Robillarda sessilis, a rare coelomycete isolated from Scots pine seedlings EUGENE YURCHENKO and DASHA BELOMESYATSEVA Laboratory of Mycology, V.F. Kuprevich Institute of Experimental Botany Akademichnaya Street 27, BY-220072 Minsk, eugene_yu@tut.by Yurchenko E., Belomesyatseva D.: Robillarda sessilis, a rare coelomycete isolated from Scots pine seedlings. Acta Mycol. 45 (1): 27–32, 2010. A coelomycete with appendage-bearing conidia, R. sessilis, was isolated three times from stems of living healthy Pinus sylvestris seedlings of the 1st year growing in a nursery in central Belarus. Macroscopic and microscopic morphology of the fungus in culture is described. Key words: Belarus, nursery, Pinus sylvestris, stem INTRODUCTION Robillarda sessilis (Sacc.) Sacc. was found during studies of the fungi inhabiting stems of healthy Pinus sylvestris seedlings in a sample nursery in central Belarus. This species is a remarkable fungus with conidia bearing three long hair-like ap- pendages. It was selected as a generic type for Robillarda, an anamorphic genus with unknown teleomorph (Nag Raj 1993). Altogether 35 species were published under this generic epithet, 12 of which were referred, according to Nag Raj (1993), to seven other genera. MATERIAL AND METHODS The inventory of the fungi was carried out by sampling plants in 1 m2 plots on a 800 m2 plantation situated in central Belarus. A first sample (taken 2 VI 2009) included 5 well-developed seedlings from a single plot located in the center of plantation. A second sample (taken 22 VI 2009) included 25 equally well-developed seedlings collected from 5 plots (5 plants per plot). Four plots were situated near corners of ACTA MYCOLOGICA Vol. 45 (1): 27–32 2010 Dedicated to Professor Barbara Gumińska on the occasion of her eighty-fifth birthday 28 E. Yurchenko and D. Belomesyatseva the plantation, one plot in the plantation center (this central plot position did not coincide with the plot in previous sampling). For isolation of the fungi, the seedlings were washed 3 min under a strong stream of tap water on a sieve. Each seedling stem, from the root neck to the foot of leaf whorl, was cut using a sterile razor blade into ca 2 mm long segments. The segments were put on malt extract agar (MEA: 1% malt extract, Amresco, USA, and 1.5% agar) with addition of tetracycline 30 μg/mL (Amresco), and incubated for 10 days at 26°C. For the control that the propagules did not originate from tap water, 1.5 mL of the water was poured in Petri dish with solid 2% MEA, in 3 replicates. Mycelia growing from stem pieces and producing pycnidia, were transferred for storing on MEA slants under mineral oil. For describing cultural morphology, isolates were taken from storage and cultured on MEA (2% malt extract, 1.5% agar) at 26°C in the dark. For describing micromorphology, preparations were mounted in 3% KOH water solution. Dry cultures were deposited in V.F. Kuprevich Institute of Experimental Botany Herbarium (MSK-F). RESULTS It the first sample from the plantation center (2 VI 2009), one isolate of R. sessilis was obtained. In the second sample (22 VI 2009) two isolates were obtained from 2 of 25 studied plants, one from central, one from a corner plot. The morphological diagnosis of R. sessilis, based on cultures, is given below. Morphological description. After 1 week: mat 25 mm in diam, more or less rounded, colorless, low felty, near 1 mm high, more or less fasciculate or tufted in periphery zone and just near the point of inoculation; margin somewhat wavy, abrupt, with scarce cilia-shaped hyphae; reverse pale apricot yellow. After 2 weeks: mat about 50 mm in diam, very pale pinkish cream, in central and middle zone floc- cose-felty; periphery zone clearly delimited, ca 10 mm wide, low velvety, with slight radial depressions; margin without aerial growth, transparent; reverse in central and middle zone brownish yellow with orange tint, with blackish or dark olive spots due to the presence of conidiomata. Conidiomata occurring about 1.5–2 weeks post inoculation, especially developing when the medium dries up, in mat periph- ery and middle zone scattered, but at the border between these zones abundant and often aggregated, forming a ring, having dark brown color in reverse (Fig. 1). Aerial hyphae moderately branched, somewhat wavy, but with even walls, smooth, hyaline or subhyaline, narrower ones (2-3.5 μm) long-celled and small-guttulate, wid- er ones (3.5-7.7 μm) with short swollen cells and rich-guttulate; thin lateral branches 0.8-1 μm wide, tapering. Repent and immersed hyphae moderately branched, with short more or less swollen cells, rich-guttulate, often disintegrating at septa, 1.7-8.7 μm wide. Marginal hyphae rather richly branched, divided into axial hyphae and their lateral branches; axial hyphae 4.3-8.2 μm wide, with short, more or less swollen, somewhat guttulate cells; lateral branches 3-4 μm wide, having blunt apices 2-3.2 μm wide (Fig. 2). Fig. 1. Two-weeks old culture of Robillarda sessilis on 2% MEA: face of the mat (above) and reverse (below). Scale bar = 1 cm. Robillarda sessilis 29 Conidiomata pycnidioid, solitary, 100-150(-500) μm in diam, or aggregated 3-10 and more and partly confluent (aggregations to 1-1.5 mm in diam), globose or irregular, flattened when large, fully or partly immersed, black, glabrous, glossy, with uneven surface, ostioles not visible; conidiomatal wall ca 25-35 μm thick, of pseudoparenchymatic cells, outer layer dark brown, 7-10 μm thick, mainly of tex- tura angularis (partly also of narrow textura prismatica) with somewhat thick-walled cells 4-13(-17.5)×(1.5-)2.2-7 μm, turning into a hyaline inner layer of thin-walled and guttulate cells 7-30×2.5-5 μm. Conidiophores reduced, mostly one-celled, in shape of somewhat elongated conidiogeneous cells, originating from pseudopa- renchyma lining the inner surface of the locule and indistinctly differring from pseudoparenchymatic cells. Conidiogeneous cells ampulliform to subcylindrical, hyaline, thin-walled, smooth, 6-11×2-3 μm. Conidiogenesis holoblastic with limited sympodial proliferation (sometimes two conidia simultaneously developing on the same cell); conidia originating at apices of conidiogeneous cells, often sitting on very small protuberances or denticles, at early developmental stages without ap- pendages; in a layer with many developing conidia, appendages situated parallel or divergent at a very sharp angle. Conidia 2-septate, consisting of a two-celled conid- ium body and an apical cell with appendages; conidium body medianly 1-euseptate, fusiform, straight or a little curved, often very little constricted at the septum, ba- sally more or less truncated, subhyaline to pale brown, usually yellowish, smooth, thin-walled, 9.3-12(-14.2)×2.7-3.5 μm; apical cell hyaline, very thin-walled, short cylindrical basally for 1-2 μm, divided into 3 hyaline, smooth, hair-like, divergent, straight or curved, more or less apically attenuated appendages 17-26 μm long and 0.5-0.6 μm wide in the middle part; older conidia in preparation often loosing api- cal cell (Fig. 3). Substrata and general distribution. R. sessilis was documented from quite variable hosts and substratum types: on stems (bark), dead branches, leaves (caus- ing leaf spot), seeds of Bischofia, Cocos, Ficus, Fragaria, Fumana, Ludwigia, Magno- lia, Paeonia, Quercus, Randia, Rosa, Rubus, Vitis. The fungus has been recorded in Europe (Hungary, Italy), Asia (India), North America (USA), Caribbeans, Africa (Angola), and on the plant material imported from Japan. The majority of records are from India (Nag Raj 1993). In 1970 this species was described from Northea seeds imported to St-Petersburg, Russia, from Indonesia, under the name Mycohyp- allage northeae (Melnik 1970). Gasich (1995) collected this fungus in Saratov oblast, Russia, where it caused leaf spot disease of Eryngium (see also Melnik 1997). So far, five localities, including the type locality in Italy and our find, but excluding the find on imported seeds, are know from Europe today. In Nebraska, USA, R. sessilis was collected on Pinus ponderosa, but details about the age of the plants or the kind of organs are unknown (Nag Raj 1993). The fungus was also isolated from soil in Aus- tralia (Matsushima 1989) and from a soil sample collected in mountain pine forest in Pakistan (Matsushima 1993). Our case indicates that R. sessilis is capable of infecting stem tissues of healthy, vigorous Pinus sylvestris seedlings as either an endophyte or phylloplane component. The fungus can be named more widely ‘associated with seedling’ because of used method of sterilization. Conidiomata were not observed on this host. Only three plants colonized by the fungus, were detected among 30 ones subjected to cultural experiment, and thus the species can be regarded as rare in the studied plantation. 30 E. Yurchenko and D. Belomesyatseva Fig. 2. Robillarda sessilis hyphae in culture: a – aerial, b – repent and immersed, c – marginal. Scale bar = 10 μm. Robillarda sessilis 31 Material examined: Belarus, Minsk oblast, Dzyarzhynsk district, near Enerhetykau settlement, Ba- sic Forest Nursery of Belarusian State Technological University, plantation of 1st year Pinus sylvestris, coll. E. Yurchenko 2 VI 2009 (MSK 7065); ibid., coll. E. Yurchenko 22 VI 2009 (MSK 7138a, b). Acknowledgements. The authors are grateful to Dr G.J.M. Verkley (Fungal Biodiversity Centre, Cen- traalbureau voor Schimmelcultures, Utrecht, the Netherlands) for critical consideration of the MS. This work is a part of the study supported financially by grant B09-126 of Belarusian Republican Foundation for Fundamental Research. Fig. 3. Robillarda sessilis: a – conidiophores and developing conidia, b – mature conidia with appendages, c – conidia that lost apical cell MSK 7065. Scale bar = 10 μm. 32 E. Yurchenko and D. Belomesyatseva REFERENCES Gasich E. L. 1995. Robillarda sessilis (Sacc.) Sacc. – the first record in Russia. Mikologiya i fitopatologiya 29 (5/6): 16–18. Matsushima T. 1989. Matsushima mycological memoirs No. 6. (In:) Matsushima mycological memoirs No. 01 (1980) – No. 10 (2001). Electronic version. September 15, 2005. Matsushima T. 1993. List of microfungi from Pakistan soils. (In:) T. Nakaike, S. Malik (eds). Cryptogamic flora of Pakistan 2: 43–63. National Science Museum, Tokyo. 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