New Record of Xylaria persicaria on Liquidambar Fruits in Korea

Abstract

Some Xylaria materials growing on the fruits of Liquidambar spp. were collected. They were identified as X. persicaria on the basis of morphological characteristics and sequence analysis of the complete ITS region (ITS1-5.8S-ITS2) of rDNA. This is the first record of this species from Korea.

Xylaria Hill ex Schrank is one of the largest genera in the Xylariaceae accommodating about 100 species (Kirk et al., 2001). Up to now, only six species, viz. X. carpophila, X. filiformis, X. hypoxylon, X. longipes, X. oxyacanthae and X. polymorpha, have been recorded in Korea (Lee and Lee, 2000).

In the course of mycofloristic investigation, Xylaria materials growing on the fruits of Liquidambar spp. were found. All collections were air-dried at room temperature and housed in the herbarium of Korea University (KUS). Morphological observation and sequence analysis of ITS rDNA (ITS1-5.8S-ITS2) were performed to identify the Xylaria materials. Sequence data used in the present study and their sources were listed in Table 1.

Table 1.

ITS sequence data used in this study

CBS - Centraalbureau voor Schimmelcultures; ATCC - American Type Culture Collection; SFC - Seoul National University Fungus Collection; IFO - Institute for Fermentation Osaka; KUS - Herbarium of Korea University, Seoul.

Each specimen was examined in the morphological characteristics of asci, ascospores, paraphyses and other structures of taxonomic value. Melzer's reagent, cotton-blue in lactic acid and distilled water were used for mounting media of microscopy. Dried materials were rehydrated in 3% aqueous KOH. Photographic works were carried out with the aid of a differential interference contrast microscope (Olympus BX51) equipped with a digital camera (ARC-CM13c).

Extraction of genomic DNA was undertaken according to the method outlined by Lee and Tayler (1990). The complete ITS rDNA regions were amplified by PCR using primers ITS1 and ITS4 (White et al., 1990). The success of the amplification was monitored by electrophoresis on 1% agarose gels, and purified using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). Purified DNAs were directly sequenced on an automatic sequencer (ABI Prism TM 377 DNA Sequencer), with primers identical for PCR.

Sequences were introduced and edited with DNAstar (DNAstar, Inc., Madison, Wis.). Phylogenetic analysis was performed according to the neighbor-joining (NJ) method in PAUP* ver. 4b10 and relative robustness of the branches was estimated by bootstrapping using 1000 replicates. Daldinia concentrica (AF163021), member of the Xylariaceae was selected as outgroup.

Results and Discussion

According to the morphological observation the fungus collected on the Liquidambar fruits was determined as Xylaria persicaria (Schwein.) Berk. & M. A. Curtis, that was characterized by its ascospores with a long spiral germination slit and ecological habit. Since Schweinitz (1822) originally described Sphaeria persicaria (= X. persicaria) as an inhabitant of buried peach seeds, it has been more frequently collected on Liquidambar fruits (Rogers, 1979; San Martin and Rogers, 1989). Xylaria carpophila (Pers.) Fr. is externally similar to the present species, but it has ascospores with a straight germination slit and occurs on fallen Fagus fruits (Rogers, 1979).

In phylogenetic tree inferred from NJ method, our materials formed a segregated clade with X. persicaria (AY909021) and this grouping was supported by high bootstrap value of 100% (Fig. 2). Phylogenetic relationship of some Xylaria spp. was studied by Lee et al. (2000), in which Xylaria spp. were classified into three groups based on the morphological and molecular similarity, viz. X. apiculata, X. arbuscula and X. mali in Group A; X. acuta, X. castorea, X. cornu-damae, X. enteroleuca, X. fioriana and X. longipes, in Group B; X. hypoxylon and X. polymorpha in Group C. X. persicaria clade was nested in the Group A with 57% bootstrap supporting in NJ analysis. But morphological common features of Group A such as a straight germ slit of ascospores were not observed in our materials.

Fig. 2.

Phylogenetic tree of Xylaria species inferred from NJ method on the basis of complete ITS region (ITS1-5.8S-ITS2). Bootstrap values are represented above the branches (1000 replication, values smaller than 50% not shown). The numbers of nucleotide changed among taxa are represented by branch length and scale bar equals the number of nucleotide substitution per site. Asterisks indicate the sequences obtained from the present study.

Seed and fruit inhabiting Xylaria species have been generally reported to be highly host-specific. Rogers et al. (2002) separated Xylaria on Liquidambar fruit from X. persicaria and proposed a new species X. liquidambaris J. D. Rogers, Y. M. Ju & F. San Martin. However, They did not provided appreciable morphological differences between X. liquidambaris and X. persicaria because the type specimen of the latter was immature (Ellis and Everhart, 1892) and lacking ascospores (Rogers et al., 2002). The validation of X. liquidambaris remains uncertain until further morphological and molecular work is carried out. We decided the Korean materials to place in X. persicaria (Schwein.) Berk. & M. A. Curtis, non Rogers et al. (2002).

Description

Xylaria persicaria (Schwein.) Berk. & M. A. Curtis, Pungnamukongggoturibeoseot (Fig. 1).

Fig. 1.

Xylaria persicaria (KUS-F50694). A. Stromata on the fruit of Liquidambar styraciflua. B. Cross-section of fertile head part of a stroma. C. 8-spored ascus. D. Bluing apical pore in Melzer's reagent. E. Ascospores with a spiraling germination slit. Scale bars: 1 cm for A; 1 mm for B; 20 µm for C; 10 µm for D and E.

Stromata erect, up to 9 cm high, exterior black to brownish black, solid, divided into fertile head and sterile stalk. Head parts clavate to cylindric-clavate, 0.4~2.8 cm high and 0.1~0.3 cm wide, with longitudinally fine wrinkles, apex commonly pointed like a needle. Stalk parts irregularly twisted and crooked, often thickened toward the base, glabrous to pubescent, somewhat flattened on one side, deeply furrowed in longitudinal direction, 0.5~3.0 cm high, concolorous or paler than head parts, occasionally branched. Perithecia embedded in a stroma, black. Interior flesh white. Growing singly or in clusters. Asci 8-spored, cylindric, long stipitate, hyaline, apical pore bluing in Melzer's reagent, 135~150 × 5~6 µm. Ascospores elliptical to bean-shaped, commonly flatten on one side, with spiraling germination slit, uniseriate, hyaline when immature stage, becoming dark brown with one distinct oil-drop, 12~15 × 4~6 µm. Paraphyses cylindric, hyaline, abundant.

Distribution

Florida, USA (Rogers et al., 2002), Mexico (Rogers, 1979; San Martin and Rogers, 1989), China (Rogers, 1979), and Korea.

Specimens examined

Korea, Namhae, Mt. Geumsan, 3 Jun 2004, on the fruits of Liquidambar styraciflua (KUS-F50693) and L. formosana (KUS-F50694); Korea, Jinju, Gajwa Arboretum, 17 Oct 2004, on the fruits of L. styraciflua (KUS-F50748) and L. formosana (KUSF50749); Korea, Jinju, Gajwa Arboretum, 29 Jun 2006, on the fruits of L. formosana (KUS-F51475).