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.

Linkage mapping of the Mediterranean cypress, Cupressus sempervirens, based on molecular and morphological markers.

Gene mapping for a Cupressus species is presented for the first time. Two linkage maps for the Mediterranean cypress (Cupressus sempervirens) varieties, C. sempervirens var. horizontalis and C. sempervirens var. pyramidalis, were constructed following the pseudo-testcross mapping strategy and employing RAPD, SCAR and morphological markers. A total of 427 loci (425 RAPDs, two SCARs) representing parents and F(1) progeny were screened for polymorphism with 32 random decamer and two SCAR primers. A morphological marker defined as "crown form" was also included. Of 274 polymorphic loci, the 188 that presented Mendelian inheritance formed the mapping dataset. Of these loci, 30% were mapped into seven linkage groups for the horizontalis (maternal) and four linkage groups for the pyramidalis (paternal) map. The putative "crown form" locus was included in a linkage group of both maps. The horizontalis and the pyramidalis maps covered 160.1 and 144.5 cM, respectively, while genome length was estimated to be 1696 cM for the former variety and 1373 cM for the latter. The four RAPD markers most tightly linked to crown form were cloned and converted to SCARs. Each of the cloned RAPD markers yielded two to three different sequences behaving as co-migrating fragments. Two SCAR markers, SC-D05(432) and SC-D09(667), produced amplified bands of the expected sizes and maintained linkage with the appropriate phenotype, but to a lesser extent compared to their original RAPD counterparts. These linkage maps represent a first step towards the localization of QTLs and genes controlling crown form and other polygenic traits in cypress.

Is Cupressus sempervirens native in Italy? An answer from genetic and palaeobotanical data.

This study represents the first large-scale analysis using nuclear molecular markers to assess genetic diversity and structure of Cupressus sempervirens L.. Genetic and fossil data were combined to infer the possible role of human activity and evolutionary history in shaping the diversity of cypress populations. We analysed 30 populations with six polymorphic nuclear microsatellite markers. Dramatic reductions in heterozygosity and allelic richness were observed from east to west across the species range. Structure analysis assigned individuals to two main groups separating central Mediterranean and eastern populations. The two main groups could be further divided into five subgroups which showed the following geographical distributions: Turkey with the Greek islands Rhodes and Samos, Greece (Crete), Southern Italy, Northern Italy, Tunisia with Central Italy. This pattern of genetic structure is also supported by SAMOVA and Barrier analyses. Palaeobotanical data indicated that Cupressus was present in Italy in the Pliocene, Pleistocene and Holocene. Furthermore, our molecular survey showed that Italian cypress populations experienced bottlenecks that resulted in reduced genetic diversity and allelic richness and greater genetic differentiation. Recent colonization or introduction may also have influenced levels of diversity detected in the Italian populations, as most individuals found in this range today have multilocus genotypes that are also present in the eastern range of the species. The data reveal a new interpretation of the history of cypress distribution characterized by ancient eastern populations (Turkey and Greek islands) and a mosaic of recently introduced trees and remnants of ancient, depauperate populations in the central Mediterranean range.

Differential Localization of Antioxidants in Maize Leaves.

The aim of this work was to determine the compartmentation of antioxidants between the bundle-sheath and mesophyll cells of maize (Zea mays L.) leaves. Rapid fractionation of the mesophyll compartment was used to minimize modifications in the antioxidant status and composition due to extraction procedures. The purity of the mesophyll isolates was assessed via the distribution of enzyme and metabolite markers. Ribulose-1,5 bisphosphate and ribulose-1,5-bisphosphate carboxylase/oxygenase were used as bundle-sheath markers and phosphoenolpyruvate carboxylase was used as the mesophyll marker enzyme. Glutathione reductase and dehydroascorbate reductase were almost exclusively localized in the mesophyll tissue, whereas ascorbate, ascorbate peroxidase, and superoxide dismutase were largely absent from the mesophyll fraction. Catalase, reduced glutathione, and monodehydroascorbate reductase were found to be approximately equally distributed between the two cell types. It is interesting that, whereas H2O2 levels were relatively high in maize leaves, this oxidant was largely restricted to the mesophyll compartment. We conclude that the antioxidants in maize leaves are partitioned between the two cell types according to the availability of reducing power and NADPH and that oxidized glutathione and dehydroascorbate produced in the bundle-sheat tissues have to be transported to the mesophyll for re-reduction to their reduced forms.

Dermatofibrosarcoma protuberans occuring in a child.

Dermatofibrosarcoma protuberans (DFSP) usually occurs in adults, however it can also occur in infancy and childhood. Diagnosis of DFSP in children is quite difficult given the rarity of the tumor and its variegated appearance. The behavior of this neoplasm is of borderline or intermediate malignancy with a high rate of recurrence but limited metastatic potential. We present the case of an eleven-year old boy who presented with a slightly raised, not tender lesion on his right shoulder. It had appeared two years before as a red-brown plaque and since then it had gradually grown to a nodule. An excisional biopsy was performed, and the diagnosis of DFSP was made based on the histological and immunohistochemical findings.

Separation of plant membrane lipids by multiple solid-phase extraction.

Plant membrane lipids were separated by multiple solid-phase extraction (SPE) in a single run. Elution was performed continuously through the modulated stationary phase employing only non-aqueous solvent systems. At the different stages of the glycerolipid separation the SPE manifold combined arninopropyl, arninopropyl/silica gel and silica gel/aminopropyl weak anion exchanger columns. The glycerolipid extract of pigment-containing plant tissues was cleared from the pigments onto the aminopropyl column. The aminopropyl column with the glycerolipid extract was then connected to a silica gel column from which monogalactosyldiacylglycerol, phosphatidylethanolamine, phosphatidylglycerol and digalactosyldiacylglycerol were eluted as individual fractions. The elution was performed under polarity, pH and temperature gradient conditions. To continue the separation, the aminopropyl column was discarded and the silica gel column containing the remaining glycerolipid extract was connected to an aminopropyl anion exchanger column. Individual fractions of sulfoquinovosyldiacylglycerol, phosphatidylcholine and phosphatidylinositol were now eluted. The separation process was supported by ammonium counter ions and by the polarity gradient of the elution systems used. The membrane lipids were isolated from pigment-containing (rice and maize leaves and rice leafy stems) and pigment-free (rice roots) tissues. The repeatability for a standard glycerolipid mixture was 2-6% (n=7), and for rice leaf lipid extracts, 3-7% (n=5). Glycerolipid recovery was 87-95%.

Determination of glycerolipid composition of rice and maize tissues using solid-phase extraction.

Currently available techniques for the separation and characterization of different glycerolipids are complicated and/or time consuming. By modulating the stationary phase in a solid-phase extraction (SPE) manifold, efficient and rapid separation of plant membrane lipids was achieved. The glycerolipids from rice and maize tissues were separated into seven classes (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylethanolamine, phosphatidylcholine, sulphoquinovosyldiacylglycerol, phosphatidylinositol and phosphatidylglycerol). The pigments present in the rice and maize leaves and rice stems were successfully removed from the total lipid extracts. Pigment-free plant tissue (rice roots) was also analysed. The fatty acid profile of each lipid class isolated by SPE agreed well with those obtained by other separation techniques. The recovery of glycerolipids was at least 87%.