MYCOTAXON. Volume 91, pp January March Dactylella coccinella sp. nov., an anamorphic species. 1

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1 MYCOTAXON Volume 91, pp January March 2005 Dactylella coccinella sp. nov., an anamorphic species YING YANG 1,2 & XING ZHONG LIU 1 liuxz@sun.im.ac.cn 1 Key Laboratory of Systematic Mycology & Lichenology Institute of Microbiology, Chinese Academy of Sciences P.O. Box 2714, Beijing , China 2 Graduate School of the Chinese Academy of Sciences Abstract Dactylella coccinella, isolated from the teleomorphic fungus Orbilia coccinella, is described as a new anamorphic species. Diagnostic characters that distinguish this anamorphic fungus from other Dactylella species are a simple conidiophore that bears a single, cylindrical, 1-7-septate conidium with a round distal end, a monoblastic conidiogenous cell, cylindrical and absence of nematode trapping capability. Key words anamorph species, ITS sequence Introduction The anamorphic genus Dactylella Grove is a heterogeneous group that has not yet been systematically studied. In our taxonomic research of Dactylella, an isolate derived from Orbilia coccinella (Sommerf.) Fr. by Benedict in October 1972, was studied for details. Comparison of its morphological and biological characters and analysis of sequence data of the rdna in ITS region suggest that the fungus is a separate species. Therefore, we describe it as a new anamorphic species, Dactylella coccinella. Materials and methods FUNGAL STRAINS The fungal strain Dactylella coccinella CBS , originally isolated by Benedict in October 1972 from Orbilia coccinella (Sommerf.) Fr., was kindly provided by Dr. Walter Gams from Centraalbureau voor Schimmelcultures. Dactylella clavata Gao, Sun & Liu (CBS ) was originally isolated by our lab and preserved in CBS. The cultures were maintained according to their specific requirements. GROWTH, MORPHOLOGY AND PREDACITY Fungi were cultured on Potato Dextrose Agar (PDA) and Cornmeal Agar (CMA) in the dark at 23ºC. Morphological characteristics and the growth rate were measured after incubation of two weeks. Trapping organs were induced by inoculation of challenging nematodes Panagrellus redivivus into a mm square slot at the margins of the colony where the agar was removed, and observed directly under a compound microscope after 1-3 days (Gao et al. 1996). Observation, measurement and photograph were carried out in water or

2 128 cotton blue lacto-phenol mounts under Olympus B51 microscope with differential interference contrast (DIC). All microscopic characteristics were measured from more than 50 individuals in water mounts. DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING Fresh mycelium was collected from the cultures growing on cellophane membrane on the PDA surface in Petri dish plates. Genomic DNA was extracted from the mycelium according to the method described by Doyle & Doyle (1987). The ITS region including the intervening 5.8S rdna, were amplified with the standard primers of ITS1 and ITS4 (White et al. 1990). The reactions were carried out in a thermal cycles with following parameters: initial denaturation at 95 C for 3 min, followed by 34 cycles consisting of denaturation at 94 C for 1 min, annealing at 54 C for 40 s and extension at 72 C for 1 min. A final extension at 72 C for 10 min followed. Primary PCR products were purified by Go3S PCR Product Purification Kit (Shenergy Biocolor BioScience & Technology Company, Shanghai, China) and sequenced on both directions with an ABI automated Sequencer (ABI 377) (Perkin Elmer) using ye-dideoxynucleotide chain-termination method. The edited sequences were deposited in GenBank (Table 1). PHYLOGENETIC ANALYSIS The determined ITS sequences were submitted to molecular phylogenetic analysis. Additional sequences (Table 1) were obtained from GenBank using direct queries for the genera involved, and using Gliocladium roseum Bainier as an outgroup (Hagedorn & Scholler 1999). An alignment of the rdna sequences was generated by the program package Clustal-X 1.81 (Thompson et al. 1994; 1997), and then it was manually realigned using BioEdit version (Tom Hall, Department of Microbiology, North Carolina State University, Raleigh, NC 27695). Phylogenetic analysis was performed using PAUP* 4.0 beta 10 (Swofford 2001) with gaps treated as missing data and all characters having equal weight. Taxonomy Dactylella coccinella sp. nov. FIGURE 1 Etym.: the species is named after its teleomorphic species name. Coloniae in agaro PDA effusae, post 14 dies 23 C 28 mm diam., pallide brunneae, hyphis aeriis sparsis. Hyphae superficiales ramosae, intertextae, frequenter anastomosantes, septatae, ad septa non constrictae, 2-3 µm latae, leves, hyalinae vel subhyalinae. Conidiophora hyalina, recta, simplicia, solitaria, septata, laevia, µm longa, basi 2-3 µm crassa, ad apicem angustata µm crassa. Conidia singularia, cylindrica, ad extremum distale rotundata vel obtusa, 1-7-septata, plerumque triseptata usque quinqueseptata, laevia, hyalina, µm longa, µm lata. Teleomorphus Orbilia coccinella (Sommerf.) Fr. Colonies when grown on PDA at 23 C for 14 days reach 28 mm in diameter, effuse, slightly brown, felty with sparse aerial mycelium and brown on the reverse. Mycelium compose of hyaline, septate, branched hyphae; hypae 2-3 µm wide. Conidiophores erect, unbranched, hyaline, septate, µm long, 2-3 µm wide at the base, and tapering to µm at the apex, bearing with only one holoblastic conidium. Conidiogenous cells monoblastic, integrated, terminal, cylindrical, hyaline, smooth.

3 129 Fig. 1. Dactylella coccinella (Holotype HMAS 87787). A-B. Conidiophores with attached conidia. C. Conidia. D. Magnification of conidia showing septa. Scale bar = 10 µm. Conidia cylindrical, straight or slightly curved, smooth, round to blunt at distal end, 1-7- transverse septate (mainly 3-5), µm. No chlamydospore has observed. No traps and capture ability has been detected in pure culture when induced by P. redivivus. Holotype: Specimen on microscope slide was deposited in Herbarium Mycologicum, Institute Microbiologici, Academiae Sinicae, HMAS Living culture CBS Discussion Dactylella Grove was first established in In the original description, the type species of the genus D. minuta Grove was incapable to prey nematodes (Rubner 1996). But this genus has been emended to include both non-predacious and predacious fungi based on their similar morphological characters (Subrammanian 1963, Schenck et

4 130 al. 1977, de Hoog & Oorschot 1985, Zhang et al. 1994). Rubner (1996) revised the generic characteristics of Dactylella and noted that, when the predacious capability was considered in partition of the genus, Dactylella must be restricted to non-predacious species. Scholler et al. (1999) accepted this amendment, and only placed non-predacious species in Dactylella according to their rdna sequence analysis. So we assign this nonpredacious fungus in Dactyella. D. coccinella is characterized by cylindrical, 1-7-septate conidia with round distal ends, simple conidiophore bearing only one conidium at apex, and no predacity. In conidia shape and number of septa, this fungus resembles Dactylella cylindrospora (Cooke) Rubner, Dactylella clavata Gao, Sun & Liu, Dactylellina asthenopaga (Drechsler) Scholler, Hagedorn & Rubner, Dactylella tenius Drechsler and Dactylella heptameres Drechsler, but can be distinguished from them via the following characteristics: D. cylindrospora has polyblastic conidiogenous cells and 1-4-septate conidium; D. clavata has branched conidiophores and clavate conidia with 1-7-septa; Da. asthenopaga produces obconical to clavate conidia with 3-septa and stalked knobs to trap nematode; D. tenius has clavate conidia with 1-4-septa; D. heptameres has polyblastic conidiogenous cells with geniculate proliferation bearing 5-10 conidia. By amplifying the ITS region, D. coccinella generated a 553 bp amplicon, and D. clavata a 534 bp amplicon. A Blast similarity search (Altschul et al. 1990) of D. coccinella on the consensus sequence of the ITS region demonstrated a close relationship with the genus Orbilia. A cladogram based on phylogenetic analysis of the ITS regions (Fig. 2) shows that D. coccinella is clustered together with all non nematode-trapping Dactylella and Fig. 2. Most parsimonious analysis with related species inferred from ITS DNA sequence data in PAUP * 4.0 beta 10. The number associated with each branch represents the percentage of 1000 bootstrap supporting that branch, with only bootstrap values >50% shown. Consistency index (CI) = , and Retention index (RI) = NCBI accession numbers are explained in table 1. K = adhesive knob; NC = non-constricting ring; C = constricting ring; N = adhesive network.

5 131 Orbilia species, while all nematode-trapping species form its own group which agrees with Scholler et al. (1999). This result also supports the inclusion of this new fungus in the genus Dactylella. Further more D. coccinella is clustered with Orbilia alnea (whose anamorph was Dactylella sp. without capture activity (Pfister 1997)) in a monophyletic clade with three bases different in 5.8s rdna regions, this indicates that the two species are phylogentically closely related and distant with other Dactylella species. Table 1. Species used in phylogenetic analysis Anamorph Predacious GenBank Teleomorph (following Scholler et al. 1999) devices Acc. # Dactylellina formosana Unknown Knob U51956 Dactylellina copepodii Unknown Knob U51964 Dactylellina huisuniana Unknown Knob U51965 Dactylellina candida Unknown Acknowledgements Knob & nonconstricting ring U51963 Dactylellina lysipaga Unknown Knob & nonconstricting ring U51959 Drechslerella polybrocha Unknown Constricting ring U51973 Arthrobotrys superba Orbilia fimicola Networks U72599 Arthrobotrys cladodes var. macroides Orbilia auricolor Networks U72592 Arthrobotrys gampsospora Unknown Networks U51960 Dactylella oxyspora Unknown None AF Dactylella cylindrospora Unknown None AF Dactylella clavata (CBS ) Unknown None AY Non-sporulating (DHP 146) Orbilia luteorubella None U72607 Non-sporulating (CBS ) Orbilia vinosa None U51981 Dactylella sp. Orbilia alnea None U72601 Dactylella coccinella (CBS ) Orbilia coccinella None AY Dicranidion sp. Orbilia delicatula None U72595 Gliocladium roseum Bionectria ochroleuca None AF We are grateful to Dr. Gams from Centraalbureau voor Schimmelcultures (CBS), Barrn, the Netherlands for providing the fungus; Drs W.Y. Zhuang and J.Y. Zhuang from Institute of Microbiology, the Chinese Academy of Science, Beijing, Dr W.P. Wu, from Novo Nordisk China, Beijing, and Dr S.D. Li from Biological Control Institute, Chinese Academy of Agricultural Science, Beijing for reviewing the manuscript and providing precious suggestions on this paper. This project is jointly supported by the National Natural Science Foundations of China (no and ).

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