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Field and pot experiments were conducted in Greece to study the occurrence of resistance in silky windgrass to acetolactate synthase (ALS)- and acetyl-CoA synthase (ACCase)-inhibiting herbicides. Twenty-four populations of silky windgrass were examined in whole-plant response experiments. High levels of field-evolved resistance to chlorsulfuron (0% to 28% control in terms of fresh weight reduction) with the recommended field rates were confirmed in most silky windgrass populations. However, other ALS inhibitors, such as pyroxsulam and a premix of mesosulfuron-methyl and iodosulfuron, provided adequate control (76% to 100% in terms of fresh weight reduction) of most populations, except eight silky windgrass populations that were found to be cross-resistant to all ALS-inhibiting herbicides tested (i.e., chlorsulfuron, commercial mixture of mesosulfuron-methyl plus iodosulfuron, and pyroxsulam). Conversely, most silky windgrass populations were controlled effectively (90% to 100% in terms of fresh weight reduction) with the recommended field rates of ACCase inhibitors cycloxydim, clethodim, and pinoxaden, but five populations were also found to be resistant to clodinafop-propargyl (10% to 68% control in terms of fresh weight reduction). The ALS gene sequencing of the eight silky windgrass populations, with cross-resistance to ALS inhibitors, revealed a point mutation at the Pro-197 position, causing amino acid substitution by Ser or Thr in the ALS enzyme. Overall, chlorsulfuron and clodinafop-propargyl were selecting agents of field-evolved multiple resistance to ALS- and ACCase-inhibiting herbicides in five silky windgrass populations. As the available postemergence-applied chemistries/modes of action registered for grass weed control in cereals are rather limited, adopting integrated management practices and implementing proactive and reactive measures to delay the evolution of resistant populations is essential.

Plant growth promoting rhizobacteria (PGPR) are able to provide cross-protection against multiple stress factors and facilitate growth of their plant symbionts in many ways. The aim of this study was to isolate and characterize rhizobacterial strains under natural conditions, associated with naturally occurring representatives of wild plant species and a local tomato cultivar, growing in differently stressed Mediterranean ecosystems. A total of 85 morphologically different rhizospheric strains were isolated; twenty-five exhibited multiple in vitro PGP-associated traits, including phosphate solubilization, indole-3-acetic acid production, and 1-aminocyclopropane-1-carboxylate deaminase activity. Whole genome analysis was applied to eight selected strains for their PGP potential and assigned seven strains to Gammaproteobacteria, and one to Bacteroidetes. The genomes harboured numerous genes involved in plant growth promotion and stress regulation. They also support the notion that the presence of gene clusters with potential PGP functions is affirmative but not necessary for a strain to promote plant growth under abiotic stress conditions. The selected strains were further tested for their ability to stimulate growth under stress. This initial screening led to the identification of some strains as potential PGPR for increasing crop production in a sustainable manner.

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3-V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.

Beans are one of the most important staple crops in the world. Runner bean (Phaseolus coccineus L.) is a small-scale agriculture crop compared to common bean (Phaseolusvulgaris). Beans have been introduced to Europe from the Central America to Europe and since then they have been scattered to different geographical regions. This has resulted in the generation of numerous local cultivars and landraces with distinguished characters and adaptive potential. To identify and characterize the underlying genomic variation of two very closely related runner bean cultivars, we performed RNA-Seq with de novo transcriptome assembly in two landraces of P. coccineus, ‘Gigantes’ and ‘Elephantes’ phenotypically distinct, differing in seed size and shape. The cleaned reads generated 37,379 and 37,774 transcripts for ‘Gigantes’ and ‘Elephantes,’ respectively. A total of 1896 DEGs were identified between the two cultivars, 1248 upregulated in ‘Elephantes’ and 648 upregulated in ‘Gigantes.’ A significant upregulation of defense-related genes was observed in ‘Elephantes,’ among those, numerous members of the AP2-EREBP, WRKY, NAC, and bHLH transcription factor families. In total, 3956 and 4322 SSRs were identified in ‘Gigantes’ and ‘Elephantes,’ respectively. Trinucleotide repeats were the most dominant repeat motif, accounting for 41.9% in ‘Gigantes’ and 40.1% in ‘Elephantes’ of the SSRs identified, followed by dinucleotide repeats (29.1% in both cultivars). Additionally, 19,281 putative SNPs were identified, among those 3161 were non-synonymous, thus having potential functional implications. High-confidence non-synonymous SNPs were successfully validated with an HRM assay, which can be directly adopted for P. coccineus molecular breeding. These results significantly expand the number of polymorphic markers within P. coccineus genus, enabling the robust identification of runner bean cultivars, the construction of high-resolution genetic maps, potentiating genome-wide association studies. They finally contribute to the genetic reservoir for the improvement of the closely related and intercrossable Phaseolus vulgaris.

The genetic variability of Lotus corniculatus , a common and important fodder legume, was studied in relation to habitat type and to species diversity at a local level. The study was conducted in Cholomontas mountain, northern Greece, at altitudes of 760–870 m. Genetic material was selected from four forested, at the edge of forest, and four open grassland sites, and was studied with the aid of ISSR molecular markers. The plant cover at each study site was measured and the floristic composition was estimated. The percentage of graminoides increased in grassland sites with high grazing intensity, while the abundance of legumes, including L. corniculatus , forbs and woody species, increased in forested sites with low grazing intensity. Gene diversity H E within the studied populations of L. corniculatus ranged from 0.167 to 0.213 and Shannon index (I) from 0.269 to 0.340. Genetic differentiation was detected between habitats as well as among the populations in each habitat, although it was low, 3% and 7% respectively. However, genetic differentiation was significant within the populations (90%). Genetic diversity of L. corniculatus was not correlated with species diversity (richness and evenness) either within or between populations, while was negatively correlated with L. corniculatus abundance.

Thirty landraces were selected from a Gene Bank collection comprising of pepper ( Capsicum annuum L.) landraces from across Greece. The genetic variability among the 30 landraces and one commercial greek cultivar, intended for industrial use, was assessed by Inter-Simple Sequence Repeat (ISSR), Start Codon Targeted (SCoT) and Expressed Sequence Tag (EST) - Simple Sequence Repeat (SSR) molecular markers. All genotypes were clearly distinguished in the dendrograms produced, with the ISSR and SCoT dendrograms being the most consistent. Three landraces were grouped together in all dendrogams revealing a possible phylogenetic relationship. Furthermore, EST-SSR markers were combined with High Resolution Melting (HRM) analysis by which the more adequate and confident separation of the landraces was accomplished due to the unique HRM profiles produced. Genotyping the rich Greek pepper germplasm by molecular markers will aid the breeding process towards well adopted Greek pepper cultivars of higher quality and productivity.

Tree nuts are nutritionally important foods, being rich sources of vitamins, minerals, healthy fats and proteins and having cardio protective effects. Despite their healthy attributes, some tree nuts are also known for triggering adverse immune responses to individuals. Thus, an ongoing challenge in molecular biology is to develop rapid, accurate and reliable techniques to identify organisms and their processed foods, particularly those which could be contaminated with allergenic compounds. Lately, in addition to different chemical and immunological methods used, DNA based methods were also applied for this purpose. We describe here a novel, specific and highly sensitive High Resolution Melting analysis based method, using chloroplast barcoding regions (Bar-HRM) in order to a) obtain barcoding information for the major tree nut species (we have also included peanuts and sesame in our study) and b) to quantitatively identify one such allergenic tree nut species, like hazelnut in processed foods. The results show that the proposed method i) is capable of distinguishing among different tree nut species, ii) can be applied to processed tree nut products such as biscuits and iii) can detect a ratio of 0.01% of hazelnut in biscuits. In conclusion, it is demonstrated that BAR-HRM is suitable for the rapid, closed-tube and accurate screening of large numbers of tree nuts and/or of their processed foods and for the identification of allergenic tree nut species contained in processed foods.

Lentil ( Lens culinaris ) is an important legume crop worldwide, consumed as dried seeds. Correct identification of lentil varieties is important in order to ensure food quality, safety, authenticity and health for consumers as well as high price from elite varieties for farmers and industry. Recently, DNA-based methods like the molecular markers microsatellites (SSRs) for nuclear DNA or the DNA barcoding which uses chloroplast or nuclear DNA have been developed for plant species or variety identification, for genotyping and for identification of their ingredients in the final food products. Here we have integrated High Resolution Melting (HRM) analysis, coupled with five SSR markers in parallel with rpoC1 chloroplast DNA barcode targeting region, in order to facilitate the identification of Protected Geographical Indication (PGI) lentil variety ‘Eglouvi’. The five SSR loci used were informative and generated a unique melting curve profile of microsatellites for each of the ten varieties tested. SSRs enabled the distinction and identification of the “Eglouvi” lentil PGI variety and furthermore they allowed the traceability of “Eglouvi” and the identification of lentil varieties admixtures of 50%. In addition, the application of the Barcode DNA High Resolution Melting (Bar-HRM) method on the species specific plant DNA barcoding region rpoC1, allowed not only the identification of adulterations but also the quantification of the most common lentil admixture. Bar-HRM detected Vicia sativa adulterants in Lens esculentum pure seed mix as low as 1:100. Hence, these assays provided flexible, cost-effective, and closed-tube SSR-HRM and Bar-HRM genotyping methods, well suited to identify adulterants in variety and species level and to food forensic uses in food products.

Legumes and common bean (Phaseolus vulgaris L.), in particular, are important crops worldwide, consumed either as dried seeds or fresh fruits. Correct identification of common bean varieties is important, in order to ensure food quality, safety and authenticity for consumers. Recently, DNA based methods, including molecular markers like microsatellites (SSR), have been developed for plant species or variety identification genotyping and for identification of their ingredients in the final food products. Here, we have applied High Resolution Melting (HRM) analysis coupled with four microsatellite markers to facilitate the identification of protected geographic indication (PGI) common bean variety ‘Plake Megalosperma Prespon’ (‘PMP’). The four microsatellite loci used were informative and were used to generate a unique melting curve profile of microsatellites for each variety tested. These microsatellite markers enabled the distinction and identification of the PGI (common bean variety ‘PMP’). Hence, this assay provided a flexible, cost-effective and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in common beans.