Fungal Diversity Observations on Aspergilli in Santa Rosa National Park, Costa Rica Jon D. Polishook', Fernando Pehiez2, Gonzalo Platas2, Francisco J. Asensio2 and Gerald F. Billsl* INatural Products Drug Discovery, Merck Research Laboratories, p.a. Box 2000, Rahway, New Jersey, 07065, D.S.A.; * e-mail: [email protected] 2Centro de Investigaci6n B<isica, Merck, Sharp and Dohme de Espafla, Josefa Valcarcel 38, 28027-Madrid, Spain Polishook, J.D., Pelaez, F., Platas, G., Asensio, F.J. and Bills, G.F. (2000). Observations on Aspergilli in Santa Rosa National Park, Costa Rica. Fungal Diversity 4: 81-100. Species of Aspergillus and their sexual states, Emericella, Eurotium, and Neosartorya, were isolated from soil or collected on natural substrata in Santa Rosa National Park in northwestern Costa Rica. Fungi were recovered by soil dilution plating, direct plating of soil on cyclosporine-containing media and direct plating on media used to recover osmotolerant and osmophilic fungi. We examined their distribution in soils collected at I-km intervals along an Il-kilometer transect on the Santa Elena Peninsula. A large and diverse group of Aspergillus species were isolated. Nine or more species were found at more than half of the sampling points along the transect. Some of the same species described from Costa Rica by Raper and Fennell were also recovered inthis study. In addition to morphological identification, representatives of all Aspergillus species were subjected to DNA sequencing using the ITSI-5.8S-ITS2 regions and the 28S 01-02 regions. Sequencing selected regions of ribosomal DNA were an effective technique in the identification of Aspergillus. In 29 out of the 35 isolates sequenced, the molecular data agreed with the macro- and micromorphology at least at the group level, and in 12of these cases, to the species level. Four species could only be recognized by reference to the molecular data. In a few cases, especially with members of the A. ochraceus group, molecular data appeared to be misleading and was indirect conflict with our morphological evaluations. Fallen fruits of Enterlobium cyclocarpum and Guazuma ulmifolia were infested with viable sclerotia of at least 4 species of Aspergillus. Emericella variecolor, infrequently cited in the literature, was abundant on fruits of E. cyclocarpum as well as in soils. Aspergillus aeneus occurred as masses of HOlle cells on litter fragments on the forest floor, in addition to being isolated from soils. Key words: Aspergillus, dry forests, ITS, ribosomal large subunit, sclerotia, soil fungi. Introduction This report presents information on species of Aspergillus and their Emericella, Eurotium and Neosartorya sexual states, isolated from soils or collected on natural substrata in Santa Rosa National Park in northwestern 81 Costa Rica. Few studies have focused on Aspergillus in Costa Rica. Thirteen species were reported in a check list of Costa Rica fungi (Covington, 1980). The majority of those reports were based on two studies of Panamanian and Costa Rican soil fungi where isolates of agricultural and forest soils from southern Costa Rica were enumerated (Farrow, 1954; Goos, 1960). The potential diversity of Aspergilli in Costa Rica and Central America was revealed by Raper and Fennell their monograph of Aspergillus (1965) when they described 12 new species from soil isolates. Subsequently, during a monographic revision of the black Aspergilli, A. helicothrix Al-Musallam, was reported from Costa Rica (Al-Musallam, 1980). During the course of a cooperative project with the Instituto Nacional de Biodiversidad (INBio), we made numerous collections of soil and litter fungi in the Guanacaste Conservation Area (GCA), including Santa Rosa National Park. The high frequencies of Aspergilli encountered in soils and the numerous observations of Aspergilli on fruits and plant debris, especially in the dry forests, intrigued us. We investigated the distribution of Aspergillus species along an ll-km transect on the Santa Elena Peninsula of Santa Rosa National Park to better understand which species might be widespread in the soils of a typical tropical dry forest. We identified these Aspergillus isolates using classical morphological identification (Raper and Fennell, 1965; Al-Musallam, 1980; Christensen, 1982; Christensen and States, 1982). In addition, we extracted, amplified and sequenced the DI-D2 regions of the large subunit (LSU) ribosomal RNA (rDNA) and the internal transcribed spacer (ITS 1-5.8S ITS2) region. These wild type sequences were analyzed and compared to sequences derived from authenticated Aspergillus strains, including type specimens. This set of sequences was generated by S.W. Peterson, U.S.D.A., Peoria, IL, U.S.A. who analyzed the sequences of the Dl-D2 regions from 215 known taxa of Aspergillus and made them available through GenBank (Peterson,2000). Aspergilli are important inhabitants of seeds and fruits of domesticated plants and their sclerotia are an adaptation to the seed habitat (Wicklow, 1985), but little is known about the natural history of wild seed- and fruit-inhabiting Aspergilli. We, therefore, report on specific and widespread infestations of fallen fruits of two major tree species of the tropical dry forest by cleistothecia and sclerotia of Aspergilli. Materials and methods Study Area The GCA encompasses three National Parks; Santa Rosa, Guanacaste, and Rincon de la Vieja, as well as Horizontes Experimental Station and the 82 Fungal Diversity Wildlife Refuge of Bahia Junquillal. The geology and the characteristics of the tropical dry forests of the region have been summarized previously (Janzen, 1983). A portion of the Santa Elena Peninsula lies within Sector Murcielago of Santa Rosa National Park and is one of the most arid regions of the GCA. Dry forests of the region are characterized by harsh dry seasons of approximately 6 months and wet seasons with 1-3 m of rainfall. A large proportion of the canopy trees are deciduous or semi-deciduous. As a result, in the dry season, sunlight often penetrates to the forest floor and the litter and upper soil horizons are desiccated to the point that decomposition may cease. Frequency and identification ofAspergilli During June, 1997, soil samples were collected in the Santa Elena Peninsula along the Murcielago road at I-km intervals starting at 0 km at the beach- forest transition at Playa Blanca and proceeding eastward until kilometer 10. At each kilometer interval, 2 soil samples were collected, 1 from each side of the one lane road for a total of 22 soil samples. Soils were collected from the first few cm below the litter layer with a soil-immersed knife and stored in Tyvek soil collection bags. The soil samples were transported at ambient temperatures to Merck Research Laboratories, Rahway, NJ and stored at 4 C until processed. The 22 soil samples were processed using 3 qualitative techniques to isolate Aspergilli: 1. Direct plating. A small amount of soil (approximately 0.1-0 .2g) is sprinkled onto MYE (malt extract, 1%; yeast extract, 0.2%) agar plates with 10 mg/L cyclosporine to retard fast-growing fungi. One plate was examined per sample. 2. Direct plating. A small amount of soil (approximately 0.1-0.2g) was sprinkled onto DG-18 (Oxoid) agar plates to select for osmotolerant and osmophilic fungi. One plate was examined per sample. 3. Soil suspensions. One ml of 1/500, 1/2500 and 1/5000 dilutions (w/w) in 0.2% carboxymethyl cellulose were spread with a bent glass rod onto DG 18 agar plates. All plates were incubated at 23 C at 80% relative humidity and a 12 h light/dark cycle. One plate was examined per each dilution per each soil sample. In total, 5 plates for each sampling point and 110 plates for the length of the transect were examined. Streptomycin sulfate and chlortetracycline were added at 50 mg/l to all isolation media. After 7 days incubation, the plates were examined by eye and with a dissecting microscope every 2-3 days for up to 28 days. Morphologically unique colonies were transferred to MYE agar slants and 83 incubated until substantial growth occurred. For identification, cultures of Aspergillus species were transferred to Blakeslee' s malt-extract agar, DG-18 and Czapek's sucrose agar (Raper and Fennell, 1965; Pitt, 1979) for identification. Nomenclature for species groups follows that designated by Raper and Fennell (1965) because their classification system is integrated into the only comprehensive identification guide for Aspergillus. For correlations with molecular analyses we referred to an updated infrageneric classification for Aspergillus (Gams et al., 1985). Representative strains described herein were preserved in 10% glycerol at -80 C at Merck Research Laboratories, Rahway, NJ, D.S.A. DNA extraction and peR 288 rDNA: Forty-two cultures deemed to be representative of the morphological groups encountered were grown on MYE plates with a 2-inch square of cellophane (Flexel Corp, Indiana) for 10-14 days. Ribosomal DNA was extracted from the fungal mycelia using Invitrogen Easy-DNA kit (Carlsbad, CA, D.S.A., kit 45-0424) following "Protocol #3-Small amounts of cells, tissues, or plant leaves". Primers used to amplify the large subunit ribosomal DNA were D1 and D2 (Peterson, 2000) supplied by GibcoBRL, Gaithersburg, MD, D.S.A. or Amersham Pharmacia Biotech, Piscataway, NJ, D.S.A. The PCR products were extracted and purified from agarose using QIAquick gel extraction kit (Qiagen, Santa Clarita, CA, D.S.A., kit 28704). For the ITS regions, the procedures followed for culture growth, DNA extraction and amplification of the ITSl-5.8S-ITS2, are described previously (Bills et al., 1999). The primers used to amplify the internal transcribed sequences were ITS1F (Gardes and Bmns, 1993) and ITS4 (White et al., 1990). DNA sequencing and phylogenetic analysis Sequencing reactions used ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit protocol performed on a PE Applied Biosystems model ABI 377 sequencer. Sequences were analyzed and contigs assembled with Factura and AutoAssembler software (PE Applied Biosystems). Resulting sequences were searched in GenBank using the FASTA protocol. Sequence alignment was performed manually. In the case of the internal transcribed spacers, the final rearrangement among groups of sequences was performed using CLDSTALW software. The phylogenetic and cladistic analysis of the aligned sequences was performed using the maximum parsimony analysis with the branch-and-bound algorithm of the PADP 3.1.1 software (Swofford, 1993). 84 Fungal Diversity Results and discussion Aspergilli recovered from transects One hundred and forty-four isolates of Aspergillus, representing 42 colony types (Table 1) were recovered from soils collected along an ll-kilometer transect in a tropical dry forest in the Santa Elena Peninsula. Based on morphological and microscopic characteristics and molecular analyses, 29 distinct species or groups were established (Table 2). Strains of A. niger and A. ochraceus groups, and A. tamarii, A. jlavipes, A. japonicus, Emericella variecolor, A. pulvinus, A. puniceus and A. auricomis were the most widespread Aspergilli along the ll-km transect (Table 2). Species of the A. niger group and A. nidulans group were the most commonly isolated groups in the ll-km transect. No correlation was obvious between sampling points along the transect and preference of Aspergillus species or groups. The highest numbers of species or groups (12) were recovered from kilometers 0, 3 and 6, while the lowest numbers (5) were recovered from kilometer 7. With classical identification, it was possible to classify most of the strains into a particular morphological group (Raper and Fennel!, 1965), and the majority of types could be identified to species or species complex. This level of species richness is comparable to other studies of Aspergilli in tropical and subtropical forests (Christensen and Tuthill, 1985). Among the 16 species noted in Costa Rica by Raper and Fennell (1965), we recovered A. aeneus, A. eburneo-cremeus, A. japonicus, A. niger, A. pulvinus and A. puniceus. Identification of Aspergilli using sequencing of rDNA regions DNA extractions or PCR amplifications failed or sequences were unreadable in a significant portion of the 42 morphological types. We obtained sequences of the D1-D2 regions of the large subunit ribosomal RNA from 29 of 42 strains. The sequences of the ITSI-5.8S-ITS2 region were obtained from 24 isolates. Therefore, for 17 strains we obtained both the sequences of the Dl D2 region as well as the ITS1-5.8S-ITS2, and for 36 of the 42 total types at least one of the two regions was sequenced (Table 1). The sequences were compared with the data available in GenBank using FASTA. The sequences giving the best matches in each case were aligned manually and the percentage of similarity were calculated based on the number of positions conserved. As a rule, we observed a reasonable agreement between the sequence data and the morphological classification (Table 1). In 29 out of the 36 isolates the molecular data agreed with the morphology at least at the group level, and in 14 of these cases, at the species level. Both rDNA regions sequenced showed similar capabilities to be used for the identification of wild isolates. Thus, a 85 Table 1. Morphological and molecular identification of 41 Aspergillus isolates. Morphological identifications are in two levels of resolution, the species group of Raper and Fennell (1965), followed by species, species complex, or distinguishing morphological features. Molecular identifications were derived from FASTA searches of GenBank using the 28s LSU region and the ITS1-5.8s-ITS4 region. ND indicates DNA extraction or peR amplifications failed. .) GNFOOFlllircaaadnhvuvuarucultaasuusAcOEEnssesusmruparseenorrgtigiceuielAmSllutslpoapseAANNENNk(ebc9arssuilRRDDt7gepanpras)RRiceaeomlAAAN,IN(AEN1krrlgtLLT9uigsga7ssuDaDBDu8mpsSppiir6rwmll)0ei4ee8Iol7loiu-rau%0rr49t7r5giggses83p6u.aiiinU48hlmllsmlsll1(Asoic2ucuuiAA9m-4ssala9sssFIo8tsseTeri5e0ppg)llul4lSafa0eeayNortblr94arrt4(eatdiruRu9ggtms9vuayss9tiiRu3(mmrlla)92lls1irLruuc9it8eiosst)igsuuIi1sGodLn8neeSo3nnmU5tBi7faiiDucnsakI-tiDoa2ncceresgsiioonns I 2] (AausaScmnuPliress)irteSceorotltiromiaaadt65,i.i,e,nasmsctiGroirncoitdluyipa"l ENNuRRrRRohtLLieu2am54d75s98c5Uh2ev9Aa5Fl4i0e7r2i7(89691) AA2ANN((A(N(A99991sssRRRBB9719pp6p)))RRR0ee1e)0rr0rLLL0ggg8U8iii4lll1224lll11uu3u95294ss1s850330237f88snlpa(U(o88AavmA(l0u1r97FF)ss3)9i0uu0)12ss007v428a986r3S0i0ecolor 7 Flavus Aspergillus tamarii ND Aspergillus tamarii with sclerotia AB008420 (99) Aspergillus flavus AB008414 (97) Aspergillus parasiticus AB008418 (97) 'Pecentage similarities in parentheses are based on similarity to GenBank accession numbers. 2Groups correspond to species groups in Raper and Fenell (1965). 86 NNOUFVFFFllliiclseaaaddathrvvvuvuusriiuisuallppcaascseeAAAAoAAYSnnesslssssssmssuoeppppppslraeeeeleeolrrrrrrlwggggggiiiiiiAyllllllSllllllesuuuuuumplpsssslsseNNeoaANNFN(NAc9rswseRDjjisRDDgpsR9alleppneaaiuR)RseRelensAEAAAvvFIENAAAnLLALLuEAA,lAnrLrLTeuLiii1susssgeusse7777msgppDBFBlcBosSppppppm8pnpliup6666ieee0ilfe040eeeel20ee3eneseal2ss7777ulo1r30rrrru70rr%00rnup5enoj04444siggggr-3ggg8ls7888clisu8ifp585656aliciii4ciii45e414lillllllh.1lvsiv3allllll1l8l181liuuuuoi8luutu(tili2jN((A((m8Aspa4a8Uanil8lsssslss9988s-ulaoesmsmuD9AIi4677iA2mpsvpglTus)))))jaa9njapFivpeveyF(lmtlSprr(8iara8eaar((ear00id9ie4i99gr3gvtm9rrvri0ji0uy7ses78saiua5aiu)4l4aihll)i))ssvsslcla2cr99rruiuiaeoton8t2itl2sosingiiillss9cLcc9ooiiuuhcGor7rpLmsusin6ueiSmu7nAlELEAAUvl5Bm7mtBiBB5ina6cee0D0u0n7orr00s0kl4lii8-8occ83D4ee4r4all1122llc(8aa409ce4rse)nhsg(i(ei(d8i9o9toeu48n9nrl))s)ao'nthsallica Fungal Diversity Table 18911I11.634(continued). (Ap(HasS=acvPUlrAee)anlSrSlsaoetiPcrttii3aceacie01u]1un2l)0s5lss,GrwohuipteL NNNAE(A(NFNANN99smsesRRRRRRR89pppne))RRRRRRReeensrrrrLLLeLLLLcigggcll4liiieeilll622225rlal3lll9uouu966600a9stss500072jil6a68as6UU6UtvUtttaeaUri222p2rmmiAA8828are9aea8808tFFs8aurr3585004iis9223i0i05244(9(9((999339969228))) AAAE(A(((uL(A999996sssms7B79993pppp56e)))))e0eee37rrrr0r14iggggc806iiiie4(llllll8lll1luuuu(4a4sss8s)4)jhpuelea(als8ltvtrieu4paurs)tssoiicttLhiuca7sul6lsi7c4a487 Identification TableNNNNNOCNO1iiirii.cicggggdeghh(eeemeuecrrrrrrrlaaoeaAccAMSEBnLSuneestcscmssaluuiopaplanressedcegenurrkreretoeiggrcdtaisiiesslR)ltaSplcoll.euluopellaesusdrejaAAAAAANk2ENNjNrlubcdblaoa1sssslassmilDRDisDvatenppppepp6sseiutesRhaeeeeacee1niANNAAAAN,rsNANINoFLrrrrhrrg(LtTigggg8sgg,nssseace7DDBDDBDDmUmpSpppiiii9iinenNw6llllll00e2eee2)Illlllytllno7lauuuu-0rRruu%0rrr39caer45gagggessss8ss89pe8Rl5.ieniiil44l86h0llllsiLgislll1laa2psou8icenuuuuieAm(-8sj0shlblmUissss9rIlaotgs5oauTiei(52pvgel1e9vrlSa)a9Nperylatnn1iv7ara4(prt(8aeceii3)RoN8t8gNmcte4rruoay-88iisRasR7l-Rals))iesl2(LcrrRuRai9t8eiotostiL9iLgslucLoiGsuoLr7asne6vSne7UuBn46a5a5Dcn3ekI0-uD9sa2c(c9e7res)sgiioonns I 221147 (AjjcavplaSaobaipPgsnurocm)iinnSehddceieticraoonnaunlgtt22i1os1rtne35a9r8newegGsheirsirot,ieupl"arge NEA5cNi(s9r0msRoRe8p4lmeR)Rae2CrsaPtreLLiceeigrcuUlnreieesiklN22a31al5rllm20ou6l51aRNsts552icmRR0a01lcveL4yRdoarUcUiloLrmoe(4Iti29rlie5o5iae8c94u4r1d8o)7m9p71l71ho83ircus UsANNAUN((3U91c2ssIRR2Rl702pp65e9)0RRR8ee5r8)orr9U0LLL3ggt10i242iia2ll284ll1u8u87((9(s8s9116594602946)0aa))UUne(9ntU22h9e99lo)u788ds942e166s6m((99i88s Identification 88 i .).) NTNNONUFFVuleiiiciseaddggrmthrvruuueesreiiisrrallpugcaacAAAAAAeDosannesstssslssssueuoppppppnssreeeeeesrrrrrreggggggiiiiiillllAASllmSlllllluuuucuusspaspplsssssefseAcAN(UANNU,leeNcs1rarrrsssfieoR2OO2ep0vRtnleggpnnppstea98muu0sRiiiieiieeRrAgvEANNNAA89lladgLLNL(INL,sn)rUrrellrLo9TieL31uesguuimsssgg777uepOOO7OOcf3r6pmrSppp42lissii666aseN6aeel)ull5eeeeI5bls777llo7urn1-sru3rrrRuu%1s444i(5rN(4sgssggg7s1csse1p92R655y.5iiii9e0nR80lh51llldsLlllllts0o)piluoeRuuu,tU(AN(((A-maAEe)blhUsw(sss8899ILossr258seTuomi7747i2pprgplo817rieS))))ane8eeeyeuvevs4n1)4nru4irrr8raeles6ei37diliggsgtcrr1troic77uyuiisspiti8e-ll3llosiistllla2lccriuruu(ltecn8ooay9Lssosgusslla9oo7is)Gorr6apLccnf7ceauliSasrn4NEenUcivS4sismDhacitaeeeate0nolrutrl1ka-uis-cs0e2allcacersehsgeiiotoennrso'thallica Fungal Diversity Table 2"126".7(continued). (AfcwcHuSoomiPtnntih)liiSlgidedatilriotioacug32p33233ipesh18nh40952lhtloosrp,rGeeisnnrsokoup" NANN(AN(N191s9sRRRRR90pp7)0RRRRRee4s)rrLLLLLcggUliiell4222arl1luou79-3681sts67905i49a8428U6au7UUUes29nt(8222u9e8999su9U3887s)N922589678R6(((9R9958991L) ANUANAEN(3u19s2nss9meR2R9ppp6io79se)RReees48errrraLULri2gggcriU3i2iiatelllo822l5tllluuluru8395(ayssss91973a42584)eN6nubcfUUis(reRhg9tun(u22ieR99nnns98r)8nLue84)ellNN47laoi420RRtay-a2(eeRR97l9LlL3ow rough Identification 89 Table 1.(continued). Identification Strain Group" Species, morphology % similarity to GenBank accessions I (ASP) 28s LSU DI-D2 region ITSI-5.8s-ITS4 region ~ Nidulans Emericella Emericella variecolor ND variecolor NRRL 1954 U29846 (lOO) Emericella astellata NRRL 2396 U29847 (99) 37 Nidulans Dense masses of Aspergillus eburneo Aspergillus versicolor Hlille cells, no cremeus NRRL 4773 L76745 (87) conidiophores U29834 (lOO) Emericella nidulans Aspergillus aeneus L76746 (87) NRRL 4769 U29826 (98) 38 Nidulans Aspergillus aeneus Aspergillus elongatus ND NRRL 5176 U29799 (97) Aspergillus raperi NRRL 2640 U29820 (97) 39 Not Non-Aspergillus Geosmithia Penicillium trachysporium determined cylindrospora strain Ll4516 (97) NRRL 2673 AF033386 Talaromyces gossypii (91) Ll4523 (96) Geosmithia argillacea NRRL 5177 AF033389 (89) 40 Versicolor Aspergillus cf Aspergillus ustus NRRL Aspergillus versicolor versicolor 1974 U29786 (98) L76745 (98) Long yellow Aspergillus puniceus Emericella nidulans conidiophores NRRL 1852 U29788 L76746 (96) (98) 41 Nidulans Emericella astellata Aspergillus versicolor NRRL 2396 U29847 L76745 (95) (99) Emericella nidulans Emericella variecolor L76746 (94) NRRL 1954 U29846 (98) 42 Ustus ND Emericella heterothallica L76743 (95) Aspergillus ustus L76744 (93) 90