Dactylella pseudobrevistipitata, a new species from China

Abstract

An anamorphic fungus was isolated from fresh specimens of Orbilia species collected in Yunnan Province, China. The fungus was characterized by very short conidiophores and 1–5 septate cylindrical conidia, and most closely related phylogenetically to Dactylella vermiformis that produces branched conidiophores and 0–1 septate conidia, but the calculated similarity of ITS sequences was only 83% between the two fungus species. Considering the morphological characteristics and the calculated similarity value, a new species, Dactylella pseudobrevistipitata, was described with holotype YMF 1.03504.

Introduction

The genus Dactylella was set up by Grove in 1884 with the type species D. minta Grove. Main characters of the genus are saprophytic, conidiophores erect, simple, conidia borne singly at the apex of conidiophores, ellipsoidal or fusoid or cylindrical, one-celled at first, later 2– to many septate, hyaline (Grove 1884). In the type strain, the predacious character was not mentioned. Later, Drechsler (1937, 1950) described many new taxa of nematode-trapping species with similar conidia following this concept. Then, circumscription of the genus was emended several times by other authors (Subramanian 1963, 1977; Schenck et al. 1977; Rubner 1996), in which Ruber’s genus concept is widely accepted (Scholler et al. 1999; Li et al. 2005; Yu et al. 2007a, b; Chen et al. 2007a, b, c). Chen et al. (2007a, b, c) further emended this genus and transferred those species with short conidiophores to Vermispora Deighton & Pirozynski and Brachyphoris J Chen, LL Xu, B Liu & XZ Liu. The latter is characterized by very short, simple conidiophores, and spindle-shaped, filiform or elongate fusoid conidia.

In our study of Orbilia and anamorphs from Yunnan Province, China, a strain was isolated from germinated ascospores of Orbilia. The Orbilia specimen was not identified in detail because there were two apothecia which were used to isolate the anamorph. The conidiophores of this anamorph are shorter than the conidia. Phylogenetic analysis inferred from ITS indicated that this species belongs to Dactylella, although it looks like Brachyphoris in having very short conidiophores; we described it as a new species of Dactylella, D. pseudobrevistipitata.

Materials and methods

Collection of teleomorph, isolation and characterization of the anamorph

Fresh specimens of an Orbilia species were collected on decaying bark of a broad-leaved tree from near Thermal Spring (500 m altitude), Yuanjiang County, Yunnan Province, China, in September 2009 by Li Qin. To isolate its anamorph, two fresh apothecia were fixed to the lid of a Petri-dish with their hymenia upside down so that ascospores were deposited on the surface of CMA (20 g corn meal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1,000 ml distilled water). For other isolating details, see Yu et al. (2007a). Microscopic characters of anamorph were observed and measured with an Olympus B51 microscope with differential interference contrast. Conidial size, conidial septate and conidiophores size were calculated by measuring more than 50 samples. Trapping organs were induced by adding about 100 nematodes (Panagrellus redivivus Goodey) to a 1 × 1 cm slot at the margins of the colony created by removing the agar.

DNA extraction, PCR amplification and sequencing

Fresh mycelium was collected from the cultures growing on cellophane menbrane on the PDA plate surface. The total DNA was extracted from fresh mycelium as described by Yu et al. (2007b). The ITS region was amplified with the standard primers of ITS4 and ITS5 (White et al. 1990). The parameters for PCR amplification were as follows: 3 min at 95°C, followed by 30 cycles of 94°C for 1 min, annealing at 50°C for 1 min and extension at 72°C for 1 min. A final extansion at 72°C for 10 min followed. The PCR products were purified with a commercial Kit (TaKaRa Biotechnology), and sequenced on both strands using the same primers that were used for amplification, with an ABI 3730 DNA analyzer automatic sequencing system, using cycle sequencing with the Thermo Sequenase-kit.

Phylogenetic analysis

We performed a phylogenetic analysis using ITS sequences of Dactylella, Vermispora and Brachyphoris used in Chen et al. (2007a), with Dactylaria purpurella as the outgroup. In addition, two similar species morphologically similar to D. pseudobrevistipitata, D. vermiformis ZF Yu, Y Zhang & KQ Zhang and D. coccinella Y Yang & XZ Liu (Yang and Liu 2005) were included.

DNA sequences were aligned using Clustalx 1.83. Cladistic analyses using the neighbor-joining method were performed with MEGA version 3.1. The neighbor-joining tree was constructed with Kimura 2-parameter model, including transitions and transversions and with pairwise deletion of gaps; bootstrap repetition was 500.

Results and discussion

A neighbor-joining tree was constructed based on 35 ITS sequences, which clustered into three different clades, Dactylella, Vermispora and Brachyphoris (Fig. 1). D. pseudobrevistipitata together with other three species, D. tenuis Drechsler, D. vermiformis, D. coccinella, formed a subclade within the Dactylella clade, which separated from most species of Dactylella. Among three species phylogenetically related to D. pseudobrevistipitata, D. vermiformis was most closely to D. pseudobrevistipitata, but the similarity of both ITS sequences was only 83%.

Fig. 1

A neighbor-joining tree inferred from the ITS DNA sequences. The number associated with each branch represents the percentage of 500 bootstraps supporting that branch. GenBank accession numbers are shown following each taxon name

The conidiophores of D. pseudobrevistipitata were shorter than the conidia, which was in accordance with one of the basic characters of the Brachyphoris genus (Fig. 2). However, D. pseudobrevistipitata differed from all members of Brachyphoris in having cylindrical conidia. In the phylogenetic analysis based on ITS, it fell into the Dactylella clade with a high bootstrap number, so D. pseudobrevistipitata is the only species with shorter conidiophores than conidia in the present accepted species of Dactylella (Chen et al. 2007a). To discover whether the species with such short conidiophores should be separated from Dactylella, it will be necessary to collect other sibling species to carry out further investigations.

Fig. 2

Dactylella pseudobrevistipitata. Holotype YMF 1.03504. a–d Conidiophores with conidia. e–n Conidia

The phylogenetic analysis showed that D. pseudobrevistipitata clustaled with D. vermiformis, but their morphological characters were entirely different. The latter had branched conidiophores and 0–1 septate conidia. D. pseudobrevistipitata was most similar to D. coccinella in conidia shape, but the conidiophores of the latter were not longer than its conidia, but longer than those of D. pseudobrevistipitata. Furthermore, the conidia of D. pseudobrevistipitata were narrower than those of D. coccinella, and the latter had 1–7 septate conidia. Their ITS similarity was only 70%. The two species cannot trap nematodes.

Besides D. coccinella, D. clavata RH Gao, MH Sun and XZ Liu, D. cylindrospora (RC Cooke) A Rubner, D. heptameres Drechsler and D. tenuis were somewhat similar to D. pseudobrevistipitata in conidia shape. However, the first three were separated from D. pseudobrevistipitata phylogenetically, and the septate number of conidia of these species was different from D. pseudobrevistipitata: D. clavata was 1–7, D. cylindrospora was 3–7, D. heptameres was 3–6 and D. tenuis was 1–4.

Interestingly, in the subclade formed by D. pseudobrevistipitata and other three species, another two species, D. vermiformis and D. coccinella, were also isolated from teleomorphs; only D. tenuis has not been connected with teleomorph. Whether Dactylella spp. isolated from teleomorphs are separated from other Dactylella spp. phylogenetically, will need other isolations to confirm.

Dactylella pseudobrevistipitata L. Qin, M. Qiao & Z.F. Yu, sp. nov. Fig. 2

Coloniae in agaro CMA, post 15 dies 25°C 30 mm diam. Mycelium sparsum, hyphis septatis, 3–4 μm latis. Conidiophora erecta, simplices vel ramose, 2.0–11.3 μm longa, 2.5 μm lata ad basim, 1.9–3.2 μm lata ad apicem. Conidia hyalina, clavata, 13.3–39.0 × 3.5–5.6 μm, 1–5 septata.

Colonies white, growing slowly on PDA, reaching 50 mm at 25°C after 15 days. Colonies white, aerial mycelium sparse on CMA, reaching 30 mm at 25°C after 10 days. Vegetative hyphae hyaline, branched and septate, 3–4 μm wide. Producing a large number of conidia in natural light after 7 days at room temperature. Conidiophores septate, unbranched, initially erect, collapse on the agar surface in less than 2 days, 2.0–11.3 μm high, Conidia cylindrical, straight or slightly curved, smooth, 1–5 transverse septate, the proportion of conidia with 2, 3, or 4 septate is 13.3, 51.1 and 26.7% respectively, 13.3–39.0 × 3.5–5.6 μm. No traps and capture ability have been detected in pure cultures when induced by P. redivivus.

Etymology: The species was named after its short conidiophores, but the name of Dactylella brevistipitata was used in Liu et al. (2005), which was transferred to Brachyphoris, we adopted Dactylella pseudobrevistipitata.

Holotype: YMF1.03504, permanent slide, near Thermal Spring, Yuan-Jiang Country, Yunnan Province, P. R. China, alt. 500 m, collected by Li Qin on Sep. 18, 2009.