Biological and chemical diversity of cytotoxin-producing symbiotic marine fungi in intertidal zone of Dalian

ZHANG Yi hubeizhangyi@163,com 0 1 2 MU Jun ) 1 FENG Yan 1 LI HeNan 1 DONG XueWei 1 0 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences , Qingdao 266071, China 1 School of Environmental and Chemical Engineering, Dalian Jiaotong University , Dalian 116028, China 2 School of Life Science and Biotechnology, Dalian University of Technology , Dalian 116024, China In this study, the biological and chemical diversity of 8 symbiotic marine fungal strains, with strong cytotoxicity against brine shrimp larvae, were investigated by nucleotide sequencing, morphology and cluster analysis of HPTLC fingerprint. These strains were identified by ITS rDNA sequencing, phylogenic analysis, and morphology to be Hypocrea lixii, Chaetomium globosum, Aspergillus fumigatus, Asp. clavatus and Alternaria sp. Their differences in secondary metabolites were shown by cluster analysis of digitalized colors of HPTLC spots, a newly developed method, which produced a similar dendrogram with that of ITS cluster analysis. Furthermore, this method can fully display intraspecific differences and even the remarkable difference in Aspergillus strains which goes beyond the boundary between genera. Their biological-chemical diversity may be the basis of their potent cytotoxicity and implies their potential in producing diversified antitumor or pesticidal constituents. - Symbiotic fungi live on the surface or in the inner tissue of their hosts. Some terrestrial symbiotic fungi have been found to produce toxins or anti-feedants to protect their hosts from predators and grazers [1]. Some of these compounds can be used as antitumor or pesticidal agents. Symbiotic marine fungi have been isolated from seaweeds, sponges, corals, mangroves and sea grasses, also showing taxonomicall diversity and producing numerous active compounds [2]. The intertidal coastline of Dalian possesses diversified natural and artificial habitats and also high biodiversity of marine plants, invertebrates, and microorganisms. In our screening for useful cytotoxins from local symbiotic marine fungi using brine shrimp lethality test, a widely used bifunctional preliminary screening model to discover antitumor drugs and pesticides from the sea [35], eight strains with potent activities were discovered. Herein, we report the study on the biological and chemical diversity of these bioactive strains by ITS rDNA sequence analysis, morphology, and metabolite fingerprinting using a new cluster method. Materials and methods Bioactive strains under investigation The eight symbiotic fungal strains, both epiphytes and endophytes, were isolated from the marine flora and fauna samples using the method previously reported [6]. The samples were collected from the intertidal zone of Fujiazhuang beach (1213613.82E, 384836.66N) in Dalian City, China, in October of 2008 and May of 2009. These strains were statically fermented for 30 d in 200 mL of PSB (potato sucrose broth) containing 2% natural sea salt at 28C. Mycelia were extracted with methanol and fermentation broth was extracted by ethyl acetate. The two extracts were combined to obtain crude organic extract, The Author(s) 2012. This article is published with open access at Springerlink.com which was finally dissolved in 3 mL of methanol after rotary evaporation [6]. A total of 51 strains were screened for their remarkable cytotoxicity against brine shrimp larvae in the following bioassay. The brine shrimp (Artemia parthenogenetica) larvae were hatched and collected using the method similar to that by Micheal et al. [7] and Lu et al. [8]. Fungal extracts and controls were respectively added into the microplates, and then dried in vacuum oven at room temperature. Afterwards, 200 L of instar II-III A. parthenogenetica nauplii suspension containing 2030 vivid larvae was added into each well. Then the microplates were incubated under fluorescent lamp in the incubator at 28C for 24 h without cover. Pure water and methanol were respectively used as blank controls; taxol, adriamycin, and trichlorphon at final concentration of 5 g/mL were used as positive controls. The results were observed and counted under a binocular dissecting microscope. The corrected average lethality rate of each sample was calculated according to Abbott Formula [9]. The bioassay consisted of 4 rounds of screenings with serially reduced dosage of extract and incubation time to select the strongest cytotoxin-producing fungal strains. For the four screenings, they were 40 L/well for 24 h, 5 L/well for 24 h, 1 L/well for 24 h, and 1 L/well for 4 h, respectively. The information about the origin, crude extract content, and bioactivity of the eight strains is listed in Table 1. Molecular taxonomy DNA extraction of the strongest active fungal strains was performed using the Plant Genomic DNA Kit DP305 (Tiangen) according to the manufacturers protocol. PCR was then performed using TaKaRa Ex Taq polymerase (TaKaRa) and the fungal universal primer pair ITS1 and ITS4, in a Takara PCR Thermal cycler Dice TP600 with the method of White et al. [10]. Then the PCR product mixture was analyzed by DNA electrophoresis on agarose gel, purified using TaKaRa DV805A Agarose Gel DNA Purification Table 1 The origin, crude extract yield and bioactivity of the 8 fungal strainsa) Kit, and sequenced by an ABI PRISMTM 3730XL DNA sequencer (TaKaRa) with primer ITS1. The sequence data had been submitted to and deposited at GenBank with the accession numbers shown in Table 2. The ITS1-5.8 S-ITS2 (internal transcribed spacer, ITS) rDNA sequences were used to search the GenBank database with the BlastN 2.2.19+ algorithm for the closest matches in the ITS1-5.8 S-ITS2 rDNA sequences of known species. Sequences were aligned with representative fungal ITS1-5.8 S-ITS2 rDNA sequences using Clustal X (version 1.81); a neighbour-joining phylogenetic tree was constructed using the MEGA 4.0 [11,12]. Morphological characterization Morphological characterization of the fungal isolates was carried out according to standard taxonomic key including colony diameter, texture, color, and the dimensions as well as the morphology of hyphae and conidia [13]. Cluster analysis of HPTLC fingerprint For the strongest active strains, 1 L of each extract was applied on high-performance thin-layer chromatographic plates (HPTLC silica gel 60 F254, Merck, Darmstadt, Germany) with a capillary. Then the HPTLC plate was developed twice by a mixture of CHCl3/MeOH (20:1, v/v). After air drying, the spots of the fungal metabolites on the plate were photographed under UV lamp at 254 and 365 nm, and then scanned after coloration by H2SO4-anisaldehyde agent at 105C for 2 min. To display the relationship and difference in secondary metabolism between these strains quantitatively, the metabolite fingerprints, i.e. the spots colors on the HPTLC plates, were digitalized in the following section and analyzed by cluster method. Since TLC plate after che (...truncated)