Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Blackwell Science, 20024696702Original ArticleInfluence of SDBS on endophytic mycobiota of beechR. Danti et al.

Environmental Microbiology (2002) 4(11), 696–702

Decline in diversity and abundance of endophytic

fungi in twigs of Fagus sylvatica L. after experimental
long-term exposure to sodium dodecylbenzene
sulphonate (SDBS) aerosol

Roberto Danti,1 Thomas N. Sieber,2* Giovanni parts of beech to SDBS can affect the amount and
Sanguineti,1 Paolo Raddi1 and Vincenzo Di Lonardo1 composition of endophytic fungal communities of lig-
1
C. N. R. , Istituto per la Patologia degli Alberi Forestali, nified twigs. Degradation of the leaf epicuticular wax
Piazzale delle Cascine 28, I-50144 Firenze, Italy. layer and changes of the assimilation capacity and
2
Swiss Federal Institute of Technology, Department of leaf water content (transpiration) of the crowns are
Forest Sciences, Forest Pathology and Dendrology, presumed to be responsible for the reduction of endo-
ETH-Zentrum, CH-8092 Zürich, Switzerland. phytic fungi detected in twigs of SDBS-treated plants.

Summary Introduction

Sodium dodecylbenzene sulphonate (SDBS) is an Atmospheric pollutants can cause damage to trees
anionic synthetic detergent found in polluted sea directly by degrading the anatomical surface structure of
aerosol and is known for its harmful effects on leaf needles and leaves, with consequences on physiological
surface ultrastructure on conifers and broadleaved processes such as assimilation capacity and transpiration
trees. Four-year-old saplings of European beech were of the crowns, and indirectly, by changing the nutritional
sprayed weekly for three consecutive growing sea- characteristics of the soil. Direct and indirect changes can
sons with either a 50 mg l−1 solution of SDBS in affect the microflora of the phyllosphere (Helander et al.,
deionized water or with pure deionized water 1996).
(control). Two- to three- year-old twigs were collected The harmful effects of pollutants on the structural state
from SDBS-treated and control plants during the of leaf surfaces have been reported by various authors on
growing season one year after the last treatment to conifers and broadleaved trees, in the field, in greenhouse
isolate endophytic fungi. The frequency of coloniza- and growth chamber studies (Cape and Fowler, 1981;
tion by endophytic fungi was significantly lower on Huttunen and Laine, 1983; Rinallo et al., 1986; Rinallo
SDBS-treated plants (63.8%) than on control plants and Raddi, 1989). Changes in the epicuticular wax
(85.4%). Multiple colonization of twigs occurred more layer and stomatal rims by air pollutants can affect the
frequently and diversity of endophyte species was number and relative frequency of fungal species, both
higher in control plants than in SDBS-treated plants. epiphytes and endophytes, on and in the aerial parts of
Thirty-six fungal species were isolated from 360 plants (phyllosphere) (Heagle, 1973; Fenn et al., 1989;
twigs. Cladosporium cladosporioides, Coryneum Kirkwood et al., 1989; Helander and Rantio-Lehtimäki,
compactum, Phialocephala dimorphospora, and a 1990; Ranta, 1990; Magan and McLeod, 1991a,b;
species each of Mycosphaerella and Phomopsis were Helander et al., 1996). Changes in the microhabitat of
the most abundant endophytes with frequencies of the germinating fungal spores can have an effect on the
colonization of more than 5%. The abundance of mechanisms which control hyphal penetration into the
the Phomopsis species proved to be significantly inner tissues of plants (Chapela et al., 1991; 1993; Toti
reduced by the SDBS treatment. Within the limits of et al., 1992; Toti, 1993; Viret et al., 1993). The degree of
the indoor experimental conditions, the obtained humidity on the leaf surface and hence the persistence of
results suggest that long-term exposure of aerial canopy wetness may also undergo changes and this may
affect spore viability and hyphal growth (Allen et al., 1991).
Decline of coastal vegetation is observed in many
areas of the Mediterranean region and Australia. The
Received 22 May, 2002; accepted 3 September, 2002. *For
correspondence. E-mail thomas.sieber@fowi.ethz.ch; Tel. (+41) decline is a result of the presence of surfactants in marine
1632 5521; Fax (+41) 1632 1380. aerosols, a consequence of sea pollution by detergents

© 2002 Blackwell Science Ltd

 Influence of SDBS on endophytic mycobiota of beech 697
(Bussotti et al., 1995; Moodie et al., 1986). Sodium dode-
cylbenzene sulphonate, an anionic synthetic detergent
which is often present in polluted sea aerosols, is known
for its harmful effects on leaf surface structures on both
conifers and broadleaves, causing the degradation of epi-
cuticular leaf wax and damaging the stomatal rims
(Rinallo and Raddi, 1989; Raddi et al., 1991; 1992;
Moricca et al., 1994). These changes in turn have a neg-
ative impact on transpiration, leaf water content and tem-
perature (Devéze and Sigoillot, 1978; Truman and
Lambert, 1978; Gellini et al., 1985). There is also an
adverse effect on tree physiology because SDBS dis-
solves the lipids of the cellular membranes and destroys
the cellular organs (Bussotti et al., 1997). The harmful
effects of SDBS on leaf surface structure have been also
quantitatively assessed on different Italian beech prove-
nances (Moricca et al., 1994).
Endophytic fungi are an important group of phyllo-
sphere symbionts in the tissues of many plant species
with which they interact in many different ways. They can
be dormant saprophytes, latent pathogens or mutualistic
symbionts, and their relationship with the host may
change during their life cycle (Stone et al., 2000; Wilson,
2000; Sieber, 2002). Endophytes can produce toxins
against insects, growth substances of use to the plant and
can occupy the microhabitat of some pathogenic fungi
(Carroll, 1986; Miller, 1986; Petrini, 1986; Clay, 1989;
1991). Some fungi that are potentially pathogenic can
spend part of their life cycle as neutral endophytes, giving
rise to symptoms only when ecological and physiological
conditions of the host are modified, as for example in
response to environmental stresses.
Various studies were done on endophytic communities
of different forest species in declining stands (Sieber and
Hugentobler, 1987; Sieber, 1988; 1989; Barengo et al.,
2000), but only a few have experimentally examined the
effect of pollutants on endophytes in aerial plant parts
(Helander et al., 1996). A series of studies have been
conducted on beech endophytes (Danti et al., 2002), also
in declining stands, but investigations on phyllosphere
microorganisms on beech trees experimentally exposed
to atmospheric pollutants are still missing. The aim of the
present study was to determine whether long-term expo-
sure of beech saplings to SDBS causes changes to the
endophytic fungal communities of the twigs.
Results
Macroscopically, no symptoms of leaf or plant growth
deterioration were visible at any time during the experi-
ment either on SDBS-treated or control plants. Overall,
63.8% and 85.4% of the twig segments of SDBS-sprayed
and control plants, respectively, were colonized by at least
one fungal species (Table 1). Most twigs were colonized
© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 696–702
Table 1. Percentage of twigs of SDBS-treated and water-sprayed
(control) beech saplings subdivided by the number of endophytic
fungal species isolated
Frequency (%) of colonized twigs
Number of Water-sprayed SDBS-sprayed
endophyte species plants (control) plants
>3 5.5 1.2
3 13.3 5.5
2 23.3 16.1
1 43.3 41.1
0 14.6 36.1
Total 100.0 100.0
by only one species on both SDBS-treated (41.1%) and
control plants (43.3%). Twigs with multiple colonization, i.e.
colonization by two or more species, were more frequently
observed on control plants than on SDBS-sprayed plants,
and this difference in frequency tended to increase
progressively with increasing species diversity on a twig.
Twigs without any fungus were much more frequent on
SDBS-sprayed (36.1%) plants than on the controls
(14.6%). The frequency of colonization of twigs of SDBS-
sprayed plants by endophytic fungi was significantly lower
(P = 0.00066) than that of the controls.
A total of 36 species of endophytic fungi were isolated,
31 species from the controls and 21 from the SDBS-
treated plants. The frequencies of colonization by each
species are shown in Table 2. Cladosporium cladosporio-
ides, Coryneum compactum, Mycosphaerella sp., Phialo-
cephala dimorphospora and Phomopsis sp. 1 showed
frequencies greater than 5% and can be considered the
main endophytes in the beech twigs examined. Other spe-
cies occurred sporadically, often only once on a single
twig. A considerable percentage of fungi failed to sporulate
in pure culture (sterile mycelia) and hence could not be
identified.
Dots representing control (marked C) and SDBS-
treated plants (marked SDBS) of the same sampling date
are closer to each other (except those of the September
sampling) than dots representing the same treatment at
different sampling dates on the map resulting from corre-
spondence analysis (Fig. 1). This implies that differences
in the frequency of colonization by the most commonly
isolated fungi were more pronounced among sampling
dates than between the two treatments. The dots repre-
senting Phialocephala dimorphospora and Mycosphaer-
ella sp. are farthest apart along principal axis 1 because
the frequency of these two species varied the most
among sampling dates. Phialocephala dimorphospora
was frequently isolated from samples collected in March
(first sampling) and September (third sampling) but only
rarely from those collected in July (second sampling)
(Fig. 2). In contrast, Mycosphaerella sp. was most fre-
698 R. Danti et al.
Table 2. Number and frequency of twigs of SDBS-treated and water-sprayed (control) beech saplings colonized by endophytic fungi. Fungi are
listed according to the state formed in culture.

Number (percentages in brackets) of colonized twigs

Water-sprayed plants SDBS-sprayed plants

Fungal taxa (control) (n = 180) (n = 180)

Ascomycetes
Coniochaeta sp. 1 (0.6) –
Mollisia sp. 6 (3.3) 5 (2.8)
Mycosphaerella punctiformis 4 (2.2) 3 (1.7)
Mycosphaerella sp. 12 (6.7) 10 (5.6)
Pezicula acericola 5 (2.8) 2 (1.1)
Deuteromycetes
Coelomycetes
Asteroma sp. 3 (1.7) –
Aposphaeria sp. 1 1 (0.6) –
Coryneum compactum 23 (12.8) 9 (5.0)
Cryptosporiopsis grisea anam. of Pezicula cinnamomea 2 (1.1) 1 (0.6)
Cryptosporiopsis sp. 2 3 (1.7) –
Discula umbrinella anam. of Apiognomonia errabunda 3 (1.7) 1 (0.6)
Melanconium sp. 1 (0.6) –
Neohendersonia kickxii 1 (0.6) –
Phomopsis sp. 1 66 (36.7) 38 (21.1)
Phomopsis sp. 2 4 (2.2) 3 (1.7)
Phomopsis sp. 3 – 1 (0.6)
Pseudoseptoria sp. 5 (2.8) 4 (2.2)
Hyphomycetes
Alternaria sp. 3 (1.7) –
Arthrinium sp. 1 (0.6) –
Aureobasidium pullulans 3 (1.7) 3 (1.7)
Cladosporium cladosporioides 31 (17.2) 18 (10.0)
Corynespora proliferata 1 (0.6) –
Cystodendron sp. 1 (0.6) –
Endophragmia sp. 2 (1.1) 7 (3.9)
Epicoccum purpurascens 15 (8.3) 7 (3.9)
Geniculosporium serpens 1 (0.6) 2 (1.1)
Petrakia echinata 1 (0.6) –
Phialocephala cf. compacta – 1 (0.6)
Phialocephala dimorphospora 24 (13.3) 19 (10.6)
Phialophora sp. 1 (0.6) –
Pithomyces sp. – 1 (0.6)
Polyscytalum fagicola 1 (0.6) 1 (0.6)
Polyscytalum sp. – 1 (0.6)
Ramichloridium sp. 1 (0.6) –
Taeniolella sp. 1 (0.6) –
sterile mycelia 46 (25.6) 32 (17.8)

Dashes indicate that the species was not detected.

quently isolated in July but not at all in September.
Correspondingly, dots representing the July samples
were close to the point representing Mycosphaerella sp.
whereas dots representing the March and September
samples were close to the point representing P. dimor-
phospora in respect to principal axis 1. As a conse-
quence of the strong influence of the sampling date,
statistical evaluation of the treatment’s influence onto the
frequency of colonization by the five most frequently iso-
lated endophytes had to be performed for each sampling
date separately. Although the total number of twigs colo-
nized by the five most frequently isolated endophytes
was consistently greater for the control than the SDBS
treatment (Fig. 2), differences were statistically significant
only for Phomopsis sp. 1 for samples collected in
September (P = 0.00041).
Discussion
The twigs examined in this study were most frequently
colonized by Phomopsis sp. 1, Cladosporium clado-
sporioides, Phialocephala dimorphospora, Coryneum
compactum and an unknown Mycosphaerella species.
Cladosporium cladosporioides and P. dimorphospora
were found as endophytes ubiquitously on a variety of
hosts at various frequencies with no apparent preference
for beech in earlier studies (Kowalski and Kehr, 1996).
Species of Phomopsis and Mycosphaerella are frequent

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 696–702

 Influence of SDBS on endophytic mycobiota of beech 699
inoculum may determine which fungi become dominant.
Fenn et al. (1989) examined the effect of some pollutants
on the phyllosphere mycoflora of different forest species
in open-top chambers and found that trees maintained in
the chambers host less fungi compared to trees grown
outdoors. This reveals the limits of indoor tests, in which
solar radiation levels, air flows, temperature, humidity and
probably also insect presence and the deposition of fungal
inoculum from surrounding plants differ from a natural
environment. These factors may affect the activity of fungi
of the phyllosphere and modify the effect of pollutants
(Magan and McLeod, 1991a,b).
Endophytic fungi were less abundant in twigs from
plants sprayed with a 50 mg l−1 SDBS solution for three
consecutive growing seasons than in those from water-
sprayed control plants. Both the number of fungal species
isolated and the number of twigs colonized by two or more
species were lower in SDBS-sprayed plants. Correspond-
ingly, the percentage of twigs without any fungal colonies
was more than twice as high on SDBS-sprayed trees
Fig. 1. Map resulting from correspondence analysis of the frequen-
cies of colonization of twigs of SDBS-treated (SDBS) and water- (36.1%) as on control trees (14.6%). The frequency of
sprayed (C) beech saplings by the five most abundant endophytic colonization of each of the five most common fungi tended
fungi detected in samples collected in March (SDBS1, C1), July also to be higher on control than on SDBS-treated plants,
(SDBS2, C2) and September (SDBS3, C3). CLA, Cladosporium cla-
dosporioides; COR, Coryneum compactum; MYC, Mycosphaerella although the differences were statistically significant only
sp.; PHI, Phialocephala dimorphospora; PHO, Phomopsis sp. 1. for Phomopsis sp. 1 at one sampling date. Scots pine
needles exposed for three consecutive years to simulated
acid rain were colonized consistently less frequently by
colonizers of aerial plant parts, and some species are host
specific. It was, however, not possible to decide whether
the species found in this study also are host specific
because identification to the species level was not
possible.
Species such as Botryosphaeria quercuum, Discula
umbrinella, Neohendersonia kickxii, or Asterosporium
asterospermum were frequently isolated as endophytes
from beech twigs in earlier studies (Sieber and
Hugentobler, 1987; Toti et al., 1993; Kowalski and Kehr,
1996; Danti et al., 2002), but showed low frequencies of
colonization or did not occur at all in the twigs examined
in this study. There are many possibilities to explain this
finding. The age of the trees certainly had an influence.
The trees were only seven years old in this study, whereas
in other studies mature beech trees were examined. The
observed composition of the endophytic mycobiota in the
beech saplings also reflects the artificial growing condi-
tions under a cover of greenhouse film. In addition, mature
beech trees were present only in a distance of 5 km from
the experimental plot. Thus, inoculum was probably not
always sufficiently abundant to warrant successful infec-
Fig. 2. Effect of SDBS treatment on the number of twigs of beech
tion. Kowalski and Kehr (1996) who studied the endo- saplings colonized by the five most abundant endophytic fungi
phytic colonization of the basal woody part of branches detected in samples collected in March, July and September 1995.
from various forest tree species reported that the degree CLA, Cladosporium cladosporioides; COR, Coryneum compactum;
MYC, Mycosphaerella sp.; PHI, Phialocephala dimorphospora;
of colonization may depend on the species diversity of the PHO, Phomopsis sp. 1. SDBS: SDBS-treated plants (n = 180); C:
surrounding vegetation and that the amount of fungal water-sprayed control plants (n = 180).

© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 696–702

700 R. Danti et al.
endophytic fungi than the irrigated controls (Helander
et al., 1996). Other studies (Kirkwood et al., 1989;
Helander and Rantio-Lehtimäki, 1990; Ranta, 1990) on
conifers and broadleaves in open-top chambers and field
sites have found that total fungal populations were signif-
icantly lower on leaves sprayed with acidified water than
on leaves sprayed with untreated water. All these studies
have shown the negative effects of atmospheric pollutants
on fungal populations of tree leaves. Helander et al.
(1996) suggested that these effects could become perma-
nent under long-term exposure to pollutants.
We are aware that a randomized block design would
have been more adequate to avoid pseudo-replication
(Hurlbert, 1984). However, space available for this exper-
iment was very limited. The risk of SDBS contamination
of control plants adjacent to SDBS-treated blocks would
have been too high using a block design. Efforts were,
thus, made to get the edaphic and light conditions as
homogenous as possible throughout the study plot.
Within the limits of the indoor experimental conditions
and the nursery type of tree cultivation, the results of the
present study suggest that the prolonged application of
SDBS on the aerial parts of beech may affect the endo-
phytic mycobiota of twigs. Subtle physiological and bio-
chemical host-specific mechanisms are known to be
involved in recognition and establishment of endophytic
symbioses (Chapela et al., 1991; 1993; Toti et al., 1992;
Toti, 1993; Viret et al., 1993). Surrounding living host
tissues provide protection against the direct action of
environmental factors once a fungus successfully has
established an endophytic thallus. However, changes pro-
duced on the aerial parts of the plants by long-term expo-
sure to SDBS probably interfere with spore attachment
and germination, with hyphal growth on plant surfaces and
subsequent penetration of the fungus into the inner
tissues, so affecting the amount and composition of
endophytic fungal communities of the twigs. In addition,
changes in the assimilation capacity and transpiration of
SDBS-treated plants as well as SDBS-mediated induced
resistance can be supposed to be involved in the reduc-
tion of endophytic fungi.
Experimental procedures
The experiment was conducted at the regional nursery of
Camporgiano (Province of Lucca), located in the Northern
Apennines at an altitude of 500 m above sea level. Four-year-
old Fagus sylvatica saplings were used for the experiment.
They had been transplanted from pots one year before the
start of the experiment at 0.5 m intervals in two parallel rows
(30 plants per row) on a wind-protected plot. The distance
between the rows was 2.5 m. The plot was covered with
transparent greenhouse film 3 m above ground with the sides
of the ‘greenhouse’ covered with the same film to a height of
30 cm above soil. The minimum distance of the ‘greenhouse’
© 2002 Blackwell Science Ltd, Environmental Microbiology, 4, 696–702
to the surrounding mixed forest was 30 m. The homogenized
soil contained 30.4% sand, 47.1% silt and 22.5% clay and
had a pH of 6.5. The cation exchange capacity was 33.1 meq
per 100 g of soil, the content of organic matter 2.18% and
that of lime <0.1%. Residuals of pesticides (herbicides, insec-
ticides, acaricides and fungicides), growth hormones were
lower than 0.01 mg per kg of soil. Irrigation occurred by
means of channels along each of the two rows. The same
amount of water was applied weekly to each of the rows. A
solution (50 mg l−1) of SDBS (sodium dodecylbenzene sul-
phonate) in deionized water was sprayed once a week on one
of the rows during three consecutive growing seasons starting
one year after the plants had been transplanted. A total of 25
sprayings per growing season were carried out from April to
September. The control trees of the other row were sprayed
in the same way and frequency with deionized water. Plants
were sprayed until the liquid started to drip from leaves and
twigs. On average, 500 ml of SDBS solution, deionized water,
respectively, were applied per plant and week during the first
growing season whereas approximately 1000 ml were
needed during the third growing season because of the
growth increment of the plants.
Samples were collected in March, July and September
1995 when the trees were seven years old by randomly
removing sixty 2 to 3-year-old twigs from each row of trees at
each sampling date. From each twig a segment approximately
10 cm long was cut. The segments were surface-sterilized by
dipping first for 1 min in 96% ethanol, then for 5 min in
sodium hypochlorite with 4% available chlorine, then rinsing
again for 30 s in 96% ethanol (Sieber and Hugentobler,
1987). Four bark fragments about 5 × 5 mm in size were cut
from each segment, placed in 90 mm Petri dishes containing
2% malt extract agar (20 g l−1 Difco malt extract, 20 g l−1 agar),
and incubated at 17°C in the dark for 30–60 days depending
on the colony growth rate. The fungi that developed from the
fragments were transferred singly to glass test tubes
(16 × 160 mm) containing 1% malt extract agar (10 g l−1 Difco
malt extract, 20 g l−1 agar), and incubated at 18°C under NUV
light with a photoperiod of 12 h, to promote sporulation of the
fungi.
Differences between SDBS-treated and control plants in
regards to the number of twigs colonized by endophytic fungi
in general and by the most frequently isolated species were
tested by means of the Chi square test. A correspondence
analysis was performed using the software package Statistica
(version 5.1 for Windows) on a reduced matrix that included
the frequency of colonization of SDBS-sprayed and control
plants by the most frequently isolated taxa at each sampling
date to visualize the various sources (treatment and sampling
date) of variation.
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