Diversity of Norway spruce needle endophytes in various mixed and pure Norway spruce stands

Mycol. Res. 102 (10) : 1183–1189 (1998)

1183

Printed in the United Kingdom

Diversity of Norway spruce needle endophytes in various mixed and pure Norway spruce stands

M I C H A E L M. M U> L L E R A N D A N N A-M A I J A H A L L A K S E L A The Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland

Norway spruce needles were sampled from two series of stand areas located in southern Finland. Both series consisted of five sampling areas in mature managed stands and one in a mature virgin stand. The proportion of spruce varied from 8 to 100 % of the basal tree area and the major other species were pubescent birch and Scots pine. From each sampling area (some of which consisted of several sites) 40 mature spruces were randomly chosen and healthy looking needles of the third age class were sampled from heights of 5–8 m and incubated on water agar for isolation of endophytic fungi. The majority of isolates were identified by their combined fatty acid and sterol profiles (FAST-profiles) as Lophodermium piceae. Tiarasporella parca was less common and Rhizosphaera kalkhoffii, Sclerophoma pythiophila, Lirula macrospora and Thysanophora penicillioides occurred occasionally. For calculations of fungal diversity all isolates were classified by their FAST-profiles into 81 groups (¯ operational chemotaxonomic units) according to a defined upper variation limit, i.e. an upper FAST-profile mismatch limit. The highest percentage of endophytically infected needles was found in pure spruce stands and dense virgin stands. Location of the stand, its proportion of spruce and total basal area of trees (i.e. tree density) explained 82 % of the variation of the overall infection rate. The effect of location was probably a consequence of differences in air quality between the various sampling areas. The endophyte diversity, expressed as the number of FAST-groups per 40 spruces of each sampling area, correlated positively and statistically significantly with the percentage of needles infected or with the proportion of spruce in the stand. The highest endophytic diversity, expressed as FAST-groups per tree, was found in pure spruce and mixed virgin stands.

The endophytic fungi of trees have attracted increasing interest after the discovery that some of them affect insects attacking the phyllosphere (Carroll, 1988). Probably all trees hide various endophytic fungi in their phyllosphere (Petrini, 1991). Some conifer needle endophytes have been shown to act antagonistically against insect herbivory, e.g. Rhabdocline parkeri Sherwood, J. K. Stone, & G. C. Carroll in Pseudotsuga menziesii (Mirb.) Franco (Carroll, 1986). On the other hand, other endophytic fungi may increase insect vitality in their host ; sycamore aphids grow larger and show higher fecundity in the sycamore tree (Acer pseudoplatanus L.) which has been infected by Rhytisma acerinum (Pers.) Fr. (Gange, 1996). Fungi dwelling in spruce needles have earlier been suspected of contributing to the needle cast observed in various parts of Europe but evidence supporting this assumption has not been obtained (Sieber, 1988 ; Suske & Acker, 1989 ; Livsey, 1995). Instead, for instance Lophodermium piceae (Fuckel) Ho$ hn., the most common endophyte of Norway spruce needles, seems to be sensitive to air pollutants and occurs more frequently in young, healthy stands than in stressed and diseased ones (Barklund, 1987). The species richness of the endophytic mycota of conifer needles is rather high : up to 110 (mean value 60) species for

a given host could be identified in a number of extensively sampled conifers (Petrini, 1986). Sieber (1988) detected 100 endophytic species from Norway spruce needles collected in Switzerland from 21 sites. Carroll (1988) proposed that short-cycle (one or several generations each year) endophytes increases the resistance potential of long-living trees against the effects of short-cycle insects. It can be assumed that the diversity of the endophytic microflora is important to the function of the forest ecosystem because at least some of the endophytic mycota affect the palatability of their host to insects. Little is known of the effects of forest management on the amount and diversity of phyllosphere endophytes. Tree density and their species composition probably affect the microclimatic conditions in the canopy and may thus be important factors directing the amount and diversity of phyllosphere endophytes. The advantages and disadvantages of mixed versus onespecies forests are continuously debated from various ecological and economical point of views. The aim of this study was to characterize the diversity of endophytic needle fungi of Norway spruce in southern Finland and to find out how the tree species composition affects this diversity. Additionally, our aim was to find out whether the needle

Diversity of endophyte needle fungi

1184

endophytic mycota of virgin spruce stands differs from that in managed stands. The identification of endophytic fungi is often difficult (Petrini, 1986) and the population structure of the morphologically defined species living in Norway spruce needles is poorly known. Additionally, all species have possibly not yet been described. A chemotaxonomic method was therefore, chosen for their classification in this study. The application of FAST-profiles provided several advantages compared to morphological classification. FAST-profiles allow e.g. division of fungi (including isolates of species not yet described) into operational chemotaxonomic units with an upper variation limit (Mu$ ller & Hallaksela, 1998).

MATERIAL AND METHODS Sampling areas Two series consisting of six sampling areas each were chosen for the sampling of Norway spruce needles. One is in Tuusula and Sipoo, adjacent rural districts approx. 15 km from the coast of the Gulf of Finland (near Helsinki), and the other in the rural districts of Padasjoki and Kuhmoinen, 120 km north from Tuusula and Sipoo. The sampling areas within both series are situated at a maximal distance of 20 km from each other. Both series included one virgin forest and five managed forest areas, all of the Myrtillus-type (Cajander, 1949) and in topographically flat areas (maximum slope 5 %). The managed

Table 1. Stand characteristics : amount, age and growth of trees of the sample areas. For range of height and number of injured and dead standing trees see text. Dominant tree species % in bold Basal area (%)

Norway spruce

Site

Trees sampled (no.)

Norway spruce

Scots pine

Pubescent European Grey birch aspen alder

Common alder m# ha−"

Age (y)

Growth dm$ tree−" 5 y−"

Tuusula & Sipoo P H D A B CA" CA # CA $ CA % CB " CB

40­10# 40 40 40 40 7 7 2 4 8 12

56 100 90 72 43 6 8 2 10 9 16

30 0 3 13 36 89 92 97 85 91 84

14 0 7 15 21 5 0 1 5 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

41±7 38±4 23±6 28±1 26±8 30±7 25±4 24±5 24±0 17±5 15±7

122 70 97 83 75 83 62 56 78 99 107

41 111 72 71 99 124 165 128 198 123 90

40

8

90

2

0

0

0

23±0

81

138

20 20

56 45

30 48

14 5

0 2

0 0

0 0

42±0 44±9

165 150

63 41

V " V

40 20 20

51 100 97

39 0 0

10 0 3

1 0 0

0 0 0

0 0 0

43±4 22±4 27±9

157 95 96

52 64 55

K " K

40 20 20

98 63 48

0 27 48

2 10 4

0 0 0

0 0 0

0 0 0

25±1 30±7 23±6

95 95 106

60 109 88

M " M

40 20 20

55 28 21

38 70 71

7 2 8

0 0 0

0 0 0

0 0 0

27±1 23±6 23±1

101 73 100

99 93 50

T " T

40 20 20

24 26 21

71 6 13

5 61 65

0 4 0

0 1 1

0 2 0

23±4 22±0 25±4

87 77 69

72 100 89

N " N

40 20 20

24 15 13

9 72 84

63 13 1

2 0 0

1 0 2

1 0 0

23±7 27±3 26±8

73 84 96

95 139 110

40

14

78

7

0

1

0

27±1

90

125

Sampling area

#

C Padasjoki & Kuhmoinen X " X #

X #

V #

K #

M #

T #

N

" Growth of spruce was determined by measuring growth of the last five years from incremental cores and using the formula 61±2 given by Laasasenaho (1982). # Results obtained from 50 trees were used for results presented in Fig. 2, for other calculations data obtained from 40 trees were used. $ From left : Picea abies Karst., Pinus sylvestris L., Betula pubescens Ehrh., Populus tremula L., Alnus incana L., and A. glutinosa (L.) Gaertn. Only trees above 10 cm diam. were measured.

M. M. Mu$ ller and Anna-Maija Hallaksela forest areas were chosen in stands as similar as possible with the exception of their proportion of spruce (Table 1). Each sampling area consisted of one, two or six round sites with a radius of 25 m with the exception of sites CB, T and N (area codes refer to Table 1) in which the radius was 30 m. The Csites are in two clusters situated 8 km from each other and within them the sampling sites were at a distance of ca 20–100 m from each other. The height of the dominant trees varied from 20 to 27 m. The number of trees thicker than 10 cm (at 120 cm trunk height from the ground) varied from 325 to 575 ha−". On most sampling sites some spruces were damaged : 0–25 trees per site had polypores, a canker or crack with resin bleeding. Additionally, in the virgin sites there were 5–9 dead spruces and 10–12 dead trees of other species standing. The corresponding values for the managed forest sites were 0–2 of spruce and 0–4 of other tree species.

1185 randomly chosen and further inoculated for FAST-characterization. Because of high infection levels in Padasjoki & Kuhmoinen only isolates from odd numbered needles were taken for FAST-characterization. The FAST-profile data were treated according to a two step statistical approach (Mu$ ller & Hallaksela, 1998). Briefly, the isolates were first grouped into taxonomic species using a discrimination model based on well identified ‘ model ’ isolates. These groups, together with one group including those isolates which remained unidentified with the discriminant model, were further divided into operational chemotaxonomic units (or FAST-groups) using a FAST-profile mismatch threshold. The mismatch threshold used in this study was 9±2 which corresponds to the average within-species mismatch of the six most common fungal species on fallen Norway spruce needles collected from the sampling areas of this study (Mu$ ller & Hallaksela, 1998). RESULTS

From each sampling area at least forty healthy looking spruces (diameter over 18 cm) were randomly chosen and sampled during September–November. In Tuusula & Sipoo this was carried out in 1994 and in Padasjoki & Kuhmoinen in 1995. One branch pointing to the centre of the site was cut from each of these trees at a height of 5–8 m. Two needle segments (one from the leading shoot and one from a side shoot) of ageclass three were cut and stored at 4 °C until further processing. Twig segment sequences delimited by a set of terminal bud scars produced in previous years were counted for the determination of needles age classes. Thus, the current season’s growth at the tips of the branches was considered to be age-class 1 foliage. Needles attached to twig segments below the first set of terminal bud scars were considered as age-class 2 needles and the next set were age-class 3. The needle segments were surface sterilized within 24 h after collection by shaking them for 1 min in 70 % ethanol, 4 min in 3–5 % sodium hypochlorite and for 1 min in 70 % ethanol. They were then rinsed twice in sterile water and 10 green healthy looking needles were removed from each segment. Four, approximately 0±5 mm thick slices, were cut from the needle base after removal of the petiole. These needle slices were incubated on water agar for one month at 20° in darkness. Fungal growth appearing in the water agar was inoculated from the edge of the colony to MOS-agar (modified orange serum agar, Mu$ ller, Kantola & Kitunen, 1994), incubated for 3–4 wk and evaluated visually for purity. Small inocula from the edge of impure looking colonies were further transferred to fresh MOS-agar. If necessary, the isolates were further purified by inoculation on water agar for 1–2 mo and then single hyphal tips were transferred to fresh MOS-agar with the aid of a microscope and a modified Pasteur pipette. FAST-analysis of isolates and statistical calculations The isolates were cultivated and analysed for their fatty acids and sterols (FAST-profiles) as described earlier (Mu$ ller et al., 1994 ; Mu$ ller & Hallaksela, 1998). If fungal growth appeared from more than one slice}needle, only one of them was

The majority of all isolates were identified by their FASTprofiles as Lophodermium piceae which were further divided into 47 mismatch-groups (Table 2). Approximately one third of all L. piceae isolates fell into one mismatch-group (Fig. 1). Only seven mismatch-groups, all identified as L. picea, were found in all sampling areas. A considerable proportion, 32 out of all 81 mismatchTable 2. Distribution of endophytic isolates according to a discriminant model (Mu$ ller & Hallaksela, 1998). Using mismatch grouping the isolates were further divided into 81 FAST-groups i.e. operational chemotaxonomical units Isolates

FAST-groups

Lophodermium piceae (Fuckel) Ho$ hn. Tiarosporella parca (Berk. & Broome) H. S. Whitney, J. Reid & Piroz. Thysanophora penicillioides (Roum.) W. B. Kendr, Rhizosphaera kalkhoffi Buba! k Lirula macrospora (R. Hartig) Darker Sclerophoma pythiophila (Corda) Ho$ hn. Unknown

1593 40

47 7

37

8

4 3 3 50

4 2 3 10

Total

1730

81

600 500 Number of isolates

Isolation of the fungi

400 300

L. piceae (all grey columns)

200

T. penicillioides T.parca Unknown T.parca

100 0

1

3 2

5 4

7 6

9 8

11 13 15 17 19 21 23 25 10 12 14 16 18 20 22 24 26 FAST-group

Fig. 1. Number of isolates of those FAST-groups with more than 10 isolates.

Diversity of endophyte needle fungi

1186

Table 3. Number of isolates of endophytic needle fungi, overall infection rate and their diversity (no. of FAST-groups per sampling area, per sample tree and the Shannon–Wiener index) FAST-groups Number Number of isolates Sampling area

Site

Sum

Tuusula & Sipoo P — H — D — A — B — CA – 42 "% VB – 38 "# Padasjoki & Kuhmoinen X 496 " X 347 # V 352 " V 445 # 200 K " K 343 # M 345 " M 331 # T 299 " T 220 # N 315 " N 158 #

Sum Average

— —

Needles infected (%)

Number of isolates investigated"

Per area

Per tree

Shannon– Wiener index#

Forest type (managed areas are listed according to decreasing proportion of spruce)

376 381 310 94 130

54 54 35 15 20

217 217 138 61 81

39 35 27 25 24

4±00a 4±15a 2±68b 1±53c 1±60c

2±79 2±68 2±35 2±88 2±39

80

17

68

20$

1±48c

2±54

Spruce dominated, virgin Pure spruce, managed Spruce dominated, managed Spruce dominated, managed Spruce dominated, managed Pine dominated, managed Pine dominated, managed

843

83

165

28

3±43d

2±64

Spruce dominated, virgin

797

85

169

36

3±30de

2±68

Pure spruce, managed

543

64

128

31

2±75e

2±79

Spruce dominated, managed

676

73

145

29

3±18de

2±73

Pine dominated, managed

519

68

135

30

2±78de

2±53

Birch dominated, managed

473

65

130

29

2±75de

2±61

Pine dominated, managed

5222 —

— 53

1654 —

81 30

— 3±03

— 2±63

—

" Only one isolate}needle was determined. # Krebs (1989). $ This value has been extrapolated (see Fig. 2). abc de

Values for Tuusula & Sipoo without a common letter differ (P ! 0±01, Kruskal-Wallis). Values for Padasjoki & Kuhmoinen without a common letter differ significantly (P ! 0±01, Kruskal-Wallis).

Table 4. Regression model for the logarithm of the overall infection rate Y

X

Coefficient

P (2-tail)

Loge % infected needles

Location (latitude) Proportion of spruce (% of basal area) Total basal area of trees (m#) Constant

1±413 0±009 0±114 ®82±04

0±000 0±010 0±080 0±000

n ¯ 19, r# ¯ 0±816 and P ¯ 0±000

groups, were found only once or twice. The average withingroup mismatch values of the five most common FASTgroups (groups 1–5, Fig. 1) varied between seven and eight (a result not shown in Tables). The mean proportion of needles infected endophytically was 53 % but varied considerably between the sampling areas (Table 3). In general, the Tuusula & Sipoo spruce needles were less infected (33 %) than those in Padasjoki & Kuhmoinen (73 %). In both area series the highest percentage of infection was found in virgin and pure spruce stands. Geographical location, spruce proportion and tree density (expressed as basal area of trees) explained over 80 % of the variation of the logarithm of the percentage of needles infected (Table 4). The geographical location alone explained 61±2 % of the variation

(a result not shown in Tables). The geographical location expressed as latitude gave a higher r# than when expressed as a dummy variable. The highest amount of FAST-groups}area was found in the virgin stand P (39 groups, Table 3). The cumulative number of FAST-groups plotted as a function of the number of sampled trees in Fig. 2 can be expressed by a model of the following type : Y ¯ a®bX (−cX−d), where X is the amount of trees sampled and a, b, c and d are sampling area-specific parameters (as given for areas P and C in the legend of Fig. 2). In addition to the 39 FAST-groups found from 40 sampled trees in the virgin area P, sampling of 10 additional trees resulted in only two more FAST-groups. Estimating by the model, 45 FASTgroups should have been found if all 87 spruces of area P

M. M. Mu$ ller and Anna-Maija Hallaksela

1187

50

45 P r = 0.962** Area P

30 20

FAST-groups

FAST-groups

40 Y = a - b*X(-c*x-d) Area C

35 D A

25

10 0

H

B

C 0

10

20 30 40 Number of sample trees

50

15

60

0

30

60

Fig. 2. Cumulative no. of FAST-groups as a function of number of trees sampled. Area P was the most diverse and area C the least diverse stand expressed as the number of FAST-groups per 40 sampled trees. For area C the number of FAST-groups per 40 trees was extrapolated as shown in the figure. Values of the model given in the figure : Area P ; a, 45±6 ; b, 43±7 ; c, 0±009 ; d, 0±116. Area C ; a, 21±9 ; b, 18±9 ; c, 0±013 ; d, 0±064.

would have been sampled. Thus, the 39 FAST-groups actually found from the 40 trees sampled represent 100¬(39}45) ¯ 87 % of the predicted number of FAST-groups. Twenty four FAST-groups were found from sampling area C. This result, however, is not comparable with the results of the other sampling areas of the Tuusula & Sipoo series since there was a considerable distance (8 km) between the two site clusters (sites CA – and CB – , Table 1) and the site "% "# characteristics (e.g. total base area of the trees per site) differed. These two site clusters are, therefore, treated separately in the diversity calculations. In both site clusters 20 trees were sampled and the mean values of the cumulative numbers of FAST-groups per site cluster were plotted in Fig. 2. With the previously mentioned model (Y ¯ a®bX(−cX−d) the value of 19±7 FAST-groups. corresponding to sampling from 40 trees, was obtained by extrapolation (Fig. 2 and Table 3). The variation of Shannon–Wiener indices (Krebs, 1989) was low between the sampling areas (Table 3). The virgin area in the Padasjoki & Kuhmoinen series had the lowest number of FAST-groups (28) and it was also much lower than that of the virgin area in Sipoo (39, Table 3). This is surprising, since the areas are rather similar according to their stand characteristics (partly given in Table 1). The number of FAST-groups calculated per tree in area X was, however, highest for the Padasjoki & Kuhmoinen series. The combined results of both virgin areas show a higher

Fig. 3. Number of FAST-groups of the sampling areas in Tuusula & Sipoo as the function of percentage of needles infected. Symbols refer to sampling areas, see Table 3.

number of FAST-groups and a higher amount of rare FASTgroups (found only on these areas) than the combined results of pure spruce or pine dominated managed areas (Table 5). Even the combination of results of three managed areas, dominated by Scots pine, gave a lower number of FASTgroups compared to that of the virgin areas. The number of FAST-groups in Tuusula & Sipoo correlated statistically significantly with the percentage of needles infected (Fig. 3). The variation of the percentage of needles infected was low in Padasjoki & Kuhmoinen (64–85 %, Table 3) and did not correlate significantly with the number of FAST-groups. The highest diversity was, however, also found in the area with the highest proportion of infections (Area V, Table 3). Multivariate regression analysis revealed a significant effect of spruce proportion (expressed as % of the total basal area) on the diversity of needle endophytes of managed stands (expressed as the number of FAST-groups per 40 sampling trees) (Fig. 4). The effect of all other recorded stand characteristics (total basal area, age, growth rate, height, number of injured trees and number of dead trees standing) remained insignificant in the analysis. If the virgin stands were also included in the regression analysis, the effect of basal area became significant in the Tuusula & Sipoo data but not in the Padasjoki & Kuhmoinen data. This is due to the fact that sampling area X has an exceptionally low amount of FASTgroups compared to its high proportion of spruce and high total basal area.

Table 5. Number of all FAST-groups and those which were not found from other areas listed in this table FAST-groups

Norway spruce dominated, virgin Pure Norway spruce, managed Scots pine dominated, managed Scots pine dominated, managed

90

Needles infected (%)

Sampling areas

Trees sampled (no.)

All

Only found in these areas

P, X H, V C, M C, M, N

80 80 80 120

47 44 37 44

11 6 6 8

Diversity of endophyte needle fungi

1188

Tuusula & Sipoo

40 35

H

30

r = 0.881*

25

D A

Overall infection rate

Number of FAST-groups

B 20 C 0

20

40

60

80

100

Padasjoki & Kuhmoinen 40

V

35 r = 0.972*

30

25 0

T N M

endophytic community, while the majority of the observed FAST-groups are rare (Fig. 1), supports earlier results on the distribution of taxonomic species (Sieber, 1988) and seems to be a general feature of endophytic communities living in spruce needles.

K

20 60 80 100 40 Proportion of spruce (%)

Fig. 4. Number of FAST-groups of the managed forest areas as a function of the proportion of spruce (% of the basal area). Symbols refer to sampling areas, see Table 3.

DISCUSSION Species and within-species diversity According to the FAST-profiles the endophytic mycota of Norway spruce is dominated by L. piceae in the 12 sampling areas. Tiarosporella parca was the second commonest species which is in accordance with earlier investigations in several other European countries (Suske & Acker, 1987 ; Barklund, 1987 ; Solheim, 1994). The number of Thysanophora penicillioides detected is noteworthy as this fungus was not mentioned in the comprehensive investigations of Sieber (1988) in Switzerland and Livsey (1995) in Sweden. Rack & Butin (1984) found T. penicillioides regularly from fallen Norway spruce needles in Germany. Identification of the species listed in Table 2 other than L. piceae and T. parca were not confirmed by morphological characterization. The phenotypic variation of all six species found was rather high as judged by the number of FAST-groups produced by the mismatch grouping (Table 2). Even low numbers of isolates split into several FAST-groups. The mismatchthreshold of 9±2 used in the mismatch-grouping corresponds to an average mismatch-value of these species isolated from fallen needles (Mu$ ller & Hallaksela, 1998). The average mismatch value of the most common FAST-groups was slightly lower (between six and seven) than the mismatchthreshold used. This observed high number of FAST-groups is in accordance with earlier observations concerning the number of genotypes of other endophytic conifer needle fungi (McCutcheon & Carroll, 1993 ; Wilson et al., 1994). Our observation that just a few FAST-groups highly dominate the

The regression analysis revealed (Table 4) how the variation of the percentage of infected needles is ruled by differences between distant forests that are not explainable with any of the recorded stand characteristics. Sieber (1988) and Livsey (1995) also found a high variation between infection rates of various Norway spruce stands. The observed difference between the infection levels of our two sampling area series may be caused by variation in air quality. According to a Finnish survey (Ympa$ risto$ katsaus, 1996) the air in Tuusula and Sipoo is more polluted than in the more nothern sampling areas, Padasjoki and Kuhmoinen. Tuusula is rather close to Helsinki and its air has a higher content of e.g. nitric oxide than the air in Padaskoki and Kuhmoinen, Sipoo being intermediate. Our assumption is also supported by earlier investigations which have shown that the frequency of L. piceae infections decrease in forests disposed to heavier air pollution (Barklund & Rowe, 1983 ; Sieber, 1988). The climate is different between districts, being more continental in Padasjoki and Kuhmoinen than in Tuusula and Sipoo since the latter are close to the Gulf of Finland. The somewhat more continental climate in the Padasjoki & Kuhmoinen district could, however, be expected to decrease fungal dispersal rather than increase it. We regard it unlikely, therefore, that the difference in climate was the reason for the observed difference in the level of infection between the Padasjoki & Kuhmoinen and the Tuusula & Sipoo stands. According to the regression model in Table 4 increasing spruce proportion and increasing forest density (expressed as basal area) favour endophytic infection. Visually estimated, Norway spruce has a considerably larger shading effect than birch and pine in our sampling areas. Thus an increasing proportion of spruce will probably reduce the amount of direct sunlight reaching the lower parts of the forest canopy and increase humidity in a similar manner to increasing basal area (i.e. increasing forest density). Also previous investigations on endophyte colonization of conifer needles have shown a positive effect for humidity (Carroll & Carroll, 1978 ; Sieber, 1988) and stand density (Sieber-Canavesi & Sieber, 1987 ; Helander et al., 1994). An increasing density of spruce of course also promotes the probability of released spores of endophytic fungi to adhere to susceptible needles. Variation of endophyte diversity between stands The models given in Fig. 2 suggest that the number of spruces sampled (40) in this study was sufficient to discover the majority of FAST-groups of the sampling areas with the applied sampling methodology (two twig segments per tree, five needles per segment etc.). As needles were only sampled from one height, however, and only the needle base (representing 15–20 % of the total length of the needle) was

M. M. Mu$ ller and Anna-Maija Hallaksela investigated, the actual number of endophytic FAST-groups of Norway spruce needles in the areas is perhaps higher. Additionally, our method favoured fast growing endophytes and, moreover, we do not know whether all endophytic fungi are culturable on the media used. Several results obtained in this investigation suggest that the virgin spruce dominated stands have a more diverse endophytic mycota than corresponding mature, managed forests. The average number of FAST-groups per tree (Table 3) and the number of FAST-groups calculated from the data after combination according to forest type (Table 5) support this statement. The result for virgin Area X, however, appears confusing since this area showed the highest diversity per tree but the lowest diversity per sampling area in the Padasjoki & Kuhmoinen series (Table 3). Unfortunately no information on the genetic variation among trees in area X in relation to that of trees in the other areas is available. The low variation of the Shannon–Wiener indices of the FAST-groups between the sampling areas (Table 3) is a consequence of the strong dominance of just one FAST-group in all of the areas. A considerable proportion of the other FAST-groups were found only once or twice in this survey. The variation of FAST-group-diversity correlates with the percentage of needles infected (Fig. 3) and seems to be influenced by the same stand characteristics as the percentage of needles infected, i.e. proportion of spruce and tree density. Within managed forests pure spruce stands have the highest diversity of endophytes per tree and per stand (i.e. per 40 sampling trees) (Table 3 and Fig. 4). As discussed earlier, increasing spruce density probably promotes a more humid microclimate in the lower canopy and thus increases the density of infection sources and targets promoting the probability of infection of susceptible needles. We are grateful to Mrs Rauni Valjakka for the cultivation of the fungi and extraction of fatty acids and sterols to Mr Matti Kaivos, Mr Pekka Helminen, Mrs Minna Terho, Mrs Katja Sorri, Mrs Nina Tuomivaaraa and Mrs Salla Pelkonen for their skilful technical assistance during different phases of this study. Also we want to thank Drs Jarkko Hantula, Kari Korhonen and Kiti Mu$ ller for constructive comments on the manuscript.

REFERENCES Barklund, P. (1987). Occurrence and pathogenicity of Lophodermium piceae appearing as an endophyte in needles of Picea abies. Transactions of the British Mycologiclal Society 89, 307–313. Barklund, P. & Rowe, J. (1983). Endophytic fungi in Norway spruce – possible use in bioindication of vitality. Aquilo Series Botanica 19, 228–232. Cajander, A. K. (1949). Forest types and their significance. Acta Forestalia Fennica 56, 21–38. (Accepted 29 December 1997 )

1189 Carroll, C. (1988). Fungal endophytes in stems and leaves : from latent pathogen to mutualistic symbiont. Ecology 69, 2–9. Carroll, G. C. & Carroll, F. E. (1978). Studies on the incidence of coniferous needle endophytes in the Pacific Northwest. Canadian Journal of Botany 56, 3034–3043. Carroll, G. C. (1986). The biology of endophytism in plants with particular reference to woody perennials. In Microbiology of the Phylloplane (ed. N. J. Fokkema & J. van den Heuvel), pp. 205–222. Cambridge University Press : Cambridge, U.K. Gange, A. C. (1996). Positive effects of endophyte infection on sycamore aphids. Oikos 75, 500–510. Krebs, C. J. (1989). Ecological Methodology. HarperCollins Publishers : New York. Helander, M. L., Sieber, T. N., Petrini, O. & Neuvonen, S. (1994). Endophytic fungi in Scots pine needles : spatial variation and consequences of simulated acid rain. Canadian Journal of Botany 72, 1108–1113. Laasasencho, J. (1982). Taper curve and volume fractions for pine, spruce and birch. Communicationes Instituti Forestalis Fenniae 180, 1–74. Livsey, S. (1995). Ecology of Endophytic Microfungi in Norway Spruce Crowns. Dissertation. Swedish University of Agricultural Sciences, Uppsala, Sweden. McCutcheon, T. L. & Carroll, G. C. (1993). Genotype diversity in populations of a fungal endophyte from Douglas fir. Mycologia 85, 180–186. Mu$ ller, M. M. & Hallaksela, A.-M. (1998). A chemotaxonomical method based on FAST-profiles for the determination of phenotypic diversity of spruce needle endophytic fungi. Mycological Research 102, 1190–1197. Mu$ ller, M. M., Kantola, R. & Kitunen, V. (1994). Combining sterol and fatty acid profiles for the characterization of fungi. Mycological Research 98, 593–603. Petrini, O. (1986). Taxonomy of endophytic fungi of aerial plant tissues. In Microbiology of the Phylloplane (ed. N. J. Fokkema & J. van den Heuvel), pp. 175–187. Cambridge University Press : Cambridge, U.K. Petrini, O. (1991). Fungal endophytes of tree leaves. In Microbial Ecology of Leaves (ed. J. H. Andrews & S. S. Hirano), pp. 179–197. Springer-Verlag : New York & Berlin. Rack, K. & Butin, H. (1984). Experimenteller Nachweis nadelbewohnender Pilze bei Koniferen I. Fichte (Picea abies). European Journal of Forest Pathology 14, 302–310. Sieber, T. (1988). Endophytische Pilze in Nadeln von gesunden und gescha$ digten Fichten (Picea abies (L.) Karsten). European Journal of Forest Pathology 18, 321–342. Sieber-Canavesi, F. & Sieber, T. N. (1987). Endophytische Pilze in Tanne (Abies alba Mill.). – Vergleich zweier Standorte im Schweizer Mittelland (Naturwald-Aufforstung). Sydowia 40, 250–273. Solheim, H. (1994). Occurrence of the Norway spruce needle endophytes Lophodermium piceae and Tiarosporella parca at the permanent plots of the Norwegian monitoring programme for forest damage. In Shoot and Foliage Diseases in Forest Trees (ed. P. Capretti, U. Heiniger & R. Stephan), pp. 17–21. Proceedings of a Joint Meeting of the Working Parties Canker and Shoot Blight of Conifers and Foliage Diseases, Vallombrosa, Firenze, Italy June 6–11, 1994. Suske, J. & Acker, G. (1987). Internal hyphae in young, symptomless needles of Picea abies : electron microscopic and cultural investigation. Canadian Journal of Botany 65, 2098–2103. Suske, J. & Acker, G. (1989). Endophytic needle fungi : culture, ultrastructural and immunocytochemical studies. In Ecological Studies (ed. E.-D. Schulze, O. L. Lange & R. Oren), vol. 77, pp. 121–136. Springer-Verlag : Berlin and Heidelberg. Wilson, R., Wheatcroft, R., Miller, J. D. & Whitney, N. J. (1994). Genetic diversity among natural populations of endophytic Lophodermium pinastri from Pinus resinosa. Mycological Research 98, 740–744. Ympa$ risto$ katsaus (1996). Suomen Ympa$ risto$ keskuksen katsaus no. 3. The Finnish Environment Institute, Report no. 3 (in Finnish), Helsinki, Finland. pp. 1–24.