First Report of Phytophthora Cinnamomi Causing Ink Disease on Castanea Sativa in Greece

Journal of Plant Pathology (2014), 96 (2), 415-417 Edizioni ETS Pisa, 2014 Tziros and Diamandis 415

Short Communication

FIRST REPORT OF PHYTOPHTHORA CINNAMOMI CAUSING INK DISEASE ON CASTANEA SATIVA IN GREECE

G.T. Tziros and S. Diamandis

Hellenic Agricultural Organization, Forest Research Institute, 57006, Vassilika, Thessaloniki, Greece

SUMMARY from the stem base (Jung et al., 1996). After collection, soil samples were moistened with sterile distilled water and In October 2012 a heterothallic Phytophthora was iso- kept at 20°C. About 200 ml of soil were flooded with 500 lated from soil and roots of European chestnut (Castanea ml of distilled water in plastic containers (Jung et al., 1996) sativa) trees from three orchards in Northern Greece. The and subjected to rhododendron leaf baiting (Themann and pathogen was identified as Phytophthora cinnamomi on the Werres, 1998). Newly well developed leaves of Rhododen- basis of morphology and ITS sequence analysis. A patho- dron catawbiense cv. Cunningham’s White were floated genicity test, following the soil infestation method, was over the flooded soil and, after 2 to 8 days, tissue pieces performed and P. cinnamomi was re-isolated confirming around the developed necrotic spots were plated onto Koch’s postulates. This is the first report of P. cinnamomi PARBHy medium. Petri dishes with PARBHy medium as the cause of ink disease in Greece. were then incubated at 20°C in the dark and examined daily under the microscope for developing Phytophthora Key words: chestnut, ITS sequence analysis, pathogenic- colonies which were then sub-cultured on V8-juice agar ity, nursery stock, invasive plant pathogens (V8A). Hyphal cultures of the isolates grown for 10 days on V8A in 90 mm Petri dishes at 20°C in darkness were used for species identification. Phytophthora isolates were Castanea sativa Mill. is one of the most important for- maintained on V8A at 20°C in darkness and sub-cultured est trees in Greece. It is cultivated in coppice forests with at 4 weeks intervals. relatively short rotation for the production of highly valued The colonies on V8A were characterized by a coral- timber and in orchards for the production of nuts on an loid mycelium and clusters of thin-walled chlamydospores. annual basis. Sporangia were produced by placing a mycelial disk from a In October 2012, approximately 20% of eight to ten- 7-day-old culture grown on V8A in non-sterile soil-extract year-old chestnut trees in three orchards in the vicinity of water. Sporangia usually formed after 3 to 4 days of incu- Orma (village in the Prefecture of Pella, Central Macedo- bation at 20°C. Soil extracts were prepared according to nia, Northern Greece) showed disease symptoms includ- Chee and Newhook (1965) by mixing 10 g of soil with 100 ing decline of the crown, dead leaves and burrs which ml of distilled water, then allowing it to rest for one day remained attached to the trees, and dark necrosis of the to let soil particles settle before filtering the supernatant inner bark below the collar with flame-shaped margins. In through a Whatman No. 1 filter paper. Morphology was some cases wilting was followed by a progressive or quick observed by light microscopy, and the length and width of death of the diseased trees. 100 sporangia were measured for each isolate. Sporangia For pathogen isolation root and soil samples containing were ovoid to ellipsoid, persistent, non-papillate, 42 to 75 the rhizosphere and fine roots of declining C. sativa plants μm long and 25 to 38 μm wide. were collected (Table 1). Roots from diseased plants were Since the isolates did not produce oogonia in single cul- rinsed in running water overnight, then tissue fragments ture two different mating tests were performed to deter- were excised and plated directly onto PARBHy selective mine whether the isolates were heterothallic and to which medium (10 mg pimaricin, 250 mg ampicillin, 10 mg ri- mating type they belonged. In the first test, a V8A plug of fampicin, 15 mg benomyl, 50 mg/l hymexazol, 20 g agar, each isolate was placed directly on a microscope slide in 200 ml V8 juice and 800 ml H2O). Each soil sample, con- contact with a V8A plug of an A1 or A2 tester strain from taining the fine chestnut roots, consisted of four monoliths a heterothallic Phytophthora species (Vettraino et al., 2001, of soil (20x30x30 cm) which were collected at the four 2005). As A1 tester strain a Phytophthora cambivora isolate compass points around the tree at a distance of 50-100 cm (21/95-KII) from Chamaecyparis lawsoniana from Italy was used, while four P. cambivora isolates from C. sativa Corresponding author: G.T. Tziros Fax: +30.2310461341 from Greece (SK2, SK3, M3, PYR1; Vettraino et al., 2005) E-mail: [email protected] acted as A2 mating tester strains. Slides were incubated 416 Phytophthora cinnamomi in chestnut in Greece Journal of Plant Pathology (2014), 96 (2), 415-417

Table 1. Soil and root samples taken in the three tested chest- from Quercus cerris in South Africa) thus confirming the nut orchards and number of Phytophthora cinnamomi isolates morphological identification. recovered by baiting from soil and direct isolation from roots Pathogenicity of these six P. cinnamomi isolates was tested by soil infestation tests (Jung et al., 1996) in a Number of samples Number of positive isolations growth chamber at 22°C using 15 two-year-old potted Soil Roots Baiting Direct isolation seedlings per isolate, and 15 non-inoculated plants as con- Orchard 1 4 2 1 2 trols. P. cinnamomi inoculum was prepared by growing Orchard 2 4 3 3 3 the above six cultures for 4 weeks at 20°C on sterilized Orchard 3 6 4 3 4 millet seeds moistened with V8 broth (200 ml V8 juice, Total number 14 9 7 9 3 g CaCO3 and 800 ml H20). Each seedling was inoculated with 30 ml/l of the inoculum, while in control plants at 20°C in the dark and the presence of oogonia in one of sterilized non-inoculated millet seeds were added to the the pairings indicated the mating type of the tested isolate. potting mixture. All plants were flooded for 24 h at two Morphology was then studied observing 100 oogonia for weeks intervals. Five weeks post inoculation, all inoculated each isolate under a light microscope. The sexual behavior seedlings showed wilting, collar and root rot symptoms, of the isolates was confirmed with the sandwich method while control plants remained symptomless. P. cinnamomi (Erwin and Ribeiro, 1996), according to which the agar was re-isolated from artificially inoculated plants, fulfilling plug of the A1 or A2 tester strain and the agar plug from Koch’s postulates. the unknown isolate were made into a sandwich by placing P. cinnamomi has been reported as the causal agent of a non-inoculated V8A plug between them. After six days ink disease of C. sativa from many countries of the world of incubation at 20°C in the dark, the plugs were sepa- (Farr and Rossman, 2013). In Greece, ink disease of chest- rated and the central one was observed microscopically. nut trees has previously been associated with P. cambivora Oogonia had plerotic oospores (35 μm average diameter) (Chitzanidis and Kouyeas, 1970) and P. cryptogea (Perlerou and amphigynous antheridia. Mating tests revealed that et al., 2010). To our knowledge, this is the first report of all isolates belonged to the A2 mating type, in accordance P. cinnamomi causing ink disease on chestnut in Greece. with Vettraino et al. (2005) who reported that all the test- P. cinnamomi and P. cambivora are considered as the ed isolates of P. cinnamomi from chestnut belong to the two species most commonly associated with ink disease worldwide dominant mating type A2. in Europe and most aggressive to chestnut (Vannini and The morphological and morphometric traits of the fun- Vettraino, 2001; Vettraino et al., 2005). In Greece, P. cam- gal structures agreed with the description of Phytophthora bivora is the prevailing species in chestnut orchards and cinnamomi Rands (Erwin and Ribeiro, 1996; Gallegly and natural stands (Vannini and Vettraino, 2001). Nonetheless, Hong, 2008). as P. cinnamomi is more aggressive than P. cambivora to Mycelial DNA of six isolates was extracted using the European chestnut (Vettraino et al., 2001), it constitutes a DNeasy plant mini kit (Qiagen, USA) according to the potential major threat to C. sativa in Greece. In fact, in the manufacturer’s instructions. Template DNA (8 μl), 0.5 μl present case, circumstantial evidence of aggressiveness was of each primer (reverse primer ITS4 and forward primer provided by the severity of the disease, as the trees were ITS6), 5 μl of a commercial red reaction buffer, contain- killed within one growing season, without showing typical symptoms like small-sized foliage, thin crown and attached ing a mixture of PCR Buffer, MgCl2 and dNTPs (Bioline, burrs from the previous season. Furthermore, as P. cin- USA) one unit of Taq polymerase and bi-distilled H2O were added to each reaction tube to make a final volume namomi has a wide range of hosts, it could be a potential of 25 μl. PCR was performed with a thermocycler (Master- threat to horticultural, ornamental and forestry plant spe- cycler, Eppendorf, USA) with the following amplification cies with economic and aesthetic importance. conditions (Cooke and Duncan,1997): initial denaturation In a previous Europe-wide study P. cinnamomi was not step at 95°C for 2 min, 30 cycles of annealing at 55°C for detected in declining chestnut stands in Greece (Vettraino 30 sec, extension at 72°C for 1 min and denaturation at et al., 2005). Most likely, P. cinnamomi was introduced 95°C for 30 sec with final extension at 72°C for 10 min. more recently in the three orchards object of this study, PCR products were purified with PureLinkTM PCR puri- subsequently in the surrounding area, through transport fication kit (Invitrogen, USA) and custom sequenced (Ce- of infected nursery stock, confirming the importance of mia, Greece). The sequences were initially visualized by the nursery pathway in the spread of aggressive invasive ChromasLite (Technelysium, Australia), visually aligned plant pathogens (Jung and Blaschke, 2004; Jung et al., and deposited in GenBank under the accession Nos. 2009). KF559324-KF559329. A BLAST comparison with sequences retrieved from GenBank revealed 100% homology with those of various P. cinnamomi isolates (e.g. with sequence GU799635 of the P. cinnamomi strain CMW33386 isolated Journal of Plant Pathology (2014), 96 (2), 415-417 Tziros and Diamandis 417

ACKNOWLEDGEMENTS Species by Morphology and DNA Fingerprinting. APS Press, St Paul, MN, USA. Study funded by the project SEE-ERAplus-138-PHY- Jung T., 2009. Beech decline in Central Europe driven by the SEE “Diversity of invading Phytophthora spp. plant patho- interaction between Phytophthora infections and climatic gens in agro and forest ecosystems in Southeast Europe”. extremes. Forest Pathology 39: 73-94. The authors gratefully acknowledge Dr. A.M. Vettraino Jung T., Blaschke M., Neumann P., 1996. Isolation, identifica- (University of Tuscia, Viterbo, Italy) for kindly providing tion and pathogenicity of Phytophthora species from declin- the A1 tester strain and the anonymous reviewer for the ing oak stands. European Journal of Forest Pathology 26: 253- valuable comments and suggestions. 272. Jung T., Blaschke M., 2004. Phytophthora root and collar rot of alders in Bavaria: distribution, modes of spread, and possible management strategies. Plant Pathology : 197-208. REFERENCES 53 Perlerou C., Tziros G., Vettraino A.M., Diamandis S., 2010. Chee K.H., Newhook F.J., 1965. Improved methods for use in Phytophthora cryptogea causing ink disease of Castanea sativa studies on Phytophthora cinnamomi Rands and other Phy- newly reported in Greece. Plant Pathology 59: 799. tophthora species. New Zealand Journal of Agriculture Re- Themann V.K., Werres S., 1998. Use of Rhododendron leaves search 8: 88-95. to detect Phytophthora species in root and soil samples. Chitzanidis A., Kouyeas H., 1970. Notes on Greek species of Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 50: 37- Phytophthora II. Annals of the Benaki Phytopathological In- 45. stitute (New Series) 9: 267-274. Vannini A., Vettraino A.M., 2001. Ink disease in chestnuts: im- Cooke D.E., Duncan J.M., 1997. Phylogenetic analysis of Phy- pact on the European chestnut. Forest Snow and Landscape tophthora species based on the ITS1 and ITS2 sequences of Research 76 (3): 345-350. ribosomal DNA. Mycological Research 101: 667-677. Vettraino A.M., Natili G., Anselmi N., Vannini A., 2001. Re- Erwin D.C., Ribeiro O.K., 1996. Phytophthora Diseases World- covery and pathogenicity of Phytophthora species associated wide. APS Press, St Paul, MN, USA. with a resurgence of ink disease in Castanea sativa in Italy. Plant Pathology : 90-96. Farr D.F., Rossman A.Y., 2013. Fungal Databases, Systematic 50 Mycology and Microbiology Laboratory, ARS, USDA. Re- Vettraino A.M., Morel O., Perlerou C., Robin C., Diamandis S., trieved December 17, 2013 from http://nt.ars-grin.gov/ Vannini A., 2005. Occurrence and distribution of Phytoph- fungaldatabases/. thora species in European chestnut stands, and their asso- ciation with Ink Disease and crown decline. European Jour- Gallegly M.E., Hong C., 2008. Phytophthora, Identifying nal of Plant Pathology 111: 169-180.

Received December 17, 2013 Accepted February 7, 2014