Zuccaro A, Schoch CL, Spatafora JW, Kohlmeyer J, Draeger S, Mitchell JI.. Detection and identification of fungi intimately associated with the brown seaweed Fucus serratus. Appl Environ Microbiol 74: 931-941

Abstract

The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2008, p. 931–941 Vol. 74, No. 4
0099-2240/08/$08.00⫹0 doi:10.1128/AEM.01158-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Detection and Identification of Fungi Intimately Associated with the
Brown Seaweed Fucus serratus
䌤
†
Alga Zuccaro,
1
* Conrad L. Schoch,
2
Joseph W. Spatafora,
2
Jan Kohlmeyer,
3
Siegfried Draeger,
1
and Julian I. Mitchell
4
Institute of Phytopathology and Applied Zoology, University of Giessen, D-35392 Giessen, Germany
1
; Department of Botany and
Plant Pathology, Oregon State University, Corvallis, Oregon 97331
2
; Institute of Marine Sciences, University of North Carolina,
Morehead City, North Carolina 28557
3
; and School of Biological Sciences, University of Portsmouth,
Portsmouth PO1 2DY, United Kingdom
4
Received 23 May 2007/Accepted 1 December 2007
The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year
period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel
electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands ob-
tained from healthy algal thalli belonged to the Lindra,Lulworthia,Engyodontium,Sigmoidea/Corollospora
complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery
was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched
unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype,
which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci.
To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron
3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in
amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was
detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence
types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which
suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environ-
mental sequences and the sequences of isolates and type species indicated that the environmental sequences
were novel in the Dothideomycetes.
Coastal macrophytes are part of highly productive ecosys-
tems and have essential functions in nutrient cycling and
habitat structuring (8, 30). Helgoland intertidal marine sea-
weed populations are dominated by fucoids with diverse
associated biocenoses. The complex communities that de-
velop in these systems provide models for investigating alga-
fungus interactions in a natural environment. Pathogens and
parasites are the predominant fungi in seaweed communi-
ties that have been described (47); however, most of these
organisms cannot be cultured in the laboratory and are
known only from herbarium specimens (26, 39). Other algi-
colous fungi include saprobes and mycobionts, and there is
little information on the autecology of these organisms.
Studies of the interactions between these fungi and their
algal hosts, therefore, can be effectively undertaken only by
using a molecular approach to detect and differentiate be-
tween environmental signal sequences.
In a preliminary study of fungi associated with Fucus
serratus, Zuccaro et al. (48) developed and described a PCR-
denaturing gradient gel electrophoresis (DGGE) system,
using novel fungus-specific primers that amplified the sec-
ond domain (D2) of the nuclear large rRNA region. Fungal
sequences retrieved from algal tissue matched sequences
from ascomycetous groups known to be active in marine
environments, as well as sequences from a group of isolates
belonging to the genus Emericellopsis and their mitosporic
form, the genus Acremonium (49). These organisms are pri-
marily recognized as fungi that are active in terrestrial en-
vironments and include known endophytes and pathogens
(9, 16, 34). The current study examined the consistency of
fungal associations with F. serratus over 1 year, and this
paper describes a real-time PCR detection system based on
sequences of intron 3 of the beta-tubulin gene. It also ad-
dresses questions related to the seasonal occurrence and
tissue localization of these fungi. In addition, sequences
derived from environmental samples, isolates, and a herbar-
ium specimen were combined in phylogenetic analyses to
provide a basis for assessing the identities of novel marine
fungal lineages. In particular, the fungi belonging to the
Dothideomycetes, which contains many of the algal para-
sites, pathogens, and mycobionts (47), were targeted.
MATERIALS AND METHODS
Sampling site and collection of algae. The sampling site and sampling strate-
gies used have been described previously in detail (48). Submerged healthy-
looking and decaying F. serratus tissues were collected on five independent
sampling occasions over the course of 1 year (April 2002, July 2002, October
2002, January 2003, and April 2003) from a rocky-shore site on the northeastern
side of Helgoland Island, Germany.
Herbarium specimen. Specimens of Didymella fucicola on Fucus vesiculosus
were kept frozen in seawater from September 1971 until September 2005 and
* Corresponding author. Mailing address: Justus-Liebig Universita¨t
Giessen, Institute of Phytopathology and Applied Zoology, Heinrich-
Buff-Ring 26-32, D-35392 Giessen, Germany. Phone: 49 641 99-37497.
Fax: 49 641 99-37499. E-mail: Alga.Zuccaro@agrar.uni-giessen.de.
† Supplemental material for this article may be found at http://aem
.asm.org/.
䌤
Published ahead of print on 14 December 2007.
931

then air dried (United Kingdom: Cornwall: West Looe, 17 September 1971, J.
Kohlmeyer [J.K.2932] [Institute of Marine Science-IMS]).
Fungal isolation, identification, genomic DNA extraction, and PCR amplifi-
cation. Fungi were isolated from algal parts in pure culture by mycelial transfer
onto agar plates and, where possible, by single-conidium isolation. For conven-
tional isolation from different parts (receptacles, growing tips, and blade and
holdfast tissues) of healthy F. serratus, approximately 2,100 segments were sur-
face sterilized with bleach and 1,000 segments were rinsed with sterile water;
approximately 400 segments from decaying material were cleaned with sterile
water. Segments were plated on different media as described by Zuccaro et al.
(48). Emerging fungi were isolated in pure cultures and identified on the basis of
morphology when possible. The genomic DNA of isolates selected on the basis
of morphological characteristics for further phylogenetic analysis, including my-
celia sterilia, was extracted using a FastDNA spin kit for soil (Bio 101 Systems or
Q-Bio gene) by following the company’s protocol. DNA was amplified using
primers NL209 and NL912, purified with a Geneclean III kit (Q-Bio gene), and
sequenced using the fluorescent method and a Li-COR 4200 DNA sequencer
(Amodia Bioservice GmbH, Braunschweig, Germany).
Extraction of DNA from the dried herbarium specimen, PCR amplification,
cloning, and sequencing for phylogenetic analysis. DNA was extracted from a
35-year-old herbarium specimen of the marine obligate parasite D. fucicola. Five
ascomata were picked off the decaying midribs of the brown algal host F. vesicu-
losus, and DNA was extracted using a FastDNA spin kit from Q-Bio gene. PCR
amplification of the large-subunit (LSU) rRNA gene was performed using a
seminested approach with the fungus-specific primers NL209 and NL912, fol-
lowed by primers NL359 and NL912GC, as previously described (48). Internal
transcribed spacer (ITS) regions were amplified using primers ITS1f (14) and
NL209r (5⬘-CTGTTGGTTTCTTTTCCTCCGCTT-3⬘) under the following con-
ditions: 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and
1 min at 72°C and a final extension for 7 min at 72°C. PCR products from the
herbarium specimen were purified with a Geneclean III kit (Q-Bio gene) and
then ligated into the vector pCR 2.1 (TA cloning kit; Invitrogen). DNA from
seven plasmids was extracted with a QIAprep spin miniprep kit (Qiagen, Hilden,
Germany) and was sequenced using primers M13f and M13r.
Environmental DNA extraction. Total environmental DNA was extracted
from 21 10-g samples of healthy F. serratus thalli which were previously sectioned
into different parts (blade, receptacles, holdfast, and growing tips) and six 10-g
samples of decaying algal material. The extraction procedure, including a CsCl
centrifugation step, was performed using the protocol previously described by
Zuccaro et al. (48). The environmental DNA was then diluted to a final con-
centration of 5 ␮g/␮l.
PCR amplification and DGGE conditions. A total of 57 PCR amplifications,
consisting of two or three replicates for each independent DNA sample, were
performed using a seminested approach with primers NL209 and NL912, fol-
lowed by primers NL359 and NL912GC, and the products were separated on
LSU rRNA gene DGGE gels with the Bio-Rad D-Code system (Bio-Rad Lab-
oratories, Hercules, CA). Detailed descriptions of the primer efficiency, PCR
conditions, DGGE gel reagents, denaturant range, and running and gel staining
conditions have been provided elsewhere (48).
Cloning and sequencing of 28S rRNA gene PCR products from decaying
seaweed. PCR products from decaying algal material, obtained using primers
NL209 and NL912, were purified with a Geneclean III kit (Q-Bio gene) and then
ligated into the vector pCR 2.1 (TA cloning kit; Invitrogen). Extracted plasmids
were reamplified using primers NL209 and NL912 and were sequenced using
primer NL912 and the fluorescent method with a Li-COR 4200 DNA sequencer
(Amodia Bioservice GmbH, Braunschweig, Germany). The reamplified inserts
were then subjected to seminested amplification using primers NL359 and
NL912GC, and the products were electrophoresed in a DGGE gel together with
the original sample to identify the corresponding environmental bands.
Real-time quantitative PCR. (i) Design of TaqMan primers and probe. Prim-
ers and a probe were designed for a TaqMan real-time quantitative PCR
assay targeting the intron 3 region of the beta-tubulin gene from Acremonium
fuci (GenBank accession number AY632690) using the Primer Express v2.0
software (Applied Biosystems, Foster City, CA). The Emericellopsis/Acremo-
nium-like forward primer TUB1F (5⬘-GCGTCTACTTCAACGAGGTGAG
T-3⬘) and reverse primer TUB2R (5⬘-ATGCTCATCCTCGCAGGC-3⬘) am-
plified a 68-bp fragment from base 108 to base 175. The 25-bp TaqMan probe
AFP1 (5⬘-CGTCCGGAACAATGATACCCTAGCA-3⬘) was between bases
132 and 156 of this region. The probe was labeled with the fluorescent
reporter dye 6-carboxyfluorescein at the 5⬘end and with the quencher dye
6-carboxytetramethylrhodamine at the 3⬘end. The probe was obtained from
Applied Biosystems, United Kingdom, and the primers were obtained from
Invitrogen Life Technologies, United Kingdom. The specificities of the prim-
ers and probe were verified experimentally by using the marine fungi Sig-
moidea marina and Lindra obtusa and the closely related organism Emericel-
lopsis minima, all of which had been isolated from F. serratus samples.
Additionally, a BLAST search (National Center for Biotechnology Informa-
tion) was performed with the primer and probe sequences.
(ii) Real-time PCR protocol. The environmental samples were subjected to
amplification using real-time quantitative PCR. The PCRs were performed in
MicroAmp optical 96-well plates using an automated ABI Prism 7700 sequence
detector (PE Applied Biosystems). Each 25-␮l (total volume) reaction mixture
contained TaqMan universal PCR master mixture, No AmpErase uracil-N-gly-
cosylase (Applied Biosystems, Roche Molecular Systems, Inc., Branchburg, NJ),
the primers at a final concentration of 900 nM, the probe at a final concentration
of 200 nM, and 500 ng/␮l of environmental DNA extracted from algal material.
A standard curve was prepared for each run, using serially diluted genomic DNA
extracted from A. fuci (8, 3, 1.5, 0.8, 0.3, 0.15, and 0.003 ng). The PCR cycling
parameters were 50°C for 3 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s
and annealing at 60°C for 1 min. Data acquisition and threshold cycle values for
each PCR were automatically calculated and analyzed by using the ABI Prism
sequence detection system software (version 1.6; Applied Biosystems). A pre-
amplification step was included for the healthy algal DNA samples using 0.5 ␮M
primer T10 (31) and 0.5 ␮M primer Bt2b (15), which amplified an approximately
300-bp fragment. Amplification was performed using the following cycling con-
ditions: 5 min at 95°C, followed by 25 cycles of 30 s at 95°C, 55 s at 55°C, and 45 s
at 72°C, and a final extension for 10 min at 72°C. One microliter of the reaction
mixture, including negative controls, was used as the substrate for the nested
real-time PCR as described above.
Phylogenetic analysis. Isolates and environmental sequences used for phy-
logenetic analysis are listed in Table 1. The phylogenetic position of the
obligate marine parasite D. fucicola on F. vesiculosus obtained from a 35-
year-old dried herbarium specimen was determined using partial LSU DNA
sequences in multigene phylogenies of related terrestrial and marine plant
parasites and environmental sequences obtained from F. serratus. Herbarium
material was used because the type material, D. fucicola, and living specimens
from later collections (21, 24, 46) were unavailable for study. A neotype for
this species was proposed by Kohlmeyer (21) and was fully illustrated by
Kohlmeyer and Kohlmeyer (23).
A data matrix of 66 taxa representing a selection of major lineages in the
Dothideomycetes was used (Table 1). We combined DNA sequence data ob-
tained as part of the Assembling the Fungal Tree of Life (AFTOL) project (29)
from ribosomal as well as protein-encoding genes. DNA sequences were ob-
tained for the nuclear small subunit and LSU, as well as the sequence between
domains 5 and 7 of the second largest subunit of RNA polymerase II (RPB2) and
the transcription elongation factor 1 alpha gene (EF) (35). We chose 36 repre-
sentative taxa following the Dothideomycetes phylogeny of Schoch et al. (35)
in order to accurately place the environmental isolates obtained in this study.
The data obtained were combined with a number of sequences obtained from
GenBank. The final matrix used was deposited at treeBASE (http://www
.treebase.org/treebase/index.html) under reference number SN3178. In order to
incorporate differently weighted character sets, we used maximum likelihood as
performed with RAxML-VI-HPC, version 2.2.0 (40), using the GTRMIX setting
(applying a GTRCAT approximation of evolution with 25 rate categories but
determining final likelihood values according to a gamma distribution with four
rate categories). The data set was divided into eight parts, including nuclear small
subunit, nuclear LSU, and each codon position of both RPB2 and EF. Nodal
support in RAxML analyses was determined by 500 nonparametric bootstrap
repetitions. Similarly, Metropolis coupled Markov chain Monte Carlo (B-
MCMCMC) analyses were conducted using MrBayes 3.1.2 (http://mrbayes.csit
.fsu.edu/index.php) with the same partition that was used with RAxML-VI-HPC
and using the GTR model with a gamma distribution approximated with four
categories and a proportion of invariable sites. Searches were conducted using
four chains with trees sampled every 100 generations. Two independent 5 mil-
lion-generation analyses were conducted, and likelihood values were examined
to verify a “burn-in” parameter. The Bayesian analyses converged on the same
topology and plateau of log likelihoods with harmonic mean values of
⫺26,913.78 and ⫺26,914.64 for runs 1 and 2, respectively. These runs were
combined, and 10,000 trees were discarded as “burn in,” yielding a single 50%
majority rule tree with the proportions of trees in the final set of 90,000 expressed
as percent posterior probabilities.
A second data matrix, comprising ITS-5.8S rRNA gene sequences, was con-
structed and represented 17 taxa that had a high level of homology with D.
fucicola; these taxa included Didymella species and mitosporic forms (Table 1).
Maximum parsimony and likelihood analyses were performed using this matrix.
The maximum parsimony settings included heuristic searches with random se-
932 ZUCCARO ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. Fungal strains and clones used in the molecular analyses
AFTOL no. Taxon Source
a
Accession no.
b
Small-subunit
rRNA gene
LSU rRNA
gene RPB2 EF ITS
Isolates
267 Allewia eureka DAOM 195275 DQ677994 DQ678044 DQ677938 DQ677883
1610 Alternaria alternata CBS 916.96 DQ678031 DQ678082 DQ677980 DQ677927
931 Bimuria novae-zelandiae CBS 107.79 AY016338 AY016356 DQ470917 DQ471087
946 Botryosphaeria dothidea CBS 115476 DQ677998 DQ678051 DQ677944 DQ767637
1586 Botryosphaeria tsugae CBS 418.64 AF271127 DQ767655 DQ767644 DQ677914
939 Capnodium coffeae CBS 147.52 DQ247808 DQ247800 DQ247788 DQ471089
1289 Cladosporium cladosporioides CBS 170.54 DQ678004 DQ678057 DQ677952 DQ677898
54 Cochliobolus heterostrophus CBS 134.39 AY544727 AY544645 DQ247790 DQ497603
Colispora elongata F-08382 AY148102
1379 Coniothyrium palmarum CBS 400.71 DQ678008 DQ767653 DQ677956 DQ677903
1568 Cucurbitaria elongata CBS 171.55 DQ678009 DQ678061 DQ677957 DQ677904
1591 Davidiella tassiana (as
anamorph Cladosporium
herbarum)
CBS 399.80 DQ678022 DQ678074 DQ677971 DQ677918
Decorospora gaudefroyi pp4723 EF1778459
1599 Delitschia winteri CBS 225.62 DQ678026 DQ678077 DQ677975 DQ677922
995 Dendryphiella arenaria CBS 181.58 DQ471022 DQ470971 DQ470924 DQ677890
Didymella bryoniae AF297228
Didymella cucurbitacearum IMI 373225 AY293779 AY293792
Didymella cucurbitacearum AY293804
2111 Didymella exigua CBS 183.55 EF177845 EF192139
Didymella fabae af1 DQ383952
Didymella lentis AL1 AY131201
Didymella pinodes AY152551
Didymella rabiei DQ383949
Didymella sp. hka9 DQ092504
Didymella sp. Hkb1 DQ092514
919 Dothidea hippophae¨s DAOM 231303 U42475 DQ678048 DQ677942 DQ677887
274 Dothidea sambuci DAOM 231303 AY544722 AY544681 DQ522854 DQ497606
1359 Dothiora cannabinae CBS 737.71 DQ479933 DQ470984 DQ470936 DQ471107
Epicoccum andropogonis AJ400905
Epicoccum nigrum CBMAI 65 DQ123608
1618 Guignardia bidwellii CBS 237.48 DQ678034 DQ678085 DQ677983
1608 Herpotrichia juniperi CBS 200.31 DQ678029 DQ678080 DQ677978 DQ677925
277 Leptosphaeria maculans DAOM 229267 DQ470993 DQ470946 DQ470894 DQ471062
1081 Magnaporthe grisea Broad AB026819 AB026819 Genome Genome
1734 Montagnula opulenta CBS 168.34 AF164370 DQ678086 DQ677984
1615 Mycosphaerella graminicola CBS 292.38 DQ678033 DQ678084 DQ677982
942 Mycosphaerella punctiformis CBS 113265 DQ471017 DQ470968 DQ470920 DQ471092
1078 Neurospora crassa Broad X04971 AF286411 XM_324476 Genome
1569 Ophiosphaerella herpotricha CBS 620.86 DQ678010 DQ678062 DQ677958 DQ677905
1595 Ophiosphaerella herpotricha
(as synonym Ophiobolus
herpotrichus)
CBS 240.31 DQ767650 DQ767656 DQ767645 DQ767639
280 Phaeosphaeria avenaria DAOM 226215 AY544725 AY544684 DQ677941 DQ677885
Phaeosphaeria nodorum Broad Genome Genome Genome Genome
Phaeosphaeria olivacea CBS 118420
2206 J.K.5540Q EF177847
1441 Phaeosphaeria orae-maris J.K.4730 EF179158
1575 Phoma herbarum CBS 276.37 DQ678014 DQ678066 DQ677962 DQ677909
1600 Pleomassaria siparia CBS 279.74 DQ678027 DQ678078 DQ677976 DQ677923
CBS 118380
2205 Pleospora avicenniae J.K.5326A EF177846
940 Pleospora herbarum var.
herbarum
CBS 541.72 DQ247812 DQ247804 DQ247794 DQ471090
CBS 118219
2207 Pleospora sp. J.K.5184D EF177848
283 Pyrenophora phaeocomes DAOM 222769 DQ499595 DQ499596 DQ497614 DQ497607
Sarcosomataceae species sd2bN1c AY465503
1594 Scorias spongiosa CBS 325.33 DQ678024 DQ678075 DQ677973 DQ677920
Sporidesmium obclavatulum HKUCC 10834 DQ408556
1256 Sporormiella minima CBS 524.50 DQ678003 DQ678056 DQ677950 DQ677897
Strumella griseola CBS433.59 AF485078
1300 Sydowia polyspora CBS 116.29 DQ678005 DQ678058 DQ677953 DQ677899
Continued on following page
VOL. 74, 2008 PCR-MEDIATED DETECTION OF MARINE FUNGI 933

quence addition (10 to 50 replicates) using the tree bisection-reconnection al-
gorithm, while the maximum likelihood analysis used the GTR ⫹G⫹I model
with estimates of the nucleotide frequency, substitution rate matrix, among-site
variation, and shape parameter from the matrix.
RESULTS
Fungal isolates from F. serratus.The fungal reference library
obtained from F. serratus contained 336 isolates representing
35 genera of the Ascomycota and Zygomycota (Table 2). A
total of 56 strains had sterile mycelia and could not be identi-
fied morphologically (Table 2). The most commonly encoun-
tered isolates belonged to the Ascomycota, in agreement with
our previous study (48). Representatives of the following five
taxa were the predominant organisms in this study: S.marina,
with 56 strains; Acremonium spp., with 36 strains, 29 of which
were identified as A. fuci (49); Cladosporium spp., with 31
strains; Dendryphiella salina, with 26 isolates; and Fusarium
spp., with 19 representative isolates. S. marina was isolated
only from healthy surface-sterilized samples, whereas the other
taxa were present in surface-sterilized and water-treated tis-
sues, as well as in segments from decaying F. serratus (Fig. 1).
Members of additional genera were isolated, but the numbers
were lower and the organisms were isolated mainly from wa-
ter-treated samples; these organisms included representatives
of Trichoderma,Alternaria,Phoma,Penicillium, and Paecilomy-
ces (Table 2). Corollospora angusta,Corollospora intermedia,
and L. obtusa were obtained sporadically from water-treated
living and decaying samples. Microascus,Chaetomium, and Ar-
thrinium spp. were isolated only from surface-sterilized living
F. serratus (Table 2 and Fig. 1).
Isolates were recovered from all thallus parts. Fewer isolates
were cultured from growing tips and receptacles than from
blades and holdfast tissues. S. marina was the predominant
isolate obtained from receptacles and growing tips (23 strains
recovered), followed by D. salina (three isolates) and a single
A. fuci isolate (data not shown).
PCR-DGGE analysis of the nuclear LSU rRNA gene in
fungal sequences obtained from whole and sectioned algal
thalli sampled seasonally. Analyses of replicates of an indi-
vidual sample generally resulted in similar profiles, although
occasionally one or two additional bands were observed,
indicating that there was a very small amount of DNA for
some of the fungi detected. The profiles obtained for all of
the living thalli of F. serratus tissues comprised a total of 87
bands, representing seven different ribotypes with one to
six bands per sample (Fig. 2 and 3). Bands corresponding to
bands amplified from S. marina were the bands that were
observed most frequently, accounting for 34.5% of the total,
followed by bads from Emericellopsis/Acremonium (19.5%),
L. obtusa (17.2%), C. angusta (ca. 8%), Engyodontium sp.
(ca. 8%), and the molecular ribotype for Lulworthia sp. (ca.
8%) (Fig. 2). One extra ribotype was recovered twice on one
sampling occasion in January, and it was identified as an
Iodophanus-like sequence after BLAST searches. S. marina
TABLE 1—Continued
AFTOL no. Taxon Source
a
Accession no.
b
Small-subunit
rRNA gene
LSU rRNA
gene RPB2 EF ITS
Tumularia aquatica MUCL28096 AY265337
Uncultured ascomycete dfmo0690_230 AY969660
Uncultured fungus from cow
rumen
AY464875
1037 Westerdykella cylindrica CBS 454.72 AY016355 AY779322 DQ470925 DQ497610
Sterilia mycelia MC340 TUB340 EF177836
from F. MC363 TUB363 EF177837
serratus MC541 TUB541 EF177838
MC545 TUB545 EF177839
MC555 TUB555 EF177840
MC556 TUB556 EF177841
MC564 TUB564 EF177842
MC565 TUB565 EF177843
MC190 TUB190 EF177844
Clones hclone2, Didymella fucicola Herbarium J.K.2932 EF177850
hclone5, Didymella fucicola Herbarium J.K.2932 EF177851
hclone9, Didymella fucicola Herbarium J.K.2932 EF177852
hcloneITS, Didymella
fucicola
Herbarium J.K.2932 EF192138
eclone7 Environment EF177832
eclone15 Environment EF177833
eclone20 Environment EF177834
eclone24 Environment EF177835
a
Abbreviations for culture collections, herbarium, and database: ATCC, American Type Culture Collection, Manassas, VA; CBS, Centraalbureau voor Schimmel-
cultures, Utrecht, The Netherlands; DAOM, National Mycological Herbarium, Department of Agriculture, Ottawa, Ontario, Canada; J.K., culture collection of Jan
Kohlmeyer and Brigitte Volkmann-Kohlmeyer, Department of Marine Sciences, University of North Carolina, Morehead City; pp, culture collection of Portsmouth
School of Biological Sciences, University of Portsmouth, Portsmouth, United Kingdom; TUB, culture collection of Technische Universita¨t Braunschweig Department
of Microbiology, Braunschweig, Germany; Broad, Broad Institute, Cambridge, MA.
b
Bold type indicates that data were obtained in this study.
934 ZUCCARO ET AL. APPL.ENVIRON.MICROBIOL.

and Emericellopsis/Acremonium ribotypes were obtained at
all five sampling times over the course of the year, whereas
the other ribotypes were recovered sporadically. An analysis
of variance (P⬎0.05, Kruskal-Wallis test) revealed no sea-
sonal patterns for these signals but a significant prevalence
of S. marina sequences compared with those retrieved spo-
radically (P⫽0.003, pairwise multiple comparison proce-
dure, Holm-Sidak method). This indicated that there was a
predominant association between this fungus and F. serratus
over the year (Fig. 2).
The molecular methods used did not detect an association
between any particular ribotype and a specific part of the
thallus. A significant difference (P⫽0.049, Tukey’s test) in
the number of bands associated with the tissue types was
observed, however, and the highest number of bands was
obtained for the DNA extracted from blades and the lowest
number of bands was obtained for the DNA extracted from
the growing tips. This observation is consistent with obser-
vations made in the culture study, where, except for S.
marina, fewer isolates were recovered from apex tissues
(data not shown).
The molecular profiles obtained for the decaying tissues
contained a total of 25 bands. L. obtusa was the predomi-
nant ribotype (25% of the bands), followed by C. angusta
(20%), S. marina (16%), Emericellopsis/Acremonium (12%),
and a Lulworthia-like sequence (8%). Since some of the
bands resolved by DGGE were too diffuse to be analyzed
further, PCR products from decaying material were cloned
and sequenced. Phoma,Mycosphaerella,Pleospora, and
Didymella-like ribotypes resulted from this cloning-sequenc-
ing analysis (data not shown).
Design, specificity, and sensitivity of A. fuci-specific primers
and probe. The real-time PCR system was developed in
order to differentiate between the environmental signals for
the Emericellopsis and Acremonium sequences. The frag-
ments amplified with primers TUB1F and TUB2R were in
the range expected based on the sequence data for A. fuci.
No amplification from DNA of L. obtusa or S. marina was
observed. Amplification was obtained for the closely related
organism E. minima, as expected from the BLASTn search,
even though the reaction efficiency was lower than that for
A. fuci. In the real-time PCR, all dilutions of DNA from A.
fuci tested gave strong positive fluorescent signals after 20 to
26 cycles with 8, 3, and 1.5 ng of DNA and after 40 cycles
with 0.003 ng of DNA. No signal was detected for the other
fungi tested using these DNA concentrations.
DNA from E. minima gave a weak fluorescent signal after
40 cycles with higher concentrations of genomic DNA, but
the reaction never reached exponential amplification (see
Fig. SA2 in the supplemental material). The AFP1 probe,
therefore, proved to be specific or highly enhanced for the
A. fuci sequence.
Detection of A. fuci beta-tubulin sequences in environmental
samples. The routine retrieval of a sequence belonging to
Emericellopsis/Acremonium using the 28S rRNA gene PCR-
DGGE system and the high isolation ratio of A. fuci were in
general agreement with the real-time PCR results. Two of
six environmental decaying F. serratus samples gave strong
positive amplification using the 28S rRNA gene system for
the Emericellopsis/Acremonium ribotype with an intense
DGGE band (Fig. 3a). The same samples resulted in strong
positive fluorescent signals after 32 and 36 cycles using real-
time PCR. The four other environmental samples were neg-
ative for this ribotype using both methods. Of the 21 sam-
ples of healthy F. serratus analyzed, 12 resulted in positive
amplification using the 28S rRNA gene system, which was
visualized as low-intensity DGGE bands (Fig. 3b). Two of
these samples gave weak fluorescent amplification signals
after 40 cycles for A. fuci using the beta-tubulin real-time
PCR system. At higher concentrations of DNA (600 to 1,000
ng/␮l algal DNA) or when a nested approach was used with
primers T10 and Bt2b followed by primers TUB1F and
TUB2R, positive amplification of A. fuci was obtained with
some of the living algal samples.
TABLE 2. Fungi isolated from specimens of decaying and
submerged, attached, healthy F. serratus thalli on
five different sampling occasions
Taxon
No. of isolates obtained from surface-
sterilized and water-treated F. serratus
Living disks
(total no., 3,100)
Decaying disks
(total no., 400)
Acremonium sp. 5
Acremonium murorum 1
Acremonium fuci 21 8
Acremonium tubakii 1
Acroconidiella sp. 1
Alternaria sp. 5 6
Arthrinium sp. 3
Aspergillus sp. 4 1
Asteromyces cruciatus 11
Botrytis cinerea 2
Chaetomium funicola 3
Chaetomium sp. 2
Cladosporium sp. 17 14
Coniothyrium sp. 3
Corollospora angusta 23
Corollospora intermedia 11
Dendryphiella salina 18 8
Emericellopsis minima 1
Epicoccum purpurascens 1
Epicoccum sp. 1 2
Fusarium sp. 15 4
Geomyces sp. 2
Gliocladium sp. 1
Humicola fuscoatra 1
Lindra obtusa 15
Microascus sp. 3
Mucor sp. 2
Mycosphaerella sp. 1
Nodulisporium sp. 1
Oidiodendron sp. 1
Paecilomyces sp. 7
Penicillium sp. 11 3
Periconia sp. 1
Phialophora sp. 2
Phoma sp. 8 2
Phomopsis sp. 2
Scopulariopsis sp. 3
Sigmoidea marina 56
Tetracladium maxilliformis 1
Trichoderma sp. 7 1
Verticillium cinnabarinum 11
Sterilia mycelia 37 19
Total 252 84
VOL. 74, 2008 PCR-MEDIATED DETECTION OF MARINE FUNGI 935

Phylogenetic analysis of environmental isolates and signal
sequences retrieved from healthy, decaying, and herbarium
Fucus thalli. To better characterize the Dothideomycetes se-
quences obtained from the diversity study, a four-gene combined
phylogenetic analysis was performed. Five clades in the Pleospo-
rales were identified (Fig. 4). The first clade is a well-supported
group of isolates clustered around Didymella and Phoma species
and includes all of the clones obtained from the herbarium sam-
ple of D. fucicola. The clade labeled Leptosphaeria is poorly sup-
ported and consists of disparate species. In contrast, the Pleospo-
raceae clade is well supported and contains one environmental
clone (Eclone15 in Fig. 4) that did not cluster closely with any
known species. This sequence could not be identified accurately
after BLAST searches. More than 100 matches with E values of
0 were obtained for taxa, including species of Phoma,Pleospora,
Setosphaeria,Cochliobolus,Leptosphaeria,Phaeosphaeria, and
Dendryphiella. The greatest number of matching hits, however,
was for sequences representing the Pleosporaceae. Likewise, the
Phaeosphaeriaceae is a well-supported clade containing two en-
vironmental sequences, one of which (clone 24) shows a strong
affinity to Ophiosphaerella herpotrichia and Phaeosphaeria orae-
maris. Clone 20 grouped with other Phaeosphaeria members but
at an uncertain position. In general, the marine species in this
group formed a clade with good support, which was separated
from the terrestrial Phaeosphaeria species. The fifth clade com-
prised only nuclear LSU sequences belonging to the four envi-
ronmental isolates that formed a supported clade with Sporides-
mium (37). The latter organism was isolated from leaf litter and
often occurs on dead branches of woody plants (B. D. Shenoy,
personal communication). Within the Capnodiales one environ-
mental clone (Eclone7 in Fig. 4) showed a very close relationship
with the ubiquitous Cladosporium species (Davidiellaceae).
The nuclear LSU rRNA gene analysis of D. fucicola revealed
that this species was closely related to other members of Didy-
mella. In order to confirm this, the ITS region was amplified from
sectioned ascoma of herbarium material and sequenced. BLAST
homology searches of the sequences indicated that the closest
matches were with uncultured ascomycetes (E ⫽1e106) or mi-
totic species, such as Tumularia aquatica (E ⫽5e84) and Stru-
mella griseola (E ⫽5e84). The greatest number of hits recorded
FIG. 1. Proportions of fungi isolated from surface-sterilized (gray bars) and water-treated (black bars) F. serratus thalli.
FIG. 2. Proportions of the predominant Ascomycetes phylotypes recovered from F. serratus tissues. Some of the April 2002 data were obtained
from reference 48.
936 ZUCCARO ET AL. APPL.ENVIRON.MICROBIOL.

was with sequences from members of the Dothideomycetes (881/
1,500 hits) within the Leptosphaeriaceae. To further clarify the
phylogenetic placement of this taxon, maximum parsimony and
likelihood analyses were performed using a matrix of 17 taxa
comprising sequences from Didymella species and the closest
matches. The alignment revealed a strong similarity between the
ITS1 and ITS2 regions of the Didymella species sampled; none-
theless, the sequences from D. fucicola had greater similarity with
the sequences from T. aquatica and S. grisolus (see Fig. SA3 in the
supplemental material).
Maximum parsimony analysis produced three trees with a
length of 335 (consistency index, 0.81; retention index, 0.87;
number of parsimony informative characters, 126) (Fig. 5).
The topology of this trees was similar to the topology obtained
from the maximum likelihood analysis (data not shown). In
both analyses the Didymella and Epicoccum species, including
two Didymella isolates cultured from coral tissue (Didymella sp.
strains HKA9 and HKB1), formed a strongly supported clade
(bootstrap value ⫽100%). D. fucicola was located at the base
of this clade, followed by two environmentally derived se-
quences (Sarcosomataceae strain Sd2bN1c and uncultured as-
comycete isolate dfmo0690_230) and the mitotic species T.
aquatica,Colispora elongata, and S. griseola.
DISCUSSION
The culture-based study and LSU rRNA PCR-DGGE analysis
of healthy and decaying thalli revealed the presence of a number
of fungal groups associated with F. serratus populations. These
groups included isolates and rRNA gene ribotypes representative
of the Halosphaeriaceae, Lulworthiaceae, Hypocreales, and Do-
thideomycetes, along with two distinct ribotypes of S. marina and
Emericellopsis/Acremonium that were detected extensively
throughout the year.
The hyphomycete S. marina is linked molecularly with the
Halosphaeriaceae via its connection to Corollospora (48). In this
study these organisms were consistently cultured after surface
sterilization from all healthy tissue types, even in the winter (Jan-
uary) when the average water temperature was 2°C (17). At this
time the S. marina environmental signal sequence represented
60% of the DGGE bands detected. It was found molecularly and
culturally on the growing tips of the alga, which represented the
youngest algal tissue. This suggests that there is systemic growth
of the fungus within the algal tissues. This behavior resembles that
of Mycophycias ascophylli, an endophytic mycophycobiont that
grows mutually within its hosts, Ascophyllum nodosum and Pelve-
tia canaliculata (1, 22, 24). This endophyte remains associated
with its algal host throughout its life cycle (41), has differential
hyphal densities within the algal thallus (12), protects the photo-
biont from desiccation (13), and has a nutritional dependence on
its partner (20). The failure to recover isolates of S. marina from
decaying material in this study suggests that this fungus is a hemi-
biotroph that cannot thrive in the environment without the pro-
tection of the alga (Table 2).
In contrast, the Emericellopsis/Acremonium LSU signal se-
quence and isolates were retrieved from both living and de-
caying F. serratus fronds (Table 2 and Fig. 2). Three closely
related isolates, A. fuci,Acremonium tubakii, and E. minima,
produced sequences that matched this signal, although only A.
fuci was routinely isolated in culture. All of these organisms are
related to the Bionectriaceae within the Hypocreales, but their
positions are uncertain (33). In order to distinguish between
these sequences, the real-time PCR system was designed based
on beta-tubulin sequence information from 22 related isolates
(49). This gene has previously been used to estimate fungal
phylogenies, including those of Stanjemonium and Emericel-
lopsis (11, 49), and to detect species-specific transcripts in the
environment (6). Intron 3 of Emericellopsis and related Acre-
FIG. 3. DGGE gels of fungi associated with decaying and sectioned living algal tissues. (a) Separation of PCR products generated by
NL359-NL912GC amplification of genomic DNA extracted from decaying thalli of F. serratus in a 38 to 60% denaturant gradient gel. Lane M,
marker DNA consisting of NL359-NL912GC amplicons from A. fuci,Lindra cf. obtusa,Verticillium cinnabarinum, and S. marina, from top to
bottom; lane 1, April 2002 collection; lane 2, July 2002 collection; lane 3, October 2002 collection; lane 4, January 2003 collection. (b) DGGE
profiles of amplified 28S rRNA gene fragments (obtained with primers NL359 and NL912GC) of DNA extracted from sectioned living algal thalli
collected in April 2002. Lanes M, marker DNA comprising NL359-NL912GC fragments from A. fuci,Lindra cf. obtusa,S. marina, and C. angusta,
from top to bottom; lanes 1 and 2, profiles for holdfasts of F. serratus; lanes 3 to 7, profiles for blades of F. serratus; lane 8, profile for growing tips
of F. serratus; lanes 9 and 10, profiles for receptacles of F. serratus.
VOL. 74, 2008 PCR-MEDIATED DETECTION OF MARINE FUNGI 937

monium types is characteristically short, but it contained
enough information to design a 25-bp hybridization probe that,
unlike the ITS region, distinguished between the isolates (see
Fig. SA1 in the supplemental material). A. fuci was chosen as
the target organism after a series of physiological tests indi-
cated that there was an interaction between the fungus and the
brown algae (49).
The TaqMan primers and probe could detect A. fuci envi-
ronmental sequences in decaying algal material without a
nested PCR. The absence of a signal from healthy fronds
contradicts the results obtained using the LSU rRNA gene
PCR-DGGE system. The contradiction, however, can be ex-
plained by the higher algal DNA/fungal DNA ratio expected
for healthy fronds than for decaying fronds and the predicted
lower copy number associated with the beta-tubulin gene com-
pared to the copy number for the rRNA genes. When a pre-
FIG. 4. Phylogenetic tree showing the relationship between the environmental sequences, the sequence from D. fucicola herbarium specimen
J.K.2932, and the isolates recovered from F. serratus thalli. A 50% majority rule for 90,000 trees obtained by Bayesian inference was used. Nodes
with ⬎95% posterior probability and ⬎70% bootstrap support are indicated by thick branches. For the other nodes the percentages of posterior
probability are indicated below the nodes and the RAxML maximum likelihood bootstrap values are indicated above the nodes. Nodes with
bootstrap values less than 50% are indicated by a minus sign, and nodes resolved differently in the RAxML consensus tree are indicated by an
asterisk. Clades containing sequences obtained in this study are highlighted and named.
938 ZUCCARO ET AL. APPL.ENVIRON.MICROBIOL.

amplification step or increased concentrations of environmen-
tal DNA were used, a signal was obtained for some living
samples. These results confirmed that this fungus was associ-
ated with the thallus but that the amounts were small (⬍1⫻
10
⫺5
ng). The difference in signal detection levels between
living and dead algae suggests that this fungus is latent in
healthy tissues. This may represent an adaptive strategy of the
saprobe for rapid colonization of the decaying material. The
life history strategies of A. fuci may parallel those of endo-
phytic fungi in higher plants (36). Some endophytes grow dis-
cretely in a healthy host, resuming saprophytic growth only
during senescence of the host (e.g., Rhabdocline parkeri in
needles of Pseudotsuga menziesii) (2, 42).
The other fungal LSU rRNA gene signals retrieved from
living and decaying host tissue after DGGE analysis included
signals for L. obtusa (Lulworthiaceae), Engyodontium album-
like species (Clavicipitaceae), and C. angusta (Halosphaeri-
aceae) and a signal for an Iodophanus-like organism (Peziza-
ceae) (48). Further environmental sequences representing
Dothideomycetes were amplified from decaying alga material
using primers NL209 and NL912 and were separated by mo-
lecular cloning. Reamplified fragments from these clones were
not resolved under the DGGE conditions that we employed.
The absence of DGGE bands representing Dothideomycetes,
therefore, does not reflect a primer bias (48) but most likely an
electrophoresis band resolution problem.
The majority of the environmental signals and rRNA genes
from sterile mycelia falling within the Dothideomycetes exhib-
ited similarities with sequence representatives of families be-
longing to the Pleosporales. The majority of marine Pha-
eosphaeria species have been obtained from beach grass and
salt marsh plants (27) but not from seaweeds. As the host is a
phylogenetically important characteristic in defining species
within this fungal group (7), the environmental signals may
reflect undescribed lineages representing novel organisms.
An additional environmental sequence (represented by
clone 7) belonging to a Cladosporium species was detected.
Commonly, Cladosporium isolates can be cultured from many
marine substrates (24, 25, 32). Some Cladosporium species
exhibit physiological adaptations to saline conditions (19),
while others can cause fish diseases (38) and are important
producers of bioactive molecules (4, 18). Algal tissues have
provided substrate material for Cladosporium algarum (24) and
a large number of unidentified Cladosporium isolates from this
study. The recovery of an environmental signal for this group
obtained from decaying material is therefore not surprising.
The other cloned sequences included in this study were the
sequences derived from ascocarps of D. fucicola (herbarium
specimen J.K.2932). This fungus is an obligate parasite whose
ascocarps are embedded in the central midribs of damaged
lower side branches of living Fucus spiralis and F. vesiculosus
and vegetative thalli of P. canaliculata, and it is often associ-
ated with the bases of Elachista clandestina and Elachista fuci-
cola (24). The D. fucicola ITS sequences, although exhibiting a
degree of similarity to the ITS sequences of other Didymella
species, exhibited more similarity to the ITS sequences of T.
aquatica,C. elongata, and S. griseola and unpublished environ-
mental signals in the GenBank database. T. aquatica and C.
elongata are aquatic mitosporic species (10) whose molecular
lineage is uncertain. Bussaban et al. (5) noted that the ITS
sequences of T. aquatica were similar to those of Pyricularia
variabilis, which is separate from other Pyricularia species in
the Magnaporthaceae. The teleomorphic form of T. aquatica is
Massarina aquatica (44, 45), suggesting a link to the Pleospo-
FIG. 5. Phylogram showing the relationship between Didymella
species, based upon ITS and 5.8S rRNA gene sequences: one of three
most parsimonious trees with 335 steps (consistency index, 0.81; re-
tention index, 0.71; homoplasy index, 0.19) generated from a single
tree island. A similar phylogram was produced after maximum likeli-
hood analysis (⫺ln L [likelihood value] ⫽2,314.347). Bootstrap values
are indicated above the branches, and the values generated by a max-
imum likelihood analysis are in parentheses. Values less than 50% are
not shown.
VOL. 74, 2008 PCR-MEDIATED DETECTION OF MARINE FUNGI 939

rales, although the exact placement of the anamorph is unclear
(3). S. griseola is another mitosporic species, but it has an
affiliation with the Sarcosomataceae of the Pezizales. The tax-
onomic position of this species, however, is questionable. It
lacks the ability to produce the hexaketide galiellalactone,
which is believed to be a chemotaxonomic marker for the
family sensu stricto (28). All of these mitosporic forms, there-
fore, represent nontypical members of their respective taxa. It
is to this heterogeneous group that D. fucicola appears to be
related based on ITS1 and the 5.8S rRNA gene data, although
the nuclear LSU rRNA gene analysis identified it as a sister
taxon of members of Didymella.
All of the nonsporulating isolates except one were members
of the Pleosporales, but none of the environmental signals
representing the Dothideomycetes exactly matched the signals
amplified from these isolates or the herbarium specimen of D.
fucicola. Therefore, their presence may reflect the existence of
novel marine dothideomycete lineages, although it should be
noted that many species belonging to this class have not been
studied yet at the molecular level (43). Furthermore, the pres-
ence of these signals associated with decaying seaweeds sug-
gests a change in fungal populations that could be related to
the release of nutrients resulting from tissue breakdown.
Our current understanding of alga-fungus relationships is
quite limited, yet a few algal ecological studies have included
fungal associations as a significant research component. The
adoption of large-scale projects, such as the AFTOL project
(http://www.aftol.org) and the international barcoding initia-
tives (http://www.bolnet.ca/rp_fungi.php), which are rapidly
improving the representation of known fungal lineages in se-
quence databases, provides a framework to link organisms to
the processes that they control and to the molecular signals
present in the environment. This is important from taxonomic,
phylogenetic, and ecological points of view as the proportion of
fungi that have been found to be actively associated with ma-
rine substrates is greater than previously thought.
ACKNOWLEDGMENTS
Part of the DNA analysis used for the phylogenetic study was sup-
ported by the National Science Foundation through the AFTOL
project (DEB-0228725). We thank Hans-Ju¨rgen Aust for financial
support.
We thank Katja Bo¨hme and Andreea Munteanu for excellent tech-
nical contributions. Andreas Wagner of the Biological Institute of
Helgoland, AWI, is thanked for his support during collection of ma-
terial at Helgoland Island. Stefan Wagner is gratefully acknowledged
for his help with the statistical analysis. Barbara Schulz is acknowl-
edged for reading the manuscript.
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