Emmanouil A. Markakis

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl−1) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.

Phenolic Responses of Resistant and Susceptible Olive Cultivars Induced by Defoliating and Nondefoliating verticillium dahliae Pathotypes

Verticillium wilt is the most serious olive disease worldwide. The olive-infecting Verticillium dahliae pathotypes have been classified as defoliating (D) and nondefoliating (ND), and the disease is mainly controlled in olive orchards by using resistant or tolerant cultivars. Limited information is available about the nature of resistance in most of the olive cultivars. In the present study, the phenolic responses of the susceptible to V. dahliae olive cv. Amfissis and the resistant cv. Koroneiki upon D and ND V. dahliae infection were monitored in relation to the fungal DNA levels in the vascular tissues with the purpose to explore the defense mechanisms of olive trees against V. dahliae. Quantitative polymerase chain reaction revealed that the decrease in symptom severity shown in Koroneiki trees was associated with significant reduction in the growth of both V. dahliae pathotypes in the vascular tissues compared with Amfissis. In Koroneiki trees, the levels of o-diphenols and verbascoside were positively associated with the DNA levels of the D and ND pathotypes. In addition, a positive association was observed between the levels of verbascoside and the fungal DNA level in Amfissis trees, whereas a negative association was revealed between the fungal DNA level and the total phenols and oleuropein content in both cultivars. The levels of verbascoside were clearly higher in Koroneiki trees compared with Amfissis trees, indicating for the first time in the literature the involvement of verbascoside in the defense mechanism of olive trees against V. dahliae.

Interaction between Bactrocera oleae (Diptera: Tephritidae) infestation and fruit mineral element content in Olea europaea (Lamiales: Oleaceae) cultivars of global interest

Olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae) infestation levels were quantified in rainfed adult olive (Olea europaea L.) (Lamiales: Oleaceae) trees of seven cultivars originating from Spain, Italy, Greece, Portugal and France to determine their relationships with fruit length, width, weight, oil and mineral element content and to investigate the effects of infestation on fruit properties. Fruit from the cultivars Koroneiki, Mastoidis, Picholine, Manzanilla, Arbequina, Branquita and Leccino was collected in November 2013 in Greece. Marked genotypic variation was observed for both total and alive fruit infestation with Manzanilla being the most susceptible and Arbequina the most resistant among the cultivars studied. Marked differences were recorded in the fruit mineral element content between the cultivars studied. B. oleae infestation was positively correlated with the fruit length, width, fresh weight, and K and Fe content. Also, B. oleae infestation caused significant changes in the P, K, Fe and Mg concentration in fruits, while an overall decreasing trend was observed for N. B. oleae infestation caused no significant changes in the fruit oil content of the studied cultivars. Results presented in this study supplemented with targeted integrated research on breeding resistant genotypes and developing improved pest control tools could contribute to important savings of resources as well as improvement of yields and food quality and safety.

Phytopathogenic, morphological, genetic and molecular characterization of a Verticillium dahliae population from Crete, Greece

A population of 84 V. dahliae isolates mainly originating from Crete, Greece, was characterized in terms of pathogenicity and virulence on different hosts, in parallel with morphological/physiological characterization, vegetative compatibility grouping and mating type determination. Tomato race 2 was found to have supplanted race 1 and was more virulent on a tomato-susceptible cultivar than race 1. Using a differential host classification system which tests pathogenicity to tomato, eggplant, sweet pepper and turnip, 59 isolates were assigned to tomato, 19 to eggplant, one to sweet pepper and five to tomato-sweet pepper pathogenicity groups. All isolates from Crete fell into VCG subgroups 2A, 2B and 4B, while a remarkably high incidence of bridging isolates (compatible with two or more VCGs) was recorded. The tomato-sweet pepper pathogenicity group was morphologically quite distinct from the others, while conidial length and pigment intensity were discriminatory parameters among VCGs 2A, 2B and 4B. PCR-based molecular marker Tr1/Tr2 was reliable in race prediction among tomato-pathogenic isolates, except for members of VCG 4B, while the application of markers Tm5/Tm7 and 35-1/35-2 was highly successful for tomato-pathogenic isolates. E10 marker was related to VCG 2B, rather than to pathogenicity groups. A single nucleotide polymorphism in the ITS2 region, and two novel molecular markers, M1 and M2, proved useful for the fast and accurate determination of major VCGs 2A, 2B and 4B, and can be used for high-throughput population analyses in future studies. The mating type was unrelated to VCG classification and probably does not control heterokaryon incompatibility in V. dahliae.

Incidence and pathogenicity of races and isolates of Verticillium dahliae in Crete, southern Greece

Classification of 32 Verticillium dahliae isolates originating from 19 plant species in eight different botanical families to races and determination of host range pathogenicity were carried out. The physiological races of isolates were identified using the two differential tomato cultivars ‘Belladonna’ (susceptible to both races 1 and 2 of V. dahliae) and ‘Ace 55VF’ (resistant to race 1, susceptible to race 2 of V. dahliae). Among these isolates, 14 were race 2 (43.8%), 12 race 1 (37.5%) and six nonpathogenic (18.7%) on tomato. The host range pathogenicity of isolates was determined using four differential hosts (eggplant, turnip, tomato (Ve−) and sweet pepper). Among isolates, five were pathogenic to both eggplant and turnip (15.6%), 21 to eggplant, turnip and tomato (65.6%), five to eggplant, turnip, tomato and sweet pepper (15.6%) and one was pathogenic to eggplant, turnip and sweet pepper (3.2%). The pathogenicity of isolates on the aforementioned five hosts was investigated on the basis of external symptoms and by calculating the relative areas under disease progress curves (relative AUDPC). Results showed that eggplant was the most susceptible, followed by turnip and tomato cv. Belladonna, while sweet pepper and tomato cv. Ace 55VF were less susceptible to all the isolates used. The pathogenicity of isolates varied from highly to mildly virulent on eggplant and turnip while on Belladonna, Ace 55VF and sweet pepper it varied from highly virulent to nonpathogenic. Belladonna exhibited a similar level of susceptibility to races 1 and 2 of V. dahliae, but was more susceptible than Ace 55VF to race 2. Interestingly, the isolates originating from eggplant were clearly more virulent than those originating from tomato and black nightshade on all solanaceous plants tested.

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 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 pathogens 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 Kochs 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 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 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 Kochs 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 Bacterial Apical Necrosis of Mango Caused by Pseudomonas syringae pv. syringae in Greece

The mango, Mangifera indica L., native to India and Southeast Asia, is grown throughout the tropics and subtropics and marketed as fresh or processed fruit contributing greatly to those countries’ income. According to FAO database, from 2007 to 2012 the mango production in Greece reached the 438 tonnes, an increase of 338% indicating a strong economic potential for the cultivation. During spring of 2014 and 2015, symptoms similar to that of the bacterial apical necrosis disease caused by Pseudomonas syringae pv. syringae (Pss) were observed on commercial mango orchards planted with cv Irwin throughout two growing areas in Chania distinct, Crete, Greece. The disease was characterized by rapidly expanding necrotic lesions on leaves, buds, stems and floral panicles, whereas fruits were seemed to be un-affected. Lesions on leaves started as interveinal, angular, water soaked spots which may coalesce, becoming black and slightly raised (Figure 1). Isolations were made from affected buds, leaf petioles, and nec...