- Email: [email protected]
Airborne fungal colonisation of coarse woody debris in North Temperate Picea abies forest: impact of season and local spatial scale
Mycol. Res. 109 (4): 487–496 (April 2005). f The British Mycological Society
487
doi:10.1017/S0953756204002084 Printed in the United Kingdom.
Airborne fungal colonisation of coarse woody debris in North Temperate Picea abies forest : impact of season and local spatial scale
Rimvydas VASILIAUSKAS1*, Vaidotas LYGIS1, Karl-Henrik LARSSON2 and Jan STENLID1 1
Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07 Uppsala, Sweden. 2 Botanical Institute, Go¨teborg University, Box 461, SE-405 30 Go¨teborg, Sweden. E-mail : [email protected] Received 27 August 2004; accepted 24 November 2004.
Coarse woody debris is important for mycodiversity in forest ecosystems, but its availability in managed stands is reduced. Leaving dead wood during felling is suggested as an option to sustain and restore the diversity. However, little is known what fungi would colonise freshly cut wood left on managed sites, and how the colonisation process is influenced by ecological factors. During summer and autumn, 120 freshly cut Picea abies stem sections over 8 cm in diameter were placed upright in mapped locations over two discrete plots separated by 100 m in a north-temperate forest. After seven weeks the sections were collected, and isolation and identification of fungi was done from their upper surfaces. In all 943 fungal strains were isolated, representing 97 species. Species richness in the summer survey was 42.5 % higher than during the autumn survey. Low species similarity characterized the different seasons (Sorensen indices : SS=0.36 and SN=0.34) and for 21 species (22 %) observation frequency was significantly affected by season. As a result, community structures in summer and autumn differed notably (z-test ; P<0.001). Species richness between the two plots differed by less than 10 %, but there were 64 species (66 %) found only in one of them, thus qualitative similarity was low (SS=0.49). Quantitative similarity was higher (SN=0.63), indicating that the dominant species colonised wood to a similar extent in both areas. Fungal community structure differed significantly among the two plots (z-test ; P<0.001). Our data showed that freshly cut CWD contributed to mycodiversity in managed north-temperate forest, providing habitats for numerous individuals from over 100 species. The fungal community within a single stand differed markedly both across small distances and over the seasons. In order to sustain and enhance mycodiversity in managed stands, coarse wood should always be left during harvesting. This study also demonstrates the importance of molecular identification and ITS sequence databases for exploring fungal diversity in natural communities.
INTRODUCTION Coarse woody debris (CWD) includes various types of dead wood, e.g. snags, logs, large branches and roots. The size used to define CWD varies among studies, but generally it is supposed to exceed 7 cm diam (Harmon et al. 1986, Samuelsson, Gustafsson & Ingelo¨g 1994). The importance of CWD for the function of forest ecosystems and to biodiversity of a broad range of organisms (e.g. mammals, birds, amphibians, invertebrates, plants, lichens, and saprobic fungi) is now widely recognized (Maser & Trappe 1984, Harmon et al. 1986, Samuelsson et al. 1994, McComb & Lindenmayer 1999). In particular, CWD is of vital importance to lignicolous fungi that play a crucial role in wood decomposition and nutrient cycling within the * Corresponding author.
ecosystem. Many studies have shown that species richness of wood-inhabiting fungi and non-vascular plants (threatened species in particular) increases with the amount of CWD in a stand (Bader, Jansson & Jonsson 1995, Ohlson et al. 1997, Lindblad 1998, Humphrey et al. 2000, Kruys 2001, Sippola, Lehesvirta & Renvall 2001). The presence of logs with high degree of decay and of large dimensions was initially thought to be one of the strongest determinants of fungal diversity within a given forest site (Bader et al. 1995, Høiland & Bendiksen 1996, Kruys et al. 1999). However, more recent studies have shown the importance also of smaller logs (Kruys & Jonsson 1999, Czederpiltz 2001, Heilmann-Clausen 2003). Intensively managed forest stands are characterized by uniform tree species, size, age and spacing, and absence of CWD (Hansen et al. 1991). Such reduced availability (or complete absence) of suitable substrates
Fungi in coarse woody debris inevitably leads to a decrease in species richness of many organisms, including lignicolous fungi (Siitonen 2001, Edman 2003). Therefore, one possible option to sustain or even to restore the mycodiversity in managed forest areas could be leaving certain amounts of dead wood during forest operations. For temperate stands of central Europe, suggested amounts comprise 1–2 % of the total number of trees in mature stands, and 5–10 m3 hax1 in other stands (Ammer 1991, Utschick 1991). However, little is known about which fungi would colonise freshly cut wood left on managed forest sites, and how the colonisation process is influenced by ecological factors, such as season or spatial scale. The structure of the primary decayer community is an important determinant for subsequent fungal succession and for species richness at later stages of wood decomposition (Hudson 1968, Cooke & Rayner 1984, Renvall 1995). The primary aims of the present work are to investigate fungal community in freshly cut CWD, artificially distributed in managed forest of Norway spruce (Picea abies), and to estimate the impact of season and spatial scale during the early stages of community establishment. In addition the distribution of mycelia inside the wood was examined. To date, distribution of fungal individuals in freshly exposed wood has been extensively studied only in logs of Fagus sylvatica (Coates & Rayner 1985c). Related studies on P. abies CWD concerned mapping of individual mycelia in older and well-decayed substrates mainly (Gustafsson 2002, Kauserud & Schumacher 2002, 2003).
MATERIALS AND METHODS Study area and fieldwork The study was carried out in the experimental area S9-Ramsjo¨n of the Swedish Forest Research Institute (Skogforsk), located in central Sweden about 150 km north-west of Stockholm (Thor 1997). It comprises a 40 old plantation of Picea abies that was thinned once, 4 yr prior to our study. The experiment was conducted in two discrete 20r10 m plots separated by about 100 m. It included 120 freshly cut sound-looking spruce stem sections (baits) over 8 cm diam cut from one tree on each occasion. Average parameters of the baits are presented in Table 1. The fieldwork was repeated twice with identical settings : in Oct.–Nov. (autumn) 2000, and in July–Aug. (summer) 2001. During each season, 60 baits were placed upright in each of two plots (35 and 25, respectively). Their exact locations were mapped in relation to the number of a neighbouring stump (all trees and stumps within the area are permanently numbered). Consequently, all them were placed in about the same location during both seasons. The bottom part of each bait was marked, and wrapped into double plastic sheets in order to protect it from soil contact. After 7 wk in the field, the baits were collected, individually placed in plastic bags, brought to the
488
Fig. 1. Patterns of fungal colonisation of Picea abies stem sections (baits) following 7 wk exposure in north-temperate forest ; (a) cross-section of the bait, showing the presence of diverse fungal individuals (separated from each other by dark lines) and one sampling locality (indicated by an arrow); (b) longitudinal section of the bait, showing penetration depth of discoloration beyond the exposed surface (black arrow), and sound-looking wood that remained encased by the bark (white arrow). Sizes of white quadrats in the background are 3r3 cm.
laboratory and stored at +5 xC for another seven weeks. Isolation and identification of fungal strains After storage, pieces of wood (about 5r5r10 mm) were cut off with a knife directly from the surface of exposed wood at the upper cross-section of the baits (Fig. 1). From each disc at least six samples were taken, sampling its central, intermediate and outer parts, and including possibly more diverse areas of colonised wood. When a high fungal diversity was observed in exposed wood, additional samples were taken from distinctly colonised areas. Wood samples were surface sterilised under a flame and placed onto an agar medium in 9 cm diam Petri dishes. Procedures of fungal isolation from the wood samples and sub-culturing of fungal strains were similar to those performed in earlier studies (Vasiliauskas & Stenlid 1998a, Lygis, Vasiliauskas & Stenlid 2004a). The extent of sampling,
R. Vasiliauskas and others isolation, and strains obtained are shown in Table 1. Isolated strains were either identified by observing morphological characteristics of the mycelium or by comparing nuclear ribosomal ITS sequences with a library of sequences from identified fruit bodies. Morphological identification was partly done by the Centraal bureau voor Schimmelcultures (CBS, Utrecht). All strains isolated are deposited in the collection of fungus cultures of the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, Uppsala. The molecular identification included DNA extraction, PCR amplifications and DNA sequencing ; and followed protocols already established at our lab (Ka˚ren et al. 1997). The ribosomal ITS region was sequenced using two primers (ITS1 and ITS4) for every specimen (White et al. 1990). Sequences obtained were checked against those available in our databases (Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, and Botanical Institute, Go¨teborg University). The two latter databases consist of sequences of woodinhabiting fungi, obtained either directly from fruit bodies or from spore cultures of known species. In addition, the NCBI BLAST database (Altschul et al. 1997) was checked. Distribution of fungal mycelia in wood Distribution of fungal mycelia was examined in a wood section SK-93 that, for this part of the work, was randomly selected from among the other baits. It was approx. 13 cm in length and 8.5 cm diam, nine samples from its surface were taken previously during the initial sampling. The section was stored in a plastic bag at 5 x for another six months, making a total of almost nine months in storage. The analysis consisted of : (1) additional sampling and isolation from the surface of the bait and from its deeper layers, and mapping the isolates ; (2) tracking columns of discoloured wood inside the bait, drawing their configurations, and modelling them in threedimensional (3-D) shape; (3) vegetative compatibility tests among the strains of the same species. At first, an additional 14 samples were taken at the top surface (depth 0–0.4 mm) of the bait. Then, the bait was dissected into 24 discs 1.5 mm thick, starting from the top. Thus, a 72 mm long section of the bait, comprising about half of the total length, was sectioned into discs. Fungal isolations were carried out from patches of discoloured wood on six discs at different depths, taking 5 samples from each disc (Table 2). The shape of discoloured columns was analysed in 23 wood discs (top disc was discarded). Prior to the analyses, they were washed in running water and wrapped into moist paper towels. Then, borders of discoloured areas on each disc were encircled with a marker by hand and photographed using digital camera JVC 3-CCD (light source from below the
489 discs). Obtained images were processed by PC software Image-Pro1 Plus (version 4.0). Marked areas and volume of continuous discoloration columns throughout the examined discs were calculated by software Image-Pro1 Plus (version 4.0). Computerised 3-D reconstruction and visualization of discoloration columns were carried out at the Wood Ultrastructure Research Centre, Department of Wood Science, Swedish University of Agricultural Sciences ; 3-D reconstructions were generated from the stack of images obtained from the 23 serial sections of the block. The images were processed and integrated in a CAD-based computerized modelling system using Non-Uniform Rational B-Splines (NURBS) for the extraction of shapes (Bardage 2001). Vegetative compatibility tests were carried out as in one of our earlier studies (Vasiliauskas & Stenlid 1998b). Statistical analyses The aim of statistical analyses was to elucidate possible differences in species richness and structure of fungal communities in two pairs of datasets : autumn vs summer, and plot 1 vs plot 2. Species richness was analysed by calculating species accumulation curves (SACs), that show the relationship between the cumulative number of species found and the sampling intensity (Colwell & Coddington 1994). SACs were calculated using R computer language (Ihaka & Gentleman 1996). Community structure was compared in two ways : (1) by calculating qualitative (SS) and quantitative (SN) Sorensen similarity indices (Magurran 1988); (2) by means of paired z-test, designed to compare number of pairvise observations from two communities influenced by a common factor (Eason, Coles & Gettinby 1986). Furthermore, the occurrence of each particular species in autumn vs summer baits, and in plot 1 vs plot 2 was compared using chi-squared test ; the presence/absence data was calculated from actual no. of strains observed among the total no. of samples taken in respective set of baits (Fowler, Cohen & Jarvis 2001).
RESULTS Among 1063 samples taken (8.9 per bait on average), 715 (67.3 %) yielded fungal cultures (the remaining 32.7 % were either surface-contaminated or sterile). From those, we subcultured 943 fungal strains representing 97 species, 82 (84.5%) of which were identified at least to genus (Table 1). The surface of the baits was usually colonized by many different fungal genotypes (Fig. 1a). Their penetration downwards into the wood was not extensive, and was limited to 3 cm at most according to visual estimates from five split summer baits. Wood that was encased by the bark appeared sound (Fig. 1b). The season had a pronounced impact on species richness of fungi colonizing freshly cut CWD on our
Fungi in coarse woody debris
490
Table 1. Fungi isolated from freshly cut stem sections (baits) distributed for 7 wk in two discrete plots (separated by about 100 m) within a Picea abies stand. Observation frequency: % in total amount of isolated strains/% of colonized sectionsa
Fungi Zygomycetes Mortierella gamsii M. isabellina M. ramanniana Unidentified sp.272 Unidentified sp.285 Unidentified sp.331 Unidentified sp.448 All Zygomycetes Ascomycetes and conidial fungi Apiospora montagnei Beauveria bassiana Capnodium sp.506 Chalara sp.400 Cistella acuum Cladosporium herbarum C. tenuissimum Cladosporium sp.452 Cylindrocarpon didymum C. lucidum Cytospora sp.554 Dipodascus sp.159 Epicoccum nigrum Geotrichum sp.180 Gibberella avenacea Glarea sp.537 Gyoerffyella sp.434 Hormonema dematioides Hypocrea sp.509 Hypoxylon serpens Lecytophora hoffmannii Lecytophora sp.13 Lecytophora sp.22 Leptodontidium elatius Mariannaea elegans Monilinia sp.269 Nectria fuckeliana N. radicicola N. viridescens Nectria sp.156 Nectria sp.170 Neurospora pannonica Ophiostoma cucullatum O. piceae Penicillium spinulosum Penicillium sp.446 Penicillium sp.514 Penicillium sp.545 Pesotum fragrans Phialocephala sp.35 Phialophora fastigiata P. malorum Phialophora sp.201 Phialophora sp.501 Phoma glomerata P. herbarum Phoma sp.552 Rosellinia desmazieressii Scleroconidioma sphagnicola Thysanopora penicillioides
GenBank accession no.
In plots 1 & 2 during October–November
July–August
Plot 1
Plot 2
Both seasons and plots
AY805541 AY805542 – AY805543 AY805544 – AY805545
0.2/1.7 3.9/21.7 5.1/31.7 0.2/1.7 0.2/1.7 –/– –/– 10.0/48.3
–/– 2.7/20.0 2.9/21.7 –/– –/– 0.8/6.7 0.6/5.0 7.0/36.7
0.2/1.4 3.2/24.3 4.5/32.9 0.2/1.4 0.2/1.4 0.7/5.7 0.2/1.4 9.1/51.4
–/– 3.5/16.0 2.9/18.0 –/– –/– –/– 0.6/4.0 7.1/30.0
0.1/0.8 3.3/20.8 3.9/26.7 0.1/0.8 0.1/0.8 0.4/3.3 0.3/2.5 8.4/42.5
AY805546 AY805547 AY805548 AY805549 AY805550 AY805551 AY805552 AY805553 AY805554 AY805555 AY805556 AY805557 AY805558 AY805559 AY805560 AY805561 AY805562 AY805563 AY805564 AY805565 AY805566 AY805567 AY805568 AY805569 AY805570 AY805571 AY805572 AY805573 AY805574 AY805575 AY805576 AY805577 AY805578 AY805579 AY805580 AY805581 AY805582 – AY805583 AY606297 AY805584 AY805585 AY805586 AY805587 AY805588 AY805589 AY805590 AY805591 AY805592 AY805593
–/– –/– –/– –/– 0.2/1.7 0.5/3.3 0.5/3.3 –*/– 6.3***/31.7 0.2/1.7 –/– 0.2/1.7 0.5/3.3 0.5/3.3 0.2/1.7 –/– –/– –**/– –/– 0.5/3.3 0.5/1.7 0.2/1.7 –/– 0.2/1.7 0.2/1.7 0.2/1.7 0.5/3.3 0.5/3.3 22.7***/83.3 1.2*/8.3 0.2/1.7 –/– –/– 2.6/15.0 –**/– –/– –/– –/– –**/– –/– 2.8*/16.7 0.2/1.7 0.5/3.3 –/– 0.2/1.7 0.9/3.3 –/– 6.3***/36.7 –***/– –/–
0.2/1.7 0.4/1.7 0.2/1.7 0.2/1.7 –/– –/– 0.2/1.7 1.0/5.0 0.4/3.3 –/– 0.2/1.7 –/– 0.2/1.7 –/– 0.4/3.3 0.2/1.7 0.2/1.7 1.6/13.3 0.8/6.7 1.0/6.7 –/– –/– 0.4/3.3 –/– 0.6/5.0 –/– 1.2/6.7 –/– 6.4/40.0 –/– –/– 0.2/1.7 0.6/5.0 3.9/18.3 1.6/11.7 0.8/5.0 0.2/1.7 0.2/1.7 1.6/6.7 0.4/3.3 0.8/6.7 0.6/5.0 –/– 0.2/1.7 –/– 0.2/1.7 0.2/1.7 0.4/3.3 2.5/20.0 0.4/3.3
0.2/1.4 –/– 0.2/1.4 0.2/1.4 –/– 0.2/1.4 0.2/1.4 0.8/4.3 1.2***/10.0 0.2/1.4 –/– 0.2/1.4 0.3/2.9 0.3/2.9 0.2/1.4 –/– 0.2/1.4 1.0/8.6 0.3/2.9 1.2/8.6 0.3/1.4 0.2/1.4 0.2/1.4 0.2/1.4 –**/– 0.2/1.4 0.5/4.3 0.3/2.9 13.4/61.4 0.7/5.7 0.2/1.4 –/– –*/– 3.3/18.6 0.7/4.3 0.7/4.3 0.2/1.4 –/– 0.8/2.9 –/– 1.2/8.6 0.7/5.7 0.3/2.9 0.2/1.4 0.2/1.4 0.8/4.3 –/– 3.0/21.4 1.5/11.4 0.2/1.4
–/– 0.6/2.0 –/– –/– 0.3/2.0 0.3/2.0 0.6/4.0 –/– 6.5/28.0 –/– 0.3/2.0 –/– 0.3/2.0 –/– 0.6/4.0 0.3/2.0 –/– 0.6/4.0 0.6/4.0 –/– –/– –/– 0.3/2.0 –/– 1.2/8.0 –/– 1.5/6.0 –/– 14.7/62.0 0.3/2.0 –/– 0.3/2.0 0.9/6.0 3.2/14.0 1.2/8.0 –/– –/– 0.3/2.0 0.9/4.0 0.6/4.0 2.6/16.0 –/– –/– –/– –/– –/– 0.3/2.0 3.2/18.0 1.2/8.0 0.3/2.0
0.1/0.8 0.2/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.2/1.7 0.3/2.5 0.5/2.5 3.1/17.5 0.1/0.8 0.1/0.8 0.1/0.8 0.3/2.5 0.2/1.7 0.3/2.5 0.1/0.8 0.1/0.8 0.8/6.7 0.4/3.3 0.7/5.0 0.2/0.8 0.1/0.8 0.2/1.7 0.1/0.8 0.4/3.3 0.1/0.8 0.8/5.0 0.2/1.7 13.9/61.7 0.5/4.2 0.1/0.8 0.1/0.8 0.3/2.5 3.3/16.7 0.8/5.8 0.4/2.5 0.1/0.8 0.1/0.8 0.8/3.3 0.2/1.7 1.7/11.7 0.4/3.3 0.2/1.7 0.1/0.8 0.1/0.8 0.5/2.5 0.1/0.8 3.1/20.0 1.4/10.0 0.2/1.7
During both seasons
R. Vasiliauskas and others
491
Table 1. (Cont.) Observation frequency: % in total amount of isolated strains/% of colonized sectionsa
Fungi Trichoderma polysporum Truncatella sp.511 Verticillium sp.199 Verticillium sp.438 Zalerion sp.460 Unidentified sp.305 Unidentified sp.457 Unidentified sp.488 Unidentified sp.490 Unidentified sp.538 Unidentified sp.543 Unidentified sp.555 All ascomycetes and conidial fungi Basidiomycetes Amylostereum chailletii Bjerkandera adusta Ceratobasidium sp.257 Collybia butyracea Coniophora arida Exidia pithya Heterobasidion spp. Hypholoma capnoides Hypochnicium geogenium Megacollybia platyphylla Mycena epipterygia M. galopus Mycena sp.480 Peniophora incarnata Phanerochaete magnoliae P. sordida Phlebia subserialis Phlebiella vaga Phlebiopsis gigantea Pholiota spumosa Resinicium bicolor Sistotrema brinkmannii S. sernanderi Sistotremastrum sp.558 Unidentified sp.499 Unidentified sp.512 Unidentified sp.529 Unidentified sp.547 All basidiomycetes No. of stem sections (baits) analysed Diameter of the stem sections (baits), mean¡S.D. (cm) Length of the stem sections (baits), mean¡S.D. (cm) No. of wood samples taken (per bait) No. (%) of samples from which fungi were isolated No. of strains isolated (per bait) No. of species defined a
GenBank accession no.
In plots 1 & 2 during October–November
July–August
Plot 1
Plot 2
Both seasons and plots
AY805594 AY805595 AY805597 AY805596 AY805598 AY805599 AY805600 AY805601 – AY805602 AY805603 –
9.5/45.0 –/– 0.2/1.7 –**/– –/– –**/– –/– –/– –/– –/– –/– –/– 58.9**/95.0
9.2/50.0 0.2/1.7 –/– 2.0/15.0 0.2/1.7 2.1/16.7 0.2/1.7 0.2/1.7 0.2/1.7 0.2/1.7 0.2/1.7 0.6/3.3 45.5/93.3
8.6/45.7 0.2/1.4 0.2/1.4 1.3/10.0 0.2/1.4 1.3/10.0 0.2/1.4 0.2/1.4 0.2/1.4 –/– –/– –*/– 48.4**/92.9
10.6/50.0 –/– –/– 0.6/4.0 –/– 0.9/6.0 –/– –/– –/– 0.3/2.0 0.3/2.0 0.9/4.0 57.4/96.0
9.3/47.5 0.1/0.8 0.1/0.8 1.1/7.5 0.1/0.8 1.2/8.3 0.1/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.3/1.7 51.6/94.2
AY805604 AY805605 AY805606 AY805607 AY805608 AY805609 – AY805610 AY805611 AY805612 AY805613 AY805614 AY805615 AY805616 AY805617 AY805618 AY805619 AY805620 – AY805621 AY805622 AY805623 AY805624 AY805625 AY805626 AY805627 – –
–/– –**/– 0.2/1.7 –*/– –/– –/– 0.2**/1.7 20.4***/71.7 1.2*/5.0 –/– –/– –*/– –/– 0.5/1.7 –/– –***/– –/– –/– –***/– 0.2/1.7 –***/– 7.9***/41.7 0.5/3.3 –/– –/– –/– –/– –/– 31.1***/91.7
0.2/1.7 1.4/10.0 0.2/1.7 1.2/8.3 0.6/3.3 0.2/1.7 1.6/10.0 2.5/21.7 –/– 0.6/3.3 0.2/1.7 1.2/6.7 0.4/3.3 –/– 0.2/1.7 4.5/31.7 0.2/1.7 0.4/1.7 13.9/55.0 0.2/1.7 14.5/68.3 2.3/16.7 –/– 0.2/1.7 0.2/1.7 0.4/3.3 0.2/1.7 0.2/1.7 47.5/96.7
–/– 1.2/8.6 0.2/1.4 1.0/7.1 0.5/2.9 0.2/1.4 1.0/7.1 12.4/55.7 0.8/4.3 0.5/2.9 –/– 0.5/2.9 0.3/2.9 0.3/1.4 0.2/1.4 1.8/12.9 0.2/1.4 0.3/1.4 8.1/31.4 0.2/1.4 8.1/35.7 4.1/30.0 –/– –/– 0.2/1.4 0.3/2.9 –/– –/– 42.5/95.7
0.3/2.0 –/– 0.3/2.0 –/– –/– –/– 0.9/4.0 7.6/34.0 –/– –/– 0.3/2.0 0.9/4.0 –/– –/– –/– 3.5/20.0 –/– –/– 6.5/22.0 0.3/2.0 7.4/32.0 6.2/28.0 0.6/4.0 0.3/2.0 –/– –/– 0.3/2.0 0.3/2.0 35.6/92.0
0.1/0.8 0.7/5.0 0.2/1.7 0.6/4.2 0.3/1.7 0.1/0.8 1.0/5.8 10.7/46.7 0.5/2.5 0.3/1.7 0.1/0.8 0.6/3.3 0.2/1.7 0.2/0.8 0.1/0.8 2.4/15.8 0.1/0.8 0.2/0.8 7.5/27.5 0.2/1.7 7.8/34.2 4.9/29.2 0.2/1.7 0.1/0.8 0.1/0.8 0.2/1.7 0.1/0.8 0.1/0.8 40.0/94.2
60
60
70
50
120
10.8¡1.4
11.1¡1.7
12.1¡0.7
9.5¡1.1
11.0¡1.6
13.5¡0.6
13.5¡1.0
13.7¡0.8
13.3¡0.8
13.5¡0.8
511 (8.5)
552 (9.2)
707 (10.1)
356 (7.1)
1063 (8.9)
333 (65.2)
382 (69.2)
460 (65.1)
255 (71.6)
715 (67.3)
431 (7.2) 42
512 (8.5) 76
603 (8.6) 78
340 (6.8) 52
943 (7.9) 97
During both seasons
Actual no. of strains of each species observed in total no. of samples (and not the percentages in total amount of strains, presented in the Table) were compared by chi-squared tests in two datasets: autumn vs summer, and plot 1 vs plot 2. Underlined percentage value indicates that the test was statistically significant for given species within the respective dataset. The levels of significance are shown as: *–P<0.05; **–P<0.01; ***–P<0.001.
Fungi in coarse woody debris
492
Table 2. Isolation of fungi from wood section (bait) SK-93 as a result of increased sampling effort and depth. Additional isolation, depth (cm) Fungi Bjerkandera adusta Gloeoporus taxicola Hormonema dematioides Nectria fuckeliana Ophiostoma piceae Paecilomyces lilacinus Peniophora piceae Phanerochaete sordida Phialophora fastigiata Phlebiopsis gigantea Phoma cava Resinicium bicolor Sarea difformis Stereum sanguinolentum Trametes versicolor Trichoderma polysporum Unidentified sp.19 Unidentified sp.90 No. of isolations attempted
GenBank accession no. AY805628 AY805630 AY805637 AY805639 – AY805631 AY805634 AY805629 AY805636 – AY805638 – AY805640 AY805632 AY805633 – AY805635 =
Initial isolation, depthf0.4 cm
f0.4
+*
+ +*
+*
+*+
0.9
1.8
3.0
4.5
6.9 +
+ + ++*
++ + +*
+*
+ + +++*
+*
+ +
++ +
+
+
++*
9
14
5
+* + + 5
+ +* + 5
+ +
5
5
Note: Underlined species were not isolated during the initial sampling, and are new for the whole community (not present in Table 1). +, isolation of single strain of a given fungus; *, strains vegetatively compatible with each other and representing one genetic individual of the fungus.
study sites. Thus, 511 samples taken in the autumn yielded 42 different species, whereas 552 taken in summer yielded 76 (Table 1, Fig. 2a). Decreasing sampling effort in summer baits to the same figure as in autumn baits (511) would give a calculated 73 different species (Fig. 2a). The difference between summer and autumn baits is statistically significant (chi-squared test ; P= 0.004). Thus our data indicate that the diversity of fungi colonizing wood within the same area of a northtemperate forest during summer months can be almost twice that observed during the late autumn (42.5 % higher). Autumn and summer communities differed considerably in species composition. As a result, qualitative and quantitative Sorensen similarity indices were only SS=0.36 and SN=0.34, respectively when different seasons are compared (Fig. 2a). A certain number of species were found only during one of the seasons. Thus we found 55 unique ‘ summer species’ and 21 ‘autumn species ’ (Table 1). Most conspicuous among the ‘summer species’ were the basidiomycetes Resinicium bicolor, Phlebiopsis gigantea, and Phanerochaete sordida, which dominated the community during July– Aug., but were completely absent during Oct.–Nov. (Table 1). Although other species, were present during both seasons, season had a significant impact on their frequency\ies of occurrence. Thus, the basidiomycetes Hypholoma capnoides and Sistotrema brinkmannii, together with the ascomycetes, Nectria viridescens and Rosselinia desmazieressii, and the conidial fungus Cylindrocarpon didymum, dominated in the community during the autumn months, but were seldom observed in summer. In contrast, the colonization frequency of
root rot basidiomycetes in the genus Heterobasidion decreased significantly in Oct.–Nov. (Table 1). In total, there were 21 species (22 %), the colonization frequency of which was significantly affected by season (Table 1). As a result, the community structures of fungi colonizing wood in summer and autumn differed significantly (z-test ; P<0.001). The species richness in plots 1 and 2, separated within the investigated plantation by about 100 m, was more or less the same. When sampling effort is kept to 356 in both plots, the estimated number of species recorded in plot 1 is 57 while 52 species were recorded in plot 2, a difference in species richness of less than 10 % (Fig. 2b). However, 64 species (66 % of the total) were found only in one of the plots (Table 1). As a result, qualitative Sorensen similarity index (SS) among the communities in plot 1 and plot 2 was only 0.49. The quantitative index, on the other hand, was higher (SN=0.63), indicating that the dominant species in the plots colonised wood to a similar extent (Fig. 2b). The results show that communities of wood-inhabiting fungi may differ significantly (z-test ; P<0.001) at a moderately local scale. Extended sampling and isolation from the bait SK-93 (39 new samples down to 7 cm depth) revealed 18 species, 14 of which were new for that particular bait, and nine of which were new for the whole community. This raises the total number of species encountered during the investigation to 106. The result is quite expected but also indicated by the raising species accumulation curves (Fig. 2). Even with the extended sampling of bait SK-93 our sampling is not at all exhaustive and certainly many other species of fungi
R. Vasiliauskas and others
493
80
No. of fungal species
70
(a) Summer
60 50 40
Autumn
30 20
SS=0.36 SN=0.34
10 0
0
100
200 300 400 No. of samples taken
500
80
(b)
70 No. of fungal species
Plot 1 60 50 Plot 2
40 30
SS=0.49 SN=0.63
20 10 0
0
100
200 300 400 500 No. of samples taken
600
700
Fig. 2. Sampling effort and species richness of fungi in Picea abies stem sections (baits) following 7 week exposure in north-temperate forest. The datasets are compared from the baits that were (a) exposed during July–August (summer) vs. October–November (autumn), and (b) distributed in two discrete plots (plot 1 and plot 2) separated by a distance of about 100 m. The structure of fungal communities is compared by qualitative (SS) and quantitative (SN) Sorensen indices of similarity.
are likely to inhabit baits at these plots in addition to those presented in the Table 1. Sectioning of the bait has revealed a high number of patchy areas of discoloured wood, which, however, were hard to map. Only three discrete decay columns were found to expand continuously throughout the examined half of the bait (Fig. 3). Several species were present inside each of the columns, but the isolates of the same species (e.g. Peniophora piceae, Trichoderma polysporum) were always vegetatively compatible (Fig. 3, Table 2). When originating from different discoloured areas inside the bait, the isolates were always incompatible. Initial and extended sampling yielded the same fungal genotypes of Hormonema dematioides, Ophiostoma piceae, and Phlebiopsis gigantea. (Table 2).
DISCUSSION We demonstrate that season and short distances inside a forest stand might have a profound impact on the
Fig. 3. Computerised three-dimensional picture of three discrete discoloration columns inside Picea abies stem section (approx. 8.5 cm in diameter) starting from the top. P.p., Peniophora piceae ; T.v., Trametes versicolor ; T.p., Trichoderma polysporum ; P.l., Paecilomyces lilacinus ; O.p., Ophiostoma piceae ; R.b., Resinicium bicolor ; S.s., Stereum sanguinolentum ; and B.a., Bjerkandera adusta. Approximate level from which each species was isolated within respective columns is indicated by the bars.
establishment of airborne wood-inhabiting fungi in CWD on north-temperate Picea abies forest sites. The much higher species richness observed during the summer months (76 species in summer vs 42 in autumn) probably reflects more colonisers of fresh wood sporulating in summer than in mid- to late autumn. Nevertheless, our data also revealed the presence of typical late season colonizers. We do not know if such species really are adapted to fruiting during the cooler period of the year, or if they are simply out-competed in summertime. While season had an impact on both species richness and similarity (Fig. 2a), local distance (ca 100 m) influenced community structure and not species richness, which remained more or less constant across the stand (Fig. 2b). The distinct difference in species composition between our plots indicates that the local spore source is an important determinant of fungal community structure. A study of Heterobasidion spp., common wood-decay polypore in the temperate zone (recorded also during the present work ; Table 1), demonstrated that the spore deposition gradient is very steep within the first 100 m from a fruit body (Stenlid 1994). A similar result was obtained in a study of corticioid wood-decomposer, Phlebia centrifuga (Norde´n & Larsson 2000). Recent work demonstrates the importance of landscape composition on spore deposition of
Fungi in coarse woody debris wood-inhabiting fungi, although on a larger (1–3 km) spatial scale (Edman et al. 2004a, b). Our data suggest that even freshly cut CWD contributes to mycodiversity on managed north temperate sites. However, our study was not designed to show how soon and to what extent (if at all) freshly cut wood could help in restoring primeval mycodiversity. An expanded long-term study close to old-growth sites with a known flora of saprophytic fungi would be desirable. Studies in tropical forest have shown that species richness of wood-inhabiting fungi returns to the level of the primary forest when the woody debris is retained and there is a patch of original forest in the vicinity (Lindblad 2002). In the present work the community structure of fungi within a single stand was found to differ significantly both over small distances and over different seasons. This implies that in order to sustain and enhance fungal diversity in managed stands some coarse wood should always be left wherever and whenever harvesting takes place. Freshly cut wood sustains a highly diverse fungal community after just a few weeks of exposure to airborne colonisation (Fig. 1a). Different species of basidiomycetes were found to co-exist in close proximity within the same small column of discolored wood (Fig. 3). In a similar experiment, high diversity was observed in aerial cut surfaces of Fagus sylvatica logs, where up to 30 mutually antagonistic fungal individuals of several species were found cmx3 (Coates & Rayner 1985a). Buried cross-sections, however, were colonised by a less diverse community (Coates & Rayner 1985b). Similarly, hundreds of fungal species were reported from stumps of spruce some weeks after they were cut and available for airborne colonisation (Woods 1996, Woodward 2003, Vasiliauskas et al. 2004). Stumps are in most cases the only source of CWD in managed stands but it should be remembered that a freshly cut stump is ecologically different from a stem section, since stumps remain attached to the root system for a considerable time (Redfern & Stenlid 1998). We demonstrated the ability of Mycena spp. and Collybia spp. to abundantly colonise freshly cut CWD (Table 1). In another study, Mycena galopus was found to inhabit living stems of pine (Lygis et al. 2004b). These findings contribute to a new picture of the ecology of those fungi. They were previously known primarily as decomposers of litter and of well decayed residues of wood, on which their fruiting bodies are commonly observed (Rodionova 1970, Stepanova 1975, Hansen & Knudsen 1992, Ryman & Holmasen 1998). We also discovered that several ‘combative ’ species are already present in freshly exposed substrate. According to Cooke & Rayner (1984), so called combative species have a secondary resource capture strategy : they arrive late, displace from the wood species that were already present and take over their previously colonised domain. The following species from Table 1 could be classified as combative : Hypholoma capnoides, Phanerochaete spp., Resinicium
494 bicolor, Sistotrema brinkmannii, and Trametes versicolor (Holmer & Stenlid 1996). It is likely that those species will be able to persist in baits for several years. In general, the basidiomycete macrofungi reported in this study are common throughout the forests of North Europe (Hansen & Knudsen 1992, Ryman & Holma˚sen 1998). Among 74 species of microfungi reported in the present work (Tables 1–2), many related species (belonging to at least the same genera) have been previously encountered in woody substrates in northtemperate and boreal forests. Thus, 44 (59 %) of those were found to inhabit stumps, snags and living trees of spruce, pine and birch in Sweden and Lithuania (Lygis et al. 2004a, b, Vasiliauskas et al. 2004, 2005). Among 60 microfungi that were identified (Tables 1–2), 27 (45 %) related species were isolated from spruce stumps in Scotland (Woods 1996), and thirty-seven (or 62%) from decomposing logs of Douglas-fir, spruce and aspen in North America (Crawford, Carpenter & Harmon 1990, Lumley, Gignac & Currah 2001). About 15% of species found remained unidentified (Tables 1–2). In other studies, where the identification was based on morphological characters of the mycelium, unidentified species comprised 25–35 % (Woods 1996, Lumley et al. 2001). This indicates that morphological identification of fungal cultures in many cases is difficult, but also that sequence coverage in GenBank and our local sequence databases is far from exhaustive. We can expect that some of these unidentified sequences represent unknown species especially among micromycetes with inconspicuous fruit bodies. It is estimated that the majority of fungi have not yet been isolated and identified (Kennedy & Clipson 2003). An example concerning wood-inhabiting species is a recent survey of microfungi from rotting wood in Canada, during which 49 species of ascomycetes were isolated, 15 of which were new for Canada, and seven were new for North America ; 20 species had not previously been reported from wood (Lumley, Abbot & Currah 2000, Sigler, Lumley & Currah 2000). During recent work in our laboratory four previously unknown species of dark septate fungi (Phialocephala spp.) have been detected in woody substrates (Menkis et al. 2004). The availability of comprehensive and well-documented sequence databases will markedely increase the efficacy of mycodiversity studies, in particular when analysing fungal DNA directly from environmental samples (Johannesson & Stenlid 1999, Vainio & Hantula 2000, Kennedy & Clipson 2003).
ACKNOWLEDGEMENTS We thank Olov Pettersson for technical assistance, and Stig Bardage for help in detailed wood analyses. This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Foundation for Strategic Environmental Research (MISTRA).
R. Vasiliauskas and others REFERENCES Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402. Ammer, U. (1991) Konsequenzen aus den Ergebnissen der Totholzforschung fur die forstliche Praxis. Forstwissenschaftliches Centrablatt 110: 149–157. Bader, P., Jansson, S. & Jonsson, B. G. (1995) Wood-inhabiting fungi and substratum decline in selectively logged boreal spruce forests. Biological Conservation 72: 355–362. Bardage, S. L. (2001) Three-dimensional modelling and visualization of whole Norway spruce latewood tracheids. Wood and Fiber Science 33: 627–638. Coates, D. & Rayner, A. D. M. (1985a) Fungal population and community development in cut beech logs. I. Establishment via the aerial cut surface. New Phytologist 101 : 153–171. Coates, D. & Rayner, A. D. M. (1985b) Fungal population and community development in cut beech logs. II. Establishment via the buried cut surface. New Phytologist 101 : 173–181. Coates, D. & Rayner, A. D. M. (1985c) Fungal population and community development in cut beech logs. III. Spatial dynamics, interactions and strategies. New Phytologist 101: 183–198. Colwell, R. K. & Coddington, J. A. (1994) Estimating terrestrial biodiversity through extrapolation. Philosophical Transactions of the Royal Society of London, Series B 345: 101–118. Cooke, R. C. & Rayner, A. D. M. (1984) Ecology of Saprotropic Fungi. Longman, London. Crawford, R. H., Carpenter, S. E. & Harmon, M. E. (1990) Communities of filamentous fungi and yeast in decomposing logs of Pseudotsuga menziesii. Mycologia 82: 759–765. Czederpiltz, D. L. L. (2001) Forest management and the diversity of wood-inhabiting polyporoid and corticioid fungi. PhD thesis, University of Wisconsin-Madison. Eason, G., Coles, C. W. & Gettinby, G. (1986) Mathematics and Statistics for the Bio-Sciences. John Wiley, New York. Edman, M. (2003) Dispersal ecology of wood-decaying fungi – implications for conservation. PhD thesis, Umea˚ University. Edman, M., Gustafsson, M., Stenlid, J. & Ericson, L. (2004a) Abundance and viability of fungal spores along a forestry gradient – responses to habitat loss and isolation? Oikos 104: 35–42. Edman, M., Gustafsson, M., Stenlid, J., Jonsson, B. G. & Ericson, L. (2004b) Spore deposition of wood-decaying fungi: importance of landscape composition. Ecography 27 : 103–111. Fowler, J., Cohen, L. & Jarvis, P. (2001) Practical Statistics for Field Biology. John Wiley and Sons, Chichester. Gustafsson, M. H. (2002) Distribution and dispersal of wood-decaying fungi occurring on Norway spruce logs. PhD thesis, Swedish University of Agricultural Sciences, Uppsala. Hansen, A. J., Spies, T. A., Swanson, F. J. & Ohmann, J. L. (1991) Conserving biodiversity in managed forests. BioScience 41: 382–392. Hansen, L. & Knudsen, H. (eds) (1992) Nordic Macromycetes. Vol. 2. Polyporales, Boletales, Agaricales, Russulales. Nordsvamp, Copenhagen. Harmon, M. E., Franklin, J. F., Swanson, F. J., Sollins, P., Gregory, S. V., Lattin, J. D., Anderson, N. H., Cline, S. P., Aumen, N. G., Sedell, J. R., Lienkaemper, G. W., Cromack, K. J. & Cummins, K. W. (1986) Ecology of coarse woody debris in temperate ecosystems. Advances of Ecological Research 15: 133–302. Heilmann-Clausen, J. (2003) Wood-inhabiting fungi in Danish deciduous forests – diversity, habitat preferences and conservation. PhD thesis, The Royal Danish Veterinary and Agricultural University, Lyngby. Høiland, K. & Bendiksen, E. (1997) Biodiversity of wood-inhabiting fungi in a boreal coniferous forest in Sor-Trondelag County, central Norway. Nordic Journal of Botany 16: 643–659.
495 Holmer, L. & Stenlid, J. (1996) Diffuse competition for heterogenous substrate in soil among six species of wood-decomposing basidiomycetes. Oecologia 106 : 531–538. Hudson, H. J. (1968) The ecology of fungi on plant remains above the soil. New Phytologist 67: 837–874. Humphrey, J. W., Newton, A. C., Peace, A. J. & Holden, E. (2000) The importance of conifer plantations in northern Britain as a habitat for native fungi. Biological Conservation 96: 241–252. Ihaka, R. & Gentleman, R. (1996) R: A language for data analysis and graphics. Journal of Computer Graphics and Statistics 5: 299–314. Johannesson, H. & Stenlid, J. (1999) Molecular identification of wood-inhabiting fungi in an unmanaged Picea abies forest in Sweden. Forest Ecology and Management 115: 203–211. Ka˚ren, O., Ho¨gberg, N., Dahlberg, A., Jonsson, L. & Nylund, J. E. (1997) Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytologist 136: 313–325. Kauserud, H. & Schumacher, T. (2002) Population structure of the endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota). Canadian Journal of Botany 80: 597–606. Kauserud, H. & Schumacher, T. (2003) Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum. Mycologia 95: 416–425. Kennedy, N. & Clipson, N. (2003) Fingerprinting the fungal community. Mycologist 17 : 158–164. Kruys, N. (2001) Bryophytes and fungi on dead spruce: integrating conservation with forest management planning. PhD thesis, Umea˚ University. Kruys, N., Fries, C., Jonsson, B. G., Lamas, T. & Stahl, G. (1999) Wood-inhabiting cryptogams on dead Norway spruce (Picea abies) trees in managed Swedish boreal forests. Canadian Journal of Forest Research 29: 178–186. Kruys, N. & Jonsson, B. G. (1999) Fine woody debris is important for species richness on logs in managed boreal spruce forests of northern Sweden. Canadian Journal of Forest Research 29: 1295–1299. Lindblad, I. (1998) Wood-inhabiting fungi on fallen logs of Norway spruce: relations to forest management and substrate quality. Nordic Journal of Botany 18 : 243–255. Lindblad, I. (2002) Wood-inhabiting fungi in primary and secondary seasonally dry tropical forest, Costa Rica. In Book of Abstracts of the 7th International Mycological Congress, Oslo 11–17 August 2002: 164–164. University of Oslo, Oslo. Lumley, T. C., Abbott, S. P. & Currah, R. S. (2000) Microscopic ascomycetes isolated from rotting wood in the boreal forest. Mycotaxon 74: 395–414. Lumley, T. C., Gignac, D. & Currah, R. S. (2001) Microfungus communities of white spruce and trembling aspen logs at different stages of decay in disturbed and undisturbed sites in the boreal mixedwood region of Alberta. Canadian Journal of Botany 79: 76–92. Lygis, V., Vasiliauskas, R. & Stenlid, J. (2004a) Planting Betula pendula on pine sites infested by Heterobasidion annosum: disease transfer, silvicultural evaluation, and community of woodinhabiting fungi. Canadian Journal of Forest Research 34 : 120–130. Lygis, V., Vasiliauskas, R., Stenlid, J. & Vasiliauskas, A. (2004b) Silvicultural and pathological evaluation of Scotts pine afforestations mixed with trees to reduce the infections by Heterobasidion annosum. Forest Ecology and Management 201: 275–285. Magurran, A. E. (1988) Ecological Diversity and Its Measurement. Princeton University Press, Princeton. Maser, C. & Trappe, J. M. (1984) The seen and unseen world of the fallen tree. General Technical Report of the USDA Forest Service GTR-PNW-164: 1–56. McComb, W. & Lindenmayer, D. (1999) Dying, dead, and down trees. In Maintaining Biodiversity in Forest Ecosystems (M. L. Hunter, ed.): 335–372. Cambridge University Press, Cambridge, UK.
Fungi in coarse woody debris Menkis, A., Allmer, J., Vasiliauskas, R., Lygis, V., Stenlid, J. & Finlay, R. (2004) Ecology and molecular characterization of dark septate fungi from roots, living stems, coarse and fine woody debris. Mycological Research 108 : 965–973. Norde´n, B. & Larsson, K. H. (2000) Basidiospore dispersal in the oldgrowth forest fungus Phlebia centrifuga. Nordic Journal of Botany 20: 215–219. Ohlson, M., Soderstrom, L., Hornberg, G., Zackrisson, O. & Hermansson, J. (1997) Habitat qualities versus long-term continuity as determinants of biodiversity in boreal old-growth swamp forests. Biological Conservation 81 : 221–231. Redfern, D. B. & Stenlid, J. (1998) Spore dispersal and infection. In Heterobasidion annosum: biology, ecology, impact and control (S. Woodward, J. Stenlid, R. Karjalainen & A. Hu¨ttermann, eds): 105–124. CAB International, Wallingford. Renvall, P. (1995) Community structure and dynamics of woodrotting Basidiomycetes on decomposing conifer trunks in northern Finland. Karstenia 35: 1–51. Rodionova, S. V. (1970) [Fungal flora of myrtillus type spruce stands of south Karelia, and its role in mineralization of needle litter.] PhD thesis, University of Petrozavodsk. [In Russsian.] Ryman, S. & Holmasen, I. (1998) Svampar: en fa¨lthanbok. 2nd edn. Interpublishing, Stockholm. Samuelsson, J., Gustafsson, L. & Ingelog, T. (1994) Dying and Dead Trees: a review of their importance for biodiversity. Swedish Threatened Species Unit, Uppsala. Sigler, L., Lumley, T. C. & Currah, R. S. (2000) New species and records of saprophytic ascomycetes (Myxotrichaceae) from decaying logs in the boreal forest. Mycoscience 41: 495–502. Siitonen, J. (2001) Forest management, coarse woody debris and saproxylic organisms: Fennoscandian boreal forests as an example. Ecological Bulletins 49: 11–41. Sippola, A.-L., Lehesvirta, T. & Renvall, P. (2001) Effects of selective logging on coarse woody debris and diversity of wooddecaying polypores in eastern Finland. Ecological Bulletins 49: 243–254. Stenlid, J. (1994) Regional differentiation in Heterobasidion annosum. Proceedings of the Eight International Conference on root and butt rots, Sweden and Finland, August 9–16, 1993 (M. Johansson & J. Stenlid, eds): 243–248. Swedish University of Agricultural Sciences, Uppsala.
496 Stepanova, O. A. (1975) [Fungi on felling residues in spruce forests of Leningrad district II.] Mikologiya i Fitopatologiya 9: 15–20. [In Russian]. Thor, M. (1997) Stump treatment against Heterobasidion annosum – techniques and biological effect in practical forestry. Licentiate’s thesis, Swedish University of Agricultural Sciences, Uppsala. Utschick, H. (1991) Beziehungen zwischen Totholzreichtum und Vogelwelt in Wirtschaftswaldern. Forstwissenschaftliches Centrablatt 110 : 135–148. Vainio, E. & Hantula, J. (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycological Research 104: 927–936. Vasiliauskas, R., Larsson, E., Larsson, K.-H. & Stenlid, J. (2005) Persistence and long-term impact of Rotstop biological control agent on mycodiversity in Picea abies stumps. Biological Control 32: in press. Vasiliauskas, R., Lygis, V., Thor, M. & Stenlid, J. (2004) Impact of biological (Rotstop) and chemical (urea) treatments on fungal community structure in freshly cut Picea abies stumps. Biological Control 31: 405–413. Vasiliauskas, R. & Stenlid, J. (1998a) Fungi inhabiting stems of Picea abies in a managed stand in Lithuania. Forest Ecology and Management 109 : 119–126. Vasiliauskas, R. & Stenlid, J. (1998b) Influence of spatial scale on population structure of Stereum sanguinolentum in northern Europe. Mycological Research 102 : 93–98. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 315–322. Academic Press, San Diego. Woods, C. M. (1996) The fungal ecology of Sitka spruce stumps. PhD thesis, University of Aberdeen. Woodward, S. (2003) Diversity in monocultures : the Sitka spruce stump. Root and Butt Rots of Forest Trees: Proceedings of the IUFRO Working Party 7.02.01, Quebec, Canada, September 16–22, 2001 (G. Laflamme, J. A. Berube & G. Bussieres, eds): 47–54. Laurentian Forestry Centre, Quebec. Corresponding Editor: D. J. Lodge
487
doi:10.1017/S0953756204002084 Printed in the United Kingdom.
Airborne fungal colonisation of coarse woody debris in North Temperate Picea abies forest : impact of season and local spatial scale
Rimvydas VASILIAUSKAS1*, Vaidotas LYGIS1, Karl-Henrik LARSSON2 and Jan STENLID1 1
Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, SE-750 07 Uppsala, Sweden. 2 Botanical Institute, Go¨teborg University, Box 461, SE-405 30 Go¨teborg, Sweden. E-mail : [email protected] Received 27 August 2004; accepted 24 November 2004.
Coarse woody debris is important for mycodiversity in forest ecosystems, but its availability in managed stands is reduced. Leaving dead wood during felling is suggested as an option to sustain and restore the diversity. However, little is known what fungi would colonise freshly cut wood left on managed sites, and how the colonisation process is influenced by ecological factors. During summer and autumn, 120 freshly cut Picea abies stem sections over 8 cm in diameter were placed upright in mapped locations over two discrete plots separated by 100 m in a north-temperate forest. After seven weeks the sections were collected, and isolation and identification of fungi was done from their upper surfaces. In all 943 fungal strains were isolated, representing 97 species. Species richness in the summer survey was 42.5 % higher than during the autumn survey. Low species similarity characterized the different seasons (Sorensen indices : SS=0.36 and SN=0.34) and for 21 species (22 %) observation frequency was significantly affected by season. As a result, community structures in summer and autumn differed notably (z-test ; P<0.001). Species richness between the two plots differed by less than 10 %, but there were 64 species (66 %) found only in one of them, thus qualitative similarity was low (SS=0.49). Quantitative similarity was higher (SN=0.63), indicating that the dominant species colonised wood to a similar extent in both areas. Fungal community structure differed significantly among the two plots (z-test ; P<0.001). Our data showed that freshly cut CWD contributed to mycodiversity in managed north-temperate forest, providing habitats for numerous individuals from over 100 species. The fungal community within a single stand differed markedly both across small distances and over the seasons. In order to sustain and enhance mycodiversity in managed stands, coarse wood should always be left during harvesting. This study also demonstrates the importance of molecular identification and ITS sequence databases for exploring fungal diversity in natural communities.
INTRODUCTION Coarse woody debris (CWD) includes various types of dead wood, e.g. snags, logs, large branches and roots. The size used to define CWD varies among studies, but generally it is supposed to exceed 7 cm diam (Harmon et al. 1986, Samuelsson, Gustafsson & Ingelo¨g 1994). The importance of CWD for the function of forest ecosystems and to biodiversity of a broad range of organisms (e.g. mammals, birds, amphibians, invertebrates, plants, lichens, and saprobic fungi) is now widely recognized (Maser & Trappe 1984, Harmon et al. 1986, Samuelsson et al. 1994, McComb & Lindenmayer 1999). In particular, CWD is of vital importance to lignicolous fungi that play a crucial role in wood decomposition and nutrient cycling within the * Corresponding author.
ecosystem. Many studies have shown that species richness of wood-inhabiting fungi and non-vascular plants (threatened species in particular) increases with the amount of CWD in a stand (Bader, Jansson & Jonsson 1995, Ohlson et al. 1997, Lindblad 1998, Humphrey et al. 2000, Kruys 2001, Sippola, Lehesvirta & Renvall 2001). The presence of logs with high degree of decay and of large dimensions was initially thought to be one of the strongest determinants of fungal diversity within a given forest site (Bader et al. 1995, Høiland & Bendiksen 1996, Kruys et al. 1999). However, more recent studies have shown the importance also of smaller logs (Kruys & Jonsson 1999, Czederpiltz 2001, Heilmann-Clausen 2003). Intensively managed forest stands are characterized by uniform tree species, size, age and spacing, and absence of CWD (Hansen et al. 1991). Such reduced availability (or complete absence) of suitable substrates
Fungi in coarse woody debris inevitably leads to a decrease in species richness of many organisms, including lignicolous fungi (Siitonen 2001, Edman 2003). Therefore, one possible option to sustain or even to restore the mycodiversity in managed forest areas could be leaving certain amounts of dead wood during forest operations. For temperate stands of central Europe, suggested amounts comprise 1–2 % of the total number of trees in mature stands, and 5–10 m3 hax1 in other stands (Ammer 1991, Utschick 1991). However, little is known about which fungi would colonise freshly cut wood left on managed forest sites, and how the colonisation process is influenced by ecological factors, such as season or spatial scale. The structure of the primary decayer community is an important determinant for subsequent fungal succession and for species richness at later stages of wood decomposition (Hudson 1968, Cooke & Rayner 1984, Renvall 1995). The primary aims of the present work are to investigate fungal community in freshly cut CWD, artificially distributed in managed forest of Norway spruce (Picea abies), and to estimate the impact of season and spatial scale during the early stages of community establishment. In addition the distribution of mycelia inside the wood was examined. To date, distribution of fungal individuals in freshly exposed wood has been extensively studied only in logs of Fagus sylvatica (Coates & Rayner 1985c). Related studies on P. abies CWD concerned mapping of individual mycelia in older and well-decayed substrates mainly (Gustafsson 2002, Kauserud & Schumacher 2002, 2003).
MATERIALS AND METHODS Study area and fieldwork The study was carried out in the experimental area S9-Ramsjo¨n of the Swedish Forest Research Institute (Skogforsk), located in central Sweden about 150 km north-west of Stockholm (Thor 1997). It comprises a 40 old plantation of Picea abies that was thinned once, 4 yr prior to our study. The experiment was conducted in two discrete 20r10 m plots separated by about 100 m. It included 120 freshly cut sound-looking spruce stem sections (baits) over 8 cm diam cut from one tree on each occasion. Average parameters of the baits are presented in Table 1. The fieldwork was repeated twice with identical settings : in Oct.–Nov. (autumn) 2000, and in July–Aug. (summer) 2001. During each season, 60 baits were placed upright in each of two plots (35 and 25, respectively). Their exact locations were mapped in relation to the number of a neighbouring stump (all trees and stumps within the area are permanently numbered). Consequently, all them were placed in about the same location during both seasons. The bottom part of each bait was marked, and wrapped into double plastic sheets in order to protect it from soil contact. After 7 wk in the field, the baits were collected, individually placed in plastic bags, brought to the
488
Fig. 1. Patterns of fungal colonisation of Picea abies stem sections (baits) following 7 wk exposure in north-temperate forest ; (a) cross-section of the bait, showing the presence of diverse fungal individuals (separated from each other by dark lines) and one sampling locality (indicated by an arrow); (b) longitudinal section of the bait, showing penetration depth of discoloration beyond the exposed surface (black arrow), and sound-looking wood that remained encased by the bark (white arrow). Sizes of white quadrats in the background are 3r3 cm.
laboratory and stored at +5 xC for another seven weeks. Isolation and identification of fungal strains After storage, pieces of wood (about 5r5r10 mm) were cut off with a knife directly from the surface of exposed wood at the upper cross-section of the baits (Fig. 1). From each disc at least six samples were taken, sampling its central, intermediate and outer parts, and including possibly more diverse areas of colonised wood. When a high fungal diversity was observed in exposed wood, additional samples were taken from distinctly colonised areas. Wood samples were surface sterilised under a flame and placed onto an agar medium in 9 cm diam Petri dishes. Procedures of fungal isolation from the wood samples and sub-culturing of fungal strains were similar to those performed in earlier studies (Vasiliauskas & Stenlid 1998a, Lygis, Vasiliauskas & Stenlid 2004a). The extent of sampling,
R. Vasiliauskas and others isolation, and strains obtained are shown in Table 1. Isolated strains were either identified by observing morphological characteristics of the mycelium or by comparing nuclear ribosomal ITS sequences with a library of sequences from identified fruit bodies. Morphological identification was partly done by the Centraal bureau voor Schimmelcultures (CBS, Utrecht). All strains isolated are deposited in the collection of fungus cultures of the Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, Uppsala. The molecular identification included DNA extraction, PCR amplifications and DNA sequencing ; and followed protocols already established at our lab (Ka˚ren et al. 1997). The ribosomal ITS region was sequenced using two primers (ITS1 and ITS4) for every specimen (White et al. 1990). Sequences obtained were checked against those available in our databases (Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Uppsala, and Botanical Institute, Go¨teborg University). The two latter databases consist of sequences of woodinhabiting fungi, obtained either directly from fruit bodies or from spore cultures of known species. In addition, the NCBI BLAST database (Altschul et al. 1997) was checked. Distribution of fungal mycelia in wood Distribution of fungal mycelia was examined in a wood section SK-93 that, for this part of the work, was randomly selected from among the other baits. It was approx. 13 cm in length and 8.5 cm diam, nine samples from its surface were taken previously during the initial sampling. The section was stored in a plastic bag at 5 x for another six months, making a total of almost nine months in storage. The analysis consisted of : (1) additional sampling and isolation from the surface of the bait and from its deeper layers, and mapping the isolates ; (2) tracking columns of discoloured wood inside the bait, drawing their configurations, and modelling them in threedimensional (3-D) shape; (3) vegetative compatibility tests among the strains of the same species. At first, an additional 14 samples were taken at the top surface (depth 0–0.4 mm) of the bait. Then, the bait was dissected into 24 discs 1.5 mm thick, starting from the top. Thus, a 72 mm long section of the bait, comprising about half of the total length, was sectioned into discs. Fungal isolations were carried out from patches of discoloured wood on six discs at different depths, taking 5 samples from each disc (Table 2). The shape of discoloured columns was analysed in 23 wood discs (top disc was discarded). Prior to the analyses, they were washed in running water and wrapped into moist paper towels. Then, borders of discoloured areas on each disc were encircled with a marker by hand and photographed using digital camera JVC 3-CCD (light source from below the
489 discs). Obtained images were processed by PC software Image-Pro1 Plus (version 4.0). Marked areas and volume of continuous discoloration columns throughout the examined discs were calculated by software Image-Pro1 Plus (version 4.0). Computerised 3-D reconstruction and visualization of discoloration columns were carried out at the Wood Ultrastructure Research Centre, Department of Wood Science, Swedish University of Agricultural Sciences ; 3-D reconstructions were generated from the stack of images obtained from the 23 serial sections of the block. The images were processed and integrated in a CAD-based computerized modelling system using Non-Uniform Rational B-Splines (NURBS) for the extraction of shapes (Bardage 2001). Vegetative compatibility tests were carried out as in one of our earlier studies (Vasiliauskas & Stenlid 1998b). Statistical analyses The aim of statistical analyses was to elucidate possible differences in species richness and structure of fungal communities in two pairs of datasets : autumn vs summer, and plot 1 vs plot 2. Species richness was analysed by calculating species accumulation curves (SACs), that show the relationship between the cumulative number of species found and the sampling intensity (Colwell & Coddington 1994). SACs were calculated using R computer language (Ihaka & Gentleman 1996). Community structure was compared in two ways : (1) by calculating qualitative (SS) and quantitative (SN) Sorensen similarity indices (Magurran 1988); (2) by means of paired z-test, designed to compare number of pairvise observations from two communities influenced by a common factor (Eason, Coles & Gettinby 1986). Furthermore, the occurrence of each particular species in autumn vs summer baits, and in plot 1 vs plot 2 was compared using chi-squared test ; the presence/absence data was calculated from actual no. of strains observed among the total no. of samples taken in respective set of baits (Fowler, Cohen & Jarvis 2001).
RESULTS Among 1063 samples taken (8.9 per bait on average), 715 (67.3 %) yielded fungal cultures (the remaining 32.7 % were either surface-contaminated or sterile). From those, we subcultured 943 fungal strains representing 97 species, 82 (84.5%) of which were identified at least to genus (Table 1). The surface of the baits was usually colonized by many different fungal genotypes (Fig. 1a). Their penetration downwards into the wood was not extensive, and was limited to 3 cm at most according to visual estimates from five split summer baits. Wood that was encased by the bark appeared sound (Fig. 1b). The season had a pronounced impact on species richness of fungi colonizing freshly cut CWD on our
Fungi in coarse woody debris
490
Table 1. Fungi isolated from freshly cut stem sections (baits) distributed for 7 wk in two discrete plots (separated by about 100 m) within a Picea abies stand. Observation frequency: % in total amount of isolated strains/% of colonized sectionsa
Fungi Zygomycetes Mortierella gamsii M. isabellina M. ramanniana Unidentified sp.272 Unidentified sp.285 Unidentified sp.331 Unidentified sp.448 All Zygomycetes Ascomycetes and conidial fungi Apiospora montagnei Beauveria bassiana Capnodium sp.506 Chalara sp.400 Cistella acuum Cladosporium herbarum C. tenuissimum Cladosporium sp.452 Cylindrocarpon didymum C. lucidum Cytospora sp.554 Dipodascus sp.159 Epicoccum nigrum Geotrichum sp.180 Gibberella avenacea Glarea sp.537 Gyoerffyella sp.434 Hormonema dematioides Hypocrea sp.509 Hypoxylon serpens Lecytophora hoffmannii Lecytophora sp.13 Lecytophora sp.22 Leptodontidium elatius Mariannaea elegans Monilinia sp.269 Nectria fuckeliana N. radicicola N. viridescens Nectria sp.156 Nectria sp.170 Neurospora pannonica Ophiostoma cucullatum O. piceae Penicillium spinulosum Penicillium sp.446 Penicillium sp.514 Penicillium sp.545 Pesotum fragrans Phialocephala sp.35 Phialophora fastigiata P. malorum Phialophora sp.201 Phialophora sp.501 Phoma glomerata P. herbarum Phoma sp.552 Rosellinia desmazieressii Scleroconidioma sphagnicola Thysanopora penicillioides
GenBank accession no.
In plots 1 & 2 during October–November
July–August
Plot 1
Plot 2
Both seasons and plots
AY805541 AY805542 – AY805543 AY805544 – AY805545
0.2/1.7 3.9/21.7 5.1/31.7 0.2/1.7 0.2/1.7 –/– –/– 10.0/48.3
–/– 2.7/20.0 2.9/21.7 –/– –/– 0.8/6.7 0.6/5.0 7.0/36.7
0.2/1.4 3.2/24.3 4.5/32.9 0.2/1.4 0.2/1.4 0.7/5.7 0.2/1.4 9.1/51.4
–/– 3.5/16.0 2.9/18.0 –/– –/– –/– 0.6/4.0 7.1/30.0
0.1/0.8 3.3/20.8 3.9/26.7 0.1/0.8 0.1/0.8 0.4/3.3 0.3/2.5 8.4/42.5
AY805546 AY805547 AY805548 AY805549 AY805550 AY805551 AY805552 AY805553 AY805554 AY805555 AY805556 AY805557 AY805558 AY805559 AY805560 AY805561 AY805562 AY805563 AY805564 AY805565 AY805566 AY805567 AY805568 AY805569 AY805570 AY805571 AY805572 AY805573 AY805574 AY805575 AY805576 AY805577 AY805578 AY805579 AY805580 AY805581 AY805582 – AY805583 AY606297 AY805584 AY805585 AY805586 AY805587 AY805588 AY805589 AY805590 AY805591 AY805592 AY805593
–/– –/– –/– –/– 0.2/1.7 0.5/3.3 0.5/3.3 –*/– 6.3***/31.7 0.2/1.7 –/– 0.2/1.7 0.5/3.3 0.5/3.3 0.2/1.7 –/– –/– –**/– –/– 0.5/3.3 0.5/1.7 0.2/1.7 –/– 0.2/1.7 0.2/1.7 0.2/1.7 0.5/3.3 0.5/3.3 22.7***/83.3 1.2*/8.3 0.2/1.7 –/– –/– 2.6/15.0 –**/– –/– –/– –/– –**/– –/– 2.8*/16.7 0.2/1.7 0.5/3.3 –/– 0.2/1.7 0.9/3.3 –/– 6.3***/36.7 –***/– –/–
0.2/1.7 0.4/1.7 0.2/1.7 0.2/1.7 –/– –/– 0.2/1.7 1.0/5.0 0.4/3.3 –/– 0.2/1.7 –/– 0.2/1.7 –/– 0.4/3.3 0.2/1.7 0.2/1.7 1.6/13.3 0.8/6.7 1.0/6.7 –/– –/– 0.4/3.3 –/– 0.6/5.0 –/– 1.2/6.7 –/– 6.4/40.0 –/– –/– 0.2/1.7 0.6/5.0 3.9/18.3 1.6/11.7 0.8/5.0 0.2/1.7 0.2/1.7 1.6/6.7 0.4/3.3 0.8/6.7 0.6/5.0 –/– 0.2/1.7 –/– 0.2/1.7 0.2/1.7 0.4/3.3 2.5/20.0 0.4/3.3
0.2/1.4 –/– 0.2/1.4 0.2/1.4 –/– 0.2/1.4 0.2/1.4 0.8/4.3 1.2***/10.0 0.2/1.4 –/– 0.2/1.4 0.3/2.9 0.3/2.9 0.2/1.4 –/– 0.2/1.4 1.0/8.6 0.3/2.9 1.2/8.6 0.3/1.4 0.2/1.4 0.2/1.4 0.2/1.4 –**/– 0.2/1.4 0.5/4.3 0.3/2.9 13.4/61.4 0.7/5.7 0.2/1.4 –/– –*/– 3.3/18.6 0.7/4.3 0.7/4.3 0.2/1.4 –/– 0.8/2.9 –/– 1.2/8.6 0.7/5.7 0.3/2.9 0.2/1.4 0.2/1.4 0.8/4.3 –/– 3.0/21.4 1.5/11.4 0.2/1.4
–/– 0.6/2.0 –/– –/– 0.3/2.0 0.3/2.0 0.6/4.0 –/– 6.5/28.0 –/– 0.3/2.0 –/– 0.3/2.0 –/– 0.6/4.0 0.3/2.0 –/– 0.6/4.0 0.6/4.0 –/– –/– –/– 0.3/2.0 –/– 1.2/8.0 –/– 1.5/6.0 –/– 14.7/62.0 0.3/2.0 –/– 0.3/2.0 0.9/6.0 3.2/14.0 1.2/8.0 –/– –/– 0.3/2.0 0.9/4.0 0.6/4.0 2.6/16.0 –/– –/– –/– –/– –/– 0.3/2.0 3.2/18.0 1.2/8.0 0.3/2.0
0.1/0.8 0.2/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.2/1.7 0.3/2.5 0.5/2.5 3.1/17.5 0.1/0.8 0.1/0.8 0.1/0.8 0.3/2.5 0.2/1.7 0.3/2.5 0.1/0.8 0.1/0.8 0.8/6.7 0.4/3.3 0.7/5.0 0.2/0.8 0.1/0.8 0.2/1.7 0.1/0.8 0.4/3.3 0.1/0.8 0.8/5.0 0.2/1.7 13.9/61.7 0.5/4.2 0.1/0.8 0.1/0.8 0.3/2.5 3.3/16.7 0.8/5.8 0.4/2.5 0.1/0.8 0.1/0.8 0.8/3.3 0.2/1.7 1.7/11.7 0.4/3.3 0.2/1.7 0.1/0.8 0.1/0.8 0.5/2.5 0.1/0.8 3.1/20.0 1.4/10.0 0.2/1.7
During both seasons
R. Vasiliauskas and others
491
Table 1. (Cont.) Observation frequency: % in total amount of isolated strains/% of colonized sectionsa
Fungi Trichoderma polysporum Truncatella sp.511 Verticillium sp.199 Verticillium sp.438 Zalerion sp.460 Unidentified sp.305 Unidentified sp.457 Unidentified sp.488 Unidentified sp.490 Unidentified sp.538 Unidentified sp.543 Unidentified sp.555 All ascomycetes and conidial fungi Basidiomycetes Amylostereum chailletii Bjerkandera adusta Ceratobasidium sp.257 Collybia butyracea Coniophora arida Exidia pithya Heterobasidion spp. Hypholoma capnoides Hypochnicium geogenium Megacollybia platyphylla Mycena epipterygia M. galopus Mycena sp.480 Peniophora incarnata Phanerochaete magnoliae P. sordida Phlebia subserialis Phlebiella vaga Phlebiopsis gigantea Pholiota spumosa Resinicium bicolor Sistotrema brinkmannii S. sernanderi Sistotremastrum sp.558 Unidentified sp.499 Unidentified sp.512 Unidentified sp.529 Unidentified sp.547 All basidiomycetes No. of stem sections (baits) analysed Diameter of the stem sections (baits), mean¡S.D. (cm) Length of the stem sections (baits), mean¡S.D. (cm) No. of wood samples taken (per bait) No. (%) of samples from which fungi were isolated No. of strains isolated (per bait) No. of species defined a
GenBank accession no.
In plots 1 & 2 during October–November
July–August
Plot 1
Plot 2
Both seasons and plots
AY805594 AY805595 AY805597 AY805596 AY805598 AY805599 AY805600 AY805601 – AY805602 AY805603 –
9.5/45.0 –/– 0.2/1.7 –**/– –/– –**/– –/– –/– –/– –/– –/– –/– 58.9**/95.0
9.2/50.0 0.2/1.7 –/– 2.0/15.0 0.2/1.7 2.1/16.7 0.2/1.7 0.2/1.7 0.2/1.7 0.2/1.7 0.2/1.7 0.6/3.3 45.5/93.3
8.6/45.7 0.2/1.4 0.2/1.4 1.3/10.0 0.2/1.4 1.3/10.0 0.2/1.4 0.2/1.4 0.2/1.4 –/– –/– –*/– 48.4**/92.9
10.6/50.0 –/– –/– 0.6/4.0 –/– 0.9/6.0 –/– –/– –/– 0.3/2.0 0.3/2.0 0.9/4.0 57.4/96.0
9.3/47.5 0.1/0.8 0.1/0.8 1.1/7.5 0.1/0.8 1.2/8.3 0.1/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.1/0.8 0.3/1.7 51.6/94.2
AY805604 AY805605 AY805606 AY805607 AY805608 AY805609 – AY805610 AY805611 AY805612 AY805613 AY805614 AY805615 AY805616 AY805617 AY805618 AY805619 AY805620 – AY805621 AY805622 AY805623 AY805624 AY805625 AY805626 AY805627 – –
–/– –**/– 0.2/1.7 –*/– –/– –/– 0.2**/1.7 20.4***/71.7 1.2*/5.0 –/– –/– –*/– –/– 0.5/1.7 –/– –***/– –/– –/– –***/– 0.2/1.7 –***/– 7.9***/41.7 0.5/3.3 –/– –/– –/– –/– –/– 31.1***/91.7
0.2/1.7 1.4/10.0 0.2/1.7 1.2/8.3 0.6/3.3 0.2/1.7 1.6/10.0 2.5/21.7 –/– 0.6/3.3 0.2/1.7 1.2/6.7 0.4/3.3 –/– 0.2/1.7 4.5/31.7 0.2/1.7 0.4/1.7 13.9/55.0 0.2/1.7 14.5/68.3 2.3/16.7 –/– 0.2/1.7 0.2/1.7 0.4/3.3 0.2/1.7 0.2/1.7 47.5/96.7
–/– 1.2/8.6 0.2/1.4 1.0/7.1 0.5/2.9 0.2/1.4 1.0/7.1 12.4/55.7 0.8/4.3 0.5/2.9 –/– 0.5/2.9 0.3/2.9 0.3/1.4 0.2/1.4 1.8/12.9 0.2/1.4 0.3/1.4 8.1/31.4 0.2/1.4 8.1/35.7 4.1/30.0 –/– –/– 0.2/1.4 0.3/2.9 –/– –/– 42.5/95.7
0.3/2.0 –/– 0.3/2.0 –/– –/– –/– 0.9/4.0 7.6/34.0 –/– –/– 0.3/2.0 0.9/4.0 –/– –/– –/– 3.5/20.0 –/– –/– 6.5/22.0 0.3/2.0 7.4/32.0 6.2/28.0 0.6/4.0 0.3/2.0 –/– –/– 0.3/2.0 0.3/2.0 35.6/92.0
0.1/0.8 0.7/5.0 0.2/1.7 0.6/4.2 0.3/1.7 0.1/0.8 1.0/5.8 10.7/46.7 0.5/2.5 0.3/1.7 0.1/0.8 0.6/3.3 0.2/1.7 0.2/0.8 0.1/0.8 2.4/15.8 0.1/0.8 0.2/0.8 7.5/27.5 0.2/1.7 7.8/34.2 4.9/29.2 0.2/1.7 0.1/0.8 0.1/0.8 0.2/1.7 0.1/0.8 0.1/0.8 40.0/94.2
60
60
70
50
120
10.8¡1.4
11.1¡1.7
12.1¡0.7
9.5¡1.1
11.0¡1.6
13.5¡0.6
13.5¡1.0
13.7¡0.8
13.3¡0.8
13.5¡0.8
511 (8.5)
552 (9.2)
707 (10.1)
356 (7.1)
1063 (8.9)
333 (65.2)
382 (69.2)
460 (65.1)
255 (71.6)
715 (67.3)
431 (7.2) 42
512 (8.5) 76
603 (8.6) 78
340 (6.8) 52
943 (7.9) 97
During both seasons
Actual no. of strains of each species observed in total no. of samples (and not the percentages in total amount of strains, presented in the Table) were compared by chi-squared tests in two datasets: autumn vs summer, and plot 1 vs plot 2. Underlined percentage value indicates that the test was statistically significant for given species within the respective dataset. The levels of significance are shown as: *–P<0.05; **–P<0.01; ***–P<0.001.
Fungi in coarse woody debris
492
Table 2. Isolation of fungi from wood section (bait) SK-93 as a result of increased sampling effort and depth. Additional isolation, depth (cm) Fungi Bjerkandera adusta Gloeoporus taxicola Hormonema dematioides Nectria fuckeliana Ophiostoma piceae Paecilomyces lilacinus Peniophora piceae Phanerochaete sordida Phialophora fastigiata Phlebiopsis gigantea Phoma cava Resinicium bicolor Sarea difformis Stereum sanguinolentum Trametes versicolor Trichoderma polysporum Unidentified sp.19 Unidentified sp.90 No. of isolations attempted
GenBank accession no. AY805628 AY805630 AY805637 AY805639 – AY805631 AY805634 AY805629 AY805636 – AY805638 – AY805640 AY805632 AY805633 – AY805635 =
Initial isolation, depthf0.4 cm
f0.4
+*
+ +*
+*
+*+
0.9
1.8
3.0
4.5
6.9 +
+ + ++*
++ + +*
+*
+ + +++*
+*
+ +
++ +
+
+
++*
9
14
5
+* + + 5
+ +* + 5
+ +
5
5
Note: Underlined species were not isolated during the initial sampling, and are new for the whole community (not present in Table 1). +, isolation of single strain of a given fungus; *, strains vegetatively compatible with each other and representing one genetic individual of the fungus.
study sites. Thus, 511 samples taken in the autumn yielded 42 different species, whereas 552 taken in summer yielded 76 (Table 1, Fig. 2a). Decreasing sampling effort in summer baits to the same figure as in autumn baits (511) would give a calculated 73 different species (Fig. 2a). The difference between summer and autumn baits is statistically significant (chi-squared test ; P= 0.004). Thus our data indicate that the diversity of fungi colonizing wood within the same area of a northtemperate forest during summer months can be almost twice that observed during the late autumn (42.5 % higher). Autumn and summer communities differed considerably in species composition. As a result, qualitative and quantitative Sorensen similarity indices were only SS=0.36 and SN=0.34, respectively when different seasons are compared (Fig. 2a). A certain number of species were found only during one of the seasons. Thus we found 55 unique ‘ summer species’ and 21 ‘autumn species ’ (Table 1). Most conspicuous among the ‘summer species’ were the basidiomycetes Resinicium bicolor, Phlebiopsis gigantea, and Phanerochaete sordida, which dominated the community during July– Aug., but were completely absent during Oct.–Nov. (Table 1). Although other species, were present during both seasons, season had a significant impact on their frequency\ies of occurrence. Thus, the basidiomycetes Hypholoma capnoides and Sistotrema brinkmannii, together with the ascomycetes, Nectria viridescens and Rosselinia desmazieressii, and the conidial fungus Cylindrocarpon didymum, dominated in the community during the autumn months, but were seldom observed in summer. In contrast, the colonization frequency of
root rot basidiomycetes in the genus Heterobasidion decreased significantly in Oct.–Nov. (Table 1). In total, there were 21 species (22 %), the colonization frequency of which was significantly affected by season (Table 1). As a result, the community structures of fungi colonizing wood in summer and autumn differed significantly (z-test ; P<0.001). The species richness in plots 1 and 2, separated within the investigated plantation by about 100 m, was more or less the same. When sampling effort is kept to 356 in both plots, the estimated number of species recorded in plot 1 is 57 while 52 species were recorded in plot 2, a difference in species richness of less than 10 % (Fig. 2b). However, 64 species (66 % of the total) were found only in one of the plots (Table 1). As a result, qualitative Sorensen similarity index (SS) among the communities in plot 1 and plot 2 was only 0.49. The quantitative index, on the other hand, was higher (SN=0.63), indicating that the dominant species in the plots colonised wood to a similar extent (Fig. 2b). The results show that communities of wood-inhabiting fungi may differ significantly (z-test ; P<0.001) at a moderately local scale. Extended sampling and isolation from the bait SK-93 (39 new samples down to 7 cm depth) revealed 18 species, 14 of which were new for that particular bait, and nine of which were new for the whole community. This raises the total number of species encountered during the investigation to 106. The result is quite expected but also indicated by the raising species accumulation curves (Fig. 2). Even with the extended sampling of bait SK-93 our sampling is not at all exhaustive and certainly many other species of fungi
R. Vasiliauskas and others
493
80
No. of fungal species
70
(a) Summer
60 50 40
Autumn
30 20
SS=0.36 SN=0.34
10 0
0
100
200 300 400 No. of samples taken
500
80
(b)
70 No. of fungal species
Plot 1 60 50 Plot 2
40 30
SS=0.49 SN=0.63
20 10 0
0
100
200 300 400 500 No. of samples taken
600
700
Fig. 2. Sampling effort and species richness of fungi in Picea abies stem sections (baits) following 7 week exposure in north-temperate forest. The datasets are compared from the baits that were (a) exposed during July–August (summer) vs. October–November (autumn), and (b) distributed in two discrete plots (plot 1 and plot 2) separated by a distance of about 100 m. The structure of fungal communities is compared by qualitative (SS) and quantitative (SN) Sorensen indices of similarity.
are likely to inhabit baits at these plots in addition to those presented in the Table 1. Sectioning of the bait has revealed a high number of patchy areas of discoloured wood, which, however, were hard to map. Only three discrete decay columns were found to expand continuously throughout the examined half of the bait (Fig. 3). Several species were present inside each of the columns, but the isolates of the same species (e.g. Peniophora piceae, Trichoderma polysporum) were always vegetatively compatible (Fig. 3, Table 2). When originating from different discoloured areas inside the bait, the isolates were always incompatible. Initial and extended sampling yielded the same fungal genotypes of Hormonema dematioides, Ophiostoma piceae, and Phlebiopsis gigantea. (Table 2).
DISCUSSION We demonstrate that season and short distances inside a forest stand might have a profound impact on the
Fig. 3. Computerised three-dimensional picture of three discrete discoloration columns inside Picea abies stem section (approx. 8.5 cm in diameter) starting from the top. P.p., Peniophora piceae ; T.v., Trametes versicolor ; T.p., Trichoderma polysporum ; P.l., Paecilomyces lilacinus ; O.p., Ophiostoma piceae ; R.b., Resinicium bicolor ; S.s., Stereum sanguinolentum ; and B.a., Bjerkandera adusta. Approximate level from which each species was isolated within respective columns is indicated by the bars.
establishment of airborne wood-inhabiting fungi in CWD on north-temperate Picea abies forest sites. The much higher species richness observed during the summer months (76 species in summer vs 42 in autumn) probably reflects more colonisers of fresh wood sporulating in summer than in mid- to late autumn. Nevertheless, our data also revealed the presence of typical late season colonizers. We do not know if such species really are adapted to fruiting during the cooler period of the year, or if they are simply out-competed in summertime. While season had an impact on both species richness and similarity (Fig. 2a), local distance (ca 100 m) influenced community structure and not species richness, which remained more or less constant across the stand (Fig. 2b). The distinct difference in species composition between our plots indicates that the local spore source is an important determinant of fungal community structure. A study of Heterobasidion spp., common wood-decay polypore in the temperate zone (recorded also during the present work ; Table 1), demonstrated that the spore deposition gradient is very steep within the first 100 m from a fruit body (Stenlid 1994). A similar result was obtained in a study of corticioid wood-decomposer, Phlebia centrifuga (Norde´n & Larsson 2000). Recent work demonstrates the importance of landscape composition on spore deposition of
Fungi in coarse woody debris wood-inhabiting fungi, although on a larger (1–3 km) spatial scale (Edman et al. 2004a, b). Our data suggest that even freshly cut CWD contributes to mycodiversity on managed north temperate sites. However, our study was not designed to show how soon and to what extent (if at all) freshly cut wood could help in restoring primeval mycodiversity. An expanded long-term study close to old-growth sites with a known flora of saprophytic fungi would be desirable. Studies in tropical forest have shown that species richness of wood-inhabiting fungi returns to the level of the primary forest when the woody debris is retained and there is a patch of original forest in the vicinity (Lindblad 2002). In the present work the community structure of fungi within a single stand was found to differ significantly both over small distances and over different seasons. This implies that in order to sustain and enhance fungal diversity in managed stands some coarse wood should always be left wherever and whenever harvesting takes place. Freshly cut wood sustains a highly diverse fungal community after just a few weeks of exposure to airborne colonisation (Fig. 1a). Different species of basidiomycetes were found to co-exist in close proximity within the same small column of discolored wood (Fig. 3). In a similar experiment, high diversity was observed in aerial cut surfaces of Fagus sylvatica logs, where up to 30 mutually antagonistic fungal individuals of several species were found cmx3 (Coates & Rayner 1985a). Buried cross-sections, however, were colonised by a less diverse community (Coates & Rayner 1985b). Similarly, hundreds of fungal species were reported from stumps of spruce some weeks after they were cut and available for airborne colonisation (Woods 1996, Woodward 2003, Vasiliauskas et al. 2004). Stumps are in most cases the only source of CWD in managed stands but it should be remembered that a freshly cut stump is ecologically different from a stem section, since stumps remain attached to the root system for a considerable time (Redfern & Stenlid 1998). We demonstrated the ability of Mycena spp. and Collybia spp. to abundantly colonise freshly cut CWD (Table 1). In another study, Mycena galopus was found to inhabit living stems of pine (Lygis et al. 2004b). These findings contribute to a new picture of the ecology of those fungi. They were previously known primarily as decomposers of litter and of well decayed residues of wood, on which their fruiting bodies are commonly observed (Rodionova 1970, Stepanova 1975, Hansen & Knudsen 1992, Ryman & Holmasen 1998). We also discovered that several ‘combative ’ species are already present in freshly exposed substrate. According to Cooke & Rayner (1984), so called combative species have a secondary resource capture strategy : they arrive late, displace from the wood species that were already present and take over their previously colonised domain. The following species from Table 1 could be classified as combative : Hypholoma capnoides, Phanerochaete spp., Resinicium
494 bicolor, Sistotrema brinkmannii, and Trametes versicolor (Holmer & Stenlid 1996). It is likely that those species will be able to persist in baits for several years. In general, the basidiomycete macrofungi reported in this study are common throughout the forests of North Europe (Hansen & Knudsen 1992, Ryman & Holma˚sen 1998). Among 74 species of microfungi reported in the present work (Tables 1–2), many related species (belonging to at least the same genera) have been previously encountered in woody substrates in northtemperate and boreal forests. Thus, 44 (59 %) of those were found to inhabit stumps, snags and living trees of spruce, pine and birch in Sweden and Lithuania (Lygis et al. 2004a, b, Vasiliauskas et al. 2004, 2005). Among 60 microfungi that were identified (Tables 1–2), 27 (45 %) related species were isolated from spruce stumps in Scotland (Woods 1996), and thirty-seven (or 62%) from decomposing logs of Douglas-fir, spruce and aspen in North America (Crawford, Carpenter & Harmon 1990, Lumley, Gignac & Currah 2001). About 15% of species found remained unidentified (Tables 1–2). In other studies, where the identification was based on morphological characters of the mycelium, unidentified species comprised 25–35 % (Woods 1996, Lumley et al. 2001). This indicates that morphological identification of fungal cultures in many cases is difficult, but also that sequence coverage in GenBank and our local sequence databases is far from exhaustive. We can expect that some of these unidentified sequences represent unknown species especially among micromycetes with inconspicuous fruit bodies. It is estimated that the majority of fungi have not yet been isolated and identified (Kennedy & Clipson 2003). An example concerning wood-inhabiting species is a recent survey of microfungi from rotting wood in Canada, during which 49 species of ascomycetes were isolated, 15 of which were new for Canada, and seven were new for North America ; 20 species had not previously been reported from wood (Lumley, Abbot & Currah 2000, Sigler, Lumley & Currah 2000). During recent work in our laboratory four previously unknown species of dark septate fungi (Phialocephala spp.) have been detected in woody substrates (Menkis et al. 2004). The availability of comprehensive and well-documented sequence databases will markedely increase the efficacy of mycodiversity studies, in particular when analysing fungal DNA directly from environmental samples (Johannesson & Stenlid 1999, Vainio & Hantula 2000, Kennedy & Clipson 2003).
ACKNOWLEDGEMENTS We thank Olov Pettersson for technical assistance, and Stig Bardage for help in detailed wood analyses. This work was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Foundation for Strategic Environmental Research (MISTRA).
R. Vasiliauskas and others REFERENCES Altschul, S. F., Madden, T. L., Scha¨ffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402. Ammer, U. (1991) Konsequenzen aus den Ergebnissen der Totholzforschung fur die forstliche Praxis. Forstwissenschaftliches Centrablatt 110: 149–157. Bader, P., Jansson, S. & Jonsson, B. G. (1995) Wood-inhabiting fungi and substratum decline in selectively logged boreal spruce forests. Biological Conservation 72: 355–362. Bardage, S. L. (2001) Three-dimensional modelling and visualization of whole Norway spruce latewood tracheids. Wood and Fiber Science 33: 627–638. Coates, D. & Rayner, A. D. M. (1985a) Fungal population and community development in cut beech logs. I. Establishment via the aerial cut surface. New Phytologist 101 : 153–171. Coates, D. & Rayner, A. D. M. (1985b) Fungal population and community development in cut beech logs. II. Establishment via the buried cut surface. New Phytologist 101 : 173–181. Coates, D. & Rayner, A. D. M. (1985c) Fungal population and community development in cut beech logs. III. Spatial dynamics, interactions and strategies. New Phytologist 101: 183–198. Colwell, R. K. & Coddington, J. A. (1994) Estimating terrestrial biodiversity through extrapolation. Philosophical Transactions of the Royal Society of London, Series B 345: 101–118. Cooke, R. C. & Rayner, A. D. M. (1984) Ecology of Saprotropic Fungi. Longman, London. Crawford, R. H., Carpenter, S. E. & Harmon, M. E. (1990) Communities of filamentous fungi and yeast in decomposing logs of Pseudotsuga menziesii. Mycologia 82: 759–765. Czederpiltz, D. L. L. (2001) Forest management and the diversity of wood-inhabiting polyporoid and corticioid fungi. PhD thesis, University of Wisconsin-Madison. Eason, G., Coles, C. W. & Gettinby, G. (1986) Mathematics and Statistics for the Bio-Sciences. John Wiley, New York. Edman, M. (2003) Dispersal ecology of wood-decaying fungi – implications for conservation. PhD thesis, Umea˚ University. Edman, M., Gustafsson, M., Stenlid, J. & Ericson, L. (2004a) Abundance and viability of fungal spores along a forestry gradient – responses to habitat loss and isolation? Oikos 104: 35–42. Edman, M., Gustafsson, M., Stenlid, J., Jonsson, B. G. & Ericson, L. (2004b) Spore deposition of wood-decaying fungi: importance of landscape composition. Ecography 27 : 103–111. Fowler, J., Cohen, L. & Jarvis, P. (2001) Practical Statistics for Field Biology. John Wiley and Sons, Chichester. Gustafsson, M. H. (2002) Distribution and dispersal of wood-decaying fungi occurring on Norway spruce logs. PhD thesis, Swedish University of Agricultural Sciences, Uppsala. Hansen, A. J., Spies, T. A., Swanson, F. J. & Ohmann, J. L. (1991) Conserving biodiversity in managed forests. BioScience 41: 382–392. Hansen, L. & Knudsen, H. (eds) (1992) Nordic Macromycetes. Vol. 2. Polyporales, Boletales, Agaricales, Russulales. Nordsvamp, Copenhagen. Harmon, M. E., Franklin, J. F., Swanson, F. J., Sollins, P., Gregory, S. V., Lattin, J. D., Anderson, N. H., Cline, S. P., Aumen, N. G., Sedell, J. R., Lienkaemper, G. W., Cromack, K. J. & Cummins, K. W. (1986) Ecology of coarse woody debris in temperate ecosystems. Advances of Ecological Research 15: 133–302. Heilmann-Clausen, J. (2003) Wood-inhabiting fungi in Danish deciduous forests – diversity, habitat preferences and conservation. PhD thesis, The Royal Danish Veterinary and Agricultural University, Lyngby. Høiland, K. & Bendiksen, E. (1997) Biodiversity of wood-inhabiting fungi in a boreal coniferous forest in Sor-Trondelag County, central Norway. Nordic Journal of Botany 16: 643–659.
495 Holmer, L. & Stenlid, J. (1996) Diffuse competition for heterogenous substrate in soil among six species of wood-decomposing basidiomycetes. Oecologia 106 : 531–538. Hudson, H. J. (1968) The ecology of fungi on plant remains above the soil. New Phytologist 67: 837–874. Humphrey, J. W., Newton, A. C., Peace, A. J. & Holden, E. (2000) The importance of conifer plantations in northern Britain as a habitat for native fungi. Biological Conservation 96: 241–252. Ihaka, R. & Gentleman, R. (1996) R: A language for data analysis and graphics. Journal of Computer Graphics and Statistics 5: 299–314. Johannesson, H. & Stenlid, J. (1999) Molecular identification of wood-inhabiting fungi in an unmanaged Picea abies forest in Sweden. Forest Ecology and Management 115: 203–211. Ka˚ren, O., Ho¨gberg, N., Dahlberg, A., Jonsson, L. & Nylund, J. E. (1997) Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytologist 136: 313–325. Kauserud, H. & Schumacher, T. (2002) Population structure of the endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota). Canadian Journal of Botany 80: 597–606. Kauserud, H. & Schumacher, T. (2003) Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum. Mycologia 95: 416–425. Kennedy, N. & Clipson, N. (2003) Fingerprinting the fungal community. Mycologist 17 : 158–164. Kruys, N. (2001) Bryophytes and fungi on dead spruce: integrating conservation with forest management planning. PhD thesis, Umea˚ University. Kruys, N., Fries, C., Jonsson, B. G., Lamas, T. & Stahl, G. (1999) Wood-inhabiting cryptogams on dead Norway spruce (Picea abies) trees in managed Swedish boreal forests. Canadian Journal of Forest Research 29: 178–186. Kruys, N. & Jonsson, B. G. (1999) Fine woody debris is important for species richness on logs in managed boreal spruce forests of northern Sweden. Canadian Journal of Forest Research 29: 1295–1299. Lindblad, I. (1998) Wood-inhabiting fungi on fallen logs of Norway spruce: relations to forest management and substrate quality. Nordic Journal of Botany 18 : 243–255. Lindblad, I. (2002) Wood-inhabiting fungi in primary and secondary seasonally dry tropical forest, Costa Rica. In Book of Abstracts of the 7th International Mycological Congress, Oslo 11–17 August 2002: 164–164. University of Oslo, Oslo. Lumley, T. C., Abbott, S. P. & Currah, R. S. (2000) Microscopic ascomycetes isolated from rotting wood in the boreal forest. Mycotaxon 74: 395–414. Lumley, T. C., Gignac, D. & Currah, R. S. (2001) Microfungus communities of white spruce and trembling aspen logs at different stages of decay in disturbed and undisturbed sites in the boreal mixedwood region of Alberta. Canadian Journal of Botany 79: 76–92. Lygis, V., Vasiliauskas, R. & Stenlid, J. (2004a) Planting Betula pendula on pine sites infested by Heterobasidion annosum: disease transfer, silvicultural evaluation, and community of woodinhabiting fungi. Canadian Journal of Forest Research 34 : 120–130. Lygis, V., Vasiliauskas, R., Stenlid, J. & Vasiliauskas, A. (2004b) Silvicultural and pathological evaluation of Scotts pine afforestations mixed with trees to reduce the infections by Heterobasidion annosum. Forest Ecology and Management 201: 275–285. Magurran, A. E. (1988) Ecological Diversity and Its Measurement. Princeton University Press, Princeton. Maser, C. & Trappe, J. M. (1984) The seen and unseen world of the fallen tree. General Technical Report of the USDA Forest Service GTR-PNW-164: 1–56. McComb, W. & Lindenmayer, D. (1999) Dying, dead, and down trees. In Maintaining Biodiversity in Forest Ecosystems (M. L. Hunter, ed.): 335–372. Cambridge University Press, Cambridge, UK.
Fungi in coarse woody debris Menkis, A., Allmer, J., Vasiliauskas, R., Lygis, V., Stenlid, J. & Finlay, R. (2004) Ecology and molecular characterization of dark septate fungi from roots, living stems, coarse and fine woody debris. Mycological Research 108 : 965–973. Norde´n, B. & Larsson, K. H. (2000) Basidiospore dispersal in the oldgrowth forest fungus Phlebia centrifuga. Nordic Journal of Botany 20: 215–219. Ohlson, M., Soderstrom, L., Hornberg, G., Zackrisson, O. & Hermansson, J. (1997) Habitat qualities versus long-term continuity as determinants of biodiversity in boreal old-growth swamp forests. Biological Conservation 81 : 221–231. Redfern, D. B. & Stenlid, J. (1998) Spore dispersal and infection. In Heterobasidion annosum: biology, ecology, impact and control (S. Woodward, J. Stenlid, R. Karjalainen & A. Hu¨ttermann, eds): 105–124. CAB International, Wallingford. Renvall, P. (1995) Community structure and dynamics of woodrotting Basidiomycetes on decomposing conifer trunks in northern Finland. Karstenia 35: 1–51. Rodionova, S. V. (1970) [Fungal flora of myrtillus type spruce stands of south Karelia, and its role in mineralization of needle litter.] PhD thesis, University of Petrozavodsk. [In Russsian.] Ryman, S. & Holmasen, I. (1998) Svampar: en fa¨lthanbok. 2nd edn. Interpublishing, Stockholm. Samuelsson, J., Gustafsson, L. & Ingelog, T. (1994) Dying and Dead Trees: a review of their importance for biodiversity. Swedish Threatened Species Unit, Uppsala. Sigler, L., Lumley, T. C. & Currah, R. S. (2000) New species and records of saprophytic ascomycetes (Myxotrichaceae) from decaying logs in the boreal forest. Mycoscience 41: 495–502. Siitonen, J. (2001) Forest management, coarse woody debris and saproxylic organisms: Fennoscandian boreal forests as an example. Ecological Bulletins 49: 11–41. Sippola, A.-L., Lehesvirta, T. & Renvall, P. (2001) Effects of selective logging on coarse woody debris and diversity of wooddecaying polypores in eastern Finland. Ecological Bulletins 49: 243–254. Stenlid, J. (1994) Regional differentiation in Heterobasidion annosum. Proceedings of the Eight International Conference on root and butt rots, Sweden and Finland, August 9–16, 1993 (M. Johansson & J. Stenlid, eds): 243–248. Swedish University of Agricultural Sciences, Uppsala.
496 Stepanova, O. A. (1975) [Fungi on felling residues in spruce forests of Leningrad district II.] Mikologiya i Fitopatologiya 9: 15–20. [In Russian]. Thor, M. (1997) Stump treatment against Heterobasidion annosum – techniques and biological effect in practical forestry. Licentiate’s thesis, Swedish University of Agricultural Sciences, Uppsala. Utschick, H. (1991) Beziehungen zwischen Totholzreichtum und Vogelwelt in Wirtschaftswaldern. Forstwissenschaftliches Centrablatt 110 : 135–148. Vainio, E. & Hantula, J. (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycological Research 104: 927–936. Vasiliauskas, R., Larsson, E., Larsson, K.-H. & Stenlid, J. (2005) Persistence and long-term impact of Rotstop biological control agent on mycodiversity in Picea abies stumps. Biological Control 32: in press. Vasiliauskas, R., Lygis, V., Thor, M. & Stenlid, J. (2004) Impact of biological (Rotstop) and chemical (urea) treatments on fungal community structure in freshly cut Picea abies stumps. Biological Control 31: 405–413. Vasiliauskas, R. & Stenlid, J. (1998a) Fungi inhabiting stems of Picea abies in a managed stand in Lithuania. Forest Ecology and Management 109 : 119–126. Vasiliauskas, R. & Stenlid, J. (1998b) Influence of spatial scale on population structure of Stereum sanguinolentum in northern Europe. Mycological Research 102 : 93–98. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a guide to methods and applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 315–322. Academic Press, San Diego. Woods, C. M. (1996) The fungal ecology of Sitka spruce stumps. PhD thesis, University of Aberdeen. Woodward, S. (2003) Diversity in monocultures : the Sitka spruce stump. Root and Butt Rots of Forest Trees: Proceedings of the IUFRO Working Party 7.02.01, Quebec, Canada, September 16–22, 2001 (G. Laflamme, J. A. Berube & G. Bussieres, eds): 47–54. Laurentian Forestry Centre, Quebec. Corresponding Editor: D. J. Lodge