First Report of Powdery Scab, Caused by Spongospora subterranea f. sp. subterranea, on Potatoes in Crete, Greece.

In February 2013, severe symptoms of the powdery scab canker type were observed during harvesting at the Mallia area of Heraklio, Crete, Greece, on approximately 20% of the tubers of two commercial smooth skinned potato (Solanum tuberosum L.) field crops (cv. Spunta), of which the seeds had been imported from the Netherlands. Initially, small (up to 2 mm diameter), sunken, purple-brown lesions appeared before harvest on the tubers, followed by pimple-like swellings, which increased in size. As lesions matured, they broke through the tuber skin and developed into shallow depressions. Lesions often expanded in depth and width, forming deep pits and open cankers, eventually destroying considerable amounts of tuber tissue. Light microscopic preparations showed the presence of spongy spore balls with a honeycomb-like spore wall structure. Spore balls were golden brown to brown, oval, circular, elongate, pyriform or irregular in shape, and 54.2 ± 0.93 (36.3 to 79.9) × 47.8 ± 0.82 (31.5 to 72.6) µm. These were composed of aggregates of hundreds of spores that were circular to polygonal in shape, and 4.5 ± 0.06 (3.5 to 5.6) µm in diameter. The morphological characteristics closely resembled those reported for the powdery scab pathogen Spongospora subterranea (Wallr.) Lagerheim f. sp. subterranea Tomlinson. Pathogenicity was confirmed by a lab-based bioassay (3), where spore balls scraped from tuber scab lesions were used to inoculate 20 tomato (cv. Earlypak) bait seedlings, while 20 other seedlings were employed as non-inoculated controls. Zoosporangia of S. subterranea were observed microscopically in root hairs and epidermal cells of all inoculated seedlings but not of any control plants 2 weeks after inoculation. The identity of the pathogen was also confirmed in the original potato samples employing Sss AgriStrip (BIOREBA AG, Reinach, Switzerland), an immunological assay specific for S. subterranea resting spores. In addition, DNA was extracted from infected tubers and the region corresponding to the internal transcribed spacers (ITS) and the ribosomal DNA (rDNA) gene regions was amplified (1). PCR products were purified, direct sequenced, and deposited in GenBank (Accession No. KF208654). In a BLASTn search, the 18S (partial)-ITS1-5.8S-ITS2 (partial) sequence stretch showed 95% similarity to a 509-bp sequence (AF102820.1) of the corresponding S. subterranea gene in GenBank (query coverage 98% and E value 0.0). Based on morphological characteristics, pathogenicity test, immunological assay, and molecular data, it was concluded that the pathogen on potato in Crete is S. subterranea f. sp. subterranea. Powdery scab of potatoes by S. subterranea had been reported earlier in some areas of Greece (Argolida, Attica, Kefallinia, Messenia, and Salonika) (2,4), but without any data confirming the identity of the pathogen. To our knowledge, this is the first record in the island of Crete, and the first confirmed report of this pathogen in Greece. Because S. subterranea f. sp. subterranea is a destructive pathogen of potatoes, its presence in Crete could be of significant concern for commercial growers. Consequently seed import regulations should be strictly enforced. References: (1) K. S. Bell et al. Eur. J. Plant Path. 105:905, 1999. (2) C. D. Holevas et al. Ann. Inst. Phytopath. Benaki 19:52, 2000. (3) U. Merz. Bull. OEPP 19:585, 1989. (4) M. E. Pantidou. Fungus-host index for Greece. Benaki Phytopath. Inst., Athens, 1973.

First Record of White Rust, Caused by Albugo occidentalis, on Spinach in Greece.

In early December 2012 and February 2013, severe symptoms of white rust were observed on several commercial crops of the spinach (Spinacia oleracea L.) cvs. Tahiti and Rembrandt in the Aghia Pelaghia and Elia areas, respectively, of Heraklio, Crete, Greece. Initially, small, chlorotic lesions developed on the upper side of the leaves. As disease progressed, small, glassy white pustules developed on the underside of each leaf, frequently in concentric rings. The pustules were blister-like and oval, irregularly oval, or elongated, ranging from 0.5 to 2.0 mm in diameter and up to 3 mm in length. Lesions often coalesced, and the pustules could cover the entire lower leaf surface, rendering them unmarketable. The dome-like epidermis of host tissue covering the white rust pustules ruptured and exposed a white, chalky 'dust' of numerous powdery spores in small, circular to elongate sori. The sporangia were arranged in basipetal chains, were globose to oval, with a smooth wall that was uniformly thick and measured 19.1 ± 1.93 (14.1 to 23.5) µm × 15.3 ± 1.49 (12.9 to 20.0) µm when hydrated. Oospores were absent from the leaves. The morphological characteristics closely resembled those reported for the white rust pathogen, Albugo occidentalis Wilson (4), as well as those of A. occidentalis measured from dried leaves of Chenopodium spp. and Monolepis nuttalliana (Schult.) Greene (Herb. IMI96980, IMI351202, and IMI26345, respectively), kindly loaned by the Royal Botanic Gardens, Kew, United Kingdom. Pathogenicity was confirmed by spraying a suspension of 105 sporangia/ml on 10 healthy 20-day-old potted spinach plants of cv. Tahiti. Inoculated plants were covered with polyethylene bags for 3 days and incubated in a growth chamber at 16 to 20°C with a 10-h photoperiod. White rust symptoms were observed on the lower surface of the leaves 10 days after inoculation. Ten control plants sprayed similarly with distilled water and maintained under the same conditions as inoculated plants showed no symptoms. The fungus present on the inoculated plants was morphologically identical to that originally observed on diseased plants, fulfilling Koch's postulates. Genomic DNA from spinach leaf sori was extracted, and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), ITS1-5,8S-ITS2, as well as the cytochrome oxidase subunit II (COX2) mitochondrial gene, were amplified. PCR products were sequenced and deposited in GenBank (KC676794 and KC676795, respectively). In a BLAST search, the ITS1-5,8S-ITS2 and COX2 sequences showed 99% similarities to 684 bp (AJ553900.1) and 599 bp (AY286220.1) sequences of the corresponding A. occidentalis genes in GenBank, respectively. Based on morphological characteristics, pathogenicity tests, and molecular sequencing data, it was concluded that the pathogen on spinach in Crete is A. occidentalis. This is an economically important pathogen of spinach in the United States, that has also has been recorded in Iran (2) and India on a Chenopodium sp. (IMI351202), and in Canada on M. nuttalliana (IMI26345). To our knowledge, this is the first report of this pathogen in Greece, and the first record on spinach in Europe. A voucher specimen has been deposited at the Royal Botanic Gardens, Kew, United Kingdom (Accession No. K(M) 181610). References: (1) J. C. Correll et al. Eur. J. Plant Pathol. 129:193, 2011. (2) A. G. Ebrahimi and H. Afzali. Rostaniha 1:73, 2000. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN, 1989. (4) G. W. Wilson. Bull. Torrey Bot. Club 34:61, 1907.

First Report of Stemphylium solani as the Causal Agent of a Leaf Spot on Greenhouse Cucumber.

During the 2011 to 2012 crop season, a severe leaf spot disease of cucumber (Cucumis sativus) cv. Cadiz was noticed on crops in some greenhouses in the Goudouras area, Lasithi, Crete, Greece. Symptoms appeared in late winter, mainly on the leaves of the middle and upper part of the plants. Initially, small necrotic pinpoint lesions with white centers, surrounded by chlorotic halos, 1 to 3 mm in diameter, appeared on the upper leaf surfaces, and these progressively enlarged to spots that could coalesce to form nearly circular lesions up to 2 cm or more in diameter. Stemphylium-like fructifications appeared on necrotic tissue of older lesions. Severely affected leaves became chlorotic and died. No other part of the plant was affected. Small tissue pieces from the edges of lesions were surface disinfected in 0.5% NaClO for 5 min, rinsed in sterile distilled water, plated on acidified potato dextrose agar and incubated at 22 ± 0.5°C with a 12-h photoperiod. Stemphylium sp. was consistently isolated from diseased samples. Colonies showed a typical septate mycelium with the young hyphae subhyaline and gradually became greyish green to dark brown with age. Conidiophores were subhyaline to light brown, 3- to 10-septate, up to 200 µm in length, and 4 to 7 µm in width, with apical cell slightly to distinctly swollen, bearing a single spore at the apex. Conidia were muriform, mostly oblong to ovoid, but occasionally nearly globose, subhyline to variant shades of brown, mostly constricted at the median septum, 22.6 ± 6.22 (11.9 to 36.9) µm in length, and 15.1 ± 2.85 (8.3 to 22.6) µm in width, with 1 to 8 transverse and 0 to 5 longitudinal septa. DNA from a representative single-spore isolate was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was amplified using the universal primers ITS5 and ITS4. The PCR product was sequenced and deposited in GenBank (Accession No. JX481911). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a S. solani isolate in GenBank (EF0767501), the fungus was identified as S. solani. Pathogenicity tests were performed by spraying a conidial suspension (105 conidia ml-1) on healthy cucumber (cv. Knossos), melon (C. melo, cv. Galia), watermelon (Citrullus lanatus cv. Crimson sweet), pumpkin (Cucurbita pepo, cv. Rigas), and sponge gourd (Luffa aegyptiaca, local variety) plants, at the 5-true-leaf stage. Disease symptoms appeared on cucumber and melon only, which were similar to those observed under natural infection conditions on cucumber. S. solani was consistently reisolated from artificially infected cucumber and melon tissues, thus confirming Koch's postulates. The pathogenicity test was repeated with similar results. In 1918, a report of a Stemphylium leaf spot of cucumber in Indiana and Ohio was attributed to Stemphylium cucurbitacearum Osner (4), but that pathogen has since been reclassified as Leandria momordicae Rangel (2). That disease was later reported from Florida (1) and net spot was suggested as a common name for that disease. For the disease reported here, we suggest the name Stemphylium leaf spot. This is the first report of a disease of cucumber caused by a species of Stemphylium. References: (1) C. H. Blazquez. Plant Dis. 67:534, 1983. (2) P. Holliday. Page 243 in: A Dictionary of Plant Pathology. Cambridge University Press, Cambridge, UK, 1998. (3) B. S. Kim et al. Plant Pathol. J. 15:348, 1999. (4) G. A. Osner. J. Agric. Res. 13:295, 1918.

Characterization of Fusarium oxysporum Isolates Obtained from Cucumber in China by Pathogenicity, VCG, and RAPD.

Thirty-four isolates of Fusarium oxysporum, obtained in China from cucumber plants showing either Fusarium wilt (F. oxysporum f. sp. cucumerinum) or root and stem rot (F. oxysporum f. sp. radicis-cucumerinum) symptoms, were characterized by pathogenicity, vegetative compatibility, and random amplified polymorphic DNA (RAPD). Of these, 23 isolates were identified by pathogenicity as F. oxysporum f. sp. cucumerinum, and one as F. oxysporum f. sp. radicis-cucumerinum, while 10 isolates were avirulent on cucumber, melon, sponge gourd, and pumpkin. The Chinese isolates of F. oxysporum f. sp. cucumerinum were assigned to RAPD groups III and XXI and to vegetative compatibility group (VCG) 0183, four new VCGs, 0184 to 0187, and a single-member VCG included in the artificial VCG 018-. The Chinese isolate of F. oxysporum f. sp. radicis-cucumerinum was assigned to RAPD group I and bridging VCG 0260/0261. The occurrence of F. oxysporum f. sp. radicis-cucumerinum on cucumber is reported for the first time in China.

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.

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.

Effect of common food preservatives on mycelial growth and spore germination of Fusarium oxysporum.

The growth and spore germination inhibition of Fusarium oxysporum f.sp. radicis-cucumerinum by the common food additives: acetic acid, formic acid potassium sorbate, propionic acid, sorbic acid, and the fungistatic agent sec-butylamine was examined in vitro. The inhibitory efficacy of these chemicals decreased in the following order: sorbic acid, potassium sorbate, propionic acid, acetic acid, sec-butylamine and formic acid. At pH 6.4, the ED50 value for mycelium growth was: 976 ppm for sorbic acid, 1292 ppm for potassium sorbate, 2435 ppm for propionic acid, 3805 ppm for acetic acid, 3962 ppm for sec butylamine and 4668 ppm for formic acid. The ED50 value for spore germination was: 225 ppm for potassium sorbate, 1201 ppm for sorbic acid, 1402 ppm for propionic acid, 1600 ppm for sec-butylamine, 1957 ppm for acetic acid and 2485 ppm for formic acid.

Effect of novel water-soluble polymeric forms of sorbic acid against Fusarium oxysporum f.sp. radicis-cucumerinum.

New controlled release water-soluble formulations of sorbic (2,4-hexadienoic) acid were prepared and their inhibitory activity on mycelium growth of Fusarium oxysporum f.sp. radicis-cucumerinum was evaluated. The new products are epoxidized polymers of polyvinylpyrrolidone (PVP) containing covalently bonded sorbic acid (polymeric esters of sorbic acid) and complexes of PVP with hydrogen bonded sorbic acid, characterized by controlled release of sorbic acid. It was shown that the polymeric complexes of sorbic acid with PVP were more effective fungicidal agents than sorbic acid polymeric esters. In all cases the activity of polymeric derivatives (esters and complexes) was increased by lowering the molecular weight of the polymeric carriers. Controlled release formulations of these polymeric derivatives are new promising products due to their low toxicity, wide range of efficient concentrations for application and ability to regulate lyophilicity. Our data contribute to the understanding of the action mechanism of various polymeric sorbic acid formulations and can result in products which are particularly suitable for food and feed protection applications.

Genetic Diversity of Fusarium oxysporum Isolates from Cucumber: Differentiation by Pathogenicity, Vegetative Compatibility, and RAPD Fingerprinting.

ABSTRACT A total of 106 isolates of Fusarium oxysporum obtained from diseased cucumber plants showing typical root and stem rot or Fusarium wilt symptoms were characterized by pathogenicity, vegetative compatibility, and random amplified polymorphic DNA (RAPD). Twelve isolates of other formae speciales and races of F. oxysporum from cucurbit hosts, three avirulent isolates of F. oxysporum, and four isolates of Fusarium spp. obtained from cucumber were included for comparison. Of the 106 isolates of F. oxysporum from cucumber, 68 were identified by pathogenicity as F. oxysporum f. sp. radicis-cucumerinum, 32 as F. oxysporum f. sp. cucumerinum, and 6 were avirulent on cucumber. Isolates of F. oxysporum f. sp. radicis-cucumerinum were vegetatively incompatible with F. oxysporum f. sp. cucumerinum and the other Fusarium isolates tested. A total of 60 isolates of F. oxysporum f. sp. radicis-cucumerinum was assigned to vegetative compatibility group (VCG) 0260 and 5 to VCG 0261, while 3 were vegetatively compatible with isolates in both VCGs 0260 and 0261 (bridging isolates). All 68 isolates of F. oxysporum f. sp. radicis-cucumerinum belonged to a single RAPD group. A total of 32 isolates of F. oxysporum f. sp. cucumerinum was assigned to eight different VCGs and two different RAPD groups, while 2 isolates were vegetatively self-incompatible. Pathogenicity, vegetative compatibility, and RAPD were effective in distinguishing isolates of F. oxysporum f. sp. radicis-cucumerinum from those of F. oxysporum f. sp. cucumerinum. Parsimony and bootstrap analysis of the RAPD data placed each of the two formae speciales into a different phylogenetic branch.