Plant Protection Science Vol. 55, 2019, No. 3: 191–201 https://doi.org/10.17221/38/2018-PPS Characterisation and pathogenicity of Cryphonectria parasitica on sweet chestnut and sessile oak trees in Serbia Dragan Karadžić 1, Zlatan Radulović 2, Katarzyna Sikora 3, Zoran Stanivuković 4, Vesna Golubović Ćurguz 5, Tomasz Oszako 3, Ivan Milenković 1,6* 1Department of Forestry, Faculty of Forestry, University of Belgrade, Belgrade, Serbia; 2Institute of Forestry, Belgrade, Serbia; 3Department of Forest Protection, Forest Research Institute – IBL, Sękocin Stary, Poland; 4Department of the Integral Protection of Forest Ecosystems, Faculty of Forestry, University of Banja Luka, Banja Luka, Bosnia and Herzegovina; 5Department of Landscape Architecture and Horticulture, Faculty of Forestry, University of Belgrade, Belgrade, Serbia; 6Phytophthora Research Centre, Mendel University in Brno, Brno, Czech Republic *Corresponding author: [email protected] Citation: Karadžić D., Radulović Z., Sikora K., Stanivuković Z., Golubović Ćurguz V., Oszako T., Milenković I. (2019): Characterisation and pathogenicity of Cryphonectria parasitica on sweet chestnut and sessile oak trees in Serbia. Plant Protect. Sci., 55: 191–201. Abstract: The presence of Cryphonectria parasitica (Murrill) M.E. Barr was studied in six natural and planted stands of sweet chestnut in Serbia. The fungus was detected on the sweet chestnut in five localities and on the sessile oak in one locality. In total, 77 isolates from the sweet chestnut and five isolates from the sessile oak were obtained. Based on the culture morphology, all the obtained isolates were proven to be free from Cryphonectria (Saccardo) Saccardo & D. Saccardo hypovirus. The isolates of C. parasitica from the sweet chestnut were compatible with three different vegetative compatibility types, EU-12, EU-2, and EU-1, while the isolates from the sessile oak belonged to EU-12. After inoculation in laboratory conditions, the isolate from the sweet chestnut and sessile oak caused the decline in 88 and 76% of the sweet chestnut plants, respectively. In the case of the sessile oak, both isolates caused the decline in 52% of the plants. In field conditions, both isolates were aggressive to sessile oak trees after previous bark wounds and they were statistically significantly different compared to the control trees. The isolate from the sweet chestnut caused significantly larger cankers compared to both the isolate from the sessile oak and the control. Keywords: chestnut blight fungus; oak decline; sequencing; vegetative compatibility type diversity; aggressiveness Cryphonectria parasitica (Murrill) M.E. Barr be- blight. The blight of the American chestnut (Castanea longs to the family Cryphonectriaceae, and according dentata (Marsh) Borkhausen), caused by C. parasitica, to Kirk et al. (2008), this family includes a total of represents one of the greatest botanical catastrophes 26 species within 12 genera. This fungus is a strong in the history of humankind. The first trees infected pathogen known worldwide as a cause of chestnut with this fungus were recorded in the New York Zoo Supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Projects No. TR 37008 and No. TR 31070, and by the “Phytophthora Research Centre”, funded by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund, Grant No. CZ.02.1.01/0.0/0.0/15_003/0000453. 191 Vol. 55, 2019, No. 3: 191–201 Plant Protection Science https://doi.org/10.17221/38/2018-PPS in 1904 (Griffin 1986). Chestnut blight caused by species (Figure 1 and Table 1). In two stands, sweet C. parasitica was found in Europe for the first time chestnut appears naturally, building autochthonous in 1938 in Italy, from which it has spread to most communities, while this species was artificially intro- European countries (Griffin 1986). duced in four stands (Table 1). Planted sweet chestnut Apart from species of the Castanea Miller genus, trees in the natural sessile oak forests near the Vršac other hosts of C. parasitica belong to several families, Mountains, part of the Carpathian basin in Serbia were including Aceraceae, Betulaceae, Fagaceae, Anacar- sampled and analysed together with sessile oak trees diaceae, Juglandaceae and Magnoliaceae (Nash & in the same locality (Table 1). Another planted forest Stambaugh 1982; Turchetti et al. 1991; Dalla- established on the natural stand of Q. frainetto was valle & Zambonelli 1999; Radócz & Tarcali sampled in the area near Kruševac in central Serbia 2005). C. parasitica is occurring as a saprotroph for (Table 1). Two natural and two planted stands of sweet most of the species listed above, with the exception chestnut trees were sampled in southern Serbia in of the oak (Quercus spp.), for which it is parasitic different localities near Vranje, including Kozji Dol, (Radócz & Tarcali 2005; Adamčíková et al. 2010; Milivojce, Muhovac and Sobina, respectively (Table 1), Tziros et al. 2015). where this species composes one of the rarely found According to Krstić (1950), cankers on the bark autochthonous communities in Serbia. Q. petraea or of the sweet chestnut in the former Yugoslavia were Q. frainetto naturally appear in the mixtures in the recorded for the first time in Slovenia in the “Panovac” latter four stands, and they were also inspected for forest. The presence of C. parasitica in Serbia was the presence of the symptoms of the disease. recorded for the first time in 1975 in the province In each locality, 30–50% of the trees were randomly of Kosovo and Metohija (Marinković & Karadžić chosen and closely inspected to confirm the presence 1985). Later field studies and monitoring of sweet of the diseases (Table 1). Approximately 10–20% of chestnut stands have confirmed the decline of sweet chestnut trees all over Serbia (personal observation, Castanea sativa – positive on CP unpublished). Generally, C. parasitica is present in Castanea sativa – negative on CP the most important natural stands of sweet chestnut Quercus petraea – positive on CP in Serbia (in the area near Vranje and Prijepolje) as Quercus petraea – negative on CP Quercus frainetto – negative on CP well as in most of the localities where individual sweet chestnut trees occur (Radulović 2013). Addition- ally, C. parasitica has been recorded on sessile oak trees in Serbia (Karadžić & Milenković 2013). Due to the lack of data about the occurrence and the characteristics of this parasitic fungus on different host trees in Serbia, our study is aimed at (i) deter- mining and confirming the presence of this fungus on sweet chestnut and oak trees, (ii) determining the vegetative compatibility (VC) types and occurrence of the hypovirus in isolates of this fungus obtained from sweet chestnut and from sessile oak trees in Serbia, and (iii) testing the pathogenicity of the se- lected isolates in the controlled and field conditions. MATERIAL AND METHODS Studied localities and sampling. The study was performed in six mixed stands of sweet chestnut and oak trees in Serbia, including three stands mixed with Figure 1. The studied localities and hosts Hungarian oak (Quercus frainetto Tenore), two stands 1 – Vršac Mountains; 2 – Kruševac/Brus; 3 – Vranje/Muhovac; mixed with sessile oak (Quercus petraea (von Mat- 4 – Vranje/Sobina; 5 – Vranje/Milivojce; 6 – Vranje/Kozji Dol; tuschka) Lieblein), and one stand mixed with both oak CP – Cryphonectria parasitica (Murrill) M.E. Barr 192 Plant Protection Science Table 1. Characteristics of the studied localities, evaluated trees and the obtained samples Age No. Presence of chestnut No. of trees (cankers) No. of isolates of the trees of observed blight symptoms sampled from the obtained from the No. Locality Year Coordinates Stand type in the stand sweet chestnut/ on the sweet Castanea sativa Miller/ sweet chestnut/ (year) oak trees chestnut/oak trees oak trees oak trees 45°07'55''N natural + 1 Vršac Mountains 2012–2013 20–40 100/172 100/17 10/5 10/52 21°23'33''E planted stand 43°25'086''N 2 Kruševac/Brus 2013–2016 planted stand 50–60 20/101 20/0 5/0 8 21°03'251''E 42°34'33''N 3 Vranje/Muhovac 2006–2012 natural stand > 120 30/51/72 30/0 10/0 18 21°51'42''E 42°33'52''N 4 Vranje/Sobina 2006–2012 natural stand > 120 50/71 49/0 20/0 31 21°50'58''E 42°31'58.49''N planted 5 Vranje/Milivojce 2015 30–40 30/31 23/0 10/0 10 21°48'47.90''E anti-erosion stand 42°20'35.13''N 6 Vranje/Kozji Dol 2016 planted stand 50–60 50/202 0/0 0 0 22°04'27.87''E 1Quercus frainetto Tenore; 2Quercus petraea (von Mattuschka) Lieblein barrageresponse. merging/ to scored visually according afterdays 14 wasexperiment repeated VCtypes twice, andthe were (22–25°C). room The temperature at indaylight days seven additional an sevenfor for then and dark days performed MEA onthe media first25°Cinthe at paired incubation ofthe donia). cultureswas The ofSkopje, FYR University Mace ofForestry, Faculty (provided EU-12 and KirilEU-10 Dr by Sotirovski, C al methods the to specified cording by parasitica isolates the description the in with comparison and cultures acteristics pure the of pycnidiachar with conidia,with the combination in peritheciaincluding with asci andascospores and bodies, infected recorded barkofthe onthe trees, based fruiting morphological the of the on features evaluated. were colour colony and hyphae the position shape, of colony pigmentation, including characteristicscolonies, older younger and the of was performed dark. 20°Cinthe at maincolony The (Merck, Germany) andMEA media, andincubation mediaagar (PDA) prepared with ofaPDA 39g/l tainedpotatoa dextrose isolatestransferred were onto cultures under a light microscope. light a under cultures four-week-old bodies andsporeswereobservedinthe 25°Cinthedark.Thehyphalcharacteristics,fruiting at was incubation performed Further biotin. mg/l 1 and Germany) andamended with 100mg/lmethionine (MEA) media MEA prepared of g/l with49 (Merck, agar extract malt a edges onto growing colonies the of the from transferred were hypha young piecesthe of dark.