979 Mycal. Res. 100 (8): 979-983 (1996) Printed in Great Britain Two leaf pathogens of Ribes spp. in North America, Quasiphloeospora saximonfanensis and Phloeosporella ribis B. c. SUTTON!, S. F. SHAMOUN 2 AND P. W. CROUS 3 International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, UK Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 West Burnside Rd, Victoria, B.c., V8Z IM5 Canada 3 Department of Plant Pathology, University of Stellenbosch, 7600 Stellenbosch, South Africa 1 2 The generic name Quasiphloeospora, type species Q. saximontanensis comb. nov., is introduced for a species associated with foliar lesions on the forest weed Ribes viscosissimum and other species of Ribes. It is compared with similar genera and species of hyphomycetes and coelomycetes, especially Phloeosporella ribis comb. nov. with which it has been confused. Competing weeds in the forest renewal sites of British Columbia (B.C.) are important reservoirs of plant pathogenic fungi. Traditionally, chemical herbicides, manual cutting and controlled burning have been used to control the growth of forest weeds, but increasing public opposition to these methods has caused much research to be directed toward development of biological control agents (mycoherbicides) (Dorworth, 1990; Wall & Shamoun, 1990; Wall, Prasad & Shamoun, 1992). In the summer of 1993, the junior author (S.F.S.) and his colleagues at the Pacific Forestry Centre conducted a field survey and collected fungi from diseased forest weeds and shrubs for screening and evaluation of their use as potential biocontrol agents against these weeds. Disease symptoms associated with a fungal infection were observed on foliage of sticky currant (Ribes viscosissimum Pursh) and the causal fungus identified as Cercospora saximontanensis Deighton. The weed is considered to be a major competitor with respect to moisture, nutrients and space in the reforestation sites of coastal and interior B.C. It also acts as an alternate host to white pine (Pinus monticola Doug!. ex D. Don) blister rust disease caused by Cronartium ribicola J. C. Fisch., and is therefore an important limiting factor to the regeneration of these trees. The aim of the present study was to characterize this species and distinguish it from other known species on this host substratum as a prerequisite to any strategies for its potential use as a biocontrol agent. MATERIALS AND METHODS Diseased leaf samples of sticky currant were collected and conidium development was examined by SEM. Leaf discs bearing conidiomata (approximately 7 x 7 mm) were fixed in glutaraldehyde, followed by 1 % osmium tetroxide in a 0'1 M phosphate buffer, dehydrated in a graded acetone series, critical point dried and mounted. Specimens were coated with gold-palladium and viewed with a JSM 6400 scanning electron microscope. All measurements were obtained by mounting specimens in lactophenol cotton blue, and observing them under the 100 x (oil) objective of a light microscope. Material was compared and contrasted with holdings in herb. 1M!. Quasiphloeospora B. Sutton, Crous & Shamoun, gen. nov. (Figs 1-7) Foliicola, laesionibus consociatus. Mycelium intemum, brunneum, ramosum, septatum. Conidiomata separata, acervularia vel sporodochialia, epidermalia vel subepidermalia, ad basim ex textura angulari brunnea et supra ex textura prismatica composita. Conidiophora brunnea, ad basim irregulariter ramosa et verruculosa, septata, cylindrica, ex cellulis superioribus conidiomatum formata. Cellulae conidiogenae in conidiophoris incorporatae, terminales vel laterales, laeves, brunneae. cylindricae, redae, proliferationibus percurrentibus et enteroblasticis et aliquot annellationibus, vel proliferationibus sympodialibus et holoblasticis. Loci conidiogeni atri et incrassati. Conidia holoblastica, pallide brunnea, laevia, cylindrica, septata, ad apicem obtusa et ad basim truncata, cicatrice basali incrassata et atra. Typus generis: Quasiphloeospora saximontanensis (Deighton) B. Sutton, erous & Shamoun. Foliicolous, associated with lesions. Mycelium internal, brown, branched, septate. Conidiomata separate, acervular to sporodochial, epidermal to subepidermal, composed of brown textura angularis at the base and textura prismatica above. Conidiophores brown, verruculose, irregularly branched at the base, septate, cylindrical, formed from the upper cells of the conidiomata. Conidiogenous cells integrated, terminal or lateral, smooth or verruculose, brown, cylindrical, straight, proliferating percurrently and enteroblastically to form annellations Leaf pathogens of Ribes spp. 980 Figs 1-4. Scanning electron micrographs of Quasiphloeospora saximontanensis. Figs 1, 2. Erumpent acervular conidiomata. Fig. 3. Conidiogenous cells with sympodial (arrow) or enteroblastic (double arrow) proliferation. Fig. 4. Cylindrical conidia with obtuse apices and truncate, thickened bases (bars, 100, 50, 1 and 10 \lm, respectively). or sympodially and holoblastically. Conidiogenous loci dark and thickened. Conidia holoblastic, pale brown, smooth, cylindricaL septate, obtuse at the apex and truncate at the base; basal scar dark and thickened. This newly described genus occupies a position intermediate between the sporodochial hyphomycetes and acervular coelomycetes, but neither Sutton (1973, 1980) nor Nag Raj (1981) consider conidiomatal structure to be of primary systematic significance in these groups. In determining the relationships of this species account should be taken of the published literature in both groups. The more compact arrangement of conidiophores suggests placement of Qunsiphloeospora in the coelomycetes rather than the hyphomycetes. However, there are many species of Pseudocercospora Speg. (a genus usually placed in the hyphomycetes) where this type of conidiomatal structure also occurs. In contrast, the nature of scars left on conidiogenous cells and those on conidial hila after secession are increasingly being recognized as vital clues to fundamental relationships in hyphomycetes, and this is especially relevant in the many genera surrounding the Cercospora complex (Luttrell, 1963; Deighton, 1973, 1976, 1979; Mangenot & Reisinger, 1976; Pons, Sutton & Gay, 1985; David, 1993). Ultrastructurally the basal scar is composed of only a Single-layered wall derived from one half of the secession septum whereas the periclinal wall is double-layered (Cole & Samson, 1980). This sort of arrangement is basic to both unthickened and thickened scars, but in the latter, additional wall material and/or melanin is laid down before, during and after the events leading to secession. The thickened scar at the conidial base in Quasiphloeospora is the character which really distinguishes this genus from others. In the coelomycetes there is no parallel at all. In genera such as Collefogloeum Petr., Phloeospora Wallr., Ahmadia Syd., Anaphysmene Bubak and others (Sutton, 1980) where there is a basic similarity in conidiogenous events and conidial morphology, all have thin, unthickened basal scars which show no differences from the periclinal walls of the conidia in examination by optical microscopy. In the hyphomycetes there are many genera in the Cercospora complex where conidial scars are thickened in different ways and Deighton (1973, 1976, 1979) has separated a number of genera such as Cercosporella Sacc. and Pseudocercosporidium Deighton (highly thickened and refractive), and Paracercospora Deighton (thickening confined to the rim), on this basis. Pseudocercospora and Paracercospora appear closely related to Quasiphloeospora, but the conidial scars in Pseudocercospora are unthickened, and in Paracercospora the thickening is restricted to the rim of the conidiogenous cell. In Cercospora Fresen., where the species on B. C. Sutton, S. F. Shamoun and P. W. Crous 981 6 associated with conidial formation as do a limited number of sympodial holoblastic proliferations. In this respect Quasiphloeospora resembles Pseudocercospora where both types of proliferation are commonly found (Sutton, Pascoe & Sharma, 1987; Sutton & Pascoe, 1988). 5 Quasiphloeospora saximontanensis (Deighton) B. Sutton, Crous & Shamoun, comb. nov. Figs 5, 6, Quasiphloeospora saximontanensis. Fig, 5. Developing conidia, conidiophores and conidiogenous cells. Fig. 6. Mature conidia. Cercospora saximontanensis Deighton, Myco!. Pap. 151: 7 (1983). Lesions amphigenous, light brown to dark red, angular, veinlimited, up to 5 mm diam., occasionally associated with chlorosis extending beyond the areas of sporulation. Mycelium immersed, consisting of smooth, olivaceous to brown interand intra-cellular, branched, septate hyphae 1'5-2'5 !Jm wide. Conidiomata epigenous and hypogenous, abundant separate, dark brown to black, acervular, sporodochial and hyphal (fasciculate), epidermal to subepidermal, composed at the base of brown textura angularis becoming paler and increasingly more like textura prismatica towards the conidiophore-bearing region, or emerging as fascicles of up to 20 through stomata, 40-130 IJffi diam. x 50-110 !Jm high (including the conidiophores). Conidiophores olivaceous to medium brown, irregularly verruculose and branched at the base, becoming less rough above, 1-4 septate, cylindricaL erect, formed from the upper cells of the conidiomata, 35-70 x 3-4'5 !Jm. Conidiogenous cells integrated, mostly terminaL occasionally lateraL irregularly verruculose, pale brown to olivaceous, cylindrical, straight or slightly flexuous, proliferating percurrently and enteroblastically to form up to 3 annellations, or sympodially and holoblastically to form 2-3 loci, 15-45 x 3-4'5 1Jffi. Conidiogenous loci non-protuberant non-geniculate, dark and thickened, 2-2'5 !Jm wide. Conidia holoblastic, pale brown to olivaceous, smooth, cylindricaL straight or gently curved, obtuse at the apex and truncate at the gradually or occasionally abruptly tapered base, eguttulate, 1-4 septate, 40-100 x 2'5-3'5 !Jm; basal scar darker and more thickened than the periclinal wall. On leaves of Ribes spp., Wyo., Idaho, Wash., Calif., U.S.A., and B.c., Canada. Fig, 7. Quasiphloeospora saximontanensis. Vertical section of a conidioma. Ribes spp. was originally described by Deighton (1983), the scars are very prominently thickened and protuberant but these features are unlike those in Quasiphloeospora. In Quasiphloeospora the very pale brown, smooth conidia are formed from verruculose conidiogenous cells. In Cercospora (Pons & Sutton, 1988) conidia are hyaline and conidiogenous cells show sympodial enteroblastic proliferation associated with conidiogenesis, but in Quasiphloeospora percurrent enteroblastic proliferation results in a series of annellations Specimens examined: On leaves of Ribes ViSCoslsslmum, Indian Paintbrush TraiL Grand Teton National Park, Wyo., U.S.A., 16 Aug. 1937, W. G. & R. Solheim & H. F. House 5369, W. G. Solheim, Mycoflora Saximontanensis Exsiccata 1191, sub nom., Cercoseptoria ribis (Davis) Deam. & House, IMI 98069, holotype; Signal Mountain, Grand Teton National Park, Wyo., U.S.A., 22 July 1955, W. G. & R. Solheim 4080, W. G. Solheim, Mycoflora Saximontanensis Exsiccata 1396, sub nom. Cercospora septoriopsis Chupp, IMI 169953; Priest River Experimental Station, Idaho, U.s.A., 3 Aug. 1931, G. G. Hedgcock, USDA Forest Pathology 54910, sub nom. Cercoseptoria ribis (Davis) Deam. & House (det. Dearness), IMI 92408; Trail to Cathedral lakes region, B. c.. Canada, 1 Aug. 1956, J. A. Calder, J. A. Parmelee & R. L Taylor 19577, DAOM 54335 sub nom. Cercospora septoriopsis Chupp, IMI 76263; Lardeau River, Benton Creek, Nelson Forest Region, B. c.. Canada, 27 Aug. 1993, A. Erickson 141, IMI 363488; on Ribes speciosum, Topanga Canyon, Santa Monica Mountains, Calif., U.S.A., 31 Mar. 1935, Southern Californian Fungi, O. A. Plunkett, sub nom. Cercospora ribicola Ellis & Everh., IMI 154326; on Ribes sanguineum, Duckabush River, Wash., Leaf pathogens of Ribes spp. 982 U.S.A., 9 Aug. 1912, E. Bartholomew, Fungi Columbiani, E. Bartholomew 4907, sub nom. Cercospora ribicola Ellis & Everh., !M! 8453. This species was originally described by Deighton (1983) from Ribes viscosissimum, R. sanguineum Pursh and R. speciosum Pursh in North America from material misidentified as Cercospora ribicola Ellis & Everh. and Cercospora septoriopsis Chupp or its synonym Cercoseptoria ribis (Davis) Dearn. & House. It only has a tenuous relationship to Cercospora in having cicatrized conidiogenous loci and filiform conidia. It differs from this genus by the pale brown conidia, the percurrently and sympodially proliferating conidiogenous cells, conidiogenesis associated with such proliferations, verruculose conidiophores and conidiogenous cells, the nonprotuberant, non-geniculate conidiogenous loci, and the acervular to sporodochial conidiomata. Quasiphloeospora saximontanensis has frequently been misidentified as what was originally known as Cylindrosporium ribis Davis, which was later placed in Cercoseptoria Petr. by Dearness & House (1940) and Cercospora by Chupp (1954). Since there are no modem accounts of this species and Cylindrosporium, Cercoseptoria and Cercospora are not appropriate placements for it, a description with revised nomenclature follows. 50jlm PhloeosporelIa ribis (Davis) B. Sutton, Crous & Shamoun, comb. nov. (Figs 8-10) Cylindrosporium ribis Davis, Trans. Wise. Acad. Sci., Arts Lett. 16: 759 (1910). Cercoseptoria ribis (Davis) Dearness & House, Circ. NY State Mus. 24: 56 (1940). Cercospora septoriopsis Chupp, A monograph of the fungus genus Cercospora: 520 (1954). Lesions amphigenous, pale to medium brown with a narrow purple-brown raised margin, circular to elliptical or irregular, not vein-limited, up to 5 mm diam. Mycelium immersed, consisting of smooth, hyaline inter- and intra-cellular, branched, septate hyphae 1'5-2'5 iJITl wide. Conidiomata mostly epigenous but a few hypogenous, abundant, separate, white to cream, acervular, epidermal to subepidermal, composed of hyaline textura angularis which is very pale brown at the base of the conidiomata, up to 50 ~m diam, x 35 m~ high (including the conidiophores). Conidiophores hyaline, smooth, branched sparingly at the base, 1-2septate, mostly cylindrical, erect, formed from the upper cells of the conidiomata, 40 x 3-4 ~m. Conidiogenous cells integrated or discrete, smooth, hyaline, cylindrical, straight or slightly flexuous, proliferating sympodially and holoblastically to form Conidiogenous loci nonup to 2 loci, 15-45 x 3-4'5 ~m. wide. Conidia protuberant, non-geniculate, hyaline, 2 ~m holoblastic, hyaline, smooth, filiform, irregularly curved, obtuse to acute at the apex and truncate at the gradually tapered base, irregularly guttulate, 1-4-septate, basal scar hyaline. 40-85 x 2-2'5 ~m; Specimens examined: On Ribes triste, La Pointe, Bayfield Co., Wis., U.S.A., 10 July 1908, J. J. Davis, ex Univ. Wise. Herb., !M! 194769, lectotype of Cylindrosporium ribis Davis; Ribes vulgare, Madison, Figs 8-10. Phloeosporella ribis. Fig. 8. Conidiogenous cells and developing conidia. Fig. 9. Conidia. Fig. 10. Vertical section of a conidioma. Wise., U.S.A., 28 July 1914, J. J. Davis, E. Bartholomew, Fungi Columbiani 4625 sub nom. Cylindrosporium ribis Davis, !M! 21475. The placement of this species in Cylindrosporium Grev. is not tenable on account of the lack of unicellular conidia formed from phialides (Sutton, 1980). Rawlinson, Sutton & Muthyalu (1978) showed that the genus is currently monotypic and restricted to C. eoneentrieum Grev., the anamorph of Pyrenopeziza brassieae B. Sutton & Rawl. Cereoseptoria, another genus in which this pathogen of Ribes spp. has been placed, is inappropriate because the conidia in Cercoseptoria are brown and often verruculose. Deighton (1976) at first accepted this genus but later (Deighton, 1987) regarded it as a synonym of Pseudoeercospora. Cereospora is also unsuitable because of its hyphomycetous habit and the fact that both conidiogenous loci and conidial scars are prominently thickened (Pons & Sutton, 1988). Cylindrosporium ribis is more correctly placed in Phloesporella Hahn. The only modem account of Pholoeosporella is by Sutton (1980) who redescribed the type species, P. eeanothi (Ell. & Everh.) Hahn. and accepted five species, four of which had been formerly placed in Cylindrosporium. Characters of the genus are the filiform hyaline septate conidia formed from holoblastic sympodially proliferating conidiogenous cells in acervular conidiomata. B. C. Sutton, S. F. Shamoun and P. W. Crous CONCLUSIONS There have been several fungi referable to the 'Cercospora complex' described from foliage of Ribes spp. (Chupp, 1954; Pollack 1987). Revision of these has been piecemeal. Chupp (1954) thought that C. magellanica Speg. described on R. magellanicum Poir. from Argentina might be classed as a Polythrincium Kunze. Braun & Rogerson (1993) dealt with C. coalescens Davis on R. inerme from Utah, U.S.A. and referred it to Phaeoramularia Munt.-Cvetk. Deighton (1983) dealt with C. septoriopsis Chupp (a nomen novum for Cylindrosporium ribis Davis) and concluded that under this name a number of collections had been misidentified. He accepted the name Cylindrosporium ribis for some material and segregated the remainder as a new taxon, Cercospora saximontanensis Deighton. It has been shown that these two taxa are indeed distinct but neither are correctly placed in Cercospora. Cylindrosporium ribis (Cercospora septoriopsis) is referred to Phloeosporella and Cercospora saximontanensis is placed in a new genus Quasiphloeospora. They differ in symptoms, conidiomatal structure, some aspects of conidiogenous events, conidial morphology and scar structure. For the purposes of quick identification Q. saximontanensis has vein-limited lesions, mostly epigenous sporulation, dark brown to black conidiomata, and the conidia are pale brown with a thickened basal scar. P. ribis has lesions surrounded by a purple raised margin, sporulation is mostly epigenous, conidiomata are white to cream, and conidia are hyaline with no thickened basal scar. REFERENCES Braun, U. & Rogerson, C. T. (1993). Phytoparasitic hyphomycetes from Utah (USA). Mycotaxon 46, 263-274. Chupp, C. (1954). A Monograph of the Fungus Genus Cercospora. Ithaca, New York. Cole, G.1. & Samson, R. A. (1980). Patterns of Development in Conidial Fungi. Pitman: London, New York, Melbourne. David, J. (1993). A revision of taxa referred to Heterosporium K10tzsch ex Cooke (mitosporic fungi). PhD Thesis, University of Reading. Dearness, j. & House, H. D. (1940). New or noteworthy New York fungi. V. Circular, New York State Museum 24, 25-60. Deighton, F. C. (1973). Studies on Cercospora and allied genera. N. Cercosporella Sacc., Pseudocercosporella gen. nov. and Pseudocercosporidium gen. nov. Mycological Papers 133, 1-62. Deighton, F. C. (1976). Studies on Cercospora and allied genera. VI. (Accepted 10 March 1996) 983 Pseudocercospora Speg., Pantospora Cif. and Cercoseptoria Petr. Mycological Papers 140, 1-168. Deighton, F. C. (1979). Studies on Cercospora and allied genera. VII. New species and redispositions. Mycological Papers 144, 1-56. Deighton, F. C. (1983). Studies on Cercospora and allied genera. VIII. Further notes on Cercoseptoria and some new species and redispositions. Mycological Papers 151, 1-13. Deighton F. C. (1987). New species of Pseudocercospora and Mycovellosiella, and new combinations into Pseudocercospora and Phaeoramularia. Transactions of the British Mycological Society 88, 365-391. Dorworth, C. E. (1990). Use of indigenous microorganisms for forest weed biocontrol - the Pacific enhancement process. In Alternatives to the Chemical Control of Weeds (ed. C. Bassett, L. j. Whitehouse & j. A. Zabkiewicz), pp. 116-119. Proceedings of an International Conference, Rotorua, New Zealand, 25-27 july 1989. Ministry of Forestry, FRI Bulletin 155. LuttrelL E. S. (1963). Taxonomic criteria in Helminthosporium. Mycologia 55, 643--673. Mangenot, F. & Reisinger, O. (1976). Form and function of conidia as related to their development. In The Fungal Spore: Form and Function (ed. D. j. Weber & W. M. Hess), pp. 789-846. j. Wiley & Sons: New York, London, Sydney, Toronto. Nag Raj, T. R. (1981). Coelomycete systematics. In Biology of Conidial Fungi 1 (ed. G. T. Cole & W. B. Kendrick), pp.43-84. Academic Press: New York. London, Toronto, Sydney, San Francisco. Pollack. F. G. (1987). An annotated compilation of Cercospora names. Mycological Memoir 12, 1-212. Pons, N. & Sutton, B. C. (1988). Cercospora and similar fungi on yams (Dioscorea species). Mycological Papers 160, 1-78. Pons, N., Sutton, B. C. & Gay, j. L. (1985). Ultrastructure of conidiogenesis in Cercospora beticola. Transactions of the British Mycological Society 85, 405-416. Rawlinson, C. J., Sutton, B. C. & Muthyalu, G. (1978). Taxonomy and biology of Pyrenoeziza brassicae sp. nov. (Cylindrosporium concentricum), a pathogen of winter oilseed rape (Brassica napus ssp. oleifera). Transactions of the British Mycological Society 71, 425-439. Sutton, B. C. (1973). Coelomycetes. In The Fungi IVA (ed. G. C. Ainsworth, F. K. Sparrow, & A. S. Sussman), pp. 513-582. Academic Press: New York. London, Toronto, Sydney. San Francisco. Sutton, B. C. (1980). The Coelomycetes. Commonwealth Mycological Institute: Kew. Sutton, B. C. & Pascoe, I. G. (1988). Pseudocercospora correicola sp. nov., another leaf pathogen of Correa species from Australia. Australian Systematic Botany 1, 87-94. Sutton, B. c., Pascoe, I. G. & Sharma, I. K. (1987). Pseudocercospora correae sp. nov., a leaf pathogen of Correa species from Australia. Australian Journal of Botany 35, 227-234. WalL R. E., Prasad, R. & Shamoun, S. F. (1992). The development and potential role of mycoherbicides for forestry. Forestry Chronicle 68, 736-741. WalL R. E. & Shamoun, S. F. (1990). Experiments on vegetation control with native pathogenic fungi in the southern interior of British Columbia. Forest Resources Development Agreement (FRDA) Report Number 134, British Columbia, Canada.