Mycologia, 95(5), 2003, pp. 955–958. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 A new species of Pseudorobillarda, an endophyte from Thuja occidentalis in Canada, and a key to the species Montréal ( JBM); ii) mature ornamental trees in the JBM; and iii) trees in a plantation of Villeray Recreation Park in Montréal. The samples were brought to the laboratory within 2 h and used for fungal isolation within 6 h of collection. From visually healthy organs, five small segments per leaf (1 3 0.5 cm) and five per bark (1 3 0.5 cm) were taken for fungal isolation (a total of 150 segments for the experiment). These segments were surface sterilized with 70% ethanol for 10 s, rinsed in sterile distilled water (SDW) for 10 s, submerged for 2 min in 2% sodium hypochlorite, washed twice in SDW for 2 min, dried on sterile filter paper and transferred to 2% potato-dextrose agar (PDA, Becton Dickinson, Maryland, U.S.A.) Petri plates incubated at 18 C for approximately 4 wk in the dark. Microscopic observations, photographs and measurement of conidiogenous cells and conidia were made in water mounts, while conidial appendages were stained using Leifson’s modified technique (Punithalingam 1989). For microscopic examination, conidiomata were obtained from PDA plates after 4 wk growth in the dark at 21 C (61). Ten conidiomata and 50 conidia from each conidioma per plate were measured. Radial increase was measured in two directions every 24 h for 1 wk, using plates incubated at 21 C (61) in the dark. Dimensions of fungal fertile structures and growth rates for different isolates (one per site) are averages from three replications. Vladimir Vujanovic1 Marc St-Arnaud Institut de recherche en biologie végétale and Département de sciences biologiques, Université de Montréal and Jardin botanique de Montréal. 4101 est, rue Sherbrooke, Montréal, Québec, H1X 2B2 Canada Abstract: Pseudorobillarda monica sp. nov. is described and illustrated. The endophyte was isolated from living leaves and bark of twigs of a Thuja occidentalis bonsai (.90 years old) at the Montréal Botanical Garden and ornamental trees in Montréal urban plantations. This pycnidial fungus is typical of the genus in morphology but clearly differs from other species in Pseudorobillarda by the distinct size of the conidiomata and the shape and size of conidia and paraphyses. Its taxonomic placement is discussed and a key to the species of Pseudorobillarda is provided. Key words: coelomycetes, endophyte, new species, systematics INTRODUCTION During studies of endophytic mycobiota biodiversity on trees in urban plantations in Montréal, Québec, Canada, an undescribed species of the coelomycetous genus Pseudorobillarda Morelet was isolated from living foliage of Thuja occidentalis (L.) Mill. Among three isolates obtained, one originated from a bonsai tree and two others were isolated from mature ornamental trees in Montréal urban plantations. The monograph by Nag Raj (1993) and a recent publication by Bianchinotti (1997) allowed the comparison of these materials with descriptions of the nine taxa accepted in the genus. RESULTS The fungus was isolated on PDA from all three sites with an average isolation frequency for all segments of 10.6%. Isolation differed among plant organs (15.1% from leaves and 6.1% from bark) and type of trees (3.9% from bonsai trees and 17.3% from ornamental trees). The colony appearance and growth rates were similar for all isolates on tested medium. Colony diameter increased 0.5 cm per day at 21 C, and mature pycnidia were produced in 4 wk. TAXONOMY MATERIALS AND METHODS Pseudorobillarda monica Vujanovic, sp. nov. Asymptomatic branches (three per tree) bearing healthy foliage were collected with a pruner, mid-November 2000, from solitary Thuja occidentalis trees. Three trees per each of these three sites were used: i) bonsai trees, more than 90 years old (No. 3099–93), at the Jardin botanique de FIGS. 1–7 [Foliicola et caulicola. Conidiomata pycnidia, solitaria vel aggregaria, immersa vel semi-immersa, unilocularia vel multilocularia, globosa vel depresse-globosa, atrobrunnea 150– 400 mm lata, ostiolata; ostiola papillata, circularia vel ovalia, 20–30 mm diam; pariete 30–60 mm crasso, ex externa textura prismatica, cellulis tenuitunicatis crassitunicatis, atrobrunneis vel brunneis, et interna textura angulari, cellulis Accepted for publication March 11, 2003. 1 Corresponding author. E-mail: vujanovv@magellan.umontreal.ca 955 956 MYCOLOGIA FIGS. 1–7. Pseudorobillarda monica from Thuja occidentalis, HOLOTY PE (MT-TO9) on PDA: 1. Papillate ostiola. Bar 5 10 mm; 2. Vertical section of conidioma. Bar 5 25 mm; 3. Conidial mass. Bar 5 10 mm; 4. Partial sectional view of hymenium showing paraphyses (left arrow) and conidial development (right arrow). Bar 5 5 mm; 5. Mature conidia showing appendages. Bar 5 5 mm; 6. Enlarged view of conidia showing truncate top (up arrow) and position of oil drops (down arrow). Bar 5 5 2.5 mm; 7. Conidia showing ramification of extracellular appendages. Bar 5 2.5 mm. pallescentibus. Conidiophora absentia. Cellulae conidiogenae discreatae, cylindricae, circum cavitatem distributia, hyalinae, cum paraphysibus mixtae, in muco involutae, 2–10 3 2–2.5 mm. Paraphyses filiformes vel iregularies, hyalinae, laeves, 20–35 mm long, 1–6 septatae, basi leniter inflata 2– 3 mm lat., apice 1–2.5 mm lat. Conidia bicellularia, subcylindrica vel fusiformia, in apice sub-acuta, base angusta et acuta, laevia, guttulata, 10–12 3 2.5–3.0 mm, (2–)3(–4) appen- dices flexibiles ad apicem ferentes, 15–25 mm longas; ratione conidii long./lat.: 4.4. Coloniae in agaro ‘‘potatodextrosum’’ crescentes, ad 0.5 cm in una die ad 21 C. Mycelium denso-aerium, marginae regulari; inverso subroseus vel brunneolus. Hyphae hyalinae, septis, 1–5 mm crassis.] Foliicolous and caulicolous. Conidiomata pycnidial, scattered or aggregated globose to depressed glo- VUJANOVIC TABLE I. AND ST-ARNAUD: PSEUDOROBILLARDA SP. NOV. 957 Features differentiating Pseudorobillarda monica, P. jaczewskii and P. setariae Characters P. jaczewskii P. monica P. setariae Conidiomata Mean width (mm) 150 250 150 Conidial Shape Number of septa Length (mm) Width (mm) Mean 1/w ratio Ellipsoidal 1 15–21 3–3.5 5.4 Subcylindrical to fusiform 1 10–12 2.5–3.0 4.4 Subcylindrical to ellipsoidal 1 14–12 3–4 5 Appendages Number Length (mm) 2–5 10–19 2–4 15–25 2–4 9–21 Paraphyses Septation Length (mm) 1 30–45 1–6 20–35 1 20–43 Host Ribes grossularia Thuja occidentalis bose, immersed to semi-immersed, 150–400 mm diam, dark brown, uni- to multilocular, wall up to 30 mm thick, composed of an outer textura prismatica with thick-walled dark brown to brown cells and an inner textura angularis with thin-walled paler cells; ostiolate, ostiole papilate, 20–30 mm diam. Conidiophores reduced to conidiogenous cells, lining the cavity of the conidioma, mixed with paraphyses and enveloped in mucus. Paraphyses filiform to irregular, unbranched, 1–6 septate, hyaline, 20–35 mm long, up to 3 mm wide at the base and 1–2 mm wide at the apex. Conidiogenous cells short, cylindrical, hyaline, smooth, 2–10 3 2–2.5 mm. Conidia bicellular, sometimes constricted at the septa, hyaline, subcylindrical to fusiform, tip subacute, base narrow and acute, smooth, guttulate, 10–12 3 2.5–3.0 mm (l/w: 4.4) bearing at one end mostly 3, occasionally 2 or 4, unbranched, flexuous, extracellular appendices 15–25 mm long. Mycelium superficial in culture, cottony, color white to pink, radial with regular margin; reverse first pinkish later becoming brownish; abundant fruiting. Hyphae septate, hyaline, 1–5 mm diam. HOLOTY PE. CANADA. QUÉBEC: Montréal, Jardin botanique de Montréal, on living foliage of Thuja occidentalis, 11. X. 2000, Vujanovic MT-TO9 (MT, Herbier Marie-Victorin of the University of Montreal) dried culture. DISCUSSION Pseudorobillarda species are known from Argentina, Cuba, India, Germany, Nigeria, Ukraine, United Kingdom and the United States (Bianchinotti 1997). This is the first report for Canada. Before this report Oryza sativa and Setaria sp. on Thuja occidentalis, Pseudorobillarda species have never been found on coniferous trees, although they are known to have a broad host ranges (Nag Raj 1993) including one broadleaf tree, Geoffroea decorticans (Gill. ex Hook. & Arn.) Burk. (Bianchinotti 1997). Whether they are pathogenic to host species remains undetermined. However, they occur on both living and dead leaves and stems. Uecker and Kulik (1986) found that stems of soybean plants inoculated with P. soyae did not show symptoms, although the fungus was re-isolated from host tissues a few weeks after inoculation. The nature of the association of P. soyae can be described as endophytic. Although preliminary, this study suggests the endophytic nature of P. monica; it was isolated from asymptomatic tree organs after rigorous surface sterilization similar to that used for isolation of endophytes from bark (Petrini 1986) and leaf (lamina and petiole) tissues (Vujanovic and Brisson 2002). Species in this genus are distinguished mainly by the presence or absence of paraphyses and by conidial features, such as size and septation (Nag Raj 1993). The new proposed species differs from all known species in the genus. The main differences are: Pseudorobillarda sojae is characterized by the absence of paraphyses; P. texana has unicellular conidia; P. indica and P. magna possess multiseptate conidia; P. agrostis and P. bambusae have conidia that measure more than 16 mm length, while P. monica conidia are less than 12 mm long. In contrast to P. bambusae, which shows minor conidiomata development on PDA, Pseudorobillarda monica fructified abundantly on this agar medium. P. monica can be distinguished also from the two most similar paraphysate species in 958 MYCOLOGIA the genus (P. jaczewskii and P. setariae) by size and septation of the paraphyses, the shape and dimension of conidia and conidiomatal size (TABLE I). Moreover, the number and length of appendages is different. These characteristics are also of diagnostic importance, as has been proposed by Bianchinotti (1997). In this genus no teleomorph connections have been made. Sutton (1980) published a key to four species. Recently, Nag Raj (1993) presented a key to eight species. Here is a key that includes the 10 species proposed to date. KEY TO SPECIES PSEUDOROBILLARDA 1. 1. 2. 2. 3. 3. 4. 4. 5. 5. 6. 6. 7. 7. 8. 8. 9. 9. Paraphyses absent . . . . . . . . . . . . . . . . . Paraphyses present . . . . . . . . . . . . . . . . Conidia unicellular . . . . . . . . . . . . . . . . Conidia multicellular . . . . . . . . . . . . . . . Conidia 1-septate . . . . . . . . . . . . . . . . . Conidia more than 1-septate . . . . . . . . . . Mean conidium length/width ratio #5.4:1 Mean conidium length/width ratio $6.5:1 Conidia 1–3 septate . . . . . . . . . . . . . . . Conidia 3–4 septate . . . . . . . . . . . . . . . Paraphyses 1-septate . . . . . . . . . . . . . . . Paraphyses 1–6 septate . . . . . . . . . . . . . Conidiomata wall of textura angularis . . Conidiomata wall of textura prismatica . . Mean conidium length/width ratio 6.5:1 . Mean conidium length/width ratio 7.8:1 . Paraphyses unbranched . . . . . . . . . . . . . Paraphyses branched . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACKNOWLEDGMENTS This research was supported financially by the NSERC and the VRQ—Plant Productivity Research Network (Quebec Environmental Diagnostic Research Centre). Thanks also are due to S. Hay and Herbier Marie-Victorin (MT) of the Université de Montréal for consultation on the English text. LITERATURE CITED Bianchinotti MV. 1997. A new species of Pseudorobillarda from a leguminous tree in Argentina. Mycol Res 101: 1233–1236. Nag Raj TR. 1993. Coelomycetous Anamorphs with Appendage-Bearing Conidia., Waterloo, Ontario, Canada: Mycologue Publications. 1101 p. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. sojae ....... 2 . . P. texana ....... 3 ....... 4 ....... 5 ....... 6 ....... 8 . . . P.indica . . P.magna ....... 7 . P. monica . P. setariae P. jaczewski ....... 9 P. phragmitis . . P. agrostis . P. bambusae Petrini O. 1986. Taxonomy of endophytic fungi of aerial plant tissues. In: Fokkema NJ, van den Heuvel J, eds. Microbiology of the phyllosphere. Cambridge, England: Cambridge University Press. p. 175–187. Punithalingam E. 1989. Techniques for staining fungal nuclei and appendages. Botanical Journal of the Linnean Society 99:19–32. Sutton BC. 1980. Coelomycetes, Fungi Imperfecti with Pycnidia, Acervuli and Stromata. Kew, Surrey, England: Commonwealth Mycological Institute. 696 p. Uecker FA, Kulik MM. 1986. Pseudorobillarda sojae, a new pycnidial coelomycete from soybean stems. Mycologia 78:449–453. Vujanovic V, Brisson J. 2002. A comparative study of endophytic mycobiota in leaves of Acer saccharum in eastern North America. Mycological Progress 1:147–154.