Bulletin OEPP/EPPO Bulletin (2015) 45 (2), 183–192 ISSN 0250-8052. DOI: 10.1111/epp.12218 European and Mediterranean Plant Protection Organization Organisation Europe´enne et Me´diterrane´enne pour la Protection des Plantes PM 7/048 (3) Diagnostics Diagnostic PM 7/048 (3) Plenodomus tracheiphilus (formerly Phoma tracheiphila) Specific scope Specific approval and amendment This Standard describes a diagnostic protocol for First approved in 2004–09. Plenodomus tracheiphilus (formerly Phoma tracheiphila).1 Revision approved in 2007–09 and 2015–04. Phytosanitary categorization: EPPO A2 list N°287; EU 1. Introduction Annex designation II/A2. Plenodomus tracheiphilus is a mitosporic fungus causing a destructive vascular disease of citrus named ‘mal secco’. 3. Detection The name of the disease was taken from the Italian words ‘male’ = disease and ‘secco’ = dry. The disease first 3.1 Symptoms appeared on the island of Chios in Greece in 1889, but the causal organism was not determined until 1929. Symptoms appear in spring as leaf and shoot chlorosis fol- The principal host species is lemon (Citrus limon), but lowed by a dieback of twigs and branches (Fig. 2A). On the the fungus has also been reported on many other citrus spe- affected twigs, immersed, flask-shaped or globose pycnidia cies, including those in the genera Citrus, Fortunella, appear as black points within lead-grey or ash-grey areas Poncirus and Severina; and on their interspecific and (Fig. 3B). On fruits, browning of vascular bundles can be intergenic hybrids (EPPO/CABI, 1997 – Migheli et al., observed in the area of insertion of the peduncle. 2009). Mal secco is present in the citrus-producing countries Colletotrichum gloeosporioides, the anamorphic state of in the Mediterranean and Black Sea areas with the exception Glomerella cingulata, is a secondary invader of withered of Spain, Portugal and Morocco (CMI map no. 155, 2004). twigs. Acervuli of C. gloeosporioides are often associated The procedure for detection and identification of with the pycnidia of P. tracheiphilus, but they can be easily P. tracheiphilus on Citrus is summarized in the flow dia- distinguished by visual inspection. The acervuli of gram in Fig. 1. C. gloeosporioides are easily visible and appear arranged in concentric rings (Fig. 3B,C), whereas the pycnidia of P. tracheiphilus are scattered and cannot easily be distin- 2. Identity guished by the naked eye, as they are immersed in the corti- Name: Plenodomus tracheiphilus (Petri) Gruyter, Avesk- cal tissues of the twig, under the epidermis (Figs 3B and 4A). amp & Verkley, comb. nov., If pycnidia are present, they can be mounted in distilled water Synonyms: Phoma tracheiphila (Petri) Kantschaveli & or lactic acid and observed under the microscope. Withered Gikashvili, Bakerophoma tracheiphila (Petri) Ciferri, twig pieces may be incubated in a humid chamber for 12– Deuterophoma tracheiphila Petri. 24 h to stimulate the development of pycnidia. After incuba- Hyphomycetous anamorph: Phialophora sp. tion, spore tendrils (cirrhi) protruding from pycnidia can eas- Teleomorph: Leptosphaeria genus. ily be observed under the stereomicroscope. Taxonomic position: Fungi, Ascomycota, Dothideomycetes, Growth of sprouts from the base of the affected branches, Pleosporales, Leptosphaeriaceae. and of suckers from the rootstock, is a very common EPPO code: DEUTTR. response of the host to the disease. Gradually, the pathogen affects the entire tree, which eventually dies. When cutting into the twigs or after peeling off the bark of the branches 1 Use of brand names of chemicals or equipment in these EPPO Stan- and the trunk of the infected trees, characteristic salmon- dards implies no approval of them to the exclusion of others that may also be suitable. pink or orange-reddish discoloration of the wood can be ª 2015 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 45, 183–192 183 184 Diagnostics Sample Fruits Twigs or leaves Alive twigs or leaves Withered twigs or leaves Incubation in a humid chamber Pycnidia produced no Isolation Identification in pure culture yes Sporulation yes no PCR tests PCR tests Morphology of conidia (conventional PCR or real- (conventional PCR or pycnidia and phialides time PCR) real-time PCR) + - + - - + Sample positive Sample negative Sample positive Sample negative Sample positive P. tracheiphila P. tracheiphila for P. tracheiphila for P. tracheiphila for P. tracheiphila for for Fig. 1 Flow-diagram for detection and identification of Plenodomus tracheiphilus on Citrus sp. seen (Figs 2A, B and 3A); this internal symptom is In the field, samples (twigs and leaves) can be taken at associated with gum production within the xylem vessels. any time of the year. If nursery plants are grafted on a On leaves, the typical symptom is the discoloration of small susceptible rootstock, such as sour orange, the rootstock veins; epinasty of young leaves can also be seen in infected should also be inspected and tested. Samples should be pro- branches (Fig. 5). In addition to the more common form of cessed within a few days after being collected. To avoid mal secco, two different forms of the disease can be distin- desiccation, they should be kept in plastic bags wrapped in guished: ‘mal fulminante’, a rapid fatal form of the disease a damp towel or paper and stored at 8–10°C. apparently due to root infection; and ‘mal nero’, a conse- quence of chronic infection of the tree leading to a brown- 3.2 Isolation ing of the heartwood. The sampling procedure depends on the material to be The fungus can be isolated by placing pieces of infected investigated (fruits, leaves, living twigs or withered twigs). tissues taken from discoloured wood from living twigs or Twigs are chosen by visual inspection, and the edge of tis- from symptomatic leaves on Potato Dextrose Agar (PDA), sue with signs of discoloration of the xylem is sampled. Carrot Agar (CA) or Malt Extract Agar (MEA) + chloram- Following isolation, an axenic culture of the fungus is phenicol (Appendix 1). These are suitable media for both obtained on common agar media and the fungus can be isolation and culture (Fig. 2C). Czapek-Dox agar may also identified by microscopic observation of the phialoconidia. be used as a culture medium (Fig. 2D). Petri dishes are ª 2015 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 45, 183–192 Plenodomus tracheiphilus (formerly Phoma tracheiphila) 185 AB C D Fig. 2 (A) Defoliation and dieback on a lemon twig infected by mal secco. The bark of the twig has been peeled off to show the typical salmon-orange discoloration of the xylem. (B) Salmon-orange stain of the wood on the trunk of a lemon tree infected by mal secco. The bark has been peeled off to show the discoloration of the wood. (C) Isolation of Plenodomus tracheiphilus from an infected twig with the typical salmon-orange discoloration. Mycelium of a red-pigmented (chromogeneous) variant of the fungus developing from wood pieces plated on Potato-Dextrose Agar. (D) Colony morphology of three P. tracheiphilus isolates on two media: Czapek-Dox (top lane) and Potato-Dextrose Agar (bottom lane). incubated in the dark at 22–24°C for 6–8 days. On agar 6–12 days. Another suggested isolation method is to cut media, the isolates soon lose their ability to produce twig sections (5–10 mm thick), immerse them in sterile pycnidia and differentiate only phialoconidia. After succes- distilled water for 30–60 min, dry them on a sterile filter sive subculturing some isolates also lose the ability to pro- paper, then quickly flame and plate on agar medium. duce phialoconidia. Isolations can also be performed from Isolation on agar media is easy but time-consuming. It is leaf veins: the leaf is cut into small pieces (2–3 mm), sur- still the most widely-used method for routine diagnosis for face sterilized with 0.5–1% NaOCl for 40 s, rinsed in dis- plants showing symptoms. tilled water and then plated. A slight modification of the conventional isolation 3.3 Growth characteristics in culture method is to cut twig sections (2–5 mm thick), surface-ster- ilize with 0.5–1% NaOCl or 50% ethanol for 40 s to Optimal temperature for growth is 23 Æ 2°C on PDA and 2–5 min depending on the thickness and diameter of the the growth rate is 3.8–6.0 mm per day at this temperature. sections, rinse in sterile distilled water and plate on PDA. The mycelium is at first hyaline and after a few days Petri dishes are incubated in the dark at 23 Æ 2°C for becomes brown or pinkish-red (Fig. 2C). After 10–12 days, ª 2015 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 45, 183–192 186 Diagnostics ABA C B Fig. 4 (A) Hand-made tangential section of a withered lemon twig showing Plenodomus tracheiphilus pycnidia immersed in the cortex. Fig. 3 (A) Longitudinal section of a lemon twig infected by Note the necks of pycnidia emerging through the epidermis (courtesy: Plenodomus tracheiphilus showing the typical salmon orange stain of S. Grasso). (B) Phialides and phialoconidia of P. tracheiphilus the xylem (courtesy: S. Grasso). (B) Comparison between acervuli of (differential interference contrast). C. gloeosporioides (top) and pycnidia of P. tracheiphilus (bottom) on a 2–3 year old dried lemon twig (courtesy: S. Grasso). (C) Dried lemon twig with acervuli of C. gloeosporioides. 4. Identification phialoconidia are produced and should be mounted in dis- When sporulation occurs, identification is possible based on tilled water and observed under the microscope. In culture, cultural and morphological characters. In the absence of confusion is possible with Epicoccum spp., which develop sporulation, identification should be based on cultural char- a brown-red to orange, relatively dense aerial mycelium acters and a molecular method. and the agar medium gradually becomes orange-yellow to brownish in colour.