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Keywords:

  • biomass;
  • ectomycorrhiza;
  • genet distribution;
  • ISSR (inter-simple sequence repeat);
  • Pinus pentaphylla var. himekomatsu;
  • Suillus pictus

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Spatial distribution and biomass of genets of sporocarps and ectomycorrhizas of Suillus pictus were studied in a plot of 20 × 24 m established in a Pinus pentaphylla var. himekomatsu plantation.
  • • 
    The biomass of S. pictus ectomycorrhizas was evaluated based on morphotypes, and genets were identified based on the inter-simple sequence repeat (ISSR) polymorphism analysis.
  • • 
    Suillus pictus was one of the dominant ectomycorrhizal fungal species in both the sporocarp and ectomycorrhizal communities in the study plot. Four genets were identified from sporocarps and these coincided with those identified from ectomycorrhizas. Sporocarps of each S. pictus genet occurred separately from those of other genets. Spatial distributions of ectomycorrhizas of each genet were wider than those of sporocarps. The largest genet occupied c. 54% of the plot, and the area of each genet differed considerably.
  • • 
    Vegetative growth of mycelia is assumed to play a more important role in the propagation of S. pictus than colonization from spores because expansions of all the four genets ranged from 25 to 30 m and no small genets were found in this plot.

Introduction

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

The species composition of ectomycorrhizal (ECM) fungi in both sporocarp and mycorrhizal communities in various forest ecosystems has been studied extensively; these studies have revealed that species composition in below-ground communities differs greatly from that of sporocarp communities in many cases (Horton & Brun, 2001). It has been also proven that spatial distribution of sporocarps does not always reflect that of mycorrhizas in the soil because sporocarp occurrence is affected not only by the distribution of ectomycorrhizas but also by various environmental conditions (Taylor & Alexander, 1990; Gardes & Brun, 1996). Thus, to elucidate the ecology of ECM fungi, it is necessary to study both sexual states, that is sporocarps, and vegetative structure, that is mycorrhizas and mycelium in the soil (Horton & Brun, 2001).

Genet distribution has been studied to elucidate the propagative manner of ECM fungi in natural ecosystems. Somatic incompatibility has been used to identify genets of sporocarps of several ECM fungi (Fries, 1987; Dahlberg & Stenlid, 1990, 1994; Baar et al., 1994; Dahlberg, 1997). Somatic incompatibility, however, sometimes failed to identify genets in Suillus granulatus (Jacobson et al., 1993) and molecular markers have been applied to identify genets of ECM fungi in recent studies (Bastide et al., 1994; Gherbi et al., 1999; Sawyer et al., 1999; Gryta et al., 2000; Guidot et al., 2001). Based on the analysis of the genet structure of sporocarps, these studies have proved that early stage fungi such as Hebeloma and Laccaria formed many small genets and late stage fungi such as Cortinarius formed a few large genets.

Suillus pictus, which associates with five-needled pine species including Japanese white pine (Pinus pentaphylla var. himekomatsu) (Murata, 1976; Imazeki & Hongo, 1989; Wu et al., 2000) occurred dominantly in a Japanese white pine plantation in our preliminary study on the spatial distribution of sporocarps. This species forms rhizomorphs and tubercle mycorrhizas (Randall & Grand, 1986; Kikuchi & Futai, 2003), which are easily identified in the field and considered appropriate for the analysis of genet structure of ectomycorrhizas. Many studies have been conducted to examine the genet expansion and spatial distribution of Suillus species based on the analysis of sporocarps (Fries, 1987; Zhu et al., 1988; Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997; Bonello et al., 1998; Zhou et al., 1999, 2000, 2001a). However, only a few of them examined the genet of mycorrhizas simultaneously. Dahlberg & Stenlid (1994) found that ectomycorrhizas sampled in the vicinity of S. bovinus sporocarps had the same genet type as sporocarps. Zhou et al. (2001b) extensively sampled ectomycorrhizas and soil mycelia of S. grevillei in small areas around sporocarps and demonstrated their horizontal and vertical distribution. These studies showed genet distribution of ectomycorrhizas in small areas (5 m2 and 1.72 m2, respectively). On the other hand, spatial distribution of ectomycorrhizas of Suillus spp. did not always overlap with that of sporocarps (Gardes & Brun, 1996; Kikuchi & Futai, 2003) and the relationship between the genet distribution of ectomycorrhizas and sporocarps remains unclear on a large scale. The objective of this study is to examine the spatial distribution of genets of S. pictus sporocarps and ectomycorrhizas in a Japanese white pine plantation to reveal the relationship between the spatial distribution of genets of sporocarps and those of ectomycorrhizas on a large scale (480 m2).

Materials and Methods

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

Site description

The study site was located in a Japanese white pine plantation (28 yr old) on a slope (average inclination: 29.7°) facing east-north-east in the Kamigamo experimental forest station of Kyoto University forest (35°04′-N, 137°31′-E, 140 m above sea level), Kyoto, Japan. Seedlings of Japanese white pine, which was not indigenous to this area, were grown in a nursery for 3 yr and then transplanted into a clear-cut site that had formerly been a mixed forest of Pinus densiflora and Quercus spp. In April 2000, plot 1 (20 × 24 m) was established in this area, and divided into 120 subplots (2 × 2 m) for examining spatial distribution of sporocarps and ectomycorrhizas. Each subplot was identified by a column letter and a row number as shown in Fig. 1. Eighty-eight Japanese white pines (average diameter at breast height = 9.8 cm) were distributed uniformly within this plot (Fig. 1). The forest floor was covered sparsely by Sasa spp., and there were no other trees than Japanese white pine. The thickness of the litter layer, measured at 480 regularly spaced points within the plot, ranged from 0.5 to 4.0 cm. In December 2000, plot 2 (6 × 16 m) was established beside plot 1, which was divided into 96 subplots (1 × 1 m) for examining spatial distribution and biomass estimation of ectomycorrhizas. In this plot, 20 Japanese white pines of the same age as in plot 1 were distributed uniformly. The conditions of plot 2 were almost the same as those of plot 1. The density of sporocarps or mycorrhizas, etc. was expressed using surface area, and the actual areas of plots 1 and 2 are 416.9 and 86.3 m2, respectively.

image

Figure 1. Sampling methods of ectomycorrhizas of Suillus pictus for the estimation of biomass in each area. Each solid circle and star represents a sporocarp of S. pictus and a tree of Japanese white pine (Pinus pentaphylla var. himekomatsu), respectively. Shaded fields show the sampled subplots. In area D, seven subplots with sporocarps (D-1) and seven subplots without sporocarps (D-2) were sampled.

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Sporocarp sampling

In plot 1, an ECM sporocarp survey was conducted once or twice a week between April and December 2002. Sporocarps were identified in the field and their positions were recorded. All sporocarps were sampled and dried in a forced-draft oven at 60°C for > 48 h and then weighed. For DNA extraction, 79 out of a total of 117 sporocarps of S. pictus collected were used and a small amount of tissues were sampled before drying. When two sporocarps occurred adjacent to each other (i.e. two sporocarps were in contact with each other at the stipe base), we chose only one for DNA analysis (20 sporocarps). When eight sporocarps occurred gregariously in a small area (< c. 10 cm in diameter), we chose two for DNA analysis (24 sporocarps in H8). The genet types of sporocarps that could not be used for molecular analysis, for example those that were rotten, were estimated based on the pattern of the distribution of the sporocarps.

We had conducted a sporocarp survey to study the species composition in this plot over the previous 2 yr (2000 and 2001).

Soil sampling for estimation of biomass and spatial distribution of ectomycorrhizas

To identify ectomycorrhizas of S. pictus, soil samples with sporocarps of S. pictus were taken outside the study plots and ectomycorrhizas of S. pictus were carefully separated. After morphological typing, we confirmed the identification by PCR–RFLP pattern of ribosomal ITS region following the methods of Matsuda & Hijii (1999).

In December 2000, to evaluate the biomass and vertical distribution of S. pictus ectomycorrhizas, soil blocks (surface area: 20 × 20 cm) were collected at soil depths of 0–5, 5–10, 10–15, 15–20 and 20–30 cm at the center of 10 subplots that were selected randomly in plot 2. Each soil block was divided into small (100 cm2 sample) and large samples (300 cm2 sample) in order to examine the effect of sample size on the biomass estimation of mycorrhizas. All samples were washed carefully and were sorted into nonectomycorrhizal fine roots (≤ 2 mm in diameter), ectomycorrhizas of S. pictus, and other types, based on the morphotype reported by Kikuchi & Futai (2003). They were dried in a forced-draft oven at 60°C for > 48 h and weighed.

In December 2002, after the termination of ECM sporocarp occurrence, plot 1 was divided into four areas in order to examine the biomass of S. pictus ectomycorrhizas in this plot, as shown in Fig. 1. Area D, where most of sporocarps of S. pictus had occurred, was further divided into two subareas according to sporocarp occurrence, that is 17 subplots where sporocarps of S. pictus occurred were grouped into area D-1, and 13 subplots without sporocarps of S. pictus were grouped into area D-2. Two soil samples per subplot were collected using a steel sampling tube (5 cm i.d.) to a depth of 15 cm for seven randomly selected subplots in each area or subarea. As c. 80% of fine roots and ectomycorrhizas were distributed at the soil depth of 0–15 cm and S. pictus mycorrhizas were found in almost every soil samples in plot 2, a small sampling size (40 cm2 in surface area and 15 cm in depth per subplot) was employed in plot 1 in order to minimize the disturbance. Fine roots and ectomycorrhizas were measured in the same way as for the samples in plot 2.

Sampling of ectomycorrhizas for DNA analysis

Plot 1 was divided into three experimental areas: L, M and S, as shown in Fig. 2, to examine the relationship between the spatial distribution of S. pictus sporocarps and that of ectomycorrhizas precisely. Areas L, M and S were divided into 100 (2 × 2 m), 80 (1 × 1 m) and 32 (0.5 × 0.5 m) subplots, respectively. Ectomycorrhizas were sampled at 113, 99 and 45 intersection points in areas L, M and S, respectively (Fig. 2). Soil core sample of c. 10 × 10 × 5 cm depth were excavated at every intersection point in each area and one ectomycorrhiza of S. pictus per soil core was sampled based on the morphology of ectomycorrhizas. In total, 242 ectomycorrhizas were sampled for DNA extraction in November and December 2002.

image

Figure 2. Spatial distribution of Suillus pictus sporocarps and other ectomycorrhizal fungi and sampling points for the estimation of genet distribution in the study plot. One tuberculate ectomycorrhiza of S. pictus was sampled at every intersection in this figure. Ectomycorrhizas were sampled at 113, 99 and 45 intersection points in areas L, M and S, respectively. Each open circle, ‘x’ and closed circle represents a S. pictus sporocarp occurring in 2000, 2001 and 2002, respectively. Each open triangle represents a sporocarp of other ectomycorrhizal fungi occurring in 2002.

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We also compared genet types of S. pictus ectomycorrhizas at a soil depth of 5–10 cm with those at 0–5 cm at six randomly selected intersections in plot 1. Soil core samples were collected using a steel sampling tube (5 cm i.d.) inserted to a depth of 10 cm. One ectomycorrhiza of S. pictus found in upper 5 cm sample and one in lower 5 cm sample were used for the genet analysis. When we could not found any S. pictus mycorrhizas in either the upper 5 cm sample or the lower 5 cm sample, we discarded the sample and took another sample from a nearby site.

Six fine roots of c. 20 cm in length were sampled in six randomly selected points outside plot 1 to examine the genet structure on a small scale. Each fine root had 6–10 active ectomycorrhizas of S. pictus, from which DNA was extracted and analyzed.

Determination of method for analysis of genet identification

We compared PCR-RFLP, RAPD, and ISSR methods for suitability of the analysis for genet identification at first. This pretest showed that: first PCR-RFLP (IGS region) did not provide polymorphic band pattern, while RAPD and ISSR did; and second ISSR had higher sensitivity than RAPD. We decided to employ the ISSR method for the analysis of genets. We compared 14 ISSR primers for the identification of genets: (AC)13; (CAA)9; (ATG)10; (ATC)10; (GTG)7; (GAC)7; (CCA)7; (GACA)7; (GCTC)5; (AG)16; (AAG)11; (GATA)13; (GTG)5 and (GTG)9. One of the ISSR primers pretested with S. pictus sporocarps occurred in 2001, (GACA)7, provided the best polymorphic band pattern. Other primers provided fewer polymorphic band patterns than (GACA)7; all of them were seen in the (GACA)7 band patterns. So we used the ISSR primer (GACA)7 for identifying genets of S. pictus and we could discriminate four genet types using this primer. When this primer was applied to S. pictus mycorrhizas, plant DNA was amplified as well as fungal DNA. However, no band patterns were detected at < 1200 base pairs as we checked the band patterns of nonmycorrhizal roots of Japanese white pine using this primer. We were able to discriminate S. pictus genet types at < 1200 base pairs, as shown in Fig. 3. We could get identical PCR profiles at < 1200 base pairs using DNA extracted from sporocarps and from mycorrhizas of S. pictus.

image

Figure 3. Inter-simple sequence repeat (ISSR) fingerprint of Suillus pictus in the study plot using primer (GACA)7. M is phy X molecular size marker.

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We followed the methods of Matsuda & Hijii (1999) for DNA extraction from sporocarps and ectomycorrhizas. PCR amplification was performed in 12.5 µl reaction mixture, which contained 0.75 µl of template DNA, 2 mm of dNTP and 0.72 µm inter-simple sequence repeat motif primer, 1.25 µl of 10 × buffer and 1 U of Taq polymerase in a PCR thermal cycler (TP240: Takara Shuzo, Tokyo, Japan). The thermal cycling schedule was as follows: the first cycle consisted of 2 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C for annealing, 2 min at 72°C and the final cycle of 10 min at 72°C. Then the reaction mixture was cooled at 4°C for 5 min to terminate the PCR reaction and stored at 4°C until use.

PCR products, 10 µl aliquots, were mixed with 1 µl loading buffer and subjected to electrophoresis on 1.5% agarose gels. After staining with ethidium bromide, band pattern was examined on a UV transilluminator.

Estimation of genet expansion and biomass

The expansion of each genet was estimated by calculating the distance between the outermost sporocarps (genet expansion of sporocarp) or ectomycorrhizas (genet expansion of ectomycorrhizas) for each genet (Dahlberg & Stenlid, 1994). The area size of sporocarps of each genet (genet area size of sporocarps) was estimated in the same way as shown in Bonello et al. (1998). The area size of each genet of ectomycorrhizas (genet area size of ectomycorrhizas) was calculated as follows; the number of sampling points of each genet inside of each experimental area was multiplied by the unit area of subplots in each area, namely, 4, 1 or 0.25 m2 for areas L, M or S, respectively. For the sampling points on the border or on the corner of each experimental area, number of sampling points of each genet was multiplied by half or one fourth of the unit area of each experimental area, respectively. For the subplots in adjacent to the smaller area, the data of the sampling points on the border of the smaller area were also used for the calculation as shown in Fig. 4(a, b). The area size of each genet was obtained by summing up these areas for each genet.

imageimage

Figure 4. Relationship between genet distribution of sporocarps and ectomycorrhizas of Suillus pictus in (a) area L (grid size: 2 × 2 m) and area M (grid size: 1 × 1 m), and (b) area M (grid size: 1 × 1 m), and area S (grid size: 0.5 × 0.5 m). Each colored small rectangular represents one sampling point of an ectomycorrhiza and each mushroom icon identifies individual sporocarp.

The biomass of ectomycorrhizas of each genet was calculated as follows; the area size of each genet of ectomycorrhizas in areas A, B, C or D was multiplied by the average density of ectomycorrhizas (depth 0–15 cm) of S. pictus in each area (Table 1b) and they were summed up for each genet.

Table 1.  The biomass of ectomycorrhizas and fine roots of Japanese white pine (Pinus pentaphylla var. himekomatsu). (a) Vertical distribution in plot 2, (b) comparison of biomass in plots 1 and 2
(a) 100 cm2 sample
 Ectomycorrhizas (g d.wt m−2)Fine roots (g d.wt m−2)
Suillus pictusOther types
0–5 cm 4.7 ± 1.1*a** (45.2)***14.4 ± 5.1a (51.4)  71.5 ± 17.4a (59.8)
5–10 cm 3.5 ± 1.3ab (33.7) 7.1 ± 1.7ab (25.4)  24.3 ± 5.7b (20.4)
10–15 cm 1.1 ± 0.6b (10.6) 3.7 ± 1.5b (13.2)  12.7 ± 3.6b (10.6)
15–20 cm 0.5 ± 0.3b (4.8) 1.6 ± 0.7b (5.7)    4.8 ± 1.3b (4.0)
20–30 cm 0.6 ± 0.3b (5.8) 1.2 ± 0.6b (4.3)    6.1 ± 2.7b (5.1)
Total10.5 ± 2.1 (100)28.0 ± 6.4 (100)119.4 ± 21.6 (100)
300 cm2 sample
 Ectomycorrhizas (g d.wt m−2) Fine roots (g d.wt m−2)
Suillus pictusOther types
0–5 cm 3.5 ± 1.0a (31.0)12.1 ± 2.6a (43.8)51.1 ± 10.3a (53.6)
5–10 cm 2.0 ± 0.8a (17.7) 7.1 ± 1.7ab (25.7)18.2 ± 3.6b (19.2)
10–15 cm 2.7 ± 1.4a (23.9) 3.4 ± 1.2b (12.3)12.6 ± 3.7b (13.2)
15–20 cm 1.9 ± 0.8a (16.8) 3.5 ± 1.5b (12.7) 8.1 ± 2.2b (8.5)
20–30 cm 1.2 ± 0.6a (10.6) 1.5 ± 0.6b (5.4) 5.2 ± 2.0b (5.5)
Total11.2 ± 3.4 (100)27.5 ± 5.1 (100)95.2 ± 17.9 (100)
(b)
Sampling areaDry weight (g m−2)
Suillus pictusOther ectomycorrhizasFine roots
  • *

    , Means ± SE, n = 10.

  • **

    , Values with the same letter within a column are not significantly different (P = 0.05) according to Bonferroni test.

  • ***

    , Figures in parentheses show percentage to total value.

  • *

    , Means ± SE, n = 7 for plot 1 and n = 10 for plot 2.

  • **

    , Values with the same letter within a column are not significantly different (P = 0.05) according to Bonferroni test.

Plot 1 (40 cm2, depth 0–15 cm)
A8.87 ± 3.08*a**28.0 ± 4.43a 95.2 ± 6.6ab
B15.8 ± 3.31a24.7 ± 5.87a 79.5 ± 6.9ab
C17.3 ± 7.91a28.8 ± 7.02a 62.1 ± 13.2ab
D-19.70 ± 5.46a13.0 ± 2.88a 38.2 ± 13.0a
D-210.5 ± 4.17a16.0 ± 2.33a 69.2 ± 14.7ab
Plot 2 (depth 0–15 cm)
100 cm2 sample9.31 ± 1.91a25.2 ± 5.99a108.5 ± 19.7b
300 cm2 sample8.16 ± 2.55a22.6 ± 4.29a 81.9 ± 14.9ab

Data analysis

Distribution patterns of sporocarps were analyzed on the basis of the m*-m method with successive changes in quadrat size (Iwao, 1972). For calculation, the plot was divided into 1920 0.25 m2 (unit-0.25), 480 1 m2 (unit-1), 120 4 m2 (unit-4) and 30 16 m2 (unit-16) subplots successively. For each unit size the mean density (m) and mean crowding (m*) of sporocarps were calculated by using the following formulas:

  • image

where Q is the number of subplots and xi is the number of sporocarps occurring in the ith subplot. For a given quadrat size, the relation of m* on m in a series of populations having common properties can be fitted by a linear regression: m* = a + bm (Iwao, 1968). In this regression, the intercept a indicates the basic component of distribution (The basic component is called the clump, hereafter). The size of the clump indicates the extent of aggregation of sporocarp occurrence in this study. The slope b indicates how clumps are distributed over space. When the distribution of clumps is random, contagious or regular, the slope b becomes unity, larger than unity or smaller than unity, respectively (Iwao & Kuno, 1971). This analysis was also performed for the sporocarp distribution of the genets. Types B and D produced too few sporocarps to perform this analysis.

To examine the spatial relationship between the distributions of S. pictus sporocarps occurring in 3 successive years, ω index, a measure of the degree of overlapping relative to the independent distributions (Iwao, 1977), was used. The mean crowding on sporocarps in X year by ones in Y year can be given by:

  • image

and the mean crowding on sporocarps in Y year by ones in X year by:

  • image

where xXj and xYj are the numbers of sporocarps in X year and Y year in the jth subplot, respectively. Q is the total number of subplots.

Then the index of overlapping γ can be given by:

  • image

If the distribution of sporocarps in 2 years is independent, the following relationship would be expected:

  • m*XY(ind) ≈mY, m*YX(ind) ≈mX.

Substituting these relations into the equation for γ index, we have the γ expected for independent distributions as follows:

  • image

Then, as a measure of the degree of overlapping relative to the independent distributions, we have:

  • ω(+) = (γ − γ(ind))/(1 − γ(ind)) for γ = γ(ind) or ω(−) = (γ − γ(ind))/γ(ind) for γ = γ(ind).

The value of ω changes from a maximum of +1 for complete overlapping, through 0 for independent occurrence, to a minimum of −1 for complete exclusion.

The same method was used for the analysis of relationships between the spatial distribution of sporocarps of S. pictus and other mycorrhizal fungi in 2002.

The biomass of ectomycorrhizas and fine roots at each soil depth, or each sampling size (100 cm2 and 300 cm2) was analyzed using one-factorial anova; means were subsequently compared using the Bonferroni test. The same test was used for comparing the biomass of mycorrhizas and fine roots in each sampling area (areas A, B, C and D). Additionally, interaction between sampling size and depth was tested using two factorial anova. Correlation between the biomass of S. pictus mycorrhizas, other mycorrhizas and fine roots were analyzed using Pearson's correlation coefficient.

Results

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

Sporocarp production and its spatial distribution of S. pictus

A total of 17 species in seven genera were identified in this plot and a total of 282 sporocarps (596.8 g d. wt) occurred in 2002. S. pictus was the most dominant species and produced 117 sporocarps (184.9 g d. wt) in this plot. S. pictus had been one of the most dominant species in this plot for the previous 2 yr also. Sporocarps of S. pictus occurred in clumps and the clumps were distributed aggregately in 2002 (m* = 8.6m+ 4.7, Fig. 2). The spatial distribution of sporocarps of S. pictus was contagious also in 2001 (m* = 4.9m + 1.4), while their distribution in 2000 was almost random (m* = 1.3m+ 1.3).

Spatial distribution of sporocarps of S. pictus in 2002 overlapped well with those in 2000 and 2001 as analyzed using the ω index (Fig. 5).

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Figure 5. ω index between the spatial distribution of Suillus pictus sporocarps in different years and ω index between the spatial distribution of S. pictus and other ectomycorrhizal fungi in 2002.

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Strobilomyces confusus, Lactarius chrysorrheus and Tylopilus castaneiceps were other dominant species and accounted for 28.4% (in number) of the total sporocarps in 2002. Spatial distribution of sporocarps of S. pictus did not overlap with those of all the other species as analyzed using the ω index (Figs 2 and 5).

Most of the sporocarps of S. pictus occurred in the latter 10 d of September, although sporocarps of this species also occurred in June, July, and September in 2002 (Fig. 6). Sporocarps of most of the other species occurred only in a particular month or season.

image

Figure 6. Fruiting phenology of Suillus pictus belonging to each genet in 2002.

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Spatial distribution and biomass of ectomycorrhizas

The morphology of ectomycorrhizas formed by S. pictus on P. pentaphylla was almost the same as those on P. strobus and P. koraiensis (Randall & Grand, 1986; Kikuchi & Futai, 2003). S. pictus often formed aggregates of ectomycorrhizas and developed rhizomorphs connecting the base of sporocarps and ectomycorrhizas.

Table 1a shows the vertical distribution of mycorrhizas and fine roots in plot 2. Soil depth significantly influenced the biomass of mycorrhizas and fine roots. Mycorrhizas and fine roots were distributed mostly in the top soil layer (0–5 cm) and decreased gradually as soil depth increased, regardless of sampling size. No significant interaction between sampling size and depth was detected. S. pictus mycorrhizas amounted to c. 30% of total mycorrhizas and were dominant to other mycorrhizas. Most of S. pictus mycorrhizas were distributed at the soil depth of 0–15 cm (100 cm2 sample: 89.5%, 300 cm2 sample: 72.6%) and S. pictus mycorrhizas were found in all the soil samples of top soil layer. There were no significant differences in the biomass of mycorrhizas and fine roots between 100 cm2 and 300 cm2 samples. There were significant correlations between the biomass of S. pictus mycorrhizas, other mycorrhizas and fine roots (r = 0.46–0.79, P = 0.001, n = 100). The proportion of ectomycorrhizal root tips to total root tips was estimated to be c. 95% (data not shown).

The biomass of ectomycorrhizas in plot 1 is shown in Table 1b, which is somewhat underestimated because only ectomycorrhizas in the 0–15 cm depth were taken into account. S. pictus mycorrhizas amounted to 24% (area A), 39% (area B), 38% (area C), 43% (area D-1) and 40% (area D-2) of total mycorrhizas. Ectomycorrhizas of Cenococcum geophilum were abundant in areas A and B. There were no significant differences in biomass of ectomycorrhizas and fine roots among four areas and plot 2 except for the difference between the fine root biomass in plot 2 and area D. Therefore, fine roots and mycorrhizas were distributed rather uniformly on a large scale in this plantation. We could find mycorrhizas of S. pictus easily at every sampling point when we took samples for the DNA analysis, which also indicated a rather uniform distribution of mycorrhizas and fine roots in this plot. There were correlations between the biomass of S. pictus mycorrhizas, other mycorrhizas and fine roots also in plot 1 (r = 0.46–0.54, P = 0.01, n = 35). Many mycorrhizas were sampled in the area where no S. pictus sporocarps were observed and the spatial distribution of S. pictus mycorrhizas was wider than that of sporocarps.

Genet distribution of S. pictus sporocarps

Sporocarps of S. pictus were classified into four genets using ISSR marker (type A to D) as shown in Fig. 4. Two sporocarps taken from a group of sporocarps occurring gregariously in a small area always belonged to the same genet type, namely type A, as analyzed using ISSR marker. We estimated the genet types of 38 sporocarps that were not analyzed with ISSR marker on the assumption that the types were the same as those of nearby sporocarps. Most of the sporocarps belonged to types A and C, and < 10% of sporocarps belonged to types B and D, as shown in Table 2. The spatial distribution of each genet is shown in Fig. 4(a). Sporocarps of S. pictus belonging to each genet occurred separately from those of other genets and most of them occurred aggregately. Clump size of sporocarps of type A was larger than that of type C, while spatial distribution of the clumps of type C (m* = 11.5m + 4) was more contagious than that of type A (m* = 4.4m + 11.0). Sporocarps of types A and C occurred gregariously in a small area around H-8 and G-7, respectively. The genet expansions and the genet area sizes of S. pictus sporocarps are shown in Table 2. Both the genet expansion and the genet area size of type A were the largest among the four genet types. The genet area size of type C was considerably larger than types B and D, while the genet expansion of types C, B and D were almost the same. Seasonal occurrence of sporocarps of S. pictus belonging to each genet is shown in Fig. 6. Those sporocarps belonging to type A and C occurred in early summer and autumn, while types B and D produced sporocarps only in autumn. The peak of sporocarp occurrence of three genets, types A, B and C, was in late September, while type D occurred only in October. Temporal and spatial distribution of sporocarps were different among each genet type.

Table 2.  The expansion, area size and biomass of sporocarps and ectomycorrhizas of Suillus pictus for each genet type
 Genet type
Type AType BType CType D
  • *

    , Including sporocarps estimated by distribution of examined sample.

  • **

    , These values were calculated as follows; the area size of each genet of ectomycorrhizas in areas A, B, C or D was multiplied by the average density of ectomycorrhizas (depths 0–15 cm) of S. pictus in each area (Table 1b) and they were summed up for each genet.

  • ***

    , Figures in parentheses show percentage of each genet to all genet type.

No. of sporocarps examined 26  4 44 5
No. of sporocarps* 48 (41)***  4 (3.4) 59 (50.4) 6 (5.1)
Genet expansion of sporocarps (m) 21  3.4  6.4 5.8
Genet expansion of ectomycorrhizas (m) 26.1 30.0 25.328.4
Genet area size of sporocarps (m2)100.8 (84.2)  1.62 (1.3) 13.9 (11.6) 3.38 (2.8)
Genet area size of ectomycorrhizas (m2) 33 (6.9)259.5 (54.1)152.1 (31.7)35.4 (7.4)
Biomass of sporocarps (g d. wt m−2)  0.16 (42.1)  0.01 (2.6)  0.19 (50.0) 0.02 (5.3)
Estimated biomass of ectomycorrhizas (g d. wt m−2)**  0.70 (5.4)  7.22 (55.2)  4.11 (31.5) 1.04 (8.0)
Ratio of ectomycorrhizal biomass to biomass of sporocarps  4.4548.3 21.252.5

Genet distribution of ectomycorrhizas of S. pictus

Each of six fine roots sampled in 6 randomly selected points outside plot 1 to examine the genet structure on a small scale had between 6 and 10 active mycorrhizas of S. pictus. These six to 10 mycorrhizas of S. pictus formed on each fine root belonged to the same genet except for one out of six ectomycorrhizas on one fine root. Ectomycorrhizas of S. pictus at soil depths of 0–5 cm and 5–10 cm at six randomly selected points in plot 1 belonged to the same genet type. Therefore, genets of S. pictus did not generally intermingle with each other on a small scale.

A total of 242 ectomycorrhizas of S. pictus were classified into four genet types corresponding to those of sporocarps, types A to D (Fig. 3). Each genet of ectomycorrhizas showed rather patchy distribution, as shown in Fig. 4(a, b). The spatial distribution of genets of ectomycorrhizas of S. pictus in smaller subplots (area M or S) showed the same tendency as in larger subplots (Fig. 4b). Genet types that could not have been detected in larger subplots were detected in the smaller subplots in some cases. Therefore, spatial distribution of genet types that occurred at a low frequency, such as types A and D, may be wider than those shown in Fig. 4(a). Most of the patches of genet consisted of more than one sampling point, especially when examined on a smaller scale (Fig. 4b), which indicated that most of the patches of genet were > 50 cm in size. The genet expansion, genet area size and biomass of S. pictus mycorrhizas are shown in Table 2. The biomass of S. pictus mycorrhizas of each genet is somewhat underestimated because only mycorrhizas at the 0–15 cm depth were taken into account in the calculation of these values. The area sizes of each genet differed widely, while the expansions of each genet were almost the same. Type B and type C were distributed widely and occupied 54.1% and 31.7% of the plot, respectively. The biomass of mycorrhizas of type B amounted to 55.2% of total biomass of S. pictus mycorrhizas and the biomass of this type was about 10 times as much as that of type A (Table 2).

Most of the sporocarps of each genet type occurred in or around the area where the corresponding genet type of ectomycorrhizas were distributed. However, spatial distributions of ectomycorrhizas of each genet were wider by far than those of corresponding sporocarps, except for type A. The genet expansions and genet area sizes of mycorrhizas of S. pictus differed greatly from those of sporocarps. Type A, which had the largest genet area size based on the distribution of sporocarps, had the smallest genet area size of the ectomycorrhizas. By contrast with type A, type B, which had the smallest genet area size of the sporocarps, had the largest genet area size of the ectomycorrhizas. But it is possibile that types A and D are also distributed in a large area at low densities in the plot. It is difficult to detect such distributions at low density with the present sampling scheme. Production of sporocarps did not correlate with genet area size of ectomycorrhizas. For instance, type B, which produced the smallest number of sporocarps among four genets, had the largest genet area size of the ectomycorrhizas, while type C, which produced the largest number of sporocarps, had the second largest genet area size of the ectomycorrhizas (Fig. 4, Table 2).

Discussion

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

Sporocarp occurrence and ectomycorrhizal biomass of S. pictus

Suillus pictus was the ECM species forming the highest number of sporocarps in the study plot (25% of all ECM sporocarps in 2002). Kikuchi & Futai (2003) also reported that S. pictus was the most dominant species, both in the sporocarp and the ectomycorrhizal community in a Korean pine (Pinus koraiensis) plantation in the same experimental forest station. Among the ECM species, only S. pictus is specific to five-needled pine species (Murata, 1976; Wu et al., 2000); all the other species, such as, Strobilomyces confusus, Lactarius chrysorrheus and Tylopilus castaneiceps, can associate with broad-leaved trees and/or two-needled pines (Imazeki & Hongo, 1989; Palfner, 1998).

Five-needled pines including Japanese white pine are not indigenous to this area and most of the ectomycorrhizal fungi seem to have come from the nearby forests of Quercus spp. and P. densiflora (two-needled pine species). S. pictus, which is specific to five-needled pine species, might be competitive with other fungi that have a broad host range, becoming dominant in the study plot. Colonization of Japanese white pine trees by S. pictus might have occurred in the nursery and persisted even after planting in the field. In this experimental station, some other five-needled pine species such as P. wallichiana, P. koraiensis and P. strobus had been planted. Inoculum of S. pictus might have come from the plantation site of these trees and colonized Japanese white pine seedlings in the nursery. Selosse et al. (1999) confirmed the persistence of nursery inoculated Laccaria bicolor for > 10 yr after plantation in the field.

Sporocarps of S. pictus occurred in only < 20% of the subplots and showed aggregated distribution. The spatial distributions of S. pictus sporocarps and other species did not overlap and S. pictus seemed to prefer places with low litter accumulation compared with other dominant species such as S. confusus and L. chrysorrheus for fructification. The average thickness of the litter layer in places where sporocarps of S. pictus occurred (1.5 cm) was significantly thinner than in places with occurrence of other dominant ECM sporocarps (2.0–2.4 cm, t-test, P = 0.05, unpublished data). Kikuchi & Futai (2003) also reported that S. pictus sporocarps mostly occurred in the disturbed area with low litter accumulation. However, there were many places with low litter accumulation without sporocarp occurrence of S. pictus in the present study plot and spatial distribution of sporocarps of S. pictus seems to be determined by factors other than litter accumulation, spatial distribution of mycorrhizas and competition between other ECM species.

The biomass of fine roots and total mycorrhizas were 107.3 ± 19.1, and 38.6 ± 7.2 g d. wt m−2 (mean ± SE, average of 100 and 300 cm2 samples), respectively, in this plantation. These values are within the range of biomass in other reports (Vogt et al., 1983; Dahlberg & Stenlid, 1994; Kikuchi & Futai, 2003). As for the distribution of mycorrhizas, there were no significant differences among the biomass of mycorrhizas and fine roots in plot 2 and four areas in plot 1 estimated using different sample sizes, which indicated rather uniform distribution of mycorrhizas and fine roots on a large scale in the study plot. This is probably because Japanese white pines of the same age were planted uniformly and densely in the corresponding plot. Similar to the sporocarp community, S. pictus was dominant in the mycorrhizal community in the study plot, and amounted to 24–43% of total mycorrhizas. This species was also dominant in a P. koraiensis stand and occupied 75% of total mycorrhizas (Kikuchi & Futai, 2003). Most of the mycorrhizas of S. pictus were distributed in the upper soil layer as shown in Kikuchi & Futai (2003).

Suillus pictus mycorrhizas were found at almost every sampling point and it was revealed that S. pictus mycorrhizas were distributed widely, irrespective of sporocarps occurrence in the study plot. Gardes & Brun (1996) also demonstrated that the spatial distribution of sporocarps of S. pungens did not always reflect that of mycorrhizas in a Californian bishop pine forest.

Relationship between genet distribution of sporocarps and ectomycorrhizas of S. pictus

Temporal distribution of S. pictus sporocarps differed among genets. Sporocarps of type D occurred mostly in the latter part of October, while those of the other three genets occurred mostly in late September. Sporocarps of S. pictus occurred at around 20°C (the mean temperature in May, June, September and October in 2002 was 18.4°C, 22.6°C, 23.2°C and 17.0°C, respectively). Phenology of sporocarp occurrence differs between species and temperature affects it greatly in field (Wilkins & Harris, 1946). There may be differences in the response to temperature among genets, even within a species. Although sporocarps of types A and C occurred both in early summer and autumn, while types B and D produced sporocarps only during the autumn, this difference may have simply resulted from the fact that types A and C produced numerous sporocarps, thus increasing the chance of production in early summer.

Spatial distribution as well as temporal distribution of S. pictus sporocarps differed among genets. Genet area sizes of sporocarps did not correlate with those of ectomycorrhizas or with the number of sporocarps of each genet. Spatial distributions of genets of S. pictus ectomycorrhizas were wider than those of sporocarps, which suggested that the production and spatial distribution of S. pictus sporocarps did not always reflect the biomass and distribution of mycorrhizas of S. pictus either at species level or at genet level. Sporocarps of each genet type occurred in or around the patches of corresponding genet of mycorrhizas in general. Several sporocarps of type A occurred somewhat away from its mycorrhizas, as in rows no. 3, 7 and 8 (Fig. 4). There may probably be small patches of type A mycorrhizas in the vicinity of these sporocarps that were not detected by our sampling method.

Concerning those species that form fairy rings of sporocarps, the spatial distribution of sporocarps seems to overlap that of mycorrhizas (Ogawa, 1975; Last et al., 1983; Dighton & Mason, 1985). Guidot et al. (2001) employed the PCR-RFLP method using species-specific primer and demonstrated that the spatial distribution of genets of Hebeloma cylindorsporum sporocarps represented well that of ectomycorrhizas in the Pinus pinaster stands on both occasions that H. cylindrosporum formed fairy rings, and formed only small patches of a few sporocarps. Zhou et al. (2001b) also revealed that the genet distribution of sporocarps reflected that of corresponding ectomycorrhizas for S. grevillei using a species-specific SSR marker. However, they also reported that the number of sporocarps was not always consistent with the size of the subterranean part of the genet, and sporocarps were not always centered over the subterranean parts. Therefore, genet of ECM fungi should be estimated on mycorrhizas for the analysis of genet structure and propagative manner of ECM fungi, as shown in the present study.

Concerning the genet distribution of mycorrhizas on a small scale, Zhou et al. (2001b) reported that the mycorrhizal genet distribution at the upper soil layer did not always reflect that at the lower soil layer. In the present study, the same genet types were detected in the top soil layer and in 5–10 cm soil for all the six sampling points examined and we considered that genets of S. pictus scarcely intermingle with each other on a small scale in this plot. As 49–79% of S. pictus mycorrhizas were distributed at a soil depth of 0–10 cm, the genet distribution of mycorrhizas revealed in the present study is considered to reflect the actual genet distribution fairly accurately. Genet expansions of mycorrhizas were almost the same among the four genet types, while genet area sizes of mycorrhizas differed greatly among genets. Small genet area sizes observed for genet types such as types A and D, which seemed to be present at a low density, may be underestimated in the present study as we examined only one ectomycorrhiza per intersection, or a progressive replacement and fragmentation of some genets (such as type A, which could have been initially uniformly distributed) by others such as type B may have occurred. If the latter was true, the competition between genets within a species is rather intense in the study plot.

Only four genets of large genet expansion were found in this plot and no small genets were found. Genet density of S. pictus in this stand was estimated as 83.3 ha−1, which is far smaller than that of S. bovinus (667 ha−1) in a 20-yr-old Scots pine plantation (Dahlberg & Stenlid, 1994). Based on the genet distribution of sporocarps, it has been assumed that species forming many small genets mainly propagated by colonization from spores and species that form a few large genets propagated mainly by mycelial extension (Dahlberg & Stenlid, 1990, 1994; Dahlberg, 1997; Anderson et al., 1998; Bonello et al., 1998; Gherbi et al., 1999; Zhou et al., 1999). Dahlberg & Stenlid (1995) has shown that Suillus spp. established by colonization from spores at the early stage formed many small genets in a young-aged stand and, expanded by mycelia, formed a few large genets as the host tree population became mature. Zhou et al. (1999) demonstrated that S. grevillei was propagated by colonization from spores even in a mature Larix stand and Redecker et al. (2001) also revealed the importance of colonization from spores for Amanita francheti, Lactarius xanthoglactus and Russula cremonicolor in mature forests.

By contrast to these results, S. pictus seems to have propagated mainly by mycelial extension even though the age of the tees is not great (28 yr) and the trees are, on average, small (mean d.b.h. = 9.8 cm). Guidot et al. (2002) demonstrated that H. cylindrosporum formed larger genets and mainly propagated by mycelial extension under conditions with low competition between other fungi. It formed many small genets, mainly propagated by colonization of spores under conditions with high competition between other fungi even in the mature forests. In the present study plot, the competition between S. pictus and other fungi seems to be not so intense and could propagate mainly by mycelial extension. Kretzer et al. (2003) showed that Rhizopogon vesiculosus propagate mainly by mycelial extension while R. vinicolor formed small genet only. Spore colonization seemed to be important for this species in mature Douglas fir stands. Even two sister species of Rhizopogon propagated differently in the same stand. The importance of the capacity to survive and expand for a long period in the soil as a vegetative mycelium and the capacity to colonize from spores seems to be different between species and under different environmental conditions.

The estimation of mycelial growth rate of ECM fungi under natural conditions varies greatly among ECM species. The value of 0.1–0.2 m yr−1 has been estimated (Ogawa, 1975; Last et al., 1983; Dahlberg & Stenlid, 1990; Bastide et al., 1994) based on the outward growth of fairly ring or the genet expansion of sporocarps, while Gryta et al. (2000) reported that the two genet of H. cylindrosporum extended at a rate of c. 0.45–0.6 m yr−1. Bonello et al. (1998) estimated mycelial growth rate of S. pungens to be 0.5 m yr−1 and Selosse et al. (1999) estimated 1.1 m yr−1 for Laccaria bicolor.

If S. pictus grows at a similar rate to S. pungens (0.5 m yr−1) for 25 yr (after plantation), genet expansion will be 25 m, which might explain some of the present genet expansion (25.3–30.0 m). But this assumption requires that all the four genet had colonized immediately after plantation. It is more likely that S. pictus belonging to each genet colonized the Japanese white pines in the nursery, and persisted even after planting in the field, as already discussed. Another possibility is that the growth rate of S. pictus hyphae and rhizomorphs may be far faster than presumed as in the case of L. bicolor (Selosse et al., 1999). Both possibilities can explain the fact that the expansion of the genet was almost the same among four genets whose area sizes varied greatly. The differences in area size among each genet might be a result of intraspecific competition and/or colonization rate of each genet on the nursery seedlings at planting.

The ratio of biomass of sporocarps to that of mycorrhizas ranged from 4.4 to 548.3 and varied greatly among genet types (Table 1b). For instance, type C, which had the second largest estimated biomass of ectomycorrhizas, produced the largest number of sporocarps, while type B produced the smallest number of sporocarps in spite of its largest estimated biomass of ectomycorrhizas. The ratio of biomass of sporocarps to that of mycorrhizas of S. pictus in a Korean pine stand was calculated as 19.1 (Kikuchi & Futai, 2003). They established their plot along the forest edge where S. pictus sporocarps were frequently found and the distribution of sporocarps and mycorrhizas of S. pictus overlapped better than our study and they regarded all the mycorrhizas of S. pictus as involved in the production of sporocarps. However, many of the mycorrhizas included in the calculation in the present study might not have played a role in the production of sporocarps, especially types B and D. Each genet may have a different strategy for investing in the sporocarp production, or soil conditions may have determined sporocarp production mainly and most of the areas occupied by type B might be inappropriate for fructification.

Our present study revealed the relationship between the genet distribution of sporocarps and ectomycorrhizas on a large scale (480 m2) for the first time. The spatial distributions of genets estimated on mycorrhizas were, in the main, far wider than those of sporocarps. We also revealed that the ratio of biomass of sporocarps to that of ectomycorrhizas varied among genets in the same stand. We conclude that vegetative growth of mycelia played an important role in the propagation of S. pictus in this plot. At this site, spatial distribution of sporocarps in the previous 2 yr was almost the same as in 2002. Therefore, the tendency in the production of sporocarp of each genet seems to be relatively stable at least for the period of 3 yr. Long-term monitoring of genet distribution is required to elucidate the dynamics of genets of S. pictus in the soil.

Acknowledgements

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

We would like to express our sincere thanks to Dr K. Tsuda, Gifu Academy of Forest Science and Culture, and Dr T. Osono, Lab. of Forest Ecology of Kyoto University, for many valuable discussions and useful suggestions. We also thank the members of our laboratory and the staff of Kamigamo Experimental Forest Station of Kyoto University forest for their support.

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