mycological research 113 (2009) 1172–1180
journal homepage: www.elsevier.com/locate/mycres
Sphaerodes mycoparasitica sp. nov., a new biotrophic mycoparasite on Fusarium avenaceum, F. graminearum and F. oxysporum Vladimir VUJANOVIC*, Yit Kheng GOH Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A8 Canada
A new species, Sphaerodes mycoparasitica (Ascomycetes, Melanosporales), was isolated from
Received 6 April 2009
isolates of Fusarium avenaceum and Fusarium graminearum originating from wheat fields in
Received in revised form
Saskatchewan, and from Fusarium oxysporum originating from asparagus fields in Quebec,
19 July 2009
Canada. The species is characterized by a unique combination of ascospore size, shape (fu-
Accepted 20 July 2009
siform and triangular) and wall ornamentation (reticulate and smooth). Also, conidia are
Available online 1 September 2009
produced from simple phialides on the surface of ascoma peridial wall, on ascoma sur-
rounding hyphae, and on irregularly branched conidiophores arising from hyphae. The
Geoffrey Michael Gadd
closest relation of S. mycoparasitica is Sphaerodes quadrangularis, which has no detected anamorphic stage. The description of S. mycoparasitica, its phylogenetic positiondbased on
DNA sequences of large subunit ribosomal RNA gene (LSU)das well as a key for all known
Sphaerodes species are provided.
ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Fusarium Melanosporales Sphaerodes
Introduction Study of biotrophic mycoparasites of Fusarium on wheat and asparagus crops throughout Canada resulted in the isolation of a fungus belonging to genus Sphaerodes. Although, at first glance, the fungus resembles Melanospora species erected by Corda (1837), many of its taxonomical features differ from Melanospora when considering both the original description of the genus by Clements (1909) and recent revisions of allied genera by Cannon & Hawksworth (1982). Cannon & Hawksworth (1982) provided the first comprehensive classification of Melanosporalean fungi; Melanospora species were noted to posses smooth, ellipsoidal to citriform ascospores and non-tuberculate germ pores. Species having ascospores with different
shapes and wall ornamentations were segregated into Persiciosporadspecies with a delicately pitted ascospore walldand Sphaerodesdspecies with complex raised tubercle-like germ pore and a usually coarsely reticulate ascospore wall (Cannon & Hawksworth 1982; Zhang & Blackwell 2002). In earlier studies, de Bary (1887) and Jordan & Barnett (1978) noted that Melanospora species establish intimate relationships with Fusarium. Recently, Harveson & Kimbrough (2000, 2001b) reported Melanospora, Persiciospora and Sphaerodes in association with distinct formae speciales of Fusarium oxysporum. Taxonomy of Melanosporales with mycoparasitic habit was very conflicting due to their unique combination of ecological and morphological features. Melanosporalean strains
* Corresponding author. Tel.: þ1 (306) 966 5048; fax: þ1 (306) 966 8898. E-mail address: [email protected]
0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.07.018
New Sphaerodes species
had been placed in Hypocreales, Ceratostomataceae of the Sordariales, or Diaporthales. Also, previous phylogenetic studies based on rDNA sequences suggested that Melanospora was within or near Hypocreales (Spatafora & Blackwell 1994; Zhang & Blackwell 2002). However, when analyzed with more taxa of the Sordariomycetes, Melanospora formed a distinct clade outside Hypocreales (Castlebury et al. 2004). A more recent study by Zhang et al. (2006) based on a fourgene phylogeny further supported the exclusion of Melanospora from Hypocreales (Hibbett et al. 2007). Furthermore, a close relationship between Coronophorales and Melanosporales was recognized (Zhang et al. 2006). Similar morphological and ecological features of the two orders include a pseudoparenchymatous ascomatal wall, clavate, deliquescent asci, lack of paraphyses (with a few exceptions in Coronophorales) and, often, a mycoparasitic habit (Zhang et al. 2006). The fungus described in this study is unlike any of the known species of Sphaerodes, bearing a unique combination of morphological characteristics dnotably about ascospore shape and size. However, due to the particular combination of morphological traits (Miller & Huhndorf 2005), its inclusion in melanosporaceous genera was problematic (Table 1). Therefore, LSU rDNA sequences were used as alternative characters for assessing the phylogenetic importance of the distinctive morphological features in the fungus. Hence, these data together with morphological features showed that the fungus was best accommodated in Sphaerodes and here we describe it as a new species. To the best of our knowledge, this is the first report of Sphaerodes association with Fusarium avenaceum and Fusarium graminearum.
Materials and methods Sampling, fungal growth and microscopy Myclobutanil-agar (MBA) medium was used for selective isolation of various Fusarium taxa (Vujanovic et al. 2002) and associated biotrophic mycoparasites from Canadian agriculture fields. Sphaerodes was recovered occasionally from Fusarium graminearum and abundantly from Fusarium avenaceum isolates originating from wheat fields in Saskatchewan (2005); it was also isolated from Fusarium oxysporum from asparagus fields in Quebec (2003), Canada. A monosporal, single culture of the mycoparasite was obtained from each Fusarium species according to the method proposed by Harveson & Kimbrough (2001a). Single ascospore isolates were maintained on Potato dextrose agar (PDA) (Difco, Becton Dickinson Diagnostics, Sparks, Maryland) supplemented with antibiotics (100 mg l1 streptomycin sulphate and 13 mg l1 neomycin sulphate) (Sigma–Aldrich, St Louis, Missouri) and stored at 80 C in Saskatchewan Microbial Collection and Database (SMCD 2220/01-03) and in the International Depositary Authority of Canada (IDAC301008-01) collections. Fungal growth was assessed on modified Leonian’s agar (MLA) and Potato dextrose agar (PDA) media (Malloch & Cain 1971). Biotrophic interactions between Sphaerodes and Fusarium strains were examined with the slide culture method proposed by Jordan & Barnett (1978). Morphological studies of ascomata, ascospores, mycelia, and anamorphic structures were performed
after two weeks of incubation (21–22 C) under a Carl Zeiss Axioskop2 with a Carl Zeiss AxioCam ICc1 camera. Fungal materials for microscopic observation were mounted in lactofuchsin and lactophenol cotton blue dyes.
DNA extraction, amplification and sequencing Three Sphaerodes strains: SMCD 2220-01 on Fusarium avenaceum from wheat, SMCD 2220-02 on Fusarium graminearum from wheat, and SMCD 2220-03 on Fusarium oxysporum from asparagus were cultured on PDA medium at 21 C for a week prior to DNA extraction. Genomic DNA was extracted with the DNeasy Plant Mini Kit (Qiagen Inc., Mississauga, ON). LSU (large subunit) rDNA fragments were amplified using primer sets NS1/NS6 (Gardes & Bruns 1993; White et al. 1990) and LS1/LR5 (Hausner et al. 1993; Rehner & Samuels 1995; Zhang & Blackwell 2002). Target regions of fungal genomic DNA samples were amplified using polymerase chain reaction (PCR) in a 25 ml reaction mixture containing 2.5 ml of 10X buffer, 5 ml of Q buffer, 0.5 ml 10 mM dNTPs, 1 ml of each primer, 0.13 ml of 0.625 unit of Taq DNA Polymerase, 2 ml of extracted fungal DNA, and 12.87 ml of sterilized ultra-pure Millipore water. Qiagen Taq PCR core kits were purchased from Qiagen Inc., Mississauga, ON. Purified DNA PCR products were sent to Plant Biotechnology Institute (PBI), Saskatoon, SK, for sequencing.
Sequence alignment and phylogenetic analyses Sequences of LSU from this study and sequences retrieved from GenBank were aligned using Clustal X software (version 1.82) (Thompson et al. 1997), and edited in Bioedit (Hall 1999). Distance trees were produced with PAUP (Phylogenetic Analysis Using Parsimony) 4.0b10 software (Swofford 2000) using a neighbor-joining approach, and validated using bootstrap analyses with 1000 repetitions. A fungal distance tree was prepared with sequences showing bootstrap values higher than 50 %. Trees were rooted with sequence Xylaria hypoxylon U47841. LSU sequences from the specimen of Sphaerodes mycoparasitica SMCD 2220-01/ IDAC301008-01 and Sphaerodes quadrangularis CBS112763 were submitted to GenBank under accession numbers: FJ748916 and GQ354530.
Taxonomy Sphaerodes mycoparasitica Vujanovic, sp. nov. (Figs 1–5) MycoBank no.: MB500012 Coloniae in agaro potato dextrosum lentior crescents, 4.0 cm ad 7d, floccose, pallido-brunneis. Hyphis septatis, ramosis, anastomosantibus, laevibus, palide fulvis, 2.5–5.0 mm diam compositum. Ascomata superficialia vel immersa, pyriformia vel globosa, ostiolata, flavo-brunnea, 250–300 mm longa, 200–280 mm diam. Collum nul, conicum vel cylindricum, 30–75 mm longum, (0–) 50–70 mm latum ad basim. Peridium membranaceum, cellulis 8–15 mm, e 3–6 stratis, 8–15 mm crassum, textura angulari compositum. Setae rectae vel parum curvae, hyalinae vel dilute flavae, crassitunicatae, 10–40 mm longae, septatae. Asci 8-spori, ovoidei vel clavati,
Table 1 – Synopsis of main characteristics of Melanosporaceous taxa Character
Superficial to immersed
Ostile Perithecial neck
Usually absent Absent to short cellular
Absent to rarely present
Brown to dark brown
Superficial to immersed Usually absent Absent to short, cellular Absent to rarely present Clavate to ellipsoid Dark brown to black Citriform to fusiform
2 terminal strongly apiculate pores
Usually coarsely reticular
2 terminal strongly apiculate pores Usually coarsely reticular
Melanospora Absent (rarely present) Superficial Usually present Absent to long cellular Present Clavate (rarely ellipsoid) Brown Citriform to ellipsoid (to discoid) 2 not or slightly apiculate pores Smooth
Immersed to Superficial Present Short, cellular
Present Long cellular
Present Long, hyphal
Superficial to subimmersed Present Long, hyphal
Present Long, hyphal
Superficial to immersed Present Long, hyphal
Absent to very short Clavate to cylindric Brown
Absent to present Clavate
Clavate to ovoid
2 slightly apiculate pores Weakly pitted
1 sunken pores
Oblongellipsoid to quadrangular 2 wide slits
Spheroid to ovoid Hyaline to pale brown Oblong to allantoid
1 narrow slit developing late
2 terminal crateriform pores Smooth
Sphaeronaemella Syspastospora Viennotidea
1 small subterminal pore Smooth
V. Vujanovic, Y. K. Goh
New Sphaerodes species
Fig 1 – Sphaerodes mycoparasitica culture after two weeks of incubation on (A) Modified Leonian’s agar and (B) Potato dextrose agar – upper sides, (C) and (D) – down sides.
50–75 17–25 mm, superne late rotundati, brevistipitati, tenuitunicati, evanescentes. Paraphysis nullis. Ascosporae unicellulares, irregulariter biseriatae, primum hyalinae, deinde brunneae vel atrobrunneae, crassitunicatae, fusiformes, 18–24 9–12 mm, reticulatae, costis protrudentibus, e polo visae polygonales, utrinque umbonatae, foramine germinali praeditae. Phialidis hyalinis, status conidialis.
2.5–5.0 mm diam., septate, anastomosis occurred soon after ascospore germination (Fig 2). Colonies on Potato dextrose agar (PDA) spread with abundant, white to pale yellow aerial mycelium and low number of ascomata. Ascomata,
Typus: Canada: Saskatchewan, on Fusarium avenaceum from wheat, Saskatoon, SK, Canada, 10 Sep. 2005, V. Vujanovic (holotypus: SMCD 2220-01; isotypus: IDAC301008-01). Etymology: mycoparasitica; referring to fungus parasitic on another fungus. Host range: Associated with F. avenaceum and Fusarium graminearum on wheat, and Fusarium oxysporum on asparagus. Cultural characteristics: Colonies on MLA grew more rapidly than on PDA, 1.1 cm versus 0.6 cm per day (21–22 C), consisting of slightly submerged mycelium and aerial hyphae, granulose due to production of ample number of ascomata. On MLA and PDAdproducing a woolly mycelium, yellowish to pinky-brownish on both sides (Fig 1). At 37 C, no growth was registered. Hyphae were white to pale yellow,
Fig 2 – Sphaerodes mycoparasitica ascospore germination, showing single- and double-polar germination patterns as well as hyphal anastomosis (arrow) formation pattern.
V. Vujanovic, Y. K. Goh
Fig 3 – Phylogenetic tree based on LSU rDNA sequences showing the position of Sphaerodes mycoparasitica (in bold). Bootstrap values of 50 % or greater from 1000 bootstrap replications are indicated for the corresponding branches.
perithecial or cleistotecial, scattered or aggregated in small groups, superficial, pyriform to globose, ostiolate (when mature), light to dark yellowish brown, translucent, appearing black due to mass of mature ascospores, 250–300 mm high, 200–280 mm diam. Neck absent to short conical or cylindrical 25–75 mm long, 20–70 mm wide at the base, sometimes surrounded with a crown of short, upright setae, 10–40 mm long. Peridium membranaceous, 3- to 6-layered, 8–10 mm thick, translucent, pale yellow to light brown, composed by cells of 8–15 mm diam. disposed in textura angularis. Asci
8-spored, clavate, 50–75 17–25 mm, rounded at apex, without apical structures, thin-walled and evanescent when mature. Paraphyses absent. Ascospores irregularly arranged inside the asci, at first hyaline but becoming brown to dark brown, thick-walled, single-celled, fusiform to rarely triangular, 18–24 9–12 mm, reticulate to rarely smooth, with irregular transverse sections, and with a strongly umbonate germ pore at each end. Phialides hyaline, ampulliform produced directly on ascomata or on hyphae surrounding ascomata, and on irregularly branched conidiophores.
New Sphaerodes species
Fig 4 – Sphaerodes mycoparasitica: (A) Ascoma, (B) Hyaline seta arising from the neck (arrow), (C) Reticulate ascospores (arrows), (D) Smooth ascospore (arrow), (E) Triangular ascospore (arrow), (F) Phialides produced on ascoma surrounding hyphae, (G) Ampulliform phialide arising from the surface of ascoma peridial wall, (H) Formation of mature and starting ascomata, (I) Formation of hook-like structures by S. mycoparasitica parasitizing on living hypha of Fusarium oxysporum (arrows) and (J) Large view of hook-like structure on living hypha of Fusarium avenaceum. Bar scales for A and H 50 mm, for B, C, D, E, F, G and J 10 mm, for I 25 mm.
V. Vujanovic, Y. K. Goh
Fig 5 – Sphaerodes mycoparasitica ascospores showing the conspicuous wall ornamentation and prominent irregular longitudinal ribs (arrows); (B) Sphaerodes quadrangularis ascospores. Bar scales 5 mm.
Additional cultures examined: Sphaerodes quadrangularis CBS112763; GenBank accession number GQ354530.
Results and discussion Molecular phylogenetic and phenotypic analyses were conducted on Sphaerodes isolated from Fusaria in wheat and asparagus fields. Sphaerodes isolates from Fusarium avenaceum, Fusarium graminearum and Fusarium oxysporum have identical LSU-rRNA gene sequences (1266 bp). Furthermore, all three isolates demonstrate typical Sphaerodes mycoparasitica phenotypic characters (Table 1) on both MLA and PDA (Fig 1) media. Sequence alignments and parsimony analyses of the LSU sequences classify S. mycoparasitica within Sphaerodes in a well-supported clade of Melanosporales (Fig 3). The node, from which all four Sphaerodes species branch, received 95 % bootstrap support (Fig 3). Results are also in agreement with those of Zhang & Blackwell (2002), i.e. confirm that Melanosporales forms a clade with Coronophorales. Our species is typical of Sphaerodes, showing such morphological features as cleistothecial or ostiolate perithecia with short or no neck (Table 1). As other members of the Sphaerodes–Melanospora clade, necks, in S. mycoparasitica, have a cellular rather than a hyphal structure and are fringed with hyaline setae (Fig 4B) (Cannon & Hawksworth 1982). Species with smooth, ellipsoidal to citriform ascospores and non-tuberculate germ pores are classified in Melanospora; those with wall ornamentations or ascospores of other shapes are segregated into other genera (Cannon & Hawksworth 1982; Zhang & Blackwell 2002). For example (Table 1), Persiciospora species have a delicately pitted ascospore wall and Syspastospora have smooth, cylindrical ascospores, while Sphaerodes species have a complex raised tubercle-like germ pore and, usually, a coarsely reticulate ascospore wall (Cannon & Hawksworth 1982; Horie et al. 1986). The raised rim around the germ pore is also a feature of Sphaerodes and not of Melanospora (Chaudhary et al. 2006). Features of the ascomata (Table 2), the reticulate
ascospores with irregular transverse sections, and the raised rim around the germ pore placed S. mycoparasitica close to Sphaerodes quadrangularis D. Garcia, Stchigel & Guarro (Garcı´a et al. 2004). However, a unique combination of featuresdascomata height >250 mm, conical to cylindrical neck (Fig 4A), setae length