First Report of Powdery Mildew of Platanus × acerifolia and P. occidentalis Caused by Erysiphe platani in Greece.

London planetrees (Platanus × acerifolia, syn. P. × hispanica), American sycamores (P. occidentalis), and oriental planes (P. orientalis) are widely planted as urban shade trees throughout Greece and many other countries. In June 2012, typical symptoms of a powdery mildew were detected on all sycamores (10 trees) along a central avenue of Heraklion (Crete, Greece), with the disease affecting approximately 80% of the leaves of all infected trees. In August 2013, similar symptoms were observed on 20% of the leaves of all three London planes in a small grove in the Vrysses area of Lasithi (Crete, Greece). In both cases, the disease was severe, with white superficial colonies developing amphigenously on leaves, twigs, floral peduncles, inflorescences, and fruits. The colonies were initially distinct and circular but gradually enlarged and often coalesced to cover the entire leaf blade. Young leaves appeared curled and chlorotic, occasionally leading to defoliation. For the morphological description of the pathogen, samples from seven infected P. occidentalis and three P. × acerifolia trees were microscopically characterized. In all samples, the pathogen's mycelium was branched, septate, and hyaline, with lobed appressoria; conidiophores were erect, cylindrical, unbranched, and consisted of three to four (to five) cells; and conidia were single or in short chains (two to four), ellipsoid or doliiform, with a truncated base and rounded apex. Their dimensions were 24.3 to 48.6 × 15.8 to 27.9 µm (averaging 39.2 × 21.2 µm; n = 100), and their surfaces appeared reticulate. The teleomorph was never observed. Total fungal DNA was extracted from conidia harvested from affected leaves of one infected plant of each of P. occidentalis and P. × acerifolia planes, and the ITS1-5.8S-ITS2 region was PCR-amplified with universal primers 18S-ITS1 and 28S-ITS2 (2) and sequenced (GenBank Accession Nos. KM068123 and KM068124, respectively). A BLASTn search of GenBank revealed 100% identity of both samples to Erysiphe platani strains described on P. orientalis in Greece (JQ365943) and P. occidentalis in Brazil (KF499270). Based on the morphological and molecular analyses, the pathogen was identified as E. platani (Howe) U. Braun & S. Takam. (formerly known as Microsphaera platani Howe) (1). To prove pathogenicity and fulfill Koch's postulates, 10 1-year-old seedlings of each of P. occidentalis and P. × acerifolia hosts were artificially inoculated with conidia obtained from naturally infected plants of the corresponding species, with two methods: (i) five plants of each host were dusted with conidia from diseased leaves, and (ii) the remaining five seedlings of each plane were sprayed with a conidial suspension of the fungus (107 conidia ml-1), while five additional control plants of each species were treated only with sterile distilled water. All plants were maintained in the greenhouse at 25 ± 3°C, with 90% humidity. Powdery mildew symptoms, which appeared 9 and 15 days after inoculation on all dusted and sprayed plants, respectively, were similar to those observed on naturally infected trees, whereas no symptoms were observed on control plants. Although E. platani is known to infect plane species in several parts of the world (1), including oriental planes (P. orientalis and P. orientalis var. cretica) in Greece (3), this is the first report of E. platani causing disease of P. occidentalis and P. × acerifolia in Greece, underlining the need for appropriate control measures to prevent significant losses to the local ornamental industry. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, 2012. (2) I. A. Papaioannou et al. Eur. J. Plant Pathol. 136:577, 2013. (3) D. J. Vakalounakis and E. Klironomou. EPPO Bull. 25:463, 1995.

First Report of Petiole (Rachis) Blight of Washingtonia filifera Caused by Phoma glomerata in Greece.

In July 2007, a severe petiole (rachis) blight disease was observed on several California fan palms (Washingtonia filifera) in the vicinity of Heraklion (Crete), Greece. Typical symptoms included discolored (brown to reddish-brown), reversed V-shaped lesions on the petiole bases of the oldest (lowest) leaves, and elongated yellow to dark-brown stripes along the petiole. The lesions progressively expanded and penetrated the petioles, resulting in gradual discoloration (from tan to brown-black) of the internal petiole tissues, including the vascular tissue. The bases of infected petioles occasionally became fragile and burst open, while the corresponding leaf blades were characterized initially by yellowing and one-sided or uneven wilt and, later, desiccation and death with the entire leaves curving downwards. The disease gradually moved upward to younger leaves, severely debilitating but rarely killing the infected trees. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) plates from sections of diseased petioles, forming dense, dark green colonies with abundant light to dark brown, subglobose pycnidia (diameter ranging between 36.4 to 177.4 µm, and averaging 99.4 µm, n = 50) on the agar surface or immersed in the medium. Chlamydospores and numerous dictyochlamydospores were also observed, with the latter being initially light to dark brown and later becoming black. The numerous conidia were hyaline, ovoid to ellipsoid, and single-celled. Their dimensions were 5.3 to 7.3 × 2.4 to 4.9 µm, averaging 6.5 × 3.2 µm (n = 100). The ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (3), were amplified with PCR from total DNA extracted from two representative isolates, and sequenced (GenBank Accession Nos. KC802086 to KC802087). Using BLASTn, both sequences were 100% identical to Phoma glomerata ITS sequences (FJ427018, FJ427011, AF126816). Based on morphological and molecular analyses, the pathogen was identified as Phoma glomerata (Corda) Wollenw. & Hochapfel, also known as Peyronellaea glomerata (Corda) Goid. ex Togliani or Coniothyrium glomeratum Corda (1,2). To prove pathogenicity and fulfill Koch's postulates, petioles of the older leaves of eight W. filifera 2-year-old seedlings were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface), inoculated with agar discs from a 2-week-old PDA culture of the fungus, and sealed with Parafilm. For controls, sterile PDA plugs were placed on the artificial wounds of five more seedlings. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Petiole blight and leaf necrosis symptoms-identical to those observed in the infected plants-were evident 5 weeks post-inoculation, and P. glomerata was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. This is the first report of petiole blight of a palm species caused by P. glomerata in Greece. Due to the extensive use of palms as ornamentals in Greece, the occurrence of P. glomerata can potentially cause economic loss to the local ornamental industry. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) R. M. Hosford, Jr. Phytopathology 65:1236, 1975. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

First Report of Palm Rot of Phoenix spp. Caused by Neodeightonia phoenicum in Greece.

In July 2007, a severe rot was observed on Phoenix dactylifera and P. canariensis palms in the vicinity of Heraklion (Crete), Greece. Initial symptoms were pale, elongated spots that gradually turned to dark brown streaks extending along the leaf base and rachis. In early stages, the upper parts of the leaves usually remained unaffected. Eventually decay and premature death of leaves occurred, followed by terminal bud necrosis. Shoot blights and stalk rots were also observed. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) from leaf base necrotic lesions. Immersed pycnidial conidiomata on pine needles in culture were multiloculate and dark brown to black. Pycnidial paraphyses were absent. Conidiogenous cells were hyaline, cylindrical, and swollen at base. Conidia were thick-walled, ovoid to ellipsoid, with rounded apex and base; initially hyaline and aseptate, 15.2 ± 0.4 × 11.7 ± 0.3 µm, later becoming dark brown and 1-septate, 21.3 ± 0.4 × 11.8 ± 0.3 µm, with a striate appearance. Total DNA was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (1). The sequence (GenBank Accession No. JX456475) was found 99% identical to Neodeightonia phoenicum ITS sequences (GenBank Accession Nos. EU673338 to EU673340), and was clustered together as a single group with the above sequences with good support by phylogenetic analysis that included representatives of other Neodeightonia species and several other Botryosphaeriaceae members. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as N. phoenicum A. J. L. Phillips & Crous (2) (syn. Diplodia phoenicum (Saccardo) H. S. Fawcett & Klotz), formerly also known as Macrophoma phoenicum Saccardo and Strionemadiplodia phoenicum (Saccardo) Zambettakis. To prove pathogenicity, the petioles of the older leaves of seven 2-year-old seedlings of each of three palms, P. canariensis, P. theophrasti, and Washingtonia filifera were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface) and inoculated with agar discs from a 1-week-old PDA culture of the fungus. For controls, PDA discs without fungal mycelium were placed on the wounds of four seedlings of each host. Petiole rot, blight, and leaf necrosis were evident on all inoculated plants 6 weeks post inoculation and the pathogen was consistently reisolated from all three inoculated palm species, whereas no symptoms were observed on control plants. N. phoenicum has repeatedly and globally been reported on P. dactylifera (3). To the best of our knowledge, this is the first report of the occurrence of N. phoenicum infecting Phoenix species in Greece. Palms are extensively used as ornamental trees throughout Greece. A potential spread of palm rot caused by N. phoenicum might have a substantial economic impact and should be urgently addressed through appropriate disease management programs. References: (1) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (2) A. J. L. Phillips et al. Persoonia 21:29, 2008. (3) A. Zaid et al. Chapter XII in: Date palm cultivation, FAO Plant Production and Protection Paper 156 Rev. 1, 2002.

First Report of Pink Rot of Phoenix and Washingtonia Species Caused by Nalanthamala vermoesenii in Greece.

A disease resembling pink rot was first observed on Phoenix dactylifera in Heraklion (Crete, Greece) in the summer of 2007, and was later found to be relatively common in the same district on additional species (P. canariensis, P. theophrasti, Washingtonia filifera, W. robusta). Symptoms included chlorotic and necrotic leaves (dead tips), light-brown spots (1 to 2 mm in diameter) on the leaves and rachis, rot of the rachis, sheath, and trunk, and eventual death of infected plants. A pinkish-orange layer formed both on the surface and within the infected tissues. A hyphomycete was isolated from symptomatic petioles and the pinkish-orange layer of the sheath. Sixteen isolates were examined on potato dextrose agar (PDA). All formed salmon to grayish-red colonies with sparse aerial mycelium, hyaline conidiophores with penicillate branches and terminal phialides, and ovoid, single-celled conidia in long chains. Mean conidial dimensions were 3.5 (± 0.1) × 5.5 (± 0.1) µm (n = 60 each), for 1-week-old cultures of two single-spore isolates recovered from W. filifera. A BLASTn search of GenBank with sequences of rDNA ITS (JX456472 to JX456474) revealed 100% identity of three isolates to that of Nalanthamala vermoesenii (Biourge) Schroers, comb. nov. [syn. Penicillium vermoesenii Biourge; Gliocladium vermoesenii (Biourge) Thom] originating from several palm species in Spain, the Czech Republic, Australia, and the United States (GenBank AY554212 to AY554217). Therefore, our examination of morphological and molecular characteristics suggested that the fungus recovered from symptomatic trees was N. vermoesenii (3,4). Pathogenicity tests were performed on wounds (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface with a sterile scalpel) of petioles of mature leaves of eight 2-year-old seedlings each of P. canariensis, P. theophrasti, and W. filifera. A 6-mm agar plug from a 1-week-old PDA culture was placed on the artificial wound of each inoculated plant. For non-inoculated controls, sterile PDA plugs were placed on the artificial wounds of four seedlings per host. All plants were maintained in the greenhouse at 16 ± 5°C, with 95% humidity and a 12-h photoperiod. Petiole and stem rot, leaf necrosis, and production of pinkish-orange spore masses appeared at 5 weeks post-inoculation. Average lesion length was 2.75 ± 0.15, 3.28 ± 0.21, and 6.14 ± 0.53 cm for P. canariensis, P. theophrasti, and W. filifera, respectively, suggesting that the latter is more susceptible. The fungus was consistently reisolated from all three inoculated palm species, whereas no symptoms appeared on control plants. To our knowledge, this is the first report of N. vermoesenii infecting palms in Greece. The invasion of the plants by the fungus is probably favored by wounds, such as those caused by pruning or by feeding of the red palm wheevil Rhynchophorus ferrugineus Olivier, which is widespread in Greece (1). References: (1) D. C. Kontodimas et al. Entomol. Hellenica 16:11, 2006. (2) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (3) H.-J. Schroers et al. Mycologia 97:375, 2005. (4) J. Y. Uchida. Page 25 in: Compendium of Ornamental Palm Diseases and Disorders, APS Press, St. Paul, MN, USA, 2004.

First Report of Leaf Spot of Phoenix theophrasti Caused by Paraconiothyrium variabile in Greece.

In the spring of 2011, a severe leaf spot disease of Phoenix theophrasti was observed in the vicinity of Heraklion (Crete), Greece. Initial symptoms were small, round-ovoid spots of varying shades of brown on the leaves, later being transformed into oblong streaks (average dimensions 7.3 ± 1.0 × 3.3 ± 0.5 mm), surrounded by dark brown rings. As the disease progressed, the expanding streaks often coalesced to form enlarged necrotic lesions. Similar symptoms were also detected on petioles and leaf bases. Extended spotting and blighting occasionally resulted in leaf death. A filamentous fungus was consistently isolated onto potato dextrose agar plates from the periphery of the characteristic lesions, with cultures invariably producing brick to cinnamon colonies with sparse aerial mycelium, subglobose and dark brown superficial pycnidial conidiomata on pine needles, 1- to 3-celled hyaline conidiophores, and hyaline, subcylindrical to ellipsoidal, 1-celled, smooth- and thin-walled conidia, with average dimensions of 3.5 ± 0.6 × 1.7 ± 0.4 µm (n = 100). Total DNA of two isolates was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (4). Both sequences (GenBank Accession Nos. JX456476 and JX456477) were 100% identical to deposited Paraconiothyrium variabile ITS sequences (EU295640 to 48, JN983440 and 41, and JF934920), and were clustered together as a single group with these sequences with good support by phylogenetic analysis that included representatives of the relative P. brasiliense and P. africanum species. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as P. variabile Riccioni, Damm, Verkley & Crous (2). To prove pathogenicity, 10 P. theophrasti 2-year-old seedlings were sprayed with a conidial suspension of the fungus (107 conidia ml-1, 10 ml per plant), while five additional control plants were treated with sterile distilled water. All plants were maintained in the greenhouse at 15 ± 5°C, with 90% humidity. Characteristic leaf spots were evident 4 weeks post inoculation on the older leaves, and P. variabile was consistently reisolated from all inoculated plants. No symptoms were observed on control plants. Paraconiothyrium variabile has been isolated from various woody host plants such as Prunus persica, P. salicina, and Malus sp. in South Africa (1,2), Actinidia chinensis and A. deliciosa in Italy (2), Laurus nobilis in Turkey (2), and Salix matsudana in China (3). To our knowledge, this is the first report of P. variabile naturally infecting and causing a leaf spot disease on a palm species. Palms are extensively used as ornamentals throughout Greece and the occurrence of P. variabile can potentially result in economic loss to the local ornamental industry. References: (1) M. Cloete et al. Phytopathol. Mediterr. 50:S176, 2011. (2) U. Damm et al. Persoonia 20:9, 2008. (3) H. Gao et al. Afr. J. Biotechnol. 10:4166, 2011. (4) M. P. Pantou et al. Mycol. Res. 109:889, 2005.

Control of Root and Stem Rot of Cucumber, Caused by Fusarium oxysporum f. sp. radicis-cucumerinum, by Grafting onto Resistant Rootstocks.

Root and stem rot of cucumber (Cucumis sativus), caused by Fusarium oxysporum f. sp. radicis-cucumerinum, is a new catastrophic disease of greenhouse crops in Greece and some other countries. No effective disease control methods are available, apart from soil disinfestation by fumigation with methyl bromide and disinfection of internal space of greenhouses with a formaldehyde solution. However, it is anticipated that the use of methyl bromide will be phased out in Greece by 1 January 2005. Therefore, alternative measures for disease management are urgently required. In this study, the efficacy of grafting commercial Dutch type cucumber hybrids onto various cucurbits, used as rootstocks, was examined in growth chamber and greenhouse experiments. Of the nine commercial Cucurbita spp. evaluated, six, A27, Cucurbita Ficifolia, Patron F1 42.91 F1, TS-1358 F1, and TZ-148 F1, found resistant to F. oxysporum f. sp. radicis-cucumerinum, were selected to serve in subsequent greenhouse experiments as root-stocks for grafting the susceptible cv. Brunex F1. Of these, Peto 42.91 F1, TS-1358 F1, and TZ-148 F1 were found to be superior to the others due to their horticultural performance under the climatic conditions prevailing in Crete during the cucumber crop season, late October to late May. This study shows that grafting commercial Dutch type cucumber hybrids onto various resistant Cucurbita rootstocks could be used as an alternative control method to methyl bromide for root and stem rot.

Isozyme variation in Verticillium dahliae isolates from Crete.

Fifteen isolates of Verticillium dahliae (eight of race 1, seven of race 2; most from the island of Crete, Greece) were examined for isozyme and molecular variation. Among the isozyme banding patterns (zymograms) of six enzymes that were "activity-stained" after electrophoresis in 9% polyacrylamide gels, differences were observed in diaphorase, alpha-esterase, peroxidase and superoxide dismutase; 2, 2, 3 and 5 different types of zymograms were recorded, respectively. The zymograms could not be correlated with either race 1 or 2. However, all six isolates originating from the Oropedio (plateau) area of Lasithi (Crete) showed an esterase zymogram clearly distinguishable from the other isolates. No differences were observed when staining for acid phosphatase or aspartate aminotransferase ('glutamic-oxaloacetic transaminase'). Furthermore, electrophoresis of random-amplified polymorphic DNA (RAPD) in 2% agarose gels showed that three race-2 isolates from Oropedio of Lasithi could also be distinguished by the RAPD pattern generated with primer OPA-1. The variation observed possibly represents adaptation of V. dahliae to the Oropedio environment.