Fungal Diversity Dominant fungi from Australian coral reefs Sarah Morrison-Gardiner Australian Institute of Marine Science; PMB 3, TownsvilleMail Centre, Townsville, Queensland, Australia, 4810; e-mail: [email protected] Morrison-Gardiner, S. (2002). Dominant fungi from Australian coral reefs. Fungal Diversity 9: 105-121. This report describes 617 fungi isolated from coral reefs in tropical Australian marine environments. Host substrates include 62 sediments, algae (8 Rhodophyta, 9 Chlorophyta, 3 Phaeophyta) and vertebrates/invertebrates (16 Bryozoa, 21 Chordata, 16Cnidaria, 70 Porifera). Results indicate that some reef dwellers may provide a natural reservoir for fungal genera normally associated with other organisms. Taxa such as Aspergillus and Penicillium, commonly thought to originate from terrestrial run-off, were frequently isolated from offshore hosts. One hundred and twenty one isolates (19.6% of the total) sporulated, but could not be identified using the available taxonomic keys, while 99 isolates (16%) did not sporulate, and thus were classified as sterile mycelium. Some isolates, such as Cochliobolus spp., have not previously been described from marine sources, and could represent novel taxa. Slow growing marine ascomycetes were not isolated, probably because they were outgrown by faster growing taxa. Key words: coral reef, filamentous, fungi, marine. Introduction Ecosystem fertility is dependent upon microbial activity (Moore and de Ruiter, 1991), and therefore filamentous fungi are likely to play a role in the heterotrophic conversion of reef biomass to nutrients. Only 800-1000 taxa of marine fungi have been identified (Hawksworth et al., 1995; lones, 1995), compared with the thousands of species of terrestrial fungi described. The majority of these taxa have been identified from mangroves, beach sand, driftwood, shell matter or as pathogens of marine organisms of economic value (Hyde, 1996). Marine fungi are generally placed into one of the following groups: manglicolous - those found in the mangrove environment; arenicolous - fungi associated with sand grains; lignicolous - wood inhabiting fungi; caulicolous - growing on or in herbaceous stems; foliicolous - fungi growing on leaves and algicolous - inhabiting marine algae (Hughes, 1975; Hyde, 1989). Research into the fungi associated with coral reef ecosystems has predominantly been concerned with pathogenic disease symptoms in various 105 host organisms, usually in the form of tissue necrosis or biomineralisation (Rand, 2000). The majority of marine mycological research has focused on organisms of economic value, particularly those of farmed crustaceans (Alderman, 1982, 1984; Hose et al., 1984; Chen et al., 1992; Huang et al., 1996a) fish (Alderman, 1982; Rand, 2000), and to a lesser extent algae (Muehlstein, 1992). In total only 22 species of ascomycetes and mitosporic fungi have been identified from carbonaceous sources in the tropics, in association with crustose sponges, coralline algae, coral slabs, forams, molluscs and barnacles (Kohlmeyer and Volkmann-Kohlmeyer, 1992). Reports of fungal taxa in the ecological context of marine environments are scarce. Littler and Littler (1998) describe an unidentified fungal pathogen of reef-forming crustose coralline algae in American Samoa, found predominantly in shallow «20 m) waters. Fungi have also been observed inhabiting the skeleton of Porites lobata, causing local decomposition of dense skeletal material (Le Campion-Alsumard et al., 1995), with gorgonians in the Carribean (Smith et al., 1997; Geiser et al., 1998), and associated with sea cucumbers (Afiyatullov et al., 2000; Pivkin, 2000). Surveys of the biodiversity of marine fungi in Australian marine environments, and studies of fungal interactions with reef-specific organisms are even more limited, with a majority of research taking place prior to 1970 (Cribb and Herbert, 1954; Cribb and Cribb, 1955, 1956, 1960, 1969; Kohlmeyer and Volkmann-Kohlmeyer, 1991, 1992; Hyde, 1996). This paper explores the diversity and ecology of natural populations of filamentous fungi isolated from the Australian marine environment, with an emphasis on host organisms sourced from the general reef community. Materials and Methods Sample collection A wide range of sediments and marine organisms were targeted for collection in an attempt to maximise the diversity of fungal isolates obtained from marine environments around Australia. Marine organisms were collected via reef walking, snorkelling, SCUBA, and, in a few instances, trawling. Each sample was placed wet into a sterile plastic bag and maintained at the seawater temperature for transport to the laboratory. A 1 cm3 section was excised from each invertebrate or algal sample, and placed into a sterile 0.06% sodium hypochlorite solution, gently shaken and left for one minute, followed successively by two 1 minute rinses in 10 mL of sterile ocean nature artificial seawater (sASW) to ensure surface contaminants were removed prior to plating. The shaking ensures that sponge 106 Fungal Diversity vacuoles and pores were also cleaned. The sample block was removed and cut into small sections «1 mm3) with a sterile scalpel. Cut sections (5 per plate) were placed onto the following selective media: filter paper (sterile filter paper on a Petri-dish, moistened with sASW)); modified malt extract agar (MEA; 1OgDifco malt extract, 1g yeast extract, 1g Difco bacteriological peptone, 15g of Bacto agar, 1L sASW); yeast peptone agar (YPA; 19 Difco yeast extract, 19 Difco bacteriological peptone, sASW); potato carrot agar (PCA; made in 1L sASW, 15g Bacto agar). Streptomycin sulphate (10 mg/L) was incorporated into each of the agar media to minimise bacterial contamination. Sediments were collected by divers (snorkel or using SCUBA directly from the sea floor), or with the use of Smith-MacIntyre (>50 m depth) or hand held scissor grabs «50 m depth). Each sediment sample (3 cm3) was collected in a pre-sterilised sawn-off syringe, and were plated onto the isolation media within two hours of collection: Sediment (2 cm3) was collected in situ or from the centre of the resultant grab sample, to minimise extraneous contamination. The sample was added to 8 mL sASW and vortexed at high speed for 10 minutes. Two hundred fll of the resultant suspension was plated onto MEA, peA and YPA plates. Small amounts of sediment were also dropped onto individual plates of MEA, YPA, PCA and filter paper with sterile forceps. Isolation All inoculations (sediments, algae and animals) were completed within two hours of collection. Inoculated plates were sealed with parafilm and incubated at 27 C. Samples were checked daily for the emergence of fungal hyphae, which were transferred to a fresh agar plate and purified. Only hyphae emerging from the source material was sub-cultured onto media for purification. Hyphae originating from samples on the filter paper media were subcultured onto MEA. Six-hundred and seventeen fungal isolates collected between August 1995 and August 1997 were chosen for taxonomic identification. Microscopic slides were prepared from plate cultures using both the sticky tape method and teasing hyphae in lactophenol cotton blue on a glass slide (Kohlmeyer, 1979). Glass slides were covered with coverslips and left 24-48 hours before sealing with clear nail varnish. To document each isolate digital photographic images are curated within the marine bioproducts database (Australian Institute of Marine Science). Fungi were identified to genera whenever possible, using a range of taxonomic keys (Kohlmeyer, 1979; Barron, 1983; Domsch et al., 1993; Alexopolous, 1996). NID (not identified) was used to indicate that the fungi could not be taxonomically assigned using the keys aVililable, while SM (sterile mycelium) was used to indicate no spores (or spore bearing structures) as viewed under the microscope. 107 • Nearshore o Offshore Fig. 1. Map of Australian collection sites for source material collected during 80 separate collection trips between 1995 and 1998. 1403 fungi were isolated from source materials collected at near-shore and offshore locations. All of the fungi collected were cryopreserved in 30% glycerol/tryptic soy broth at -80 C and -130 C, and are curated in the marine microbial collection at the Australian Institute of Marine Science. Results One-hundred and sixty sediment samples and representatives of the following marine phylum were collected: 3 Annelida; 20 Bryozoa; 53 Chordata; 61 Cnidaria; 4 Crustacea; 8 Echinodermata; 7 Mollusca; 166 Porifera; 20 Chlorophyta; 18 Rhodophyta; 11 Phaeophyta. Source substrates were collected between June 1995 and September 1998, from a number of locations around Australia (Fig. 1), and resulted in the isolation of 1403 108 Fungal Diversity 8500 00 1760000 c;b2::/l) c~4•I:.eb:-.:.<:./) 439000 0- 20 10 o D Sediments Algae source Invertebrates Mitosporic Sterile mycelium ~ Not identified DIIII Zygomycotina Oomycotina IIIIIlIlI Ascomycotina Fig. 2. The distribution of fungal classes within sediments, algae and marine invertebrates/ chordates. filamentous fungi. Collection sites were split into near-shore or offshore locations (Fig. I), based on distance to shoreline, biodiversity and tidal influences, as inferred by the Great Barrier Reef Marine Park Authority. Of the 1403 fungi collected, 617 were studied morphologically for taxonomic identification (Table 1), resulting in the identification of 54 distinct taxa, and sterile mycelia. The majority, 41 taxa, were mitosporic fungi. Nine taxa were ascomycetes, one genus (Pythium sp.) an oomycel:e and three taxa, zygomycetes. Many (19.6%) of the isolates could not be identified, while 16% of the isolates were sterile mycelium. Twenty taxa have previously been recognised as true marine isolates (Kohlmeyer, 1979), and contributed to 17.5% (108 individuals) of the identified collection (marked with "*,, in Table 1). Algae supported a high proportion of mitosporic taxa when compared to other fungal groups. Combined algae (Chlorophyta, Rhodophyta and Phaeophyta) hosted 58% mitosporic fungi, 4% ascomycetes, 2% zygomycetes, 30% NID and 6% SM (Fig. 2). The ascomycetes isolated from algae were identified as Cochliobolus spp., a teleomorph of the mitosporic Curvularia 109 o Table 1.Taxonomic characterisation of 617 fungi collected from Australian nearshore and offshore marine sites. Sediment Algae Invertebrates Total Sediment Chlorophyta Rhodophyta Phaeophyta Bryozoa Chordata Cnidaria Porifera N** 0 NON 0 N 0 NON 0 N 0 N 0 Mitosporic fungi Acremonium spp. 3 121231657058 2 13 3 6 Alternaria* spp. 7 4 1 61 51 1 1 11 1 25 Aspergillus spp. 6 16 2 1 4 1 3 22 90 Beauveria spp. 2 1 3 Blodgettia* sp. 2 2 Camarosporium* sp. 1 Chrysosporium sp. 2 Cirrenalia* sp. 1 2 Cladosporium *spp. 4 2 I 1 1 2 37 Curvularia spp. 1 1 2 2 2 2 13 Cylindrocarpon* sp. 1 Dactylella sp. 1 Dactlyosporium* sp. 2 Diheterospora* sp. 1 Dreschlera* spp. 4 Echinobotryum sp. 1 Epicoccum *sp. 1 Exserohilum *spp. 1 3 Fusarium spp. 2 5 1 1 3 6 20 Gilmaniella sp. 1 Gliomastix spp. I 2 3 Gonatobotryum sp. 1 Humicola* spp. 4 1 2 3 14 Monilia sp. 2 Nigrospora sp. 2 4 * indicate taxa previously recognised as marine isolates; **N Nearshore, 0 Offshore. =0 =0 Fungal Diversity Table 1.(continued). - - - 00000000 I 12NN11NNNNN11117481111212658s5p0. 2CA2C1PBC311771ohhlrngyroliioadorfrdeaezorraopiata2a13a11h165yta1221111111119 2RPhhaoS2e2d1oeopdphih11myy11tteaant 1I1n11verte2brates 1T1otal 1 1 1111 11 221 11121 111 SPPPZCHTTCGPPZPSVACSTTWPcathoehhpyseoaeaararosaoeenlagsyrioiorcipcrlerecalrctutuotsirobmhroeouuiarihichladdlgrocscolcmoinlmloiyaoianoopaeuoriolospoobmll*tcmauonmyospintblihhbpogihpiunrp*cryhiaoeiuooyatnaoOiiomeoncharsscpcuumrlpa*tlretyopouieiaieismlmidsiarusdsuam.rrslisnpdul*smaeimsaaopssy-imlpaslpp*lscd.sssispapss*p..ekpepppsspp.*.pssen...spppp.p.pdss.....pp.rso.*p.ssppn... sppS.ediment N** Aniptodera* sp. - - N Table222112NNN4143311N0NN4344155200N000221111014918161321I6693051460.5029988I(351con24t138C31A1P0i2C91I1I1C6139113n3252600oh01l0756nuh62grlieoo4iadfdrreeado)rr.apaitahaytI222a422I016I 9Rho91d8op2hyta PBInhrvayeeoorztpoehabIyrtaates T1otal 42 617 ZRNMSSNTGhsyouuatiogturebzcmatomro-oainimtppdllrobdeluetyeeonassdrctmOfltpooifpfsstytooia.hpicemfnlopedasr.ylieocauortcinesa Sediment N** APbytshidiuiam s*pspp.. Sediment Fungal Diversity spp., which was isolated from all three near shore algae. A similar trend is seen in the invertebrate/ chordate group, with taxa identified as 59% mitosporic, 3% ascomycetes, 4% zygomycetes, 18% NID and 16% SM. Sediment samples showed the greatest fungal diversity supporting 35 of the 56 identified taxonomic groups, with the following composition: 56% mitosporic, 4% ascomycetes, 1% oomycetes, 2% zygomycetes, 19% not identified and 18% sterile mycelia (Fig. 2). The taxa most commonly observed (appearing from 6 or more sources) were identified as Alternaria spp., Aspergillus spp., Cladosporium spp., Cochliobolus spp., Curvularia spp., Fusarium spp., Humicola spp. and Penicillium spp., which in combination represent 46% of all taxa identified. The data collected was difficult to analyse statistically due to the inherent nature of variables in each data set, but some general trends within these taxa are apparent. One such trend is finding Cladosporium spp. in similar proportions to Aspergillus spp. and Penicillium spp. from many of the Porifera (sponge) samples. Twenty Cladosporium spp. were identified from 70 poriferan sources (28% of all poriferans collected). Similarly, Aspergillus spp. were collected in higher proportions from algae and chordates, with relatively low proportions occurring in sediments. Indeed only 44% (16/36) of offshore sediments yielded Aspergillus spp., compared to 77% (10/13) associated with off shore chordates. This trend is also seen in the nearshore collections, with 23% (6/26) originating from sediments and 75% (6/8) from chordates. Penicillium spp. were isolated consistently in higher proportions from offshore samples, excepting sediments and bryozoans. Taxa identified as Fusarium spp. appeared more commonly in near shore source materials, with 14 of the 20 identified originating from near-shore sites. Twenty-one percent of near shore bryozoans and poriferans yielded Fusarium spp., with the offshore representatives originating only from sediments (14% of off shore sediments) and sponges (2% of off shore Porifera). Acremonium spp. are common to soil, therefore it was not surprising to isolate three specimens from near shore sediment samples. Their presence associated with Porifera from offshore sites was surprising (7% of poriferans sampled), since none were recorded from the 29 near shore Porifera. Database examination revealed that all three were found in Axinellid sponges; two were identified as Carteriospongia spp., and one from an unidentified sponge. Alternaria spp. were associated with each substrate group type: sediment, alga and invertebrates, showing no preference for a specific source. The Pestalotiopsis-like sp. in this collection were mostly isolated from marine animals, and showed no preference for collection site. Cnidarians were the only source that consistently harboured this fungus in both near shore (17%) and 113 offshore (10%) locations. Forty-six of the taxa identified were found in low numbers and isolated from less than six source materials (Table 1). Many of these exhibited slow growth characteristics in comparison to some of the more dominant genera, and could have been present in higher numbers, but were overgrown or growth inhibited by other competing, fast growing fungal genera present. Isolates that produced spores but could not be identified (121), or those that produced sterile mycelium (99) were common to each host group, and in combination represented 35% of the 617 fungi examined. Sediments, chlorophytes, rhodophytes, chordates and poriferans all had greater than 30% of the total fungi identified falling into these groups, with Phaeophyta, Cnidaria and Bryozoa, hosting 15%, 18% and 22% (respectively) of these unidentified taxa. Differences between genera collected from near shore and offshore sites were compared using a student t-Test (two sample assuming unequal variances). Significantly more genera (t = 4.97, DF = 142, P=<O.OOl) were collected from near shore sites than from offshore sites. Pooling of the sources into six groups, near shore and offshore sediments, algae and animals, allowed further statistical analysis of the data. An F-test comparing the six groups indicates that the differences in the mean values among the groups is greater then would be expected by chance (F = 30.257, DF = 5, P = <0.001). Tukeys test (pair wise multiple comparison procedures) on these data sets, indicates a greater diversity of fungi collected from near shore sediments compared to offshore algae and offshore invertebrates (Q = 2, DF = 6, P = <0.05). Near shore invertebrates also showed higher diversity in the numbers of genera than offshore invertebrates (Q = 1.8, DF = 6, P = <0.05) or algae (Q = 2.3, DF = 6, P = <0.05). Discussion The most widely accepted definition of a marine fungus is one "...capable of producing successive generations by sexual and asexual means in natural oceanic waters and oceans diluted by freshwater or on intertidal substrates" (Hughes, 1975). Many fungi that are isolated from marine environments have been classified as geofungi (a fungus from terrestrial origins that has adapted to life in marine environments). Common genera identified in this study, and in a comparative study from Singapore (Huang et al., 1996b) show that these geofungi are frequently isolated from a wide range of marine hosts (Table 1) occurring both at near shore and offshore locations. Previous research into fungi in the marine environment has focussed on the isolation of obligate marine fungi, finding these species extensively in sea foam and seawater, marine plants and occasionally marine animals (Vrijmoed, 114