FIRST RECORD OF SIROCOCCUS CONIGENUS ON NORWAY SPRUCE (PICEA ABIES ) IN BULGARIA Maria Dobreva 1, Margarita Georgieva 2, Pencho Dermendzhiev 1, Vasil Velinov 1, Rumen Nachev 1, Georgi Georgiev 2 1Forest Protection Station - Plovdiv 2Forest Research Institute – Sofia Bulgarian Academy of Sciences Abstract: Damages on Norway spruce ( Picea abies (L.) Karst) seedlings, associated with a fungal pathogen Sirococcus conigenus (Pers.) Cannon et Minter (Ascomycota, Diaportales, Gnomoniaceae), were established for first time in Bulgaria. In 2016, shoot blight disease was noticed on 4-year old seedlings in a nursery of Training and Experimental Forest Range, Yundola Vill., Southwest Bulgaria. Currently, the pathogen is not widespread in Bulgaria, but it has potential to cause intense problems on the conifer species growing in nurseries, young plantations, mature stands and ornamental trees. Key words: Sirococcus conigenus, Picea abies, shoot blight disease Sirococcus conigenus (DC.) P. Cannon et Minter (Ascomycota, Diaportales, Gnomoniaceae) (syn. S. strobilinus G. Preuss) is an asexually reproducing fungus causing shoot blight and death of seedlings of many conifer hosts throughout the Northern hemisphere (Halmschlager et al., 2007). Sirococcus shoot blight disease was described for first time in 1890 in Germany on current-year shoots of Norway spruce ( Picea abies (L.) Karst.) (Hartig, 1890). Subsequently, the disease was reported on a wide range of conifer hosts from genera Picea , Pinus , Larix , Tsuga , Pseudotsuga in Europe and North America (Peace, 1962; Sutherland et al., 1987; Butin, 1995; Smith et al., 2003; Sinclair, Lyon, 2005), and recently on Picea spinulosa (Griff.) Henry in Bhutan, South Asia (Kirisits et al., 2007). In 2008, two new species from Sirococcus genus were recognized – S. piceicola described from Picea spp. in Canada and Switzerland, and S. tsugae known only from western part of North America on Cedrus spp. and Tsuga spp. (Rossmann et al., 2008). In Europe, S. tsugae was reported for first time on C. atlantica in Germany (EPPO Reporting Service, 2015) and England (Pérez-Sierra et al., 2015). The purpose of this study is to report Sirococcus shoot blight disease presence on Norway spruce seedlings that has appeared for first time in Bulgaria. In March 2016, severe symptoms of shoot blight disease were observed on 4-year old Norway spruce seedlings in a nursery of Training and Experimental Forest Range, Yundola Vill. (in the pass between Rila and the Western Rhodopes mountains, 42°03'37.63''N; 23°51'01.82''E; 1375 m a.s.l.). The total number of spruce seedlings was 2000, and the infested seedlings – 15 (0.75%). The seedlings were produced from seeds harvested in 100- year old spruce stand, located about 10 km from the nursery. All blighted spruce seedlings were eradicated and sent to Forest Protection Station - Plovdiv and Laboratory of phytopathology at Forest Research Institute - Sofia. Symptoms of wilting and dieback of the youngest terminal shoots were noticed (Fig. 1A). The infected shoots died and a characteristic hook shape of shoots without needles were observed (Fig. 1B). Parts of symptomatic shoots were surface washing with tap water for 5 min, sterilized for 1 min in 96% ethanol, subsequently rinsing them once in sterile distilled water. Sterilized parts were placed in Petri dishes on moistened filter paper and incubated at 22°C under artificial light. Isolations of fungal pathogen was achieved by placing small portions of tissue on to malt extract agar (MEA: 20 g malt extract; 16 g agar-agar; 1000 ml tap water), supplemented after autoclaving with 100 mg streptomycin sulphate to suppress bacterial growth. The shape and size of conidia were measured at magnification 40× using an ocular micrometer of Olympus CH20 and 125× Carl Zeiss NU2 light microscopes, Zeiss GSZ stereo microscope equipped with a digital camera DinoEye AM-423X. Numerous small (0.3-1.0 mm) fruit bodies (pycnidia) of conidial stage developed on the twigs. They were initially light-colored, later dark-brown at maturity. Pycnidia appeared abundantly at the base of needles (Fig. 1C). Conidia were observed with fusiform shape, with one-septa, straight or slightly curved (Fig. 1D). The measured size of spores were 11.6-14.3 (12.7) × 2.5-2.9 (2.7) µm. The size of conidia were consistent with descriptions of the fungus made by Butin (1995) and Smith et al. (2003). On malt extract agar, the fungus produced slow-growing (less than 2 mm per day) aerial mycelium with yellow-olive colour. Based on morphological characteristics and representative isolates, the pathogen Sirococcus conigenus Pers. Cannon et Minter (Ascomycota, Diaportales, Gnomoniaceae) has been observed as the agent of the disease. All examined Norway spruce seedlings were damaged by the fungus. Although individual saplings were seriously affected, the overall disease severity appeared to be low. In March 2017, no symptoms of Sirococcus shoot blight disease were observed on spruce seedlings in Yundola nursery. However, other pathogens (Phomopsis sp. and Fusarium sp.) were established on stems of individual seedlings in the nursery. The current observation of pathogen S. conigenus on Norway spruce seedlings is first report in Bulgaria. Sirococcus is a genus of asexually reproducing fungi that includes important pathogens causing shoot blight and tip dieback of conifers (Rossmann et al., 2008). S. conigenus infects seeds and the current-year shoots with conidia borne in small, black pycnidia on cone scales, needles or shoots (Lilja et al., 2010). The fungus particularly affects stands in cold, humid and low light conditions in early spring (Butin, 1995). The observed infection in could be related to the physiological stress of seedlings in early spring when significant evapotranspiration occurred and they cannot be compensated by the absorption of water by roots. According to Sutherland et al. (1981, 1987), in the nursery, the primary inoculum source of infection is from infected seed or disseminated spores by rain and wind from the infected trees nearby. After seed germination spores formed in pycnidia on diseased seedlings disseminate the disease to new seedlings. Seeds also become contaminated with S. conigenus during storage under high relative humidity. Presently, Sirococcus shoot blight disease has not appear to cause severe damage in Bulgaria yet. However, the fungus has the potential to cause intense problems on many conifer species not only in nurseries and young plantations, but also in mature stands and ornamental trees in urban areas. For this reason, monitoring on the pathogen presence should be carry out annually in Yundola region. REFERENCES Butin, H. 1995. Tree diseases and disorders. Causes, biology and control in forest and amenity trees. Oxford University Press, New York, Tokyo, 252. EPPO Reporting Service. 2015. URL: http://archives.eppo.int/EPPOReporting/2015/Rse- 1504.pdf. Halmschlager, E., A. Gabler, F. Andrae. 2000. The impact of Sirococcus shoot blight on radial and height growth of Norway spruce ( Picea abies ) in young plantations. Forest Pathology, 30, 127-133. Halmschlager, E., H. Anglberger, K. Katzensteiner, H. 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Sirococcus conigenus on Picea abies seedlings: A – typical symptoms of shoot blight disease; B – died terminal shoots; C – pycnidia; D – conidia .