo e, we hypo-
he isized ha he decomposi ion ac i i y (measu ed as CO2
p oduc ion) is co ela ed o o e all a ia ion o he ungal com-
muni y and he decomposi ion inc eases o di ec ion ha is
pa allel o inc easing species di e si y (measu ed as numbe o
de ec ed OTUs).
MATERIALS AND METHODS
WOOD SAMPLES
The s udy ma e ial was collec ed om an unmanaged o es in
Lapinjä i (Sou he n Finland, 60◦39.413N, 26◦7.352E, al i ude
50 m, empe a u e sum 1300◦C d; u he de ails a e p o ided
in Rajala e al., 2012). Wood samples we e ob ained om s em
discs sawn on si e om 46 allen No way sp uce (Picea abies)logs
(diame e >5cm a b eas heigh ). Logs we e classi ied as ea ly,
in e media e o la e s ages o decay (I, III, and V acco ding o
Mäkinen e al., 2006). Sample discs we e sawn in May 2011 and
s o ed in plas ic bags a −18◦C un il p ocessing in he labo a o y.
The p ope ies o he wood (C/N a io, lignin con en and den-
si y) o indi idual discs we e analyzed as desc ibed in Rajala e al.
(2010) and he mean alues o 14 discs om ea ly-decay s ages, 12
discs om in e media e-decay s ages and 16 discs om la e-decay
s ages we e calcula ed (Table 1).
Ba k om he ou e mos laye o each ozen sample disc was
emo ed wi h a lame-s e ilized kni e. F ozen discs we e hen
d illed wi h a lame-s e ilized and cooled bi (Ø =10 mm) om
he su ace h ough he sapwood and hea wood. Sha ings and
sawdus o eachdecays agewe epooledin oasinglesampleand
s o ed in a plas ic bag a −18◦C p io o use as a ungal inoculum.
A ew ac i e ungi migh be supp essed du ing he p ocessing o
he samples, bu species inhabi ing dead wood in bo eal o es s
a e acclima ed o a ying empe a u es and he ungal commu-
ni y ob ained om he inocula was conside ed o e lec species
composi ion in each decay phase.
The expe imen al g ow h medium (i.e., sawdus ) was p epa ed
by milling i e discs om each decay s age ha had been deba ked
and d ied a 105◦C o 48 h. D y sawdus om each s age was
pooled in o a single sample and s e ilized wice by au ocla ing
(121◦C o 20 min) wi h a 3-day in e al.
EXPERIMENTAL SET-UP
The incuba ion ook place in s e ile 100-mL glass lasks con-
aining di e en amoun s o inoculum and au ocla ed subs a e
(Table 2). S e ile dis illed wa e was added o each lask o
emois en he cul u e o a d y ma e con en simila o ha a
sampling, i.e., 77% o ea ly, 59% o in e media e and 21% o
la e decay s ages (Tables 1,2). Flasks we e hen sealed wi h ub-
be sep a and a one-way s opcock was connec ed o a 50 mm
needle. The s opcock sys em was designed o en ila e ai and
p e en anoxic condi ions in he mic ocosm. The s opcock was
opened only when he en ila ion was pe o med a e measu e-
men o CO2(see below). The ai in he lasks was en ila ed
by connec ing he caps ock o a diaph agm acuum pump ( ype
N 022 AN.18, KNF Neube ge GmbH, Ge many) o 2 min
30 s. The caps ock was hen closed and eopened o lush he
lasks o 5 min wi h mois ai p e iously channeled h ough an
au ocla ed en il e (0.2 μm, Millipo e, USA). The e acua ion-
lushing sys em p e en ed oxygen deple ion and d ough in he
mic ocosms.
PREPARATION OF MICROCOSMS AND EXPERIMENTAL DESIGN OF
DECOMPOSITION STUDY (EXPERIMENT 1)
The e ec o ungal di e si y on espi a ion ac i i y was assessed
in mic ocosms by exposing he inoculum o a dilu ion p ocedu e
(We z e al., 2007). We es ed ou le els o ungal di e si y (i.e.,
dilu ions) on iplica e subs a es o h ee di e en quali ies (i.e.,
decay s ages). Expe imen al condi ions in mic ocosms we e as
ollows: (1) undilu ed (UD) inoculum p epa ed wi h non-s e ile
sawdus ; (2) a dilu ion o app oxima ely 1g inoculum (d y ma -
e , dm) pe 10 g o subs a e (dm) was p epa ed by i s mixing
non-s e ile sawdus wi h s e ile dis illed wa e o c ea e slu ies
wi h d y ma e o 27% (i.e., ea ly), 14% (i.e., in e media e), o
3% (i.e., la e), hen adding aliquo s o 540 mg dm (ea ly), 400 mg
dm (in e media e), and 150mg dm (la e) o he co esponding
d y subs a e o yield a inal inoculum dilu ion o 10−1(Table 2);
(3) a dilu ion o app oxima ely 1 g inoculum (dm) pe 100 g o
subs a e (dm) was p epa ed simila ly, bu slu y d y ma e was
0.8% (ea ly), 0.5% (in e media e) o 0.3% (la e), and aliquo s o
15 mg (dm) o each slu y we e added o he co esponding d y
subs a e (Table 2); (4) au ocla ed sawdus was used as a con ol
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Valen ín e al. Loss o di e si y a ec s decomposi ion
Table 1 | P ope ies o No way sp uce (Picea abies) wood o di e en decay s ages (ea ly, in e media e and la e) and he numbe o pooled
discs used o p epa e he inoculum (non-s e ile sawdus ) o he au ocla ed sawdus o he mic ocosms.
Wood decay s age C/N Lignin (%) Densi y kg/dm aD y ma e (%) bWa e con en (%) No o pooled discs
inoculum Subs a e
Ea ly 352 ±41 32 ±50.42±0.02 77 ±030±1145
In e media e 328 ±63 37 ±10 0.28 ±0.01 59 ±171±3125
La e 136 ±42 51 ±19 0.15 ±0.02 21 ±1376±16 16 5
The C/N a io, lignin, diame e , and densi y (de e mined as desc ibed in Rajala e al., 2010) a e he mean alues (±SD) o 14 discs om ea ly-decay s ages, 12 discs
om in e media e-decay s ages and 16 discs om la e-decay s ages. D y ma e and wa e con en we e de e mined by d ying esh sawdus a 105◦C o 24 h.
aD y ma e (%) was calcula ed as: weigh o d y sawdus (g)/weigh o we sawdus (g) *100.
bWa e con en (%) was calcula ed as: [weigh o we sawdus (g)—weigh o d y sawdus (g)]/weigh o d y sawdus (g) *100.
Table 2 | Amoun o inoculum (non-s e ile sawdus ) o au ocla ed sawdus in d y o m added o 100-mL glass lasks o assess he e ec o
ungal di e si y on he decomposi ion ac i i ies o No way sp uce sawdus in ea ly, in e media e and la e s ages o decay.
Dilu ions o aInoculum (non-s e ile Au ocla ed sawdus Added wa e To al ini ial d y weigh o Ini ial d y
he inoculum sawdus ) he incuba ed sample ma e con en
Decay s age Amoun (g, dm) Decay s age Amoun (g, dm) (ml) (g, dm) (%)
Undilu ed
Ea ly 5.0 – – 0 5.9 79
In e media e 3.8 – – 0 3.8 59
La e 1.4 – – 0 1.4 21
Dilu ion 10−1
Ea ly 0.54 Ea ly 4.7 5.0 5.24 73
In e media e 0.4 In e media e 3.5 3.0 3.9 52
La e 0.15 La e 1.3 1.6 1.45 24
Dilu ion 10−2
Ea ly 0.013 Ea ly 4.7 5.0 4.71 66
In e media e 0.015 In e media e 3.5 3.0 3.52 45
La e 0.015 La e 1.3 1.6 1.32 23
Con ol (C l)
– – Ea ly 5.2 1.9 5.2 65
– – In e media e 3.8 3.5 3.8 46
– – La e 1.5 5.7 1.5 21
The same amoun s we e used in a pa allel expe imen o he assessmen o wood-deg ading enzymes.
aD y ma e (dm) con en o inoculum ha was added as we sawdus (wa e con en acco ding o ield condi ions, epo ed in Table 1).
(C l). The ini ial ungal di e si y o he mic ocosms was es ed
wi h he DGGE (Rajala e al., 2010 and desc ip ion below) and
he numbe o obse ed OTUs was used as a measu e o ungal
di e si y. The es showed ha he dilu ion p ocedu e educed
he numbe o obse ed ungal OTU in he inocula (Figu e 1).
All ea men s we e pe o med in iplica e, yielding a o al o 36
lasks ( ou le els o ungal di e si y ∗ h ee decay s ages ∗ h ee
eplica es =36 lasks). Sealed lasks we e incuba ed a 21◦Cin
o al da kness o 16 weeks. The cumula i e CO2was measu ed
pe iodically (see below) and he ai space o he incuba ion lasks
was en ila ed immedia ely a e CO2measu emen .
A he end o he incuba ion, he subs a e om each eplica e
was pooled in o a single sample and s o ed a –18◦Cp io o he
ex ac ion o o al DNA.
MEASUREMENT OF CO2, EVACUATION AND CALCULATION OF CARBON
LOSS
An ai sample (100 μL) was collec ed om he sealed lask
wi h a Hamil on Mic o il e ™ glass sy inge, p e iously wiped
wi h 95% e hanol, and injec ed manually in o a gas ch oma o-
g aph (Hewle -Packa d 6890, Finland) o analyse CO2(assum-
ing ha mos o he p oduced CO2was ans e ed o he
gas phase). The ch oma og aph was equipped wi h a capil-
la y column (Agilen 19095P-MS6 HP-PLOT MoleSie e 5 Å,
leng h 30 m and diame e 530 μm) and a empe a u e con-
duc o de ec o a 250◦C o de ec ingCO
2. Helium was used
as ca ie gas a a spli a io o 10:1 and low a e o 7.9 mL
min−1. Injec o and o en empe a u es we e 120 and 40◦C,
espec i ely.
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Valen ín e al. Loss o di e si y a ec s decomposi ion
FIGURE 1 | Numbe o ITS1F-GC/ITS2 ungal DNA-de i ed DGGE bands
(Ope a ional Taxonomic Uni s; OTUs) in No way sp uce pooled
subs a e samples a he beginning o incuba ion unde a ange o
inoculum dilu ions (UD, undilu ed ungal inoculum; dilu ion 10−1g
inoculum/g subs a e (dm); dilu ion 10−2g inoculum/g subs a e (dm);
C l, au ocla ed sawdus ) o ea ly, in e media e and la e s ages o
decay.
The CO2le el was measu ed weekly a he beginning o he
incuba ion and e e y 2 weeks a e week i e. Es ima ed ca bon
loss (Closs, %) om mic ocosms was calcula ed using cumula i e
CO2a he end o he incuba ion ollowing equa ions (1) and (2):
C loss(%) =cumula i e CO2(g)/Max CO2(g) ∗100 (1)
Max CO2(g) =mass sawdus (g) ∗C(%)/100 ∗MCO2
(g mol−1)/MC(g mol−1)(2)
whe e Max CO2is he maximum p oduc ion o CO2 om each
mic ocosm, mass sawdus is he amoun o sawdus in he lask (in
dm), Cis o al ca bon con en in he sawdus , and MCO2and MC
a e he molecula masses o CO2(44 g mol−1) and ca bon (12 g
mol−1), espec i ely.
PREPARATION OF MICROCOSMS AND EXPERIMENTAL DESIGN OF THE
HYDROLYTIC ENZYME STUDY (EXPERIMENT 2)
The ac i i y o se e al wood-deg ading hyd oly ic enzymes was
measu ed om lasks incuba ed in pa allel wi h he mic ocosms
in Expe imen 1. The expe imen al design was simila o ha in
Expe imen 1 (i.e., ou le els o ungal di e si y ∗ h ee decay
s ages ∗ h ee eplica es =36 lasks). In his s udy, each lask was
opened unde s e ile condi ions a e weeks 4, 6, 9, 11, 13, and
16 o collec abou 1 g (we weigh , ww) o he subs a e. Hal o
he sample was subjec ed immedia ely o enzyme ac i i y assays
and he o he hal was s o ed a −18◦Cp io oDNAex ac ion
(samples om weeks 9 and 13).
EXTRACTION OF ENZYMES AND ACTIVITY ASSAYS
Fou glycoside hyd olases [β-glucu onidase (EC 3.2.1.31),
β-xylosidase (EC 3.2.1.37), β-glucosidase (EC 3.2.1.21), and cel-
lobiohyd olase (EC 3.2.1.37)] we e eco e ed om he subs a e
samples ollowing a ecen ly desc ibed enzyme ex ac ion me hod
wi h sligh modi ica ions (Heinonsalo e al., 2012). The me hod
is based on he eco e y o ex acellula enzymes by cen i ug-
ing a small amoun o subs a e (∼155 mg, ww) placed in o a
cen i uge ube il e (0.45μm po e size; 500 μLwo king ol-
ume; Cos a ® Spin-X® CLS8162; Co ning Inc., NY, USA). The
cen i uga ion speed was 15,700 g o 30min.Toob ainenough
enzyme solu ion o un he assays, h ee sub-samples we e p e-
pa ed om each lask. The day be o e ex ac ion, 200 μLs e ile
dis illed wa e was added o each subs a e sub-sample. A e
cen i uga ion, app oxima ely 250 μL enzyme solu ion was eco -
e ed, pooled wi h ha om he o he h ee sub-samples, adjus ed
o a o al olume o 2 mL wi h wa e and used di ec ly o he
ac i i y assays. Once all he ubes we e cen i uged, he inne il e
ubewasplacedinano ena 70
◦C o 48 h o measu e subs a e
d y ma e .
Hyd oly ic enzymes we e measu ed by a luo ime ic assay
(P i sch e al., 2004, 2011) and modi ied acco ding o he equi e-
men s o his s udy. Th ee solu ions we e p epa ed: (1) incuba ion
bu e (pH 4.5) con aining 7.2 mM maleic acid, 7.3 mM ci ic
acid, 10 mM bo ic acid, 10 mM T is (2-amino-2(hyd oxyme hyl)-
1,3-p opanediol), and 48.8mM sodium hyd oxide; (2) di e en
luo ogenic subs a e solu ions based on 4-me hylumbelli e one
(MU) o he de ec ion o each glycosidic enzyme we e p e-
pa ed om 5 mM s ock solu ions in 2-me hoxye hanol. The
concen a ion o each luo ogenic subs a e in he wo king solu-
ion was 1500 μMo MU-β-D-glucu onide ( o de ec ion o
β-glucu onidase), 1500 μMo MU-β-D-xyloside ( o de ec ion
o β-xylosidase), 1500 μMo MU-β-D-glucoside ( o de ec ion o
β-glucosidase), and 1200 μMo MU-β-D-cellobioside ( o de ec-
ion o cellobiohyd olase); (3) he s op solu ion was 1 M T is
a pH 10-11 o enhance he luo escence esponse. T iplica e
assays we e pe o med in black la -bo om 96-well mic opla es,
wi h each well-con aining a eac ion mix u e o 50 μLenzyme
solu ion, 50 μL incuba ion bu e and 50 μL o he espec i e lu-
o ogenic subs a e solu ion. Pla es we e incuba ed a 20◦Con
a mic opla e shake o 30min, excep o β-glucosidase whe e
15 min was used. A he end o incuba ion, 150 μlL s op solu ion
was added o all wells and he luo escence esponse was measu ed
wi h a Wallac 1430 Vic o 3(Pe kinElme , Inc., USA) mul ilabel
pla e eade a an exci a ion wa eleng h o 364nm and emission
wa eleng h o 450 nm. Calib a ion wells we e p epa ed by adding
100 μL s op solu ion, 100 μL incuba ion bu e and 50 μLs ock
solu ion mixed wi h wa e o gi e inal MU concen a ions o 0,
0.4, 0.8, 1.2, 1.6, and 2 μM.
Enzyme ac i i ies (EA) we e exp essed as picoka als pe g am
o subs a e (in dm). A picoka al is one picomol o eac ed luo o-
genic subs a e pe second. EA we e calcula ed using he equa ion
(3). To al hyd oly ic ac i i y was es ima ed by summing ha o
he ou measu ed enzymes.
EA (pka g−1)=(sample −nega i e con ol)/(a∗ )∗Va/V∗
s1/ms
(3)
whe e sample is he luo escence esponse (coun s) o he enzyme
solu ion and nega i e con ol is he esponse wi hou enzyme
( luo ogenic subs a e wi hou sample), ais he slope o he
eg ession line o he calib a ion cu e (coun s/pmol), is he
incuba ion ime (sec), Vais he adjus ed olume o he h ee sub-
samples (2000 μL), Vsis he olume o he enzyme solu ion in he
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Valen ín e al. Loss o di e si y a ec s decomposi ion
well (50 μL), and msis he sum o he ex ac ed subs a e mass o
he h ee sub-samples (g, dm).
MOLECULAR ANALYSIS OF THE FUNGAL COMMUNITY
To al DNA was ex ac ed om subs a es (100–150 mg, ww) a he
beginning, a e weeks 9 and 13 (only om he mic ocosms sub-
jec ed o enzyme analyses) and a he end o incuba ion (week 16)
using he NucleoSpin® 96 Soil Ki (Mache ey-Nagel, Ge many).
Ex ac ed o al DNA was ampli ied using polyme ase chain eac-
ion (PCR) wi h he GC-clamped in e nal ansc ibed space 1
(ITS1F) p ime (Ga des and B uns, 1993) and he ITS2 p ime
pai (Whi e e al., 1990) ypical o ungal ibosomal DNA. PCR
was pe o med in a 50 μL eac ion con aining 25 μLMyTaq™HS
Red Mix (which included eac ion bu e , dNTP, DNA Polyme ase
and loading dye; Bioline, Ge many), 1 μLeach25μM p ime ,
2.5 μL empla e and 20.5 μL s e ile dis illed wa e . The he mal
p o ile o he PCR was: 1min a 95◦C o ini ial dena u a ion,
34 cycles o dena u a ion o 15 s a 95◦C, annealing o 15 s a
58◦C and ex ension o 10s a 72◦C, and a inal ex ension o 72◦C
o 10 s. Equal concen a ions o PCR p oduc s we e esol ed by
dena u ing g adien gel elec opho esis (DGGE) as desc ibed in
Rajala e al. (2010). B ie ly, he dena u ing g adien in an ac y-
lamide gel was 18–58% and unning condi ions we e 75 V a 60◦C
o 16 h. Gels we e s ained wi h SYBR® Gold (Molecula P obes,
Eugene, O egon) and isualized wi h blue ligh on a Sa eImage ™
ansillumina o (In i ogen, Ca lsbad, Cali o nia). The DGGE
gels we e analyzed using GelCompa II so wa e (Applied Ma hs
BVBA, Belgium). The bands a he same posi ion in he DGGE
gel we e conside ed he same ope a ional axonomic uni (OTU).
Thus, his s ep gene a ed a p esence–absence ma ix o ungal
axa (OTUs) in each mic ocosm and we used he numbe o he
de ec ed OTUs as a measu e o ungal di e si y in his expe imen .
STATISTICAL ANALYSIS
We analyzed he e ec s o inoculum dilu ion (undilu ed, 10−1
and 10−2) and decay s age on CO2p oduc ion and enzymes ac i -
i ies measu ed om samples du ing he incuba ion pe iod using
he me hods o longi udinal da a analysis (Diggle e al., 2002). We
also pe o med some addi ional analyses o cumula i e CO2p o-
duc ion du ing he en i e incuba ion pe iod and on EA a he end
o he expe imen (week 16) using ANOVA, whe e longi udinal
da a analysis was no needed. In addi ion, we es ed co ela ion
be ween he numbe o obse ed OTUs and he cumula i e CO2
p oduc ion a he end o he expe imen .
In he longi udinal analyses o consecu i e weekly CO2p o-
duc ion and log- ans o med o al hyd oly ic EA o he samples,
we used a mixed model whe e eplica es ( ) ep esen ed andom
e ec s [∼N(0, σ2
)]. The ixed pa o he model consis ed o
he decay s age and dilu ion ac o s, and hei in e ac ion, i.e.,
he e ec s we we e in e es ed in. Se ial co ela ion o epea ed
measu emen s in he ime se ies o eplica es was ea ed wi h a
con inuous au o eg essi e co a iance s uc u e AR(1).
Residuals o he i ed models we e inspec ed isually o no -
mali y and homoscedas ici y. In all cases, we ound ha he
esiduals o he CO2 i we e no mally dis ibu ed by i ed
alues, and he dis ibu ions we e also no mal and ai ly sim-
ila by explana o y a iables. Random e ec s we e dis ibu ed
app oxima ely no mally as well. To ul il hese equi emen s o
he o al hyd oly ic EA, we con e ed hem o a log-scale.
S ong in e ac ions in he ixed pa o he model can make i
di icul o in e p e he model. The e o e, i we ound signi ican
in e ac ions be ween he ixed e ec s, we simpli ied he model by
ei he eg ouping he dilu ion and decay s age o a single ac o , o
by i ing he model o dilu ion ac o by he decay-s age ac o ,
o he o he way a ound.
We also in es iga ed he ends o log- ans o med o al
hyd oly ic enzyme ac i i y, and he a io o hyd oly ic ac i i y o
CO2p oduc ion du ing he incuba ion pe iod. In hese cases, we
added a slope pa ame e o incuba ion week o he ixed pa
o he model. The ends o hese a iables we e es ed o each
decay s age sepa a ely, so ha we allowed he end o a y by dilu-
ion. Fo he a io o hyd oly ic ac i i y and CO2p oduc ion, he
esul s a e p esen ed o he ea ly decay s age by dilu ion.
Longi udinal analyses we e implemen ed using he lme
unc ion o he nlme lib a y o R, using he maximum like-
lihood me hod. Pa ame e signi icance was es ima ed by he
lme- unc ion.
When a ime-se ies app oach was no needed, we used a ype II
ANOVA wi h in e ac ion be ween he decay s age. These analyses
we e conduc ed using he ANOVA unc ion in he ca -lib a y (Fox
and Weisbe g, 2011)o R(R De elopmen Co e Team, 2011).
The same p inciples o examining he esiduals and spli ing he
model in o pa s i in e ac ions we e p esen , we e applied as wi h
he longi udinal app oach. Pai wise compa isons we e made wi h
he Tukey Hones ly Signi ican Di e ence (HSD) es .
Fungal communi y composi ion (i.e., he p esence/absence
o OTUs) was isualized by non-me ic mul idimensional scal-
ing (NMDS) using me aMDS o he egan lib a y (Oksanen
e al., 2008). We gene a ed a dissimila i y ma ix (nsample∗nsample)
o B ay-Cu is coe icien s and used NMDS o c ea e a g aph
whe e he dis ances be ween poin s ( ep esen ing samples) co e-
sponded as closely as possible o he o iginal dissimila i y ma ix.
A ows ha indica e he di ec ion o maximum co ela ion o
cumula i e CO2and OTU numbe wi h he o dina ed samples
we e supe imposed on o he o dina ion g aph. The signi icance
o hese co ela ions was assessed wi h a pe mu a ion es imple-
men ed in he en i unc ion o egan lib a y (Oksanen e al.,
2008). The NMDS was pe o med sepa a ely o each incuba-
ion ime. The analyses made o week 0 and 16 co esponded
o he expe imen whe e lasks we e closed du ing incuba ion
(Expe imen 1), whe eas hose made o week 9 and 13 co e-
sponded o he mic ocosms in which lasks we e opened o collec
samples (Expe imen 2).
RESULTS
EFFECTS OF SUBSTRATE AND REDUCED DIVERSITY ON
DECOMPOSITION
The wood decay s age was an impo an ac o o explain CO2
p oduc ion du ing he expe imen (Figu e 2, Supplemen a y
Ma e ial). When we analyzed he consecu i e CO2obse a ions
and hei associa ion wi h he decay s age wi h longi udinal anal-
ysis, we ound ha la e- and in e media e-decay subs a es we e
associa ed wi h signi ican ly highe CO2p oduc ion a es han
ea ly-decay subs a es ( =8.0, d =17, p≤0.001, and =5.7,
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Valen ín e al. Loss o di e si y a ec s decomposi ion
FIGURE 2 | E ec o dilu ion on he p oduc ion o CO2(cumula i e mg
o CO2pe g o subs a e; indm) wi hin ea ly- (A), in e media e- (B),
and la e- (C) decay s ages. Dilu ion o ungal di e si y is shown by
symbols as undilu ed ungal inoculum (squa es), dilu ion 10−1g inoculum/g
subs a e (ci cles), dilu ion 10−2g inoculum/g subs a e ( iangles), and
con ols (diamonds). E o ba s indica e he SD o he mean o
obse a ions and we e calcula ed o each decay s age and dilu ion
sepa a ely.
d =17, p<0.001, espec i ely). This e ec was p esen when we
analyzed each inoculum sepa a ely (p<0.02 o decay-s age di -
e ences wi hin inocula), al hough he 10−1inoculum esponded
signi ican ly di e en ly om o he inocula in he ull model wi h
in e ac ions.
When we analyzed he e ec o inoculum (which had di e en
numbe o ungal OTUs) acco ding o decay s age wi h longi udi-
nal analysis, we ound ha he weekly CO2p oduc ion o dilu ed
10−1gand10
−2g inocula di e ed om ha o undilu ed inocu-
lum in in e media e-decay subs a es ( =−5.5, p=0.002 and
=−3.9, p=0.008, espec i ely). The ANOVA o cumula i e
CO2a week 16 suppo ed hese indings. When ungal di e si y
(OTU) was added o he p e ious model, i was also signi ican
(F=4.4, d =1, p=0.05). A e 16 weeks, he cumula i e p o-
duc ion o CO2 om undilu ed inoculum in la e-decay subs a es
eached 65 mg CO2g−1subs a e (dm), co esponding o a C loss
o 3.1%, whe eas C loss om undilu ed in e media e- and ea ly-
decay subs a es we e 2.6 and 0.2%, espec i ely (Figu es 2C,3).
A he end o he expe imen , he cumula i e CO2p oduc ion
was co ela ed wi h he obse ed numbe o OTUs ( =0.83,
p<0.001).
EFFECTS OF SUBSTRATE AND REDUCED DIVERSITY ON ENZYME
ACTIVITY
Di e ences in o al hyd oly ic EA depended on he subs a e,
he e o e, we pe o med he analyses by subs a e and by dilu ion
(Figu e 4).
When we analyzed he consecu i e o al hyd oly ic ac i i y
o eplica es wi h longi udinal analysis, we ound ha i was
highe in p epa a ions con aining he 10−2dilu ion han in he
undilu ed inoculum in ea ly- ( =3.6, d =5, p=0.02) and
la e-decay subs a es ( =6.0, d =6, p=0.001). Mo eo e , he
hyd oly ic ac i i ies we e also signi ican ly di e en be ween ea ly
decayed and o he subs a es in undilu ed inocula (bo h >4.0,
d =6, p<0.005) and in he 10−1dilu ion (bo h >3.2, d =
5, p<0.03).
FIGURE 3 | Cumula i e ca bon loss (means o h ee eplica es) du ing
he incuba ion o ea ly, in e media e and la e s ages o decay and
ecei ing a ious dilu ions o ungal inoculum [UD, undilu ed; dilu ion
10−1g inoculum/g subs a e (dm); dilu ion 10−2g inoculum/g
subs a e (dm); C l, au ocla ed sawdus ].
When we analyzed he o al hyd oly ic enzyme ac i i y mea-
su emen s a he end o he expe imen wi h ANOVA, we also
ound ha he ac i i ies depended on he dilu ion-subs a e
combina ion (in e ac ion F=10.2, d =6, p<0.001). We hen
g ouped he inoculum and subs a e in o a compounded ac o ,
pe o med an ANOVA and conduc ed a pai -wise compa ison
o ac o s, which showed ha he mean hyd oly ic ac i i y o
he mos dilu ed (10−2) inoculum o he la e-decayed subs a e
was g ea e han ha in any o he o he inoculum decay-s age
combina ions (p<0.0001 o all pai s).
Fu he mo e, he numbe o ungal OTUs appea ed o in lu-
ence he enzyme ac i i y a he end o he expe imen (F=14.2,
d =1, p=0.09).
In all dilu ions o he in e media e-decay inoculum, a neg-
a i e end in o al hyd oly ic ac i i y was eco ded owa d he
end o he incuba ion (Figu e 4B). Acco ding o he longi udinal
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Valen ín e al. Loss o di e si y a ec s decomposi ion
FIGURE 4 | E ec o ungal communi y dilu ion on o al hyd oly ic
ac i i y (pka /g o subs a e; in dm) as he sum o
1,4-β-glucu onidase, 1,4-β-xylosidase, cellobiohyd olase, and
1,4-β-glucosidase om ea ly-decayed (A), in e media e-decayed (B),
and la e-decayed subs a e (C). The dilu ion o ungal di e si y is shown
by symbols as undilu ed ungal inoculum (squa es), dilu ion 10−1g
inoculum/g subs a e (ci cles), dilu ion 10−2g inoculum/g subs a e
( iangles), and con ols (diamonds).
analysis, he highe in e cep and s onge nega i e end o
hyd oly ic ac i i y in undilu ed inoculum han in ei he o he
dilu ed inocula ( he model in e cep di e ences in compa ison o
he undilu ed inoculum bo h had <−3.0, d =6, p<0.02 and
o he end coe icien , hey had >3.4, d =42, p<0.001,
espec i ely) we e coinciden wi h he measu ed espi a ion a es
ha s a ed a he highes alues ( <−3.9, d =6, p<0.01)
and hen dec eased he quickes ( >2.3, d =42, p<0.02) in
he undilu ed inoculum in compa ison wi h o he dilu ions in he
in e media e-decay subs a e (Figu es 2B,4B).
The a io o hyd oly ic ac i i y o espi a ion (Hyd oly ic
ac i i y/CO2) clea ly dec eased owa d he end o he incuba-
ion in he ea ly decay subs a e ha ecei ed undilu ed ( =
−3,4, d =13, p=0.005), as well as dilu ed inocula (10−1
dilu ion: =−2.9, d =9, p<0.02) (Figu e 5).Thesamephe-
nomenon did no occu in mo e decayed subs a es (da a no
shown).
FUNGAL COMMUNITY STRUCTURE AND RESPIRATION ACTIVITY IN
DIFFERENT DECAY STAGES
Fungal communi ies o di e en decay s ages di e ed a he
s a o he expe imen (Figu e 6A) and communi y s uc u es
changed du ing he incuba ion pe iod (Figu e 6). In conjunc ion
wi h obse ed di e ences be ween undilu ed and dilu ed ungal
communi ies in he in e media e decay subs a e (Figu e 6B), we
measu ed highe espi a ion ac i i y in he mic ocosm p epa ed
wi h he undilu ed inoculum, especially du ing he i s 6 weeks
(Figu e 2B). In he ea ly decay subs a e, he sligh ly dilu ed
(10−1) ungal communi y, which had he highes espi a ion a e
(Figu e 2A), appea ed o de ia e om all o he communi ies a e
13 weeks o incuba ion (Figu e 6C).
A he end o he expe imen , cumula i e CO2( =0.91, p<
0.001) and he numbe o OTUs ( =0.87, p<0.001) inc eased
in he communi ies composed o in e media e- and la e-decay
s age species, as shown by he linea i supe imposed on he
NMDS g aph (Figu e 6D).
FIGURE 5 | Rela ionship o ca bohyd a e deg ada ion (pka /g) and
weekly CO2p oduc ion (mg/g subs a e, dm) in he ea men s
ecei ing undilu ed ungal inoculum o ea ly- (ligh g ay),
in e media e- (da k g ay), and la e- (black) decay subs a e du ing he
16-week incuba ion.
DISCUSSION
EFFECTS OF FUNGAL DIVERSITY ON WOOD DECOMPOSITION
The main indings o he expe imen con i med ou hypo hesis
ha ungal di e si y a ec s decomposi ion a e and ha esis ance
o he decomposi ion p ocess o he loss o di e si y depends on
he decomposi ion s age o woody subs a es. We used inocula
ha o igina ed om ac i ely decaying wood, whe e he numbe
o OTUs is highe han in p e ious expe imen s using selec ed
species om pu e cul u es (Se älä and McLean, 2004; Tiuno
and Scheu, 2005). These s udies wi h communi ies c ea ed by
combining cul u ed ungal species ha e shown how he decom-
posi ion a e becomes asymp o ic wi h ela i ely ew decompose
species, and ou esul s on la e-decay communi ies wi h ini ially
high numbe o ungal OTUs suppo his inding. Howe e ,
we obse ed ha he unc ioning o he decompose s o he
in e media e-decay s age, whe e he ini ial numbe o OTUs was
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Valen ín e al. Loss o di e si y a ec s decomposi ion
FIGURE 6 | Non-me ic mul idimensional scaling (NMDS) o dina ion
showing he sepa a ion o ungal communi ies a he beginning
(A), a e 9 weeks (B), 13 weeks (C), and 16 weeks (D) in he
di e en dilu ions o con ols (au ocla ed sawdus ) acco ding o he
decay s age o subs a e. Symbols ep esen he dilu ion o he ungal
communi y: undilu ed (squa es), dilu ion 10−1g inoculum/g subs a e
(ci cles), dilu ion 10−2g inoculum/g subs a e ( iangles) and con ols
(diamonds). The in ensi y o he symbol indica es he decay s age.
Vec o s indica e he di ec ion o maximum co ela ion o CO2p oduc ion
and numbe o OTUs wi h o dina ed samples (p<0.01). Ellipses indica e
he mean loca ion o each decay s age: E, ea ly; I, in e media e; and
L, la e.
lowe han in he la e-decay wood, was less esis an o he loss o
di e si y. In he in e media e-decay s age, decomposi ion ac i -
i y was highes in non-dilu ed communi ies and he expe imen al
dilu ion o he ungal communi y was associa ed wi h a lowe
espi a ion a e.
This s udy showed ha CO2p oduc ion, as an indica o o
decomposi ion, inc eased in ela ion o he decay s age o No way
sp uce wood and peaked du ing he la e-decay s age, whe e ungal
di e si y (measu ed as he numbe o obse ed OTUs) was also
maximal. The obse ed inc ease in decomposi ion ac i i y wi h
inc easing OTU di e si y is in ag eemen wi h ou hypo hesis.
Ea lie s udies, whe e decay models we e i ed o empi ical ield
da a, p edic ed ha he a e o decay is highes du ing in e medi-
a e phases o decomposi ion (Ha mon e al., 2000; Mäkinen e al.,
2006; Tuomi e al., 2011), which coincides wi h he pe iod when
whi e- and b own- o ungi a e a hei peak p e alence (Rajala
e al., 2011, 2012). Howe e , we sugges ha decomposi ion ac i -
i y is also main ained in he la e-decay s age wood ha migh be
unde - ep esen ed in p e ious s udies (i.e., hey used a ela i ely
small numbe o la e-decay samples, which a e di icul o da e).
In addi ion, ou esul s indica e ha lowe ing ungal di e si y,
especially in he in e media e-decay s age, has a di ec impac on
ungal communi y unc ion, and he eby on he decomposi ion
o woody li e .
We showed ha in he mos di e se ungal communi y inhab-
i ing he la e-decay subs a e, lowe ing he di e si y wi h dilu ion
o he inocula had only a sligh impac on decomposi ion a e.
This sugges s ha he communi y ha emained a e dilu ion
was su icien ly di e se o p o ide he same ecological unc ion as
ha o he undilu ed sys em, which ag ees well wi h ea lie s ud-
ies o he di e si y- unc ion ela ionship in soil mic obes (We z
e al., 2006), whe eas he less di e se communi ies we e mo e
a ec ed by andom selec ion. Consequen ly, we p edic a s onge
esilience o he di e se ungal communi y ound in ad anced
s ages o wood decay.
In he less di e se communi y inhabi ing he ea ly decay sub-
s a e, a 10−1dilu ion caused an unexpec ed esul in ha CO2
p oduc ion inc eased. I appea s ha loss o di e si y in a low-
densi y communi y a ec s compe i i e in e ac ions and decom-
posi ion in unp edic able ways. The esponse is highly sensi i e
o he ac i i y o he emaining species and he unc ional edun-
dancy in he ungal communi y o ea ly-s age decompose s is
lowe han ha ound in la e s ages.
The use o indigenous ungal communi ies as inocula o s udy
he ela ionship be ween di e si y and unc ion o e es ial un-
gal communi ies is no el and was applied o he i s ime o
wood-inhabi ing ungi in his s udy. P e ious labo a o y s udies
ha e assessed he abili y o cul u ed ungi o deg ade woody sub-
s a es (Boddy e al., 1989; Tiuno and Scheu, 2005; Toljande
e al., 2011) and compe e wi h na u al wood-inhabi ing ungi
(Holme and S enlid, 1997; Holme e al., 1997). Acco ding
o he me hod p oposed by We z e al. (2006), hedilu ion
p ocedu e educed he numbe o ungal axa de ec ed in he
inoculum. P io o ou expe imen , we es ed di e en a ios
o dilu ions and ound ha a ela i ely high (1/100) dilu ion is
needed o a ema kable educ ion o OTUs in highly di e se
inocula. Howe e , o a mode a e educ ion in he OTU di e -
si y, we chose o apply a dilu ion a io (1/10) ha was op imal o
he in e media e decay s age, bu comp omised la e decay, whe e
he esul ing educ ion in OTU di e si y was only sligh .
As expec ed, he numbe o de ec ed ungal species inc eased
in all ea men s wi h he p og ession o he incuba ion, since
he applied PCR-DGGE me hod canno iden i y in equen ungi
and only a e eplica ion o DNA du ing he incuba ion hey
became isible. Ou s e iliza ion p ocedu e o he con ol sub-
s a e was appa en ly su icien o des oy mos o he ungal
DNA, as OTUs we e no de ec able ia DGGE a he s a o he
expe imen (Figu e 1). Howe e , sligh CO2p oduc ion du ing
he incuba ion indica ed ha some mic obes su i ed au ocla -
ing (Figu e 2), as i is known ha some spo es migh be esis an
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Valen ín e al. Loss o di e si y a ec s decomposi ion
o his o m o s e iliza ion (Cheng e al., 2008). This was he ea-
son ode ec a ew ungalOTUsin hecon olsa e 9weeks(da a
no shown). Howe e , NMDS showed he communi y s uc-
u e in con ols o be dis inc om ha o o he mic ocosms.
This sugges s ha he key species in ol ed in decomposi ion
did no su i e au ocla ing, al hough we canno o ally deny
ha some species o igina ed om au ocla ed sawdus . Howe e ,
CO2p oduc ion in con ols was a lowe han in any o he
ea men s and he e o e we belie e ha e ec o con aminan
species in dilu ion ea men s is mino . The ac ha s e iliza-
ion o wood, soil o o he subs a e by au ocla ing is p ac ically
impossible should s ill be conside ed in his kind o dilu ion
p ocedu es.
ENZYME ACTIVITY DURING WOOD DECOMPOSITION IN RESPONSE TO
FUNGAL DIVERSITY
Con a y o ou hypo hesis, he mos dilu ed (10−2)inocu-
lum was associa ed wi h a highe ac i i y o hyd oly ic enzymes
han ea men s o he same subs a e ecei ing inoculum wi h a
highe ungal ichness (Figu e 4). This migh be explained by less
compe i ion in he dilu ed communi ies in ol ing species spe-
cializing in he p oduc ion o ca bohyd a e-deg ading enzymes
(e.g., b own- and so - o ungi). We also ound ha he a io o
hyd oly ic ac i i y and p oduc ion o CO2pe weekinea ly-decay
subs a es was ini ially high and g adually dec eased. The nega i e
slope o he ela ionship be ween hyd oly ic ac i i y and weekly
CO2p oduc ion sugges s ha ungal axa ha a e ac i e du -
ing he ea ly phases o decomposi ion mus p oduce hyd oly ic
enzymes o elease he wide a ie y o ca bohyd a es ha a e
me abolized in la e s ages.
Theenzyme eco e yme hod,basedona il e cen i uga ion
app oach, has been alida ed in aga media supplemen ed wi h
di e en o ganic p oduc s (Heinonsalo e al., 2012), bu has ne e
been applied o a mo e complex subs a e such as one composed
o decomposing sp uce wood. A s anda d and uni e sal p o o-
col o ex ac ing and measu ing enzymes om en i onmen al
samples does no exis (Bald ian, 2009). Common p ac ice is o
soak he samples in bu e (e.g., ace a e o sul a e bu e s) and
ex ac he enzymes by agi a ing he slu y, bu such ea men
migh nega i ely a ec EA (Vepsäläinen, 2001). In ou s udy,
he le el o enzyme ac i i y was in he ange 10–100 pka g−1
d y subs a e. P e ious s udies ha epo ed ac i i ies as high as
1000 pka g−1(dm) in No way sp uce needles used pu e ungal
inoculum om a cul u ed isola e (Ži èáko á e al., 2011). Ou
esul s indica e ha ex acellula EA in sp uce wood ha has no
ecei ed a pu e and concen a ed inoculum a e much lowe . I is
known ha sp uce wood is a complex subs a e o he eco e y o
wood-deg ading enzymes, since wa e -soluble cons i uen s such
as phenols, suga s, o ganic acids, xylo-oligosaccha ides and p o-
eins all ac as inhibi o s o hyd oly ic enzymes (e.g., Lagae e al.,
2009; Kim e al., 2011). Ne e heless, he e ec o wood ex ac s
on enzyme eco e y in his s udy was expec ed o be minimal as
was p e iously epo ed by Valen ín e al. (2010).
CONCLUSIONS
Dilu ion o ungal inocula eco e ed om Picea abies logs in
h ee di e en decay s ages e ealed a s age-dependen esponse
in decomposi ion a e. Reduced ungal di e si y was associa ed
wi h lowe espi a ion a es du ing he in e media e s ages o
decay, bu no di e si y e ec s we e de ec ed in la e s ages,
whe e ini ial numbe o de ec ed species was high. This sug-
ges ed ha he highly di e se communi y o he la e-decay s age
was mo e esis an o he loss o di e si y han less di e se
communi ies o ea ly decompose s. In ea ly-decay communi-
ies, dilu ion caused unexpec ed changes o he decomposi ion
p ocess, p obably due o he s ong s ochas ic e ec o dilu-
ion on less di e se communi ies. The esul s o his s udy also
showed ha he decomposi ion a e and he ungal di e si y
inc eased as decay ad anced. We sugges ha ungal ac i i y and
he unc ional edundancy o he ungal communi y in decaying
wood inc ease du ing he ungal succession om in e media e- o
la e-decay s ages.
ACKNOWLEDGMENTS
We a e g a e ul o he o es and labo a o y pe sonnel o he
Finnish Fo es Resea ch Ins i u e o hei assis ance in ield wo k
and he p epa a ion o samples o chemical, enzyma ic and
molecula analyses. The s udy was unded by he Academy o
Finland (p ojec numbe s 121630 and 257701).
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound online
a : h p://www. on ie sin.o g/jou nal/10.3389/ micb.2014.
00230/abs ac
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