COST action FP1401 A global network of nurseries as early warning system against alien tree pests- Global warning Training school Fungal taxonomy and fungal identification using traditional (i.e. not molecular) techniques Identification of Botryosphaeriaceae associated with the die-back of ornamental trees in the Western Balkans Dr Milica Zlatković Warsaw, 2016 1
During the course of last decade extensive die-back and mortality of various trees and shrubs has been observed in Serbia, Montenegro, Bosnia and Herzegovina External symptoms: Necrotic lesions A. hippocastanum Q. robur C. atlantica Cankers A. concolor C. sempervirens C. atlantica 2
Resin bleeding C. lawsoniana C. atlantica T. occidentalis C. sempervirens P. nigra A. concolor Top die-back C. lawsoniana C. sempervirens C. atlantica 3
Death of the whole trees C. sempervirens C. atlantica T. occidentalis P. tremula P. abies Internal wood symptoms: Circular or irregular browning or reddening of the vascular tissue Brown vascular streaking visible as spots in cross sections S. giganteum C. sempervirens P. laurocerasus P. nigra T. gigantea 4
The cause of the die-back appeared to be associated with extreme weather conditions ( Heat waves, Cold waves, severe droughts, storms, heavy snowfall, large scale flooding) And Botryosphaeriaceae infections Wound-infecting, opportunistic canker pathogens Exist as endophytes in asymptomatic plant tissues Bot associated diseases emerge after trees have been subjected to stress Recent increased pathogenic activity and geographic range expansion of the Bot has been linked to warmer climate and extreme weather Luchi et al. 2016. Appl Microbiol Biotechnol 100:5189-5204 www.weatherquestions.com 5
Bot have a broad host range The majority of Bot infect multiple hosts, which may or may not be closely related to each other Move from native to non native species and vice versa Bot have mostly been found associated with angiosperms Angiosperms appear to have been major group of plants on which these fungi evolved and only later colonized and specialized on gymnosperms Malus domestica, Delgado-Cerrone et al. 2013 Pseudotsuga menziesii Jami et al. 2014. Fungal biology 118: 168-179 Vitis vinifera, Abreo et al. 2013 Some Bot predominantly infect conifers-diplodia sapinea 6
Bot are difficult to identify The taxonomy of these species has been confused for years Large number of phylogenetically closely related and morphologically similar cryptic species Differentiation of species mostly relies on molecular phylogenetic analyses of multiple gene regions The family currently comprises 22 genera Phillips et al. 2013. Studies in mycology 76: 51-167 2. Aim of the study To clarify the identity of the Botryosphaeriaceae associated with symptoms of dieback, cankers and abundant resin production on ornamental trees in the Western Balkans 7
3. Materials and methods Disease surveys, sample collection Disease surveys were conducted between 2009 and 2014 in Serbia, Montenegro, Bosnia and Herzegovina and Greece Urban forests, public greens, parks, private gardens in 15 cities, 3 villages, 8 forest stands, 3 forest plantations and 2 ornamental nurseries 219 trees (40 species) 15 shrubs (5 species) P. menziesii F. europaea A. concolor C. lawsoniana C. sempervirens P. abies 3. Materials and methods Botryosphaeriaceae isolation Bot were isolated from the edges of necrotic lesions, resinous chips of xylem tissues, discolored wood, resinous twigs, needles or cone petioles, asymptomatic tissues and fruit bodies Small pieces of tissues were washed in tap water, surface disinfected, rinsed in sterile distilled water, dried on sterile paper towels, flamed and placed onto 2% MEA complemented with lactic acid (AMEA) 8
3. Materials and methods Botryosphaeriaceae isolation Petri dishes were sealed with Parafilm and incubated at room temperature in the dark for 2 weeks Bot-like isolates (mycelium greenish-grey, fast growing) were transferred aseptically to new Petri dishes containing MEA Isolates were purified by hyphal tip transfers and stored on half strength MEA slants at 4 C 3. Materials and methods DNA extraction, PCR and sequencing Combination of nuclear (ITS, LSU) and mitochondrial genes (TEF-1-α, β- TUB, RPB2) GCPSR Molecular identification of fungi, 2010. Y. Gherbawy, V. Kersin (Eds.), Springer-Verlag, Berlin 9
3. Materials and methods Morphological characterization To induce sporulation cultures were grown on WA overlaid with triple sterilized pine needles or Lawson cypress twigs and incubated under near UV light at 25 C for 6 weeks Morphological observations were made with Carl Zeiss stereo and light microscopes using Axiocam digital camera and Axiovision imaging software 3. Materials and methods Morphological characterization Fruit bodies were sectioned using Jung tissue freezing medium and Leica cryotome or smashed by hand and mounted in sterilized distilled water 50 measurements were made of the lengths and widths of conidia and microconidia. Dimensions of other fungal structures were given as the range of least 20 measurements 10
3. Materials and methods Morphological characterization Shape, color, presence and absence of septa and a mode of conidiogenesis were recorded 3. Materials and methods Growth studies Isolates were used to study colony morphology and determine the effect of temperature on radial colony growth in culture For each isolate 6 diam. mycelial plugs were taken from the edges of 1- week old cultures and transferred to the centers of 90 mm diam. Petri dishes containing 2% MEA Plates were incubated at 9 temperatures: 5-40 C for 2 weeks in the dark 11
3. Materials and methods Growth studies Two measurements of colony diameter at right angles to each other were taken daily at a fixed time for 3 days The optimal temperature was defined as the temperature that produced maximum mycelial growth Molecular phylogenetic identification Phylogenetic analyses revealed six major and strongly supported clades, each representing a separate genus, including: Dothiorella, Neofusicoccum, Botryosphaeria, Phaeobotryon, Sphaeropsis and Diplodia Nine known species were identified, including Do. sarmentorum, Do. omnivora, B. dothidea, D. seriata, D. sapinea, D. mutila, S. visci, P. cupressi and N. parvum One additional species was recognized in Dothiorella 12
Taxonomy Dothiorella sp. Pycnidia with long necks (200-1000µm diam., 250-3000µm high) Annelidic and phialidic conidium development Rough-walled conidia Taxonomy Dothiorella sarmentorum Conidia remained hyaline long after discharge from the pycnidium and became 1-3 septate and rough walled after ageing Diplodia seriata Conidia became rough-walled and septate with age or septate without becoming dark 13
Taxonomy Dichomera syn-asexual morphs of Do. omnivora, B. dothidea, N. parvum and D. sapinea Conidia were oval, ovoid or ellipsoid, doliiform, muriform, golden brown with transversal, longitudinal and oblique septa, in D. sapinea rarely bearing 1-3 appendages Or they were hayaline, ellipsoid or pyriform to limoniform, aseptate Taxonomy Botryosphaeria dothidea Hyaline fusiform conidia readily formed 1-3 septa after ageing Dark-walled spores were observed while still attached to conidiogenous cells Neofusicoccum parvum Conidia became 1-3 septate after ageing 14
Taxonomy Diplodia sapinea Conidia were mostly 1-4 septate, sometimes also with 3 horizontal and 1 oblique septum Isolate from C. atlantica formed flat mycelial mat Taxonomy Diplodia mutila Conidia were sometimes cylindrical, strongly constricted at the septum with 1 white cell and 1-2 dark brown cells Hyaline conidia were often 1-2 septate becoming dark and up to 2 septate with age 15
Taxonomy Sphaeropsis visci Pycnidia were immersed or partially erumpent Conidia were much larger then previously described (up to 56 x 36 µm) becoming dark brown and 1-2 septate with age Taxonomy Phaeobotryon cupressi Isolate formed conidiophores that have not been observed in previous collections Conidiogenous cells were broad ampuliform, narrowing to form a long neck, proliferating internally and percurrently giving rise to annellations Conidia formed up to 2 septa 16
Growth studies Bot species grew over a wide range of temperatures (5-40 C) Optimum temperature for the majority of species was 25 C D. sapinea CMW39338 and B. dothidea had the optimal temperature of 30 C 5. Outputs 17
Acknowledgements 18