Under Pressure: A Comparative Study of Botrytis cinerea Populations from Conventional and Organic Farms in Cyprus and Greece.

The highly heterogeneous nature of Botrytis cinerea provides adaptive benefits to variable environmental regimes. Disentangling pathogen population structure in anthropogenic agroecosystems is crucial to designing more effective management schemes. Herein, we studied how evolutionary forces exerted in different farming systems, in terms of agrochemicals-input, shape B. cinerea populations. In total, 360 B. cinerea isolates were collected from conventional and organic, strawberry and tomato farms in Cyprus and Greece. The occurrence and frequency of sensitivities to seven botryticides were estimated. Results highlighted widespread fungicide resistance in conventional farms since only 15.5% of the isolates were sensitive. A considerable frequency of fungicide-resistant isolates was also detected in the organic farms (14.9%). High resistance frequencies were observed for boscalid (67.7%), pyraclostrobin (67.3%), cyprodinil (65.9%), and thiophanate-methyl (61.4%) in conventional farms, while high levels of multiple fungicide resistance were also evident. Furthermore, B. cinerea isolates were genotyped using a set of seven microsatellite markers (simple sequence repeat [SSR] markers). Index of association analyses (Ia and rBarD) suggest asexual reproduction of the populations, even though the mating-type idiomorphs were equally distributed, indicating frequency-dependent selection. Fungicide resistance was correlated with farming systems across countries and crops, while SSRs were able to detect population structure associated with resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. The expected heterozygosity in organic farms was significantly higher than in conventional, suggesting the absence of selective pressure that may change the allelic abundance in organic farms. However, genetic variance among strawberry and tomato populations was high, ranking host specificity higher than other selection forces studied.

Characterization of Phytophthora infestans Populations in Cyprus, the Southernmost Potato-Producing European Country.

Cyprus is the southernmost island country of Europe, located in the Mediterranean. Despite its limited area, potato production is considered an integral source of the national agricultural revenue. During 2010-2012, a late blight epidemic period for the country, the population structure of Phytophthora infestans was analyzed via a sample of 539 isolates collected from all of the main potato-cultivating regions of Cyprus. We determined mating type, mefenoxam sensitivity, and genetic polymorphism at 12 simple sequence repeat (SSRs) loci. Although both mating types were detected in the country, a gradual but dynamic shift toward A2 dominance was manifested over time. The pathogen population also demonstrated reduced sensitivity to the phenylamide fungicide, since 96.2% of the tested isolates had high (70.3%) and intermediate (25.9%) resistance to mefenoxam, which suggests that it should be replaced with other active ingredients in local disease management strategies. The genotypic analysis also revealed the predominance of the highly aggressive mefenoxam-insensitive EU_13_A2 lineage across the country, with a frequency of 79.2%. Other samples comprised an older lineage EU_2_A1 (19.5%), a very low proportion of EU_23_A1 (0.37%), and others that did not match any known lineage (0.92%). SSRs data supported triploid genomes among the dominant lineages, and patterns of their asexual population history were also apparent. A high subclonal variation of the 13_A2 population was detected, which suggested introduction events of this widespread genotype to Cyprus from major tuber-exporting countries. Present data indicate the severe impact of inoculum migration to the structure of the local population; thus, current phytosanitary procedures should be reconsidered and possibly attuned. This is the first comprehensive study to elucidate the diversity of P. infestans in Cyprus and could serve as a baseline for future monitoring of this highly adaptive plant pathogen, given that late blight management strategies should be constantly refined according to the traits of the dominant genotypes of P. infestans.

Structural Diversity and Highly Specific Host-Pathogen Transcriptional Regulation of Defensin Genes Is Revealed in Tomato.

Defensins are small and rather ubiquitous cysteine-rich anti-microbial peptides. These proteins may act against pathogenic microorganisms either directly (by binding and disrupting membranes) or indirectly (as signaling molecules that participate in the organization of the cellular defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. In the current study, we screened the Solanum lycopersicum genome for the identification of defensin genes, predicted the relating protein structures, and further studied their transcriptional responses to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus, and Potato Virus Y infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The immunodetection of plant defensin 1 protein was achieved only in the roots of plants inoculated with V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to substantially induce the gene transcription suggest that these factors are probably not primarily targeted by the tomato defensin network.

A Novel Sorbitol-Based Flow Cytometry Buffer Is Effective for Genome Size Estimation across a Cypriot Grapevine Collection.

Flow cytometry (FCM) is a widely used technique to study genome size (C-value), but recalcitrant metabolites in grapevines often hinder its efficiency in grapevine research. The aim of the present study was (i) to develop a novel buffer tailormade for the nuclei isolation of grapevines and (ii) to characterize a Cypriot germplasm collection based on C-values. A local cultivar "Xinisteri" was used as a pilot test to evaluate a Sorbitol-based buffer, while sprouting, young, and fully matured leaves were examined to evaluate the developmental parameter. The novel Sorbitol buffer was shown to have a coefficient of variation (CV) of 4.06%, indicating improved properties compared to other commonly used FCM buffers [WPB (7.69%), LB01 (6.69%), and LB (7.13%), respectively]. In addition, a significant variation in genome size between genotypes was found in a comprehensive application with 24 grape varieties. Nucleic content (2C) ranged from 0.577/1C pg for the "Assyrtiko" cultivar up to 0.597/1C pg for the "Spourtiko" cultivar, revealing a 17.6/1C Mbp difference. The lowest coefficient of variation (CV) across all entries was found in the variety "Ofthalmo" (2.29%), while the highest was observed in "Pinot Noir" (3.44%). Anova analysis revealed several distinct clusters, showing that in several cases, C-values can be used as a simple method to distinguish grapevine cultivars.

Minimally invasive transapical aortic valve implantation and simultaneous major pulmonary resection.

We report a 71-year-old male who underwent successful minimally invasive transapical aortic valve implantation to treat severe aortic stenosis, with simultaneous pulmonary resection for the treatment of lung cancer. At five-year follow-up the patient remains free of symptoms (NYHA I) and recurrence.

Genome Size Variation across a Cypriot Fabeae Tribe Germplasm Collection.

DNA content is an important trait linked to the evolutionary routes of taxa and often connected to speciation. In the present study, we studied C-values variation across the Cypriot Fabeae gene pool. Several hundred plants (Vicia spp., Lens spp., Pisum spp.) were sampled across Cyprus. Accurate estimates were established by flow cytometry and propidium iodine staining for 155 discrete populations/accessions. A ten-fold variation was detected across lineages with 1C DNA content varying from 1.584 pg for V. cretica (ARI02420) to 13.983 pg for V. faba (ARI00187). In general, flow cytometry was precise for the characterization of species, even though there were instances of genome overlapping across taxa. Most analyses in the current work refer to species that have not been characterized before by flow cytometry (or any other DNA content estimation method). Still, a correlation to C-values previously reported in Kew Plant DNA C-values database was attempted. A high degree of correlation except for V. dalmatica was established. The evaluation of genome size trait in relation with the Fabeae phylogeny, revealed that Pisum and Lens genera were rather homogenous, but an astonishing fluctuation was shown for Vicia spp. Moreover, it was established that genome up- or down-scaling was not directly linked to speciation drivers. The genomic size measurements presented here could deliver extra quality control for the identification and characterization of taxa in germplasm collections, particularly in cases where species share morphological characters.

Automated Grapevine Cultivar Identification via Leaf Imaging and Deep Convolutional Neural Networks: A Proof-of-Concept Study Employing Primary Iranian Varieties.

Extending over millennia, grapevine cultivation encompasses several thousand cultivars. Cultivar (cultivated variety) identification is traditionally dealt by ampelography, requiring repeated observations by experts along the growth cycle of fruiting plants. For on-time evaluations, molecular genetics have been successfully performed, though in many instances, they are limited by the lack of referable data or the cost element. This paper presents a convolutional neural network (CNN) framework for automatic identification of grapevine cultivar by using leaf images in the visible spectrum (400-700 nm). The VGG16 architecture was modified by a global average pooling layer, dense layers, a batch normalization layer, and a dropout layer. Distinguishing the intricate visual features of diverse grapevine varieties, and recognizing them according to these features was conceivable by the obtained model. A five-fold cross-validation was performed to evaluate the uncertainty and predictive efficiency of the CNN model. The modified deep learning model was able to recognize different grapevine varieties with an average classification accuracy of over 99%. The obtained model offers a rapid, low-cost and high-throughput grapevine cultivar identification. The ambition of the obtained tool is not to substitute but complement ampelography and quantitative genetics, and in this way, assist cultivar identification services.

Automated In Situ Seed Variety Identification via Deep Learning: A Case Study in Chickpea.

On-time seed variety recognition is critical to limit qualitative and quantitative yield loss and asynchronous crop production. The conventional method is a subjective and error-prone process, since it relies on human experts and usually requires accredited seed material. This paper presents a convolutional neural network (CNN) framework for automatic identification of chickpea varieties by using seed images in the visible spectrum (400-700 nm). Two low-cost devices were employed for image acquisition. Lighting and imaging (background, focus, angle, and camera-to-sample distance) conditions were variable. The VGG16 architecture was modified by a global average pooling layer, dense layers, a batch normalization layer, and a dropout layer. Distinguishing the intricate visual features of the diverse chickpea varieties and recognizing them according to these features was conceivable by the obtained model. A five-fold cross-validation was performed to evaluate the uncertainty and predictive efficiency of the CNN model. The modified deep learning model was able to recognize different chickpea seed varieties with an average classification accuracy of over 94%. In addition, the proposed vision-based model was very robust in seed variety identification, and independent of image acquisition device, light environment, and imaging settings. This opens the avenue for the extension into novel applications using mobile phones to acquire and process information in situ. The proposed procedure derives possibilities for deployment in the seed industry and mobile applications for fast and robust automated seed identification practices.

Morphological Diversity, Genetic Characterization, and Phytochemical Assessment of the Cypriot Tomato Germplasm.

Tomato (Solanum lycopersicum L.) is considered one of the most valuable and versatile vegetable crops globally and also serves as a significant model species for fruit developmental biology. Despite its significance, a severe genetic bottleneck and intense selection of genotypes with specific qualitative traits have resulted in the prevalence of a restricted number of (geno)types, also causing a lack of diversity across widespread cultivated types. As a result, the re-emergence of landraces as well as traditional and heirloom varieties is largely acknowledged as a countermeasure to restore phenotypic, phytochemical and genetic diversity while enriching the aroma/taste tomato palette. On those grounds, the Cypriot tomato germplasm was assessed and characterized. Ten landrace accessions were evaluated under greenhouse conditions and data were collected for 24 IPGRI discrete phenotypic traits. Grouping of accessions largely reflected the fruit shape and size; four different fruit types were recorded across accessions (flattened, heart-shaped, rounded and highly rounded). Moreover, a single run panel consisting of ten SSRs was developed and applied in order to genetically characterize 190 Cypriot genotypes and foreign heirloom varieties. Based on genetic indexes it was established that tomato landraces have a rather low level of heterogeneity and genetic variation. Finally, mineral and phytochemical analyses were conducted in order to estimate biochemical attributes (total phenolics, ascorbic acid, lycopene, ß-carotene, total soluble content, titratable acidity) across genotypes; thus, ascertaining that the Cypriot panel has a high nutritional value. Due to the thermo-drought adaptation and tolerance of these genotypes, the current study serves as a roadmap for future breeding efforts in order to incorporate desirable traits or develop novel tomato lines combining resilience and alimentary value.

The Cypriot Indigenous Grapevine Germplasm Is a Multi-Clonal Varietal Mixture.

Cypriot vineyards are considered as one among the earliest niches of viticulture and a pivotal hub for the domestication and dissemination of grapevine. The millennial presence of Vitis spp. in this Eastern Mediterranean island has given rise to a plethora of biotypes that have not been adequately characterized, despite their unique attributes and stress tolerance. This ancient germplasm also has an additional value since it survived the phylloxera outbreak; hence, it possesses a large amount of genetic diversity that has been unnoticed. In order to provide useful insights to the lineage of Cypriot vineyards, a two-year-spanning collection of centennial grapevine cultivars mostly regarded to belong to four indigenous variety clusters ("Mavro", "Xynisteri", "Maratheftiko", and "Veriko") was initiated. There were 164 accessions across the broader Commandaria wine zone sampled and characterized using a universal microsatellite primer set. Genetic analysis indicated that considered indigenous Cypriot germplasm has a polyclonal structure with a high level of heterozygosity. Moreover, several lineages or unexplored varieties may exist, since a larger than considered number of discrete genotypes was discovered. Furthermore, it was established that grapevine lineages in Cyprus were shaped across eras via clonal, as well as, sexual propagation. The special attributes of the Cypriot landscape are discussed.

The Role of Proteases in Determining Stomatal Development and Tuning Pore Aperture: A Review.

Plant proteases, the proteolytic enzymes that catalyze protein breakdown and recycling, play an essential role in a variety of biological processes including stomatal development and distribution, as well as, systemic stress responses. In this review, we summarize what is known about the participation of proteases in both stomatal organogenesis and on the stomatal pore aperture tuning, with particular emphasis on their involvement in numerous signaling pathways triggered by abiotic and biotic stressors. There is a compelling body of evidence demonstrating that several proteases are directly or indirectly implicated in the process of stomatal development, affecting stomatal index, density, spacing, as well as, size. In addition, proteases are reported to be involved in a transient adjustment of stomatal aperture, thus orchestrating gas exchange. Consequently, the proteases-mediated regulation of stomatal movements considerably affects plants' ability to cope not only with abiotic stressors, but also to perceive and respond to biotic stimuli. Even though the determining role of proteases on stomatal development and functioning is just beginning to unfold, our understanding of the underlying processes and cellular mechanisms still remains far from being completed.

Polyamine homeostasis in tomato biotic/abiotic stress cross-tolerance.

Adverse conditions and biotic strain can lead to significant losses and impose limitations on plant yield. Polyamines (PAs) serve as regulatory molecules for both abiotic/biotic stress responses and cell protection in unfavourable environments. In this work, the transcription pattern of 24 genes orchestrating PA metabolism was investigated in Cucumber Mosaic Virus or Potato Virus Y infected and cold stressed tomato plants. Expression analysis revealed a differential/pleiotropic pattern of gene regulation in PA homeostasis upon biotic, abiotic or combined stress stimuli, thus revealing a discrete response specific to diverse stimuli: (i) biotic stress-influenced genes, (ii) abiotic stress-influenced genes, and (iii) concurrent biotic/abiotic stress-regulated genes. The results support different roles for PAs against abiotic and biotic stress. The expression of several genes, significantly induced under cold stress conditions, is mitigated by a previous viral infection, indicating a possible priming-like mechanism in tomato plants pointing to crosstalk among stress signalling. Several genes and resulting enzymes of PA catabolism were stimulated upon viral infection. Hence, we suggest that PA catabolism resulting in elevated H2O2 levels could mediate defence against viral infection. However, after chilling, the activities of enzymes implicated in PA catabolism remained relatively stable or slightly reduced. This correlates to an increase in free PA content, designating a per se protective role of these compounds against abiotic stress.

Stomatal behavior following mid- or long-term exposure to high relative air humidity: A review.

High relative air humidity (RH ≥ 85%) is frequent in controlled environments, and not uncommon in nature. In this review, we examine the high RH effects on plants with a special focus on stomatal characters. All aspects of stomatal physiology are attenuated by elevated RH during leaf expansion (long-term) in C3 species. These include impaired opening and closing response, as well as weak diel oscillations. Consequently, the high RH-grown plants are not only vulnerable to biotic and abiotic stress, but also undergo a deregulation between CO2 uptake and water loss. Stomatal behavior of a single leaf is determined by the local microclimate during expansion, and may be different than the remaining leaves of the same plant. No effect of high RH is apparent in C4 and CAM species, while the same is expected for species with hydropassive stomatal closure. Formation of bigger stomata with larger pores is a universal response to high RH during leaf expansion, whereas the effect on stomatal density appears to be species- and leaf side-specific. Compelling evidence suggests that ABA mediates the high RH-induced stomatal malfunction, as well as the stomatal size increase. Although high RH stimulates leaf ethylene evolution, it remains elusive whether or not this contributes to stomatal malfunction. Most species lose stomatal function following mid-term (4-7 d) exposure to high RH following leaf expansion. Consequently, the regulatory role of ambient humidity on stomatal functionality is not limited to the period of leaf expansion, but holds throughout the leaf life span.

Genetic variability in landraces populations and the risk to lose genetic variation. The example of landrace 'Kyperounda' and its implications for ex situ conservation.

Genetic characterization enhances the development of rational conservation strategies and the utilization of germplasm to plant breeding programs. In the present study, 19 microsatellite markers were employed to evaluate the genetic diversity and the genetic affiliations across 20 Cypriot durum wheat (Triticum turgidum L. subsp. durum) landraces, 13 landraces from the broader Mediterranean basin and 22 modern varieties. Cluster analysis depicted a clear separation among modern varieties and landraces, regardless of their origin. Landraces presented the highest genetic variation (average discriminating power of 0.89) and a high number of private alleles (131) was detected; underlying the unique genetic mark-up of this genepool. AMOVA revealed that the highest variability was detected within the landraces originating from Cyprus and landraces from the broader Mediterranean basin. The Cypriot landrace 'Kyperounda' was selected for further evaluation of its' intra-genetic variation and it was determined that genetic diversity was higher in accessions conserved as sublines (He 0.643-0.731) than bulks (He 0.384-0.469). Bayesian analysis revealed substantial admixture within 'Kyperounda' accessions, depicted also by Principal Coordinate Analysis. The findings of the current manuscript emphasize that high intra-genetic diversity is retained when landraces are conserved as sublines in ex situ collections, while landraces that are conserved as bulks have a higher risk of bottleneck. Hence, a more exhausting diversity evaluation is needed in order to fully utilize landraces in breeding schemes and to prevent the loss of genetic variation.

Benefits of the Use of Lactic Acid Bacteria Starter in Green Cracked Cypriot Table Olives Fermentation.

Table olives are one of the most established Mediterranean vegetables, having an exponential increase consumption year by year. In the natural-style processing, olives are produced by spontaneous fermentation, without any chemical debittering. This natural fermentation process remains empirical and variable since it is strongly influenced by physicochemical parameters and microorganism presence in olive drupes. In the present work, Cypriot green cracked table olives were processed directly in brine (natural olives), using three distinct methods: spontaneous fermentation, inoculation with lactic acid bacteria at a 7% or a 10% NaCl concentration. Sensory, physicochemical, and microbiological alterations were monitored at intervals, and major differences were detected across treatments. Results indicated that the predominant microorganisms in the inoculated treatments were lactic acid bacteria, while yeasts predominated in control. As a consequence, starter culture contributed to a crucial effect on olives fermentation, leading to faster acidification and lower pH. This was attributed to a successful lactic acid fermentation, contrasting the acetic and alcoholic fermentation observed in control. Furthermore, it was established that inhibition of enterobacteria growth was achieved in a shorter period and at a significantly lower salt concentration, compared to the spontaneous fermentation. Even though no significant variances were detected in terms of the total phenolic content and antioxidant capacity, the degradation of oleuropein was achieved faster in inoculated treatments, thus, producing higher levels of hydroxytyrosol. Notably, the reduction of salt concentration, in combination with the use of starter, accented novel organoleptic characteristics in the final product, as confirmed from a sensory panel; hence, it becomes obvious that the production of Cypriot table olives at reduced NaCl levels is feasible.

Diverticulum of the left ventricle: etiology and surgical treatment.

Congenital left ventricular (LV) diverticula are rare findings, particularly when first diagnosed in adulthood. We describe successful surgical repair of an isolated congenital apical LV diverticulum associated with an abnormal submitral apparatus in a young adult who received his diagnosis following a peripheral embolism. We intraoperatively identified an abnormally originating belly of the posteromedial papillary muscle, which arose from the interventricular septum, causing partition of the LV cavity and forming an apical aneurysmal communicating chamber. This anatomy of abnormal papillary muscle bifurcation was possibly the mechanism for the development of the apical diverticulum and to the best of our knowledge has not previously been described. Reattachment of the abnormal belly to its normal position preserved mitral valve competence, and an endoventricular patch repair restored LV volume and geometry.

Genetic diversity of Avena ventricosa populations along an ecogeographical transect in Cyprus is correlated to environmental variables.

Avena ventricosa Balansa ex Coss. is considered the C-genome donor of the cultivated hexaploid oat and is a 'priority' species for conservation, since it has limited geographic distribution and the only recorded populations in Europe are present in Cyprus. The current study attempts to characterize the genetic structure and fragmentation of the species via the application of genotypic markers. It was revealed that the genetic variety was mainly allocated among the populations collected, since clustering obtained was according to the geographic origin of the samples and the habitat. Species distribution modeling showed that the most important climatic variable defining A. ventricosa distribution is the mean diurnal temperature. Furthermore, significant association of the genetic structure to environmental variables was detected; overall, a negative association to precipitation was confirmed, while significant correlations of genetic structure and the temperature at the time of anthesis and germination were established. The safeguarding of this valuable genetic resource is discussed.

Monumental olive trees of Cyprus contributed to the establishment of the contemporary olive germplasm.

Even though Cyprus was an important crossing point for the westward spread of olive, and one of the primary regions of domestication, its genetic recourses remain uncharted at a great extent. Throughout the centuries, a number of ancient olive trees remain in the same orchards, contributing to Cypriot oleiculture and society. In an attempt to explore this monumental genetic pool, a survey was conducted to identify centennial olive trees in rural provinces of Cyprus. Microsatellites were employed in order to study their genetic composition (including rootstocks when feasible) and to establish possible associations among genotypes. High numbers of specific alleles, suggestive of the distinctiveness of this germplasm, were detected, and both grafting and rootstock propagation was verified. Moreover, it was determined by Bayesian structural and network reticulate analysis that centennial olives can be divided in two discrete genetic clusters having intermediate admixed accessions. Furthermore, it was determined that all contemporary Cypriot cultivars, that were included in the present study, were highly affiliated exclusively to one genetic group, a strong evidence of selection among elite clones. The information acquired from the current study reveals the genetic rareness of this material and its contribution to the current olive germplasm.

Genetic structure and population diversity of eleven edible herbs of Eastern Crete.

BACKGROUND: The present work aimed to investigate the genetic structure of 11 edible herbs grown in the wild of eastern Crete that are becoming vulnerable due to habitat destruction and unregulated harvesting. Thirty three populations (268 individuals) of Reichardia picroides, Scolymus hispanicus, Scandix pecten-veneris, Leontodon tuberosus, Cichorium spinosum, Sonchus asper ssp. glaucescens, Urospermum picroides, Prasium majus, Hypochoeris radicata, Centaurea raphanina ssp. raphanina and Anagallis arvensis were collected and identified from nine regions with distinct microclimate (Lassithi prefecture), and their genetic composition was studied by means of RAPD markers. RESULTS: A total of ten primers per population were used to detect genetic diversity and bootstrap analysis was conducted for clustering the samples. High levels of heterogeneity were revealed while the Analysis of Molecular Variance documented that variance was allocated mainly within populations and at a lesser extent among populations. Fst values among regions were moderate to high, suggesting partial population fragmentation. Bayesian structure analysis revealed fine genetic composition and substantial admixture between species present in different regions, although clustering was mainly geographically related. CONCLUSIONS: High altitude regions, with little residential and agricultural development (Kefala, Agrilos, Ziros and Tziritis), were the areas where high biodiversity was detected. On the other hand, coastal regions had lower biodiversity, probably due to degradation of their habitat.

Temporary assist device for postcardiotomy cardiac failure.

Postcardiotomy left ventricular failure (LVF) complicating a cardiac surgical procedure is usually managed with intraaortic balloon pump (IABP) counterpulsation. We report two cases of postcardiotomy LVF unresponsive to inotropic support and IABP counterpulsation that were managed successfully with the use of the TandemHeart percutaneous ventricular assist device (pVAD) (CardiacAssist, Pittsburgh, PA). The TandemHeart pVAD appears to be safe and effective as a bridge to recovery in the treatment of postcardiotomy cardiac failure.