Xylaria sp. The Candle Snuff Fungus from West Java

Keyword: Ascomycota Bean-shaped Herbarium Bogoriense Stromata Xylaria 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 Article history: Received: 30/03/2021 Revised: 09/06/2021 Accepted: 11/06/2021

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 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 1 st 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.

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.
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. 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)  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.

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. As a single gene identification using LSU region is not enough and strong to identify BO 24426 into a species among Xylaria. Moreover, BO 24426 has potential as a new species, because the clade of them showed the branch length that separated with other species.