Breeding increases the efficacy of Chondrostereum purpureum in the sprout control of birch

RESEARCH ARTICLE Breeding Increases the Efficacy of Chondrostereum purpureum in the Sprout Control of Birch Leena Hamberg*, Henna Vartiamäki, Jarkko Hantula Finnish Forest Research Institute, Vantaa Research Unit, Vantaa, Finland *[email protected] Abstract We tested whether the pairing of selected isolates could be used to increase the efficiency of a decay fungus Chondrostereum purpureum (Pers. Ex Fr.) Pouzar to control hardwood sprouting in Finland. We paired C. purpureum strains efficient in sprout control or highly active in laccase production, and tested the efficacy of their progeny in spout control experiments. This procedure resulted in a strain with an efficacy superior to that of the parental strains. The mortality of birch (Betula pendula Roth. and B. pubescens Ehrh.) 1 cm in stump diameter was 78%, 56% and 9% for the best progeny, the best parental strain and the control, respectively. Mortality was only slightly higher for B. pendula than for B. pubescens but no significant differences were found between the number or maximum height of stump sprouts. Our results showed that cross breeding of this decay fungus is a good alternative in attempts to produce efficient biocontrol agents against hardwood sprouting. Introduction In Finland, sprouting of broad-leaved trees is a hindrance in spruce (Picea abies [L.] H. Karst.) and pine (Pinus sylvestris L.) regeneration areas, alongside roads and railways, under electric power lines and above gas pipe lines. In regeneration areas, broad-leaved species such as silver and downy birch (Betula pendula Roth. and B.pubescens Ehrh.), decrease the growth of more commercially valuable conifers, and therefore non-crop species are typically cleaned, preferably at an early stage when trees are about 1 m in height [1,2,3]. Next to roads and railways, broadleaved trees form a threat to the safety of traffic as they restrict visibility, cover traffic signs and tempt moose, and are therefore regularly removed. Electric power lines are kept open to ensure continuous electric transmission and gas pipe lines marked with visible signs are frequently cleared in order to avoid unintended excavations. Sprout control of broad-leaved trees costs more than 60 million euros annually in Finnish forest regeneration areas, along roads and railways, and at electric power and gas pipe lines (information gathered from UPM Forest Ltd., The Finnish Transport Agency, Fingrid Ltd. and Gasum Ltd.). Herbicides were previously successfully used in sprout control [4,5], but the use of chemicals is no longer recommended in Finnish groundwater areas due to their harmful effects [6,7]. PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 1/20 a11111 OPEN ACCESS Citation: Hamberg L, Vartiamäki H, Hantula J (2015) Breeding Increases the Efficacy of Chondrostereum purpureum in the Sprout Control of Birch. PLoS ONE 10(2): e0117381. doi:10.1371/journal.pone.0117381 Academic Editor: Petr Karlovsky, Georg-AugustUniversity Göttingen, GERMANY Received: October 3, 2014 Accepted: December 23, 2014 Published: February 12, 2015 Copyright: © 2015 Hamberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data sets and figures are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.f5s6r. Funding: The study was funded by the Finnish Funding Agency for Technology and Innovation (http://www.tekes.fi/en/, project number 265/31/2012), Verdera Ltd. (http://verdera.fi/en/front-page/), Metsäteho Ltd. (http://www.metsateho.fi/aboutus/ presentation), Fingrid Ltd. (http://www.fingrid.fi/en/ Pages/default.aspx), The Finnish Transport Agency (http://portal.liikennevirasto.fi/sivu/www/e/), UPMKymmene Ltd. (http://www.upm.com/en/Pages/ default.aspx), Marjatta and Eino Kolli Foundation (http://www.kollinsaatio.com/, project number 1041), Also, public opinion is strongly against their usage. On the other hand, mechanical cutting alone is not effective due to the vigorous sprouting of broad-leaved trees [8,9]. Therefore, new methods for sprout control are needed. One promising option is to utilize a natural pathogen of broad-leaved trees, the silver leaf fungus, Chondrostereum purpureum (Pers. ex Fr.) Pouzar [10,11], which has been shown to restrict the sprouting of several tree species [8,12,13,14]. Therefore it can be considered a ‘nature friendly’alternative to chemicals. C.purpureum is a basidiomycete commonly found on wounded broad-leaved wood in boreal and temperate vegetation zones in Europe [15,16,17]. The fungus is widespread in nature due to its efficient basidiospore production resulting in frequent new infections [15,18]. In nature, monokaryotic spores landing on wood first germinate, after which the hyphae from different spores hybridize and form dikaryotic mycelia which then colonize the host [18]. However, in man-made sprout control, dikaryotic fungal hyphae are directly spread onto freshly cut stumps [9,12,13]. The use of C.purpureum in sprout control is based on its ability to grow inside a stump and finally decay it. During the invasion, C.purpureum penetrates through starch granules and cell walls enzymatically, and induces the occlusion of tree vessels [15,19,20,21]. The resulting dehydration combined with fungal toxins may finally cause mortality of the host. White rot fungi, such as C.purpureum, are efficient in breaking down lignin [22,23]. In woody cell walls, lignin surrounds the cellulose which is the actual carbon and energy source for the fungus, and possibly the ultimate reason for lignin degradation [23,24]. Laccases are the main oxidative enzymes in this decay process in addition to lignin and manganese peroxidases [13,23]. The efficacy of C.purpureum in sprout control depends on the host tree species [5,8,9,13,14]. Also, an increase in the diameter of an inoculated stump has been shown to affect stump mortality [9,14]. Furthermore, considerable variation exists in the ability of different C.purpureum strains to prevent sprouting [4,5,13], but the possibility of increasing control efficiency by breeding has not been tested before. Breeding among sexually propagating fungi has previously been used in industrial applications such as the chemical industry (e.g. enzymes for bioethanol), and in wine production in order to increase the yield of cultivated organisms and economic benefit [25,26]. Moreover, the biocontrol ability of a saprophytic fungus Phlebiopsis gigantea (Fr.) Jül. against a root rot fungus Heterobasidion parviporum Niemelä & Korhonen was improved by traditional breeding [27]. Therefore, breeding based on the natural variation of C.purpureum can be expected to provide an efficient way to increase the ability of this fungus to control sprouting. The main aim of this study was to test whether breeding can be used to increase the efficacy of the decay fungus C.purpureum in preventing sprouting of small birch stumps (Betula pendula and B.pubescens). We hypothesized that pairing of strains efficient in (i) sprout control and (ii) laccase production could result in a superior combination of genes producing at least one progeny strain better than the parental strains. In addition, we tested whether the ability of C.purpureum to control sprouting differs between the birch species. Here we show that breeding can successfully be used to improve the biocontrol ability of C.purpureum but no significant differences between the two birch species investigated were found. Materials and Methods The study was composed of four phases (Fig. 1): 1) pairing of the best strains in terms of laccase activity and progeny testing in the laboratory, 2) pairing of the most efficient parental strains available [13] and progeny testing in the field, 3) pairing of the best progenies from Phases 1 and 2 and progeny testing in the field, and 4) final testing of the efficacy of the best progenies Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 2/20 the Finnish Cultural Foundation (http://www.skr.fi/en, project number 00081430) and the Finnish Forest Research Institute (http://www.metla.fi/index-en.html). LH received salary from the Finnish Forest Research Institute which obtained funding from the abovementioned sources. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Verdera Ltd. is interested in developing a commercial product including C. purpureum strain R53, Fingrid Ltd. and UPMKymmene Ltd. provided sites for the study, and Metsäteho Ltd. together with other funders (except Marjatta and Eino Kolli Foundation, the Finnish Cultural Foundation, and the Finnish Funding Agency for Technology and Innovation) are potential users of the commercial biocontrol product, and therefore the authors have cooperated with them in order to pay attention to critical points in sprout control. This does not alter the authors’adherence to PLOS ONE policies on sharing data and materials. from Phases 2 and 3. All data sets and figures are available from the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.f5s6r Ethics Statement Permission for the field experiments of Phases 2–4 was granted by land owners, Fingrid Ltd., UPM-Kymmene Ltd. and the Finnish Forest Research Institute. Endangered or protected species were not used in this study. Fungal strains of C.purpureum have been deposited in the culture collection of the Finnish Forest Research Institute. Laccase activity tests Laccase activity of the isolates was studied as it has been shown that laccase activity of C.purpureum isolates correlates with the efficacy of their sprout control [13]. In order to find efficient laccase producers, altogether 69 heterocaryotic C.purpureum isolates were collected from birch (Betula pendula and B.pubescens) stumps in July-October 2009. These isolates were collected from different parts of Finland, in Alavus (3 isolates), Vantaa (40), Hyytiälä (1), Juupajoki (2), Mäntsälä (16) and Järvenpää (8) (Table 1). We used ABTS (2,2’-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid]) plates whose color reaction reveals the laccase activity of C.purpureum (0 = no color reaction, i.e., low laccase activity to 3 = strong dark-green color reaction, i.e., high laccase activity, see [13]). The ABTS plates (modified from [28]) were prepared by mixing 1.0 g glucose (Acros Organics), 2.0 g KH 2 PO 4 (Merck), 0.5 g MgS0 4 ×7H 2 O (Fluka), 0.13 g CaCl 2 ×2H 2 O (Merck), 0.5 g (NH 4 ) 2 -tartrate (Alfa Aesar), 1.78 g dimethylsuccinic acid (Merck), 0.2 g yeast extract (Becton, Dickinson and Company) and 25 g agar (Becton, Dickinson and Company) with 1000 ml deionized water. All elements except agar were suspended in water and pH adjusted to 5.0 with 1.0 M NaOH. Agar was added and the substrate was autoclaved at 121°C for 15 min. Altogether 250 mg ABTS (Applichem) diluted with 99% ethanol was added to the medium when the temperature was 48–50°C. All C.purpureum isolates were grown on potato dextrose agar Petri plates (PDA: 24 g potato dextrose broth and 15 g agar with 1000 ml deionized water; Becton, Dickinson and Company), and a 6-mm diameter agar plug at the periphery of the grown mycelium was transferred to the middle of an ABTS plate using a sterilized Pasteur pipette and a scalpel. Two replicate plates Fig 1. The Breeding Process of the Study. doi:10.1371/journal.pone.0117381.g001 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 3/20 Table 1. Chondrostereum purpureum Isolates Collected for Phase 1. No. Isolate Laccase activity a Municipality/locality Collected by Date of isolation 1 AL1 1 Alavus, Murronneva H. Vartiamäki 2009–07–19 2 AL2 2 Alavus, Murronneva H. Vartiamäki 2009–07–19 3 AL3 2 Alavus, Murronneva H. Vartiamäki 2009–07–19 4 KY1 1 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 5 KY2 1 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 6 KY3 3 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 7 KY4 1 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 8 KY5 3 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 9 KY6 2 Vantaa, Kylmäoja H. Vartiamäki 2009–08–07 10 KY7 2 Vantaa, Kylmäoja H. Vartiamäki 2009–08–17 11 KY8 2 Vantaa, Kylmäoja H. Vartiamäki 2009–08–17 12 KY9 3 Vantaa, Kylmäoja H. Vartiamäki 2009–08–17 13 KY10 2 Vantaa, Kylmäoja H. Vartiamäki 2009–08–17 14 KY11 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 15 KY12 1 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 16 KY13 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 17 KY14 1 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 18 KY15 2 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 19 KY16 1 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 20 KY17 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 21 KY18 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 22 KY19 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 23 KY20 2 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 24 KY21 1 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 25 KY22 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 26 KY23 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 27 KY24 2 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 28 KY25 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 29 KY26 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–02 30 KY27 2 Vantaa, Kylmäoja H. Vartiamäki 2009–09–14 31 KY28 3 Vantaa, Kylmäoja H. Vartiamäki 2009–09–14 32 JU1 2 Juupajoki, Hyytiälä A. Uotila 2009–08–26 33 OR1 3 Orivesi A. Uotila 2009–09–14 34 HA1 2 Mäntsälä, Kortistonkulma L. Hamberg 2009–10–06 35 HA2 1 Mäntsälä, Kortistonkulma L. Hamberg 2009–10–06 36 HA3 2 Mäntsälä, Kortistonkulma L. Hamberg 2009–10–06 37 OH1 1 Mäntsälä, Kivistönkulma L. Hamberg 2009–10–06 38 OH2 2 Mäntsälä, Kivistönkulma L. Hamberg 2009–10–06 39 JÄ1 2 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 40 JÄ2 3 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 41 JÄ3 2 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 42 JÄ4 1 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 43 JÄ5 1 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 44 JÄ6 1 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 45 JÄ7 1 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 46 JÄ8 1 Järvenpää, Paavonpolku L. Hamberg 2009–10–06 47 PI1 3 Mäntsälä, Pirjola L. Hamberg 2009–10–11 (Continued) Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 4/20 per isolate were incubated at 25°C for 4 d. Enzymatic activity was visually estimated as the strength of the color reaction on a plate. The two isolates with the highest laccase activity were selected and paired (see below) with each other (see Tables 1and 2, Phase 1). Laccase activities of the 24 progeny isolates were also investigated after 4 d on the ABTS plates as described above, and the best producer was named E+ 1 . Pairings First, the best two C.purpureum strains in terms of laccase activity (see Table 1) were paired (Table 2, Phase 1). The isolates to be paired were cultured on PDA Petri plates, one isolate per plate, for ca. 3–4 weeks until they formed fruiting bodies. A fruiting body was cut from a plate with a sterilized scalpel and transferred onto a new PDA plate lid. One drop of sterilized water was pipetted just next to the fruiting body to activate spore release. One day later, when spores had started to germinate on a PDA Petri plate, 24 spores per isolate JÄ2 and 20 spores per isolate PI7 were successfully isolated from the plates with a modified and sterilized Pasteur pipette and transferred to new PDA plates, one spore per plate. After the occurrence of hyphae their morphology was investigated with a microscope to verify that single spore isolations were successful and that the hypha was homokaryotic (no visible clump connections). A single hyphal tip was further isolated from each plate to verify a homokaryotic state. These isolates were Table 1. (Continued) No. Isolate Laccase activity a Municipality/locality Collected by Date of isolation 48 PI2 2 Mäntsälä, Pirjola L. Hamberg 2009–10–11 49 PI3 1 Mäntsälä, Pirjola L. Hamberg 2009–10–11 50 PI4 3 Mäntsälä, Pirjola L. Hamberg 2009–10–11 51 PI5 1 Mäntsälä, Pirjola L. Hamberg 2009–10–11 52 PI6 1 Mäntsälä, Pirjola L. Hamberg 2009–10–11 53 PI7 3 Mäntsälä, Pirjola L. Hamberg 2009–10–11 54 PI8 2 Mäntsälä, Pirjola L. Hamberg 2009–10–11 55 PI9 2 Mäntsälä, Pirjola L. Hamberg 2009–10–11 56 PI10 1 Mäntsälä, Pirjola L. Hamberg 2009–10–11 57 PI11 3 Mäntsälä, Pirjola L. Hamberg 2009–10–11 58 RU1 3 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 59 RU2 3 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 60 RU3 2 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 61 RU4 3 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 62 RU5 1 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 63 RU6 2 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 64 RU7 1 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 65 RU8 2 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 66 RU9 1 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 67 RU10 2 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 68 RU11 2 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 69 RU12 1 Vantaa, Ruskeasanta L. Hamberg 2009–10–19 All C.purpureum strains were isolated from fruiting bodies on birch stumps (Betula pendula and B.pubescens) in Finland and were heterokaryotic. The two best strains in the laccase activity test are in bold. a 0: No color reaction, i.e., low laccase activity; 1: slight color reaction; 2: intermediate color reaction; 3: strong color reaction, i.e., high laccase activity on Petri plates. doi:10.1371/journal.pone.0117381.t001 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 5/20 Table 2. Breeding Process of the Study. Phase 1 Phase 2 Phase 3 Progeny Parent strain/ spore number Laccase activity a Progeny Parent strain/ spore number p b Progeny Parent strain/ spore number 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 Breeding phase, C.purpureum strain and its spore number in each breeding phase are presented. The best progenies in terms of laccase activity (the strongest color reaction, Phase 1) or in terms of efficacy in sprout control in the field (Phases 2 and 3) three months after C.purpureum application are in bold (see Fig. 2). During Phase 2 the best parent strains based on field experiments were bred and in Phase 3 the best progenies from Phases 1 and 2 were bred. Note: the subscript of a progeny indicates the breeding phase. a 0: No color reaction, i.e., low laccase activity; 1: slight color reaction; 2: intermediate color reaction; 3: strong color reaction, i.e., high laccase activity on a Petri plate. b Statistical difference in the number of stump sprouts between the control (cutting only) and the C.purpureum treatment, and the liquid control (inoculum medium spread without C.purpureum) and the C.purpureum treatment, respectively. P-values indicating statistically significant differences are based on generalized linear mixed models. P-values <0.05 are in bold and those between 0.05 and 0.10 have been underlined. doi:10.1371/journal.pone.0117381.t002 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 6/20 paired according to Table 2 (Phase 1), i.e., two spores from different isolates were placed on a single PDA Petri plate to allow them to form heterokaryotic mycelium. After one week, an interaction zone between the isolates developed between the inocula, from which hyphae were transferred to a new PDA Petri plate. Successful pairing was verified microscopically by confirming clump connections. A single heterokaryotic hyphal tip was further transferred to a new PDA plate for further use. The three most efficient natural strains tested by Vartiamäki et al. [13], isolates HY4, P4 and 3.11, were originally collected from birches (Betula pendula and B.pubescens) in Juupajoki in 2003, Vantaa in 2004 and Helsinki in 2001, respectively, and used for the second set of pairings. Altogether 10 spores per isolate HY4 and P4, and 9 spores per isolate 3.11 were successfully isolated from the plates and paired as described above (see Table 2, Phase 2). Third, the best progenies from Phases 1 and 2 were paired according to Table 2 (Phase 3), as described above. Inoculum for field experiments The inoculum medium for the field experiments was prepared as follows: 24 g potato dextrose broth (Becton, Dickinson and Company) and 20 g Sipernat 22S (Evonik Degussa) per 1000 ml deionized water was autoclaved in an Agarmatik machine at 121°C for 15 min. Erlenmayer flasks, 250 ml in volume were also autoclaved at 121°C for 15 min. Altogether, 150 ml cooled autoclaved inoculum medium was added to the flasks and C.purpureum hyphae from one PDA cellophane plate (per isolate, Phase 2) was transferred to the flask with a sterilized scalpel. This inoculated medium was incubated in the dark at 18°C for 7–10 d on a rotator shaker (100 rpm). The inoculum was homogenized by Ultra Turrax apparatus for 1.5 min and diluted 1:10 with tap water before treatments in the field. For Phases 3 and 4, the fungal inoculum was prepared as in Phase 2 except that the weight of hyphae added to the Erlenmayer bottles was equal (0.120 ± 0.024 g and 0.167 ± 0.018 g, mean ± SD, respectively) in each treatment (i.e., for each fungal isolate used in the experiment). All strains were cultivated for 10 d in a shaker at 20°C. Experiments in the field The efficacy of all progenies of Phases 2, 3 and 4 was investigated in field experiments. The first experiment (see Table 2, Phase 2) was established on 9 and 10 June 2009 under electric power lines in Porvoo, Hinthaara, southern Finland. Both days were cloudy with a temperature of ca. 15°C. However, 9 June was partly drizzly. The site included plenty of naturally growing birches (Betula pendula and B.pubescens) with a basal diameter (at ca. 15 cm above soil surface) of 2.2 ± 1.4 cm (mean ± SD). The efficacy of 15 different progenies and their parental strains HY4, 3.11 and P4 were tested in this field experiment (see Table 2). Furthermore, sample plots for controls (cutting only) and liquid controls (procedural controls: inoculum medium without C.purpureum) were also established. Altogether, 80 circular sample plots, four per isolate or control treatment, were established randomly on the site. Each sample plot included ca. 20 birch stumps. Thus, altogether 1583 birch stumps were investigated in this experiment (Betula bendula 49%, B.pubescens 49% and unrecognized birches 2%—the proportion of tree species was investigated three months after the treatments and therefore some were already dead). In the C.purpureum sample plots, fungal inoculum was spread on stumps immediately after cutting. Treatments in the liquid control sample plots were similar, but only inoculum medium without any mycelium was sprayed on the stumps. Viability of the fungal inoculum was confirmed before and after field applications by squirting inoculum to PDA Petri plates. All of the C.purpureum isolates were viable before and after the experiment. Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 7/20 Three and a half months later, after one growing season, in September 2009, the number of living sprouts per birch stump and basal diameters of stumps (mm) were measured. Based on the number of sprouts, the two best C.purpureum progenies were chosen for further investigation. The two best ones had the lowest number of stump sprouts per stump and the statistical difference was the highest (although usually not significant) when compared to the control stumps. These progenies were named V1 2 and V2 2 (see Table 2). In the second field experiment, the efficacy of the progenies and their parental strains in Phase 3 (Table 2) were tested in the field. The efficacy of different C.purpureum strains was tested in three different regeneration areas of spruce in Turenki, southern Finland, including lots of naturally grown birches (Betula pendula and B.pubescens) 1.0 ± 0.4 cm (mean ± SD) in basal diameter (at ca. 15 cm above soil surface). At each site, the efficacy of 12 progenies and the parental strains from different phases, i.e., HY4, P4, 3.11 and V1 2 ,V2 2 ,E+ 1 , was tested. Furthermore, sample plots for controls (cutting only) and liquid controls (inoculum medium spread without C.purpureum) were established. One sample plot per treatment was randomly placed within each site. Each sample plot included ca. 30 birch stumps. Thus, 60 sample plots including 1796 birch stumps were included in the study (Betula pendula 46%, B.pubescens 53%, and unrecognized birches 1%). Sample plots were treated on 15 and 16 June 2010. 15 June was partly cloudy, sunny and rainy with 15°C whereas 16 June was sunny but windy with 17°C. All C.purpureum strains were viable before and after the experiment. Three months later, in September 2010, the number of stump sprouts per birch stump and basal diameters of stumps (mm) were measured, and the three best C.purpureum progenies were chosen for further investigation. The three best strains had the lowest numbers of stump sprouts and statistical differences were the highest (although usually not significant) when compared to the control stumps. In the third field experiment we tested the efficacy of the best C.purpureum strains from Phases 2 and 3 for three growing seasons. The best strains from Phase 3, R3 3 (progeny strain from the pairing between V1 2 and E+ 1 ), R5 3 (V1 2 ×E+ 1 ) and R9 3 (V2 2 ×E+ 1 ), together with strains V1 2 and V2 2 from Phase 2 were included in the experiment. Furthermore, HY4, the best parental strain originally collected from the field (based on the study by Vartiamäki et al. [13]), and a liquid control (inoculum medium without C.purpureum) were included. Eight regeneration areas of spruce with a high frequency of birches (Betula pendula and B.pubescens) were chosen for the study (Table 3). Four of the sites were located in Lapinjärvi, and four in Turenki, both in southern Finland. At each site, circular sample plots including ten birch saplings per treatment were established. The order of the sample plots was randomized within each site. Altogether 560 birch saplings, 80 saplings per treatment, were included in the study (Betula pendula 12%, B.pubescens 75% and unrecognized birches 13%). The mean basal diameter of birches was 1.1 ± 0.4 cm (mean ± SD, ca. 15 cm above soil surface). The experiment was established on 10 to 13 May 2011. The weather was sunny with a temperature of 14–21°C. The mortality of stumps, the number and maximum height of stump sprouts in living stumps and the occurrence of fruiting bodies were investigated one, two and three growing seasons (2011, 2012 and 2013) after the treatments. Furthermore, the basal area of stumps was measured (mm), and the number of other saplings (cut or uncut), and retention trees (i.e., mature trees left on sites, diameter at breast height 5 cm, m 3 ha -1 ) around an investigated stump were measured as these have an effect on sprouting [9]. The number of other stumps and saplings around an investigated sapling was measured within a circular subsample plot 0.5 m in radius whereas the diameters of retention trees (cm) at breast height were measured within a circular sample plot 10 m in radius. Tree volume was calculated using the models by Laasasenaho [29]. The occurrence of moose browsing was recorded per investigated stump to take this into account as it has an effect on the height of stump sprouts. Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 8/20 Statistical analyses Generalized linear mixed models (GLMMs) were used to investigate differences between the treatments. In Phases 2 and 3 the effects of different treatments (control, liquid control and different C.purpureum strains) on the number of stump sprouts were investigated using a Poisson model with log link function in package lme4 in the statistical program R [30,31]. Thus, the number of stump sprouts per stump three months after the treatments was the response variable and treatments (as a factor) and the basal diameter of a stump were explanatory variables in the models. In Phase 2, the sample plot was used as a random factor to take into account pseudoreplication, i.e., the fact that 20 stumps within each sample plot may be—due to environmental conditions—more similar than randomly chosen saplings. In Phase 3, site (logging unit) and sample plot were treated as nested random factors to take into account the fact that several stumps within the same site and sample plot were investigated. Both in Phase 2 and 3, the control treatment (cutting only) and the liquid control (inoculum medium without a fungus) were compared to the other treatments. The best fungal strains were chosen for further investigation based on the biggest differences in the number of stump sprouts per stump between the control treatments and the fungal treatments (lower in the fungal treatment) and the lowest p-values. The GLMMs were also used to analyze data relating to the final field experiment, which lasted for three growing seasons from 2011 to 2013 (Phase 4). We estimated the models separately for each year. The package lme4 in R was used to investigate the effects of controls (inoculum without C.purpureum spread on cut stumps) and different C.purpureum strains on the investigated stumps. Effects on the mortality of investigated stumps and the probability of occurrence of fruiting bodies on stumps were analyzed using a binomial model with logit link function [30,31]. Effects on the number of stump sprouts per stump were investigated using a Poisson model with log link function. The effect of different treatments on the maximum height of stump sprouts per stump was investigated using function lme in the nlme package in R[31,32] assuming a normal distribution. All stumps were included in the mortality models whereas only living stumps were included when differences between the number and maximum height of stump sprouts were investigated. Explanatory variables in the models were 1) treatment (as a factor with seven levels; however, the liquid control was excluded from the fruiting body model), 2) the basal diameter of a stump (mm), 3) the number of other stumps Table 3. Description of the Study Sites Included in the Field Experiment in Phase 4. Site Site type a Topography Soil Clear-cutting (year) Soil cultivation Spruce saplings planted (year) Lapinjärvi 67 MT Partly sloping Sand moraine 2003 Mounding 2004 Lapinjärvi 58 MT Even Gravel moraine/stony 1998 No 1999 Lapinjärvi 245 OMT Even Clay/gravel moraine 1999 Disc trenching 2000 Lapinjärvi 349 OMT Even Clay/gravel moraine/peatcovered 2006 b Disc trenching 2007 Turenki 93 OMT Even Gravel moraine 2002 Mounding 2003 Turenki 116 OMT Even Gravel moraine 2004 No 2004 Turenki 220 OMT Even Gravel moraine 2003 Mounding 2004/2007 Turenki 241.2 OMT Even Peat 2003 Mounding 2004–2005 a MT = Myrtillus type forest, OMT = Oxalis—Myrtillus type forest. b Windfall spruces on this site. doi:10.1371/journal.pone.0117381.t003 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 9/20 in the control in every investigated year. However, in terms of the maximum height of sprouts (in living stumps), hardly any differences between the treatments were found (see also [13]). Our results revealed that an increasing volume of trees around an investigated stump increased stump mortality. This is in accordance with earlier findings as shading of neighboring trees has been shown to have a profound effect on shoot growth due to a greater proportion of shoot buds dying under heavy shading [37,38]. Furthermore, our results showed that an increase in the number of surrounding saplings decreased the number and maximum height of stump sprouts (see also [39]). On the other hand, high mortality with C.purpureum inoculated stumps (especially with R5 3 ) may provide more growing space for those stumps that are still living. This may be the reason why significant differences between the treatments were not observed in stump numbers and height. All C.purpureum strains investigated were originally collected from birch stumps in southern and middle Finland (see Methods section and Table 1). However, investigations from Finland, Canada and New Zealand showed that C.purpureum strains are genetically diverse, and are not associated with a specific host species or ecological region [11,17,18]. Thus, it is possible that the most efficient strain, R5 3 , is efficient also against the sprouting of other tree species and in other geographical areas. Table 6. Differences in the Height of Stump Sprouts of Living Birches between the C.purpureum Strain R5 3 and Other Treatments. Explanatory Response variables variables Stump sprout height in 2011 Stump sprout height in 2012 Stump sprout height in 2013 n= 454 n= 317 n= 279 Coeff. ±SE pCoeff. ±SE PCoeff. ±SE p Intercept 46.065 ±4.588 <0.001 75.190 ±10.645 <0.001 112.429 ±16.939 <0.001 Treatment -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 Stump basal diameter (mm) 1.466 ±0.205 <0.001 2.403 ±0.454 <0.001 3.598 ±0.725 <0.001 Saplings around -0.180 ±0.180 0.316 -0.718 ±0.396 0.071 -1.692 ±0.611 0.006 Tree volume (m 3 ha -1 ) -0.163 ±0.071 0.027 -0.153 ±0.156 0.335 -0.553 ±0.229 0.021 Browsing -6.459 ±1.649 <0.001 1.263 ±3.413 0.712 3.365 ±6.634 0.613 The effect of different treatments (C.purpureum strain or liquid control (LC)), the basal area of investigated stumps, the number of saplings and the volume of trees around an investigated sapling, and browsing on the maximum height of stump sprouts of birch (Betula pendula and B.pubescens)in eight regeneration areas of spruce (Picea abies) three months (in 2011), one year (in 2012) and two years (in 2013) after the treatments (linear mixed model results). Living stumps have been included in the models. Statistically significant p-values (p<0.05) for the model coefficients are in bold and indicative results have been underlined (0.05 p0.10). The sign of a coefficient indicates whether an explanatory variable has an increasing (+) or decreasing (-) effect on the maximum height of stump sprouts. See Figs. 3c and 4c. Note: the subscript of a C.purpureum progeny relates to the breeding phase (see Table 2). a Liquid control i.e., cut stumps were spread with inoculum medium without C.purpureum. b The best parent stain based on an earlier study [13]. c The best progenies from Phase 2. d The best progenies from Phase 3. doi:10.1371/journal.pone.0117381.t006 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 16 / 20 Furthermore, earlier studies have indicated that a single genotype can be used as inoculum without the risk of introducing genes that differ significantly from local populations [11,17]. The best strain R5 3 was developed by traditional breeding (via consequent pairings of selected mycelia), i.e., similarly like any other C.purpureum strain in the nature. In that sense this strain does not differ from its natural counterparts. Moreover, as the fungus has no asexual stage, the genetic combination of a single exceptionally efficient biocontrol fungus breaks up in meiosis before the spores are released. Thus, the same C.purpureum genotype applied on cut stumps cannot spread further in forest ecosystems, and thus there is no risk for explosion of the biocontrol strain. Classical breeding experiments with biocontrolling fungi are scarce. However, in addition to ours, Wall et al. [10] compared the efficacy of C.purpureum monokaryons and their dikaryon progenies in causing wood tissue mortality, but could not find any differences. However, their experiment was not tested under field conditions. Another biocontrol fungus, Phlebiopsis gigantea, was bred to improve the efficacy against Heterobasidion root rot in forests [27]. Results of that study indicated that progeny strains had better properties against H.parviporum than the parental strains. Furthermore, classical pairings were successfully used to improve temperature tolerance of fermenting yeasts using a backcross approach [25]. Thus, these studies (including the present one) show that improving by breeding may help in developing more efficient fungal strains for different purposes. Table 7. Differences in the Occurrence of Fruiting Bodies in Birch Stumps between the C.purpureum Strain R5 3 and Other Treatments. Explanatory Response variables variables Occurrence of fruiting bodies in 2011 Occurrence of fruiting bodies in 2012 Occurrence of fruiting bodies in 2013 n= 480 n= 479 n= 480 Coeff. ±SE pCoeff. ±SE pCoeff. ±SE p Intercept -2.339 ±0.752 0.002 -2.382 ±0.570 <0.001 -- Treatment -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 - - Stump basal diameter (mm) 0.258 ±0.040 <0.001 0.145 ±0.032 <0.001 -- Saplings around -0.014 ±0.031 0.640 0.007 ±0.023 0.752 - - Tree volume (m 3 ha -1 ) 0.012 ±0.010 0.249 0.001 ±0.007 0.910 - - The effect of different treatments (C.purpureum strain), the basal area of investigated stumps, the number of saplings and the volume of trees around an investigated sapling on the probability of occurrence of fruiting bodies on birch (Betula pendula and B.pubescens) stumps in eight regeneration areas of spruce (Picea abies) three months (in 2011) and one year (in 2012) after the treatments (generalized linear mixed model results). The model for year 2013 was not estimated because the occurrence of fruiting bodies was too low (4%). All stumps except those in the liquid control have been included in the models. Statistically significant p-values (p<0.05) for the model coefficients are in bold and indicative results have been underlined (0.05 p0.10). The sign of a coefficient indicates whether an explanatory variable has an increasing (+) or decreasing (-) effect on the occurrence of fruiting bodies. See Fig. 4d. Note: the subscript of a C.purpureum progeny relates to the breeding phase (see Table 2). a The best parent stain based on an earlier study [13]. b The best progenies from Phase 2. c The best progenies from Phase 3. doi:10.1371/journal.pone.0117381.t007 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 17 / 20 However, breeding studies with field experiments may be time consuming as seen in our study. Time for follow-up is usually limited, which forces us to choose the best strains for subsequent steps after a short time period. For example, in our study, we had to choose progeny isolates for the next pairings after the first growing season (both in 2009 and 2010) based only on the number of stump sprouts in different C.purpureum treatments (a low mortality did not allow for mortality analyses). Moreover, as seen also in the present study, the efficacy of C.purpureum is not tightly associated with the parents, because each parent can produce relatively virulent as well as avirulent strains [10]. This is not surprising as Wall et al. [10] have stated that the inheritance of efficacy is probably multifactorial and complex and may be subject to different modifying factors under field and laboratory conditions. Thus, final efficacy should always be investigated in field experiments. This view was supported by our results as the best strain in terms of laccase activity in the laboratory, E+ 1 , was not efficient in sprout control (see Fig. 2b) although some of its progenies (R5 3 and R3 3 ) in the end showed the best ability to prevent sprouting of broad-leaved trees in the field. Conclusions We showed that traditional breeding can increase the efficacy of C.purpureum in the sprout control of birch. Mortality of the treated birch stumps was highest when the progeny strain R5 3 was used to control sprouting. This resulted in 78% mortality in birch stumps 1 cm in diameter. The efficacy of strain R5 3 was significantly higher than that of the investigated parental strain HY4 (56%). The effect of sprout control was especially pronounced when the volume of trees and the number of surrounding saplings was high. However, breeding experiments that aim at increasing the efficacy of sprout control are time consuming because efficacy has to be investigated in long-lasting field experiments. Acknowledgments We thank Fingrid Ltd., UPM-Kymmene Ltd. and the Finnish Forest Research Institute for providing forest sites, and Antti Uotila for providing C.purpureum strains for the study. Markku Rantala, Erkki Piiroinen, Kaarina Pynnönen, Asko Harju, Marja-Leena Santanen, Ritva Vanhanen, Pekka Hämäläinen and Juha Honkaniemi helped in the field. Sonja Sarsila and Minna Sinkkonen are acknowledged for preparing plates for the study. We also thank Marina Brandtberg for valuable comments on the manuscript. Anne Siika modified the figures, and Johan Kotze checked the language. Author Contributions Conceived and designed the experiments: LH HV JH. Performed the experiments: LH HV JH. Analyzed the data: LH. Contributed reagents/materials/analysis tools: LH HV JH. Wrote the paper: LH HV JH. References 1. Finnish Forest Research Institute (1995) National forest inventory data. Vantaa, Finland. 2. Valkonen S, Ruuska J (2003) Effect of Betula pendula admixture on tree growth and branch diameter in young Pinus sylvestris stands in southern Finland. Scand J For Res 18: 416–426. 3. Uotila K, Saksa T (2014) Effects of early cleaning on young Picea abies stands. Scand J For Res 29: 111–119. 4. Harper GJ, Comeau PG, Hintz W, Wall RE, Prasad R, et al. (1999) Chondrostereum purpureum as a biological control agent in forest vegetation management. II. Efficacy on Sitka alder and aspen in western Canada. Can J For Res 29: 852–858. Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 18 / 20 5. Pitt DG, Dumas MT, Wall RE, Thompson DG, Lanteigne L, et al. (1999). Chondrostereum purpureum as a biological control agent in forest vegetation management. I Efficacy on speckled alder, red maple and aspen in eastern Canada. Can J For Res 29: 841–851. 6. FSC Standard for Finland (2010) Finnish FSC association. 46 p. Available: http://ic.fsc.org/forcedownload.php?file=136. Accessed 2014 Jun 25. 7. Äijälä O, Koistinen A, Sved J, Vanhatalo K, Väisänen P, editors (2014) Metsänhoidon suositukset [Recommendations for forestry]. Metsätalouden kehittämiskeskus Tapion julkaisuja. ISBN 978–952–6612– 32–4 Available: http://www.tapio.fi/files/tapio/metsanhoitosuositukset/Metsanhoidon_suositukset_ ver2_netti.pdf. (In Finnish) Accessed 2014 Jun 25. 8. Wall RE (1990) The fungus Chondrostereum purpureum as a silvicide to control stump sprouting in hardwoods. North J Appl For 7: 17–19. 9. Hamberg L, Malmivaara-Lämsä M, Löfström I, Hantula J (2014) Effects of a biocontrol agent Chondrostereum purpureum on sprouting of Sorbus aucuparia and Populus tremula after four growing seasons. BioControl 59: 125–137. 10. Wall RE, Macey DE, Sela E (1996) Virulence and interfertility of Chondrostereum purpureum isolates. Biol Control 7: 205–211. 11. Gosselin L, Jobidon R, Bernier L (1995) Assessment of genetic variation within Chondrostereum purpureum from Québec by random amplified polymorphic DNA analysis. Mycol Res 100: 151–158. 12. Vandenbroucke JE, Gaucher C, Major N (2005) Biological vegetation management: an alternative to chemical pesticides. J Arboric 31: 251–256. 13. Vartiamäki H, Maijala P, Uotila A, Hantula J (2008a) Characterization of growth and enzyme production of Chondrostereum purpureum isolates and correlation of these characteristics with their capability to prevent sprouting of birch in field. Biol Control 47: 46–54. 14. Roy V, Dubeau D, Auger I (2010). Biological control of intolerant hardwood competition: Silvicultural efficacy of Chondrostereum purpureum and worker productivity in conifer plantations. For Ecol Manage 256: 1571–1579. 15. Butler EJ, Jones SG (1949) Silver leaf disease of plum, Stereum purpureum (Fr.) Fr. In: Plant Pathology. London: MacMillian & Co., pp. 763–767. 16. Scheepens PC, Hoogerbrugge A (1989) Control of Prunus serotina in forests with the endemic fungus Chondrostereum purpurem. In: Delfosse ES, editor. Proceedings of the VII International Symposium on Biological Control of Weeds, Rome: Ist Patol Veg (MAF). pp. 545–551. 17. Vartiamäki H, Uotila A, Vasaitis R, Hantula J (2008b) Genetic diversity in Nordic and Baltic populations of Chondrostereum purpureum: a potential herbicide biocontrol agent. For Pathol 38: 381–393. 18. Spiers AG, Brewster DT, Slade A, Gardiner SE (2000) Characterization of New Zealand isolates of Chondrostereum purpureum with regard to morphology, growth, pathogenicity and RAPD banding patterns. Mycol Res 104: 395–402. 19. Spiers AG, Edwards WRN, Hopcroft DH (1987) Effects of silverleaf infection on ultrastructure of foliage of Prunus,Rosa, and Populus. NZ J Bot 25: 411–423. 20. Spiers AG, Brewster DT, Bus VG, Hopcroft DH (1998) Seasonal variation in susceptibility of xylem tissue of Malus,Pyrus, and Salix species to Chondrostereum purpureum in New Zealand. Mycol Res 102: 881–890. 21. Strunz GM, Bethell R, Dumas MT, Boyonoski N (1997) On a new synthesis of sterpurene and the bioactivity of some related Chondrostereum purpureum sesquiterpene metabolites. Can J Chem 75: 742–753. 22. Kirk TK, Farrell RL (1987) Enzymatic “combustion”: the microbial degradation of lignin. Annu Rev Microbiol 41: 465–505. PMID: 3318677 23. ten Have R, Teunissen PJM (2001) Oxidative mechanisms involved in lignin degradation by white-rot fungi. Chem Rev 101: 3397–3413. PMID: 11749405 24. Leatham GF (1986) The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium. Appl Microbiol Biotechnol 24: 51–58. 25. Marullo P, Mansour C, Dufour M, Albertin W, Sicard D, et al. (2009) Genetic improvement of thermo-tolerance in wine Saccharomyces cerevisiae strains by a backcross approach. FEMS Yeast Res 9: 1148–1160. doi: 10.1111/j.1567-1364.2009.00550.x PMID: 19758333 26. Amorim HV, Lopes ML, deCastro Oliveira JV, Buckeridge MS, Goldman GH (2011) Scientific challenges of bioethanol production in Brazil. Appl Microbiol Biotechnol 91: 1267–1275. doi: 10.1007/ s00253-011-3437-6 PMID: 21735264 Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 19 / 20 27. Sun H, Korhonen K, Hantula J, Asiegbu FO, Kasanen R (2009) Use of breeding approach for improving biocontrol efficacy of Phlebiopsis gigantea strains against Heterobasidion infection of Norway spruce stumps. FEMS Microbiol Ecol 69: 266–273. doi: 10.1111/j.1574-6941.2009.00711.x PMID: 19496817 28. Steffen KT, Hofrichter M, Hatakka A (2000) Mineralization of 14 C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi. Appl Microbiol Biotechnol 54: 819–825. PMID: 11152075 29. Hyvämäki T (2002) Tapion taskukirja [The handbook of Tapio]. Helsinki: Kustannusosakeyhtiö Metsälehti. 555 p. (in Finnish) 30. Bates D, Maechler M, Bolker B (2012) lme4: Linear mixed-effects models using S4 classes. R package version 0.999999–0. Available: http://CRAN.R-project.org/package = lme4 31. R Core Team (2012) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3–900051–07–0, URL Available: http://www.R-project.org/. doi: 10.1002/jcc.22917 PMID: 22278855 32. Pinheiro J, Bates D, DebRoy S, Sarkar D (2012) nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1–104. Available: http://CRAN.R-project.org/package = nlme 33. Vartiamäki H, Hantula J, Uotila A (2009) Effect of application time on the efficacy of Chondrostereum purpurem treatment against the sprouting of birch in Finland. Can J For Res 39: 731–739. 34. Kaila S, Kiljunen N, Miettinen A, Valkonen S (2006) Effect of timing of precommercial thinning on the consumption of working time in Picea abies stands in Finland. Scand J For Res 21:496–504. 35. Jobidon R (2000) Density-dependent effects of northern hardwood competition on selected environmental resources and young White spruce (Picea glauca) plantation growth, mineral nutrition, and stand structural development—a 5-year study. For Ecol Manage 130: 77–97. 36. Wall (1997) Fructification of Chondrostereum purpurem on hardwoods inoculated for biological control. Can J Plant Pathol 19: 181–184. 37. Jones M, Harper JL (1987) The influence of neighbours on the growth of trees I. The demography of buds in Betula pendula. Proc R Soc Lond B Biol Sci 232: 1–18. 38. Atkinson MD (1992) Betula pendula Roth. (B.verrucosa Ehrh.) and B.pubescens Ehrh. Biological Flora of the British Isles. J Ecol 80: 837–870. 39. Hamberg L, Vartiamäki H, Malmivaara-Lämsä M, Löfström I, Valkonen S, et al. (2011) Short-term effects of mechanical and biological treatments on Sorbus aucuparia L. sprouting in mesic forests in Finland. Scand J For Res 26: 505–514. Breeding of C. purpureum for Sprout Control PLOS ONE | DOI:10.1371/journal.pone.0117381 February 12, 2015 20 / 20