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Wood-disk traps provide a robust method for studying spore dispersal of wood-decaying basidiomycetes

     Department of Ecology and Environmental Science, Umeå University, SE-901 87 Umeå, Sweden

Mårten Gustafsson

     Department of Forest Mycology and Pathology, Swedish Agricultural University, Box 7026, SE-750 07 Uppsala, Sweden

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS LITERATURE CITED

 

Spore traps consisting of disks containing monokaryotic mycelia as bait were tested to find a robust, long-time sampling method for studying dispersal of wood-decaying basidiomycetes. In total, 288 disks, 48 for each of six fungal species, were exposed 2 wk at 12 sites in northern Sweden. Both common and rare fungi were used, and the longest distance to a potential dispersal source exceeded 3 km. After 3–16 wk of incubation in the laboratory, the disks were investigated for spore hits. These were detectable both microscopically, by the presence of hyphal clamps, and macroscopically, by mycelial incompatibility zones. Spore traps resisted rain and freezing temperatures well, and spore hits from all species were found at all 12 sites. We argue that lengthy sampling makes it possible to detect low rates of spore deposition, aiding in the study of long-distance dispersal and dispersal of rare species. In addition, because several spore hits can be recognized in the same trap, spore deposition of wood-decaying fungi can be characterized with quantitative data.

Key words: disks, dispersal, monokaryotic mycelia, somatic incompatibility, wood-decaying basidiomycetes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS LITERATURE CITED

 
Dispersal is an important determinant of regional abundance and persistence of species. Thus, information about dispersal abilities is important in the understanding of species distribution patterns, genetics and population dynamics. Species' ability to disperse spores is crucial to the establishment of new populations and to preventing loss of genetic variation in small, geographically isolated populations (Young et al 1996). Consequently, knowledge of dispersal abilities is necessary when designing conservation and restoration plans for threatened species. In addition, data on dispersal are needed when studying pathogenic fungi. However, the long-distance dispersal ability (>1 km) of many basidiomycetes still is poorly known, mainly due to insufficient sampling methods. One problem when studying long-distance dispersal is the lack of a method to detect low concentrations of airborne spores.

Studies of spore dispersal fall into three main categories, where capture of specimens is concerned: those in which spores are caught on a sticky surface (e.g., Hirst 1952, Lacey 1996); those in which spores are collected on disks (Risbeth 1959, Kallio 1970, Möykkynen et al 1997); and those in which spores are collected with species-specific monokaryotic mycelia (Adams et al 1984, Williams et al 1984, Vilgalys and Sun 1994, Nordén and Larsson 2000, Hallenberg and Küffer 2001). The first category requires that spores are easily identified by their appearance, which is rarely possible with basidiospores. Similary, disks can be used only for species with easily recognized mycelia (Risbeth 1959, Kallio 1970, Möykkynen et al 1997). In the case of some wood-decaying basidiomycetes, spore dispersal instead has been studied, using monokaryotic mycelia grown on agar as bait (Adams et al 1984, Williams et al 1984, Nordén and Larsson 2000, Hallenberg and Küffer 2001). The latter method assumes that actively growing monokaryotic mycelium will limit the establishment and growth of spores from other species, while allowing species-specific spores to establish and mate with the monokaryotic mycelium, giving rise to a dikaryotic mycelium (Adams et al 1984). This holds true, providing that the mycelia are of different mating types. With this method, a spore hit is indicated by the formation of a dikaryotized mycelium, which is identified by the presence of clamps or other morphological differentiation of the mycelium. This method was first described by Adams et al (1984) and since has been used to describe spore deposition and small-scale dispersal patterns of several wood-decaying basidiomycetes (Adams et al 1984, Williams et al 1984, Vilgalys and Sun 1994, Nordén and Larsson 2000).

However, using monokaryotic mycelia grown on agar has some major restrictions when studying long-distance dispersal. First, since the number of deposited spores will decline with increasing distance from the parent, mire exposure time is needed. However, agar spore traps are contaminated easily when exposed for lengthy periods (Vilgalys and Sun 1994). Second, the agar is sensitive to rain, drought and frost, factors that also restrict its use, especially in relation to the time of exposure (pers obs). In fact, agar's sensitivity to frost renders it extremely difficult to use in boreal areas during the main period of spore release. Owing to these factors, this method has been used only to study the deposition of spores up to 1 km from the source, with a maximum time of exposure of 48 h (see however Hallenberg and Küffer 2001). The agar method is useful in that spatial and temporal parameter. However, sensitivity to rain, drought, frost and contamination requires the daily exchanges of agar plates, which results in the use of a large number of agar plates during lengthy studies. Furthermore, it often is difficult to separate different spore hits directly from the agar, which implies that results from agar spore traps usually are reported as qualitative data, i.e., presence or absence of spores. The only exception that we are aware of is that of Williams et al (1984). Still, by pairing hyphal subcultures from dikaryotized agar spore traps, it is possible to identify separate spore hits as indicated by somatic incompatibility (Vilgalys and Sun 1994, Adams et al 1984). However, this requires extensive laboratory work and might be cumbersome for large field experiments.

The purpose of this study was to test spore traps consisting of monokaryotic mycelia grown on disks instead of agar. This was done to find a robust method that can be used for sampling spores over long time periods and for studies of long-distance dispersal of wood-decaying basidiomycetes.

	MATERIALS AND METHODS

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Spore traps and species tested – Fresh, healthy trees of Norway spruce [Picea abies (L.) Karst.] were cut into 0.9 cm thick disks with a diameter of 8 cm. The disks were soaked with de-ionized water and autoclaved 2 x 30 min at 120 C. Under sterile conditions, each disk was, put in a 9-cm-diameter Petri dish and inoculated with a piece (ca 0.5 x 0.5 cm) of Hagem agar (Stenlid 1985) with a 2-wk-old culture of a monokaryotic mycelium. Petri dishes, holding the inoculated disks, were put in sterile plastic bags and incubated 2 mo at room temperature in the dark. The cultures of monokaryotic mycelia originated from spores collected from fruit bodies in Västerbotten county, northern Sweden. Only one monokaryotic mycelium per species was used to avoid the risk of inadvertent dikaryotization due to contamination during laboratory and field procedures. The monokaryotic mycelia used were the ones that most easily dikaryotized when pairing several monokaryotic strains in the laboratory (cf. Williams et al 1984).

Six species of wood-decaying basidiomycetes growing on dead conifers were used for the tests (Table I). Only species that form clamps during dikaryotization were chosen. To get a large range in the number of spore hits, both common and rare fungi were used. Fomitopsis pinicola is a common generalist fungus, whereas Gloeoporus taxicola is considered fairly common in northern Europe (Ryvarden and Gilbertson 1993). Fomitopsis rosea, Trichaptum laricinum, Cystostereum murraii and Phlebia centrifuga are rare and considered threatened in several northern European countries (Larsson 1997, Gärdenfors 2000). These four fungi are found almost exclusively in old-growth forests (Larsson 1997).

Fomitopsis pinicola, F. rosea and P. centrifuga developed dense monokaryotic aerial mycelia that mostly covered the surface of the disk after 2 mo incubation. Trichaptum laricinum and C. taxicola developed looser aerial mycelia that did not totally cover the surface of the disk. The loosest aerial mycelium was developed by C. murraii and was hardly visible, but still, the disks were decayed, as indicated by a clear, dark change in color.

Field methods and laboratory techniques – To obtain a range in the number of spore hits and to increase the probability of trapping spores from the rare species, several well-separated sites were sampled. On Aug. 11, 1999, spore traps were placed at 12 sites in Västerbotten county, northern Sweden. The sites were separated by a minimum distance of 6 km and located within mature forests. The amount of coniferous forest >80 yr old within 2 km radius (12, 6 km²) of the sites ranged between 0.2 and 3.9 km² (that is 1.6–30.9%). To avoid over representation of closely located fruit bodies, all dying and dead trees within the nearest 400 m radius of the sites were investigated carefully for fruit bodies of the studied species. Only those sites without such fruit bodies were accepted. In addition, in the three most deforested landscapes in the study, all spruce within a 3-km radius of the sampling sites were carefully searched for F. rosea, P. centrifuga and C. murraii. No fruit bodies of these three species were found in those three landscapes. At each of the 12 sites, four spore traps were used for each species, for total of 48 traps per species. The disks were placed on plastic nets (3 x 3 cm mesh) that covered open plastic boxes (90 x 18 x 12 cm). To reduce the risk of drying, the plastic boxes were half-filled with water. Further, to prevent flooding, holes were drilled in the upper part of the plastic box.

After 2 wk the disks were collected and placed in 9 cm Petri dishes. Deionized water was added to disks that were desiccated, and all Petri dishes were put in sterile plastic bags and incubated at room temperature in the dark. After 4–16 wk, aerial mycelia on the disks were scored for the presence of dikaryotic mycelia. This was done both microscopically and macroscopically. Using a light microscope (x 300), small samples of mycelia were searched for clamps. Mycelia were visually inspected for somatic incompatibility, as inferred from mycelial incompatibility zones (Worral 1997).

	RESULTS

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Spore hits – Spore hits, as indicated by dikaryotic mycelia, were found for all six species at all 12 sites. Accordingly, spore hits of the rare fungi F. rosea, P. centrifuga and C. murraii were found at all three sample sites where the distance to the nearest fruit body of these species exceeded 3 km. The percentage of disks that were hit ranged between 79–100% depending on species. The total number of spore hits per disk ranged between 0–40 (Table I). Incompatibility zones between different mycelia, originating from different spores, were mostly distinct and easily recognized (Fig. 1). However, in the case of both T. laricinum and C. murraii a maximum of only one successful establishment per disk was recognized. In the case of C. murraii, it is likely that incompatibility zones were missed due to loose aerial mycelia. T. laricinum is rare, which partly might explain the low number of spore hits and the absence of mycelial incompatibility zones. Although not tested in this study, isolation and subsequent pairings of the dikaryotized mycelia could be examined to determine the number of dikaryons actually formed by C. murraii and T. laricinum. Most spore hits were detected for the common generalist fungus F. pinicola, with a mean of 19.5 hits per disk. This is probably an underestimation of the total number of spore hits because of a likely limit in the number of successful spore establishments per disk. Thus the probability of successful establishment will decline under high spore densities. In addition, when large numbers of spores land and germinate on a woody surface, the lines of antagonisms might be suppressed. Thus, the accuracy of the estimates of spore deposition per disk probably will decline with increasing number of spore hits. One way to control the problem with spore-hit saturation is to vary the time of exposure among batches of spore traps. Another reason why the visible spore hits represent a minimum number is that a monokaryotic mycelium might not dikaryotize with all spores due to genetic incompatibility. In addition, incompatibility usually is weaker and less frequently expressed among closely related dikaryons (Worral 1997). Thus, using several monokaryons of different mating types should improve the interpretation of data in field studies.

View larger version (105K): [in this window] [in a new window]   FIG. 1. Wood-discs with mycenai incompatibility zones indicating different spore-hits for P. centrifuga (left) and F. rosea (right). Two spore-hits are identifiable for P. centrifuga and seven for F. rosea.

Contamination – The disks resisted the effects of drought and rain well. Although exposed 2 wk, the percentage of the disks that were contaminated was low and ranged between 0–21%, depending on species (Table I). Contamination exclusively consisted of molds, which grew on the basidiomycete mycelia. Because molds might affect dikaryotization of the basidiomycete, contaminated disks were excluded. The degree of contamination seemed to be linked to the density and vitality of the aerial mycelia. Cystostereum murraii, which developed loose aerial mycelia, was the species with the highest degree of contamination (Table I). In contrast, F. rosea, F. pinicola and P. centrifuga, which developed dense aerial mycelia covering the disk, rarely were contaminated (Table I). Trichaptum laricinum and C. taxicola developed fairly dense aerial mycelia and displayed intermediate contamination.

Potential applications – Disks containing monokaryotic mycelia constitute a robust method for studying different aspects of dispersal in wood-decaying fungi. The method permits a lengthy sampling, which is needed to quantify low concentrations of depositing spores. Thus, the method is suitable for studies of long-distance dispersal and dispersal of rare species. In addition, the benefits of lengthy sampling and, therefore, the opportunity to easily recognize several hits on the same trap assist in the collection of quantitative data that integrates variation in deposition over time. By varying the time of exposure of the disks depending on species studied, it should be possible to account for the effects of spore-hit saturation.

Combined with inventories of the spatial distribution of different species at a landscape scale, the quantitative data from spore-trapping experiments might be used to create dispersal models for populations at a landscape level. Another possibility is the use of wood-disk spore trapping as a complement to the collection of extensive species inventories and for monitoring species abundance over time. As suggested by Vilgalys and Sun (1994), spore trapping also might be used to sample genetic diversity and to study gene flow in natural populations.

	FOOTNOTES

Accepted for publication November 21, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS LITERATURE CITED

 
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Risbeth M., 1959 Dispersal of Fomes annosus Fr. and Peniophora gigantea (Fr.) Masse. Trans Brit Mycol Soc 42:243-260

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Stenlid J., 1985 Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility and isoenzyme patterns. Can J Bot 63:2268-2273

Vilgalys R, Sun B L., 1994 Assessment of species distributions in Pleurotus based on trapping of airborne basidiospores. Mycologia 86:270-274

Williams END, Todd NK, Rayner ADM., 1984 Characterization of the spore rain of Coriolus versicolur and its ecological significance. Trans Br Mycol Soc 82:323-326

Worral JJ., 1997 Somatic incompatibility in basidiomycetes. Mycologia 89:24-36

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