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: [email protected]
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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