RESEARCH ARTICLE
B eeding Inc eases he E icacy o
Chond os e eum pu pu eum in he Sp ou
Con ol o Bi ch
Leena Hambe g*, Henna Va iamäki, Ja kko Han ula
Finnish Fo es Resea ch Ins i u e, Van aa Resea ch Uni , Van aa, Finland
*leena.hambe g@me la. i
Abs ac
We es ed whe he he pai ing o selec ed isola es could be used o inc ease he e iciency
o a decay ungus Chond os e eum pu pu eum (Pe s. Ex F .) Pouza o con ol ha dwood
sp ou ing in Finland. We pai ed C. pu pu eum s ains e icien in sp ou con ol o highly ac-
i e in laccase p oduc ion, and es ed he e icacy o hei p ogeny in spou con ol expe i-
men s. This p ocedu e esul ed in a s ain wi h an e icacy supe io o ha o he pa en al
s ains. The mo ali y o bi ch (Be ula pendula Ro h. and B. pubescens Eh h.) 1 cm in s ump
diame e was 78%, 56% and 9% o he bes p ogeny, he bes pa en al s ain and he con-
ol, espec i ely. Mo ali y was only sligh ly highe o B. pendula han o B. pubescens bu
no signi ican di e ences we e ound be ween he numbe o maximum heigh o s ump
sp ou s. Ou esul s showed ha c oss b eeding o his decay ungus is a good al e na i e in
a emp s o p oduce e icien biocon ol agen s agains ha dwood sp ou ing.
In oduc ion
In Finland, sp ou ing o b oad-lea ed ees is a hind ance in sp uce (Picea abies [L.] H. Ka s .)
and pine (Pinus syl es is L.) egene a ion a eas, alongside oads and ailways, unde elec ic
powe lines and abo e gas pipe lines. In egene a ion a eas, b oad-lea ed species such as sil e
and downy bi ch (Be ula pendula Ro h. and B.pubescens Eh h.), dec ease he g ow h o mo e
comme cially aluable coni e s, and he e o e non-c op species a e ypically cleaned, p e e ably
a an ea ly s age when ees a e abou 1 m in heigh [1,2,3]. Nex o oads and ailways, b oad-
lea ed ees o m a h ea o he sa e y o a ic as hey es ic isibili y, co e a ic signs and
emp moose, and a e he e o e egula ly emo ed. Elec ic powe lines a e kep open o ensu e
con inuous elec ic ansmission and gas pipe lines ma ked wi h isible signs a e equen ly
clea ed in o de o a oid unin ended exca a ions. Sp ou con ol o b oad-lea ed ees cos s
mo e han 60 million eu os annually in Finnish o es egene a ion a eas, along oads and ail-
ways, and a elec ic powe and gas pipe lines (in o ma ion ga he ed om UPM Fo es L d.,
The Finnish T anspo Agency, Fing id L d. and Gasum L d.).
He bicides we e p e iously success ully used in sp ou con ol [4,5], bu he use o chemicals
is no longe ecommended in Finnish g oundwa e a eas due o hei ha m ul e ec s [6,7].
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 1/20
a11111
OPEN ACCESS
Ci a ion: Hambe g L, Va iamäki H, Han ula J (2015)
B eeding Inc eases he E icacy o Chond os e eum
pu pu eum in he Sp ou Con ol o Bi ch. PLoS ONE
10(2): e0117381. doi:10.1371/jou nal.pone.0117381
Academic Edi o : Pe Ka lo sky, Geo g-Augus -
Uni e si y Gö ingen, GERMANY
Recei ed: Oc obe 3, 2014
Accep ed: Decembe 23, 2014
Published: Feb ua y 12, 2015
Copy igh : © 2015 Hambe g e al. This is an open
access a icle dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any
medium, p o ided he o iginal au ho and sou ce a e
c edi ed.
Da a A ailabili y S a emen : All da a se s and
igu es a e a ailable om he D yad Digi al
Reposi o y: h p://dx.doi.o g/10.5061/d yad. 5s6 .
Funding: The s udy was unded by he Finnish
Funding Agency o Technology and Inno a ion
(h p://www. ekes. i/en/, p ojec numbe 265/31/2012),
Ve de a L d. (h p:// e de a. i/en/ on -page/),
Me sä eho L d. (h p://www.me sa eho. i/abou us/
p esen a ion), Fing id L d. (h p://www. ing id. i/en/
Pages/de aul .aspx), The Finnish T anspo Agency
(h p://po al.liikenne i as o. i/si u/www/e/), UPM-
Kymmene L d. (h p://www.upm.com/en/Pages/
de aul .aspx), Ma ja a and Eino Kolli Founda ion
(h p://www.kollinsaa io.com/, p ojec numbe 1041),
Also, public opinion is s ongly agains hei usage. On he o he hand, mechanical cu ing
alone is no e ec i e due o he igo ous sp ou ing o b oad-lea ed ees [8,9]. The e o e, new
me hods o sp ou con ol a e needed.
One p omising op ion is o u ilize a na u al pa hogen o b oad-lea ed ees, he sil e lea
ungus, Chond os e eum pu pu eum (Pe s. ex F .) Pouza [10,11], which has been shown o e-
s ic he sp ou ing o se e al ee species [8,12,13,14]. The e o e i can be conside ed a ‘na u e
iendly’al e na i e o chemicals.
C.pu pu eum is a basidiomyce e commonly ound on wounded b oad-lea ed wood in bo eal
and empe a e ege a ion zones in Eu ope [15,16,17]. The ungus is widesp ead in na u e due o
i s e icien basidiospo e p oduc ion esul ing in equen new in ec ions [15,18]. In na u e, mono-
ka yo ic spo es landing on wood i s ge mina e, a e which he hyphae om di e en spo es hy-
b idize and o m dika yo ic mycelia which hen colonize he hos [18]. Howe e , in man-made
sp ou con ol, dika yo ic ungal hyphae a e di ec ly sp ead on o eshly cu s umps [9,12,13].
The use o C.pu pu eum in sp ou con ol is based on i s abili y o g ow inside a s ump and
inally decay i . Du ing he in asion, C.pu pu eum pene a es h ough s a ch g anules and cell
walls enzyma ically, and induces he occlusion o ee essels [15,19,20,21]. The esul ing dehy-
d a ion combined wi h ungal oxins may inally cause mo ali y o he hos . Whi e o ungi,
such as C.pu pu eum, a e e icien in b eaking down lignin [22,23]. In woody cell walls, lignin
su ounds he cellulose which is he ac ual ca bon and ene gy sou ce o he ungus, and possi-
bly he ul ima e eason o lignin deg ada ion [23,24]. Laccases a e he main oxida i e enzymes
in his decay p ocess in addi ion o lignin and manganese pe oxidases [13,23].
The e icacy o C.pu pu eum in sp ou con ol depends on he hos ee species
[5,8,9,13,14]. Also, an inc ease in he diame e o an inocula ed s ump has been shown o a ec
s ump mo ali y [9,14]. Fu he mo e, conside able a ia ion exis s in he abili y o di e en
C.pu pu eum s ains o p e en sp ou ing [4,5,13], bu he possibili y o inc easing con ol e i-
ciency by b eeding has no been es ed be o e.
B eeding among sexually p opaga ing ungi has p e iously been used in indus ial applica-
ions such as he chemical indus y (e.g. enzymes o bioe hanol), and in wine p oduc ion in
o de o inc ease he yield o cul i a ed o ganisms and economic bene i [25,26]. Mo eo e , he
biocon ol abili y o a sap ophy ic ungus Phlebiopsis gigan ea (F .) Jül. agains a oo o un-
gus He e obasidion pa ipo um Niemelä & Ko honen was imp o ed by adi ional b eeding
[27]. The e o e, b eeding based on he na u al a ia ion o C.pu pu eum can be expec ed o
p o ide an e icien way o inc ease he abili y o his ungus o con ol sp ou ing.
The main aim o his s udy was o es whe he b eeding can be used o inc ease he e icacy
o he decay ungus C.pu pu eum in p e en ing sp ou ing o small bi ch s umps (Be ula pen-
dula and B.pubescens). We hypo hesized ha pai ing o s ains e icien in (i) sp ou con ol
and (ii) laccase p oduc ion could esul in a supe io combina ion o genes p oducing a leas
one p ogeny s ain be e han he pa en al s ains. In addi ion, we es ed whe he he abili y o
C.pu pu eum o con ol sp ou ing di e s be ween he bi ch species. He e we show ha b eed-
ing can success ully be used o imp o e he biocon ol abili y o C.pu pu eum bu no signi i-
can di e ences be ween he wo bi ch species in es iga ed we e ound.
Ma e ials and Me hods
The s udy was composed o ou phases (Fig. 1): 1) pai ing o he bes s ains in e ms o laccase
ac i i y and p ogeny es ing in he labo a o y, 2) pai ing o he mos e icien pa en al s ains
a ailable [13] and p ogeny es ing in he ield, 3) pai ing o he bes p ogenies om Phases 1
and 2 and p ogeny es ing in he ield, and 4) inal es ing o he e icacy o he bes p ogenies
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 2/20
he Finnish Cul u al Founda ion (h p://www.sk . i/en,
p ojec numbe 00081430) and he Finnish Fo es
Resea ch Ins i u e (h p://www.me la. i/index-en.h ml).
LH ecei ed sala y om he Finnish Fo es Resea ch
Ins i u e which ob ained unding om he
abo emen ioned sou ces. The unde s had no ole in
he s udy design, da a collec ion and analysis,
decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: Ve de a L d. is in e es ed in
de eloping a comme cial p oduc including C.
pu pu eum s ain R53, Fing id L d. and UPM-
Kymmene L d. p o ided si es o he s udy, and
Me sä eho L d. oge he wi h o he unde s (excep
Ma ja a and Eino Kolli Founda ion, he Finnish
Cul u al Founda ion, and he Finnish Funding Agency
o Technology and Inno a ion) a e po en ial use s o
he comme cial biocon ol p oduc , and he e o e he
au ho s ha e coope a ed wi h hem in o de o pay
a en ion o c i ical poin s in sp ou con ol. This does
no al e he au ho s’adhe ence o PLOS ONE
policies on sha ing da a and ma e ials.
om Phases 2 and 3. All da a se s and igu es a e a ailable om he D yad Digi al Reposi o y:
h p://dx.doi.o g/10.5061/d yad. 5s6
E hics S a emen
Pe mission o he ield expe imen s o Phases 2–4 was g an ed by land owne s, Fing id L d.,
UPM-Kymmene L d. and he Finnish Fo es Resea ch Ins i u e. Endange ed o p o ec ed spe-
cies we e no used in his s udy. Fungal s ains o C.pu pu eum ha e been deposi ed in he cul-
u e collec ion o he Finnish Fo es Resea ch Ins i u e.
Laccase ac i i y es s
Laccase ac i i y o he isola es was s udied as i has been shown ha laccase ac i i y o
C.pu pu eum isola es co ela es wi h he e icacy o hei sp ou con ol [13]. In o de o ind
e icien laccase p oduce s, al oge he 69 he e oca yo ic C.pu pu eum isola es we e collec ed
om bi ch (Be ula pendula and B.pubescens) s umps in July-Oc obe 2009. These isola es we e
collec ed om di e en pa s o Finland, in Ala us (3 isola es), Van aa (40), Hyy iälä (1), Juu-
pajoki (2), Män sälä (16) and Jä enpää (8) (Table 1).
We used ABTS (2,2’-azino-bis[3-e hylbenzo hiazoline-6-sulphonic acid]) pla es whose colo
eac ion e eals he laccase ac i i y o C.pu pu eum (0 = no colo eac ion, i.e., low laccase ac i -
i y o 3 = s ong da k-g een colo eac ion, i.e., high laccase ac i i y, see [13]). The ABTS pla es
(modi ied om [28]) we e p epa ed by mixing 1.0 g glucose (Ac os O ganics), 2.0 g KH
2
PO
4
(Me ck), 0.5 g MgS0
4
×7H
2
O (Fluka), 0.13 g CaCl
2
×2H
2
O (Me ck), 0.5 g (NH
4
)
2
- a a e (Al a
Aesa ), 1.78 g dime hylsuccinic acid (Me ck), 0.2 g yeas ex ac (Bec on, Dickinson and Compa-
ny) and 25 g aga (Bec on, Dickinson and Company) wi h 1000 ml deionized wa e . All elemen s
excep aga we e suspended in wa e and pH adjus ed o 5.0 wi h 1.0 M NaOH. Aga was added
and he subs a e was au ocla ed a 121°C o 15 min. Al oge he 250 mg ABTS (Applichem) di-
lu ed wi h 99% e hanol was added o he medium when he empe a u e was 48–50°C.
All C.pu pu eum isola es we e g own on po a o dex ose aga Pe i pla es (PDA: 24 g po a o
dex ose b o h and 15 g aga wi h 1000 ml deionized wa e ; Bec on, Dickinson and Company),
and a 6-mm diame e aga plug a he pe iphe y o he g own mycelium was ans e ed o he
middle o an ABTS pla e using a s e ilized Pas eu pipe e and a scalpel. Two eplica e pla es
Fig 1. The B eeding P ocess o he S udy.
doi:10.1371/jou nal.pone.0117381.g001
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 3/20
Table 1. Chond os e eum pu pu eum Isola es Collec ed o Phase 1.
No. Isola e Laccase ac i i y
a
Municipali y/locali y Collec ed by Da e o isola ion
1 AL1 1 Ala us, Mu onne a H. Va iamäki 2009–07–19
2 AL2 2 Ala us, Mu onne a H. Va iamäki 2009–07–19
3 AL3 2 Ala us, Mu onne a H. Va iamäki 2009–07–19
4 KY1 1 Van aa, Kylmäoja H. Va iamäki 2009–08–07
5 KY2 1 Van aa, Kylmäoja H. Va iamäki 2009–08–07
6 KY3 3 Van aa, Kylmäoja H. Va iamäki 2009–08–07
7 KY4 1 Van aa, Kylmäoja H. Va iamäki 2009–08–07
8 KY5 3 Van aa, Kylmäoja H. Va iamäki 2009–08–07
9 KY6 2 Van aa, Kylmäoja H. Va iamäki 2009–08–07
10 KY7 2 Van aa, Kylmäoja H. Va iamäki 2009–08–17
11 KY8 2 Van aa, Kylmäoja H. Va iamäki 2009–08–17
12 KY9 3 Van aa, Kylmäoja H. Va iamäki 2009–08–17
13 KY10 2 Van aa, Kylmäoja H. Va iamäki 2009–08–17
14 KY11 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
15 KY12 1 Van aa, Kylmäoja H. Va iamäki 2009–09–02
16 KY13 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
17 KY14 1 Van aa, Kylmäoja H. Va iamäki 2009–09–02
18 KY15 2 Van aa, Kylmäoja H. Va iamäki 2009–09–02
19 KY16 1 Van aa, Kylmäoja H. Va iamäki 2009–09–02
20 KY17 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
21 KY18 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
22 KY19 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
23 KY20 2 Van aa, Kylmäoja H. Va iamäki 2009–09–02
24 KY21 1 Van aa, Kylmäoja H. Va iamäki 2009–09–02
25 KY22 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
26 KY23 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
27 KY24 2 Van aa, Kylmäoja H. Va iamäki 2009–09–02
28 KY25 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
29 KY26 3 Van aa, Kylmäoja H. Va iamäki 2009–09–02
30 KY27 2 Van aa, Kylmäoja H. Va iamäki 2009–09–14
31 KY28 3 Van aa, Kylmäoja H. Va iamäki 2009–09–14
32 JU1 2 Juupajoki, Hyy iälä A. Uo ila 2009–08–26
33 OR1 3 O i esi A. Uo ila 2009–09–14
34 HA1 2 Män sälä, Ko is onkulma L. Hambe g 2009–10–06
35 HA2 1 Män sälä, Ko is onkulma L. Hambe g 2009–10–06
36 HA3 2 Män sälä, Ko is onkulma L. Hambe g 2009–10–06
37 OH1 1 Män sälä, Ki is önkulma L. Hambe g 2009–10–06
38 OH2 2 Män sälä, Ki is önkulma L. Hambe g 2009–10–06
39 JÄ1 2 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
40 JÄ2 3 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
41 JÄ3 2 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
42 JÄ4 1 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
43 JÄ5 1 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
44 JÄ6 1 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
45 JÄ7 1 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
46 JÄ8 1 Jä enpää, Paa onpolku L. Hambe g 2009–10–06
47 PI1 3 Män sälä, Pi jola L. Hambe g 2009–10–11
(Con inued)
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 4/20
pe isola e we e incuba ed a 25°C o 4 d. Enzyma ic ac i i y was isually es ima ed as he
s eng h o he colo eac ion on a pla e. The wo isola es wi h he highes laccase ac i i y we e
selec ed and pai ed (see below) wi h each o he (see Tables 1and 2, Phase 1). Laccase ac i i ies
o he 24 p ogeny isola es we e also in es iga ed a e 4 d on he ABTS pla es as desc ibed
abo e, and he bes p oduce was named E+
1
.
Pai ings
Fi s , he bes wo C.pu pu eum s ains in e ms o laccase ac i i y (see Table 1) we e pai ed
(Table 2, Phase 1). The isola es o be pai ed we e cul u ed on PDA Pe i pla es, one isola e pe
pla e, o ca. 3–4 weeks un il hey o med ui ing bodies. A ui ing body was cu om a pla e
wi h a s e ilized scalpel and ans e ed on o a new PDA pla e lid. One d op o s e ilized wa e
was pipe ed jus nex o he ui ing body o ac i a e spo e elease. One day la e , when spo es
had s a ed o ge mina e on a PDA Pe i pla e, 24 spo es pe isola e JÄ2 and 20 spo es pe iso-
la e PI7 we e success ully isola ed om he pla es wi h a modi ied and s e ilized Pas eu pipe e
and ans e ed o new PDA pla es, one spo e pe pla e. A e he occu ence o hyphae hei
mo phology was in es iga ed wi h a mic oscope o e i y ha single spo e isola ions we e suc-
cess ul and ha he hypha was homoka yo ic (no isible clump connec ions). A single hyphal
ip was u he isola ed om each pla e o e i y a homoka yo ic s a e. These isola es we e
Table 1. (Con inued)
No. Isola e Laccase ac i i y
a
Municipali y/locali y Collec ed by Da e o isola ion
48 PI2 2 Män sälä, Pi jola L. Hambe g 2009–10–11
49 PI3 1 Män sälä, Pi jola L. Hambe g 2009–10–11
50 PI4 3 Män sälä, Pi jola L. Hambe g 2009–10–11
51 PI5 1 Män sälä, Pi jola L. Hambe g 2009–10–11
52 PI6 1 Män sälä, Pi jola L. Hambe g 2009–10–11
53 PI7 3 Män sälä, Pi jola L. Hambe g 2009–10–11
54 PI8 2 Män sälä, Pi jola L. Hambe g 2009–10–11
55 PI9 2 Män sälä, Pi jola L. Hambe g 2009–10–11
56 PI10 1 Män sälä, Pi jola L. Hambe g 2009–10–11
57 PI11 3 Män sälä, Pi jola L. Hambe g 2009–10–11
58 RU1 3 Van aa, Ruskeasan a L. Hambe g 2009–10–19
59 RU2 3 Van aa, Ruskeasan a L. Hambe g 2009–10–19
60 RU3 2 Van aa, Ruskeasan a L. Hambe g 2009–10–19
61 RU4 3 Van aa, Ruskeasan a L. Hambe g 2009–10–19
62 RU5 1 Van aa, Ruskeasan a L. Hambe g 2009–10–19
63 RU6 2 Van aa, Ruskeasan a L. Hambe g 2009–10–19
64 RU7 1 Van aa, Ruskeasan a L. Hambe g 2009–10–19
65 RU8 2 Van aa, Ruskeasan a L. Hambe g 2009–10–19
66 RU9 1 Van aa, Ruskeasan a L. Hambe g 2009–10–19
67 RU10 2 Van aa, Ruskeasan a L. Hambe g 2009–10–19
68 RU11 2 Van aa, Ruskeasan a L. Hambe g 2009–10–19
69 RU12 1 Van aa, Ruskeasan a L. Hambe g 2009–10–19
All C.pu pu eum s ains we e isola ed om ui ing bodies on bi ch s umps (Be ula pendula and B.pubescens) in Finland and we e he e oka yo ic. The
wo bes s ains in he laccase ac i i y es a e in bold.
a
0: No colo eac ion, i.e., low laccase ac i i y; 1: sligh colo eac ion; 2: in e media e colo eac ion; 3: s ong colo eac ion, i.e., high laccase ac i i y on
Pe i pla es.
doi:10.1371/jou nal.pone.0117381. 001
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 5/20

Table 2. B eeding P ocess o he S udy.
Phase 1 Phase 2 Phase 3
P ogeny Pa en s ain/ spo e
numbe
Laccase
ac i i y
a
P ogeny Pa en s ain/ spo e
numbe
p
b
P ogeny Pa en s ain/ spo e
numbe
p
b
R2
1
JÄ2/2 × PI7/2 3 R1
2
3.11/1 × HY4/1 0.312 /
0.213
R1
3
V2
2
/1 × E+
1
/1 0.474 /
0.332
R3
1
JÄ2/3 × PI7/3 3 R2
2
3.11/2 × HY4/2 0.282 /
0.191
R2
3
V1
2
/2 × E+
1
/2 0.376 /
0.529
R4
1
JÄ2/4 × PI7/4 3 R3
2
3.11/3 × HY4/3 0.374 /
0.262
R3
3
V1
2
/3 × E+
1
/3 0.323 /
0.214
R5
1
JÄ2/5 × PI7/5 3 R4
2
3.11/4 × HY4/4 0.204 /
0.134
R4
3
V1
2
/4 × E+
1
/4 0.799 /
0.611
R6
1
JÄ2/6 × PI7/6 3 R5
2
3.11/5 × HY4/5 0.185 /
0.120
R5
3
V1
2
/5 × E+
1
/5 0.304 /
0.199
R7
1
=E
+
1
JÄ2/7 × PI7/7 3 R6
2
3.11/6 × P4/1 0.823 /
0.990
R6
3
V1
2
/6 × E+
1
/6 0.272 /
0.175
R8
1
JÄ2/8 × PI7/8 2 R7
2
3.11/7 × P4/2 0.771 /
0.600
R7
3
V2
2
/1 × E+
1
/7 0.817 /
0.627
R9
1
JÄ2/9 × PI7/9 2 R8
2
3.11/8 × P4/3 0.299 /
0.204
R8
3
V2
2
/2 × E+
1
/8 0.959 /
0.759
R10
1
JÄ2/10 × PI7/10 3 R9
2
3.11/9 × P4/4 0.425 /
0.302
R9
3
V2
2
/3 × E+
1
/9 0.153 /
0.092
R11
1
JÄ2/11 × PI7/11 2 R10
2
3.11/1 × P4/5 0.114 /
0.070
R10
3
V2
2
/4 × E+
1
/10 0.596 /
0.433
R12
1
JÄ2/12 × PI7/12 3 R11
2
HY4/6 × P4/6 0.327 /
0.225
R11
3
V2
2
/5 × E+
1
/11 0.614 /
0.803
R14
1
JÄ2/14 × PI7/14 3 R12
2
=
V1
2
HY4/7 × P4/7 0.109 /
0.067
R12
3
V2
2
/6 × E+
1
/12 0.198 /
0.302
R15
1
JÄ2/15 × PI7/15 1 R13
2
HY4/8 × P4/8 0.813 /
0.637
R16
1
JÄ2/16 × PI7/16 2 R14
2
HY4/9 × P4/9 0.361 /
0.251
R17
1
JÄ2/17 × PI7/17 3 R15
2
=
V2
2
HY4/10 × P4/10 0.071 /
0.042
R18
1
JÄ2/18 × PI7/18 1
R19
1
JÄ2/19 × PI7/19 3
R20
1
JÄ2/20 × PI7/20 3
R21
1
JÄ2/21 × PI7/21 3
R22
1
JÄ2/22 × PI7/1 3
R24
1
JÄ2/24 × PI7/3 1
R25
1
JÄ2/25 × PI7/4 3
R26
1
JÄ2/26 × PI7/5 2
R27
1
JÄ2/27 × PI7/7 3
B eeding phase, C.pu pu eum s ain and i s spo e numbe in each b eeding phase a e p esen ed. The bes p ogenies in e ms o laccase ac i i y ( he
s onges colo eac ion, Phase 1) o in e ms o e ficacy in sp ou con ol in he field (Phases 2 and 3) h ee mon hs a e C.pu pu eum applica ion a e in
bold (see Fig. 2). Du ing Phase 2 he bes pa en s ains based on field expe imen s we e b ed and in Phase 3 he bes p ogenies om Phases 1 and 2
we e b ed. No e: he subsc ip o a p ogeny indica es he b eeding phase.
a
0: No colo eac ion, i.e., low laccase ac i i y; 1: sligh colo eac ion; 2: in e media e colo eac ion; 3: s ong colo eac ion, i.e., high laccase ac i i y on
a Pe i pla e.
b
S a is ical di e ence in he numbe o s ump sp ou s be ween he con ol (cu ing only) and he C.pu pu eum ea men , and he liquid con ol (inoculum
medium sp ead wi hou C.pu pu eum) and he C.pu pu eum ea men , espec i ely. P- alues indica ing s a is ically significan di e ences a e based on
gene alized linea mixed models. P- alues <0.05 a e in bold and hose be ween 0.05 and 0.10 ha e been unde lined.
doi:10.1371/jou nal.pone.0117381. 002
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 6/20
pai ed acco ding o Table 2 (Phase 1), i.e., wo spo es om di e en isola es we e placed on a
single PDA Pe i pla e o allow hem o o m he e oka yo ic mycelium. A e one week, an in-
e ac ion zone be ween he isola es de eloped be ween he inocula, om which hyphae we e
ans e ed o a new PDA Pe i pla e. Success ul pai ing was e i ied mic oscopically by con-
i ming clump connec ions. A single he e oka yo ic hyphal ip was u he ans e ed o a new
PDA pla e o u he use.
The h ee mos e icien na u al s ains es ed by Va iamäki e al. [13], isola es HY4, P4 and
3.11, we e o iginally collec ed om bi ches (Be ula pendula and B.pubescens) in Juupajoki in
2003, Van aa in 2004 and Helsinki in 2001, espec i ely, and used o he second se o pai ings.
Al oge he 10 spo es pe isola e HY4 and P4, and 9 spo es pe isola e 3.11 we e success ully iso-
la ed om he pla es and pai ed as desc ibed abo e (see Table 2, Phase 2).
Thi d, he bes p ogenies om Phases 1 and 2 we e pai ed acco ding o Table 2 (Phase 3), as
desc ibed abo e.
Inoculum o ield expe imen s
The inoculum medium o he ield expe imen s was p epa ed as ollows: 24 g po a o dex ose
b o h (Bec on, Dickinson and Company) and 20 g Sipe na 22S (E onik Degussa) pe 1000 ml
deionized wa e was au ocla ed in an Aga ma ik machine a 121°C o 15 min. E lenmaye
lasks, 250 ml in olume we e also au ocla ed a 121°C o 15 min. Al oge he , 150 ml cooled
au ocla ed inoculum medium was added o he lasks and C.pu pu eum hyphae om one
PDA cellophane pla e (pe isola e, Phase 2) was ans e ed o he lask wi h a s e ilized scalpel.
This inocula ed medium was incuba ed in he da k a 18°C o 7–10 d on a o a o shake (100
pm). The inoculum was homogenized by Ul a Tu ax appa a us o 1.5 min and dilu ed 1:10
wi h ap wa e be o e ea men s in he ield.
Fo Phases 3 and 4, he ungal inoculum was p epa ed as in Phase 2 excep ha he weigh
o hyphae added o he E lenmaye bo les was equal (0.120 ± 0.024 g and 0.167 ± 0.018 g,
mean ± SD, espec i ely) in each ea men (i.e., o each ungal isola e used in he expe imen ).
All s ains we e cul i a ed o 10 d in a shake a 20°C.
Expe imen s in he ield
The e icacy o all p ogenies o Phases 2, 3 and 4 was in es iga ed in ield expe imen s. The i s
expe imen (see Table 2, Phase 2) was es ablished on 9 and 10 June 2009 unde elec ic powe
lines in Po oo, Hin haa a, sou he n Finland. Bo h days we e cloudy wi h a empe a u e o ca.
15°C. Howe e , 9 June was pa ly d izzly. The si e included plen y o na u ally g owing bi ches
(Be ula pendula and B.pubescens) wi h a basal diame e (a ca. 15 cm abo e soil su ace) o 2.2
± 1.4 cm (mean ± SD). The e icacy o 15 di e en p ogenies and hei pa en al s ains HY4,
3.11 and P4 we e es ed in his ield expe imen (see Table 2). Fu he mo e, sample plo s o
con ols (cu ing only) and liquid con ols (p ocedu al con ols: inoculum medium wi hou
C.pu pu eum) we e also es ablished. Al oge he , 80 ci cula sample plo s, ou pe isola e o
con ol ea men , we e es ablished andomly on he si e. Each sample plo included ca. 20
bi ch s umps. Thus, al oge he 1583 bi ch s umps we e in es iga ed in his expe imen (Be ula
bendula 49%, B.pubescens 49% and un ecognized bi ches 2%— he p opo ion o ee species
was in es iga ed h ee mon hs a e he ea men s and he e o e some we e al eady dead). In
he C.pu pu eum sample plo s, ungal inoculum was sp ead on s umps immedia ely a e cu -
ing. T ea men s in he liquid con ol sample plo s we e simila , bu only inoculum medium
wi hou any mycelium was sp ayed on he s umps. Viabili y o he ungal inoculum was con-
i med be o e and a e ield applica ions by squi ing inoculum o PDA Pe i pla es. All o he
C.pu pu eum isola es we e iable be o e and a e he expe imen .
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 7/20
Th ee and a hal mon hs la e , a e one g owing season, in Sep embe 2009, he numbe o
li ing sp ou s pe bi ch s ump and basal diame e s o s umps (mm) we e measu ed. Based on
he numbe o sp ou s, he wo bes C.pu pu eum p ogenies we e chosen o u he in es iga-
ion. The wo bes ones had he lowes numbe o s ump sp ou s pe s ump and he s a is ical
di e ence was he highes (al hough usually no signi ican ) when compa ed o he con ol
s umps. These p ogenies we e named V1
2
and V2
2
(see Table 2).
In he second ield expe imen , he e icacy o he p ogenies and hei pa en al s ains in
Phase 3 (Table 2) we e es ed in he ield. The e icacy o di e en C.pu pu eum s ains was
es ed in h ee di e en egene a ion a eas o sp uce in Tu enki, sou he n Finland, including
lo s o na u ally g own bi ches (Be ula pendula and B.pubescens) 1.0 ± 0.4 cm (mean ± SD) in
basal diame e (a ca. 15 cm abo e soil su ace). A each si e, he e icacy o 12 p ogenies and
he pa en al s ains om di e en phases, i.e., HY4, P4, 3.11 and V1
2
,V2
2
,E+
1
, was es ed.
Fu he mo e, sample plo s o con ols (cu ing only) and liquid con ols (inoculum medium
sp ead wi hou C.pu pu eum) we e es ablished. One sample plo pe ea men was andomly
placed wi hin each si e. Each sample plo included ca. 30 bi ch s umps. Thus, 60 sample plo s
including 1796 bi ch s umps we e included in he s udy (Be ula pendula 46%, B.pubescens
53%, and un ecognized bi ches 1%). Sample plo s we e ea ed on 15 and 16 June 2010. 15
June was pa ly cloudy, sunny and ainy wi h 15°C whe eas 16 June was sunny bu windy wi h
17°C. All C.pu pu eum s ains we e iable be o e and a e he expe imen .
Th ee mon hs la e , in Sep embe 2010, he numbe o s ump sp ou s pe bi ch s ump and
basal diame e s o s umps (mm) we e measu ed, and he h ee bes C.pu pu eum p ogenies
we e chosen o u he in es iga ion. The h ee bes s ains had he lowes numbe s o s ump
sp ou s and s a is ical di e ences we e he highes (al hough usually no signi ican ) when
compa ed o he con ol s umps.
In he hi d ield expe imen we es ed he e icacy o he bes C.pu pu eum s ains om
Phases 2 and 3 o h ee g owing seasons. The bes s ains om Phase 3, R3
3
(p ogeny s ain
om he pai ing be ween V1
2
and E+
1
), R5
3
(V1
2
×E+
1
) and R9
3
(V2
2
×E+
1
), oge he wi h
s ains V1
2
and V2
2
om Phase 2 we e included in he expe imen . Fu he mo e, HY4, he
bes pa en al s ain o iginally collec ed om he ield (based on he s udy by Va iamäki e al.
[13]), and a liquid con ol (inoculum medium wi hou C.pu pu eum) we e included. Eigh e-
gene a ion a eas o sp uce wi h a high equency o bi ches (Be ula pendula and B.pubescens)
we e chosen o he s udy (Table 3). Fou o he si es we e loca ed in Lapinjä i, and ou in
Tu enki, bo h in sou he n Finland. A each si e, ci cula sample plo s including en bi ch sap-
lings pe ea men we e es ablished. The o de o he sample plo s was andomized wi hin
each si e. Al oge he 560 bi ch saplings, 80 saplings pe ea men , we e included in he s udy
(Be ula pendula 12%, B.pubescens 75% and un ecognized bi ches 13%). The mean basal diam-
e e o bi ches was 1.1 ± 0.4 cm (mean ± SD, ca. 15 cm abo e soil su ace). The expe imen was
es ablished on 10 o 13 May 2011. The wea he was sunny wi h a empe a u e o 14–21°C.
The mo ali y o s umps, he numbe and maximum heigh o s ump sp ou s in li ing
s umps and he occu ence o ui ing bodies we e in es iga ed one, wo and h ee g owing sea-
sons (2011, 2012 and 2013) a e he ea men s. Fu he mo e, he basal a ea o s umps was
measu ed (mm), and he numbe o o he saplings (cu o uncu ), and e en ion ees (i.e., ma-
u e ees le on si es, diame e a b eas heigh 5 cm, m
3
ha
-1
) a ound an in es iga ed s ump
we e measu ed as hese ha e an e ec on sp ou ing [9]. The numbe o o he s umps and sap-
lings a ound an in es iga ed sapling was measu ed wi hin a ci cula subsample plo 0.5 m in a-
dius whe eas he diame e s o e en ion ees (cm) a b eas heigh we e measu ed wi hin a
ci cula sample plo 10 m in adius. T ee olume was calcula ed using he models by Laasase-
naho [29]. The occu ence o moose b owsing was eco ded pe in es iga ed s ump o ake his
in o accoun as i has an e ec on he heigh o s ump sp ou s.
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 8/20
S a is ical analyses
Gene alized linea mixed models (GLMMs) we e used o in es iga e di e ences be ween he
ea men s. In Phases 2 and 3 he e ec s o di e en ea men s (con ol, liquid con ol and di -
e en C.pu pu eum s ains) on he numbe o s ump sp ou s we e in es iga ed using a Poisson
model wi h log link unc ion in package lme4 in he s a is ical p og am R [30,31]. Thus, he
numbe o s ump sp ou s pe s ump h ee mon hs a e he ea men s was he esponse a i-
able and ea men s (as a ac o ) and he basal diame e o a s ump we e explana o y a iables
in he models. In Phase 2, he sample plo was used as a andom ac o o ake in o accoun
pseudo eplica ion, i.e., he ac ha 20 s umps wi hin each sample plo may be—due o en i on-
men al condi ions—mo e simila han andomly chosen saplings. In Phase 3, si e (logging uni )
and sample plo we e ea ed as nes ed andom ac o s o ake in o accoun he ac ha se e al
s umps wi hin he same si e and sample plo we e in es iga ed. Bo h in Phase 2 and 3, he con-
ol ea men (cu ing only) and he liquid con ol (inoculum medium wi hou a ungus) we e
compa ed o he o he ea men s. The bes ungal s ains we e chosen o u he in es iga ion
based on he bigges di e ences in he numbe o s ump sp ou s pe s ump be ween he con ol
ea men s and he ungal ea men s (lowe in he ungal ea men ) and he lowes p- alues.
The GLMMs we e also used o analyze da a ela ing o he inal ield expe imen , which
las ed o h ee g owing seasons om 2011 o 2013 (Phase 4). We es ima ed he models sepa-
a ely o each yea . The package lme4 in R was used o in es iga e he e ec s o con ols (inoc-
ulum wi hou C.pu pu eum sp ead on cu s umps) and di e en C.pu pu eum s ains on he
in es iga ed s umps. E ec s on he mo ali y o in es iga ed s umps and he p obabili y o oc-
cu ence o ui ing bodies on s umps we e analyzed using a binomial model wi h logi link
unc ion [30,31]. E ec s on he numbe o s ump sp ou s pe s ump we e in es iga ed using a
Poisson model wi h log link unc ion. The e ec o di e en ea men s on he maximum
heigh o s ump sp ou s pe s ump was in es iga ed using unc ion lme in he nlme package in
R[31,32] assuming a no mal dis ibu ion. All s umps we e included in he mo ali y models
whe eas only li ing s umps we e included when di e ences be ween he numbe and maxi-
mum heigh o s ump sp ou s we e in es iga ed. Explana o y a iables in he models we e
1) ea men (as a ac o wi h se en le els; howe e , he liquid con ol was excluded om he
ui ing body model), 2) he basal diame e o a s ump (mm), 3) he numbe o o he s umps
Table 3. Desc ip ion o he S udy Si es Included in he Field Expe imen in Phase 4.
Si e Si e
ype
a
Topog aphy Soil Clea -cu ing
(yea )
Soil
cul i a ion
Sp uce saplings plan ed
(yea )
Lapinjä i 67 MT Pa ly
sloping
Sand mo aine 2003 Mounding 2004
Lapinjä i 58 MT E en G a el mo aine/s ony 1998 No 1999
Lapinjä i
245
OMT E en Clay/g a el mo aine 1999 Disc enching 2000
Lapinjä i
349
OMT E en Clay/g a el mo aine/pea -
co e ed
2006
b
Disc enching 2007
Tu enki 93 OMT E en G a el mo aine 2002 Mounding 2003
Tu enki 116 OMT E en G a el mo aine 2004 No 2004
Tu enki 220 OMT E en G a el mo aine 2003 Mounding 2004/2007
Tu enki 241.2 OMT E en Pea 2003 Mounding 2004–2005
a
MT = My illus ype o es , OMT = Oxalis—My illus ype o es .
b
Wind all sp uces on his si e.
doi:10.1371/jou nal.pone.0117381. 003
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 9/20
in he con ol in e e y in es iga ed yea . Howe e , in e ms o he maximum heigh o sp ou s
(in li ing s umps), ha dly any di e ences be ween he ea men s we e ound (see also [13]).
Ou esul s e ealed ha an inc easing olume o ees a ound an in es iga ed s ump in-
c eased s ump mo ali y. This is in acco dance wi h ea lie indings as shading o neighbo ing
ees has been shown o ha e a p o ound e ec on shoo g ow h due o a g ea e p opo ion o
shoo buds dying unde hea y shading [37,38]. Fu he mo e, ou esul s showed ha an in-
c ease in he numbe o su ounding saplings dec eased he numbe and maximum heigh o
s ump sp ou s (see also [39]). On he o he hand, high mo ali y wi h C.pu pu eum inocula ed
s umps (especially wi h R5
3
) may p o ide mo e g owing space o hose s umps ha a e s ill
li ing. This may be he eason why signi ican di e ences be ween he ea men s we e no ob-
se ed in s ump numbe s and heigh .
All C.pu pu eum s ains in es iga ed we e o iginally collec ed om bi ch s umps in sou h-
e n and middle Finland (see Me hods sec ion and Table 1). Howe e , in es iga ions om Fin-
land, Canada and New Zealand showed ha C.pu pu eum s ains a e gene ically di e se, and
a e no associa ed wi h a speci ic hos species o ecological egion [11,17,18]. Thus, i is possible
ha he mos e icien s ain, R5
3
, is e icien also agains he sp ou ing o o he ee species
and in o he geog aphical a eas.
Table 6. Di e ences in he Heigh o S ump Sp ou s o Li ing Bi ches be ween he C.pu pu eum S ain R5
3
and O he T ea men s.
Explana o y Response a iables
a iables S ump sp ou heigh in 2011 S ump sp ou heigh in 2012 S ump sp ou heigh in 2013
n= 454 n= 317 n= 279
Coe . ±SE pCoe . ±SE PCoe . ±SE p
In e cep 46.065 ±4.588 <0.001 75.190 ±10.645 <0.001 112.429 ±16.939 <0.001
T ea men
-LC
a
4.330 ±3.557 0.230 3.155 ±7.444 0.674 5.965 ±11.204 0.598
-HY4
b
-1.704 ±3.620 0.640 2.906 ±7.900 0.715 -5.446 ±11.974 0.652
-R12
2
=V1
2c
0.717 ±3.668 0.846 -0.057 ±7.785 0.994 1.679 ±11.857 0.888
-R15
2
=V2
2c
0.766 ±3.580 0.832 -9.614 ±7.727 0.221 -9.347 ±11.767 0.432
-R3
3d
-2.646 ±3.692 0.478 -8.554 ±8.040 0.294 -21.540 ±12.295 0.088
-R9
3d
2.173 ±3.644 0.554 -7.848 ±7.736 0.317 -15.882 ±11.896 0.190
S ump basal diame e (mm) 1.466 ±0.205 <0.001 2.403 ±0.454 <0.001 3.598 ±0.725 <0.001
Saplings a ound -0.180 ±0.180 0.316 -0.718 ±0.396 0.071 -1.692 ±0.611 0.006
T ee olume (m
3
ha
-1
) -0.163 ±0.071 0.027 -0.153 ±0.156 0.335 -0.553 ±0.229 0.021
B owsing -6.459 ±1.649 <0.001 1.263 ±3.413 0.712 3.365 ±6.634 0.613
The e ec o di e en ea men s (C.pu pu eum s ain o liquid con ol (LC)), he basal a ea o in es iga ed s umps, he numbe o saplings and he
olume o ees a ound an in es iga ed sapling, and b owsing on he maximum heigh o s ump sp ou s o bi ch (Be ula pendula and B.pubescens)in
eigh egene a ion a eas o sp uce (Picea abies) h ee mon hs (in 2011), one yea (in 2012) and wo yea s (in 2013) a e he ea men s (linea mixed
model esul s). Li ing s umps ha e been included in he models. S a is ically significan p- alues (p<0.05) o he model coe ficien s a e in bold and
indica i e esul s ha e been unde lined (0.05 p0.10). The sign o a coe ficien indica es whe he an explana o y a iable has an inc easing (+) o
dec easing (-) e ec on he maximum heigh o s ump sp ou s. See Figs. 3c and 4c. No e: he subsc ip o a C.pu pu eum p ogeny ela es o he b eeding
phase (see Table 2).
a
Liquid con ol i.e., cu s umps we e sp ead wi h inoculum medium wi hou C.pu pu eum.
b
The bes pa en s ain based on an ea lie s udy [13].
c
The bes p ogenies om Phase 2.
d
The bes p ogenies om Phase 3.
doi:10.1371/jou nal.pone.0117381. 006
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 16 / 20

Fu he mo e, ea lie s udies ha e indica ed ha a single geno ype can be used as inoculum
wi hou he isk o in oducing genes ha di e signi ican ly om local popula ions [11,17]. The
bes s ain R5
3
was de eloped by adi ional b eeding ( ia consequen pai ings o selec ed myce-
lia), i.e., simila ly like any o he C.pu pu eum s ain in he na u e. In ha sense his s ain does
no di e om i s na u al coun e pa s. Mo eo e , as he ungus has no asexual s age, he gene ic
combina ion o a single excep ionally e icien biocon ol ungus b eaks up in meiosis be o e he
spo es a e eleased. Thus, he same C.pu pu eum geno ype applied on cu s umps canno sp ead
u he in o es ecosys ems, and hus he e is no isk o explosion o he biocon ol s ain.
Classical b eeding expe imen s wi h biocon olling ungi a e sca ce. Howe e , in addi ion o
ou s, Wall e al. [10] compa ed he e icacy o C.pu pu eum monoka yons and hei dika yon
p ogenies in causing wood issue mo ali y, bu could no ind any di e ences. Howe e , hei
expe imen was no es ed unde ield condi ions. Ano he biocon ol ungus, Phlebiopsis
gigan ea, was b ed o imp o e he e icacy agains He e obasidion oo o in o es s [27]. Re-
sul s o ha s udy indica ed ha p ogeny s ains had be e p ope ies agains H.pa ipo um
han he pa en al s ains. Fu he mo e, classical pai ings we e success ully used o imp o e
empe a u e ole ance o e men ing yeas s using a backc oss app oach [25]. Thus, hese s ud-
ies (including he p esen one) show ha imp o ing by b eeding may help in de eloping mo e
e icien ungal s ains o di e en pu poses.
Table 7. Di e ences in he Occu ence o F ui ing Bodies in Bi ch S umps be ween he C.pu pu eum S ain R5
3
and O he T ea men s.
Explana o y Response a iables
a iables Occu ence o ui ing bodies
in 2011
Occu ence o ui ing bodies
in 2012
Occu ence o ui ing
bodies in 2013
n= 480 n= 479 n= 480
Coe . ±SE pCoe . ±SE pCoe . ±SE p
In e cep -2.339 ±0.752 0.002 -2.382 ±0.570 <0.001 --
T ea men
-HY4
a
-0.970 ±0.496 0.051 0.248 ±0.350 0.480 - -
-R12
2
=V1
2b
-0.959 ±0.511 0.061 0.022 ±0.350 0.950 - -
-R15
2
=V2
2b
-0.698 ±0.490 0.154 0.759 ±0.346 0.028 --
-R3
3c
-0.678 ±0.489 0.166 0.031 ±0.355 0.931 - -
-R9
3c
-0.584 ±0.481 0.225 0.209 ±0.349 0.549 - -
S ump basal diame e (mm) 0.258 ±0.040 <0.001 0.145 ±0.032 <0.001 --
Saplings a ound -0.014 ±0.031 0.640 0.007 ±0.023 0.752 - -
T ee olume (m
3
ha
-1
) 0.012 ±0.010 0.249 0.001 ±0.007 0.910 - -
The e ec o di e en ea men s (C.pu pu eum s ain), he basal a ea o in es iga ed s umps, he numbe o saplings and he olume o ees a ound an
in es iga ed sapling on he p obabili y o occu ence o ui ing bodies on bi ch (Be ula pendula and B.pubescens) s umps in eigh egene a ion a eas o
sp uce (Picea abies) h ee mon hs (in 2011) and one yea (in 2012) a e he ea men s (gene alized linea mixed model esul s). The model o yea 2013
was no es ima ed because he occu ence o ui ing bodies was oo low (4%). All s umps excep hose in he liquid con ol ha e been included in he
models. S a is ically significan p- alues (p<0.05) o he model coe ficien s a e in bold and indica i e esul s ha e been unde lined (0.05 p0.10).
The sign o a coe ficien indica es whe he an explana o y a iable has an inc easing (+) o dec easing (-) e ec on he occu ence o ui ing bodies. See
Fig. 4d. No e: he subsc ip o a C.pu pu eum p ogeny ela es o he b eeding phase (see Table 2).
a
The bes pa en s ain based on an ea lie s udy [13].
b
The bes p ogenies om Phase 2.
c
The bes p ogenies om Phase 3.
doi:10.1371/jou nal.pone.0117381. 007
B eeding o C. pu pu eum o Sp ou Con ol
PLOS ONE | DOI:10.1371/jou nal.pone.0117381 Feb ua y 12, 2015 17 / 20
Howe e , b eeding s udies wi h ield expe imen s may be ime consuming as seen in ou
s udy. Time o ollow-up is usually limi ed, which o ces us o choose he bes s ains o sub-
sequen s eps a e a sho ime pe iod. Fo example, in ou s udy, we had o choose p ogeny
isola es o he nex pai ings a e he i s g owing season (bo h in 2009 and 2010) based only
on he numbe o s ump sp ou s in di e en C.pu pu eum ea men s (a low mo ali y did no
allow o mo ali y analyses). Mo eo e , as seen also in he p esen s udy, he e icacy o C.pu -
pu eum is no igh ly associa ed wi h he pa en s, because each pa en can p oduce ela i ely
i ulen as well as a i ulen s ains [10]. This is no su p ising as Wall e al. [10] ha e s a ed
ha he inhe i ance o e icacy is p obably mul i ac o ial and complex and may be subjec o
di e en modi ying ac o s unde ield and labo a o y condi ions. Thus, inal e icacy should
always be in es iga ed in ield expe imen s. This iew was suppo ed by ou esul s as he bes
s ain in e ms o laccase ac i i y in he labo a o y, E+
1
, was no e icien in sp ou con ol (see
Fig. 2b) al hough some o i s p ogenies (R5
3
and R3
3
) in he end showed he bes abili y o p e-
en sp ou ing o b oad-lea ed ees in he ield.
Conclusions
We showed ha adi ional b eeding can inc ease he e icacy o C.pu pu eum in he sp ou
con ol o bi ch. Mo ali y o he ea ed bi ch s umps was highes when he p ogeny s ain R5
3
was used o con ol sp ou ing. This esul ed in 78% mo ali y in bi ch s umps 1 cm in diame e .
The e icacy o s ain R5
3
was signi ican ly highe han ha o he in es iga ed pa en al s ain
HY4 (56%). The e ec o sp ou con ol was especially p onounced when he olume o ees
and he numbe o su ounding saplings was high. Howe e , b eeding expe imen s ha aim a
inc easing he e icacy o sp ou con ol a e ime consuming because e icacy has o be in es i-
ga ed in long-las ing ield expe imen s.
Acknowledgmen s
We hank Fing id L d., UPM-Kymmene L d. and he Finnish Fo es Resea ch Ins i u e o p o-
iding o es si es, and An i Uo ila o p o iding C.pu pu eum s ains o he s udy. Ma kku
Ran ala, E kki Pii oinen, Kaa ina Pynnönen, Asko Ha ju, Ma ja-Leena San anen, Ri a Vanha-
nen, Pekka Hämäläinen and Juha Honkaniemi helped in he ield. Sonja Sa sila and Minna
Sinkkonen a e acknowledged o p epa ing pla es o he s udy. We also hank Ma ina B and -
be g o aluable commen s on he manusc ip . Anne Siika modi ied he igu es, and Johan
Ko ze checked he language.
Au ho Con ibu ions
Concei ed and designed he expe imen s: LH HV JH. Pe o med he expe imen s: LH HV JH.
Analyzed he da a: LH. Con ibu ed eagen s/ma e ials/analysis ools: LH HV JH. W o e he
pape : LH HV JH.
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