Kretzschmaria quercicola sp. nov., an Undescribed Fungus from Living Oak in Mt. Daeryong, Korea.

We encountered an unfamiliar ascomycete fruiting body, fitting characteristics of the genus Kretzschmaria, which features in a stipitate ascigerous stroma with carbonaceous interior and disintegrating perithecia. In this study, we report and characterize a new species of the decaying fungus. Compared to other species, one of the notable features of this specimen (TPML150908-046) is its stromatal size (up to 15 cm). Although TPML150908-046 is morphologically similar to K. milleri and K. sandvicensis, it differs sharply from both species in apical ring size (TPML150908-046, 6.5~10.5 µm; K. milleri, 11~16 µm) and ascospore width (TPML150908-046, 10.5~17 µm; K. sandvicensis, 8.5~11.5 µm). Phylogenetic trees based on β-tubulin, ITS, and RPB2 sequences showed that our collection clustered with K. sandvicensis, with the respective similarities for these sequences being 95.6%, 91.3%, and 97.7%, signifying it as another species. With these results, we report it as a new species, which we call Kretzschmaria quercicola sp. nov.

Kretzschmaria species are found throughout the temperate and tropical regions of the world, and are known to cause white rots and root rots [1]. These fungi are also the key agents of wood decomposition, playing a central role in the ecology of forests, and are likely to have a tremendous impact on regulating energy and nutrient fluxes [23]. The genus Kretzschmaria was well revised by Rogers and Ju [1]. According to their study, this genus is divided into the taxa Kretzschmarioid and Ustulinoid. Stromata of the former are stipitate or sessile, with their fertile parts and/or stipes often fused, usually with entire margins, while stromata of the latter are more or less sessile, but often attached by rhizoid-like processes or narrow connectives, usually with crenate margins. A detailed molecular taxonomic study of the genus Kretzschmaria has not yet been reported. The studies on family Xylariaceae [45] showed that the genus Kretzschmaria has been considered closely related to the genus Xylaria. Although 43 species of Kretzschmaria have been recorded worldwide, the genus was recently revised by Rogers and Ju [1] to include some taxa previously placed under Ustulina, and 16 taxa were recognized and described. Subsequently, two additional species have been reported [67]. In the study on wood decay fungi, one Kretzschmaria species could not be assigned to any known species; in this study, we report this species as a new species and also discuss its molecular taxonomy.

The morphological character of TPML150908-046 (Fig. 1) belongs to the Ustulinoid taxa. The species most similar to TPML150908-046 are K. milleri, having almost identical stromata shapes and overlapping sizes of ascospores, and K. sandvicensis which has similar stromata shapes and a slightly overlapping length size of ascospores (Table 1) [167]. However, TPML150908-046 differs sharply from K. milleri and K. sandvicensis in the size of its apical ring, which does not overlap at all (TPML150908-046, 6.5~10.5 µm; K. milleri, 11~16 µm), as well as in having different ostioles. Additionally, TPML150908-046 differs sharply from K. sandvicensis in having a much broader width of ascospore (TPML150908-046, 10.5~17 µm; K. sandvicensis, 8.5~11.5 µm) (Table 1) [1]. Sizes of apical ring and ascospore are important keys in Kretzschmaria taxonomy [1678].

Fig. 1

Colony and morphological characters of Kretzschmaria quercicola sp. nov. TPML150908-046. A, Colony on oat meal agar (2%) after 31 days of inoculation; B, Stomata on substrate; C, Vertical section of a stroma showing several immersed perithecia; D, Stromatal surface showing polygonal scales and ostiolar openings; E, Asci with ascospores; F, Apical rings bluing in Melzer's iodine reagent (scale bars: B = 8 cm, C, D = 8 mm, E, F = 26.1 µm).

Table 1

Morphological characters of the genus Kretzschmaria [167]

The specimen (TPML150908-046) used in this study was collected on September 8, 2015 from the Mt. Daeryong in Chuncheon. Morphological characters were examined by stereo (SMZ 1500; Nikon, Tokyo, Japan) and optical (COOLPIX 4500; Nikon) microscopy, following the methods described by Rogers and Ju [1]. For observations of cultural characteristics, the pure culture was isolated from specimens and was incubated on oatmeal agar at 20℃ for more than 20 days. The isolate has been deposited in the Korean agricultural culture collection (KACC) as KACC 54398 (exo-type). To extract genomic DNA, the isolate (TPML150908-046) was cultured in potato extract broth. After harvesting the mycelium, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen 69106; Qiagen, Hilden, Germany) according to the manufacturer's instructions. β-Tubulin (using T1 and T22) [9], partial nuclear internal transcribed spacer (ITS) (ITS1 and ITS4 primers) [10], and the second largest subunit of RNA polymerase II (RPB2) (fRPB2-5F and fRPB2-7cR) [11] regions were amplified under conditions described in references [91011] and sequenced to determine the phylogenetic position of this species. Reference sequences (Table 2) were obtained from the study of Hsieh et al. [12]. The data were analyzed using the Tamura-Nei parameter distance calculation model, and the distances were used to construct the Neighbor-Joining tree with MEGA ver. 7.0.14 [13].

Table 2

Sequences used in this study and their information

aEF, GQ, and GU numbers [11] were obtained from the NCBI GenBank homepage (https://www.ncbi.nlm.nih.gov/genbank/) and KX numbers were sequenced in this study.

Each species was well distinguished in individual phylogenetic trees generated by using sequences of β-tubulin, ITS, and RPB2 (data not shown). The combined phylogenetic tree (Fig. 2) based on the β-tubulin-ITS-RPB2 sequences dataset showed that TPML150908-046 was closely related to K. sandvicensis, belonging to the same clade. The homologies of TPML150908-046 with K. sandvicensis JDR 113 were 95.6% (β-tubulin), 91.3% (ITS), and 97.7% (RPB2), respectively. No significant incongruence was detected among β-tubulin, ITS, and RPB2 datasets in each phylogenetic tree, hence we present a combined dataset. When we consider homology values of individual comparisons, the homology values of TPML150908-046 with K. sandvicensis JDR 113 can also signify a different species. Based on morphological and molecular characters, TPML150908-046 represents a new species in the genus Kretzschmaria and it is named as Kretzschmaria quercicola sp. nov. We describe its taxonomic characteristics below.

Fig. 2

A phylogenetic tree generated by using the neighbor-joining method for the β-tubulin-ITS-RPB2 dataset. Xylaria cranioides, X. teberoides, and X. hypoxylon were used as outgroups. Numbers at the nodes indicate bootstrap values (> 60). Bootstrap analysis was performed with 1,000 replications.

Taxonomic description.

J. H. Yun & D. H. Kim, sp. nov. (Table 1, Fig. 1).

Etymology: quercicola (Lat.), living on oak.

MycoBank No.: MB 817221.

Stromata pulvinate, discrete, densely aggregated or fused, up to 15 cm × 1~5 mm, with narrow connectives, usually with crenate, sloped blackish margins; surface dark brown, copper brown to blackish brown, with black ash like spots, often with reticulate cracks and polygonal scales, interior blackish, carbonaceous; tissue between and beneath perithecia fused with substrate, black. Perithecia obovoid to globose, 0.6~0.8 × 1.2~1.8 mm, ostioles finely papillate to papillate. Asci fragmentary, ca. 275 µm × 11~15 µm, with apical ring, 6.5~10.5 µm × 4~5.5 µm, bluing in Melzer's iodine reagent. Ascospores brown to dark brown, unicellular, fusoid-inequilateral or more or less subellipsoid with smooth ends, 35~46 µm × 10.5~17 µm, with straight germ slit less than spore-length.

Anamorph not observed on stromata. Cultures on 2% oatmeal agar (Difco, Detroit, MI, USA) plate at 20℃ in darkness covering 7.5 cm after a month incubation, seeming never reach the edge of the agar plate even after prolonged incubation times; at first white to gray, velvety, zonate, furrowed, distorted, and tend to separate from the plate, turning blackish from center outwards. Reverse becoming blackish from center. Anamorph not observed on culture.

Habitat: Oak-dominated deciduous forest on Quercus mongolica Fisch trunk.

Holotype: Korea, Kangwon Province, Chuncheon-si, Mt. Daeryong, coll. Yun & Lee, 8 Sep 2015, deposited to Korea National Arboretum Herbarium (KA15-0891-1).

Isotype: Korea, Kangwon Province, Chuncheon-si, Mt. Daeryong, coll. Yun & Lee, 8 Sep 2015, deposited to Korea National Arboretum Herbarium (KA15-0891-2).

Notes: K. quercicola sp. nov. and K. sandvicensis have similar lengths of ascospore but K. quercicola sp. nov. has a much broader width of ascospore (K. quercicola sp. nov., 10.5~17 µm; K. sandvicensis, 8.5~11.5 µm). Although K. quercicola sp. nov. has ascospore size more or less similar to that of K. milleri in terms of both length and width, the apical ring size of K. quercicola sp. nov. does not overlap with the that of K. milleri (K. quercicola sp. nov., 6.5~10.5 × 4~5.5 µm; K. milleri, 11~16 µm × 7~8.5 µm) and their ostiolar papillae are very different.

Currently, taxonomic studies on Kretzschmaria dealing with phylogenetic analysis have not yet been conducted. DNA sequences of Kretzschmaria that are available online are not from studies focused only on Kretzschmaria, but are only part of a study [12] on the overall Xylariaceae family. Hence, we used the sequences from the study of Hsieh et al. [12] for our comparisons. Ideally, sequences of K. milleri, if available, would provide clearer phylogenetic relationships with our specimen, but morphological characteristics were sufficient to divide the two species of K. quercicola sp. nov. and K. milleri. According to the study of Rogers and Ju [1], a number of these species seem to be morphologically distinguishable mainly on ascospore size, and the ascospores have ranges of lengths that only slightly overlap. Besides, judging by the fact that apical ring and ostioles were used to distinguish between K. sandvicensis and K. milleri, they also serve as identification keys [1]. The genus Kretzschmaria is not a large family, and has not been studied in detail. Until date, most species have been largely distinguishable based on their microscopic features. Recently, Mugambi et al. [7] reported a new species of Kretzschmaria, and Recio Herrera and Maldonado Gonzalez [8] revised the genus Kretzschmaria in Cuba, based on only morphological characteristics. In short, in taxonomy, data from morphology has been recognized to be the only key of identification. Our study is the first to use genetic information of Kretzschmaria, and we believe it should be further utilized in taxonomic studies of the genus.