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Keywords:

  • birch;
  • dispersal;
  • endophytes;
  • fragmentation;
  • fungi;
  • island biogeography;
  • trees

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
  • • 
    The effect of environmental fragmentation on the species distribution and frequency of horizontally transmitted endophytic fungi in Betula pubescens and Betula pendula leaves was studied in an archipelago in southwestern Finland.
  • • 
    The study system consisted of 14 islands, ranging in size and distance to the mainland, and five mainland sites. Endophytic fungi were grown out from surface-sterilized leaves.
  • • 
    The frequency of endophytic fungi mainly depended on the size of the island, explaining 32–35% of the variation, and the distance to the mainland explaining 29–35% of the variation. The birch trees on the largest islands near the mainland had the highest endophyte frequencies. Fusicladium betulae, Gnomonia setacea and Melanconium betulinum were the most commonly isolated fungi.
  • • 
    Foliar endophytes of birch trees are able to disperse to fairly fragmented areas, but their frequencies seem to depend on environmental isolation and size of the island.

Introduction

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

Microfungi, along with other microorganisms including bacteria, form a highly diverse and abundant community of organisms within the plants. Many microorganisms, for example, mycorrhizal fungi and nitrogen-fixing bacteria, form associations with the host plant below ground in the root system. The above-ground parts of the plants are inhabited by epiphytic microorganisms on plant surfaces, and endophytic microorganisms that live at least part of their life cycle inside plant tissues without causing visible symptoms to the host (Wilson, 1995). The taxonomy of the endophytic fungi suggests that they are often closely related to pathogenic fungi (Clay, 1988; Gennaro et al., 2003). In some cases, these symbionts may benefit the host plant, for example by producing herbivore-deterrent mycotoxins. Some endophytes may, however, turn out to be pathogens, if the environmental factors, or genotypic or phenotypic condition of the host plant or their interactions are favorable for disease expression. Thus, the symbiotic endophytic fungi represent a continuum from antagonistic to mutualistic interactions (Saikkonen et al., 1998, 2004, 2006).

Foliar tree endophytes are nonsystemic and transmitted horizontally by spores (Helander et al., 1993; Wilson & Carroll, 1994). Tree leaves are endophyte-free when they are unfurling from buds; however, they soon become infected with airborne, rainborne or insect-vectored fungal spores, and the frequency of infections increases towards the end of the growing season (Helander et al., 1993; Ahlholm et al., 2002a), and in perennial plant organs it increases continuously over the years (Helander et al., 1994; Carroll, 1995; Hata et al., 1998; Kumaresan & Suryanarayanan, 2002). Although many tree endophytes are known to be host-specific, such as Rhabdocline parkeri in Douglas fir needles (Stone, 1987), Lophodermium piceae in Norway spruce needles (Barklund, 1987), and Neohendersonia kickxii in beech twigs (Danti et al., 2002), mutualistic relationships, such as in systemic and vertically transmitted grass endophytes and their hosts (Clay & Schardl, 2002; Saikkonen et al., 2004, 2006), should not be expected between woody plants and their foliar endophytes, because the fungus is not tightly coupled with and highly dependent on the host.

Abiotic and biotic environmental factors modify the frequency and composition of horizontally transmitted endophytic fungi in host plants (Helander, 1995; Saikkonen et al., 1996; Ahlholm et al., 2002a,b). In addition to availability of the spores, biotic environmental factors include, for example, the host plant phenotype and genotype, interactions with other microorganisms in the plant, and the herbivores using the same host plant and/or acting as vectors of the endophytes (Ahlholm et al., 2002a,b). The most important abiotic factors influencing the frequency and composition of endophytic fungi in the plant foliage are temperature and humidity (Colhoun, 1973). Germination of fungal spores occurs only over a limited range of temperature and requires access to water or high air humidity. In addition, release and dispersal of fungal spores are modified by weather conditions, especially by rain and wind (Fitt et al., 1989). Coincidence of viable spores and infection sites is another requirement for successful establishment of endophytic fungal thalli.

Islands provide an excellent opportunity to study factors affecting the dispersal ability of endophyte species and their relative frequencies in fragmented environments. According to the theory of island biogeography (MacArthur & Wilson, 1967), the size of an island and its distance from the mainland, relating to the immigration and extinction rates of species, are the two main predictors determining the number of species inhabiting it. An ‘archipelago’ for endophytic fungi is, however, hierarchically structured, comprising individual leaves (‘islands’ for single spore origin infections) and trees (genets) distributed across true islands. The number of infection sources and microclimatic conditions of an ‘archipelago’ are largely determined by growth form of individual trees (monocormic, polycormic or clonally growing cluster of trees) and whether individual tree genets grow solitarily or in clusters of trees in open areas, homogenous forests, or mixed forests (K. Saikkonen, unpublished). In this study Betula pendula and Betula pubescens were chosen as host species and the island-rich Finnish Archipelago Sea as the fragmented environment. Specifically, we wanted to examine if the number of fungal endophyte species is dependent on size and remoteness of the island; and if the common foliar endophyte species differ in their ability to disperse and infect birch leaves, depending on the birch species and environmental isolation.

Materials and Methods

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

Study area

Our study area is located in the island-rich area of the Archipelago Sea, which is part of the brackish Baltic Sea. The archipelago consists of an expansive mosaic of more than 22 000 islands with a surface area of almost 10 000 km2 (Granöet al., 1999). The islands and rocky islets extend towards the sea for 100 km along the southwest coast of Finland. The landscape in the area varies from large islands and verdant islets to bald rock islets in the outer archipelago.

We chose to sample birch leaves from five birch populations on the coast of the mainland and from 14 islands, ranging in size and distance to the mainland (Fig. 1, Table 1), at the beginning of August 1998. Sampling included both downy birch (Betula pubescens Ehrh.) and silver birch (Betula pendula Roth), if both species were available on the specific island (Table 1). The two most remote islands (Isokari and Isokari 2), were located almost 20 km from the mainland, where B. pendula trees are not growing (Fig. 1, Table 1).

image

Figure 1. Location of the study area in southwestern Finland. The five mainland sampling sites are marked M, the islands are numbered from one to 14, and the two isolated islands are called Isokari and Isokari 2. The corresponding codes for the sampling sites are used in Table 1.

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Table 1.  Characteristics of the study sites (the corresponding codes for the sampling sites are used in Fig. 1)
CodeLand area (km2)aDistance from the mainland (km)Number of islands within a distance of ≤ 1.5 km of the study plotMean openness indexMean distance to the next birch tree (m)Number of B. pendula trees examined (number of fungal species detected)Number of B. pubescens trees examined (number of fungal species detected)Total number of fungal species detected
  • M, mainland; *, the two isolated Isokari islands.

  • a

    Measured as the land area within a radius of 750 m from the sampling site.

M11.25 0 03 4.44 (14)5 (17)25
M21.35 0 01.9 5.44 (7)5 (9)11
M31.05 0 42.7 44 (8)7 (10)11
M41.02 0 92 2.55 (12)1 (8)13
M50.61 0 32.3 4.96 (12)4 (15)19
10.65 1.6162.4 3.75 (13)013
20.6 0.7122.8 35 (16)016
30.74 1111.4 5.45 (7)0 7
40.72 1112.312.51 (6)3 (8) 9
50.61 1132 4.35 (12)012
60.69 0.9 52.6174 (7)5 (15)15
70.84 1.8 3220.34 (8)1 (8) 9
80.78 0.4 62 65 (12)5 (15)19
90.16 1.2 52.4 9.25 (7)5 (7)10
100.03 2 61.640.12 (10)5 (12)13
110.06 4.2 01.7 0.1 (1)2 (7) 7
120.12 6.9 312002 (8) 8
13*0.6417.5 21.313.605 (12)12
14*0.9918.1 41.2 8.205 (12)12

Sampling of leaves, and isolation and identification of endophytes

Birch dwarf shoots were cut at a height of 1.5 m from cardinal points of the compass (four shoots from a tree) and transported in water vials to the laboratory in early August. Four leaves were detached from each shoot for further analyses (16 leaves per tree).

Leaves were surface-sterilized by dipping them into 75% ethanol (30 s), 4% Na-hypochlorite (1 min) and 75% ethanol (15 s). After air drying (5 min), five 5-mm-diameter discs (0.2 cm2 surface area) were cut aseptically from each leaf and placed on 2% (w/v) malt extract agar in Petri dishes (no antibiotics added). The surface sterilization procedure was carried out in a laminar hood to avoid contaminations. The five discs were taken from the tip, middle and base of the midrib and from the lamina on both sides of the midrib, to take into account within-leaf variation in endophyte frequencies (Helander et al., 1993). Petri dishes were sealed with Parafilm and stored at room temperature. The outgrowing endophyte colonies were counted and subcultured 1, 2, 4 and 8 wk after incubation at 20°C and identified as soon as sporulation occurred. Tree-specific means of the number of endophyte colonies per five discs (1 cm2 in area) were used in statistical analyses.

We studied closely the three most frequently isolated fungal endophyte species of birch trees, as follows:

In Finland the three frequently isolated fungal endophytes in birch foliage are symptomless during their life in green leaves. However, all of them have either been reported to cause symptoms in other host species or/and are close relatives of pathogen species.

All the other endophyte species were isolated only infrequently, and thus they were included only in total endophyte frequencies and the number of fungal species.

Environmental characters

The distance from islands to the mainland was measured to the nearest seashore. The land area around each sampling site was measured within the radius of 750 m using the MapInfo system (http://www.mapinfo.fi/), and the number of islands within a radius of 1.5 km from the sampling place were counted. To take into account the microclimatic conditions of a study site, the density of the vegetation with regard to the light conditions around each sampled tree was ranked on a scale from 1 to 3, and the distance from the sampled tree to the nearest birch tree was measured.

Statistical analyses

We conducted an analysis of variance using the GLM procedure of SAS to examine whether there are interactions between tree species and islands in their endophyte species number, enabling us to understand if the intertree species differences are consistent.

Other statistical analyses were carried out separately for the birch species, because B. pubescens was distributed across the study area (except for islands 1, 2, 3, and 5) but B. pendula trees were not detected in the Isokari islands (and island 12). In the case of B. pubescens, the statistical analyses were performed in two ways, with and without the two isolated Isokari islands (Fig. 1, Table 1), because the combined land area of these two islands is distinctly larger than the other islands, and they are located approx. 10 km away from the other islands which form a continuous distance gradient within the fragmented aggregate of islands located within 7 km of the mainland (Fig. 1). Correlation analyses among the environmental characteristics of the study sites (distance to the mainland, land area, number of nearby islands) or study trees (distance to the nearest birch tree, openness of the vegetation) were carried out using Spearman's correlation analysis of the SAS statistical package (version 8.02). To analyze the effect of distance to the mainland, land area, and number of islands on endophyte species number, total endophyte frequencies and the three common birch endophytes (F. betulae, G. setacea, M. betulinum) individually, we used simple linear regression analyses of SAS.

Results

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

Island geography and birch distribution

The smallest islands were located in the outer archipelago, with the exception of Isokari island, which was the largest and the farthest from the mainland. Thus the distance from the mainland and land area of the study site were negatively correlated (n = 17, r = –0.6926, P < 0.0021), if the two Isokari islands were excluded from the analyses. Distance from the mainland was also negatively correlated with the openness of the vegetation (n = 124, r = –0.36549, P < 0.0001), indicating that the study sites with dense vegetation were located close to the mainland. In addition, the density of birch trees was higher in large islands, that is, land area was negatively correlated with the distance to the nearest birch tree (n = 118, r = –0.2839, P < 0.0018). In short, the density of birch trees decreased from the mainland to the outer archipelago where the islands were smaller, except for Isokari island, which is large enough to support a dense B. pendula population. B. pubescens was absent from the island, however, indicating that it is not able to grow in the harsh environmental conditions of the outer archipelago.

Distribution of endophyte species

Altogether we identified 24 fungal endophytes to species/genus level. In addition, 12 nonsporulating morphotypes were detected. The two birch species differed in their fungal composition of the three most frequently isolated fungal species (Fig. 2). Fusicladium betulae was the most commonly isolated fungus in both B. pendula and B. pubescens, consisting of 66 and 36% of all the isolates, respectively. Gnomonia setacea was isolated frequently from B. pubescens leaves (25% of all fungal isolates), but was rare in B. pendula foliage (3% of all fungal isolates). Sixteen per cent of the fungal isolates from B. pubescens leaves and 11% of the isolates from B. pendula leaves belonged to M. betulinum. Fungi isolated from less than 1% of the leaves were Acremonium sp., Aposphaeria sp., Asteroma microsperma, Asteromella sp. Aureobasidium sp., Coniochaeta velutina, Coniothyrium fuckelii, Cytospora sp., Diplodina sp., Fusicoccum betulae, Geniculosporium sp., Gliocladium sp., Hormonema sp., Nodulisporium sp., Paecilomyces sp., Periconia sp., Pezicula sp., Phialophora sp., Phoma cava, Trimmatostroma betulinum and Xylariaceae o. Sclerotiniaceae.

image

Figure 2. Proportions of the three commonly isolated endophytic fungi, Fusicladium betulae, Gnomonia setacea and Melanconium betulinum, on leaves of Betula pendula and Betula pubescens growing in the 14 islands and five mainland populations in southwestern Finland. Total number of trees studied: B. pubescens, 60; B. pendula, 65.

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We found the total number of endophyte species to vary between seven and 25 among study sites. The number of endophyte species per study site varied between seven and 17, and between one and 16 for B. pubescens and B. pendula, respectively. The land area, distance to mainland and number of nearby islands were not correlated with the number of endophyte species detected in an island or the total number of species isolated from either B. pubescens or B. pendula. In addition, we did not find significant interaction between tree species and island when the number of isolated endophyte species was used as a dependent variable ( d.f. = 11, F = 0.76, P < 0.6772), confirming that intertree species differences were consistent in a specific island.

Endophyte frequencies

Microclimatic conditions (density of the vegetation and distance to the nearest birch tree) did not correlate with total endophyte frequencies or any of the studied endophyte species.

Birch leaves from the mainland, the large islands near the mainland and the Isokari islands were most frequently colonized by endophytic fungi. If the two remote Isokari islands were excluded from the analyses, distance to mainland explained 35 and 29%, and land area 35 and 32% of the variation in total endopyte frequencies of B. pubescens and B. pendula, respectively (Fig. 3). However, if the Isokari islands (where only B. pubescens was detected) were included in the analyses, the distance to mainland explained only 1% of the variation, indicating that large remote islands are comparable to mainland areas in their total endophyte frequencies.

image

Figure 3. The relationship between the density (CFU, colony-forming units) of colonization of birch (Betula pubescens and Betula pendula) leaves by the three most abundant endophytes (Fusicladium betulae, Melanconium betulinum and Gnomonia setacae) and total endophyte frequencies, and the distance of the study site from the mainland or the land area within a radius of 750 m of the study site. The two isolated Isokari islands (only B. pubescens) are marked with black triangles. Lines are fitted by simple linear regression. Differences are significant at the following: *, 5%; **, 1%; ***, 0.1%.

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The frequency of F. betulae increased with increasing land area of the island for both B. pubescens and B. pendula, explaining 25–38% of the variation (Fig. 3). In B. pendula foliage, the F. betulae infections were highest in the mainland and gradually decreased towards the outer archipelago (Fig. 3), explaining 28% of the variation. The same pattern was seen in B. pubescens, if the two Isokari islands were excluded from the analysis.

Frequencies of commonly isolated endophyte species, however, did not always follow the pattern that the number of species found on an island is determined by two factors, the effect of distance from the mainland and the effect of island size. M. betulinum endophyte infections were absent from the small islands and their frequency decreased from the mainland to the outer archipelago, but distance to the mainland explained only 4–6% of the variation (Fig. 3). However, surprisingly, the two Isokari islands (large and smaller) both had higher frequency of M. betulinum endophytes than any other sampled site. G. setacea infection frequencies were scarce on B. pendula foliage (Fig. 3). None of the measured parameters explained the occurrence of G. setacea infections in the islands.

Discussion

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

In concordance with the theory of island biogeography (MacArthur & Wilson, 1967) and empirical evidence with pathogenic fungi (Carlsson-Granér & Thrall, 2002), overall frequencies of endophytic fungi in birch leaves depend on the size of the island and its distance from the mainland. The largest islands close to the mainland had the highest overall foliar endophyte infection frequencies, and the frequencies decreased with increasing distance to the mainland and decreasing size of the island. This might be because of the low availability of inocula, as the number of host plants is sparse and they are infrequently distributed in small and distant islands. Because some of the studied islands are fairly small and far from the nearest potential inoculation source, the seasonally infected birch leaves need to receive new inocula by air from other islands. In the autumn the deciduous birch trees shed their endophyte-infected leaves and the new spores are formed in the fallen leaves during the following spring. The new inocula are usually spread by rain splash, insect vectors or wind to the unfurling leaves during the following growing season. However, in remote islands with sparse vegetation and harsh climatic conditions, it is likely that the fallen leaves will be wiped out under the birch trees during the winter, and thus the endophyte infections in the following season are highly dependent on immigration rate and adaptive radiation of the fungi.

The proportional frequencies of common endophyte species changed along the distance gradient and varied between the two birch species. The distribution of endophytic fungal species, however, appears not to be restricted in fragmented environment in terms of the size of the island and the number of nearby islands, indicating that the windborne endophyte spores are able to colonize the distant islands.

The three most frequently isolated fungal species in birch foliage in archipelago area, similarly to other studies conducted elsewhere (Helander et al., 1993; Elamo et al., 1999; Lappalainen et al., 1999), were F. betulae, G. setace and M. betulinum. F. betulae is the most abundant endophyte in birch leaves throughout the studied environments from the arctic tree line in northern Finland to natural and managed boreal forests in southern Finland (Helander et al., 1993; Elamo et al., 1999; Lappalainen et al., 1999; Helander et al., 2006) and it is believed to be widespread throughout the distribution range of Betula spp. (Barr, 1968). The species is abundant in B. pendula and B. pubescens as well as in several birch hybrids (Saikkonen et al., 2003). It was also the most frequent endophyte in leaves of B. pubescens collected a few weeks after bud burst in Switzerland (Barengo et al., 2000). The Venturia spp. type ascospores (teleomorphic state of F. betulae) are commonly detected from the air samples after rainy periods and their occurrence is correlated with the increase of endophyte infections in birch trees (M. Helander, unpublished). The airborne ascospores are easily dispersed over large areas, and the success of the infection is assured by humid conditions after rain.

In accordance with previous studies (Lappalainen et al., 1999; Saikkonen et al., 2003), G. setacea was frequently isolated from B. pubescens leaves (25% of all the isolates), but only rarely from B. pendula foliage (3% of all the isolates). B. pendula may have some morphological or chemical barriers that prevent the success of G. setacea infections on its leaves. However, there may be some genetically different strains of G. setacea that are able to overcome these barriers. A recent study by Helander et al. (2006) showing that G. setacea was frequently isolated from B. pendula leaves in natural and managed forests, but only rarely from trees growing in sapling sites, suggests that the genetic background of the host plant or some environmental factors may also affect the success of infection. However, in the present study, only 2–6% of the variation in G. setacea frequencies was explained by distance to the mainland or size of the land area around the study site (Fig. 3). Thus, it seems that G. setacea is able to survive from year to year or effectively re-immigrate to a certain area in a fragmented environment. This may hold true, because G. setacea is also able to infect shoots (Kessler, 1978) where the fungus is able to survive the winter and other harsh environmental conditions.

Similarly to G. setacea, less than 6% of the variation in M. betulinum frequencies was explained by distance to the mainland or size of the land area around the study site (Fig. 3). Thus, in contrast to island biogeography theory (MacArthur & Wilson, 1967), the distribution of G. setacea and M. betulinum endophytes is not predominantly determined by distance to mainland and size of the island. Proportional infection frequencies of M. betulinum were highest in the two Isokari islands, which are isolated from the other islands approx. 18 km from the mainland. One island is fairly large, while the other is a small island in the vicinity of the larger one. Proportionally high infection frequencies of M. betulinum compared with other common endophytes may reflect adaptive differences among the fungal species to environmental conditions in the outer archipelago. In addition to the availability and dispersal ability of the fungal inocula, the early moments of the germination of the fungus are also critical for the realization of the infection. Successful infection requires, for example, the correct temperature, windiness and humidity.

In conclusion, the foliar endophytes of birch trees are able to disperse to fairly fragmented areas, but their frequencies seem to depend on the environmental isolation and size of the island rather than microclimatic conditions. However, fungal species show differences in their adaptive ability to persist in harsh conditions in the outer archipelago. Since many of these fungal endophytes or their close relatives are dormant saprophytes or latent pathogens (Saikkonen et al., 2004), recognizing the driving forces of patterns and frequencies of endophytic fungi enables us to understand changes in mycoflora caused by fragmenting previously continuous habitat through forest practices. Contrary to the conventional wisdom of negative effects of habitat fragmentation on biodiversity and ecosystem functions, forest fragmentation appears to decrease pathogen risk.

References

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