CSIRO PUBLISHING www.publish.csiro.au/journals/app Australasian Plant Pathology, 2005, 34, 255–258 SHORT RESEARCH NOTES Species of Pestalotiopsis and related genera occurring on grapevines in Australia V. SergeevaA,C , M. PriestB and N. G. NairA A Centre for Horticulture and Plant Sciences, University of Western Sydney, Locked Bag 1797, South Penrith DC, NSW 1797, Australia. B Orange Agricultural Institute, Forest Road, Orange, NSW 2800, Australia. C Corresponding author. Email: v.sergeeva@uws.edu.au Abstract. Descriptions of several appendage-bearing coelomycetes such as Pestalotiopsis and related genera occurring on grapevines in Australia are given. Species isolated included Pestalotiopsis uvicola, P. menezesiana, Seimatosporium hysterioides, Truncatella angustata, and Sporocadus rhododendri. Infection studies showed that berries of grapevines were infected by P. uvicola more readily at later stages of berry development than at the earlier stages. Additional keywords: Pestalotiopsis uvicola, Pestalotiopsis menezesiana, Seimatosporium hysterioides, Truncatella angustata, Sporocadus rhododendri, Seimatosporium sp., bleached canes, internal wood rot, Semillon, Shiraz. The occurrence of various fungi associated with infection of canes and trunks of grapevines (Vitis vinifera) in Australia has been reported recently by Sergeeva et al. (2001), Castillo-Pando et al. (2001) and Edwards and Pascoe (2002). In general, those authors reported on a number of known, woody-tissue-invading fungi such as Botryosphaeria, Eutypa lata, Phaeomoniella and Phaeoacremonium. However, little is known in Australia about the incidence, distribution or role of various appendage-bearing coelomycetes such as Pestalotiopsis Steyaert, Seimatosporium Corda and Truncatella Steyaert observed on grapevines. Furthermore, evidence of pathogenicity for these fungi is lacking. Published reports of the occurrence of these fungi on grapevines in Australia are few; the earliest being that of Pestalotia uvicola Speg., recorded on leaves and bark in Queensland (Bailey 1888 in Simmonds 1966). Pestalozzia monochaetioides Sacc. & Ellis was reported from Victoria in 1940 (Washington and Nancarrow 1983). This fungus has had a chequered nomenclatural history being renamed Monochaetia ellisiana Sacc. & D. Sacc., then subsequently suggested by Guba (1961) to be a synonym of Cryptostictis lonicerae (Cooke) Sacc. (= Seimatosporium lonicerae (Cooke) Shoemaker) but currently placed by Nag Raj (1993) as a synonym of Seimatosporium hysterioides (Fuckel) Brockmann. Shivas (1989) listed Seimatosporium © Australasian Plant Pathology Society 2005 lichenicola (Corda) Shoemaker & Mueller associated with dead-arm and S. lonicerae (Cooke) Shoemaker on bark of grapevines in Western Australia. The specimens associated with these two records (in Herb. IMI) were not examined in the present study. In South Australia, Seimatosporium lichenicola was listed by Cooke and Dubé (1989). This record, based on the specimen ADW 3623, was originally given as Amphichaeta europaea Grove but the specimen has since been examined by Nag Raj (1993) and re-identified as Seimatosporium hysterioides (Fuckel) Brockman. There are no published reports of the occurrence of any of these species on grapevines in New South Wales, only specimen records in the Plant Pathology Herbarium which have been examined and are dealt with below. More recently in Australia, the occurrence of Pestalotiopsis on canes, berries, flowers and leaves of grapevines, and Seimatosporium from canes was reported by Sergeeva et al. (2001). Castillo-Pando et al. (2001) also reported isolating a Pestalotiopsis sp. from the woody tissues of grapevines cv. Semillon from Hunter Valley, New South Wales, Australia. However, in neither case was the specific identity of the fungus given. In this paper, we present descriptions of several appendaged Coelomycetes recently isolated from, but not necessarily implicated in, a range of symptoms on grapevines in Australia, including Pestalotiopsis menezesiana 10.1071/AP05009 0815-3191/05/020255 256 Australasian Plant Pathology (Bres. & Torr.) Bissett, P. uvicola (Speg.) Bissett, Seimatosporium hysterioides (Fuckel) Brockmann, Sporocadus rhododendri (Schw.) Morelet and Truncatella angustata (Pers. ex Link) Hughes. In isolating fungi from grapevines, samples of dormant canes, leaves or flowers were surface sterilised by immersing them in 1% NaOCl for 1 min followed by rinsing three times in sterile distilled water. The sterilised samples were then placed on moistened paper towel in seed germination trays and incubated in a humid environment at 25◦ C for 3–5 days. Pure cultures of fungi were obtained by transferring conidia from exuding cirrhi of developed conidiomata to potato-dextrose agar (PDA) in Petri plates using a sterile needle under aseptic conditions. The inoculated plates were incubated at 25◦ C. Additionally, small pieces of darkened wood from trunk and arms of grapevines exhibiting symptoms of dieback were removed using a sterile scalpel, surface sterilised with 70% ethanol, plated onto acidified PDA (pH 5.6, Difco) and incubated for 5 days at 25◦ C. Subsequent mycelial growth was transferred onto normal PDA in plates under aseptic conditions and incubated at 25◦ C. For the identification of fungi, isolates were subcultured onto carnation-leaf agar (CLA) (water agar containing pieces of gamma-irradiated carnation leaf) and incubated for 7 days at 23◦ C before transferring them to an alternating temperature and light regime (17◦ C day/24◦ C night with a 12 h photoperiod under a light bank consisting of two 40 W cool fluorescent tubes and a single 36 W black light tube). Morphological characteristics of conidia were studied only from CLA cultures according to Guba (1961), Sutton (1980), Bissett (1982) and Nag Raj (1986, 1993). Infection studies with P. uvicola (DAR 75797) isolated from grape berries were carried out on berries of grapevines cv. Chardonnay at pea size (7 mm diameter) and veraison (15 mm diameter) stages. Infection studies were carried out only with P. uvicola because this appeared to be the dominant species occurring on grapevines. Fifty berries of cv. Chardonnay from ten bunches were collected at pea size and veraison stages at random from a vineyard in NSW. The berries were surface sterilised using NaOCl as above, wounded with a sterile needle, and placed on wet paper towel in plastic trays. They were then inoculated by spraying 1 mL of spore suspension (1 × 103 spores/mL) of P. uvicola, covered with lids, and incubated at 25◦ C for 5 days. Non-inoculated berries sprayed with sterile water were used as control. The berries were examined under a light microscope (400×) for the presence of P. uvicola. Stock cultures of the fungus were prepared by placing the conidia from infected berries on PDA. The cultures were stored at 0◦ C. P. uvicola infected the berries at the veraison stage after 5 days incubation at 25◦ C. However, we observed that infection of pea size berries occurred only when they were incubated for a further period of 14 days at 27◦ C V. Sergeeva et al. wrapped in wet cotton wool for increased humidity and wetness. The results showed that berries of grapevines were infected by P. uvicola more readily at later stages of berry development than at the earlier stages. The delay in infection of berries at the pea size stage of growth may indicate that the fungus undergoes a quiescent phase soon after flower infection. Descriptions of the fungi are as follows: 1. Pestalotiopsis uvicola (Spegazzini) Bissett, Can. J. Bot. 60: 2572 (1982) ≡ Pestalotia uvicola Spegazzini, Riv. di Viticoltura ed Enologia, Conegliano, 2: 340, (1878). Colonies on PDA white, cottony, with little aerial mycelium, reverse pale yellow-brown to dark grey-brown. Fructifications scattered on the agar, black. Conidiomata acervular. Conidia fusiform to narrow-ellipsoidal, 4-septate, often constricted at the septa, straight, occasionally slightly curved and measuring 18–25 × 5.5–7.5 µm; the three intermediate cells being mostly concolorous, pale greybrown, the middle coloured cell occasionally darker and contrasting with the upper and lower cells. The lower coloured cell often with a rugose wall seen under high magnification. Apical and basal cells are hyaline and conic in shape, with (2–) 3 apical appendages, divergent, long and filiform, hyaline, (10–) 15–26 µm long, basal appendage when present, hyaline and up to 7 µm long. Isolated from Vitis vinifera bleached canes, internal wood rot, leaf spots, flower rachises and berries (Sergeeva et al. 2001). Specimens examined: all on Vitis vinifera in New South Wales; Rooty Hill, Feb.1963 (DAR 8057); Pokolbin, Jan.1989 (DAR 76455); Cessnock, 2000 (DAR 75797); Ebeneezer, Aug. 2001 (DAR 75819); cv. Chamborson, Tyalgum, Jan. 2002 (DAR 76640); cv. Shiraz, Pokolbin, June 2002 (DAR 76453), Pokolbin, Oct. 2002 (DAR 76454). Pestalotia uvicola Speg. was transferred to the genus Pestalotiopsis by Bissett (1982) without examination of the type material. However, Nag Raj (1986) gave a detailed description of the type collection and noted some features not provided by Bissett (1982), including the coloured cells with rugose walls. All of the Australian collections examined exhibit the concolorous median cells and rugose cell walls. In addition, the size of the conidia and the number and length of the appendages are consistent with the measurements given by Guba (1961), Nag Raj (1986) and Xu et al. (1999). On the basis of the morphological characters and habit on Vitis, the collections here are placed under the name of P. uvicola. The often slightly contrasting nature of the middle coloured cell of P. uvicola has not been previously noted as a specific character, but is clearly evident in the photomicrographs of the conidia of P. uvicola presented by Nag Raj (1986) and in several conidia figured by Xu et al. (1999). P. uvicola was originally described from V. vinifera in Italy but has since been reported from V. vinifera and V. indivisa Pestalotiopsis on grapevines Australasian Plant Pathology in Europe, the United States and Brazil by Guba (1961) and from Japan (Xu et al. 1999). P. uvicola is often isolated from bleached canes, a condition normally associated with Phomopsis viticola Sacc., and is also found in association with other wood infecting fungi such as Botryosphaeria spp. and Greeneria uvicola in a complex of fungal species associated with woody tissue disease. In Japan, P. uvicola was shown to be a cause of a fruit rot in both the field and on post-harvest fruit (Xu et al. 1999). 2. Pestalotiopsis menezesiana (Bres. & Torr.) Bissett, Can. J. Bot. 60: 2570 (1982) ≡ Pestalotia menezesiana Bres. & Torr., Broteria ser. Bot. 8: 142 (1909). Colonies on PDA cottony with very little aerial mycelium. Reverse pale creamy-white, occasionally exhibiting an amber-brown to black discoloration of the agar in concentric rings. Fructifications scattered on the agar, black. Conidiomata acervular. Conidia clavate to fusiform, straight, 4-septate, occasionally constricted at the septa, the three median cells coloured, distinctly versicoloured, upper two cells darker at the septum and opaque, 22–28 × 7–8 (–8.5) µm. Apical cell with mostly three, rarely two or four hyaline cellular appendages, filiform, 10–15(–25) µm long and the basal cell with a single, short hyaline, cellular appendage 3–5 µm long. Specimens examined: all on Vitis vinifera in New South Wales; cv. Chardonnay, Pokolbin, Jan. 1985 (DAR 51177); cv. Verdelho, Denman, June 2002 (DAR 76456); Pokolbin, Nov. 2002 (DAR 76457); cv. Chardonnay, Cambewarra, Aug. 2002 (DAR 76639); Port Macquarie, Aug. 2002 (DAR 76637). Bissett (1982) transferred P. menezesiana to the genus Pestalotiopsis after studying material isolated from wilting cuttings of grape ivy (Cissus rhombifolia Vahl.) in glasshouses in Canada. The fungus was identified as P. menezesiana on the basis of the versicolored median cells and the tendency for extensive pigment to be laid down at the septum of the upper two cells. The Australian collections examined agree with the descriptions given by Guba (1961), Bissett (1982) and Xu et al. (1999). In India, P. menezesiana has been implicated as causing severe defoliation of grapevines (Mundkur and Thirumalachar 1946) and a rot of berries (Mishra et al. 1974). P. menezesiana together with P. uvicola were shown to cause fruit rotting in Japan (Xu et al. 1999). 3. Seimatosporium hysterioides Sydowia 28: 331 (1976) (Fuckel) 257 Conidiomata acervular. Conidia mostly clavate, straight, occasionally curved, 3-septate, smooth-walled, the two median cells mid-brown, end cells pale brown, with dark septa, often collapsing between septa, 24–20 × 5–6.5 µm and when present, a single basal and apical appendage up to 15 µm long, hyaline, unbranched and flexuous. Specimens examined: on Vitis vinifera: dead twigs, Adelaide Hills, South Australia, Nov. 1953 (ADW 3623) as Amphichaeta europaea Grove; on dead stems of cv. Shiraz, Australian Capital Territory, May 2002 (DAR 51223); on cankered canes, Orange, NSW, Nov. 2003 (DAR 76503). Seimatosporium hysterioides was placed as a synonym of S. lichenicola (Corda) Shoemaker & Mueller by many authors including Sutton (1980) but Brockman (1976) regarded them as separate species on the basis of the total lack of appendages in S. lichenicola v. a mixture of setose and non-setose conidia in S. hysterioides. On the basis of the lack of appendages, S. lichenicola belongs in the genus Sporocadus Corda. S. hysterioides was originally described from grapevines in Germany and has since been recorded widely throughout Europe (Brockman 1976; Shoemaker 1964), usually associated with dead stems. The records of S. lichenicola in Western Australia (Shivas 1989) and Pestalozzia monochaetioides in Victoria (Washington and Nancarrow 1983) probably refer to this species. 4. Truncatella angustata (Pers.: Link) Hughes, Can. J. Bot. 36: 822,1958 ≡ Stilbospora angustata Persoon, Syn. Meth. Fung. 96, 1801. Conidiomata acervular. Conidia fusiform, 3-septate, 14–22 × 5–7 µm, with a truncate base, median cells midto dark brown, smooth to rough-walled and concolorous, apical and basal cells very pale brown to almost hyaline, with apical appendages which are filiform, hyaline, flexuous and irregularly branched up to 25 µm long, basal appendage absent. Specimen examined: on dormant canes cv. Shiraz, Australian Capital Territory, May 2002 (DAR 51222). T. angustata has a world-wide distribution on many hosts including grapevines and has also been isolated from soil (Sutton 1980; Domsch et al. 1993; Nag Raj 1993). No pathogenicity to grapevine has been reported. Brockman, 5. Sporocadus rhododendri (Schw.) Morelet, Ann. Soc. Sci. Nat. Archeol. Toulon and Var. 37(4): 234 (1985) ≡ Cryptostictus hysterioides Fuckel, Fungi Rhenani, No.1838, fasc. IV, 1866. For further synonymy see Nag Raj (1993). Colonies on PDA grey-brown to reddish-brown with little aerial mycelium, reverse reddish-brown to dark grey. Fructifications scattered on the agar, dark brown. ≡ Coryneum rhododendri Schweinitz, Trans. Amer. Phil. Soc. 2A: 307 (1832). ≡ Seimatosporium rhododendri (Schw.) Pirozynski & Shoemaker, Can. J. Bot. 48: 202 (1970). Conidiomata acervular. Conidia 15–18 (–22) × 5–7.5 µm, 3-septate, clavate, appendages absent, middle cells 258 V. Sergeeva et al. Australasian Plant Pathology mid-brown, apical and basal cells paler, basal cell nearly hyaline. Specimen examined: on Vitis vinifera canes, Orange, New South Wales, 2002 (DAR 76638) The genus Sporocadus has been recognised by Brockman (1976) and Nag Raj (1993) as a distinct genus from Seimatosporium due to the lack of appendages of the conidia. Sporocadus rhododendri is characterised by its clavate conidia which markedly taper to a narrow base. It is separated from Sporocadus lichenicola (which has been recorded on grapevines under both the names; Seimatosporium lichenicola and S. hysterioides) by the wider conidia, and the lack of either thickened septa or the collapsing cell walls seen in S. lichenicola. In conclusion, our work has shown that several appendage-bearing coelomycetes such as Pestalotiopsis, Seimatosporium and Truncatella are associated with grapevines in Australia. Infection studies carried out with P. uvicola indicated that berries of grapevines were infected more readily at later stages of berry development than at the earlier stages. Acknowledgements We thank the Centre for Horticulture and Plant Sciences, University of Western Sydney, for providing the facilities to carry out the work. References Bissett J (1982) Pestalotiopsis menezesiana on greenhouse plantings of Cissus rhombifolia with notes on related fungi occurring on Vitaceae. Canadian Journal of Botany 160, 2570–2574. Brockman I (1976) Unterschungen uber die gattung Discostroma Clements (Ascomycetes). Sydowia 28, 275–338. Castillo-Pando M, Somers A, Green CD, Priest M, Sriskanthadas M (2001) Fungi associated with dieback of Semillon grapevines in the Hunter Valley of New South Wales. 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(Commonwealth Mycological Institute: Kew, Surrey, England) Washington WS, Nancarrow RJ (1983) List of diseases recorded on fruit and vegetable crops in Victoria before 30 June 1980. Victorian Department of Agriculture, Technical Series No. 66. Xu L, Kusakari S, Hosomi A, Toyoda H, Ouchi S (1999) Postharvest disease of grapes caused by Pestalotiopsis species. Annals of the Phytopathological Society of Japan 65, 305–311. Received 16 May 2004, accepted 14 October 2004 http://www.publish.csiro.au/journals/app