ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2007 Vol. 50, [t. 1: 5–18
Sprejeto (accepted): 2007-11-26
Fungal colonization of the roots of selected halophytes from Sečovlje salterns
Glivna kolonizacija korenin izbranih halofi tov iz Sečoveljskih solin
Silva SONJAK1, Tamara GLAVINA2, Metka UDOVIČ3, Marjana REGVAR1*
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 
Ljubljana, Slovenia; Tel: +386-1-4233388, Fax: +386-1-2573390; E-mail (*Corresponding author): 
marjana.regvar@bf.uni-lj.si
2Monteko d.o.o. Šmarje, Šmarje 10, SI-6274 Šmarje, Slovenia.
3Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101,
SI-1000 Ljubljana, Slovenia.
Abstract. To reveal their mycotrophic status, we have here analysed the fungal colonisation 
of the roots, and the identity of these fungi, of the most abundant halophytic plant species from 
Sečovlje salterns (Slovenia): Aster tripolium, Limonium angustifolium and Salicornia europaea. 
The highest frequency of fungal colonisation was seen for the roots of A. tripolium, followed by 
those of L. angustifolium and S. europaea. Hyphae and occasional microsclerotia, the assumed 
structures of dark septate endophytes, were seen in the roots of all three species, whereas arbu-
scules, as typical structures of arbuscular mycorrhiza, were seen in the roots of A. tripolium and 
in one specimen of L. angustifolium. Fungal partial small subunit ribosomal DNA (SSU-rDNA) 
fragments were amplifi ed from total root DNA extracts for further restriction fragment length 
polymorphism (RFLP) analyses of the structures of the fungal root communities. Fifteen different 
RFLP profi les were obtained that grouped into two major clusters. Two RFLP profi les strongly 
dominated in samples of all three of the plant species. Sequencing revealed that one of these 
profi les corresponds to the species Cylindrobasidium laeve (Basidiomycota), and the second 
to Capnobotryella sp. / Phaeotheca fi ssurella, putative dark septate endophytes from the class 
Dothideomycetes (Ascomycota). Intraspecifi c RFLP polymorphisms were demonstrated for both 
species. To our knowledge, this is the fi rst report on dark septate endophyte fungi occurrence in 
roots of A. tripolium, L. angustifolium and S. europaea. 
Keywords: Aster tripolium, Limonium angustifolium, Salicornia europaea, halopytes, 
mycorrhizal fungi, dark septate endophytes, restriction fragment length polymorphism 
Izvleček. Analizirali in določili smo glivno kolonizacijo korenin treh najpo gostejših halofi -
tskih rastlinskih vrst iz Sečoveljskih solin (Slovenija): Aster tripolium, Limonium angustifolium 
in Salicornia europaea, da bi ugotovili njihov mikotrofni status. Najvišjo frekvenco glivne 
kolonizacije smo opazili v koreninah vrste A. tripolium, sledili sta vrsti L. angustifolium in S. 
europaea. Hife in mikrosklerocije, verjetne strukture temnih septiranih endofi tov, smo opazili v 
koreninah vseh treh rastlinskih vrst, arbuskule, tipične strukture arbuskularne mikorize, pa le v 
koreninah vrste A. tripolium in ene rastline vrste L. angustifolium. Iz celokupne koreninske DNA 
smo pomnožili glivne DNA fragmente dela majhne ribosomske podenote (SSU-rDNA) in v na-
daljevanju analizirali polimorfi zem dolžin restrikcijskih fragmentov (RFLP) struktur koreninskih 
glivnih združb. Dobili smo petnajst različnih RFLP profi lov, ki so se združevali v dve glavni gruči. 
V vzorcih vseh treh rastlinskih vrst sta prevladovala dva RFLP profi la, sekveniranje pa je poka-
zalo, da eden ustreza vrsti Cylindrobasidium laeve (Basidiomycota), drugi pa Capnobotriyella/ 
Pheotheca fi ssurella, domnevnemu temnemu septiranemu endofi tu iz razreda Dothideomycetes 
6 Acta Biologica Slovenica, 50 (1), 2007
(Ascomycota). V nadaljevanju smo ugotovili, da pri obeh vrstah obstaja intraspecifi čni RFLP 
polimorfi zem. Kot nam je znano, je to prvo poročilo o pojavljanju temnih septiranih glivnih 
endofi tov v koreninah vrst A. tripolium, L. angustifolium in S. europaea.
Ključne besede: Aster tripolium, Limonium angustifolium, Salicornia europaea, halofi ti, 
mikorizne glive, temni septirani endofi ti, polimorfi zem dolžin restrikcijskih fragmentov 
Introduction
Salterns represent one of the most extreme of environments, where drought and salinity are the 
most important abiotic factors that limit plant growth (RUIZ-LOZANO 2003). Plants that can establish 
themselves, grow to maturity and reproduce under these conditions have developed many effi cient 
mechanisms to overcome the osmotic and ionic stresses (LARCHER 1995). Halophytes are thus recognized 
as a diverse group of vascular plants with adaptation at the morphological, anatomical and cellular 
levels that allow them to avoid these stresses or to increase their tolerance to them (BRAY 1997). In 
addition to such morpho-physiological adaptations, plants can have associated soil microorganisms that 
alleviate the stress symptoms. Symbiosis with mycorrhizal fungi has been repeatedly demonstrated to 
enhance the water and mineral nutrient supply for plants and to protect them against diverse biotic and 
abiotic stresses (SMITH & READ 1997). Many benefi cial effects of this symbiosis have been described 
for host plants and for ecosystems, including the enhancement of plant tolerance to drought and salt 
stress (SMITH & READ 1997, RUIZ-LOZANO & AZCON 2000). Among these symbiotic microorganisms, 
the cosmopolitan arbuscular mycorrhizal fungi (AMF) have been extensively studied (SMITH & READ 
1997, GUPTA & al. 2002), with their occurrence also having been confi rmed in European saline envi-
ronments (HILDEBRANDT & al. 2001, LANDWEHR & al. 2002). Dark septate endophytes (DSEs), on the 
other hand, are widespread organisms that can form mutualistic, mycorrhiza-like, associations with 
their host plants (JUMPPONEN 2001). They are especially common in stressful environments (JUMPPONEN 
& TRAPPE 1998, BARROW & AALTONEN 2001, BARROW 2003), including those that are extremely arid 
(RUOTSALAINEN & al. 2007). Despite this, little is known about their ecology and identity or about the 
effects that they have on the plants that they inhabit (JUMPPONEN & TRAPPE 1998).
Plants growing in high saline soils belong to families that are frequently reported as non-myco-
trophic; nevertheless, mycorrhizal colonization has been recorded in plant species from northern (above 
51° N) European salt marshes and that belong to the families: Chenopodiaceae, Caryophyllaceae and 
Juncaceae (HILDEBRANDT & al. 2001, LANDWEHER & al. 2002). Aster tripolium (Asteraceae), Limo-
nium vulgare (Plumbaginaceae) and Salicornia europaea (Chenopodiaceae) are among the species 
documented for their maintenance of arbuscular mycorrhizae (WANG & QIU 2006); however, there 
have been no reports regarding the mycorrhizal status of L. angustifolium, and Salicornia spp. are 
frequently reported to be non-mycorrhizal (HARLEY & HARLEY 1987). The aim of the present study was 
therefore to examine the root fungal colonization of these three halophytic plant species growing in 
Sečovlje salterns (Slovenia), and to identify their fungal endophytes using the molecular techniques 
of cloning, RFLP and sequencing.
Materials and methods
Site description and sampling
The Sečovlje salterns are situated in south-eastern Piran Bay in the Gulf of Trieste (northern Adriatic 
Sea) on the sediment of the Dragonja River. The bottom of the pans consists of fl ysch silt and clay, 
and on average the water salinity in the Gulf of Trieste reaches 3.7% (KALIGARIČ 1988). The climate is 
Mediterranean, with most of the annual precipitation falling in autumn (934 mm) (SORS 2005). The 
plants were collected in October 2002 and 2003 from the abandoned southern sections of the salterns, at 
7S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
Fontanigge. The water content of the soil in the sampling area was on average 27%, the organic matter 
3.88%, with a pH of 7.9 and soil extract conductivity of 1880 µS/cm (UDOVIČ 2004). We selected two 
plant species that have been frequently reported as non-mycorrhizal: L. angustifolium (Tausch) Degen 
[Statice serotina Rchb., L. vulgare Mill. subsp. serotinum (Rchb.) Gams] (Plumbaginaceae) and S. 
europaea L., which is considered as an aggregate of closely related species in the literature (Cheno-
podiaceae), and additionally a moderately mycorrhizal plant A. tripolium L. [Tripolium vulgare Nees] 
(Asteraceae) for comparison (MARTINČIČ & al. 2007). Six specimens (three from each year) of each 
plant species were collected for the analysis of their mycorrhizal colonization, which was estimated 
according to TROUVELOT & al. (1986). Fifteen root fragments per plant were cleaned with tap water, 
cleared in 10% KOH, and stained with trypan blue (PHILIPS & HEYMANN 1970). The frequency (F%) 
and intensity (M%) of their fungal colonisation, along with their arbuscular (A%), vesicular (V%) and 
microsclerotial (MS%) densities, were determined under light microscopy (Zeiss).
DNA extraction 
Roots of three specimens of each plant species from the two consecutive years were used for 
further DNA analysis. The roots were washed thoroughly with tap water and then sterile water, ran-
domly sampled (~150 mg of roots from each plant) and stored either at room temperature (in 2002) 
or frozen at –20 °C (in 2003) until use. The total DNA was extracted from the roots after they had 
been ground in liquid nitrogen, using the DNeasy Plant Mini Kit (Qiagen), following the manufacturer 
recommendations. After isolation, 10 μl of 20% (w/v) Chelex 100 was added to 50 μl of DNA eluents 
(to scavenge multivalent metal ions that could inhibit the PCR amplifi cation), and after an incubation 
for 1 min, the samples were centrifuged and the supernatants used for the PCR reactions.
Nested PCR amplifi cation
Amplifi cations of the partial fungal small subunit ribosomal DNAs (SSU-rDNAs) were carried 
out in a fi nal volume of 50 μl, using 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U Taq DNA 
polymerase (all from Fermentas), 0.4 μM primers and 2 μl DNA extract or PCR product. In the fi rst 
PCR reaction, a 1330 bp SSU-rDNA fragment was amplifi ed using a crude DNA extract and the MH2 
and MH4 primers (VANDENKOORNHUYSE & LEYVAL 1998), with the following cycle conditions: 95 °C 
for 2 min, followed by 33 cycles at 94 °C for 1 min, 48 °C (–0.1 °C per cycle) for 1.5 min, 72 °C 
for 2 min, and a fi nal extension at 72 °C for 8 min. The second, nested PCR was then performed on 
1 μl of the MH2/MH4 PCR product. The universal eukaryotic primer NS31 (SIMON & al. 1992) and 
an AMF-specifi c primer AM1 (HELGASON & al. 1998) were used, with the latter designed to amplify 
a 550 bp fragment of SSU-rDNA from Glomerales from colonized roots. The PCR cycle conditions 
were as follows: 95 °C for 2 min, followed by 30 cycles at 95 °C for 1 min, 62 °C for 1 min, 72 °C 
for 1 min and a fi nal extension at 72 °C for 8 min. All of the PCR amplifi cations were performed with 
a Thermal Cycler (Biozym). To check the size and quality of the PCR products, they were visualized 
under UV light after electrophoresis with 1% (w/v) agarose gels and staining with 0.5 mg/l ethidium 
bromide (Biorad). 
Cloning, RFLP, sequencing and sequence analysis
Single PCR bands of ~550 bp were excised from the agarose gels, purifi ed with the Wizard® SV 
Gel and PCR Clean-up System (Promega), and cloned using the pGEM-T Easy Vector Systems II 
with JM109 competent cells (Promega), following the manufacturer protocols. The transformants 
were selected using blue/ white screening on LB agar containing X-Gal, isopropyl-1-thio-β-D-ga-
lactopyranoside (IPTG) and ampicilin (Sigma). For the RFLP analysis, up to 20 positive clones of 
each of the SSU-rRNA gene libraries were randomly selected and re-amplifi ed (333 in all; by colony 
8 Acta Biologica Slovenica, 50 (1), 2007
PCR) with the NS31 and AM1 primers, as described above. Re-amplifi ed fragments were digested 
with the HphI, HinfI and MboI restriction enzymes (Fermentas), according to the manufacturer recom-
mendations, and analysed by 3% agarose gel electrophoresis. The restriction enzymes were selected 
through an analysis of several SSU sequences from GenBank, using the Webcutter 2.0 (HEIMAN 1997) 
and NebCutter 2.0 (VINCZE & al. 2003) programmes, and according to the literature (HELGASON & al. 
2002, VANDENKOORNHUYSE & al. 2002). The DNA restriction fragment patterns were visualized using 
a UVItec gel imaging and documentation system (UVItec Limited), and they were analysed using 
ImageQuant TL (Molecular Dynamics). Each restriction fragment was treated as a unit character and 
scored for its presence or absence. The dendrogram was constructed using the Jaccard distance equation 
and simple average clustering. Calculations were carried out with the Biodiversity pro application 
programme (The Natural History Museum, London). Representatives of most frequent RFLP patterns 
were selected for sequencing. Therefore, the plasmids were isolated using the Wizard® Plus Minipreps 
DNA Purifi cation System (Promega). The sequencing reactions were performed using the SP6 or T7 
primers and the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), following the 
manufacturer instructions, and the sequencing was carried out using an ABI prism 377 (Perkin-Elmer 
Corporation), as provided by the Omega company (Omega d.o.o, Slovenia). The sequences obtained 
were compared to the available sequences of the National Center for Biotechnology Information 
(NCBI) using the BLAST-n programme (ALTSCHUL & al. 1990, 1997). The sequences were aligned 
using CLUSTAL W (THOMPSON & al. 1994), and for the phylogenetic analysis, the neighbor-joining 
method (SAITOU & NEI 1987) was used. Data were fi rst analyzed using a Kimura 2-parameter model 
(KIMURA 1980), which was than used to construct the neighbor-joining tree with MEGA3 software 
(KUMAR & al. 2004). To determine the support for each clade, bootstrap analysis was performed with 
10,000 replications. The sequences generated through this study have been deposited with GenBank, 
and their Accession numbers are given in Table 1.
Results 
Fungal colonization of the roots
Fungi were seen to be present in the roots of all three of the selected halophytic plant species (Fig. 
1). The highest frequency of colonisation was seen for the root fragments of A. tripolium, following 
by L. angustifolium and S. europaea (Fig. 1; F%). The roots of A. tripolium were clearly colonized 
by AMF, since both vesicles and arbuscules were seen. For the roots of L. angustifolium, there were 
vesicles, whereas arbuscules were only seen in one specimen (Fig. 1; A%). Hyphae and extremely 
rare vesicles were present in the roots of S. europaea, but no arbuscules were seen. There were also 
melanized hyphae and microsclerotia of DSE fungi in the roots of all three of these halophytic plant 
species; however, microsclerotia were only seen in one specimen of each species (Fig. 1; MS%).
9S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
Figure 1: Fungal colonisation in the roots of the selected halophytic plant species Aster tripolium, Limonium 
angustifolium and Salicornia europaea, sampled in two consecutive years (2002, 2003). ■, frequency 
of colonisation (F%); ○, density of arbuscules (A%); □, density of microsclerocia (MS%).
Slika 1: Glivna kolonizacija korenin izbranih halofi tskih rastlinskih vrst Aster tripolium, Limonium angustifolium 
in Salicornia europaea, vzorčenih v dveh zaporednih letih (2002, 2003). ■, frekvenca kolonizacije (F%); 
○, gostota arbuskulov (A%); □, gostota mikrosklerocijev (MS%).
RFLP and sequence identifi cation
Only after the second (nested) PCR were adequate and equivalent amounts of the amplifi ed partial 
SSU-rDNAs obtained for all 18 of the samples. These samples were cloned, and up to 20 clones from 
each were subjected to RFLP analysis. Fifteen different RFLP profi les were obtained with the HphI 
(6) and HinfI (5) restriction enzymes. Since no additional profi les were obtained with MboI, these 
results were not included in the subsequent analyses. Profi les 1 and 12 dominated strongly and were 
obtained for each of the 18 samples, with the exception of profi le 1 in sample A4 (Fig. 2a). For each 
plant species, ~80% of all of the clones analysed resulted in these two profi les (Fig. 2b), regardless 
of the year of sampling (Fig. 2c), whereas all of the other profi les taken together represented the 
remaining 20% (Fig. 2b). 
10 Acta Biologica Slovenica, 50 (1), 2007
Figure 2: Percentages for the different RFLP profi les obtained: a) for each plant specimen; b) as means for each 
species: Aster tripolium, Limonium angustifolium and Salicornia europaea; and c) as means for each 
species within each single year (2002, 2003).
Slika 2: Procenti posameznih RFLP profi lov pridobljenih: a) za vsako rastlino; b) kot povprečje za vsako rastlinsko 
vrsto: Aster tripolium, Limonium angustifolium in Salicornia europaea; in c) kot povprečje za vsako 
vrsto v enem letu (2002, 2003).
11S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
Figure 3: Dendrogram generated from the RFLP data. Altogether, 333 clones with fungal SSU-rDNA fragments 
were obtained from 18 root samples of the halophytic plant species analysed: Aster tripolium, Limonium 
angustifolium and Salicornia europaea. The percentages of each of the RFLP profi les obtained are given 
in parenthesis.
 $, sequences of the representative clones that showed 99% similarity with Cylindrobasidium laeve; #, 
sequences of the representative clones that showed up to 99% similarity with species from the class 
Dothideomycetes.
Slika 3:  Dendrogram narejen na osnovi RFLP podatkov. V celoti je bilo pridobljenih 333 klonov z glivnimi 
SSU-rDNA fragmenti iz 18 koreninskih vzorcev analiziranih halofi tskih rastlinskih vrst: Aster tripolium, 
Limonium angustifolium in Salicornia europaea. V oklepajih je podan delež, ki ga predstavlja vsak 
posamezen RFLP profi l. 
 $, sekvence reprezentativnih klonov so kazale 99 % podobnosti z vrsto  Cylindrobasidium laeve; #, 
sekvence reprezentativnih klonov so kazale do 99 % podobnosti z vrsto iz razreda Dothideomycetes.
In the dendrogram constructed from the RFLP data, profi les 1–10 grouped into the fi rst main 
clusters, and profi les 11–15 into the second (Fig. 3). Representative clones of the dominant profi les 1 
(one clone of A. tripolium: A2-16 and three clones of S. europaea: S1-1, S4-24, S4-30) and 12 (S4-
26) were selected for sequencing. Additionally, clones were selected for sequencing from some of 
the less frequent profi les: 4 (S4-12), 6 (A1-20, S4-31) and 15 (S4-29). Furthermore, a PCR product 
was obtained by single amplifi cation with the NS31-AM1 primer pair of sample S4, and this was 
cloned, with three of these clones selected for sequencing. The sequences of the partial SSU-rDNAs 
that corresponded to profi les 1, 4 and 6 showed 99% similarity with the sequence of the fungal spe-
cies Cylindrobasidium laeve (Basidiomycota), whereas those corresponding to profi les 12 and 15 
showed 98%-99% similarity with the sequences belonging to members of the class Dothideomycetes 
(Ascomycota) Capnobotryella sp. / Phaeotheca fi ssurella (Table 1). The sequences obtained from 
sample S4 after one NS31-AM1 PCR amplifi cation were shown to be chimeric. Approximately 100 
bp of these sequences showed 96% similarity with sequences corresponding to representatives of the 
phylum Glomeromycota (SCHÜΒLER & al. 2001) (data not shown).
12 Acta Biologica Slovenica, 50 (1), 2007
Table 1: Nearest GenBank matches and percentages of similarity with the sequences obtained. The GenBank 
accession numbers of the deposited fungal sequences and the number of the corresponding RFLP profi le 
and clone are given. 
Tabela 1: Najbližji zadetki iz podatkovne baze GenBank in procenti podobnosti s pridobljenimi sekvencami. Podane 
so številke dostopanja do glivnih sekvenc deponiranih v bazo GenBank in oznake pripadajočega RFLP 
profi la in klona.
Fungal phylum 
/ class
RFLP
profi le/ clone
Accession 
number*
Nearest GenBank match %
similarity
Basidiomycota 1 / A2-16 EU189957 AF518576 Cylindrobasidium laeve 99
Agaricomycetes 1 / S1-1 EU189952 AF518576 Cylindrobasidium laeve 99
1 / S4-24 EU189953 AF518576 Cylindrobasidium laeve 99
1 / S4-30 EU189954 AF518576 Cylindrobasidium laeve 99
4 / S4-12 EU189955 AF518576 Cylindrobasidium laeve 99
6 / A1-20 EU189951 AF518576 Cylindrobasidium laeve 99
6 / S4-31 EU189956 AF518576 Cylindrobasidium laeve 99
Ascomycota 12 / S4-26 EU189958 AJ972854 Capnobotryella sp. 98
Dothideomycetes Y18697 Phaeotheca fi ssurella 98
15 /  S4-29 EU189959 AJ972854 Capnobotryella sp. 99
Y18697 Phaeotheca fi ssurella 99
Discussion
Species from hypersaline environments have frequently been reported as non-mycorrhizal, includ-
ing S. europaea (Chenopodiaceae) (HARLEY & HARLEY 1987, LANDWEHR & al. 2002, WANG & QIU 
2006). The association of plants with mycorrhizal fungi has traditionally relied on morphological 
characterisation after trypan blue staining, which appears not to be a sensitive enough method in 
cases of low colonisation levels (REGVAR & al. 2003). We saw arbuscules in A. tripolium and in one 
specimen of L. angustifolium, but not in S. europaea, and this therefore confi rms the presence of AMF 
using this traditional approach only for the fi rst two species here. In addition, although frequently 
neglected in AMF studies, melanized hyphae and microsclerotia of presumed DSE fungi were also 
seen in all three species. 
Nested PCR had to be performed to obtain enough products for our molecular analyses. The PCR-
RFLP technique has been previously applied in molecular identifi cation studies of mycorrhizal fungi 
to reduce the need for further sequencing (HELGASON & al. 2002, VANDENKOORNHUYSE & al. 2002). The 
15 different RFLP profi les that we obtained in the present study grouped into two main clusters (Fig. 
3) and were numbered consecutively from 1 to10 in cluster 1 and from 11 to 15 in cluster 2. Profi le 1 
strongly dominated in cluster 1 and profi le 12 dominated in cluster 2. The sequences of the selected 
clones showed that the RFLP profi les 1, 4 and 6 from cluster 1 represent the species Cylindrobasidium 
laeve (Basidiomycota). RFLP profi les 12 and 15 from cluster 2 correspond to species from the class 
Dothideomycetes (Ascomycota). As different RFLP profi les were obtained for the same species, this 
shows intraspecifi c RFLP polymorphisms of the fungi belonging to both of these clusters, which 
has previously been seen for some ectomycorrhizal fungi (HORTON 2002). Using the blast searching, 
two closest matches were obtained for the clones of profi les 12 and 15 with the same percentage of 
similarity, namely Capnobotryella sp. and Phaeotheca fi ssurella (Table 1). We were therefore not 
able to determine the precise taxonomic positions of the sequences obtained even after constructing 
the phylogenetic tree (Fig. 4).
13S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
Figure 4: Phylogenetic tree inferred from neighbor-joining analysis of the partial SSU-rRNA gene sequences. 
The numbers at the nodes represent the bootstrap values of >50% (out of the 10,000 replications). The 
number of nucleotide changes between the taxa is given by the branch length.
Slika 4: Filogenetsko drevo narejeno na osnovi analize združevanja najbližjega soseda delnih SSU-rRNA genskih 
sekvenc. Številke ob razvejitvah predstavljajo vrednosti >50 % (od 10000 ponovitev) statistične metode 
vezenja. Število nukleotidnih sprememb med taksoni je ponazorjeno z dolžino veje.
Except for the chimeric sequences, no other sequences showed similarities with sequences of 
species from the phylum Glomeromycota. The abundance of arbuscular mycorrrhizal fungal spores 
found in saline environments has been shown to be rather variable, although with an apparent low 
species diversity, whereas the degree of root colonisation varies with the individual plant, plant species 
and vegetation period (HILDEBRANDT & al. 2001, LANDWEHR & al. 2002). Although the AM1 primer 
(HELGASON 1998) was designed to amplify species from some of the AMF groups, its broader lack of 
specifi city was only recently reported (DOUHAN & al. 2005). There are only a few base mismatches in 
the annealing sequences of the AM1 primer that were also seen in some of the species from the phyla 
Ascomycota and Basidiomycota (DOUHAN & al. 2005). The specifi city of primers usually depends on 
the content and quality of the target DNA (ANDERSON & CAIRNEY 2004), and thus a small amount of 
AM sequences in the total DNA extract can lead to loss of specifi city of the NS31-AM1 primer pair, 
which therefore amplifi es extended non-target DNA (DOUHAN & al. 2005). Nevertheless, the NS31-
AM1 primer pair is still widely used for detection and identifi cation of AMF in planta (SANTOS & al. 
2006, SANTOS-GONZÁLEZ & al. in press). The specifi city of the amplifi cation can also be reduced using 
the nested PCR reaction (LANDWEHR & al. 2002). In addition, freezing of the AMF spores drastically 
reduces the DNA concentration in the PCR products (LANDWEHR & al. 2002), which may also apply 
for the samples in the present study, in which the root samples were dried (2002) or frozen (2003) 
prior to the DNA extraction. Thus, the higher sensitivity of the DNA of the AMF to all of the above-
mentioned processes may have contributed to the results.
14 Acta Biologica Slovenica, 50 (1), 2007
Clones with RFLP profi les related to species C. laeve and Capnobotryella sp./ P. fi ssurella were 
obtained from samples of all three of the selected halophytic plants. C. laeve is known as a wooden 
saprophyte (PHILLIPS 1994); however, it can obviously penetrate into the roots of green plants. The 
frequent detection of the corresponding RFLP profi les in our analysed root extracts from all three 
of the species indicates the extensive presence and association with plant roots of this fungus in the 
section of the Sečovlje salterns that was sampled, where the presence of decayed wooden and plant 
material supports its growth. Capnobotryella sp. and P. fi ssurella are mitosporic fungi that have 
previously been isolated from plants (ZALAR & al. 1999, HAMBELTON & al. 2003). According to some 
of their morphological features, production of microsclerotia and thick-walled conidiogenous cells 
impregnated with numerous melanin-like granules (ZALAR & al. 1999, HAMBELTON & al. 2003) and 
their relation to the identifi ed endophytic putative DSE fungi from the class Dothideomycetes (JUMP-
PONEN & TRAPPE 1998), they could be described as DSE fungi. Typical structures of DSEs, such as 
septate melanised hyphae and distinct microsclerotia, were seen in the roots of all three of our sam-
pled halophytes. The desiccation-tolerant, melanin-rich cell wall of DSE fungi is one of their main 
characteristics that allows them to tolerate unfavourable environments (JUMPPONEN & TRAPPE 1998). 
Furthermore, the intracellular DSE microsclerotia may also provide a mechanism for the fungi to 
withstand unfavourable environments and/or may serve as dispersal propagules in heavily eroded 
areas (RUOTSALAINEN & al. 2007). Extensive root colonisation by DSE fungi of these three selected 
halophytes that thrive in Sečovlje salterns therefore indicates their functional importance for those 
species in the environment where the propagules of AMF are clearly reduced and the demands for 
symbiotic associations are considerable. 
Thus, fungi from the phyla Ascomycota and Basidiomycota extensively colonise plants in extremely 
saline environments; however, the nature of these associations and the signifi cance for their hosts are 
not easily determined. Finding compatible DSE fungal symbionts of such halophytic plant species may 
be of importance for the applications of comparable symbiosis in saline agricultural environments.
Conclusions
• AMF colonisation was demonstrated for L. angustifolium and A. tripolium on the basis of micro-
scopic examinations, whereas it was not confi rmed by molecular identifi cation, apparently due to 
the combination of reduced colonisation levels, the sensitivity of the DNA of the AMF to drying/ 
freezing, and the lack of specifi city of the NS31-AM1 primer pair in the case of insuffi ciency of 
target DNA from Glomeromycota.
• Melanized hyphae and microsclerotia of DSE fungi were seen in all three of the species. Sequencing 
revealed the presence of the putative dark septate fungi Capnobotryella sp. / Phaeotheca fi ssurella 
from the class Dothideomycetes.
• Additionally, sequences related to the saprophyte Cylindrobasidium laeve were also obtained from 
all three of the plant species.
• RFLP analysis showed two dominating profi les out of the 15 profi les identifi ed. However, sequen-
cing revealed that more than one of the RFLP profi les corresponded to the same fungal species, 
indicating high intraspecifi c RFLP polymorphism.
• This is to our knowledge the fi rst report on the occurrence of DSE fungi in the roots of L. angu-
stifolium, S. europaea and A. tripolium. 
15S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
Povzetek 
Soline predstavljajo ekstremno okolje, kjer sta suša in slanost glavna dejavnika, ki omejujeta rast 
rastlin. V takem okolju lahko uspešno živijo in se razmnožujejo le rastline, ki so odporne na osmotski 
in ionski stres. Pri tem jim pomagajo tudi mikorizne glive, med katerimi so najbolj proučevane arbu-
skularno mikorizne (AM) glive. Temne septirane endofi tske (DSE) glive so zelo pogoste v stresnih 
okoljih, vendar je o njih zelo malo znanega. Rastline, ki uspevajo v ekstremno slanih okoljih, pogosto 
sodijo v družine, ki veljajo za nemikorizne. Halofi tske vrste Aster tripolium (Asteraceae), Limonium 
vulgare (Plumbaginaceae) in Salicornia europaea (Chenopodiaceae) sicer lahko vzdržujejo mikorizo, 
vendar pa ne poznamo mikoriznega statusa vrste L. angustifolium, medtem ko rod Salicornia pogosto 
opisujejo kot nemikorizen. 
V predstavljenem delu smo analizirali glivno kolonizacijo korenin omenjenih treh vrst iz Sečo velj-
skih solin. S klasičnim morfološkim pregledom pobarvanih korenin smo najvišjo frekvenco glivne 
kolonizacije opazili v koreninah vrste A. tripolium, sledili sta vrsti L. angustifolium in S. europaea. 
Temne septirane hife in mikrosklerocije, verjetne strukture DSE gliv, smo opazili v koreninah vseh treh 
rastlinskih vrst, arbuskule, tipične strukture arbuskularne mikorize, pa le v koreninah A. tripolium in 
ene rastline L. angustifolium. Z vgnezdeno PCR reakcijo smo z uporabo začetnega nukleotidnega para 
MH2-MH4 v prvi in NS31-AM1 v drugi PCR reakciji iz celokupne koreninske DNA pomnožili glivne 
fragmente SSU rDNA, pripravili gensko knjižnico in izvedli RFLP analizo z restrikcijskimi encimi 
HphI, HinfI in MboI. Dobili smo petnajst različnih RFLP profi lov, ki so se v dendrogramu združevali 
v dve glavni gruči. V primeru vseh treh rastlinskih vrst sta prevladovala dva profi la, sekveniranje pa je 
pokazalo, da eden ustreza saprofi tski vrsti Cylindrobasidium laeve (Basidiomycota), drugi pa domnevni 
DSE vrsti iz razreda Dothideomycetes (Ascomycota), za katero je fi logenetska analiza pokazala, da 
je sorodna vrstama Capnobotryella sp. in Phaeotheca fi ssurella. V nadaljevanju smo ugotovili, da pri 
obeh vrstah obstaja intraspecifi čni RFLP polimorfi zem. 
AMF kolonizacije opažene pri vrstah A. tripolium in L. angustifolium nismo uspeli potrditi z 
molekularno identifi kacijo. Začetni oligonukleotid AM1 so sicer razvili z namenom pomnoževanja 
AM gliv, vendar pa se je izkazalo, da je predvsem v primeru nezadostne količine tarčne DNA zelo 
nespecifi čen. Poleg tega je DNA AM gliv zelo občutljiva na procese sušenja oziroma zmrzovanja, kar 
zmanjšuje uspešnost pomnoževanja. Glive iz debel Basidiomycota in Ascomycota očitno predstavljajo 
zelo razširjene kolonizatorje rastlin v ekstremno slanih okoljih. Velik pomen za rastline v ekstremnih 
okoljih naj bi imeli temni septirani glivni endofi ti. Kot nam je znano, je to prvo poročilo o prisotnosti 
DSE gliv v koreninah vrst A. tripolium, L.  angustifolium in S. europaea.
Acknowledgements
This research was supported by the sponsors Mobitel d.d. and Soline d.o.o., by COST 8.38 »Manag-
ing Arbuscular Mycorrhizal Fungi for Improving Soil Quality and Plant Health in Agriculture«, as well 
as COST 8.59 »Phytotechnologies to Promote Sustainable Land Use Management and Improve Food 
Chain Safety« and by the Slovenian Research Agency as funder of the following projects: »Tolerance 
of Organisms in Stressed Ecosystems and Potential for remediation« (L1-5146), »Inventory of Sečovlje 
Salterns Flora and Optimisation of Growth of the Autochthonous Salicornia Species« (L1-7001) and 
programs: »Plant Biology« (P1-0212), Applied Botany, Genetics and Ecology (P4-0085). 
16 Acta Biologica Slovenica, 50 (1), 2007
References
ALTSCHUL S. F., T. L. MADDEN, A. A. SCHÄFFER, J. ZHANG, Z. ZHANG, W. MILLER & D. J. LIPMAN 1997: 
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic 
Acids Res. 25: 3389–3402.
ALTSCHUL S. F., W. GISH, W. MILLER, E. W. MYERS & D. J. LIPMAN 1990: Basic local alignment search 
tool. J. Mol. Biol. 215: 403–410.
ANDERSON I. C. & W. G. CAIRNEY 2004: Diversity and ecology of soil fungal communities: increased 
understanding through the application of molecular techniques. Environ. Microbiol. 6: 769–779.
BARROW J. R. 2003: Atypical morphology of dark septate fungal root endophytes of Bouteloua in arid 
southwest USA rangelands. Mycorrhiza 13: 239–247.
BARROW J. R. & R. E. AALTONEN 2001: Evaluation of the internal colonization of Atriplex canescens 
(Pursh) Nutt. Roots by dark septate fungi and the infl uence of host physiological activity. Mycor-
rhiza 11: 199–205.
BRAY E. A. 1997: Plant responses to water defi cit. Trends Plant Sci. 2: 48–54.
DOUGHAN G. W., C. PETERSEN, C. S. BLEDSOE & D. M. RIZZO 2005: Contrasting root associated fungi 
of three common oak-woodland plant species based on molecular identifi cation: host specifi city 
or non-specifi c amplifi cation? Mycorrhiza 15: 365–372.
GUPTA N., U. C. BASAK & J. DAS 2002: Arbuscular mycorrhizal association of mangroves in saline and 
non-saline soils. Mycorrhiza News 13: 14–19.
HAMBELTON S., A. TSUNEDA & R. S. CURRAH 2003: Comparative morphology and phylogenetic placement 
of two microsclerotial fungi from Sphagnum. Mycologia 95: 959–975.
HARLEY J. L. & E. L. HARLEY 1987: A check-list of mycorrhiza in the British fl ora. New Phytol. 
(Suppl.) 105: 1–102.
HEIMAN M. 1997: Webcutter 2.0. http://rna.lundberg.gu.se/cutter2/ (25.09.2007).
HELGASON T., T. J. DANIELL, R. HUSBAND, A. H. FITTER & J. P. W. YOUNG 1998: Ploughing the wood-wide 
web? Nature 394: 431.
HELGASON T., J. W. MERRYWEATHER, J. DENISON, P. WILSON, A. H. FITTER & J. P. W. YOUNG 2002: Selectivity 
and functional diversity in arbuscular mycorrhizas of co-occurring fungi and plants from temperate 
deciduous woodland. J. Ecol. 90: 371–384.
HILDEBRANDT U., K. JANETTA, F. OUZIAD, B. RENNE, K. NAWRATH & H. BOTHE 2001: Arbuscular mycorrhizal 
colonization of halophytes in central European salt marshes. Mycorrhiza 10: 175–185.
HORTON T. R. 2002: Molecular approaches to ectomycorrhizal diversity studies: variation in ITS at a 
local scale. Plant Soil 244: 29–39.
JUMPPONEN A., J. M. TRAPPE 1998: Dark septate endophytes: a review of facultative biotrophic root-
colonizing fungi. New Phytol. 140: 295–310.
JUMPPONEN A. 2001: Dark septate endophytes: are they mycorrhizal? Mycorrhiza 11: 207–211.
KALIGARIČ M. 1985–1986: Botanični sprehod po Sečoveljskih solinah. Proteus 48: 102–106.
KIMURA M. 1980: A simple method for estimating evolutionary rates of base substitutions through 
comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111–120.
KUMAR S., K. TAMURA & M. NEI 2004: MEGA3: Integrated software for molecular evolutionary 
genetics analysis and sequence alignment. Brief. Bioinform. 5:150–163.
LANDWEHR M., U. HILDEBRANDT, P. WILDE, K. NAWRATH, T. TÓTH, B. BIRÓ & H. BOTHE 2002: The 
arbuscular mycorrhizal fungus Glomus geosporum in European saline, sodic and gypsum soils. 
Mycorrhiza 12: 199–211.
LARCHER W. 1995: Physiological Plant Ecology. Ecophysiology and Stress Physiology of Functional 
Groups, 3rd ed. Springer-Verlag, New York, pp. 379–409.
17S. Sonjak, T. Glavina, M. Udovič, M. Regvar: Fungal colonization of the roots of selected halophytes …
MARTINČIČ A., T. WRABER, N. JOGAN, A. PODOBNIK, B. TURK, B. VREŠ, V. RAVNIK, B. FRAJMAN, S. STRGULC 
KRAJŠEK, B. TRČAK, T. BAČIČ, M. A. FISCHER, K. ELER & B. SURINA 2007: Mala Flora Slovenije, 4th 
ed. Tehniška založba Slovenije, d.d., Ljubljana, 967 pp.
PHILIPS J. M. & D. S. HAYMANN 1970: Improved procedures for clearing roots and staining parasitic 
and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Brit. Mycol. 
Soc. 55: 158–161.
PHILLIPS R. 1994: Mushrooms and other fungi of Great Britain & Europe, Macmillan Publishers Ltd, 
London, 288 pp.
REGVAR M., K. VOGEL, N. IRGEL, T. WRABER, U. HILDEBRANDT, P. WILDE & H. BOTHE 2003: Colonisa-
tion of pennycresses (Thlaspi spp.) of the Brassicaceae by arbuscular mycorrhizal fungi. J. Plant. 
Physiol. 160: 615–626.
RUIZ-LOZANO J. M. & R. AZCÓN 2000: Symbiotic effi ciency and infectivity of an autochthonous 
arbuscular mycorrhizal Glomus sp. from saline soils and Glomus deserticola under salinity. 
Mycorrhiza 10: 137–143.
RUIZ-LOZANO M. J. 2003: Arbuscular mycorrhizal symbiosis and alleviation of osmotic stress. New 
perspectives for molecular studies. Mycorrhiza 13: 309–317.
RUOTSALAINEN A. L., A. MARKKOLA & M. V. KOZLOV 2007: Root fungal colonisation in Deschampsia 
fl exuosa: Effects of pollution and neighbouring trees. Environ. Pollut. 147: 723–728.
SAITOU N. & M. NEI 1987: The neighbour-joining method: a new method for reconstructing phyloge-
netic trees. Mol. Biol. Evol. 4: 406–425.
SANTOS J. C.,  R. D. FINLAY & A. TEHLER 2006: Molecular analysis of arbuscular mycorrhizal fungi 
colonising a semi-natural grassland along a fertilisation gradient. New Phytol. 172: 159–168.
SANTOS-GONZÁLEZ J. C., R. D. FINLAY & A. TEHLER in press: Seasonal dynamics of arbuscular 
mycorrhizal fungal communities in roots in a semi-natural grassland. Appl. Environ. Microbiol. 
doi:10.1128/AEM.00262–07.
SCHÜΒLER A., D. SCHWARZOTT & C. WALKER 2001. A new fungal phylum, the Glomeromycota: phylogeny 
and evolution. Mycol. Res. 105: 1413–1421.
SIMON L., M. LALONDE & T. D. BURNS 1992: Specifi c amplifi cation of 18S fungal ribosomal genes 
from vesicular-arbuscular endomycorrhizal fungi colonizing roots. Appl. Environ. Microbiol. 1: 
291–295.
SORS 2005. Territory and Climate. Statistical Yearbook of the Republic of Slovenia, Statistical offi ce 
of the Republic of Slovenia, Ljubljana, 45 pp., http://www.stat.si/Letopis/2005/01-05.pdf.
SMITH S. & D. READ 1997: Mycorrhizal Symbiosis, 2nd ed. Academic Press, San Diego, California, 
605 pp. 
THOMPSON J. D., D. G. HIGGINS & T. J. GIBSON 1994: CLUSTAL W: improving the sensitivity of pro-
gressive multiple sequence alignment through sequence weighting, positions-specifi c gap penalties 
and weight matrix choice. Nucleic Acids Res. 22: 4673–4680.
TROUVELOT A., J. L. KOUGH & V. GIANINAZZI-PEARSON 1986: Mesure du taux de mycorhization VA d’ 
un systeme radiculaire. Recherche de methodes d’ estimation ayant une signifi cation fonctionnelle. 
In: GIANINAZZI-PEARSON V. & S. GIANINAZZI (eds.): Mycorrhizae, Physiology and Genetics, INRA 
Press, Paris, pp. 216–222.
UDOVIČ M. 2004: Micotrophic status of some halophytes in the marine salt works of Sečovlje. Gra-
duation Thesis. University of Ljubljana, Biotechnical Faculty, Ljubljana, 72 pp.
VANDENKOORNHUYSE P. & C. LEYVAL 1998: SSU-rDNA sequencing and PCR fi ngerprinting reveal genetic 
variation within Glomus mosseae. Mycologia 90: 791–797.
VANDENKOORNHUYSE P., T. J. DANIELL, R. HUSBAND, A. H. FITTER, J. P. W. YOUNG, J. WATSON & J. M. 
DUCK 2002: Arbuscular mycorrhizal community composition associated with two plant species in 
grassland ecosystem. Mol. Ecol. 11: 1555–1564.
18 Acta Biologica Slovenica, 50 (1), 2007
VINCZE T., J. POSFAI & R. J. ROBERTS 2003: NEBcutter: a programme to cleave DNA with restriction 
enzymes. Nucleic Acids Res. 31: 3688–3691. 
WANG B. & Y-L. QIU 2006: Phylogenetic distribution and evolution of mycorrhizas in land plants. 
Mycorrhiza 16: 299–363.
ZALAR P., G. S. DE HOOG & GUNDE-CIMERMAN N. 1999: Taxonomy of the endoconidial black yeast genera 
Phaeotheca and Hyphospora. Stud. Mycol. 43: 49–56.