biodiversity of marine fungi associated with the seagrass

Biol. Mar. Mediterr. (2011), 18 (1): 85-88
L. Panno, S. Voyron, A. Anastasi, R. Mussat Sartor*, G.C. Varese
Mycoteca Universitatis Taurinensis (MUT), Dipartimento di Biologia Vegetale, Università di Torino,
Viale Mattioli, 25 - 10125 Torino, Italia.
[email protected]
*Laboratorio di Zoologia e Biologia Marina, Dipartimento di Biologia Animale e dell’Uomo,
Università di Torino, Via Accademia Albertina, 13 - 10123 Torino, Italia.
BIODIVERSITY OF MARINE FUNGI ASSOCIATED WITH THE
SEAGRASS POSIDONIA OCEANICA: AN ECOLOGICAL AND
BIOTECHNOLOGICAL PERSPECTIVE
BIODIVERSITÁ FUNGINA ASSOCIATA ALLA FANEROGAMA
MARINA POSIDONIA OCEANICA: UNA PROSPETTIVA ECOLOGICA
E BIOTECNOLOGICA
Abstract - In this work, for the first time the quali-quantitative composition of the mycoflora
associated to the seagrass Posidonia oceanica (L.) Delile (an endemic seriously threatened seagrass of
the Mediterranean Sea) was estimated. Eighty-eight fungal species were identified by morphological and
molecular methods and the most important genera were Penicillium, Cladosporium and Acremonium.
Many species are saprotrophic but other species could have a pathosistic nutritional strategy on P.
oceanica. Most of the fungal strains, cultured at different salt concentration, were tested by means of
a quali-quantitative microtitre plate method for oxidoreductase activity and tannases activity. Several
fungal strains showed a good enzyme production, many of them exclusively at high salt concentrations.
These findings suggest that marine fungi play an important ecological role in the decomposition of
ligninocellulosic matrices in the marine environment and are a good source of novel extremoenzymes
that can be used in different biotechnological applications.
Key-words: marine fungi, seagrass, biotechnology, Posidonia oceanica.
Introduction - Marine environments are characterized by a plethora of microorganisms still unknown. A big part of this microbial diversity is made of marine
fungi, organisms extremely important from an ecological point of view encompassing
saprotrophes, parasites, mycophycobiontes and endophytes. Recent papers underline
the importance of the chemical diversity of marine fungi for biotechnological and
pharmacological applications. In biotechnology, the metabolites of marine fungi allow
the performance of industrial processes even in harsh conditions (Hyde and Pointing,
2000). In pharmacology, the chemistry of marine fungi has let to the discovery of
a surprisingly large number of novel structures possessing bioactivity with potential
pharmaceutical applications (Bugni and Ireland, 2004). The main goals of this study
were: 1) the isolation and identification of fungi associated to the seagrass Posidonia
oceanica (L.) Delile; 2) the comparison of the mycoflora associated to the different
districts of P. oceanica meadow; 3) the analysis of the effect of abiotic and biotic
parameters on the growth and sporulation of fungi; 4) the development of a new
quali-quantitative microtitre plate method for the rapid screening of oxidoreductase
(laccases, peroxidases) and tannase activity.
Materials and methods - The study area was a P. oceanica meadow localized
in the in the Riva Trigoso Bay (Liguria, Italy). A total of 9 plants and matte were
collected in March 2008 between –5 and –21 m depth. The plants were divided into
3 parts (leaves, rhizomes, roots). Five g (fresh weight) of each composite sample
were homogenized and the opportune dilutions were plated on 3 oligotrophic
cultural media. Plates were incubated at 20 °C, monitored daily for 30 days and
the number of colony forming units (CFU) per g of dry weight of plant (CFU/g dw)
was estimated for each identified fungal species. The statistical analysis conducted
L. Panno. S. Voyron, A. Anastasi, R. Mussat Sartor, G.C. Varese
86
were: the nonparametric Mann-Whitney test, several diversity indexes (Margalef,
Berger- Parker, Shannon and Simpson indexes) and multivariate analysis (BrayCurtis indexes and Principal Component Analysis - PCA). The enzymatic screening
was performed in microtitre plates, containing per well one mycelium disk (3
mm Ø) and 800 μl of Malt Extract (ME) added with 0.2 g/l Remazol Brillant
blue (RBBR, laccases), 0,2 g/l Amaranth Red (peroxidases) or 10 g/l tannic acid
(tannases) at 0, 15 and 30 g/l NaCl. The culture media were sampled after 3 and
7 days and laccase, peroxidase and tannase activity were evaluated following the
absorbance reduction of RBBR (l 596), Amaranth Red (l 522) and tannic acid (l
274), respectively.
Results - The total fungal load ranged from 1.4·102 to 1.6·103 CFU/g dw depending
on the different districts and cultural media used. Rhizomes was the district with the
highest fungal load, followed by matte, leaves and roots (Tab. 1).
Tab. 1 - Mean fungal load and standard error (CFUg-1dw ± SE) of taxa isolated in leaves, roots,
rhizomes and matte of P. oceanica on three cultural media incubated at 20 °C.
Carica fungina media ed errore standard (CFUg-1dw ± SE) dei taxa isolati dalle foglie, radici,
rizomi e dalle matte di P. oceanica sui tre terreni culturali incubati a 20 °C.
Roots
Roots
Rhizomes
Matte
Mean CFU ±
standard deviation
Mean CFU ±
standard deviation
Mean CFU ±
standard deviation
Mean CFU ±
standard deviation
CMA
1.8·102± 8.1·101 aA
2.0·102± 7.2·101 abA
1.6·103± 2.6·102 aB
1.4·103± 4.0·102 aBC
AP
2.9·10 ± 1.2·10 abA
4.8·10 ± 1.1·10 aAB
8.8·10 ± 2.2·10 abBC
1.1·103± 2.1·102 aC
GPYA
5.2·102± 2.3·102 bAB
1.4·102± 0.0 bC
4.4·102± 9.3·101 bB
1.4·103± 3.2·102 aA
2
2
2
2
2
2
Different uppercases indicate significant differences (p≤0,05, Mann-Whitney test) among the load
of the same medium obtained in different districts of P. oceanica; different lowercases indicate
significant difference among the load of the same district of P. oceanica obtained in different
media; CMA=Corn Meal Agar; AP=Agar Posidonia; GPYA=Glucose Peptone Yeast Agar.
Differenti lettere maiuscole indicano differenze di carica significative (p≤0.05, Mann-Whitney) dello stesso
terreno ottenute in differenti distretti di P. oceanica; differenti lettere minuscole indicano differenze di carica
significative dello stesso distretto ottenute in differenti terreni; CMA=Agar con farina di mais; AP=Agar
con tessuti di Posidonia oceanica omogeneizzati; GPYA=Agar con glucosio, peptone ed estratto di lievito.
The mycofloras associated to the three districts and matte have a very low
similarity. A total of 88 taxa were identified from the four districts of P.
oceanica meadow: 43 from matte, 34 from rhizomes, 20 from leaves and 14 from
roots. Considering both fungal load and number of species, roots displayed the
highest species richness, whereas the highest dominance indexes were observed in
rhizomes. The 88 fungal entities comprised 70 Ascomycetes, 4 Basidiomycetes and
14 unidentified fungi. A total of 42 genera were identified (Tab. 2). Penicillium,
Cladosporium and Acremonium were the most abundant genera. Twenty-nine
morphotypes resulted Sterile Mycelia (SM) in pure culture despite numerous
attempts to induce fruiting by various methods. As regards to the oxidoreductase
screening, many marine fungi showed a good ability to degrade RBBR and
Amaranth Red dyes and some strains degraded them exclusively at 15 and/or 30
g/l of salt. Moreover, an important production of tannases was observed in fungi
belonging to the genera Penicillium, Cladosporium and Clonostachys, particularly at
the highest salt concentration.
Biodiversity of marine fungi associated with the seagrass P. oceanica: an ecological and biotechnological perspective 87
Tab. 2 - Genera of fungi isolated from leaves, rhizome, roots and matte of P. oceanica meadow.
Generi fungini isolati dalle foglie, rizomi, radici e matte della prateria di P. oceanica.
Genera
Acremonium
Alternaria
Apiospora
Arthrinium
Aspergillus
Beauveria
Candida
Cephalotrichum
Cladosporium
Clonostachys
Cremasteria
Crocicreas
Cyclothyrium
Cylindrocarpon
L Rh Ro M
x x x x
x x x
x
x
x
x x
x x
x
x
x x x x
x
x
x
x
x
Genera
Dactylaria
Diaporthe
Didymella
Exophiala
Geotrichum
Gibellulopsis
Gliomastix
Leptosphaeria
Lophiostoma
Mycosphaerella
Myrmecridium
Myrothecium
Paraconiothyrium
Penicillium
L Rh Ro
x
x
M
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Genera
Phaeocryptopus
Phialophora
Pleurophoma
Pycnidiophora
Pyrenochaete
Radulidium
Schizophyllum
Sordariomycetes
Sporobolomyces
Stachylidium
Torula
Trichoderma
Trichosporon
Wallemia
L Rh Ro
x
x x
M
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
L=Leaves; Rh=Rhizomes; Ro=Roots; M=Matte.
L=Foglie; Rh=Rizomi; Ro=Radici; M=Matte.
Conclusions - The mycoflora of P. oceanica is very rich, both in term of
fungal load and number of species, and is higher than those found on many other
marine substrates (Holler et al., 2000; Hernàndez et al., 2007). The use of different
oligotrophic culture media, prepared using seawater, allowed the isolation of slow
growing, rare and less competitive species more closely related to their natural
host. The composition and structure of the mycoflora change significantly among
the different analyzed districts and matte. This may be due to multiple factors:
specific environmental parameters (nutrients, light, exposure to water-movement,
etc.); presence of different antagonistic microorganisms, particularly epiphytes or
herbivores; presence of toxic or repellent molecules localized in a specific district
of P. oceanica, i.e. the presence of tannic acid in the leaves. The highest values of
fungal load and species were found in rhizomes and matte, probably in consequence
of the high morphological heterogeneity of these districts that allows the formation
of micro-habitats and niches rich in organic substances. Our data confirm that fungi
belonging to the phylum Ascomycota represent the predominant mycoflora in marine
environments. Many of the species found in this study (Acremonium spp., Alternaria
spp., Aspergillus spp., Cladosporium spp., Penicillium spp. and Wallemia sp.) are well
adapted to marine environments. They perform important ecological functions,
mainly in the decomposition of organic matter, in the recycling of elements, in the
synthesis of humic compounds and in different interactions with other organisms
(Das et al., 2006). Surprisingly, the few fungi that have been already reported by
other authors associated with P. oceanica were not found in our survey (Cuomo and
Vanzanella, 1985). This result could be explained by the different sampling seasons:
our study was conducted in spring on young plants, while the other studies were
conducted in autumn and winter on senescent plants. Hence, the mycoflora associated
to P. oceanica can change significantly in the different seasons, in relation to the life
cycle of the plant. In our study we report several species isolated for the first time
from marine environments. Some of these species may have a parasitic nutritional
strategy, similar to their behaviour in the terrestrial environment. Others are known
88
L. Panno. S. Voyron, A. Anastasi, R. Mussat Sartor, G.C. Varese
for the excellent saprotrophic ability, that allows them to play an important action of
degradation of submerged wood. Thirty-three percent of isolated fungal strains grow
only as sterile mycelia (SM) in axenic conditions. This results support the hypothesis
that marine-derived fungi are able to disperse also by hyphal fragments and not only
through conidia or spores. For 10 of these SM, the molecular analysis didn’t allow
a specific identification but indicated the proximity to not cultivable endophytes
and phytopathogenic species. The growing of these fungi is an important goal of
this work that may have important implications for future taxonomic, phylogenetic,
ecological and applicative studies. As regards to the screening at different salt
concentrations, through the use of the RBBR dye we showed that 39 anamorphic
Ascomycetes have a ligninolytic activity amenable to laccase production, while 24
anamorphic Ascomycetes and 2 Basidiomycetes have a ligninolytic activity amenable
to peroxidase production. This result proves that marine anamorphic fungi are
potential lignin degraders involved in wood decay and hence, these fungi play an
important role in marine environment. Fifty-five fungal strains are able to produce
tannases. Tannins are polyphenolic compounds extremely abundant in P. oceanica,
mainly in rhizomes and leaves. In these districts “tannin cells”, very rich in tannins,
are present and are involved in a defensive function. Actually, these compounds make
the plant uneatable to most of the fauna present in the sea and have a disturbance
action against saprotrophic and parasite microorganisms. Fungi able to produce
tannases would reduce the content of tannins present in P. oceanica matrix allowing
the use of this substrate by other organisms. So, these fungi play a major role in
this ecosystem. Moreover, marine laccases, peroxidases and tannases could be of
great biotechnological interest in different sectors in which high concentration of
salts are required. These results contribute to a better knowledge of marine fungi
and show the presence of many saprotrophic and pathogenic fungal species that live
in association to P. oceanica. Future studies will clarify the ecological role of the
mycoflora associated to this seagrass, increasing our knowledge about the complex
interactions within this phytocoenosis which are essential to its preservation. The
creation of a collection of marine fungi at the MUT provides the scientific community
of a number of fungal strains that will be further investigated for production of
secondary metabolites of pharmaceutical and biotechnological interest.
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