Diversity and Antimicrobial Activity of Culturable Fungi Isolated from Six Species of the South China Sea Gorgonians

Microb Ecol (2012) 64:617–627 DOI 10.1007/s00248-012-0050-x FUNGAL MICROBIOLOGY Diversity and Antimicrobial Activity of Culturable Fungi Isolated from Six Species of the South China Sea Gorgonians Xiao-Yong Zhang & Jie Bao & Guang-Hua Wang & Fei He & Xin-Ya Xu & Shu-Hua Qi Received: 16 December 2011 /Accepted: 27 March 2012 /Published online: 15 April 2012 # Springer Science+Business Media, LLC 2012 Abstract Fungi in gorgonians are now known to cause proportion (38 %) of fungal isolates displayed distinct anti- gorgonian diseases, but little attention has been paid to the bacterial and antifungal activity, suggesting that the nature of fungal communities associated with gorgonians. gorgonian-associated fungi may aid their hosts in protection The diversity of culturable fungi associated with six species against pathogens. This is the first report comparing the of healthy South China Sea gorgonians were investigated diversity of fungal communities among the South China using a culture-dependent method followed by analysis of Sea gorgonians. It contributes to our knowledge of fungal internal transcribed spacer sequences. A total of 121 gorgonian-associated fungi and further increases the pool fungal isolates were recovered and identified using the Basic of fungi available for natural bioactive product screening. Local Alignment Search Tool search program. These belonged to 41 fungal species from 20 genera. Of these, 30 species and 12 genera are new reports for gorgonians, Introduction and the genera Aspergillus and Penicillium were the most diverse and common in the six gorgonian species. Compar- Coral reefs are widely recognized as one of the most diverse ison of the fungal communities in the six gorgonian species, ecosystems, containing large and diverse microbial commu- together with results from previous relevant studies, indicat- nities. Schimel et al. [1] reported that complex microbial ed that different gorgonian species and the same gorgonian communities were extremely important in coral ecosystems, species living in different geographic locations had different with significant influence on these ecosystems. Corals pro- fungal communities. The gorgonian Dichotella gemmacea vide a structurally and environmentally complex array of harbored the most fungal species and isolates, while Echi- habitats supporting a broad microbial diversity that influen- nogorgia aurantiaca had the least fungal diversity. Among ces both host physiology and ultimately ecosystem process- the six media used for fungal isolation, potato glucose agar es [2]. Many studies have indicated that microbial yielded the highest isolates (27 isolates), while glucose communities occupy a range of niches on corals, from peptone starch agar had the best recoverability of fungal within the surface mucus layer [3, 4] to on and within the species (15 species). The antimicrobial activity of the 121 coral tissue layers [5, 6]. Furthermore, a variety of studies fungal isolates was tested against three marine bacteria and have suggested that coral-associated microorganisms may two marine gorgonian pathogenic fungi. A relatively high be saprophytic or pathogenic or may provide other important functions for corals [7, 8]. Microorganisms found in : : : : : corals might, for instance, protect the host against pathogens X.-Y. Zhang J. Bao G.-H. Wang F. He X.-Y. Xu [9, 10] and/or supply nutrients [11]. S.-H. Qi (*) Key Laboratory of Marine Bio-resources Sustainable Utilization/ As part of our ongoing investigation of the diversity and RNAM Center for Marine Microbiology/Guangdong Key Laboratory antimicrobial activities of culturable microorganisms asso- of Marine Material Medical, South China Sea Institute of Oceanology, ciated with South China Sea gorgonians, fungi attracted our Chinese Academy of Sciences, attention because the fungal strains found exhibited relative- 164 West Xingang Road, Guangzhou 510301, China ly high diversity and antimicrobial activity. The fungal e-mail: [email protected] diversity of corals has been poorly understood [12]. Initially 618 X.-Y. Zhang et al. there were few reports of fungi associated with scleractinian umbraculum were collected from the South China Sea, near corals. Kendricks et al. [13] and Kohlmeyer et al. [14–17] Sanya City (18°11′ N, 109°25′ E), in August 2010 (Fig. 1). isolated several endolithic fungi from reef-building corals Three to five samples were obtained for each gorgonian and hydrozoans in Caribbean and Australian waters. Betis et species. Gorgonian species were identified by Dr. Hui al. [18] reported unknown fungal hyphae associated with Huang and Xiu-bao Li (South China Sea Institute of Ocean- two reef-building corals from the Pacific Ocean. Priess et al. ology, Chinese Academy of Sciences) [27]. Samples were [19] recovered an Aspergillus-like endolithic fungus from transferred directly to sterile plastic bags without seawater the reef-building corals in Mayotte and Moorea Islands. and then sent to the laboratory as soon as possible, main- Later reports have increasingly emphasized fungi associ- taining ice-cold conditions to enable fungal isolation. ated with gorgonians. Some fungi in gorgonians are now known to cause gorgonian diseases. Since Smith et al. [20] Fungal Isolation and Comparison initially reported that Aspergillus sydowii was the causal agent of the aspergillosis of gorgonians (Gorgonia ventalina Gorgonian samples (15–20 g) were rinsed three times in and Gorgonia flabellum) in the Caribbean, a few studies sterile seawater to remove transient and loosely attached have found that widespread diseases and mortality in Carib- microorganisms. The washed samples were then cut into bean and West Indian gorgonians were also caused by the 1cm3 pieces and thoroughly homogenized using a sterile fungus A. sydowii [21–23]. In addition, a recent disease mortar with the addition of 2 volumes of sterile seawater. A outbreak of Aspergillus versicolor, affecting 250 fallen gor- 10-fold dilution was made and 0.1 ml of the resulting gonians in Southeast Asia indicated that other fungal species solution was plated on agar. The inoculated plates were were susceptible to gorgonian diseases [24]. However, some cultured at 28°C for 1–3 weeks until the morphology of of the fungi that have been identified in diseased colonies the fungi could be distinguished. Fungal isolates were cho- and implicated in gorgonian diseases have also been found sen and transferred onto new corresponding agar plates on in healthy gorgonians [25]. Furthermore, some gorgonian the basis of their morphological differences based on visible diseases are believed to be caused by fungal communities examination of growth characteristics, mycelia, and diffus- and not by single pathogenic fungi [26]. These findings ible pigments. The resulting plates were incubated at 28°C highlight our ignorance of the basic fungal ecology of for pure culture. gorgonians. Investigation of the nature of fungal communi- Six isolation media were used to isolate gorgonian- ties in gorgonians is therefore vital for providing a general associated fungi; the compositions of the six media are understanding of these microbes, their diversity, distribu- shown in Table 1. To prohibit the growth of bacteria, 0.5 g tion, and ecology, as well as giving opportunities for further L−1 benzylpenicillin and 0.03 gL−1 rose bengal were added exploitation of novel fungi and bioactive natural products. to the basic media. Our aim was to investigate and compare the diversity and antimicrobial activities of culturable fungi associated with six DNA Preparation, ITS Gene Sequencing, and Identification species of South China Sea gorgonians: Dichotella gemma- of Fungi cea, Echinogorgia aurantiaca, Melitodes squamata, Muri- cella flexuosa, Subergorgia suberosa,andVerrucella For DNA extraction, the selected fungal strains were inoc- umbraculum. These six gorgonians are unevenly distributed ulated into 7 ml centrifuge tubes containing 1 ml potato in the South China Sea [27]. Six different isolation media were glucose medium and cultured at 28°C with shaking at utilized for fungal isolation. The fungi phylogenetic diversity 150 rpm for 7 days. Total genomic DNA was extracted from was analyzed based on internal transcribed spacer (ITS) all selected strains, as described by Lai et al. [28]. From the sequences of the culturable fungi associated with the six genomic DNA, nearly full-length ITS sequences were am- gorgonians. In addition, the antimicrobial activities of the plified by polymerase chain reaction using primers ITS1 (5′- fungal isolates were primarily assayed using a double-layer TCCGTAGGT GAACCTGCGG-3′)andITS4(5′- technique with five indicator microorganisms including three TCCTCCGCTTATTGAT ATGC-3′). All primers were syn- marine bacteria and two gorgonian pathogenic fungi. thesized by SBS Genetech (China). The polymerase chain reaction mixtures consisted of 12.5 μl Taq premix (TakaRa, China), 0.25 μl (10 μM) of each primer (TakaRa, China), Materials and Methods 0.75 μl DMSO, 10.25 μl water, and 1 μl template DNA. After denaturation at 94°C for 6 min, amplification was Sample Sites and Sample Collection performed with 30 cycles of 45 s at 94°C, 45 s at 53°C, 2 min at 72°C and a final extension at 72°C for 10 min. Healthy gorgonian samples including D. gemmacea, E. DNA sequencing of the selected fungal strains was car- Aurantiaca, M. squamata, M. flexuosa, S. suberosa, and V. ried out by Invitrogen (China). Sequences were corrected Diversity and Antimicrobial Activity of Culturable Fungi 619 Figure 1 Map of the South China Sea and location of the sampling site using Sequencher, and the most similar sequences in Gen- [31, 32] and two marine gorgonian pathogenic fungi (A. versi- Bank were found using Basic Local Alignment Search Tool color SCSGAF0096 and A. sydowii SCSGAF0035) [20, 24]. (BLAST) searches. When the top three matching BLAST The indicator bacteria were cultured in nutrient agar (5 g pep- hits were from the same species and were ≥95 % similar to tone, 3 g yeast extract, 20 g agar, and 1 l of seawater) at 37°C for the query sequence, this species name was assigned to the 18 h, and the indicator fungi were cultured on PDA at 30°C for selected isolate [29].

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