Jurnal Biota Vol. 7 No. 2, 2021 ISSN: 2460-7746 (online); ISSN: 2528-262X (print) | 77 Xylaria sp. The Candle Snuff Fungus from West Java Rudy Hermawan *, Yuyun Nisaul Khairillah Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Darmaga Campus, Bogor 16680, Indonesia *email: hermawan_rudy@apps.ipb.ac.id Article Info Keyword: Ascomycota Bean-shaped Herbarium Bogoriense Stromata Xylaria Article history: Received: 30/03/2021 Revised: 09/06/2021 Accepted: 11/06/2021 ABSTRACT Candle snuff fungus belongs to Xylaria group. Generally, Xylaria has a form like stick or candle or elongated fruit of shapes. Xylaria is classified into Ascomycota within Xylariaceae. This study found one species of candle-shaped mushroom in IPB Unversity. This study aimed to identified and characterized the specimen using molecular and morphological data. The specimen was collected and preservedinto FAA solution and deposited into Herbarium Bogoriense as BO 24426. Molecular analyses using Large Subunit as a region for amplification showed that the BO 24426 was classified into Xylaria sp. This species closes to Xylaria consociata. The stromata were erected, unbranched, and tapered to the apex. The texture was rigid and hard. Ascus bore 8 ascospores. The ascospores were fusiform or bean-shaped and smooth. The morphological observations supported molecular identification of BO 24426 as Xylaria sp. Other genes were needed to ensure the exact species of Xylaria Copyright © 2021 Author(s). All Right Reserved Introduction Mushroom is a macro-fungus that belongs to Ascomycota and Basidiomycota (Brundrett et al., 1996). Some mushrooms show the unique characters as the unusual shapes. In Indonesia, many unique shapes of mushrooms are found as a specific name of genus, such as puffball for Calvatia (Hermawan & Putra, 2018, 2021), Cannonball for Sphaerobolus (Hermawan & Maulana, 2020), goblet for Trichaleurina (Hermawan, Amelya, et al., 2020), fan for Telephora (Hermawan, Imaningsih, et al., 2020), coral for Ramaria (Hermawan, Imaningsih, et al., 2020), trumpet for Cantharellus (Hermawan, Imaningsih, et al., 2020), microphone-like for Lysurus (Hermawan et al., 2021), and parasol for Chlorophyllum (Hermawan, Imaningsih, et al., 2020). Others unique mushroom is Xylaria. The Xylaria is famous with name of the dead men's finger or the candle snuff fungus. Xylaria contains 819 name records (Index Fungorum, 2021). Xylaria is classified into Xylariales within Pezizomycotina (Mycobank, 2021). The traits of this genus are known as saprobic and also endophytic fungi. Xylaria as an endophyte lives inside the plant tissue without causing any disease symptoms for the plant (Petrini, 1991). A special trait of Xylaria also emerges from nest of termites (Rogers et al., 2005). This study found the Xylria stromata on the ground (without substrate). It indicates the possibility of the trait as symbiont with insect. Traditional classification and identification of fungi has relied upon microscopic features, colony characteristics on media and also biochemical reactions (Sutton & Cundell, 2004). Sometimes the traditional method http://jurnal.radenfatah.ac.id/index.php/biota 78 | Jurnal Biota Vol. 7 No. 2, 2021 cannot ensure the name of species, especially for Xylaria. The Xylaria are difficult to identify and classify using only the stromata characteristics (Whalley, 1996). Some researchers in Xylaria study using the molecular comprehensive for identifying the species (Ju et al., 2009; Ma et al., 2013; Okane et al., 2008). In Indonesia, study of Xylaria is usually conducted using only morphological character (Kristin et al., 2020). The molecular study is important to make sure the name of species and will be supported using morphology. The internal transcribed spacer (ITS) region is a good region to identify the fungi (Brandt et al., 2005; White et al., 1990). But, other region such as Large Subunit is also needed to make stronger analyses in fungi. For Xylariaceae, the sequences that popular to be analyzed are Large Subunit region (Okane et al., 2008), some ITS sequences are rarely available in GenBank. Therefore, this study used molecular study for Large Subunit sequences. Materials and Methods Mushrooms Sampling Site The stromata were collected in August 2019 and located in the Arboretum of IPB University. The fungus was found as a Xylaria stroma based on the morphology. The stroma was collected, documented, and observed the morphological characters. The observation was conducted in the mycology laboratory of Biology Department, Mathematics and Natural Sciences, IPB University, Indonesia. The apothecium was preserved in FAA (Seshagirirao et al., 2016) and deposited into Herbarium Bogoriense Indonesia. Morphological Observation The morphological data of stromata were observed and documented to confirm the genus as Xylaria. The data would be used to support the molecular analyses. The observation was conducted using an Olympus stereo and binocular microscope cs22LED. The features of macro-and micro-morphology such as asci and ascospores. The observations were about the shape, size, and ornamentation, then compared with the other publication of the species within Xylariaceae. Molecular Identification The stromata were identified using molecular analyses. The genomic DNA was extracted using the protocol as in (Hermawan, Amelya, et al., 2020). The genomic DNA quality and quantity were verified using a nanodrop spectrophotometer. The amplification was used Large Subunit (LSU) as forward LR0R (5’-GTA CCC GCT GAA CTT AAG C-3’) and reverse LR5 (5’-ATC CTG AGG GAA ACT TC-3’) primers. PCR amplification was performed in 40 µL total reaction containing 12 µL ddH2O, 2 µL of 10 pmol of each primer, 20 µL PCR mix from 2X Kappa Fast 2G, and 4 µL 100 ng template DNA. Amplification used a Thermoline PCR. The PCR condition was set as follows: initial denaturation at 94 ℃ for 2 minutes, followed by 30 cycles of denaturation at 94 ℃ for 45 seconds, annealing at 56 ℃ for 1 minute, and extension at 72 ℃ for 1 minute. Then final extension was set at 72 ℃ for 10 minutes. The amplicons were estimated on 1 % agarose gels and visualized by the Gel DocTM XR system. PCR products were sent to the 1st Base Malaysia for sequencing. Phylogenetic Analyses The sequence was deposited in GenBank. This sequence, Xylaria species in (Okane et al., 2008), some Xylaria species from GenBank, and Vamsapriya bambusicola (outgroup) were constructed into phylogenetic tree (Table 1). Sequences were aligned using Clustal X Ver. 2.1 software and saved as PHYLIP format files. All sequences were aligned using 600 base pairs of the ITS region. The phylogenetic tree of Randomized Axelerated Maximum Likelihood (RAxML) Black Box was generated on CIPRES (Stamatakis, 2014). Bootstrap analyses with 1000 replicates assessed the phylogenetic tree. Bootstrap (BS) ≥ 50 was shown on the branch. http://jurnal.radenfatah.ac.id/index.php/biota Jurnal Biota Vol. 7 No. 2, 2021 | 79 Table 1. Collection code, species, GenBank accession numbers of Internal Transcribed Spacer used in this study. Species Xylaria allantoidea Xylaria anisopleura Xylaria apiculata Xylaria arbuscula Xylaria arbuscula Xylaria arbuscula Xylaria arbuscula Xylaria aristata Xylaria aristata Xylaria badia Xylaria badia Xylaria bambusicola Xylaria bambusicola Xylaria bambusicola Xylaria bambusicola Xylaria bambusicola Xylaria coccophora Xylaria consociata Xylaria cubensis Xylaria cubensis Xylaria cubensis Xylaria cubensis Xylaria cubensis Xylaria curta Xylaria curta Xylaria enteroleuca Xylaria escharoidea Xylaria escharoidea Xylaria feejeensis Xylaria grammica Xylaria grammica Xylaria grammica Xylaria grammica Xylaria hypoerythra Xylaria hypoxylon Xylaria hypoxylon Xylaria hypoxylon Xylaria hypoxylon Xylaria juruensis Xylaria juruensis Xylaria juruensis Xylaria juruensis Xylaria juruensis Xylaria juruensis Xylaria laevis Xylaria longipes Xylaria longipes Xylaria longipes Xylaria longipes Collection code BCC 1340 BCC 17352 BCC 1136 BCC 1156 RHP 21 CBS 126416 DHK-10 BCC 1229 BCC 1260 BCC 1171 BCC 1190 BCC 22739 BCC 23628 BCC 23659 MFLUCC 11-0606 BCC 23627 BCC 1085 BCC 18196 BCC 1321 BCC 1144 BCC 1303 BCC 11027 BCC 1219 BCC 1007 BCC 1151 CBS 128357 BCC 23279 BCC 23634 BCC 1115 IHI A82 BCC 1002 BCC 1170 5084 BCC 22968 AFTOL-ID 51 CBS 126417 DM1153 DSM 108379 BCC 1232 BCC 1086 BCC 1083 BCC 1263 BCC 1234 BCC 1233 BCC 1182 19GCAS018 CBS 148.73 CBS 347.37 DSM 107183 GenBank acc. no LSU AB376730 AB376732 AB376700 AB376703 MT215561 MH875561 MN376820 AB376716 AB376722 AB376705 AB376711 AB376809 AB376821 AB376825 KU863148 AB376820 AB376688 AB376733 AB376729 AB376701 AB376725 AB376683 AB376715 AB376681 AB376702 MH876349 AB376818 AB376882 AB376696 MK408621 AB376679 AB376704 JQ862608 AB376812 AY544648 MH875562 MT773340 MK577428 AB376717 AB376689 AB376687 AB376723 AB376719 AB376718 AB376709 MW077324 MH872351 MH867427 MK408619 http://jurnal.radenfatah.ac.id/index.php/biota 80 | Jurnal Biota Vol. 7 No. 2, 2021 Table 1. Continue Species Xylaria mellissii Xylaria mellissii Xylaria multiplex Xylaria multiplex Xylaria obovata Xylaria obovata Xylaria obovata Xylaria papulis Xylaria phyllocharis Xylaria phyllocharis Xylaria piperiformis Xylaria polymorpha Xylaria psidii Xylaria psidii Xylaria schweinitzii Xylaria schweinitzii Xylaria sicula Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria tuberoides Xylaria xylarioides Xylaria xylarioides Vamsapriya bambusicola Collection code BCC 1005 BCC 1186 BCC 1177 BCC 1036 BCC 1053 BCC 18718 BCC 1100 BCC 22966 BCC 1352 BCC 1065 BCC 22987 CBS 162.22 BCC 1199 BCC 1127 BCC 1013 BCC 1001 CBS 401.58 BO 24426 BCC 1181 BCC 1133 BCC 1288 BCC 18361 CBS 127883 CBS 128018 MFLUCC 11-0577 Results and Discussion Specimen Two stromata were found that grew on the ground without substrate (Figure 1a). Sexual morphology: Stromata look like greyish candle snuff with whitish powder covered stromata from upper (Figure 1a-b). The stromata were erected, unbranched, and 6.5-9.1 cm of height. The stromata were tapered to the apex of stromata. The texture was rigid and hard. Asci were appeared inside the stromata. Ascus contains 8 ascospores (Figure 1c). The ascospore has oil globular inside, 1 or 2 oil globules appeared in the corner of ascospore, fusiform or bean-shaped and smooth (free of ornament). The size was 12.2–13.2 x 5.0–5.9 µm (Figure 1d). Specimen examined: Landscape Arboretum of IPB University, BO 24426, Rudy Hermawan. GenBank Submission: LSU: MW433688. GenBank acc. no LSU AB376680 AB376710 AB376707 AB376684 AB376685 AB376737 AB376690 AB376811 AB376731 AB376686 AB376814 MH866242 AB376713 AB376698 AB376682 AB376678 MH869355 MW433688 AB376708 AB376699 AB376724 AB376736 MH876177 MH877980 KM462836 Xylaria has the unique morphology as a candle, spoon and finger (Kirk et al., 2008). Then, Fournier et al. (2018) described the updated morphology of Xylaria species as in pulvinate to depressed-spherical shape. Xylaria BO 24426 has the morphology with candle snuff with whitish powder covering the stromata. The whitish powder does not contain spore or other sexual structure. The spore as ascospore is inside the stromata part. Rogers and Samuels (1986) described Xylaria species has dark carbonaceous stromata and pigmented ascospores as dark color with a germ slit. Xylaria BO 24426 showed the dark ascospores. The ascospores was produced inside the ascus with 8 spores. Germ slit character is really hard to be observed. The observation should use the advanced microscope with the high magnification of lens. the observation of BO 24426 has not successful observed the germ slit characteristic. http://jurnal.radenfatah.ac.id/index.php/biota Jurnal Biota Vol. 7 No. 2, 2021 | 81 Figure 1. Morphology of Xylaria BO 24426 (A; B) Stromata on substrate; (C) ascus; (D) ascospores. Scale bars: (A) 10 cm; and (C; D) 10 µm. The ascospore shape of BO 24426 has fusiform or bean-shaped. Other species of Xylaria have many shapes such as ellipsoid slightly inequilateral with broadly to narrowly rounded ends (as X. berteroi), ellipsoidinequilateral to sub-oblong with broadly rounded ends (as X. alboareolata) etc. (Fournier et al., 2018). Based on our BO 24426, the ascospores also have the oil globules inside the ascospores. Molecular work The phylogenetic tree showed that the specimen BO 24426 was classified as Xylaria sp. with 41% bootstrap value (Figure 2) making other clades from Xylaria consociata. Okane et al. (2008) mentioned the X. consociata as a saprobic Xylaria. Whereas the Xylaria sp. BO 24426 is not saprobic Xylaria. According to Figurure 2, BO 24426 has a different line length with the Xylaria consociata. This line was indicated that the evolutionary based on their sequences is different. It can be showed that Xylaria BO 24426 is not Xylaria consociata. In Okane et al. (2008) also mentioned some of Xylaria as Xylaria sp. The region of gene amplification for molecular identification within Xylaria species sometimes can’t show the strong identification. As a single gene or region in this study, BO 24426 only can be identifying until the Genus name Based on this phylogenetic tree of Xylaria BO 24426, the species can be assumed as a new species or unidentified. The BO 24426 is assumed as a new species because the BO 24426 makes other clade among other species. Or it can also be assumed as unidentified species. The other gene regions are needed to complete the differences among the species and make the perfect phylogenetic tree. This additional work will ensure the BO 24426 as a new species or not. The other functional genes for identification are Internal Transcribed Spacer, Actin, Small Sub Unit, etc. Schoch et al. (2012) mentioned the ITS region is the most useful for identification and also as a general gene for identification among fungal species. The existence of ITS as a general gene in Xylaria is not really helpful to be found in NCBI or GenBank. Okane et al. (2008) had used the LSU as additional gene for making the phylogenetic tree among Xylaria species. The phylogenetic was enough clearly to distinguish the species among Xylaria species. http://jurnal.radenfatah.ac.id/index.php/biota 82 | Jurnal Biota Vol. 7 No. 2, 2021 Figure 2. Xylaria BO 24426 phylogenetic tree based on the LR0R/LR5 region using RAxML. Bootstrap (BS)≥50 was shown on the branch. The Xylaria BO 24426 must be in bold. Conclusion Xylaria BO 24426 has been identified as Xylaria sp. with 41% BS value and closed to X. consociata. The stromata look like greyish candle snuff with whitish powder covering stromata. 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