Two Endophytic Fungi in Different Tissues of Scots Pine Buds(Pinus sylvestris L.)

A.M. Pirttila,1,2 H. Pospiech,2 H. Laukkanen,1 R. Myllyla,2 A. Hohtola1

1 Department of Biology/Botany, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland2 Department of Biochemistry, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland

Received: 17 June 2002; Accepted: 4 October 2002; Online publication: 17 December 2002

A B S T R A C T

Two fungal species were isolated with different frequencies from pine tissue cultures originating

from buds. One species was detected in 33.1% of the cultures initiated in March, and another was

present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological

characteristics these fungi were identified as Hormonema dematioides (isolated in March) and

Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides

and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R.

minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to

H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was

found to be localized in the scale tissues of the buds. Fungal endophytes have previously been

detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi

may reflect their different roles in the plant.

Introduction

Fungal endophytes are detected in the leaves of many

grasses and trees [5, 35, 40]. Endophytic fungi are well

studied in grasses, but the endophytes of trees are known

in less detail. The fungal diversity is large in tree leaves,

especially in conifers, where the needles are colonized

locally by several endophytic fungal species [11, 12, 16, 18,

32, 45, 46]. In addition to leaves, endophytic fungi are

found in other tree tissues, such as wood and bark [3, 8,

15, 22, 33]. However, plant buds have been scarcely

studied for fungal endophytic presence.

Although endophytic fungi are rarely detected from

conifer needles soon after emergence [12, 14, 17], bud

scales of some trees contain fungal endophytes [50]. In

addition, fungi are frequently detected in plant tissue

cultures that are initiated from the buds. The microbes

appearing in plant tissue cultures create a particular

problem in terms of regenerating the plant. Some mi-

crobes may also remain latent in the plant tissue and are

considered the most problematic ones, because of diffi-

culties in their detection [27, 28]. Although many of these

microbes may represent endophytes, they are usually re-

garded as contaminants originating from outside the cul-

Current address: A.M. Pirttila, Department of Botany and Plant Pathol-

ogy, Purdue University, 1155 Lilly Hall, West Lafayette, IN 47907-1155,

USA

Correspondence to: A.M. Pirttila; E-mail: AM.Pirttila@oulu.fi

Microb Ecol (2003) 45:53–62

DOI: 10.1007/s00248-002-1038-8

� 2002 Springer-Verlag New York Inc.

ture, and methods for eliminating them have been devel-

oped [7, 21, 27, 28, 41, 48].

Scots pine (Pinus sylvestris L.) is the main source of

timber in Northern Europe, but micropropagation of the

mature tree is hampered by several factors. Tissue cultures

originating from buds of Scots pine are very often occu-

pied by filamentous fungi or yeasts, and browning and

deterioration of the cultures is a typical feature [20, 24].

The browning is accompanied by lipid peroxidation and

lignification of cells, which are symptoms of high oxidative

stress and characteristic features of the plant defense re-

action [25, 26]. We have previously found that bacteria

isolated from tissue cultures of Scots pine originated from

the bud tissues [36]. In the beginning of this study,

we considered the question of whether also the fungal

contaminants of tissue cultures would originate from

the buds, regarding the possibility that endophytes were

responsible for the defense reaction occurring in the tis-

sue cultures. In this study, we isolated fungi from the

tissue cultures and investigated the origin, location, and

occurrence of two fungal isolates in bud tissues of Scots

pine.

Materials and Methods

Plant Tissue Culture and Isolation of Fungi

In March 1999, pine buds were collected from mature, healthy-

looking trees on natural stands in Oulu (65�N; 25�30¢E) (240

buds) and in Sodankyla (67�30¢N; 27�E) (328 buds) in Northern

Finland. Additionally, 180 buds were collected in June 1999 from

Oulu. The buds were surface disinfected for 1 min in 70% ethanol

and for 20 min in 6% calcium hypochlorite. After rinsing, the

brown bud scales were aseptically removed and the apex was

separated and placed on a modified Murashige and Skoog me-

dium (MS) [20, 34]. This experiment was part of a larger study,

aiming to elucidate the browning phenomenon occurring in the

pine tissues. However, frequent contamination of the tissue

cultures drew our attention to the microbes, which were mainly

fungi. When fungal growth was detected on the plant tissue,

the colony habit and cell morphology were examined, and rep-

resentatives were transferred to a fresh culture medium (Luria

Bertani, LB, or MS). The fungi that had been detected on

more than one explant (isolates L, M, T; Table 1) were studied

further.

DNA Extraction, PCR, Cloning, and Sequencing of the Fungal

18S rDNA

The 18S ribosomal DNA (rDNA) was amplified from the isolated

fungal DNA (42) by PCR (PTC200, MJ Research) in a reaction

mixture of universal primers NS1 (5¢-GTAGTCATATGCTTGT-

CTC-3¢, Saccharomyces cerevisiae positions 20–38) and NS8 (5¢-TCCGCAGGTTCACCTACGGA-3¢, positions 1788–1769) [53],

nucleotides (Pharmacia), buffer, and PCR enzyme (Dynazyme,

Finnzymes). The DNA was first denatured in 94�C for 5 min, and

the first 3 cycles of 1 min at 94�C, 1 min at 53�C, and 3 min at

72�C were performed. The cycles were then repeated 3 times with

51�C, and 30 times with 49�C as annealing temperatures, and the

final extension was carried out at 72�C for 5 min. The PCR

products were cloned into pUC19 using End Conversion Mix

(Novagen) and T4 DNA ligase (MBI Fermentas), and transformed

into Escherichia coli DH5a competent cells by using ampicillin

and the IPTG (isopropyl-ß-D-1-thiogalactopyranoside)–X-Gal (5-

bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) blue-white se-

lection system [43]. Plasmid DNA was isolated and sequenced

from three clones according to the manufacturer’s instructions

(Abi Prism BigDye Terminator Cycle Sequencing Kit and Abi

Prism 377 DNA Sequencer, PerkinElmer).

The 18S rDNA of isolate T was not amplifiable using the

primers NS1 and NS8, but a 1200-bp segment was amplified

using the primers NS8 [53] and M-sp1 (5¢-TCTGGTGCCAG-

CAGCCGC-3¢ Saccharomyces cerevisiae positions 563–580). The

DNA was first denatured at 94�C for 5 min, and the first 4 cycles

of 1 min at 94�C, 1 min at 62�C, and 3 min at 72�C were per-

formed. The cycles were repeated 4 times with 60�C and 30 times

with 58�C as annealing temperatures. The final elongation was

carried out at 72�C for 5 min. The PCR products were cloned into

pUC19 and three clones were sequenced, as described above. The

sequences were aligned with all accessible sequences obtained

Table 1. Isolation of fungi from tissue cultures originating from pine buds

Collection area LocationCollection

monthNumber of

buds

Detectiontime

(weeks)a

Allmicrobes

(%)b Fungi (%) Isolates (%)c

Oulu 65�N; 25�30¢E March 240 1–2 40 39 L, M (33.1)Sodankyla 67�30¢N; 27�E March 328 1–2 28.7 28.7Oulu 65�N; 25�30¢E June 180 2–4 8.3 6.1 T (1.7)

a Time after which visible growth was detected in the tissue culturesb The percentage of tissue cultures where any microbes were detected in relation to the number of all tissue cultures initiatedc The percentage of tissue cultures where the isolates or morphologically similar fungi were growing, in relation to the number of all tissue cultures

initiated in March (for isolates L, M) and in June (isolate T)

54 A.M. Pirttila et al.

through the Basic Local Alignment Search Tool (BLAST) [1] and

the Ribosomal Database Project (RDP) [31].

Identification of the Fungal Isolates

The fungi were identified by their morphological and/or physio-

logical characteristics at the Deutsche Sammlung von Mikro-

organismen und Zellkulturen GmbH (DSMZ, Braunschweig,

Germany).

Accession Numbers of Strains and Nucleotide Sequences

Strains M and T were deposited in DSMZ under accession

numbers DSM 14201 and DSM 14202, and the 18S rDNA se-

quences were submitted to Genbank under accession numbers

AY150054 and AY 150055, respectively.

Molecular Phylogenetic Analysis

In order to further characterize the fungal isolates, and to define

the organisms to which the 18S rDNA-based oligonucleotide

probes would be specific in the in situ hybridization, the phy-

logenetic position for the isolates was determined. Based on the

alignment data, a phylogenetic analysis for the isolates was per-

formed with the 18S rDNA sequences of close relatives of Au-

reobasidium pullulans and Rhodosporidium dacryoides, as

represented in the RDP [31]. The sequences were retrieved from

GenBank [2] and aligned by using ClustalW [49] with the fol-

lowing default parameters: gap opening penalty 10.0, gap ex-

tension penalty 0.20, and DNA weight matrix IUB. The gap

positions were excluded manually with the Unix Pico program

(Seibel M., Hubert S. & Lundblade L., University of Washington,

2001). A distance matrix was created with DNADIST of Phylip

[13], from which the tree topology was built by the neighbor-

joining method in the program NEIGHBOR. The confidence for

individual branches of the resulting tree was estimated by per-

forming 1000 bootstrap replicates by using the programs SEQ-

BOOT, DNADIST, NEIGHBOR, and CONSENSE.

Design, Synthesis, and Labeling of the Probes

To determine the localization of the two fungal species in the

pine buds, an in situ hybridization approach was applied. Oli-

gonucleotide probes complementary to the 18S rDNA of the

isolates were designed (HD13, 5¢-TCCTTCCGGACAAGGTGA-

TGAAC-3¢ for strain M, S. cerevisiae positions 1506–1484 and

RM6, 5¢-TGAGTCATTAAAAACCTCATC-3¢ for strain T, posi-

tions 1064–1044) using the programs ‘‘Sequence Match’’ and

‘‘Probe Match’’ of RDP [31]. The probes HD13 and RM6 showed

at least one mismatch with all accessible 18S rDNA sequences

obtained through the RDP and BLAST [1, 31]. The oligonucleo-

tides were labeled with digoxigenin following the manufacturer’s

protocol (DIG Oligonucleotide 3¢-End Labeling Kit, Roche).

Determination of Stringent Conditions for Hybridization

Stringent conditions for the in situ hybridization of the probes

were determined by ‘‘whole cell’’ and dot blot hybridization, as

described in Wagner et al. [51] and Pirttila et al. [36]. S. cerevisiae

L40, Hymenoscypha ericae Read 100, Phialophora finlandia

UAMH 8322, and Phialocephala fortinii SE 24 were used as ref-

erence strains, from which DNA was isolated according to Rogers

and Bendich [42]. Probes HD13 and RM6 hybridized specifically

to their targets at formamide concentrations of 45 and 40%, re-

spectively (data not shown).

Collection and Fixation of Pine Bud Specimens

Pine buds (five from separate branches of each tree, three trees

per area) from the two areas in Northern Finland, Oulu and

Sodankyla, were studied for fungal occurrence by in situ hy-

bridization. Buds were collected in February 2001, from mature,

healthy trees and surface disinfected as described above. After

rinsing, the brown bud scales were removed aseptically and buds

longer than 2 mm were cut in half longitudinally. Bud specimens

were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M

NaH2PO4/Na2HPO4 (pH 7.4), at 4�C overnight. The fixed speci-

mens were dehydrated, cleared through an ethanol t-butanol

series, and embedded in paraffin (Merck).

In situ Hybridization

Paraffin-embedded bud specimens were sectioned longitudinally.

The sections, 6 mm thick, were baked on silane-coated slides and

paraffin was removed in xylene. Altogether 30 vegetative buds

were examined by performing 1 to 4 hybridizations per bud. For

each sample, two probes, HD13 and RM6 were hybridized as

described [36], and controls of a eubacterial probe E11 [36] and

blank (no probe) were included each time. The slides were

treated prior to hybridization as described in DeLong et al. [10]

and hybridized under maximum-stringency conditions, as de-

termined. The hybridization buffer contained 3· SET (450 mM

NaCl, 60 mM Tris-HCl pH 7.5, and 3 mM EDTA), Denhardt’s

solution (0.02% Ficoll, Pharmacia, 0.02% polyvinyl pyrrolidone,

Sigma, 0.02% bovine serum albumin, Sigma), 0.02% tRNA (Sig-

ma), 0.02% polyadenylic acid (Sigma), 10% dextran sulfate

(Merck), 50 mM DTT (Calbiochem), and the probe (0.5 ng/mL).

Slides containing the hybridization mixture were placed in air-

tight chambers saturated with 3· SET and incubated at 38�Covernight. After hybridization, the slides were washed with 2·SET at room temperature for 15 min and with 0.1· SET at 53�Cfor 15 min. Detection was performed using a DIG Nucleic Acid

Detection Kit (Roche), after which the slides were rinsed with

70% ethanol, air-dried, covered with immersion oil and a cover

glass, and viewed under bright field illumination (Optiphot-2

Photomicroscope, Nikon). Negative and positive control (sense

and antisense) hybridizations with an RNA-probe targeted to-

ward the plant 25S rRNA [52] were also performed.

Fungal Endophytes of Pine Buds 55

Isolation of Fungi from Bud Scales and Microscopic Studies

Surface disinfected (1 min in 70% ethanol and 20 min in 6%

calcium hypochlorite), unpeeled pine buds were placed on MS

medium for the isolation of fungi. Preparing paraffin sections

from the scales proved to be unmanageable, as the scales are

composed of very thin and hard tissue. Besides difficulties in

sectioning, the scales would easily come off the glass slides de-

spite the type of coating used. Strong pigmentation would also

hamper hybridization and detection of the probes in the scale

tissue. Because of these problems, the presence of fungi in de-

tached pine scales was studied with a light microscope (SMZ-2T,

Nikon). Hyphae were scratched from the scales using a needle,

transferred into a drop of sterilized water on a microscopic slide,

and stained with ethidium bromide (0.5 mL of 10 mg/mL solution

was added under the cover slide) for 2 h the dark. Hyphae were

also transferred aseptically onto MS medium.

Results

Isolation of Fungi from Tissue Cultures

During our routine micropropagation, microbes were

found in 40% of tissue cultures initiated in March from

buds originating from Oulu. Bacterial growth was detected

in three of these, and fungal growth was found in 39% of

the tissue cultures after 1–2 weeks (Table 1). Based on

morphological characteristics, the fungi were identified as

a single species. Two isolates from distinct pine cultures

were obtained after transferring the fungal cells onto fresh

plant culture medium, and designated strains L and M.

Fungi morphologically similar to strains L and M were

detected in 28.7% of the tissue cultures originating from

Sodankyla (Table 1; 33.1% of all the cultures initiated in

March). Of the tissue cultures started in June, 6.1% con-

tained fungi after 2–4 weeks (Table 1). Three yeast isolates

with similar morphology were detected (1.7% of cultures

initiated in June), one of which was transferred onto fresh

plant medium and designated strain T. The isolated fungi

did not survive surface disinfection, which was tested by

deliberately contaminating bud surfaces with the fungal

isolates, and by surface disinfection, and then dipping the

buds in the medium (MS or LB; data not shown). We

therefore considered the isolates to be potential endo-

phytes and studied them further.

Identification and Phylogenetic Analysis

Isolate M was identified as Hormonema dematioides Lag-

erberg & Melin, and isolate T as Rhodotorula minuta

(Saito) F. C. Harrison at the Deutsche Sammlung von

Mikroorganismen und Zellkulturen GmbH (Braunschweig,

Germany).

Since 18S rDNA-based oligonucleotide probes were

used in the subsequent in situ hybridization studies, the

organisms to which these probes would be specific had to

be determined by phylogenetic analysis. When the fungal

18S rDNA sequences were aligned using BLAST [1] and

RDP [31], sequences L and M were identical and aligned

best with Aureobasidium pullulans 18S rDNA. Sequence T

aligned better with 18S rDNA of Rhodosporidium dacryo-

ides than with R. minuta strain JCM3777 of GenBank [2].

Therefore, a phylogenetic analysis for isolates M and T was

performed with sequences of close relatives of Aureoba-

sidium pullulans and Rhodosporidium dacryoides, as rep-

resented in the RDP [31]. According to this analysis,

isolate M formed a cluster with Aureobasidium pullulans,

Dothidea insculpta, and D. hippophaeos, supported by a

bootstrap value of 997 of 1000 repeats, and isolate T with

Rhodotorula minuta strain JCM3777 at a bootstrap value

of 999 (Fig. 1A, 1B).

Localization of Fungal Isolates in Bud Tissues of Scots Pine

When in situ hybridizations were performed on pine bud

tissues, probe RM6, targeted toward the 18S rRNA of R.

minuta strain T, hybridized to 40% of all the samples (Fig.

2). The hybridization signal was detected in buds of every

tree examined from both study areas, Sodankyla and Oulu

(Fig. 2). The signal was especially strong in the cells of

scale primordia (Fig. 3A). In addition, hybridization signal

was abundant in the epithelial cells of the resin ducts and

in the outermost cells of the meristems (Fig. 3A), although

there was less microbial rRNA inside the meristems. The

hybridization signal was also frequent in the cells of the

developing stem, directly below the meristems, but there

was little or no signal in the vascular tissues or intercel-

lular spaces. Probe HD13, targeted toward 18S rRNA of H.

dematioides strain M, did not hybridize to any of the bud

samples (Fig. 3B). There was a weak hybridization signal

detected in some samples, but this was interpreted as

background. The eubacterial probe E11 hybridized to 83%

of all the bud samples examined, which was consistent

with our previous results [36]. Control hybridizations with

a sense 25S rRNA probe [52] did not result in signal de-

tection (Fig. 3C), but the antisense 25S rRNA [52], which

served as a positive control, stained the pine bud tissue

intensively (Fig. 3D).

56 A.M. Pirttila et al.

Presence of Hormonema dematoides isolate M was

studied further in the buds of Scots pine after the finding

that fungi morphologically similar to isolate M grew from

100% of unpeeled buds on the MS medium. H. dematioides

was then postulated to be localized between the bud and its

scales, or inside the scale tissue. Using light microscopy,

structures resembling fungal hyphae were detected on the

interior of the scale, primarily at the tips of the outermost

scales (Figs 3E, 3F). The hyphae partly emanated from the

scale tissue (Fig. 3F). When the hyphae were detached

from the scales to be examined on a microscopic slide,

there was an abundancy of resin fibers present which

hampered detection of the hyphae. Therefore, the slide was

stained with ethidium bromide and hyphal structures were

detected (data not shown). To confirm that the hyphae

represented H. dematioides, they were transferred to MS

medium and fungus morphologically similar to isolate M

grew on the plate in 15 days (data not shown).

Discussion

Identification and Phylogenetic Analysis

In this study, two fungal species were found in tissue

cultures originating from buds of pine (Pinus sylvestris L.).

Isolate M was identified as Hormonema dematioides, but

the 18S rDNA sequence aligned best with Aureobasidium

pullulans 18S rDNA using BLAST [1] and RDP [31]. To

our knowledge, the phylogenetic position based on 18S

rDNA has not been determined for H. dematioides, but H.

dematioides and A. pullulans are closely related by taxo-

nomic classification [19]. A close phylogenetic relation

between these two species was expected based on the 18S

rDNA alignment data, and ascertained in the 18S rDNA

phylogenetic analysis. Although H. dematioides strain M

grouped in this analysis with Dothidea insculpta and D.

hippophaeos as well as with A. pullulans, D. insculpta and

D. hippophaeos formed a separate group and were more

divergent from H. dematioides strain M than was A.

pullulans.

Although isolate T was identified as Rhodotorula min-

uta, its 18S rDNA sequence was aligned best with Rho-

dosporidium dacryoides using BLAST [1] and RDP [31]. In

the 18S rDNA phylogenetic analysis isolate T and R.

minuta strain JCM3777 formed a group and were diver-

gent from R. dacryoides. The alignment was performed

with a shorter sequence than was the phylogenetic analy-

sis; therefore, certain parts of strain T 18S rDNA may be

more identical with R. dacryoides than with R. minuta

strain JCM3777. We were not able to amplify the complete

Fig. 1. (A and B) Phylogenetic positions of the pine bud-asso-

ciated fungal isolates M and T. The 18S rDNA sequences of the

isolates, Saccharomyces cerevisiae (outgroup), and close relatives

of Aureobasidium pullulans and Rhodosporidium dacryoides, as

represented in the RDP [31], were analyzed with the neighbor-

joining method. Values from 1000 bootstrap repeats are pre-

sented if support was >50%. The species names are followed by

the GenBank accession numbers.

Fungal Endophytes of Pine Buds 57

18S rDNA of strain T using R. minuta strain JCM3777-

specific primers (data not shown), which also showed the

divergency between these strains. The phylogenetic posi-

tion of R. minuta strain JCM3777 was consistent with an

earlier analysis [47].

Endophytic Fungi in Tissue Cultures of Scots Pine

H. dematioides was detected in bud-derived tissue cultures

initiated in March with a high frequency, but it was not

found in the June samples. When the in situ hybridization

technique was applied to study location of H. dematioides

inside the bud, it was not detected in the meristematic

tissues. Instead, using light microscopy H. dematioides

was localized in the bud scales. The location in the bud

scales explains why H. dematioides was detected in cul-

tures initiated in March but not in June, since bud scales of

elongating shoot have fallen off in June, and scales of the

new, developing bud are emerging. The reason why H.

dematioides was detected in the tissue cultures despite not

being found in the meristematic tissue by in situ hybrid-

ization is likely that some Hormonema hyphae become

detached from the scales when a bud is peeled for mi-

cropropagation. Mediated by scalpel and forceps, the hy-

phae further contaminate the inner surface of the bud,

which was detected to occur when buds were peeled under

light microscope (data not shown).

A visible growth of H. dematioides is observed on the

pine bud tissue soon after start of culture (within 1–2

weeks) and the fungus occupies the whole culture in 1–2

days. Therefore, the pine tissue usually dies shortly after

H. dematioides is detected (data not shown). Once in tis-

sue culture, H. dematioides is growing on a tissue which

is not its natural habitat, and it seems thus to have an

advantage compared to the survival of the plant. In the

beginning of this study we hypothesized that the problems

in regenerating the mature Scots pine were due to a de-

fense reaction caused by endophytes growing uncontrol-

lably in the plant tissue, because browning, lipid

peroxidation, and lignification of cells, characteristic fea-

tures of the plant defense reaction, occurred in the tissues

[25, 26]. However, when we further studied the occurrence

of H. dematioides inside the cultured plant tissue, we

found that it produces a visible growth when present and

does not stay latent in the tissue (data not shown [37]).

Because contaminated tissues are usually removed as soon

as microbial growth is detected on them, H. dematioides is

unlikely to cause the symptoms observed in the remaining

tissues [37].

R. minuta was isolated from a few cultures initiated in

June. However, according to the in situ hybridization data,

R. minuta inhabited 40% of the meristematic bud tissues

of pine. The location of R. minuta in the bud tissue, that is,

in the cells of scale primordia, in the epithelial cells of the

resin ducts, in the outermost cells of the meristems, and in

the cells of the developing stem resembles that of intra-

cellular bacteria, detected previously in pine bud tissues

[36]. As with R. minuta, the bacterial endophytes grew

infrequently out of the pine tissue [36]. In another study

we studied their presence further during tissue culture of

Scots pine, and it was detected that these endophytes grew

inside the cultured tissues latent, but more vigorously than

in the bud during its natural, active growth [37]. Based on

that study it appears that these endophytes are under strict

control in a growing pine bud, but become uncontrollable

once a tissue culture is initiated from the bud [37].

Therefore, it is possible that these endophytes are re-

sponsible for the defense reaction occurring in pine tissue

cultures [37].

Fig. 2. Detection of Rhodotorula minuta in pine buds by in situ

hybridization. Bud specimens (five per tree) were collected from

three trees in each of the two sample areas (Sodankyla and Oulu).

The graph represents the percentage of bud specimens hybrid-

izing positively with the R. minuta-specific probe RM6, from

each tree examined; S, Sodankyla; O, Oulu. The percentages for

the two areas and for all samples are also presented.

58 A.M. Pirttila et al.

Fungal Endophytes and Their Roles in Buds

The term endophyte is defined as ‘‘fungi or bacteria found

inside the plants that do not elicit symptoms or disease’’

[54]. Because H. dematioides emanated from the bud scale

tissue, it may be regarded as an endophyte. There are

earlier reports of H. dematioides as an endophyte in pine

and fir needles [11, 18, 45, 46], but it has not been detected

in bud scales before. The bud scale is metabolically less

active than the bud meristem or the leaf tissue, and per-

Fig. 3. In situ hybridization of pine (Pinus sylvestris L.) bud

tissue with digoxigenin-labeled probes, and light microscopic

studies of fungal presence in pine bud scales. For differentiation

from the positively hybridized cells, the naturally brown cells of

the pine tissue which contain tannins and other phenolic com-

pounds, are marked with t. Needle primordium (N) and scale

primordia (S), hybridized with (A) the Rhodotorula minuta-

specific probe RM6, (B) the Hormonema dematioides-specific

probe HD13, (C) the negative control of sense 25S rRNA, and (D)

the positive control of antisense 25S rRNA. (E) Interior of bud

scales, examined by light microscopy. Fungal hyphae are de-

tected on the scales (arrow). (F) Fungal hyphae on the interior of

bud scales, partly emanating the tissue (arrow). Scale bar 20 mm

in (A–D), 200 mm in (E, F).

Fungal Endophytes of Pine Buds 59

haps resembles the bark tissue in its protective function

and low metabolic activity. There are several reports of

endophytes isolated from the bark tissues [8, 15, 33], and

Discula umbrinella has been isolated from the bud scales

of beech (Fagus sylvatica), whereas it was absent from the

leaves enclosed by the scales [50]. The scale-inhabiting

fungi may represent a group of endophytes that share a

similar role in the plant. The position in the scales that are

sheltering the bud, which is the most valuable organ in the

plant in terms of regeneration and reproduction, could

indicate a protecting role against pathogens. Some Hor-

monema strains produce toxic substances, which would

support this theory [4, 38].

R. minuta has previously been found as an epiphyte on

trees [6, 29], but it has not been reported as an endophyte.

However, species closely related to R. minuta (R. glutinis

and Sporobolomyces sp.) are found as endophytes in the

wood of grapevine [44]. The observation that R. minuta is

located intracellularly in similar positions in the bud tissue

as the endophytic bacteria [36] raises the question of

whether these endophytes have a similar role in the buds.

In an another study we found that these endophytes in-

crease in number or metabolic activity prior to growth of

the bud (Pirttila et al., unpublished data). Therefore, these

microbes may have a role in plant growth. One fungal

endophyte, Colletotrichum sp., found in the stem of Ar-

temisia annua (Asteraceae) produces the plant growth

hormone indole-acetic acid in liquid culture [30]. Pro-

duction of auxins and gibberellins is also typical for many

fungi and bacteria associated with plant roots, although

the significance of these products for the plant has re-

mained ambiguous [9, 23, 39].

In this study, two fungal species, Hormonema dema-

tioides and Rhodotorula minuta were localized in bud

scales and meristematic cells of buds in Pinus sylvestris L.,

respectively. Specialization in different tissues by the two

fungal endophytes may reflect their different roles within

the plant. Endophytes are in many respects still an un-

known area for plant biologists, but they are attracting

more attention as their significance for the plant is being

unraveled. Besides their importance for the plant through

protection against pathogens, endophytes have an impact

on commercial and scientific plant tissue culture, where

contamination of the cultures is causing constant pro-

duction losses [27, 28]. Determining the location and role

of endophytes in the plant may help overcome these

problems, in addition to producing new information on

endophytes.

Acknowledgments

We thank T. Uusitalo for technical assistance, S. Mattila

for help in constructing Fig. 1, M. M. Muller and C. P.

Woloshuk for their valuable comments on the manuscript,

M.-A. Alenius and S. Rintamaki for providing the con-

taminated tissue cultures, M. Makiniemi for the S. cere-

visiae strain L 40, and A. L. Ruotsalainen for the strains of

Hymenoscypha ericae Read 100, Phialophora finlandia

UAMH 8322, and Phialocephala fortinii SE 24. This work

was supported by Societas Pro Fauna et Flora Fennica

and the Finnish Cultural Foundation.

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