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Vicia sativa L., commonly known as the common vetch, is an annual, self-pollinating legume used primarily as fodder both by livestock and wildlife. Additionally, it contributes to environmental balance through nitrogen fixation and the improvement of soil properties. The phenotypic, genetic, and metabolite variability among four advanced lines (BK45, BK29, BK23, BK27) and two commercial varieties (M-6900, BI-65) of V. sativa were evaluated in order to be used for future breeding programs aimed at producing genetically improved varieties. BK45 was the most promising line due to its high genetic polymorphism, but also because it exhibited a significant amount of seed production and high seed quality based on its metabolomics profile. A stepwise multiple regression analysis (MRA) revealed a relationship between SCoT alleles, seed, and biomass yield. Additionally, several statistically significant marker bands linked to metabolites were found using the SCoT marker analysis. Hence, data assessed via MRA may be helpful in marker-assisted breeding programs. Finally, the two commercial varieties can be further exploited in breeding programs due to their high genetic diversity.

AbstractThe present study aimed to evaluate the genetic diversity and differentiation of Pinus nigra Arnold, the European black pine, populations located in protected forested areas in three countries from the Balkan and Mediterranean region: Bulgaria, Greece, and Cyprus. Totally, 175 DNA samples from P. nigra plants collected from eight populations in these countries were analyzed using three SRAP primer pairs. The applied SRAP markers demonstrated high-resolution power, resulting in the identification of an average of 215 ± 67 polymorphic loci per SRAP primer pair. The analysis of molecular variance (AMOVA) showed that 82% of the observed variation was due to intra-population variation, while 18% was due to inter-population variation. The overall analysis of the population structure suggested low (Fst ≤ 0.01) intra-country differentiation for the populations from Bulgaria and Cyprus and moderate (0.15 ≤ Fst ≤ 0.17) inter-country differentiation. On the contrary, high differentiation between the populations (0.06 ≤ Fst ≤ 0.20) and complex genetic structure were characteristic of the three populations from Greece, relatively closely located in the area of the Pindos National Park. Analysis of the population structure also revealed that one of the Pindos populations belonged to the cluster of Bulgarian populations (Fst ≤ 0.01), showing moderate (Fst ≤ 0.11) to high (Fst ≤ 0.20) genetic differentiation from the other analyzed Pindos populations. The further use of SRAP markers for mapping the genetic diversity among P. nigra populations and identification of local populations diverted from the overall phylogeography-driven pattern in the studied region are discussed.

Phaseolus coccineus L. is a highly valuable crop for human consumption with a high protein content and other associated health benefits. Herein, 14 P. coccineus L. landraces were selected for genetic characterization: two Protected Geographical Indication (PGI) landraces from the Prespon area, namely “Gigantes” (“G”) and “Elephantes” (“E”), and 12 additional landraces from the Greek Gene Bank collection of beans (PC1–PC12). The genetic diversity among these landraces was assessed using capillary electrophoresis utilizing fluorescence-labeled Simple Sequence Repeat (SSR) and Expressed Sequence Tag (EST); Simple Sequence Repeat (SSR) is a molecular marker technology. The “G” and “E” Prespon landraces were clearly distinguished among them, as well as from the PC1 to PC12 landraces, indicating the unique genetic identity of the Prespon beans. Overall, the genetic characterization of the abundant Greek bean germplasm using molecular markers can aid in the genetic identification of “G” and “E” Prespon beans, thus preventing any form of fraudulent practices as well as supporting traceability management strategies for the identification of authenticity, and protection of the origin of local certified products.

Olive (Olea europaea L.) is a crop of enormous economic and cultural importance. Over the years, the worldwide production of olive oil has been decreasing due to various biotic and abiotic factors. The current drop in olive oil production resulting from climate change raises concerns regarding the fulfillment of our daily demand for olive oil and has led to a significant increase in market prices. In the future, there will be a higher chance that we will face a severe shortage of olive oil, which could harm both the economic sector and the food supply. As olive groves cover more than 5 million hectares in the European Union alone, the need to preserve the crop in the context of extreme climatic events is imperative. As drought is considered one of the most limiting factors in agriculture, drought-resistant varieties and sustainable irrigation strategies are being developed to mitigate the impact of drought on crop productivity and secure the future supply of olive oil. This review focuses on recently gained insights into drought stress in olive trees through omics and phenomics approaches to unravelling mechanisms that may lead to developing new varieties that are tolerant against drought elicited by changes in growing systems.

The dimorphic fungus Taphrina deformans is the causal agent of peach leaf curl disease, which affects leaves, flowers, and fruits. An RNA-seq approach was employed to gain insights into the transcriptional reprogramming of a peach cultivar during leaf inoculation with the yeast phase of the fungus across a compatible interaction. The results uncovered modulations of specific peach differentially expressed genes (DEGs) in peaches and pathways related to either the induction of host defense responses or pathogen colonization and disease spread. Expression profiles of DEGs were shown to be highly time-dependent and related to the presence of the two forms of the fungal growth, the inoculated yeast form and the later biotrophic phase during mycelial development. In parallel, this differential reprogramming was consistent with a diphasic detection of fungal load in the challenged leaves over the 120 h after inoculation (HAI) period. Leaf defense responses either occurred during the early yeast phase inoculation at 24 HAI, mediated primarily by cell wall modification processes, or more pronouncedly during the biotrophic phase at 72 HAI, as revealed by the activation of DEGs related to pathogen perception, signaling transduction, and secondary metabolism towards restraining further hypha proliferation. On the contrary, the expression patterns of specific DEGs at 120 HAI might further contribute to host susceptibility. These findings will further allow us to elucidate the molecular responses beyond the peach—T. deformans interaction.

In the last two decades, plant taxonomy has bloomed, following the development of a novel technique, namely, DNA barcoding. DNA barcodes are standardized sequences, ideally unique, coding or non-coding, either from the genome of the organism or from its organelles, that are used to identify/classify an organismal group; in short, the method includes amplification of the DNA barcode, sequencing and comparison with a reference database containing the relevant sequences from different species. In plants, the use a universal DNA barcode, such as COI, which is used in animals, has not been achieved so far. This review provides a comprehensive overview of the progress made in DNA barcoding within the field of plant taxonomy. It highlights the success of various barcode loci, the emergence of super barcodes from the chloroplast genome, and the overall impact of next-generation sequencing technologies on the field. The discussion of different approaches reflects the ongoing efforts to refine and optimize DNA barcoding techniques for plants, contributing to the advancement in our understanding of plant biodiversity.

Walnut is one of the most important nuts regarding their production and consumption. The available but uncharacterized genetic resources of walnut are important for the development and breeding of local varieties. Greece holds an important number of genetically uncharacterized walnut landraces, especially within the area of Parnon, which is considered to play a significant role as an in situ gene bank, due to its unique location traits. However, the genetic characterization and further use of these resources has been insufficient, due to the absence of genetic studies. In this study, we implemented SSR molecular markers, both to genetically characterize the walnut tree genetic diversity of the Parnon area and to identify its unique genetic structure, which will form the starting material for subsequent breeding programs. Overall, high levels of genetic variation were found among the individual walnut accessions that were collected in the Parnon mountain region.

Plants can develop stress memory as a response to various abiotic stresses, but the underlying mechanisms are not yet fully understood. Most of the knowledge concerning the mechanisms of stress memory development and inheritance in plants is primarily based on research in the model plant Arabidopsis. While shared mechanisms exist across plant species, it is crucial to expand our understanding of epigenetic regulation in crops. Stress priming, or prior exposure to mild stress, can enhance a plant’s adaptation to future stress events and the development of stress memory. During stress priming, plants undergo physiological, biochemical, molecular, and epigenetic changes that can be transient or maintained throughout their lifespan, and in some cases, these changes can also be inherited by the offspring. In this review, we present the current state of knowledge on the development of priming-induced stress memory in agronomically important crops towards stress resilience. The most prominent abiotic stresses, namely, heat, cold, salt, drought, and waterlogging, are highlighted in relation to stress cis-/trans-priming and memory development at the intra-, inter-, and transgenerational levels. The cost for developing stress memory in plants along with the duration of these memory imprints and stress memory fading are also discussed. This review is particularly important in the era of climate change, which necessitates the development of agricultural sustainability strategies.

Microalgae are a renewable and sustainable source of bioactive compounds, such as essential amino acids, polyunsaturated fatty acids, and antioxidant compounds, that have been documented to have beneficial effects on nutrition and health. Among these natural products, the demand for natural antioxidants, as an alternative to synthetic antioxidants, has increased. The antioxidant activity of microalgae significantly varies between species and depends on growth conditions. In the last decade, microalgae have been explored in livestock animals as feed additives with the aim of improving both animals’ health and performance as well as product quality and the environmental impact of livestock. These findings are highly dependent on the composition of microalgae strain and their amount in the diet. The use of carbohydrate-active enzymes can increase nutrient bioavailability as a consequence of recalcitrant microalgae cell wall degradation, making it a promising strategy for monogastric nutrition for improving livestock productivity. The use of microalgae as an alternative to conventional feedstuffs is becoming increasingly important due to food–feed competition, land degradation, water deprivation, and climate change. However, the cost-effective production and use of microalgae is a major challenge in the near future, and their cultivation technology should be improved by reducing production costs, thus increasing profitability.

Monilinia laxa, Monilinia fructicola and Monilinia fructigena are the three main causal agents of brown rot, which is one of the most important diseases of stone fruits in pre- and postharvest conditions. Nowadays, the need for the precise genotyping of these Monilinia species in terms of the genetic diversity of their populations or differences in their pathogenicity and host range is a prerequisite for any efficient disease management. In our study, the genetic structure of Monilinia populations in Serbia from three geographically distinct regions was investigated employing a high-resolution melting (HRM) analysis which is a sensitive and rapid molecular approach in fungal ge­notyping and diagnostics. Using species-specific primer pairs genotype-specific HRM melting curve profiles were generated allowing to efficiently decipher the genetic diversity of the Monilinia populations. The Monilinia genotypes could be easily distinguished according to their melting curves. The isolates from the northern region were assigned to distinct genotypes and grouped rather independently compared to the isolates of the other two regions among all three tested Monilinia spp. M. fructicola and M. fructigena showed a higher genetic diversity among their populations (44%) compared with the genetic diversity among the M. laxa populations (7%). In contrast, the genetic variance within the pathogen populations was higher in the case of M. laxa (93%). Our data revealed an absence of host specificity in the Monilinia spp. populations.

In the present study, selected cosmeceutical properties of aqueous extracts from the microalgae strain Trachydiscus minutus were assessed and compared with those obtained using three widely used Chlorella strains (C. vulgaris, C. sorokiniana, and C. minutissima). Among all extracts, T. minutus extracts showed the highest total antioxidant capacity (TAC) and inhibitory potency towards elastase, suggesting potential activity in controlling skin aging. Furthermore, the cytotoxicity, anti-inflammatory activity and UVA protection of T. minutus extract were evaluated employing normal human dermal fibroblasts (NHDF) and human keratinocyte HaCaT cells. The results showed that the T. minutus extract was able to significantly inhibit the transcription of selected marker genes involved in inflammation [interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor alpha (TNFα)]. In addition, treatment of NHDF and HaCaT cells with T. minutus extract ameliorate the UVA-induced cell damage by decreasing the accumulation of reactive oxygen species (ROS). Extracts from T. minutus were formulated into a skin care cream and an aqueous gel. Both formulas exhibited excellent compatibility and stability. Comprehensively, all these results suggest that T. minutus extract displays promising cosmeceutical properties by providing antioxidant, anti-aging, and anti-inflammatory activities, and therefore has potential for cosmeceutical use.

Local germplasm may be the basis for genetic improvement for sustainability and resilience, yet little is known about Greek peach [Prunus persica (L.) Batsch] local genetic resources. The aims of the present study were to entail a prospection in the mainland and islands of Greece for local traditional and underutilized germplasm and study their genetic and morphological variation and susceptibility to shoot blight from Cylindrocarpon destructans and Monilinia laxa. A total of 32 peach cultivars/accessions were prospected from the mainland (Imathia and Magnesia) and islands (Andros, Ikaria, Kythira, Lesvos and Samos) of Greece. Leaf and fruit morphological characterization was made in situ and ex situ using 42 qualitative and quantitative morphological descriptors. Nine clones of ‘Lemonato’, isolated in Magnesia, with differing ripening times, were found to vary in leaf and fruit phenotypic traits. The local peach genotypes were separated from 12 old foreign cultivars, in 8 out of the 42 leaf and fruit phenotypic traits studied. We observed greater lesion damages from C. destructans than M. laxa. Local cultivars had greater resistance to Cylindrocarpon destructans than foreign cultivars, with ‘Lemonato Andrea’, ‘Daggalakou’ and ‘Papagianni’ exhibiting the greatest resistance. Genetic characterization was performed in the studied local and foreign peach cultivars/accessions, using eight Inter Simple Sequence Repeats (ISSRs), resulting in a total of 404 bands. Analysis of molecular variance and principal coordinates analysis revealed moderate to low genetic diversity among the peach cultivars, and three distinct clusters were formed. Furthermore, multiple regression analysis was implemented for the association study between morphological traits and the ISSR markers, revealing several markers that are statistically and significantly correlated with fruits’ traits. The obtained results could be valuable for breeding programs and future research on peach.

Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement.

Apple scab caused by Venturia inaequalis has the most destructive effects among other phytopathogens in apple crops all over the world. The integration of resistance genes from local and domestic cultivars is a prerequisite for the efficient control of this disease and is a main target in efficient breeding approaches. Across Greece, many domestic apple cultivars are reported without deep knowledge about the presence and diversity of scab resistance genes. In this study, the presence of five resistance genes (Rvi2, Rvi4, Rvi6, Rvi8 and Rvi11) was evaluated across twenty local and domestic apple genotypes, employing twelve molecular markers closely linked to known apple scab resistance loci. Significant differences and polymorphisms among the tested cultivars were detected suggesting that some of them carry a sufficient number of resistance genes. This observed genetic diversity could be exploited in ongoing breeding approaches as a natural source of polygenic resistance against apple scab.

This article describes testing the application of SRAP (sequence-related amplified polymorphism) markers for characterisation of a small set of plants of Quercus coccifera L and samples from 25 other forest tree species. The results suggest that SRAP markers could be used as an affordable and efficient tool for the characterisation of the genetic diversity in populations of tree species as a part of the characterisation of forest biodiversity and the related decision making and management.

Capture-based aquaculture is now gaining much attention in Southeast Asia. This system was used to produce several fish species with social and economic implications, including the giant snakehead (Channa micropeltes). As wild harvesting of organisms for seed stock is one of main practices in capture-based aquaculture, abundance and distribution of the wild stock are essential for both environmental impact evaluation and stock management. Mark and recapture, visual observation and physical capture of target species are costly, ineffective, and labour intensive for fish surveys in several cases. Detection of target organisms using eDNA (environmental DNA) could be a good alternative as it has proved to be a non-invasive, rapid, and sensitive method for aquatic species monitoring and surveying. Here, we developed a TaqMan assay that targets the 16S region of giant snakehead DNA to amplify eDNA captured in water samples. 300 µl of water samples were collected from 15 sites located in the Chao Phraya River Basin (Ping, Wang, Yom, Nan, and Chao Phraya River) and filtered with 0.7 µm glass fibre membrane filter. Giant snakehead eDNA was detected in most tributaries (60%) with concentrations ranging from 74.0 copies/ml in Wang River sites to 7.4 copies/ml in Nan River sites. As intensification of capture-based aquaculture could lead to depleting of wild fish stocks, urgent management is needed. However, the existing conventional approaches for assessment of fish overexploitation, survey and monitoring have several limitations.

Vicia faba L. (faba bean) is one of the most promising pulse crops due to its nutritional value and high nitrogen fixation capacity. The aim of the present study was to compare the genetic diversity and the seed metabolite profiles of five genetic materials of faba bean. Specifically, three newly developed advanced lines (KK18, KK14 and KK10) and two commercial cultivars (POLIKARPI and TANAGRA), were evaluated for this purpose. Genetic diversity among populations was assessed by SCoT molecular markers. Through UPGMA dendrogram, genetic distances between populations were estimated. Untargeted metabolomics analysis of the seeds was performed employing GC/EI/MS. The cultivar POLYKARPI exhibited the highest polymorphism. All varieties showed a higher within-cultivars and advanced lines variability than between. POLYKARPI and KK14 had the lowest genetic distances, while KK18 and TANAGRA presented the highest ones. The advanced line KK18 displayed the best nutritional profile, the highest concentration of desirable metabolites (lactic acid and trehalose), the lowest concentration of anti-nutritional factors (oxalic acid) and the lowest concentration of saturated fatty acids (palmitic and stearic acid). According to the results of the present study, KK18 line is a very promising material for further exploration and utilization in breeding programs.

The destructive effects of Fusarium wilts are known to affect the production of many crops. The control of Fusarium oxysporum and other soilborne pathogens was mainly based on soil fumigation (methyl bromide), which has long been prohibited and, nowadays, is based on a limited number of available fungicides due to legislation restrictions on residue tolerances and environmental impacts. Alternatively, natural and environmentally safe compounds, such as essential oils, are being investigated for their efficacy in the control of soilborne diseases. The great fungicidal ability of the oregano essential oil components (carvacrol and thymol) has been reported to inhibit the germination and the mycelial development of several fungal species, including F. oxysporum. The aim of our study was to demonstrate how the metabarcoding approach can provide valuable information about the positive or negative impacts of two different doses of oregano essential oil on Fusarium oxysporum and other fungal species which were present in the studied soil samples through the amplification of the ITS1 and ITS2 regions, which were analyzed on a MiSeq platform. A higher dose of oregano essential oil decreased the abundance of F. oxysporum, along with other fungal species, but also had negative effects on Trichoderma evansii and Mortierella chlamydospora, species with possible fungicidal properties. Soil properties, essential oil properties, the fungal composition, and interactions between fungal species should be considered as factors influencing the effectiveness of essential oils as biological control agents for soilborne pathogens.

Leguminous crops play a key role in food production and agroecosystem sustainability. However, climate change and agricultural intensification have a significant impact on the available arable land, soil microbiome functions, and ultimately, crop productivity. The “Prespa bean” (Phaseolous coccineous L.) is an important leguminous crop for the agricultural economy of the rural areas surrounding the lake, Mikri Prespa, which is of significant ecological importance. The seasonal effects on soil microbiome structure, diversity and functions associated with the runner bean cultivation were investigated using 16S rRNA amplicon sequencing. The results indicated that the presence of the runner bean differentially shaped the soil microbial community structure. The runner bean was implicated in the recruitment of specific bacteria, by favouring or excluding specific classes or even phyla. Soil functions involved in nutrient availability and carbon metabolism, among other pathways, were associated with microbiome–plant interactions. The temporal relative abundance shifts could be explained by the impact of soil organic matter, the fertilization regime, and the equilibrium in carbon metabolic processes. This research has shown the effect of runner bean cultivation on the soil microbiome which, in future, may potentially contribute to research into sustainable agricultural productivity and the protection of soil ecosystem services.

Background The severe deforestation, as indicated in national forest data, is a recurring problem in many areas of Northern Thailand, including Doi Suthep-Pui National Park. Agricultural expansion in these areas, is one of the major drivers of deforestation, having adverse consequences on local plant biodiversity. Conserving biodiversity is mainly dependent on the biological monitoring of species distribution and population sizes. However, the existing conventional approaches for monitoring biodiversity are rather limited. Methods Here, we explored soil DNA at four forest types in Doi Suthep-Pui National Park in Northern Thailand. Three soil samples, composed of different soil cores mixed together, per sampling location were collected. Soil biodiversity was investigated through eDNA metabarcoding analysis using primers targeting the P6 loop of the plastid DNA trnL (UAA) intron. Results The distribution of taxa for each sample was found to be similar between replicates. A strong congruence between the conventional morphology- and eDNA-based data of plant diversity in the studied areas was observed. All species recorded by conventional survey with DNA data deposited in the GenBank were detected through the eDNA analysis. Moreover, traces of crops, such as lettuce, maize, wheat and soybean, which were not expected and were not visually detected in the forest area, were identified. It is noteworthy that neighboring land and areas in the studied National Park were once used for crop cultivation, and even to date there is still agricultural land within a 5–10 km radius from the forest sites where the soil samples were collected. The presence of cultivated area near the forest may suggest that we are now facing agricultural intensification leading to deforestation. Land reform for agriculture usage necessitates coordinated planning in order to preserve the forest area. In that context, the eDNA-based data would be useful for influencing policies and management towards this goal.

The current study aimed to produce rootstock material through micropropagation by developing efficient regeneration and Agrobacterium-mediated transformation protocols for three high quality commercial tomato hybrids (Felina, Siena and Don Jose) to overexpress the GmGSTU4 gene from Glycine max L. previously shown to enhance antioxidant activity. We investigated the plant growth regulators zeatin (Z) and 3-idoleacetic acid (IAA) to determine their best combination for an efficient regeneration protocol for each hybrid. The highest regeneration efficiency was observed in Felina (94.4%) with 1.0 mg/l Z and 0.1 mg/l IAA. In contrast, Don Jose (92.5%) and Siena (83.3%) performed better with 0.5 mg/l Z and 0.1 mg/l IAA. The three hybrids did not differ in micropropagation index, however, Felina showed the highest number of in vitro rooted and in vivo acclimatized plants. Factors such as the age of explant, days in pre- and co-culture and the concentrations of acetosyringone and thiamine on Agrobacterium-mediated genetic transformation were assessed. The transformation indices were 37.04% for the Felina, 13.8% for Siena and 8.33% for Don Jose. We conclude that targeted genotype-specific regeneration protocols will provide an efficient and cost effective genetic transformation system for rootstock production and further incorporation into micropropagation and transgrafting systems.

Abstract Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.

Foliar fungal diseases are a serious threat to winter wheat production and climate change appears to favor pathogens associated with leaf blotch and tan spot symptoms in the Mediterranean area. The present work aimed to highlight these risks and propose appropriate disease management strategies by evaluating the seed treatment with the Systiva® (BASF) fungicide as a means to protect the crop against foliar fungal infections during the early growing stages. Towards that aim, plant tissue symptoms affected by the pathogens Pyrenophora tritici-repentis and Septoria spp. were systematically recorded in a study field in the region of Larissa, central Greece for three years (2016–2018), and the findings were associated with the monthly weather anomalies. Consequently, for the growing period of 2021–2022, a field experiment was established in the same disease prone field, comparing different doses of the seed treatment with Systiva® fungicide against leaf blotch and tan spot diseases. The evaluation was made by visual disease assessments, remote sensing with an unmanned aerial vehicle (UAV) and metagenomics analysis. Parallel measurements on straw residues were also made to characterize the plant residues perithecia (pseudothecia). Visual leaf disease assessments and UAV remote sensing data showed that Systiva® treatments at doses of 125 cc and 150 cc per 100 kg of wheat seed can reduce the percentage of infected wheat plants caused by foliar fungal pathogens at wheat growth stages GS23-25 and GS30-31. Moreover, the metagenomics analyses performed on the microbial communities revealed that Systiva® can decrease the degree of infection by P. tritici-repentis and Z. tritici but do not provide sufficient protection against P. nodorum. Foliar diseases were influenced by the soil surface area covered with straw residue with a high proportion of natural inoculum (pseudothecia/ascospores).

The aim of this study was to develop three supervised self-organizing map (SOM) models for the automatic recognition of a systemic resistance state in plants after application of a resistance inducer. The pathosystem Fusarium oxysporum f. sp. radicis-lycopersici (FORL) + tomato was used. The inorganic, defense inducer, Acibenzolar-S-methyl (benzo-[1,2,3]-thiadiazole-7-carbothioic acid-S-methyl ester, ASM), reported to induce expression of defense genes in tomato, was applied to activate the defense mechanisms in the plant. A handheld fluorometer, FluorPen FP 100-MAX-LM by SCI, was used to assess the fluorescence kinetics response of the induced resistance in tomato plants. To achieve recognition of resistance induction, three models of supervised SOMs, namely SKN, XY-F, and CPANN, were used to classify fluorescence kinetics data, in order to determine the induced resistance condition in tomato plants. To achieve this, a parameterization of fluorescence kinetics curves was developed corresponding to fluorometer variables of the Kautsky Curves. SKN was the best supervised SOM, achieving 97.22% to 100% accuracy. Gene expression data were used to confirm the accuracy of the supervised SOMs.

Plant species confirmation is a crucial step in using native plant species for forest restoration. To enhance this, a hybrid method of DNA barcoding and high-resolution melting analysis (Bar-HRM) was investigated in this study. In total, 12 native plant species samples were collected from forest restoration sites in Nan, a province in Northern Thailand. Simulation HRM analysis was performed to find the most appropriate region for in vitro Bar-HRM analysis. After that, in vitro Bar-HRM was carried out to validate the performance of native plant species. Results from both simulation and in vitro analyses revealed that the nuclear ribosomal internal transcribed spacer (ITS) region can be used as a primer set that can clearly discriminate native plant species in this study. With our study, Bar-HRM was proved of use in native plant species confirmation, even if that species had no molecular data available. In this context, Bar-HRM would be useful for the identification of native plant species used in tropical forest restoration not only in Thailand but also in any areas with similar plant groups.

Vegetable grafting is extensively used today in agricultural production to control soil-borne pathogens, abiotic and biotic stresses and to improve phenotypic characteristics of the scion. Commercial vegetable grafting is currently practiced in tomato, watermelon, melon, eggplant, cucumber, and pepper. It is also regarded as a rapid alternative to the relatively slow approach of breeding for increased environmental-stress tolerance of fruit vegetables. However, even though grafting has been used for centuries, until today, there are still many issues that have not been elucidated. This review will emphasize on the important mechanisms taking place during grafting, especially the genomic interactions between grafting partners and the impact of rootstocks in scion’s performance. Special emphasis will be drawn on the relation between vegetable grafting, epigenetics, and the changes in morphology and quality of the products. Recent advances in plant science such as next-generation sequencing provide new information regarding the molecular interactions between rootstock and scion. It is now evidenced that genetic exchange is happening across grafting junctions between rootstock and scion, potentially affecting grafting-mediated effects already recorded in grafted plants. Furthermore, significant changes in DNA methylation are recorded in grafted scions, suggesting that these epigenetic mechanisms could be implicated in grafting effects. In this aspect, we also discuss the process and the molecular aspects of rootstock scion communication. Finally, we provide with an extensive overview of gene expression changes recorded in grafted plants and how these are related to the phenotypic changes observed. Τhis review finally seeks to elucidate the dynamics of rootstock-scion interactions and thus stimulate more research on grafting in the future. In a future where sustainable agricultural production is the way forward, grafting could play an important role to develop products of higher yield and quality in a safe and “green” way.

Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution.

Abstract Background Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed. Methods Sequences from the selected regions (matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products. Results The ITS1 barcode was found to be the suitable primer as melting data from the HRM assay proved to be capable of distinguishing T. crispa from its related species. The developed protocol was then employed to authenticate medicinal products in powdered form. HRM analysis of all tested samples here revealed that five out of eight products contained not only the indicated species T. crispa but also other Tinospora, that have a high level of morphological similarity. Conclusion Misrepresentation, poor packaging and inappropriate labeling of the tested medicinal herbal products are thought to be the reason of the results here. Using Bar-HRM with the ITS marker lead to success in authenticating the tested herbal products.

Biosolid application is an effective strategy, alternative to synthetic chemicals, for enhancing plant growth and performance and improving soil properties. In previous research, biosolid application has shown promising results with respect to tomato resistance against Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Herein, we aimed at elucidating the effect of biosolid application on the plant–microbiome response mechanisms for tomato resistance against Forl at a molecular level. More specifically, plant–microbiome interactions in the presence of biosolid application and the biocontrol mechanism against Forl in tomato were investigated. We examined whether biosolids application in vitro could act as an inhibitor of growth and sporulation of Forl. The effect of biosolid application on the biocontrol of Forl was investigated based on the enhanced plant resistance, measured as expression of pathogen-response genes, and pathogen suppression in the context of soil microbiome diversity, abundance, and predicted functions. The expression of the pathogen-response genes was variably induced in tomato plants in different time points between 12 and 72 h post inoculation in the biosolid-enriched treatments, in the presence or absence of pathogens, indicating activation of defense responses in the plant. This further suggests that biosolid application resulted in a successful priming of tomato plants inducing resistance mechanisms against Forl. Our results have also demonstrated that biosolid application alters microbial diversity and the predicted soil functioning, along with the relative abundance of specific phyla and classes, as a proxy for disease suppression. Overall, the use of biosolid as a sustainable soil amendment had positive effects not only on plant health and protection, but also on growth of non-pathogenic antagonistic microorganisms against Forl in the tomato rhizosphere and thus, on plant–soil microbiome interactions, toward biocontrol of Forl.

Nutrients in the form of fertilizers and/or other additives such as amino acids, dramatically influence plant development and growth, plant nutrient composition and the level of soil pollution. Moreover, the treatment of soil microbiota is emerging as a new strategy in plant breeding to achieve desirable traits. Thus, integrated study of fertilizer application and soil microbiota might lead to a better understanding of soil-plant interactions and inform the design of novel ways to fertilize plants. Herein we report metagenomics data for soil microbiota in lettuce (Lactuca sativa) treated with fertilizer, amino acids or their combinations as follows: N-fertilizer + Amino16®, Amino16®, N-fertilizer and no treatment control. Data have been deposited in the NCBI Sequence Read Archive (SRA) (accession number: PRJNA388765).

Much is known about microbes originally identified in caves, but little is known about the entrapment of microbes (bacteria) in stalactites and their possible environmental origins. This study presents data regarding the significant environmental distribution of prokaryotic bacterial taxa of a Greek stalactite core. We investigated the involvement of those bacteria communities in stalactites using a metataxonomic analysis approach of partial 16S rRNA genes. The metataxonomic analysis of stalactite core material revealed an exceptionally broad ecological spectrum of bacteria classified as members of Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and other unclassified bacteria. We concluded that (i) the bacterial transport process is possible through water movement from the upper ground cave environment, forming cave speleothems such as stalactites, (ii) bacterial genera such as Polaromonas, Thioprofundum, and phylum Verrucomicrobia trapped inside the stalactite support the paleoecology, paleomicrobiology, and paleoclimate variations, (iii) the entrapment of certain bacteria taxa associated with water, soil, animals, and plants such as Micrococcales, Propionibacteriales, Acidimicrobiales, Pseudonocardiales, and α-, β-, and γ-Proteobacteria.

The loss of forests is a major environmental, social, and economic problem. The disappearance has been occurring to an extreme degree in many parts of Southeast Asia, including Thailand. Logging and clearing of forests for agriculture, cash crops, and food production has destroyed much of the tropical forests in Thailand. Floristic inventory could provide essential information for forest conservation but species identification as a part of inventory creating could be challenging in some cases. Barcode DNA coupled with High Resolution Melting analysis (Bar-HRM) was used here in aiding species identification of plant in Dipterocarpaceae (Dipterocarpus alatus, D. costatu, D. intricatus, D. obtusifolius, Hopea ferrea, H. odorata, Shorea guiso, S. obtuse, S. roxburghii, and S. siamensis) and Fagaceae (Castanopsis echinocarpa, C. inermis, Lithocarpus wallichianus, Quercus aliena and Q. oidocarpa) families. Two main experiments were conducted including: (1) a comparing method for primer design and (2) testing the robustness of the Bar-HRM by trying to identify tree samples that did not have sequences in the GenBank. In experiment 1, the manual design primer pair was found to be the best fit for the work. Of key importance is finding the primers which give the most nucleotide variations within the generated amplicon; this is a parameter that cannot be set in any web-based tools. Next, in experiment 2, Bar-HRM using primers of ITS1 and ITS2 regions were able to discriminate all 10 tested tree species without any problem, even when there were no sequences of the samples to be analysed before performing the HRM. Here, Bar-HRM poses potential to be a game-changer in tropical forest conservation, as it will be useful for species identification.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Abstract Background A variety of plants in Acanthaceae have long been used in traditional Thai ailment and commercialised with significant economic value. Nowadays medicinal plants are sold in processed forms and thus morphological authentication is almost impossible. Full identification requires comparison of the specimen with some authoritative sources, such as a full and accurate description and verification of the species deposited in herbarium. Intake of wrong herbals can cause adverse effects. Identification of both raw materials and end products is therefore needed. Methods Here, the potential of a DNA-based identification method, called Bar-HRM (DNA barcoding coupled with High Resolution Melting analysis), in raw material species identification is investigated. DNA barcode sequences from five regions (matK, rbcL, trnH-psbA spacer region, trnL and ITS2) of Acanthaceae species were retrieved for in silico analysis. Then the specific primer pairs were used in HRM assay to generate unique melting profiles for each plants species. Results The method allows identification of samples lacking necessary morphological parts. In silico analyses of all five selected regions suggested that ITS2 is the most suitable marker for Bar-HRM in this study. The HRM analysis on dried samples of 16 Acanthaceae medicinal species was then performed using primer pair derived from ITS2 region. 100% discrimination of the tested samples at both genus and species level was observed. However, two samples documented as Clinacanthus nutans and Clinacanthus siamensis were recognised as the same species from the HRM analysis. Further investigation reveals that C. siamensis is now accepted as C. nutans. Conclusions The results here proved that Bar-HRM is a promising technique in species identification of the studied medicinal plants in Acanthaceae. In addition, molecular biological data is currently used in plant taxonomy and increasingly popular in recent years. Here, DNA barcode sequence data should be incorporated with morphological characters in the species identification.

The bacterial diversity in rhizosphere soil of pear trees (Pyrus communis L. cv. Krystalli) from an orchard at Thessaly region of Greece was characterized employing amplicon-based metagenomics analysis. Pathogenic filamentous oomycetes of the genus Phytophthora comprises more than 150 recognized species and cause highly destructive soil-borne diseases in deciduous trees crops worldwide. Moreover, the treatment of soil microbiota with amino acids is an alternative strategy to achieve desirable effects even against phytopathogenic oomycetes. In our study, samples from rhizosphere soil were collected either from naturally Phytophthora-infected trees, from completely asymptomatic ones, or from trees as above subjected also to treatments with amino acids (Amino16®) under different fertilization regimes. The interactions of bacterial communities with plant pathogenic oomycetes are crucial to determine the course of infection and the pathogenicity encompassing various functional contexts like biofilm formation. Thus, for deciphering the structure and diversity of these soil bacterial communities, we applied a 16S rRNA Illumina sequencing approach targeting the V3-V4 gene region. After quality check 478,479 sequences were obtained in the dataset comprising a total read length of 192,291,625 base pairs. Proteobacteria were the dominant phylum (46.1%) followed by Acidobacteria (13.2%) and Actinobacteria (12.4%). Different distributions of phyla were observed among our samples which is indicative of various alterations of soil bacterial communities in rhizosphere. The metagenome data from this survey are available at NCBI Sequence Read Archive (SRA) database and Biosample under accession number PRJNA542725. Keywords: Deep amplicon sequencing, Metagenomics, 16S bacteriome communities, Phytophthora, Oomycetes

Background Korean ginseng has long been famous and is one of the most well known forms of ginseng. The root of plants in the genus Panax is commonly recognized as ginseng. Different Panax species of ginseng root have been used as treatments. Although many other herbs are called ginseng, they do not contain the active compounds of ginsenosides. In Thailand, we have Thai ginseng which is of course not one of Panax species. Thai ginseng is the root from Talinum paniculatum and, due to its morphological root similarity, it is almost impossible to differentiate between them. Also, another plant species, Phytollacca americana, has significantly similar root morphology to real ginseng but its seeds and root are poisonous. Misunderstanding what true ginseng is compared to others could endanger lives and cause financial loss by buying inferior products. Methods DNA barcoding combination with High Resolution Melting (called Bar-HRM) was used for species discrimination of the Panax ginseng and others. Five regions included ITS2, matK, psbA-trnH and rbcL were evaluated in the analyses. Results The ITS2 region was found to be the most suitable primers for the analysis. The melting profile from the HRM analyses using the chosen ITS2 primers showed that Korean ginseng (Panax ginseng) could be discriminated from other Penax species. Also, other ginseng species with morphological similarity could be easily distinguished from the true ginseng. The developed Bar-HRM method poses a great potential in ginseng species discrimination and thus could be also useful in ginseng authentication.

Chloroplasts are organelles subjected to extreme oxidative stress conditions. Biomolecules produced in the chloroplasts act as signals guiding plant metabolism toward stress tolerance and play a major role in regulating gene expression in the nucleus. Herein, we used transplastomic plants as an alternative approach to expression of transgenes in the nucleus for conferring stress tolerance to abiotic stresses and herbicides. To investigate the morphophysiological and molecular mechanisms and the role of plastid expressed GSTs in tobacco stress detoxification and stress tolerance, we used transplastomic tobacco lines overexpressing a theta class glutathione transferase (GST) in chloroplasts. The transplastomic plants were tested under drought (0, 100, and 200 mM mannitol) and salinity (0, 150, and 300 mM NaCl) in vitro, and under herbicide stress (Diquat). Our results suggest that ptAtGSTT lines were tolerant to herbicide-induced oxidative and salinity stresses and showed enhanced response tolerance to mannitol-induced osmotic stress compared to WT plants. Overexpression of the Arabidopsis thaliana AtGSTT in the chloroplasts resulted in enhanced photo-tolerance and turgor maintenance under stress. Whole-genome transcriptome analysis revealed that genes related to stress tolerance, were upregulated in ptAtGSTT2a line under both control and high mannitol stress conditions. Transplastomic plants overexpressing the ptAtGSTT2a in the chloroplast showed a state of acclimation to stress, as only limited number of genes were upregulated in the ptAtGSTT2a transplastomic line compared to WT under stress conditions while at the same time genes related to stress tolerance were upregulated in ptAtGSTT2a plants compared to WT in stress-free conditions. In parallel, the metabolic profile indicated limited perturbations of the metabolic homeostasis in the transplastomic lines and greater accumulation of mannitol, and soluble sugars under high mannitol stress. Therefore, transplastomic lines seem to be in a state of acclimation to stress under stress-free conditions, which was maintained even under high mannitol stress. The results help to elucidate the role of GSTs in plant abiotic stress tolerance and the underlying mechanisms of the GSTs expressed in the chloroplast, toward environmental resilience of cultivated crops.

Native tomato landrace varieties (Solanum lycopersicum L.) are sources of high genetic diversity and are adaptable to local environmental conditions. The in vitro propagation system can be used as a tool to produce disease-free, high-quality propagation material and preserve the unique characteristics of commercial Greek tomato varieties, such as the variety Areti and the traditional landrace variety Makedonia, both of exceptional quality and economic importance. Twenty- and 27-day-old cotyledon and leaf explants were cultivated in 4 regeneration media supplemented with 0 or 0.1 mg∙L−1 indole-3-acetic acid and 0.5 or 1 mg∙L−1 zeatin. Regeneration efficiency was dependent on variety, explant type and age, and regeneration media. Areti was a more appropriate target genetic material, exhibiting a six-fold greater response to regeneration compared to Makedonia, regardless of media and explant age. The regenerated shoots of both varieties were successfully rooted (60%) and acclimatized (98%). This regeneration protocol would be valuable in the production of propagation material for commercial and conservation practices and in breeding programs for genetic improvement.

Pomegranate is one of the oldest known fruit crops, well adapted to hot and dry areas and successfully grown in Mediterranean countries. During the last two decades, numerous publications have revealed the traditionally known associations between pomegranate consumption and health benefits, which led to increased demand by consumers and expansion in cultivation areas. Pomegranate is well adapted to areas with diverse pedoclimatic conditions and local cultivars could provide an essential source of genes for breeding. In this study, fruit phenotypic and genetic variability, and relationships, were studied in 26 Greek and foreign pomegranate cultivars/accessions grown in an ex situ collection located in Naoussa Greece, using Inter Simple Sequence Repeat (ISSR) and Start Codon Targeted (SCoT) molecular markers. Results from the principal component analysis made on fruit phenotypic characters revealed five components that accounted for 74.8% of the total variance, the first being related to skin color parameters and the second to juice antioxidant contents and aril color. Clustering from phenotypic data allocated individuals into four clusters. A total of 184 bands were generated for all markers applied across the 26 pomegranate cultivars/accessions, with an average of 77 bands per ISSR markers and 82 bands for SCoT markers. Low variability in the phenotypic and genotypic level was indicated; nevertheless, results from the association study between phenotypic traits and molecular markers that were obtained using Multiple Regression Analysis (MRA) could prove valuable for marker-assisted breeding programs, especially from SCoT markers that were found to be strongly or averagely associated with the morphological traits and chemical components.

A selection of sesame (Sesamum indicum L.) landraces of different eco-geographical origin and breeding history have been characterized using 28 qualitative morpho-physiological descriptors and seven expressed sequence tag-simple sequence repeat (EST-SSR) markers coupled with a high-resolution melting (HRM) analysis. The most variable qualitative traits that could efficiently discriminate landraces, as revealed by the correlation analyses, were the plant growth type and position of the branches, leaf blade width, stem pubescence, flowering initiation, capsule traits and seed coat texture. The agglomerative hierarchical clustering analysis based on a dissimilarity matrix highlighted three main groups among the sesame landraces. An EST-SSR marker analysis revealed an average polymorphism information content (PIC) value of 0.82, which indicated that the selected markers were highly polymorphic. A principal coordinate analysis and dendrogram reconstruction based on the molecular data classified the sesame genotypes into four major clades. Both the morpho-physiological and molecular analyses showed that landraces from the same geographical origin were not always grouped in the same cluster, forming heterotic groups; however, clustering patterns were observed for the Greek landraces. The selective breeding of such traits could be employed to unlock the bottleneck of local phenotypic diversity and create new cultivars with desirable traits.

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. “Plake Megalosperma Prespon” is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3–3 and Pvgstu2–2 genes. The overexpression of Pvgstu3–3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3–3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.

A new molecular method via the high resolution melting (HRM) analysis of the Ypt1 gene non-coding regions was validated for ten Phytophthora species with a broad host range from forest trees to crop species. The melting curve analysis of the amplicons specifically grouped all species into 10 respective unique and distinct HRM curve profiles. The analysis of the normalised HRM melting curves, assigning P. nicotianae as a normalised reference genotype, revealed that the genotype similarities among all the species were adequately low, indicating that Ypt1 marker was sufficient to identify and differentiate the tested species. This HRM method is rapid and reproducible allowing the identification of Phytophthora species and the screening of eventual variants eliminating the separate steps and reducing the risk of contamination.

Pea (P. sativum L.), one of the most important legume crops worldwide, has been traditionally cultivated in Lesser Cyclades since ancient times. The commonly known traditional pea cultivar, ‘Katsouni’, is endemic to the islands of Amorgos and Schinoussa and is of great local economic importance. Despite the widespread cultivation of ‘Katsouni’ in both islands, it is still unknown whether the current Schinoussa and Amorgos pea populations are distinct landraces, and if they have common evolutionary origin. To assist conservation and breeding of the pea crop, the genetic diversity and phylogenetic relationships of 39 pea samples from Amorgos and 86 from Schinoussa were studied using DNA barcoding and ISSR marker analyses. The results indicate that both populations are different landraces with distinct geographical distribution and are more closely related to P. sativum subsp. elatius than the P. abyssinicum and P. fulvum species. Further characterization of the ‘Katsouni’ landraces for functional polymorphisms regarding pathogen resistance, revealed susceptibility to the powdery mildew (Erysiphe pisi DC.). This work represents the first investigation on the genetic diversity and population structure of the ‘Katsouni’ cultivar. Exploiting the local genetic diversity of traditional landraces is fundamental for conservation practices and crop improvement through breeding strategies.

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology.

Grafting has been used to improve yield, fruit quality and disease resistance in a range of tree and vegetable species. The molecular mechanisms underpinning grafting responses have only recently started to be delineated. One of those mechanisms involves long distance transfer of genetic material from rootstock to scion alluding to an epigenetic component to the grafting process. In the research presented herein we extended published work on heritable changes in the DNA methylation pattern of Solanaceae scion genomes, in Cucurbitaceae inter-species grafting. Specifically, we examined global DNA methylation changes in scions of cucumber, melon and watermelon heterografted onto pumpkin rootstocks using MSAP analysis. We observed a significant increase of global DNA methylation in cucumber and melon scions pointing to an epigenetic effect in Cucurbitaceae heterografting. Exploitation of differential epigenetic marking in different rootstock-scion combinations could lead to development of epi-molecular markers for generation and selection of superior quality grafted vegetables.

It is long believed that some spices may help protect against certain chronic conditions. Spices are usually parts of plants that have been powdered into small pieces. Have you ever wondered what the curry powder in your dish is made of? The aim of this work was to develop an appropriate DNA-based method for assessment of spice identity. Selecting the best marker for species recognition in the Zingiberaceae family. Six DNA regions were investigated in silico, including ITS, matK, rbcL, rpoC, trnH-psbA and trnL. Then, only four regions (ITS, matK, rbcL and trnH-psbA) were included in the simulated HRM (High-resolution Melting) analysis as the results from previous analysis showed that rpoC and trnL may not be suitable to be used to identify Zingiberaceae species in HRM analysis based on both the percentage of nucleotide variation and GC content. Simulated HRM analysis was performed to test the feasibility of Bar-HRM. We found that ITS2 is the most effective region to be used for identification of the studied species and thus was used in laboratory HRM analysis. All seven tested Zingiberaceae plants were then able to be distinguished using the ITS2 primers in laboratory HRM. Most importantly the melting curves gained from fresh and dried tissue overlapped, which is a crucial outcome for the applicability of the analysis. The method could be used in an authentication test for dried products. In the authentication test, only one of seven store-sold Zingiberaceae products that were tested contained the species listed on their labels, while we found substitution/contamination of the tested purchased products in the rest.

Livestock production in the European Union EU is highly dependent on imported soybean, exposing the livestock farming system to risks related to the global trade of soybean. Lupin species could be a realistic sustainable alternative source of protein for animal feeding. Lupinus is a very diverse genus with many species. However, only four of them—namely, L. albus, L. angustifolius, L. luteus and L. mutabilis—are cultivated. Their use in livestock farming systems has many advantages in relation to economic and environmental impact. Generally, lupin grains are characterized by high protein content, while their oil content is relatively low but of high quality. On the other hand, the presence of quinolizidine alkaloids and their specific carbohydrate composition are the main antinutritional factors that prevent their use in animal feeding. This research is mainly related to L. albus and to L. angustifolius, and to a lesser extent, to L. lauteus and L. mutabilis. The breeding efforts are mostly focused on yield stabilization, resistance to biotic and abiotic stresses, biochemical structure associated with seed quality and late maturing. Progress is made in improving lupin with respect to the seed quality, as well as the tolerance to biotic and abiotic stress. It has to be noted that modern cultivars, mostly of L. albus and L. angustifolius, contain low levels of alkaloids. However, for future breeding efforts, the implementation of marker-assisted selection and the available genomic tools is of great importance.

Botrytis cinerea, is a high-risk pathogen for fungicide resistance development. Pathogen` resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant`s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the 4 mutations located at codon 225, the N230I mutation, the 3 mutations located in codon 272 and the non mutated isolates (isolates of wild type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of antiresistance strategies implemented in crops heavily treated with botryticides.

Plant selection, input, and field management are proven strategies that produce high yields of crops bearing selected desirable characteristics for the nutraceutical and cosmeceutical industry. This study reports on the effect of substrate and light on selected quantitative and qualitative bioactive properties of two cultivars of Rubus idaeus L (‘Ruvi’ and ‘Cayuga’). Our results demonstrated that the quantitative and qualitative fruit characteristics (yield, fruit dimensions, titratable acidity, and total soluble solids contents), plant growth, biosynthesis of secondary metabolites, and total antioxidant capacity, are significantly affected by genotype, light intensity, and substrate type. Fruits from ‘Ruvi’ plants cultivated under low light conditions, on soil/peat substrate exhibited high levels of antioxidant capacity, phenolics, flavonoids, anthocyanins, and high inhibitory potency towards the skin-regulating enzymes tyrosinase and elastase. Extract derived from these fruits was formulated into a topical skin care cream. This cream exhibited excellent compatibility and stability characteristics. Our research concluded that quantity and quality of Rubus idaeus L. fruits could be efficiently managed through conventional agronomic practices. Our project determined the optimal agronomic management practices to produce desirable characteristics and maximize bioactive content that determine the nutraceutical and cosmeceutical quality of the red raspberry.

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.