Margarita Georgieva Forest Research Institute - Sofia Bulgarian Academy of Sciences. Abstract INTRODUCTION

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1 Silva Balcanica, 17(2)/2016 First record of the pathogen Botryosphaeria dothidea associeted with Sequoiadendron giganteum in Bulgaria Margarita Georgieva Forest Research Institute - Sofia Bulgarian Academy of Sciences Abstract Extensive dieback of giant sequoia (Sequoidendron giganteum) was established in Bulgaria in recent years. On the symptomatic trees, the fungus Botryosphaeria dothidea was identified as the causative agent of the dieback that is the first report from Bulgaria. Stem cankers, irregularly distributed twig and branch dieback, resin exudation, crown wilting and tree mortality were observed in trees of all ages during the period Identification of pathogen was achieved based on the morphological characteristics of its anamorph fungus Fusicoccum aesculi. This article presents general information on the dieback associated with S. giganteum and gives details on the symptoms of the newly found disease and biology of the causal fungus. Key words: dieback, Sequoidendron giganteum, Botryosphaeria dothidea, Fusicoccum aesculi INTRODUCTION Most of the exotic tree species introduced in Bulgaria over the past century were planted as ornamental trees in green urban zones and arboreta for botanical purpose or cultivated in nurseries for afforestation or landscape management. Giant sequoia, Sequoiadendron giganteum (Lindl.) Buchholz, is one of the most successfully introduced species in Bulgaria and well adapted to the site s environmental conditions, especially in the south-western part of the country (Alexandrov et al., 2002). The species is considered as a long-lived tree because of its high resistance to damage and mortality due to pests, diseases and fire (Piirto, 1994). However, during the last decades accounts of introduced invasive pathogen and insect outbreaks on nature and exotic tree species are extending throughout Europe. An increase in intensity of Botrvosphaeria dothidea (Moug.: Fr.) Ces. & De Not. dieback on S. giganteum was reported from urban green sites in Germany (Kehr, 2004), Serbia (Zlatković et al., 2010) and Eastern Austria (Cech, Tomiczek, 2013). The occurrence of Botryosphaeria dieback at giant sequoia trees is stimulated by high temperatures in the summer in connection with a drought stress caused by an additional shortage of water in dry soils (Ahimera et al., 2003). At a recent time, a dieback on S. giganteum, was found in many locations of Bulgaria both on young seedlings in nurseries and mature trees in parks and gardens. The number of reports of this emerging problem increased continuously. The disease appears 57

2 to spread too rapidly and within a few years it was widely distributed throughout almost all of the districts where the species was cultivated. The aim of this study was to identify and characterize the fungal pathogens associated with giant sequoia dieback and to give details on the symptoms of the newly found disease and on the biology of the causal fungus. MATERIAL AND METHODS The detection of fungal pathogens was carried out by using direct visual examination of symptoms to check for fungal structures and methods for their detection by inducing fungal growth and sporulation. Observations of disease symptoms and collection of materials were performed between April 2015 and August 2016 in a nursery for ornamental plants in Central Bulgaria and in the National arboretum of Forest Research Institute (FRI) - Sofia. The evolution of the disease severity was measured by symptomatic appearance on the infected trees and by monitoring the spore population. To identify the pathogen, diseased branches from infected trees were collected and carried to Laboratory of Phytopathology, FRI. The branches were surface sterilized with ethanol and placed in a damp chamber at room temperature. Sterile Petri dishes were used as damp chambers and sterile filter papers were added and moistened periodically with distilled water. The samples were checked daily for sporulation under a stereoscopic microscope NU2. The fungus mycelium was isolated from the diseased specimens and plate into 2% potato-dextrose agar (PDA). Isolates were made from pycnidia on dead or dying twigs as explained in Slippers et al. (2004). When a fungus was sporulating on the surface, the mycelium was transferred to a new PDA growth medium and cultured routinely for 5 to 7 days at C in the dark. Fungal species were identified based on traits of the anamorph (asexual stage) using microscopic methods. RESULTS Incidence of the disease on giant sequoia was established for first time in spring 2015 both on young trees in the nursery and old trees in the arboretum. Irregularly distributed dry twigs with brownish needles were noticed on the crown (Fig. 1A). In next year, an increase in disease severity was established and the highest percentage of infection was detected in the summer of The symptoms of infection on young trees in the nursery included: necrosis on the main stems, brown tips of branches and progressive dieback. Along the necrosis, significant resin exudates were visible, which was also noticeable in the cross section of the shoots and stems. The similar symptoms were observed on old trees planted in the arboretum of FRI. The disease prevalence was assessed by the presence of death branches in the crown (Fig. 58

3 1B). The dieback was associated with cankers on the branches or trunk. After removing the reddish-brown bark of dead stems, where necrosis was recognizable, the cambium was coloured in brown (Fig. 1C). Cankers girdled and killed twigs and branches. Black pycnidia containing conidia developed in the canker. Based on signs and symptoms of the disease both on young and old specimens, the causative agent of the dieback was preliminary identified as Botryosphaeria dothidea. The further observation of samples collected from both sites revealed large number of asexual structures (pycnidia). Black pycnidia were ostiolate, globose, formed in stromata that were immersed in the bark. Conidia were hyaline, fusoid to ellipsoid, aseptate, μm in size (Fig. 1D). On the basis of morphology, the pathogen was identified as Fusicoccum aesculi Corda, asexual stage of B. dothidea. Fungal colonies of F. aesculi obtained from affected host tissue developed for 7-10 days on PDA placed at 24 to 25 C in the dark. Colonies grew slowly, with pale colour firstly and elevated in dark grey mycelium (Fig. 1E). Dark brown, unilocular pycnida formed after 2 to 3 weeks in the cultures. The fruit structure (perithecia) of teleomorph stage (B. dothidea) occurred more rarely and was observed only on two samples taken in August 2016 from the arboretum of FRI at the time when the plants were dying (Fig. 1F). On the surface of the necrotic area, perithecia were formed into groups with about μm measured ascospores formed. In August 2016 the bark of died branches was also colonized by saprotrophic fungi from genera Alternaria, Phomopsis, Pestalotiopsis and Diplodia. It was established that B. dothidea has spread too rapidly and within a few years it distributed throughout the country. Only in a few specific places on the landscape it was detected single trees with good vitality without symptoms of an infection. DISCUSSION Based on the literature, this is the first report of Botrvosphaeria dothidea causing extensive dieback of giant sequoia (Sequoidendron giganteum) in Bulgaria. Bega (1964), Libby (1982), Worrall et al. (1986) have conducted studies on giant sequoia trees planted outside their native range and found some of them severely damaged by a canker fungus Botryosphaeria sp. According to Piirto (1994), Botryosphaeria spp. and Armillaria spp. appear to be two major pathogens commonly associated with disease problems in younggrowth giant sequoia trees. The pathogen B. dothidea is widely distributed in the temperate and tropical climates worldwide (Sinclair et al., 1987). It attacks woody plants in hundred genera and is a serious problem for forest, ornamental and fruit tree species. Extensive dieback and mortality of various ornamental trees and shrubs has been observed in parts of Western Balkans region during the past decade (Zlatković et al., 2016). The disease symptoms have been typical of those caused by pathogens residing in Botryosphaeriaceae. In Bulgaria, wilting of poplar seedlings caused by Botrvosphaeria sp. was observed in 2013 in Forest 59

4 Fig. 1. Botryosphaeria dothidea: A an infected tree; B typical symptoms; C coloured cambium; D conidia of anamorph stage; E mycelium; F perithecia of teleomorph stage 60

5 Nursery Kapsidata (State Forestry Asenovgrad, Central Bulgaria) (Georgieva et al., 2014). Ten years earlier serious damages of Botryosphaeria obtusa (Schwein.) Shoemaker were detected on different apple cultivars growing in the region of Kyustendil, West Bulgaria (Borovinova, 2006). B. dothidea is a typically opportunistic pathogen classified as a parasite of weakness and causes disease on plants that are stressed by drought or frost (Slippers, Wingfield, 2007). In current study it was established that the emerging infections by B. dothidea on giant sequoia and rapidly development of the disease throughout the country were consequence of the long-term drought conditions in the past few years ( ), when high temperatures and lack of precipitation during the growing seasons were recorded. The high existence of pathogen outbreaks contributed considerably to physiological weakness of established trees and they became more susceptible to attack by secondary infections of fungi of genera Phomopsis, Pestslotiopsis and Diplodia. The fruit structures of the teleomorph stage (B. dothidea) appeared late more than one year after the initial disease symptoms were noticed. According Phillips (2000), Kehr (2004), the teleomorph of fungus develops rarely and could be found on dead tissue of branches. In fresh dead tissue, large number of pycnidia of anamorph stage (F. aesculi) arose and they were used to diagnose the disease. The characteristics of conidia fall within the concept of Fusicoccum morphology gave by Phillips (2000) and Kehr (2004). Based on our results, B. dothidea remained dormant in the wood during the winter. In spring 2016, the fungus resumed activity and affected trees were repeatedly symptomatic and extend of dry branches appeared the next year since the outbreak has been first observed in The disease was spreading from symptomatic trees to initially healthy trees. The consequence of repeated damages by the pathogens is expected to continue in next years following unusually drought summer Botryosphaeria shoot blight is extremely difficult to control, especially if allowed to increase over several years. The optimum temperature for the development of this fungus is between C (Sinclair et al., 1989; Copes et al., 2004). Fungicide utilization for control of Botryosphaeria disease is inconsistent (Bester et al., 2007; Kim, Uhm, 2002). Avoiding plant stress, which predisposes plant tissue to infection and colonization by this fungal group, is the best strategy to prevent Botryosphaeria disease problems (Bush, 2009). REFERENCES Ahimera, N., Driever, G.F., Michailides, T.J Relationships among propagule numbers of Botryosphaeria dothidea, latent infection, and severity of panicle and shoot blight in Pistachio orchards, Plant Disease, The American Phytopathological Society, USA, 87, Alexandrov, A., K. Genov, H. Tsakov, E. Nozharov The introduction of Sequoiadendron giganteum (Lindl.) Buchholz in Sofia Region, Ososgovo and the Rhodopes Mountains. Forest Science, 2, Bega, R.V Diseases of sequoia. - In: Diseases of widely planted forest trees. FAO/IUFRO Symposium on internationally dangerous forest diseases and insects; Oxford, 1964 July 20-30,

6 Benson, D.M., B.I. Daughtry, R.K. Jones Botryosphaeria dieback in hybrid rhododendron, Biological and Cultural Tests, 6,108. Bester, W., Crous, P.W., Fourie, P.H Evaluation of fungicides as potential grapevine pruning wound protectants against Botryosphaeria species. Australian Plant Pathology, 36, Borovinova, M Susceptibility of apple cultivars to Botryosphaeria obtuse. Not. Bot. Hort. Agrobot. Cluj, XXXIV, Bush, E Botryosphaeria Canker and Dieback of Trees and Shrubs in the Landscape. Virginia Cooperative Extension, Virginia Polytechnic Institute and State University. vt.edu/450/ / html. Cech, T., C. Tomiczek Increasing Prevalence of Botryosphaeria-canker (Botryosphaeria dothidea) of Giant Sequoia in Eastern Austria. BWF Forstschutz Aktuell 57/58, Georgieva, M., M. Dobreva, R. Nachev, G. Georgiev Necrotic disease on poplar seedlings caused by Botryosphaeria sp. - In: Proceedings Scientific publications of Forest Research Institute, Copes, W.E., F.F. Hendrix Jr Effect of temperature on sporulation of Botryosphaeria dothidea, B. obtusa, and B. rhodina. Plant Disease 88, Jurć, D., Ogris, N., Grebenc, T., Kraigher, H First report of Botryosphaeria dothidea causing bark dieback of European hop hornbeam in Slovenia. Plant Pathology, 55 (2), 299. Kehr, R Triebschäden an Mammutbaum (Sequoiadendron giganteum) durch Botryosphaeria dothidea auch in Deutschland nachgewiesen. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes, 56 (2), Kim, D.H., Uhm, J.Y Effect of application timing of ergosterol biosynthesisinhibiting fungicides on the suppression of disease and latent infection of apple white rot cause by Botrosphaeria dothidea. J. of Genetic Plant Pathology, 68, Libby, W.J Some observations of Sequoiadendron and Calocedrus in Europe. California Forest and Forest Production 49. Berkeley, University of California, 12. Phillips, A Botryosphaeria populi sp. nov. and its Fusicoccum anamorph from poplar trees in Portugal, Mycotaxon, Mycotaxon Ltd., USA, 76, Phillips, A.J.L., A. Alves, J. Abdollahzadeh, B. Slippers, M.J. Wingfield, J.Z. Groenewald, P.W. Crous The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology, 76, doi: /sim0021. Piirto, D Giant Sequoia Insect, Disease, and Ecosystem Interactions. USDA Forest Service Genetic Technical Report PSW-151, Sinclair, W.A., H.H. Lyon, W.T. Johnson Diseases of Trees and Shrubs. Cornell University Press, London, 575. Slippers, B., P.W. Crous, S. Denman, T.A. Coutinho, B.D. Wingfield, M.J. Wingfield Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia, 96, Slippers, B., Wingfield, M.J., Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biology Reviews, 21, Zlatković, M., N. Keča, T. Milijašević Symptoms of Botryosphaeriaceae on forests and shade trees in Serbia. - In: First Serbian Forestry Congress Future with Forests, Belgrade, Serbia, November, Topic E: Protection of forests, wood and ornamental plants, Zlatković, M., N. Keča, M.J. Wingfield, F. Jami, B. Slippers Botryosphaeriaceae associated with the die-back of ornamental trees in the Western Balkans. Antonie van Leeuwenhoek, 109, 543. doi: /s Worrall, J.J., J.C Correll, A.H McCain Pathogenicity and teleomorph-anamorph connection of Botrvosphaeria dothidea on Sequoiadendron giganteum and Sequoia sempervirens. Plant Disease 70 (8), margaritageorgiev@gmail.com 62

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