North American Fungi Volume 8, Number 10, Pages 1-13 Published June 19, 2013 Vialaea insculpta revisited R.A. Shoemaker, S. Hambleton, M. Liu Biodiversity (Mycology and Botany) / Biodiversité (Mycologie et Botanique) Agriculture and Agri-Food Canada / Agriculture et Agroalimentaire Canada 960 Carling Avenue / 960, avenue Carling, Ottawa, Ontario K1A 0C6 Canada Shoemaker, R.A., S. Hambleton, and M. Liu. 2013. Vialaea insculpta revisited. North American Fungi 8(10): 1-13. doi: http://dx.doi: 10.2509/naf2013.008.010 Corresponding author: R.A. Shoemaker: [email protected]. Accepted for publication May 23, 2013 http://pnwfungi.org Copyright © Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada Abstract: Vialaea insculpta, occurring on Ilex aquifolium, is illustrated and redescribed from nature and pure culture to assess morphological features used in its classification and to report new molecular studies of the Vialaeaceae and its ordinal disposition. Tests of the germination of the distinctive ascospores in water containing parts of Ilex flowers after seven days resulted in the production of appressoria without mycelium. Phylogenetic analyses based on a fragment of ribosomal RNA gene small subunit suggest that the taxon belongs in Xylariales. Key words: Valsaceae and Vialaeaceae, (Diaporthales), Diatrypaceae (Diatrypales), Amphisphaeriaceae and Hyphonectriaceae (Xylariales), Ilex, endophyte. 2 Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 Introduction: Vialaea insculpta (Fr.) Sacc. is on oatmeal agar at 20°C exposed to daylight. a distinctive species occurring on branches of Isolation attempts from several other collections Ilex aquifolium L. Oudemans (1871, tab. v, f.9) yielded only contaminants. The extractions were illustrated the asci and ascospores as did performed using the UltraClean Microbial DNA Saccardo (1896, Tav. V. fig. 10a,b) but Saccardo isolation kit (Mo BIO Laboratories, Inc. gave a misleading illustration of perithecia Carlsbad, CA USA) according to the immersed in a surrounding stroma (fig. 10c), manufacture’s protocol. A fragment of ribosomal which may have influenced later authors in the RNA gene 18S small subunit was amplified using ordinal disposition. Cannon (1995) reviewed the a primer pair NS1 and NS6 (White et al 1990). history of the various placements of V. insculpta PCR was performed in 10 µl reactions containing as follows: Amphisphaeriaceae (Diatrypales) 0.1 mM dNTPs (Invitrogen Canada Inc. (Chadefaud 1957, 1960, 1961); ‘Vialaeacéés’ Burlington, ON), 2 pmoles of each primers, 1 µl (Diatrypales) (Schrantz 1960); Diaporthaceae 10× TitaniumTm Taq buffer, 0.1 µl 50× (Sphaeriales) (Müller & von Arx 1962); TitaniumTm Taq DNA Polymerase (BD Amphisphaeriaceae (Xylariales) (Müller & von Biosciences, Mississauga, ON). Thermal cycling Arx 1973; Barr 1978; Dennis 1978; Hawksworth conditions were set as: 95°C for 3 min followed et al. 1983; Redlin 1989; Eriksson & by 36 cycles of 95°C for 30 sec, 63°C for 20 sec, Hawksworth 1993); Hyponectriaceae and 72°C for 2 min, followed by a final extension (Xylariales) (Barr 1990). Cannon (1995) of 72°C for 8 min and hold at 10°C. PCR proposed the new family Vialaeaceae, which he products were direct-sequenced using BigDyeTm tentatively placed in Diaporthales. Kirk et al. Terminator v. 3.1 Cycle Sequencing Reaction Kit (2008) placed Vialaeaceae in an uncertain (ABI Prism/ Applied Biosystems, Streetsville, position within Sordariomycetidae. These ON) in 10 µl sequencing reactions using the PCR dispositions were made on the basis of primers. The reaction mix included 1.75 µl 5× morphology. The current molecular study based Buffer, 0.5 µl 2.5x BDT sequencing Mix and 1.6 on a culture that produced mature perithecia in pmol primer. Amplicons were purified by vitro, and a similar study based on fresh ethanol/sodium acetate precipitation and perithecia dissected from Ilex aquifolium stems analyzed by Applied Biosystems 3130×1 Genetic adds new information on the classification of the Analyzer (AppliedBiosystems, Streetsville, ON). organism. Phylogenetic analyses based on a Sequences were edited using SequencherTM ver fragment of ribosomal RNA gene small subunit 4.8 (Gene Codes Corporation, Ann Arbor, MI). suggest that the taxon belongs to Xylariales. Cultural studies have been impeded by the lack of germination of the ascospores and, in our Phylogenetic analyses: experience, by the frequent occurrence of fungus Sixty-seven DNA sequences of 18S small subunit contaminants: Phoma sp., Diaporthe sp., ribosomal RNA gene were downloaded from Pullularia sp., and Diplodia ilicicola Desm. GenBank by searching nucleotide sequences in Sordariomycetes generated by AFTOL. After preliminary analyses showing that Vialaea is Materials and Methods located in Xylariales, 26 more DNA sequences DNA extraction, PCR and sequencing: DNA from Xylariales were included. Finally ninety- was extracted from whole perithecia dissected four DNA sequences (Table 1) were included in a from twigs of specimen DAOM 240257, and parsimony analysis. Sequences were aligned from the perithecia and mycelium of vegetative with MAFFT version 6 isolate, DAOM 240860, that produced (http://mafft.cbrc.jp/alignment/server/index.ht perithecia, asci and ascospores in pure culture ml). Heuristic search settings were as follow: Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 3 starting tree obtained by random stepwise and illustrations of the microscopic features of addition, one tree held at each step during asci and ascospores were given by Oudemans stepwise addition, branch-swapping by tree- (1871, p. 30, Pl. V, Figs. 9, 10) and by Saccardo bisection-reconnection (TBR), 20 replicates, (1896, p. 67, Tab. V. fig. 10 b,c) who in fig. 10c number of re-arrangements per replicate limited illustrated several perithecia immersed in to 1,000,000. For bootstrapping analysis, 200 stroma, above the wood, with the tips of replicates were conducted with heuristic search individual beaks protruding but barely evident settings as mentioned previously. above the raised surface layer of host material. He described the structure as follows. Results “Stromatibus laxe gregariis, pustulatis, Vialaea insculpta (Fr.) Sacc., Bull. Soc. Mycol. epidermide tumidula velatis, 2 mm diam., intus France 12: 66. 1896 flavidis.” From recent examinations, the typical ≡ Sphaeria insculpta Fr.: Fr., Elench. Fung. position is intraepidermal with flush beaks 2: 95. 1828 surrounded with a well developed clypeus and ≡ Zignoëlla insculpta (Fr.: Fr.) Sacc., Syll. the naked spherical part of the perithecium Fung. 2: 225. 1883 situated below without any surrounding stroma ≡ Boydia insculpta (Fr.: Fr.) Grove, J. Bot., as shown by Redlin (1989, Fig. 2) and quite London 59: 13. 1921 unlike the stromatic Diaporthales. = Boydia insculpta forma sparsa Grove, J. Bot., London 59: 15. 1921, nom. inval. Morphology: Perithecia solitary or a few in a = Boydia remuliformis A.L. Smith, Trans. group. Clypeus surrounding the upper beak, up Brit. Mycol. Soc. 6: 151. 1921 to 400 μm diam., composed of an upwardly = Cryptospora ludwigiana Kirschst., aligned pallisade of rectangular to globoid cells Hedwigia 91: 195. 1944 beneath the cuticle and above the phellem (Fig. 1). Beak immersed in clypeus, with a wall 16-20 Symptoms: The disease of Ilex aquifolium, μm wide of 2-5 layers of 4-7 μm thin-walled English holly, is a die-back of branches with or textura globosa, 300-400 μm long, 100-130 μm without conspicuous cankers but with wide. Ostiole 60-80 μm wide, filled with numerous, small, white discs of raised outer wall numerous, hyaline, periphyses, 2-3 μm wide of the epidermis, often circumscribed by a (Fig. 11). Perithecium wall in surface view a clypeus, which appears as a brown-pigmented textura globosa of 12-20 μm thin-walled cells ring. Within the clypeus are a number of very (Fig. 2); in section, 20-50 μm thick of 3-6 layers small points, which are the flush apices of of mainly rectangular cells 15-20 x 8-12 μm; perithecium beaks. The brown circular edge of body 300-450 μm. Paraphyses not observed. the clypeus appears as though engraved under See the description and illustration of short, the upper half of the split epidermis of the bark septate, tapered paraphyses by Cannon (1995). (insculpta). The Canadian collections examined Asci free-floating at maturity, unitunicate, with usually do not include conspicuous cankers. 8 bi- to triseriate spores (Fig. 8), 70-90 x 6-8 However, Atkinson & Trelawny (1962) μm. Apical apparatus a coussinet and illustrated cankers they observed at Saanichton, manubrium 5-6 μm diam distally, slightly B. C. in a commercial planting. Dennis (1968, narrowed to 3-4 μm proximally, 6-8 μm long Fig. 23c) illustrated the usual appearance of the (Fig. 7), subtended by a flat subapical ring 3 μm fungus in branches without cankers. For some diam. IKI+ (blue) (Fig. 12). Ascospores of two years, the disease was known as Boydia canker fusoid parts joined by a narrow isthmus, curved, and attributed to Boydia remuliformis A.L. 70-90 x 6-8 μm, transversely 0-1-septate (in Smith, a later synonym. An early description isthmus), rarely with two close septa, septum 4 Shoemaker et al. Vialaea inscupta. North American Fungi 8(10): 1-13 thin, ascospore wall very thin, hyaline, cell with the peak season September to December content granular, multiguttulate, later with one and March through April, if rainfall was large guttule, surface smooth, (Fig. 3), without adequate. Adrian Leuchtmann who has studied evident appendages or sheath (in India ink). many endophytic fungi suggested that spores The clypeus that raises the outer wall of the stem may have dormancy and germinate on newly epidermis is conspicuous on diseased plants but opened buds in the spring (pers. comm. 19 Sept. absent around the long cylindrical beak of 2003). Species within the genus Ilex are perithecia formed in agar cultures. Redlin dioecious. The ascospores might be adapted to (1989) found that a clypeus could be induced to selectively infect opening female flowers perhaps form in culture in response to contact aided by pollinating bees as vectors.