VOLUME 2 DECEMBER 2018 Fungal Systematics and Evolution PAGES 45–56 doi.org/10.3114/fuse.2018.02.04 Caliciopsis pleomorpha sp. nov. (Ascomycota: Coryneliales) causing a severe canker disease of Eucalyptus cladocalyx and other eucalypt species in Australia I.G. Pascoe1*, P.A. McGee (Maher)2, I.W. Smith3, S.-Q. Dinh4, J. Edwards4 130 Beach Road, Rhyll, Victoria 3923, Australia 2RMIT University, GPO Box 2476, Melbourne VIC 3001, Australia 3Bushbury Forest Pathology Service, 31 Old Monbulk Road, Belgrave, Victoria 3160, Australia 4Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, 5 Ring Road, La Trobe University, Bundoora, Victoria 3083, Australia *Corresponding author: [email protected] Key words: Abstract: Caliciopsis pleomorpha sp. nov. is described from a severe stem canker disease of cultivated Eucalyptus cladocalyx one new taxon ‘Nana’ (dwarf sugar gum) in Australia. The fungus is a pleomorphic ascomycete (Coryneliales), with pycnidial (pleurophoma- pathogenicity like) and hyphomycetous (phaeoacremonium-like) morphs, and differs in these respects and in ITS sequences from other stem canker Caliciopsis spp. The fungus was also found associated with cankers on other Eucalyptus species growing in native habitats, taxonomy and was successfully inoculated under glasshouse conditions into a wide range of Eucalyptus species on which it caused cankers of varying severity. Published online: 13 June 2018. INTRODUCTION MATERIALS AND METHODS Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] In 1979, a stem canker disease was observed on Eucalyptus Microscopic observations cladocalyx, at Frankston, Victoria, Australia. Cankers were first seen on E. cladocalyx ‘Nana’, a dwarf selection of E. Ascomata and pycnidia were rehydrated with a drop of water cladocalyx that is widely grown as a shelter-belt and street tree. for ease of manipulation, and observed by hand sectioning Subsequently the disease was found to be extremely damaging or crushing. Specimens were mounted in water, a solution of on E. cladocalyx ‘Nana’, in urban amenity trees and rural shelter- 50 % lactic acid/50 % glycerol or a lactic acid/glycerol solution belts in many localities, in some cases virtually destroying with acid fuchsin added, and examined under the compound shelter-belt plantings on farms. microscope. Slides were sealed with nail varnish for long-term A fungus consistently found sporulating on the cankers storage and for observations using oil immersion magnification. was identified as a species of Caliciopsis (Ascomycota, Drawings were prepared using a drawing tube. Observations and Coryneliales). Comparison of the fungus with other species of descriptions are a compilation of data from all the Eucalyptus the genus Caliciopsis showed the fungus to be a new species. canker specimens listed in “Specimens Examined” below. Its morphology and taxonomy, the disease symptoms caused and experiments on pathogenicity, host range and control are Growth in culture described in detail in the MSc thesis of Maher (1993). The present paper describes the symptoms of the disease Standard mycological media used are as in Crous et al. (2009). All (Fig 1), the morphology of the various morphs of the fungus isolations from the host were derived from single ascospores or (Figs 2–7) both on host tissue and in culture, investigates the single conidia and grown initially on potato dextrose agar (PDA). phylogeny of the fungus using ITS sequencing (Fig. 8), formally Experiments were conducted to determine the best media for describes the fungus as a new species, Caliciopsis pleomorpha, vegetative growth and sporulation. Isolates were grown on and documents its pathogenicity to E. cladocalyx ‘Nana’ and 14 PDA, malt extract agar (MEA), distilled water agar (WA), corn other taxa of Eucalyptus (Fig. 9) while listing 9 Eucalyptus taxa meal agar (CMA), oatmeal agar (OMA) Czapek agar (CZA) and and 2 Corymbia taxa on which the fungus has been recorded synthetic nutrient-poor agar (SNA) (Nirenberg 1976, Crous et from field collections in 4 Australian states. al. 2009) ). In an attempt to improve in vitro production of ascomata and conidiomata, additional media were trialed which consisted of WA with added plant material, including gamma- irradiated carnation leaves (CLA), autoclaved millet seeds (MSA) and autoclaved or gamma-irradiated Eucalyptus twigs (Eucalyptus twig agar - ETA). The different plant materials were added to freshly poured, still molten WA. Fungal Systematics and Evolution is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License © 2018 Westerdijk Fungal Biodiversity Institute 45 Pascoe et al. Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Fig. 1. Symptoms of Caliciopsis canker. A. Severely diseased young shelter-belt trees at Flinders, Victoria. B. Severe cankers on twigs (1–2 cm diam) of E. cladocalyx ‘Nana’. C. Developing lesions on vigorous main stem (3–4 cm diam) of E. cladocalyx ‘Nana’, centred on Aulographina eucalypti lesions. D. Lesion on E. cladocalyx ‘Nana’ 3 mo after agar plug inoculation. E. Uninoculated lesion 3 mo after wounding. 46 © 2018 Westerdijk Fungal Biodiversity Institute Caliciopsis canker of Eucalyptus Single ascospore isolations from VPRI 16689 were plated onto with sodium hypochlorite and placed onto distilled water agar PDA to assess the growth of the fungus at a range of temperatures and incubated at 25 °C, for 1–3 wk. and with or without light. PDA plates were inoculated with 5 mm diam colonised agar plugs (2 per plate) taken from 4-wk-old PDA Pathogenicity to some commercially important taxa of cultures. To determine growth rate and optimal temperature for Eucalyptus was tested with the following 15 taxa: E. botryoides, growth, three plates per temperature were grown at 5, 10, 15, E. cladocalyx (tall form), E. cladocalyx ‘Nana’, E. delegatensis, 20, 25, 30 and 35 °C. In a second experiment to further refine E. fastigata, E. globulus, E. grandis, E. marginata, E. nitens, the optimum temperature cultures were grown at 23, 25, 28, E. obliqua, E. radiata, E. regnans, E. saligna, E. sieberi and E. Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] 30, 32 and 35 °C. Growth of the colonies in each experiment viminalis. The experiment was performed in temperature- was assessed by measuring the diameter of each colony in two controlled glasshouses with an automatic watering system using directions, perpendicular to each other. The diameters were overhead sprinklers. then averaged and the results, including descriptions of visual Five randomly chosen saplings of each species were appearance of cultures, recorded. inoculated with colonised 5 mm PDA discs, while five saplings of each species randomly chosen as controls were inoculated Molecular analysis with sterile 5 mm PDA discs. Prior to inoculation the surface of the chosen stem was sterilised by swabbing with 70 % ethanol. DNA from mycelium of pure cultures VPRI 17721 and VPRI Inoculum and control discs were inserted under a 5–6 × 2–3 mm 15646, was extracted with Bioline Plant DNA Kits following flap cut into the periderm with a sterile scalpel blade at a point the manufacturer’s instructions. The ribosomal DNA internal 150 mm above soil level. The inoculation sites were wrapped transcribed spacer region was amplified and sequenced using in Parafilm© for 2 wk for protection against desiccation of the primers ITS1F and ITS4 (White et al. 1990). Attempts to obtain LSU sapwood. Assessments of lesion development were made at 1, sequences were unsuccessful. The PCR products were purified 2.5, 4, 5, 6 and 7 mo after inoculation. Lesions were assessed and sequenced using Sanger 454 by Macrogen (Korea). The for length and width and presence or absence of pycnidia and sequence data were visually corrected and aligned (ClustalW), ascomata and rated according to their general appearance. and the Neighbour-Joining tree was built using Geneious v. 7. Pathogenicity testing RESULTS Pathogenicity tests were conducted initially with E. cladocalyx Symptoms of disease ‘Nana’. Later, additional pathogenicity tests were conducted with 15 commercially important taxa of Eucalyptus including On severely affected trees of E. cladocalyx ‘Nana’, cankers E. cladocalyx ‘Nana’. Inoculum for experiments was produced can be found on branches, twigs and the main trunk and the by inoculating PDA plates with 5 mm diam agar mycelial plugs tree gradually defoliates over a number of years and dies back colonised by the isolates VPRI 16689 (E. cladocalyx ‘Nana’ as the cankers coalesce (Fig. 1A). Severe cankers are mostly experiment) and VPRI 16907 (multi-species experiment). The seen on smaller twigs where they destroy the bark and leave inoculum plates were incubated for 4 wk at 25 °C, prior to an elongated dark brown, ragged canker (Fig. 1B) exposing experimentation. the sapwood and quickly girdling the twig. Callus production appears to be suppressed. Milder cankers are seen on larger, Pathogenicity to E. cladocalyx ‘Nana’ was investigated by vigorously growing branches (> 3 cm diam), and these develop inoculation of four randomly selected branches (each with one into lesions up to 5 cm diam (Fig. 1C) while leaving the