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
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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
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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. References BUGNI T.S., IRELAND C.M. (2004) - Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Natural Product Reports, 21: 143-163. CUOMO V., VANZANELLA F. (1985) - Fungal flora of Posidonia oceanica and its ecological significance. Transactions of the British Mycological Society, 84: 35-40. DAS S., LYLA P.S., KHAN S.A. (2006) - Marine microbial diversity and ecology: importance and future perspectives. Current Science, 90: 1325-1335. HERNÀNDEZ T.C., GONZALES B.A., VAZQUEZ O.O.E., SABAT A.M., BAYMAN P. (2007) Fungi in the sea fan Gorgonia ventalina: diversity and sampling strategies. Coral Reefs, 26: 725-730. HOLLER U., WRIGHT A.D., MATTHEE G.F., KONIG G.M., DRAEGER S., AUST H.J., SCHULZ B. (2000) - Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycological Research, 104: 1354-1365. HYDE K.D., POINTING S.B. (2000) - Marine Mycology - A Practical Approach. Fungal Diversity Research Series 1, Fungal Diversity Press, Hong Kong: 377 pp. PANNO L. (2009) - Fungal biodiversity associated with the sea grass P. oceanica. Laurea Magistrale in Biologia vegetale, Facoltà di Scienze MFN, Università degli studi di Torino, Italia: 129 pp. RAGHUKUMAR C. (2008) - Marine fungal biotechnology: an ecological perspective. Fungal Diversity, 31: 19-35.
