The Diversity and Antimicrobial Properties of Gorgonian Coral Fungi

Coral reefs are some of the most diverse and important ecosystem in the world. They are structurally and environmentally complex habitats that accommodate a range of microbial communities as that have a large number of niches, from surface layer mucus to the various tissue layers. This investigation considers the antifungal and antimicrobial properties of the culturable fungi in South China Sea gorgonian corals. Even in scleractinian corals, this was a poorly understood area with an inadequate scattering of articles throughout the literature. Present data suggests that there is a strong fungal association with Gorgonians that are noted for both their anti-pathological and pathogenic properties. For example, Aspergillus sydowii is known to cause diseases in both West Indian and Caribbean Gorgonians. However, evidence is mixed, as some species of fungus found on diseased corals have transversely also been found on healthy corals. Therefore this investigation is important as it provides further general understanding of the fungal diversity and ecology of gorgonians.

3-5 samples of healthy D. gemmacea, E. aurantiaca, M. squamata, M. felxuosa, S. suberosa and V. umbraculum were collected from the South China Sea and placed in sterile plastic bags.

These samples were then cut into pieces and homogenized. A ten-fold dilution was made and plated. These were then incubated until the morphology of the fungi could be distinguished. These fungi were then isolated and incubated on there own plates. Six different isolation media were used to isolate the fungi of the gorgonians. The growth of bacteria was prohibited using benzylpenicillin and rose Bengal.

DNA was extracted from selected fungi strains and PCR was used to amplify the ITS sequences using primers ITS1 and ITS4. These were then amplified. The sequencing was then corrected using Sequencher and BLASTed to assign a species name.

In order to determine the antimicrobial properties, isolated fungi were grown on PDA agar (potato glucose agar). These inoculated plates were then spread with five indicator microorganisms; M. luteus, P. piscida, V. alginolyticus, A. versicolor and A. sydowii. The antimicrobial/fungal activity was expressed as the diameter of growth inhibition zone. Three repeats were carried out for each isolate.

Results indicate that 208 fungal species were isolated from the six coral species, 127 of which were isolated for analysis. BLAST analysis showed that 121 isolates were from 20 genera. Penicillium was the most common genus whilst Aspergillus was the most diverse. The most abundant fungi were A. sydowii, P. citrinum and P. oxalicum however the other fungi were only isolated once. The coral D. gemmacea had the highest fungal isolates whilst E. aurantiaca had the least. Of the six isolating media tested PDA yielded the highest isolates but GYMA (glucose yeast malt agar) had the highest recovery (does anyone know the difference?). Some fungi could only be isolated on specific agar such as A. niger, A. tubingensis and F. proliferatum could only be isolated on SYA (starch yeast agar). There were many other fungal species which occur in very small numbers and were not included in this study. Lastly, 38% of the 121 strains analysed exhibited antimicrobial/fungal properties, with the majority isolated from D. gemmacea and E. aurantiaca. Most were reported to exhibit strong activity.

There are few reports of fungi associated with gorgonian corals, this is especially true in the South China Sea. Of the 20 genera isolated 12 were new reports in for marine corals. This report strengthen previous reports that gorgonians have a rich and diverse fungal community, however Apergillus and Penicillium were the most common which have also been found in scleractinian corals and marine invertabrates. This paper suggests that sample size, fragment size, the method of tissue processing and tissue collection all influence the results of fungal isolation. The communities found within the six gorgonians varied greatly between each other. It was suggested that this is due to the various gorgonian morphology. However, when these results were compared to the same coral species in Singapore, the fungal communities significantly varied from the South China Sea coral communities. This suggests that gorgonian coral species at different locations have various fungal communities and that the environment differences may provide some evidence for this variation. The different medias used yielded different results in terms of fungal isolation. Although past studies indicate that not more than 2 agar media would be sufficient in harbouring all fungal species, this paper suggests using multiple isolation media to fully isolate all present fungi. Finally, 38% of the fungi isolated were shown to produce an antimicrobial/fungal activity. This is supported by various other reports that have found new sources of biotechnological products to be used in the antifouling, anti-fungal and antimicrobial industries.

In conclusion this paper is significant as it provides evidence for an improved method in the isolation of gorgonian coral fungi, suggests that the diversity of these fungi is mostly untapped and unknown and that the microorganisms may be able to provide significant  natural products for use in the commercial world.

Zhang X, Bao J, Wang G, He F, Xu X & Qi S. 2012. Diversity and Antimicrobial Activity of Culturable Fungi Isolated from Six Species of the South China Sea Gorgonians, Microb. Ecol. 64: 617-627.