Mycosphere Caliciopsis indica sp. nov. from India Pratibha J1,2, Amandeep K3, Shenoy BD3* and Bhat DJ1* 1Department of Botany, Goa University, Taleigao Plateau, Goa - 403 206, India. [email protected] 2MykoTech Pvt. Ltd., Plot No. 12, Mapusa Industrial Estate, Mapusa, Goa – 403 507, India 3Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Sector 39A, Chandigarh – 160036, India. [email protected] Pratibha J, Amandeep K, Shenoy BD, Bhat DJ 2010 – Caliciopsis indica sp. nov. from India. Mycosphere 1, 65–72. Caliciopsis indica sp. nov. is described from leaf lesions of kokum (Garcinia indica, Clusiaceae) from the Western Ghats, India. Caliciopsis indica is morphologically similar to C. myrticola but differs in having larger ascomata, longer asci and smaller ascospores. Phylogenetic analysis of partial 28S rRNA gene sequence data has confirmed its placement within the Coryneliaceae (Coryneliales, Eurotiomycetes). The ITS/5.8S rRNA gene sequence, however, did not provide any clarity on the species delineation due to lack of reference sequences in GenBank. Key words – biodiversity – bitunicate ascomycete – forest ecology Article Information Received 12 April 2010 Accepted 15 April 2010 Published online 30 April 2010 *Corresponding author: Bhat DJ – e-mail – [email protected] and Shenoy BD –e-mail – [email protected] Introduction saccate, long-pedicellate asci that lack apical Kokum (Garcinia indica Choisy, structures. Though the fungus is morpho- Clusiaceae) is an economically important plant logically similar to Caliciopsis myrticola bearing edible fruits and indigenous to the Huguenin (Benny et al. 1985), it is described as Western Ghats of India (Korikanthimath & a novel species of Caliciopsis based on Desai 2005, Bhat et al. 2006). During our differences in dimensions of ascomata, asci and studies on foliicolous fungi on forest plants of ascospores. A note on its phylogenetic Goa and neighbouring regions of Western placement based on partial 28S rRNA gene Ghats in southern India (Pratibha et al. 2004, sequence data is also included. Pratibha & Bhat 2005, Pratibha 2006), we collected an ascomycete on leaf lesions of Methods Kokum. The leaf lesions were apparently formed by insects grazing on mature leaves. Isolates and morphology The fungus was first collected from Mashem, Fresh leaves of kokum with greyish- Canacona in Goa; additional specimens were centered brown patches or spots were collected collected from neighbouring Karnataka and taken to the laboratory in zip-lock (Ankola, Uppinangadi), indicating that the polythene bags. Fungal material found growing fungus might be distributed in most regions along the brown margin of leaf spots was where Kokum is grown. The fungus is carefully picked up with a fine-tipped needle characterized by superficial non-setose and mounted on a slide containing a drop of perithecia with a prominent stalk, and aseptate lactophenol solution or water as mountant and light brown ascospores enclosed in bitunicate, examined under a light microscope. Part of the 65 material was air-dried and placed in labelled Sequence alignment and phylogenetic paper bags along with a piece of naphthalene analysis pellet to maintain as herbarium specimens. The Sequences obtained from the respective specimens were deposited in the Botany primers were aligned using Sequencher version Herbarium of Goa University (GUBH). The 4.9 (Gene Codes Corporation) and the con- fungus was isolated in pure culture by sensus sequences were deposited in GenBank ascospore isolation method of Choi et al. with accession numbers GQ259980 (partial (1999). An ascoma was placed in a drop of 28S rRNA gene) and GQ259981 (ITS/5.8S sterile distilled water and cut open. The ascoma rRNA gene region). A dataset based on 28S debris was removed from the slide. Mature rRNA gene sequence was prepared using ascospores were streaked in malt extract agar MEGA4 (Tamura et al. 2007). Additional plates by a fine-tipped needle. Ascospores reference sequences retrieved from GenBank germinated within 24 h. Colonies developed and their accession numbers are listed in Fig. 3. from germinated ascospores were individually Phylogenetic analyses were conducted in transferred into MEA slants using a fine-tipped MEGA4 (Tamura et al. 2007). Phylogenetic needle. A culture was deposited at Fungus relationships of Caliciopsis indica were Culture Collection of Goa University analysed based on Neighbor-Joining method (GUFCC). (Saitou & Nei 1987). The evolutionary distances were computed using the Kimura 2- DNA extraction, PCR and sequencing parameter method (Kimura 1980). All positions The fungal isolate was grown on potato containing alignment gaps and missing data dextrose agar (PDA) medium for 7 days just were eliminated only in pairwise sequence before total genomic DNA was extracted using comparisons (Pairwise deletion option) ZR Fungal/Bacterial DNA Kit (Zymo Research, (Tamura et al. 2007). catalogue number D6005). DNA amplification was performed by polymerase chain reaction Taxonomy (PCR). The ITS4-ITS5 and LROR-LR5 (White et al. 1990) primer-pairs were used to amplify Caliciopsis indica J. Pratibha & Bhat, sp. nov. the internal transcribed spacers (ITS)/ 5.8S Figs 1–2 rRNA gene region and partial 28S rRNA gene, MycoBank 501254 respectively. Amplification reactions were Etymology – named for its origin country, performed in a 50 µl reaction volume as India. outlined by Shenoy et al. (2006). The PCR Ascomata perithecialis, superficialia, thermal cycle was as follows: 95°C for 3 min, pedunculati, solitaria, clavatum, atro-brunnea, followed by 34 cycles of denaturation at 95°C ostiolata, 205–300 × 35–70 μm; papilla absens; for 1 min, annealing at 52°C for 30s and setis absens; pedunculus erectus, cylindricus, elongation at 72°C for 1 min, with a final atro-brunnea; stroma absens. Filamenta extension step of 72°C for 10 min. The PCR hamathecii nulla. Asci bitunicati, octospori, products spanning approximately 900 bp (for uteriformis, ad apicem rotundus, pedunculati, 28S rRNA gene) and 600 bp (for ITS/5.8S 18–28 × 4–7 μm, apice non amyloideo, annulo rRNA gene) were checked on 1% agarose nullo. Ascosporae globosa, laevis, pallide electrophoresis gel stained with ethidium brunneae, unicellulares, 2.5–4.5 μm diam. bromide. PCR products were then purified Lesions epiphyllous, irregular, greyish in using quick spin column and buffers (washing the center, brown towards the margin, 0.2–1.5 buffer and elution buffer) according to the × 0.6–1 cm. Colonies on MEA medium slow- manufacturer's protocol (QIA quick gel growing, attaining a diameter of 0.4 cm in 7 extraction kit, catalogue number 28706). DNA days, flat, velvety, pale brown, with irregularly sequencing was performed using the above serrated margin, reverse medium brown. mentioned primers in an Applied Biosystem Mycelium immersed, composed of septate, 3130 xlanalyzer at Central DNA sequencing branched, hyaline to subhyaline, smooth, 2–4 facility of Institute of Microbial Technology, μm wide hyphae. Ascomata perithecial, Chandigarh. 66 Mycosphere Fig.1. Caliciopsis indica: Stalked ascocarp, L.S. of ascocarp, asci and ascospores. superficial, with a prominent stalk, solitary, ostiolar canal cylindrical, moderately brown, dark brown, velvety, surface rough, with an truncate at the apex, 25–55 × 40–50 μm. elongate wide ostiole at the apex, 205–300 × Stroma absent. Peridium soft, outwardly 35–70 μm; stalk erect, cylindrical, dark brown, composed of 2–3 layers of brown, thick-walled, slightly wider at the basal region, narrower polygonal cells (2–3.5 μm diam.) and inwardly below the venter (ascoma proper), 110–140 × composed of 4–5 layers of thin-walled, 35–40 μm; venter sitting on the end of the stalk, elongate cells (2–3 × 2–4.5 μm). Interthecial oval, dark brown, wider in the middle and filaments not observed. Asci bitunicate, 8- narrow on either ends, 70–100 × 45–70 μm; spored, saccate, apically rounded, pedicellate, 67 Fig. 2. Caliciopsis indica, a. leaf lesion, b. ascomata (arrowed, under stereoscope), c. stalked ascocarp, d. ascocarp, e. L.S. of ascocarp, f. asci, g. ascospores. 18–28 × 4–7 μm. Ascal apex non- Known distribution – known from Goa amyloid, lacking apical ring or any discharge and Karnataka states of India. mechanism. Ascospores globose, smooth, light Holotype – India, Goa, Canacona, brown, aseptate, 2.5–4.5 μm diam., sometimes Mashem, on leaves of Garcinia indica, 2 arranged in two rows. January 2005, P. Ashish, Herb. GUBH No. Anamorph – Not seen. P166. 68 Mycosphere 99 Capronia munkii EF413604 49 Exophiala dermatitidis DQ823100 Exophiala pisciphila DQ823101 47 100 Exophiala salmonis EF413609 82 Capronia pilosella DQ823099 85 Chaetothyriales Ramichloridium anceps DQ823102 25 Phialophora verrucosa EF413615 100 Cyphellophora laciniata EF413619 Ceramothyrium carniolicum EF413628 45 Glyphium elatum AF346420 64 Pyrgillus javanicus DQ823103 99 Pyrenula aspistea EF4110632 94 Pyrenula cruenta AF279407 Pyrenulales 99 99 Granulopyrenis seawardii EF411062 61 Pyrenula pseudobufonia AY640962 Norrlinia peltigericola AY300845 Staurothele frustulenta DQ823098697 97 Agonimia sp. DQ782913 54 53 Dermatocarpon miniatum AY584644 Verrucariales 10 Verrucaria pachyderma AF356668 65 13 Endocarpon pallidulum DQ823097 20 Polyblastia melaspora EF413601 87 Caliciopsis pinea DQ678097 100 Caliciopsis orientalis DQ470987 Coryneliales Caliciopsis indica GQ259980 99 Eupenicillium javanicum EF413621 100 Eupenicillium limosum EF411064 71 Penicillium freii AY640958 87 Eurotiales 99 Hamigera