Bulletin OEPP/EPPO Bulletin (2014) 44 (3), 338–349 ISSN 0250-8052. DOI: 10.1111/epp.12159 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/14 (2) Diagnostics Diagnostic PM 7/14 (2): Ceratocystis platani Specific scope Specific approval and amendment This Standard describes a diagnostic protocol for First approved in 2002–09. Revised in 2014–06. Ceratocystis platani.1 M.J. Wingf. & E.C.Y. Liew (from Syzygium aromaticum), Introduction C. fimbriata sensu stricto (s.s.) Ellis & Halsted (from Ipomea Ceratocystis platani causes a destructive tracheomycosis in batata), C. cacaofunesta Eng. &Harr. (from Theobroma Platanus spp. In the EPPO region, Platanus 9 acerifolia cacao), C. variospora (Davids.) C. Moreau (from Quercus (Ait.) Willd., suffers severe damage. Infected trees usually spp.), C. populicola J. A. Johnson and Harrington (from die within 3–7 years (EPPO/CABI, 1997). The disease is Populus spp.), C. caryae J.A. Johnson and Harrington (from native to the USA, and in Europe its presence is confirmed Carya spp., Ulmus spp., Ostrya virginiana), C. smalleyi J.A. in Italy, France, Switzerland and Greece. An outbreak was Johnson and Harrington (from Carya spp.), C. manginecans detected in Spain in 2010 and has been eradicated. The M. van Wyk, A Al Adawi & M.J. Wingf. (from Mangifera fungus is transmitted by contaminated pruning tools and indica), C. atrox M. van Wyk & M.J. Wingf. (from terracing machinery which causes wounding to the roots. It Eucalyptus grandis), C. tsitsikammensis Kamgan & Jol. Roux may also be transmitted by root contact (anastomosis), and (from Rapanea melanophloeos), C. neglecta M. van Wyk, Jol. infected dead plant tissue in soil which may be infectious Roux & C. Rodas (from Eucalyptus spp.), C. fimbriatomima for up to 5 years. Sawdust and wood chips from diseased M. van Wyk & M.J. Wingf (from Eucalyptus sp.). trees are highly infective. Apart from phytosanitary Phylogenetic analyses of DNA sequences clearly showed that measures which prevent the spread of the disease to new among the above cited cryptic species, C. fimbriata s.s., areas, no control methods are available. C. cacaofunesta, C. manginecans, C. fimbriatomima and Ceratocystis platani belongs to the C. fimbriata sensu C. neglecta are the closest to C. platani (Baker et al.,2005; latu (s.l.) species complex, which includes several host- Van Wyk et al., 2007b, 2009; Rodas et al., 2008). specialized cryptic species and not completely characterized Host specialization is considered a major factor driving lineages in the process of speciation or awaiting species speciation within the genus and appears to be a major description. All these groups are morphologically similar or factor which helps to define the groups/populations as (nearly) indistinguishable. Phylogenetic analyses of DNA cryptic species within C. fimbriata s.l. (Baker et al., 2003; sequences revealed three geographic clades within the Johnson et al., 2005; Baker-Engelbrecht & Harrington, C. fimbriata complex which are located respectively in 2005). In inoculation trials C. platani showed a strong host North America, Latin America and Asia. specialization and, as a host, Platanus was shown to be The list of described cryptic species within C. fimbriata s.l. strongly selective when challenged with C. fimbriata and their hosts: C. albifundus M.J. Wingf., De Beer & Morris isolates obtained from a variety of host species (Baker (from Acacia mearnsii), C. pirilliformis Barnes & M.J. et al., 2003; Baker-Engelbrecht & Harrington, 2005). To Wingf. (from Eucalyptus spp.), C. polychroma M. van Wyk, date, C. platani is the only Ceratocystis species that has been isolated from and which is able to cause a disease in 1Use of brand names of chemicals or equipment in these EPPO the genus Platanus. Thus, it is unlikely (although it cannot Standards implies no approval of them to the exclusion of others that be excluded) that Platanus will become infected by may also be suitable. C. fimbriata cryptic species other than C. platani. 338 ª 2014 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 44, 338–349 Ceratocystis platani 339 Fig. 1 Flow diagram for the detection and identification of Ceratocystis platani. A flow diagram describing the diagnostic procedure for where necrosis advances, show no callus reaction. The C. platani is presented in Fig. 1. pathogen is able to enter the vessel elements and progresses vertically. It also invades the xylem parenchyma and proceeds radially. In the latter case C. platani can reach the Identity bark (Fig. 2B) causing the appearance of typical reddish/ Name: Ceratocystis platani (J. M. Walter) Engelbr. & T. bluish spots in the external surface. After death of the tree C. Harr. or of sections of the stem, the bark becomes hazel brown Synonyms: Endoconidiophora fimbriata (Ellis & Halsted) and detaches from the vascular cylinder. Davidson f. sp. platani Walter, Ceratocystis fimbriata sp platani Detection from recently infected plant material or plant Taxonomic position: Fungus; Ascomycota; Pezizomycotina; material that has recently died Sordariomycetes; Hypocreomycetidae; Microascales; Ceratocystidaceae Light microscopy: C. platani can be detected in samples EPPO code: CERAFP collected, preferably, on the necrotic edge of an advancing Phytosanitary categorization: EPPO A2 list no. 136; EC canker by means of an increment borer. Thin hand-made Annex designation II/A2 sections are then directly prepared from cores, and the presence of the fungus can be ascertained by light microscope observation of dark diagnostic aleurioconidia inside xylem Detection vessels (200–4009) (Fig. 3). Wood sections should be prepared longitudinally (vessel-oriented) to maximise the Symptoms chance of detecting aleurioconidia. Aleurioconidia are Platanus species are the only hosts of C. platani. The present only in the necrotic tissue, not in the healthy-looking pathogen can be present in growing plants, wood, wood tissue. Aleurioconidia observed in the plant tissue may have chips, and sawdust. Plants may be affected singly or in the same shape and colour as those observed in culture. groups. Externally, the whole canopy or a single branch However, the plant tissue-inhabiting aleurioconidia can also can begin to decline showing sparse chlorotic foliage. be deformed to different degrees, depending on their location Infection and progress of the pathogen in the roots and the inside the vessels and their age. Specifically, in tissue that stem cause a greyish to brown discoloration of the wood died some time ago and in dehydrated wood, aleurioconidia and a reddish to bluish staining of the bark. These are the are rarely observable and they frequently appear as a deflated signs of necrosis and loss of function of the vascular ball in which one half incorporates the other. Detection of system, as well as of the parenchymatous tissue, cambium aleurioconidia, both in the canonical and non-canonical form, and bark (Fig. 2). In general, the edges of the lesions, is sufficient for a positive detection. If rainy or humid weather ª 2014 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 44, 338–349 340 Diagnostics A B Fig. 2 A) Typical discolored areas in a transversal section of a trunk of Platanus infected by Ceratocystis platani, B) Symptoms on bark. are placed into polyethylene bags and incubated in the dark at 20–25°C. Ceratocystis platani is detected when perithecia emerge from the inoculation points and ascospores, if present, are observed by light microscopy. The whitish/greyish mycelium typical of C. platani has also a diagnostic value if endoconidia are observed by light microscopy (Pilotti et al., 2009, 2012). • Baiting test: to detect C. platani in wood samples, soil or water a trap technique has been adapted from Grosclaude et al. (1988) and improved by ANSES (FR). This test is presented in Appendix 2. • Real-time PCR (see Appendix 3): this test is particularly valuable due to its high analytical and diagnostic sensitivity and specificity. Confirmation using real-time PCR is Fig. 3 Presence of aleurioconidia of Ceratocystis platani inside xylem recommended in case of negative results with microscope vessels. examination, moist chamber, isolation and carrot tests, as conditions persist, C. platani perithecia can also be present. these tests are reported to give false negative results. It is The presence of perithecia with typical ascospores is also recommended that a negative result of a baiting test sufficient for a positive diagnostic. on samples collected from symptomatic trees is confirmed In freshly collected samples, in the absence of perithecia using real-time PCR. Note that real-time PCR methods or aleurioconidia, the following methods can be used for have not been validated on water and soil. the detection of C. platani: When detection is based on the observation of perithecia • Moist chamber test: cores collected on the edge of lesions (freshly collected samples, moist chamber, carrot test) by means of an increment borer are cut into discs (2–3mm typical C. platani ascospores should be observed (at the top thick) and incubated in a moist chamber at 20–25°C. of the perithecium neck or inside the perithecium). Ceratocystis platani, if present, can produce endoconidia and perithecia after 3–8 days (Vigouroux, 1979); Detection from material which died a long time ago, • Isolation on media: the pathogen can be isolated from soil or water pieces of necrotic wood collected from the leading edge of a lesion on a freshly diseased plant. These are chopped From tissue that has been dead for a long time (collected into small pieces, rapidly flamed and placed on potato either from a dead tree or from a dead part of a living dextrose agar (PDA), malt agar (MA) or malt yeast tree), successful isolation on agar media is highly unlikely, extract agar MYEA. All media are supplemented with as many saprophytic fungi colonize dead wood and will streptomycin (see Appendix 1). Plates should be outcompete C.