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Keywords:

  • Armillaria cepistipes;
  • Armillaria ostoyae;
  • Picea abies (Norway spruce);
  • population structure;
  • genets;
  • spatial pattern;
  • interaction;
  • soilborne fungi

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The preferentially saprotrophic basidiomycete Armillaria cepistipes and the pathogenic Armillaria ostoyae occur sympatrically in many European forests.
  • • 
    The spatial population structure of both Armillaria species was investigated in three 1-ha plots established in managed Norway spruce (Picea abies) forests in the Swiss Alps (1400 m asl). A total of 740 Armillaria isolates, 296 from rhizomorphs in the soil and 444 from the stumps, were recovered and identified to species and genets (somatic incompatibility groups).
  • • 
    The incidence of A. cepistipes and A. ostoyae varied greatly among the plots. Two to six genets of each Armillaria species were identified and mapped in each plot. Genets of the same species overlapped rarely and only on the borders. Large spatial overlaps between A. cepistipes and A. ostoyae genets were observed in the plot with the highest incidence of fragmented genets. In five subplots (c. 0.1 ha) established along interspecific boundaries, overlaps were found in all three plots.
  • • 
    Our study suggests a strong intraspecific competition in both Armillaria species. Evidence for competition between the two species, as indicated by spatial mutual exclusion, was only observed on parts of the investigated area.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The majority of recent studies on the ecology of basidiomycetes are based on population structure analysis. Using the phenomenon of somatic incompatibility (Guillaumin et al., 1996; Worrall, 1997; Malik & Vilgalys, 1999), it is possible to identify genetically distinct secondary mycelia of the same species coexisting within a population. These mycelia are called genets and can be considered the units of a population. Knowledge about the size and spatial distribution of genets can provide important information on the processes of establishment and growth of a particular fungal population (Kirby et al., 1990; Worrall, 1994). The population structure of several basidiomycetes has been studied, including that of Heterobasidion annosum (e.g. Swedjemark & Stenlid, 1993), Phellinus weirii (e.g. Hansen & Goheen, 2000), Fomitopsis pinicola (Högberg et al., 1999), Resinicium bicolor (Kirby et al., 1990), Marasmius androsaceus (Holmer & Stenlid, 1991), Suillus bovinus (e.g. Dahlberg & Stenlid, 1994), and Armillaria spp. (e.g. Worrall, 1994).

The genus Armillaria comprises several species which are important components of the mycoflora in many forest ecosystems worldwide (Shaw & Kile, 1991). Armillaria spp. can behave as primary or secondary pathogens causing root and butt rot on numerous coniferous and broadleaved tree species in both naturally regenerated forests and plantations (Guillaumin et al., 1993; Morrison et al., 2000). As parasites, Armillaria spp. can cause significant economic losses (Morrison & Mallett, 1996) and influence the tree species composition of forests (Kile et al., 1991). In addition, all Armillaria species can survive saprotrophically in woody substrates such as roots, stumps and debris. To date, about 40 species of Armillaria are known, seven of which occur in Europe (Watling et al., 1991). The European species differ in geographical distribution, ecological behaviour, host range, and pathogenicity (Guillaumin et al., 1993).

Different species of Armillaria frequently coexist in the same forest stand (Rizzo & Harrington, 1993; Legrand et al., 1996; Bruhn et al., 2000; Marxmüller & Holdenrieder, 2000). In central Europe, the pathogenic Armillaria ostoyae (Romagnesi) Herink and the preferentially saprotrophic Armillaria cepistipes Velenovsky often occur sympatrically in mountainous forests (Legrand et al., 1996; Marxmüller & Holdenrieder, 2000). Armillaria species are probably significant competitors of the root and butt rot pathogen Heterobasidion annosum (Holdenrieder & Greig, 1998) and they also compete with each other for resources (S. Prospero et al. unpublished). Therefore, potential agents for the biological control of H. annosum and parasitic Armillaria species may be primarily saprotrophic Armillaria species and their interaction with these fungi deserves attention.

The purpose of the present study was to analyse the population structure of A. cepistipes and A. ostoyae coexisting in three managed Norway spruce (Picea abies (L.) Karst.) stands in the Alps. Specifically, we addressed the following questions: (i) What is the spatial distribution of the genets of the two species?; (ii) Are there overlaps between genets of the same or of different species?; and (iii) What are the spatial patterns of genets along interspecific boundaries? Considering these aspects, inferences are made about intra- and interspecific interactions.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study sites

The three study sites, Ludiano (46°25′26″ N, 8°56′28″ E), Lurengo (46°30′5″ N, 8°44′55″ E), and Dalpe (46°27′50″ N, 8°47′25″ E) are located in naturally regenerated and managed Norway spruce stands in the southern Swiss Alps at about 1400 m asl and 5–17 km apart. The stands are comparable in vegetation type (spruce-fir forest), tree age (140–160 yr), and past management practice.

In each stand, a 100 m × 100 m (1 ha) plot was established in summer 1999 containing numerous 1- to 3-yr-old-stumps. Tree species were recorded and all stumps over 12 cm in diameter were mapped.

Sampling

In each plot, a systematic sampling was conducted by taking a cube of soil (15 cm side) at each point in a 10 m × 10 m square grid. If no rhizomorphs were found in the systematic sample, additional soil samples (max. 4) were taken within 2 m from the grid points. The soil samples were sieved through a 9-mm square mesh to separate the roots and rhizomorphs from the soil. All rhizomorphs were collected and brought to the laboratory for isolation.

All stumps present in the plots were examined for Armillaria colonisation as follows: portions of bark were removed from the collar region of three main lateral roots using an axe and the presence of subcortical mycelial fans or rhizomorphs was recorded. For isolation, pieces of wood with mycelial fans or rhizomorphs on the surface were brought to the laboratory.

Subplots

After determining the distribution of A. cepistipes and A. ostoyae genets in the soil, five subplots (12 m × 12–14 m) were established within the three plots (one in Ludiano and two each in Lurengo and Dalpe) at the borders of two Armillaria species or where they overlapped in the soil (Fig. 1a–d). In each subplot, a systematic sampling was conducted by taking a cube of soil (15 cm side) at each point of a 1.4 m × 1.4 m square grid (61–72 samples per subplot).

image

Figure 1. Spatial distribution of genets of Armillaria cepistipes (B, blue), Armillaria ostoyae (C, red) and Armillaria gallica (E, green) in Ludiano (a), Lurengo (b), and Dalpe (c and d): triangular symbols indicate soil samples with one genet (solid triangles); square symbols indicate soil samples with two genets of the same species (squares with crosses) or with genets of different species (squares with dots inside); circular symbols indicate stumps colonised by one genet (solid circles), by two genets of the same species (open circles with crosses inside), and by genets of different species (open circles with dots inside); x, soil samples without rhizomorphs; arrow, slope direction (downhill). Black rectangles indicate the location of the subplot(s) in each plot.

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Isolation of Armillaria spp.

The collected rhizomorphs were first dipped in 50% ethanol for 15–20 s. Then, from each rhizomorph three segments of 1 cm length were surface sterilised in 30% hydrogen peroxide (H2O2) for 25–40 s (depending on the thickness of the rhizomorphs) and placed on a Petri plate (diameter 8.5 cm) containing malt extract agar (12 g l-1 malt extract; 15 g l-1 Bacto Agar) amended with 2 mg l-1 benomyl and 100 mg l-1 streptomycin (Maloy, 1974).

The pieces of wood with subcortical mycelial fans were washed under running tap water with a brush and blotted dry between paper towels. For each sample, six small pieces (2–5 mm × 2–5 mm) of mycelium were surface sterilised in sodium-hypochlorite (7% active chlorine; Chemische Fabrik Schweizerhalle, CH-4013 Basel) for 5–10 s and rinsed in sterile distilled water for 10–15 s (Prospero et al., 1998). The samples were dried between paper towels and placed on MATS plates (20 g l-1 malt extract; 15 g l-1 Bacto Agar; 230 mg l-1 thiabendazole in 1 ml concentrated lactic acid; 100 mg l-1 streptomycin) modified according to Legrand & Guillaumin (1993).

All isolation plates were incubated in the dark at 20–25°C. After one to three weeks, pure cultures were transferred to malt extract agar (15 g l-1 Bacto Agar; 20 g l-1 Diamalt, Hefefabriken AG, CH-3324 Hindelbank).

Identification of Armillaria genets and species

Genets were identified by pairing diploid isolates on Shaw & Roth's medium as described by Harrington et al. (1992). Mycelial plugs (3–4 mm side), cut from the margin of growing cultures, were placed c. 5 mm apart onto the agar surface and incubated in the dark at 20–25°C. Three pairings were performed in each Petri plate (diameter 8.5 cm). After 3 wk, somatic incompatibility reactions between the isolates were classified according to Rizzo & Harrington (1993). Each pairing was repeated twice and each isolate was also self-paired. To reduce the number of pairings, we first paired 8–10 isolates of each plot in all combinations to identify some genets. The other isolates were then paired with the nearest isolate representing an identified genet. Finally, the isolates which could not be assigned to an identified genet were paired in all combinations. Subcultures representing each genet were transferred to malt extract agar and stored at 4°C in the dark. The genets were designated as follows: plot (Lud = Ludiano, Lur = Lurengo, and Da = Dalpe), species (B = A. cepistipes, C =A. ostoyae, and E =Armillaria gallica Marxmüller & Romagnesi), and genet number.

Species identification was performed by pairing three isolates (if available) of each genet with three haploid tester strains (Korhonen, 1978) of the five European annulate Armillaria species as described by Harrington et al. (1992). The tester strains were kindly provided by J.-J. Guillaumin (INRA, Clermont-Ferrand, France). In addition, all genets were also identified to species with PCR-RFLP analysis of a portion of the intergenic spacer (IGS) region of the ribosomal DNA (Harrington & Wingfield, 1995). The PCR-products were digested using the restriction enzymes AluI, HincII, Mva 1269 I (BsmI), and NdeI (MBI Fermentas). Three isolates (if available) of each genet were analysed with the PCR-RFLP method.

Estimation of genet boundaries, sizes, and spatial overlaps

An isolate recovered from a point on the 10 m × 10 m sample grid was considered representative for a genet area of 100 m2 (corresponding to a circular area with a radius of about 5.64 m). Each stump colonised by Armillaria was considered to represent a circle with a radius of about 2.82 m (c. 25 m2), corresponding approximately to the diameter of the large root system. Two isolates of the same genet, distant ≤ 20 m, were considered to represent a contiguous (nonfragmented) genet regardless of whether there was an isolate belonging to another genet in-between. This criterion was set in respect to the 10 m × 10 m sample grid and allowed one negative grid point between two positive points without affecting the contiguity of a genet. Within the subplots, each isolate of the 1.4 m × 1.4 m grid was considered representative for a genet area of 2 m2 and only isolates of the same genet ≤ 3 m away were considered contiguous.

Genet boundaries were outlined by modifying the method of Worrall (1994). First, we drew circles around each positive soil sample point (r = 5.64 m) and around each colonised stump (r = 2.82 m) from which isolates of the same genet were recovered. Then, for each genet the shortest outline enclosing all circles was drawn as a smoothed polygon. Genet sizes were calculated using the software ArcView, Version 3.2a (Environmental Systems Research Institute, Inc., Redlands, CA, USA). The maximum linear extent of a genet was determined as the maximum spatial distance between two isolates of the same genet, even if they belong to different ramets.

To determine the extent of spatial overlaps among genets of the same or different Armillaria species, polygons were superimposed in ArcView. The size of areas occupied by more than one genet was then calculated with ArcView. In addition, overlaps were also quantified as the percentage of the total territory covered by Armillaria spp.

Statistical analysis

Statistical analysis of data was performed with the software DataDesk, Version 6 (Data Description, Inc., Ithaca, NY, USA). The quantitative variables of genets (i.e. size and linear extent) were analysed using one-way analysis of variance (anova).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Population structure

The incidences of Armillaria species and genets in the soil and on the stumps in the three study plots are summarised in Table 1. From 100 soil samples collected in each plot, 54–85 per plot contained rhizomorphs. Stumps colonised by Armillaria were detected at proportions of 39% (77 out of 199) in Ludiano, 79% (160 out of 202) in Lurengo, and 86% (115 out of 133) in Dalpe. Armillaria isolates were successfully recovered from 184 soil samples and 269 stumps. From 77 soil samples and 134 stumps more than one isolate was obtained. In total, 740 Armillaria isolates were identified to species and genets, 296 from rhizomorphs in the soil and 444 from the stumps. A. cepistipes and A. ostoyae were found in all three plots (Table 1). Both species occurred in similar proportions in Dalpe while A. ostoyae dominated in Lurengo and A. cepistipes in Ludiano. In this last plot, A. gallica was isolated from seven soil samples and 12 stumps.

Table 1.  Incidence of Armillaria species and genets in the soil and on the stumps in the three plots, Ludiano, Lurengo, and Dalpe
OriginAll samplesSamples from which more than one Armillaria isolate was recovered
NPositiveaN of samples with species ofb NN of samples with genets ofc
A. cepA. ostA. galA. cepA. ostA. galA. cep + A. cepA. ost + A. ostA. gal + A. galA. cep + A. galA. cep + A. ost
  • a

    Number of soil samples containing rhizomorphs of Armillaria and stumps colonised by Armillaria. Numbers in brackets refer to positive samples from which Armillaria could be isolated.

  • b

    Number of soil samples containing rhizomorphs of A. cepistipes (A. cep), A. ostoyae (A. ost), and A. gallica (A. gal). A sum exceeding the number of positive soil samples from which Armillaria could be isolated indicates the presence of samples with two species.

  • c

    Number of soil samples or stumps with one genet of a species (e.g. A. cep), two genets of a species (e.g. A. cep + A. cep), or two genets of different species (e.g. A. cep + A. gal).

Soil
Ludiano100 54 (50)38  8 7 2315 121013 0
Lurengo100 60 (52) 2 50 0 21 21900000 0
Dalpe100 85 (82)54 35 0 3315 802100 7
Total300199 (184)94 93 7 77322823113 7
Stumps
Ludiano199 77 (63)44  912 3524 450002 0
Lurengo202160 (111) 2110 0 47 04000600 1
Dalpe133115 (95)53 54 0 5222170100012
Total534352 (269)991731213446615160213

Most soil samples (62 out of 77) and stumps (112 out of 134) from which more than one isolate was recovered, yielded isolates of a single genet. Soil samples containing rhizomorphs of both A. cepistipes and A. ostoyae were only found in Dalpe (7 out of 33). Likewise, several stumps (12 out of 52) in Dalpe were colonised by both species. In all plots, stumps colonised by more than one Armillaria genet were located on the borders between genets or where they overlapped in the soil (Fig. 1a–d).

Using somatic incompatibility tests, two to six different genets of A. cepistipes and A. ostoyae were identified in each plot (Table 2). This corresponds to a mean density of eight Armillaria genets per hectare. More genets were detected in the soil than on the stumps. In Ludiano and Dalpe, a few small (100–200 m2) genets present in the soil (Ludiano: Lud B3, Lud C2; Dalpe: Da C4) were not found on the stumps. Fragmented genets were detected in all plots, and were particularly frequent in Dalpe (Table 2). The estimated sizes of the genets ranged from 100 (i.e. found at only one sample point) to about 4100 m2 and the maximal linear extent from 10 to 105 m. No significant (P < 0.05) differences in the genet sizes were observed (i) between A. cepistipes and A. ostoyae and (ii) among the three plots. The A. gallica genets were not included in the analysis because of their reduced number (two) and their occurrence only in Ludiano.

Table 2.  Characteristics of the Armillaria populations in the three plots
CharacteristicsLudianoLurengoDalpe
A. galA. cepA. ostA. cepA. ostA. cepA. ost
  • a

    A genet was considered fragmented if the distance between two isolates was more than 20 m.

  • b

    Percentage refers to the total area occupied by the Armillaria species indicated.

  • c

    Percentage refers to the total area occupied by the two Armillaria species indicated.

Territory occupied (m2)80041001270250707065005200
Genets (N)2342634
Genets in the soil (N)2342634
Genets on the stumps (N)2232633
Fragmented genetsa (N)1000122
Maximal linear extent (m)45744110105105105
Mean area of a genet (m2)4751425340125128523101350
Intraspecific overlaps (m2)1501801000650440200
(%)b18.84.47.909.26.83.8
Interspecific overlaps (m2) 350 202503200
(%)c7.7 0.43.537.6

In Ludiano, a total of nine genets were identified, three of A. cepistipes, four of A. ostoyae, and two of A. gallica. A. cepistipes and A. ostoyae occupied different sectors of the plot and interspecific spatial overlaps were very rare (Fig. 1a, Table 2). The two genets of A. gallica (Lud E1 and Lud E2) were confined to one edge of the plot and partially overlapped with the A. cepistipes genet Lud B2. The estimated sizes of the A. cepistipes genets ranged from 100 (Lud B3) to about 3000 m2, whereas genets of A. ostoyae and A. gallica were smaller (100–700 m2). The three Armillaria species showed little intraspecific spatial overlaps (Fig. 1a, Table 2).

In Lurengo, six genets of A. ostoyae and two genets of A. cepistipes were found (Fig. 1b). The largest genet (Lur C2) had an estimated size of 4100 m2 and a maximal linear extent of 105 m. Only one genet (Lur C3) showed a fragmented distribution. Spatial overlaps among A. ostoyae genets were rare (9.2% of the territory occupied by A. ostoyae, Table 2) and mainly limited to the border zone of the genets. The two small (100 and 150 m2) A. cepistipes genets in Lurengo were located in two different sectors occupied by the A. ostoyae genet Lur C2 (Fig. 1b).

Three genets of A. cepistipes and four genets of A. ostoyae were detected in Dalpe (Fig. 1c,d). By contrast to the other plots, in Dalpe genets of the two species overlapped considerably. These interspecific overlaps were estimated to cover an area of 3200 m2, which corresponds to 37.6% of the total territory occupied by both species. On the other hand, intraspecific overlaps were rare and limited to the borders of genets. Two genets of A. ostoyae (Da C2 and Da C3) and two genets of A. cepistipes (Da B1 and Da B3) showed a noncontiguous, fragmented distribution. The two largest genets (Da B2 and Da C1) had a maximal linear extent of 105 m and covered 4100 and 3230 m2.

Subplots

The five subplots were established in areas where a genet of A. cepistipes and A. ostoyae adjoined or overlapped in the soil (see Fig. 1a–d). The goal was to investigate the spatial patterns of genets along interspecific boundaries.

The incidence of soil samples with rhizomorphs varied from 59.7% (43 out of 72 in Lurengo 2) to 93.1% (67 out of 72 in Dalpe 1). Except in Dalpe 1 where about 90% of the territory was occupied by Armillaria, in the other four subplots contiguous areas without rhizomorphs in the soil were observed (see Fig. 2a,b for representative examples). In all subplots, positive samples contained on average two, and a maximum of four (Ludiano 1, Lurengo 2) to six (Dalpe 1, Dalpe 2) rhizomorphs. The success of Armillaria isolation from rhizomorphs ranged from 91.6% (Dalpe 1) to 97.3% (Ludiano 1). Species identification showed that the incidences of A. cepistipes and A. ostoyae varied greatly among the subplots (Table 3). A. cepistipes was dominant in Ludiano 1 and Dalpe 1, whereas A. ostoyae was dominant in the other three subplots. Most positive soil samples contained either rhizomorphs of A. cepistipes or A. ostoyae (Table 3). Nevertheless, overlaps between A. cepistipes and A. ostoyae genets, indicated by the finding of rhizomorphs of both species in a single soil sample, were detected in all five subplots (Table 3). The overlapping areas ranged from 4.2% (Lurengo 2) to 27.7% (Dalpe 2) of the total area occupied by Armillaria. Numerous genets of both Armillaria species showed a scattered and irregular distribution (e.g. Fig. 2a,b).

image

Figure 2. Spatial distribution of Armillaria cepistipes (B, blue) and Armillaria ostoyae (C, red) in the subplots Lurengo 1 (a) and Dalpe 2 (b): triangular symbols indicate soil samples with one genet (solid triangles); square symbols indicate soil samples with two genets of the same species (squares with crosses inside) or with genets of different species (squares with dots inside); x, soil samples without rhizomorphs; arrow, slope direction (downhill).

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Table 3.  Incidence of Armillaria cepistipes and Armillaria ostoyae in the soil in the subplots, established along interspecific boundaries
SubplotN of samplesN of rhizomorphs per samplebN of samples with rhizomorphs ofcArea covered by Armillaria spp.
TotalPositiveaA. cepA. ostA. cep + A. ostTotal (m2)Overlaps A. cep. +  A. ostd (%)
  • a

    Soil samples containing rhizomorphs.

  • b

    b Number of rhizomorphs (mean value and standard deviation) in the positive soil samples.

  • c

    Number of positive samples containing rhizomorphs of A. cepistipes (A. cep), A. ostoyae (A. ost) or both species (A. cep + A. ost).

  • d

    Spatial overlaps between A. cepistipes and A. ostoyae genets given as percentages of the total area covered by Armillaria.

Ludiano 1 61 391.9 ± 0.9 33  3 3 90 8.9
Lurengo 1 72 481.8 ± 1 18 27 310519.0
Lurengo 2 72 431.7 ± 1  2 39 2 95 4.2
Dalpe 1 72 672.1 ± 1.1 46  61514827.7
Dalpe 2 57 412.3 ± 1.1  9 25 7 9220.7
Total3342382.0 ± 1.11081003053017.4

New Armillaria genets were found only in the subplots of Lurengo. One new genet (Lur C7) was located in the subplot Lurengo 1 (Fig. 2a) and five genets (Lur C8, Lur C9, Lur B3, Lur B4, and Lur B5) in the subplot Lurengo 2. Four new genets (Lur B3, Lur B4, Lur B5, and Lur C9) were only found in a single soil sample, each with an estimated size of 2 m2. Two new genets were larger. They were found in five and seven soil samples and covered an area of c. 10 m2 and 14 m2.

PCR-RFLP analysis

The results of species identification with diploid-haploid pairings were confirmed with the PCR-RFLP analysis of a portion of the IGS region of the ribosomal DNA. Digestion of the PCR products with the enzyme AluI showed that 10 out of 11 A. cepistipes genets belonged to the known restriction patterns type cep 1 or cep 2 (Pérez Sierra et al., 1999). All three isolates of the A. cepistipes genet Lud B1 showed a new AluI restriction pattern with fragment sizes of 600, 340, and 200 bp. This new pattern was characterised by a sum of the fragment sizes exceeding the size of the PCR product of 920 bp. This ambiguity could be due to an incomplete digestion or a microheterogeneity in the IGS regions of the ribosomal RNA genes. Within A. ostoyae and A. gallica no intraspecific variation was detected. The observed AluI patterns corresponded with already published patterns (Harrington & Wingfield, 1995; Pérez Sierra et al., 1999). The results with the enzymes HincII, Mva 1269 I BsmI and NdeI also confirmed those obtained in previous studies (Harrington & Wingfield, 1995; Pérez Sierra et al., 1999).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We conducted an intensive sampling in the soil and stumps in three comparable spruce stands to analyse the population structure of coexisting Armillaria species. A. cepistipes and A. ostoyae were shown to be the dominant species in all investigated spruce stands.

Based on somatic incompatibility, two to six genets of each Armillaria species were detected in each 1-ha plot. The resulting densities of seven to nine Armillaria genets per hectare are similar to those observed by Worrall (1994) in North America in plots with only A. ostoyae. In France, Legrand et al. (1996) found two study sites with A. cepistipes, A. ostoyae, and A. gallica, where the density was either five or six Armillaria genets per hectare. Most other species of wood-inhabiting basidiomycetes are generally characterised by more dense populations than those of Armillaria. For example, Kirby et al. (1990) found 19 genets of the wood-decay fungus Resinicium bicolor in a spruce stand of 1250 m2 (152 genets per ha). Populations of the pathogenic H. annosum can be composed of up to 4800 genets per hectare (Piri et al., 1990; Swedjemark & Stenlid, 1993). According to Hansen & Hamelin (1999), the density of a basidiomycete population, measured as the number of genets in a stand, is affected by substrate availability, frequency of opportunities for establishment, external disturbances (e.g. forest management), and intra- and interspecific competition.

The number of genets that can be detected probably depends on the intensity of sampling. The intensive main sampling conducted in our study has provided a representative view of the density of large and medium-sized Armillaria genets. However, the detection of additional very small genets in two subplots suggests that the actual number and density of genets is higher than estimated.

All Armillaria genets found on the stumps were also found in the soil. Thus, a systematic soil sampling seems to be sufficient to determine the occurrence and minimum density of Armillaria species and genets. Nevertheless, stump investigation can provide supplementary detailed information on the sizes and spatial distribution of genets. Delimiting the exact physical extent of genets, which is known to be difficult in studies of the population structure of soil-borne basidiomycetes (Anderson & Kohn, 1995), was not a primary objective of this study. Nevertheless, the estimated sizes of the larger genets (0.3–0.4 ha) are comparable with those reported for A. cepistipes and A. ostoyae in coniferous and hardwood forests in central Europe (Legrand et al., 1996; Marxmüller & Holdenrieder, 2000). Similar sizes of A. ostoyae genets were also observed by Smith et al. (1994) in a red pine (Pinus resinosa Aiton) seedling plantation in North America. The larger genets occurring in our plots probably further extend into the surrounding stands. Therefore, sampling on a larger scale would be necessary to determine the effective size of these genets. Some genets could also be smaller than estimated. Sampling in the five subplots demonstrated that, on a small scale, considerable gaps can exist within the territory occupied by Armillaria. These observations indicate that rhizomorphs of a genet are not evenly distributed in the soil, but vary considerably in density.

Several of the identified genets showed a noncontiguous, patchy distribution. The single patches can be considered as the physically and physiologically independent units of the genets, called ramets (Dahlberg & Stenlid, 1994; Dettman & van der Kamp, 2001). Kile (1983) suggested that the probability of fragmenting increases with the size and age of a genet. The scattered genets observed in our plots could therefore represent remnants of larger genets. They could also be the result of an irregular vegetative growth from an initial point (e.g. colonised stump) through the production of rhizomorphs or spread via root contacts. Another possible explanation is that noncontiguous genets are inbred siblings (Kile, 1983), which are not always distinguished by somatic incompatibility tests (Guillaumin et al., 1991). The highly scattered A. ostoyae genet Da C3 in Dalpe could have been created by past management operations. By dragging stems downhill it is possible that rhizomorphs or colonised woody debris were displaced and became established in another sector of the stand. Fragmented genets have also been observed in other rhizomorph- and cord-producing fungi, such as Marasmius androsaceus (Holmer & Stenlid, 1991), Tricholomopsis platyphylla (Thompson & Rayner, 1982), as well as in the ectomycorrhizal fungus Suillus bovinus (Dahlberg & Stenlid, 1990). Fragmentation could be beneficial for the survival of a genet by impeding the spread of deleterious cytoplasmic elements. Fragmented genets could be the result of a famine-induced thinning of the rhizomorph network or mycelium because of insufficient disposability of resources (Smith et al., 1994; Anderson & Kohn, 1995).

The assessment of genet fragmentation is strongly affected by the specific criteria employed to identify noncontiguity between neighbouring isolates. In our study, two isolates of the same genet, distant ≤ 20 m, were considered to represent a contiguous genet. Decreasing this arbitrary distance would certainly increase the number of fragmented genets identified in our three plots. The quantitative estimates of spatial overlaps between genets also depend on the criteria adopted to define the limits of genets.

In our study, intraspecific spatial overlaps were rare and were generally limited to the borders of genets as has been observed in previous studies (Rizzo & Harrington, 1993; Worrall, 1994; Rizzo et al., 1995; Legrand et al., 1996). This suggests that genets of the same species are strongly antagonistic, probably because of somatic incompatibility and use of the same ecological strategy. Thus, the spatial distribution of a genet would be mainly affected by the position of adjacent genets of the same species (Rizzo & Harrington, 1993; Smith et al., 1994).

In Ludiano, no spatial overlaps between A. cepistipes and A. ostoyae were observed. The two species occupied different sectors of the plot and there is probably little interspecific interaction. By contrast, a genet of A. cepistipes partially overlapped with the two genets of A. gallica. The detection of A. gallica in Ludiano was unexpected, because this species typically occurs in hardwood forests at low altitudes (Guillaumin et al., 1993). Both A. cepistipes and A. gallica are preferentially saprotrophs and spatial overlaps among genets of the two species have been rarely observed (Legrand et al., 1996). In Lurengo, the two small genets of A. cepistipes were located within the territory occupied by an A. ostoyae genet, which could suggest a recent establishment. Whether these two A. cepistipes genets will gain size in the future at the cost of the A. ostoyae genet needs to be determined.

The third plot (Dalpe) was characterised by large overlaps between A. cepistipes and A. ostoyae. About 38% of the total territory covered by Armillaria was occupied by both species. Dalpe was also the plot with the highest incidence of fragmented and irregularly distributed genets. Therefore estimates of overlapping areas in this plot will strongly depend on the criteria adopted to define the boundaries and contiguity of a genet. Detailed sampling in the subplots, however, showed that spatial overlaps along interspecific boundaries are common in all three plots. Considerable overlaps between preferentially saprotrophic and pathogenic Armillaria species have been observed in other studies and have been assumed to indicate different colonisation strategies and resource partitioning between the species (Rizzo & Harrington, 1993; Smith et al., 1994; Legrand et al., 1996; Baumgartner & Rizzo, 2001). In our plots, this hypothesis is supported by the finding that A. ostoyae is more efficient in primary stump capture than A. cepistipes (S. Prospero et al., unpublished).

Our study shows that, in comparable Norway spruce stands where A. cepistipes and A. ostoyae coexist, the incidence of each species, as well as the spatial patterns and sizes of genets, can vary considerably. The factors determining this variation are not known. It can be assumed that site characteristics (e.g. exposition, soil, and topographical heterogeneity) and stand characteristics (e.g. history, management practise, and species composition) could influence the population dynamics of Armillaria. A general type of interaction between A. cepistipes and A. ostoyae is difficult to deduce from our results. On parts of the investigated areas, both species seem to exclude each other indicating competition, probably because of a similar ecological strategy. However, on other parts (e.g. in Dalpe) there were considerable interspecific overlaps, which could be explained by a more neutralistic coexistence with both species ignoring each other. Our study only gives a snapshot of a dynamic system. Further studies, focusing on the dynamic and experimental manipulation of local Armillaria populations are needed to examine the degree of competition between A. cepistipes and A. ostoyae.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to C. Cattaneo, E. Cereghetti, F. Fibbioli, and C. Matter for the technical assistance in the field. We would also like to thank Ursula Heiniger for critical feedback on the manuscript, and Silvia Dingwall for English correction. We greatly appreciated the helpful comments and criticism made by the reviewers.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Anderson JB, Kohn LM. 1995. Clonality in soilborne, plant-pathogenic fungi. Annual Review of Phytopathology 33: 369391.
  • Baumgartner K, Rizzo DM. 2001. Ecology of Armillaria spp. in mixed-hardwood forests of California. Plant Disease 85: 947951.
  • Bruhn JN, Wetteroff Jr JJ, Mihail JD, Kabrick JM, Pickens JB. 2000. Distribution of Armillaria species in upland Ozark Mountain forests with respect to site, overstory species composition and oak decline. European Journal of Forest Pathology 30: 4360.
  • Dahlberg A, Stenlid J. 1990. Population structure and dynamics in Suillus bovinus as indicated by spatial distribution of fungal clones. New Phytologist 115: 487493.
  • Dahlberg A, Stenlid J. 1994. Size, distribution and biomass of genets in populations of Suillus bovinus (L. Fr.) Roussel revealed by somatic incompatibility. New Phytologist 128: 225234.
  • Dettman JR, Van Der Kamp BJ. 2001. The population structure of Armillaria ostoyae in the southern interior of British Columbia. Canadian Journal of Botany 79: 612620.
  • Guillaumin J-J, Anderson JB, Korhonen K. 1991. Life cycle, interfertility, and biological species. In: ShawCG, KileGA, eds. Armillaria root disease. Agricultural handbook no. 691. Washington DC, USA: USDA Forest Service, 1020.
  • Guillaumin J-J, Anderson JB, Legrand P, Ghahari S, Berthelay S. 1996. A comparison of different methods for the identification of genets of Armillaria spp. New Phytologist 133: 333343.
  • Guillaumin J-J, Mohammed C, Anselmi N, Courtecuisse E, Gregory SC, Holdenrieder O, Intini M, Lung B, Marxmüller H, Morrison D, Rishbeth J, Termorshuizen AJ, Tirro A, Van Dam B. 1993. Geographical distribution and ecology of the Armillaria species in western Europe. European Journal of Forest Pathology 23: 321341.
  • Hansen EM, Goheen EM. 2000. Phellinus weirii and other native root pathogens as determinants of forest structure and process in western North America. Annual Review of Phytopathology 38: 515539.
  • Hansen EM, Hamelin RC. 1999. Population structure of basidiomycetes. In: WorrallJJ, ed. Structure and dynamics of fungal populations. Dordrecht, The Netherlands: Kluwer Academic Publishers, 251281.
  • Harrington TC, Wingfield BD. 1995. A PCR-based identification method for species of Armillaria. Mycologia 87: 280288.
  • Harrington TC, Worrall JJ, Baker FA. 1992. Armillaria. In: SingletonLL, MihailJD, RushCM, eds. Methods for research on soilborne phytopathogenic fungi. St. Paul, MN, USA: APS Press, 8185.
  • Högberg N, Holdenrieder O, Stenlid J. 1999. Population structure of the wood decay fungus Fomitopsis pinicola. Heredity 83: 354360.
  • Holdenrieder O, Greig BJW. 1998. Biological methods of control. In: WoodwardS, StenlidJ, KarjalainenR, HüttermannA, eds. Heterobasidion annosum: biology, ecology, impact and control. New York, USA: CAB International, 235258.
  • Holmer L, Stenlid J. 1991. Population structure and mating system in Marasmius androsaceus Fr. New Phytologist 119: 307314.
  • Kile GA. 1983. Identification of genotypes and the clonal development of Armillaria luteobubalina Watling & Kile in Eucalypt forests. Australian Journal of Botany 31: 657671.
  • Kile GA, McDonald GI, Byler JW. 1991. Ecology and disease in natural forests. In: ShawCG, KileGA, eds. Armillaria root disease. Agricultural handbook no. 691. Washington DC, USA: USDA Forest Service, 102121.
  • Kirby JJH, Stenlid J, Holdenrieder O. 1990. Population structure and responses to disturbance of the basidiomycete Resinicium bicolor. Oecologia 85: 178184.
  • Korhonen K. 1978. Interfertility and clonal size in the Armillariella mellea complex. Karstenia 18: 3142.
  • Legrand P, Ghahari S, Guillaumin J-J. 1996. Occurrence of genets of Armillaria spp. in four mountain forests in central France: the colonization strategy of Armillaria ostoyae. New Phytologist 133: 321332.
  • Legrand P, Guillaumin J-J. 1993. Armillaria species in the forest ecosystems of the Auvergne (Central France). Acta Oecologica 14: 389403.
  • Malik M, Vilgalys R. 1999. Somatic incompatibility in fungi. In: WorrallJJ, ed. Structure and dynamics of fungal populations. Dordrecht, The Netherlands: Kluwer Academic Publishers, 123138.
  • Maloy OC. 1974. Benomyl-malt agar for the purification of cultures of wood decay fungi. Plant Disease Reports 58: 902904.
  • Marxmüller H, Holdenrieder O. 2000. Morphologie und Populationsstruktur der beringten Arten von Armillaria mellea s.1. Mycologia Bavarica 4: 932.
  • Morrison D, Mallett K. 1996. Silvicultural management of Armillaria root disease in western Canadian forests. Canadian Journal of Plant Pathology 18: 194199.
  • Morrison DJ, Pellow KW, Norris DJ, Nemec AFL. 2000. Visible versus actual incidence of Armillaria root disease in juvenile coniferous stands in the southern interior of British Columbia. Canadian Journal of Forest Research 30: 405414.
  • Pérez Sierra A, Whitehead DS, Whitehead MP. 1999. Investigation of a PCR-based method for the routine identification of British Armillaria species. Mycological Research 103: 16311636.
  • Piri T, Korhonen K, Sairanen A. 1990. Occurrence of Heterobasidion annosum in pure mixed spruce stands in southern Finland. Scandinavian Journal of Forest Research 5: 113125.
  • Prospero S, Rigling D, Giudici F, Jermini M. 1998. Détermination des espèces d’armillaire responsables du pourridié-agaric de la vigne au Tessin. Revue Suisse de Viticulture Arboriculture Horticulture 30: 315319.
  • Rizzo DM, Blanchette RA, May G. 1995. Distribution of Armillaria ostoyae genets in a Pinus resinosa-Pinus banksiana forest. Canadian Journal of Botany 73: 776787.
  • Rizzo DM, Harrington TC. 1993. Delineation and biology of clones of Armillaria ostoyae, A. gemina and A. calvescens. Mycologia 85: 164174.
  • Shaw CG III, Kile GA, eds.1991. Armillaria root disease. Agricultural handbook no. 691. Washington DC, USA: USDA. Forest Service.
  • Smith ML, Bruhn JN, Anderson JB. 1994. Relatedness and spatial distribution of Armillaria genets infecting red pine seedlings. Phytopathology 84: 822829.
  • Swedjemark G, Stenlid J. 1993. Population dynamics of the root rot fungus Heterobasidion annosum following thinning of Picea abies. OIKOS 66: 247254.
  • Thompson W, Rayner ADM. 1982. Spatial structure of a population of Tricholomopsis platyphylla in a woodland site. New Phytologist 92: 103114.
  • Watling R, Kile GA, Burdsall Jr HH. 1991. Nomenclature, Taxonomy, and Identification. In: ShawCG, KileGA, eds. Armillaria root disease. Agricultural handbook no. 691. Washington DC, USA: USDA Forest Service, 19.
  • Worrall JJ. 1994. Population structure of Armillaria species in several forest types. Mycologia 86: 401407.
  • Worrall JJ. 1997. Somatic incompatibility in basidiomycetes. Mycologia 89: 2436.