EuropeanBlackwell Publishing Ltd and Mediterranean Plant Protection Organization PM 7/86 (1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes Diagnostics Diagnostic Diaporthe vaccinii Specific scope Specific approval and amendment This standard describes a diagnostic protocol for Diaporthe Approved in 2008-09. vaccinii1. Introduction Diaporthe vaccinii Shear (anamorph Phomopsis vaccinii Shear) is recorded on stems, shoots and leaves of cultivated Vaccinium corymbosum L. (blueberry), V. macrocarpon Aiton (American cranberry), V. vitis-idaea L. (cowberry) and autochtonous species of European V. myrtillus L., (blueberry), V. oxycoccus L. (cranberries). D. vaccinii causes phomopsis canker and dieback, twig blight, viscid rot (fruit rot). It is common in temperate climate areas of North America: Canada (Nova Scotia), USA (in 11 States). There are a few reports of this fungus on plants in Europe: in Romania, UK (eradicated) and Lithuania. Identity Name: Diaporthe vaccinii Shear Anamorph: Phomopsis vaccinii Shear Fig. 1 (A) Symptoms of Phomopsis/Diaporthe vaccinii on twigs of Taxonomic position: Fungi: Ascomycota: Diaporthales Vaccinium corymbosum. (B) Conidiomata on stem of blueberry. EPPO computer code: DIAPVA Phytosanitary categorization: EPPO A1 list no. 211, EU in two months, killing single twigs and often entire plants of a Annex designation: II/A1 susceptible cultivar. On stems, D. vaccinii causes a brown discoloration of the xylem below wilt symptoms. Conidiomata Detection appear on lesions on 1–2 year old twigs (Fig. 1B), and ascomata on 2–3 year old twigs. The fungus also infects leaves, buds, and Blueberries can be killed by D. vaccinii within a few months. fruits of cranberries (Fig. 2A, Fig. 3). Berries become brownish The first symptoms appear on the tips of non-woody shoots red, inflated and shiny. (Fig. 1A). Infected succulent, current-years shoots wilt in 4–6 Similar disease symptoms and morphological features can be days and become covered with minute lesions. The fungus associated with other fungi of the genus Phomopsis described spreads downward through vascular vessels on average 5.5 cm on Vaccinium L., for example P. columnaris D.F. Farr & Castlebury, P. myrtilli Petr. and P. conorum Sac. (Died.), however these species show differences in the size of conidiomata and in 1 Use of names of chemicals or equipment in these EPPO Standards implies the shape of conidia (Table 1). Another Phomopsis species no approval of them to the exclusion of others that may also be suitable. regularly encountered on Vaccinium corymbosum in the 18 © 2009 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 39, 18–24 Diaporthe vaccinii 19 Fig. 2 (A) Symptoms caused by Phomopsis/Diaporthe vaccinii on twig and Fig. 4 (A) Typical lesions caused by Fusicoccum putrefaciens (×10). leaves of Vaccinium macrocarpon. (B) Conidiomata on stem of cranberry (B) Conidia (×400). (damp chamber test). of Diaporthe/Phomopsis vaccinii morphology and its morpho- logical overlap with other Diaporthe species it should be confirmed by internal transcribed spacer (ITS) amplicon sequencing. A flow diagram describing the appropriate tests necessary for a positive diagnosis is given in Fig. 5. Morphology Morphological identification in vivo Direct examination If suspicious symptoms of Phomopsis spp., e.g. fruiting bodies, are observed on diseased parts of Vaccinium spp., a preliminary Fig. 3 Cranberries with symptoms of viscid rot caused by Phomopsis/ diagnosis is possible by direct examination. Spores from Diaporthe vaccinii (right, artificial inoculation) and healthy cranberries fruiting bodies can be removed with the tip of a sterile needle (left). and placed directly in a drop of distilled water on a microscope slide for examination under a compound microscope. Spores Netherlands, is P. viticola (Sacc.) Sacc but its conidia are bigger should also be transferred to an agar medium for isolation in (Table 1). pure culture as described in the section ‘Morphological Symptoms similar to Phomopsis dieback can be associated identification in vitro’. with other fungal pathogens such as Godronia cassandrae Peck (anamorph Fusicoccum putrefaciens Shear) (Fig. 4), Colleto- Damp chamber test trichum spp., Fusarium spp. and Botryosphaeria dothidea (Moug.: Fr) Ces. & de Not (anamorph Fusicoccum aesculi In the absence of fruiting bodies on diseased twigs, leaves or Corda). In the presence of reproductive structures, these fungi fruits, the specimens should be incubated in damp chambers can in the first instance be separated from Phomopsis spp. by with damp filter papers to induce production of fruiting bodies, checking for sporulating structures under a stereoscopic micro- namely conidiomata (pycnidia) (Fig. 2B). Pycnidia are usually scope (20×) or a compound microscope (400×–1000×). In the produced in 4–7 days. Incubation may be prolonged up to absence of sporulating structures, incubation in a damp chamber 30 days to induce the production of ascomata (perithecia) (see below) may stimulate formation of fruiting bodies. although this stage has only been observed in America. Portions of twigs (5–7 cm long, containing healthy parts as well as lesions) should be excised with a scalpel, then washed in distilled water Identification before incubation in damp chambers at 22–25°C in a 12/12 h The identification of the fungus is possible on the basis of light/dark period. The chambers should be periodically morphology but in case of uncertainty given the variable nature moistened and examined daily under a stereoscopic microscope © 2009 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 39, 18–24 20 Diagnostics Table 1 Morphology and diagnostic features of Phomopsis vaccinii, P. conorum, P. myrtilli, P. columnaris and P. viticola Host Conidiomata Conidia Species (country of observation) (measured on host plant) (measured on host plant) Culture on PDA P. vaccinii Shear Vaccinium macrocarpon Pycnidia 200 μm high and 500 μm Conidia of two types: Alpha Mycelium not compact, (Jovaijien1, 2005) (Lithuania) wide (average), dark, subcuticular, conidia 6.0–10.5 × 2.2–3.2 μm, radiate growth pattern, slightly scattered to confluent, spherical, hyaline, unicellular, fusiform, floccose, zonate, white, after large with broader base, unilocular, pointed at both ends, straight, 3 weeks sometimes greyish ostiole single. Conidiophores short, biguttulate.2 white around agar plug in septate and branched. Conidiogenous some strains. cells 12 μm enteroblastic, phialidic. P. conorum Sacc. Vaccinium myrtillus Pycnidia 290–300 μm wide, dark, Conidia 6.5–8.0 × 2.3–3.2 μm, Mycelium white from reverse, (Died.) (Jovaijien1 (Lithuania) scattered, subcuticular, spherical, hyaline, elongated oval, some with equally cottony, minutely & Kaçergius 2008) unilocular, ostiole single. one end obtuse, some slightly floccose. curved, with two at the ends or one large oil globule. P. myrtilli Petr. Vaccinium myrtillus Pycnidia 250–350 × 100–150 μm, Conidia 8.5–14.5 × 3.1–4.0 μm, (Farr et al., 2002b) (Austria, Czech scattered, black, subcuticular, fusiform, sometimes obovate or Republic) conical, ostiole central, oval, tapering towards base, hyaline, 30 μm, uniloculate or occasionally non-septate, smooth, apex slightly multiloculate. Conidiophores absent. rounded, base narrowly truncate or Conidiogenous cells laginiform to rounded, straight or slightly ampulliform, 6–9 × 3–4 μm, curved, with scattered, small 2–4 holoblastic, annellids present. oil globules. P. columnaris Vaccinium vitis-idaea Pycnidia 200–330 μm high and Conidia 6.4–9.4 × 3.6–4.9 μm, Mycelium cottony, white to Farr&Castl. (USA) 440–840 μm wide, subcuticular, ellipsoidal to oval, apex broadly olive grey, radiate growth (Farr et al., 2002b) scattered to confluent, uniloculate, rounded, base slightly truncate, pattern, reverse black to dark black, broadly spherical to flattened, hyaline, unicellular, generally brown, without zones. uniostiolate. Conidiophores thin- with one large gutulla. walled, dark brown, vertically aligned, multicellular. Conidiogenous cells 5–12 × 1.5– 2.5 μm, obclavate to cylindric, straight or curved, developing from the apex of columnar cells. P. viticola Sacc. Vitis vinifera (Australia, No measurement available. Conidia 13.0–17.0 × 2.5–3.0 μm Colony edge entire/slightly S-Africa) Vaccinium (on oatmeal agar) Hyaline, undulate; mycelium cottony, corymbosum (the unicellular, fusiform, pointed at white. Sometimes sectoring Netherlands) both ends, with many small visible. In older cultures guttulla. occurrence of orange droplets on colony surface. 2 Βeta conidia may also been seen 15.0–24.0 × 0.8–1.5 μm, hyaline, filiform, uncinate. These conidia are not used for identification. so that any sporulating structures can be prepared for 20 × 1 μm) (according to Farr et al. (2002a), α conidia are examination as described above. Spores should be transferred 6–11 × 2–4 μm; according to Guerrero & Godoy (1989), β to agar medium for isolation in pure culture as described in the conidia are 14–20 × 0.5–1 μm). section ‘Morphological identification in vitro’. Teleomorph: Diaporthe vaccinii Caruso & Ramsdell (1995) described the teleomorph on Morphological characteristics V. macrocarpon in the USA. Ascomata are localized between Anamorph: Phomopsis vaccinii bark and xylem and are sometimes near conidiomata. Perithecia Conidiomata are dark, spherical, flat near the base, partially 0.3–0.5 × 0.2–0.4 mm diameter, spherical with black, thick, subcuticular, scattered to confluent, uniloculate, uniostiolate, multilayered walls, and flat near the base. Necks of mature 0.2 × 0.5