DNA Barcoding and Fertilization Strategies in Sideritis syriaca subsp. syriaca, a Local Endemic Plant of Crete with High Medicinal Value.

Herein, we applied DNA barcoding for the genetic characterization of Sideritis syriaca subsp. syriaca (Lamiaceae; threatened local Cretan endemic plant) using seven molecular markers of cpDNA. Five fertilization schemes were evaluated comparatively in a pilot cultivation in Crete. Conventional inorganic fertilizers (ChFs), integrated nutrient management (INM) fertilizers, and two biostimulants were utilized (foliar and soil application). Plant growth, leaf chlorophyll fluorescence, and color were assessed and leaf content of chlorophyll, key antioxidants (carotenoids, flavonoids, phenols), and nutrients were evaluated. Fertilization schemes induced distinct differences in leaf shape, altering quality characteristics. INM-foliar and ChF-soil application promoted yield, without affecting tissue water content or biomass partitioning to inflorescences. ChF-foliar application was the most stimulatory treatment when the primary target was enhanced antioxidant contents while INM-biostimulant was the least effective one. However, when the primary target is yield, INM, especially by foliar application, and ChF, by soil application, ought to be employed. New DNA sequence datasets for the plastid regions of petB/petD, rpoC1, psbK-psbI, and atpF/atpH were deposited in the GenBank for S. syriaca subsp. syriaca while the molecular markers rbcL, trnL/trnF, and psbA/trnH were compared to those of another 15 Sideritis species retrieved from the GenBank, constructing a phylogenetic tree to show their genetic relatedness.

Molecular detection of bacteria in plant tissues, using universal 16S ribosomal DNA degenerated primers.

Highly specific, sensitive and rapid tests are required for the detection and identification of covert bacterial contaminations in plant tissue cultures. Current methods available for this purpose are tedious, time consuming, highly error prone, expensive, require advanced technical expertise and are sometimes ineffective. We report here the development of a sensitive polymerase chain reaction (PCR) based method for the rapid detection and identification of bacteria occurring in plant tissue cultures. A total of 121 16S ribosomal DNA (rDNA) coding regions from 14 different groups of bacteria, algae and plants, available in the Gene Bank/European Molecular Biology Laboratory databases, were aligned and several conserved DNA sequences of bacterial origin were identified. From those, five degenerated primers were designed in order to amplify only the bacterial DNA present in mixed plant/bacteria genomic DNA extracts. A known amount of bacterial suspension of either covert Pseudomonas or covert Bacillus were added to in vitro plant leaves and total plant/bacterial DNA extracted using three different methods to determine the lowest number of bacteria required to be present in order to allow their detection. The highest sensitivity of the bacterial cell detection was 2.5 × 106 cells of both Bacillus and Pseudomonas inoculums, using template DNA prepared by the MiniPrep method. Generation of PCR amplification fragments was achieved only for the 16S rDNA bacterial gene by using four combinations of degenerated primers. Successive sequence analysis of these amplified fragments led to the rapid detection and molecular identification of bacteria covertly associated with plants.

Adaptability of Wild-Growing Tulips of Greece: Uncovering Relationships between Soil Properties, Rhizosphere Fungal Morphotypes and Nutrient Content Profiles.

Wild-growing Greek tulips are protected plants but almost nothing is known about their natural nutrient status and rhizosphere fungal morphotypes in the wild, thus no insight is currently available into their growth and adaptation to their natural environment or artificial settings. To this end, several botanical expeditions were conducted with a special collection permit, and 34 tulip and soil samples were collected, representing 13 species from two phytogeographical regions of Greece (North Aegean Islands, Crete Island) and seven regions of mainland Greece. The tulips' content in essential macro- and micro-nutrients, respective physicochemical soil properties, and rhizosphere fungal morphotypes were assessed across samples, and all parameters were subjected to appropriate statistical analysis to determine their interrelationships. The results showed that soil variables played a significant role in shaping tulips' nutrient content, explaining up to 67% of the detected variability as in the case of phosphorus (P) in the above-ground plant tissue. In addition, significant correlations were observed (with an r value of up to 0.65, p < 0.001) between essential nutrients in the tulips, such as calcium (Ca) and boron (B). The principal component analysis (PCA) revealed that between the three spatial units examined, the total variability of tulips' nutrient content produced a clear distinction among sampled species, while the first two PCA axes managed to explain 44.3% of it. This was further confirmed by the analysis of variance (ANOVA) results which showed corresponding significant differences (at p < 0.05) in both the tulips' nutrient content and the studied soil properties as well (mean values of N, P, and K in the North Aegean Islands tulips' nutrient content, up to 53%, 119%, and 54% higher compared to those of the Crete Island, respectively). Our study sheds light on Greek tulips' adaptability and resilience in their original habitats, facilitating at the same time the undertaken efforts regarding their conservation and potential domestication in artificial settings.

Effect of Temperature on Breaking of Morphophysiological Dormancy and Seed Germination Leading to Bulblet Production in Two Endemic Tulip Species from Greece.

Due to botanical tulips' economic interest coupled with limited information regarding their seed germination, we investigated the effect of temperature on dormancy release and germination in two endangered local endemic tulip species of Greece (Tulipa hageri Heldr., T. orphanidea Heldr.). Their germination responses at five constant temperatures (5, 10, 15, 20, and 25 °C) were evaluated in growth chambers, while the type of seed dormancy and the temperature effect on its release were determined based on open-sourced, R-derived species-specific ecological profiles illustrating abiotic conditions of their wild habitats. The results indicated a range-specific temperature dependence in seed germination for both studied species with seed germination observed only in very low temperatures (5-10 °C). The seeds of both species after dispersal had an underdeveloped embryo. The existence of a complex morphophysiological seed dormancy was confirmed in both species by the significant embryo development only at 5 and 10 °C (almost doubled after 30 days) coupled with observed delay in germination only at low temperatures. Furthermore, to facilitate their cultivation and ex situ conservation, the germinated seeds were planted in pots to develop bulblets in greenhouse conditions resulting in bigger T. orphanidea bulblets compared to T. hageri.

Influence of Temperature on Seed Germination of Five Wild-Growing Tulipa Species of Greece Associated with Their Ecological Profiles: Implications for Conservation and Cultivation.

Although tulips are famous worldwide as ornamental plants, the knowledge about the seed germination of wild-growing species remains limited. The aim of the present study was to investigate the effect of temperature on seed germination of the local, wild-growing Greek endemics Tulipa bakeri and T. goulimyi and the sub-Balkan endemic T. undulatifolia, which are threatened with extinction, as well as the Mediterranean T. australis and the Asiatic T. clusiana naturalized on Chios Island (Greece). The germination responses at five constant temperatures (5, 10, 15, 20, and 25 °C) were assessed for all studied species in growth chambers under a 12:12 light-dark photoperiod. The ecological profile for each species was developed in R using open-source bioclimatic data; this was built to illustrate the abiotic environmental conditions of their wild habitats, to facilitate the examination of temperature effects on seed germination, and to facilitate their cultivation in artificial environments. The results indicated that the seed germination requirements of the studied species had a range-specific temperature dependence, reflecting their natural adaptation to local ecological conditions. Seed germination of T. bakeri, T. australis, and T. clusiana was observed only in a narrow range of very low temperatures (5-10 °C), whereas germination of T. undulatifolia and T. goulimyi occurred at temperatures between 5 and 15 °C. A temperature increase to 20 or 25 °C resulted in the absence of seed germination for all five Greek tulip species. The germinated seeds were planted in pots and bulblets were developed under greenhouse conditions. Seeds and bulblets constitute valuable genetic materials for the cultivation and ex situ conservation of these five Greek tulip species, three of which are threatened with extinction.

DNA Barcoding, GIS-Facilitated Seed Germination and Pilot Cultivation of Teucrium luteum subsp. gabesianum (Lamiaceae), a Tunisian Local Endemic with Potential Medicinal and Ornamental Value.

In the context of plant conservation and sustainable use of unique neglected and underutilized phytogenetic resources, this study focused on the Tunisian local endemic Teucrium luteum subsp. gabesianum (Lamiaceae). Using Geographical Information Systems and online databases, detailed taxon-specific ecological profiling was produced for the first time, which illustrated the temperature and climate conditions in its wild habitats and facilitated the investigation of how temperature affects its seed germination, thus making its cultivation in anthropogenic environments possible. Following the seed propagation first reported herein (77.5−81.25% at temperatures between 15 and 25 °C), species-specific in situ and ex situ conservation efforts or sustainable exploitation strategies can be enabled. This study also reported for the first time how chemical and integrated nutrient management (INM) fertilizers affect the growth and pilot cultivation of its seedlings (INM more advantageous). The firstly-reported herein DNA barcoding may enable its traceability, allowing future product design. The multidisciplinary approach followed has paved the way to bridge important research gaps hindering conservation efforts and/or the sustainable exploitation of this local Tunisian endemic plant to date. Based on the aforementioned results, the feasibility and readiness timescale for its sustainable exploitation was overviewed and re-evaluated herein, upgrading (>two-fold) its potential value for the medicinal-cosmetic, agro-alimentary, and ornamental-horticultural sectors.

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study.

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.

Greek Tulips: Worldwide Electronic Trade over the Internet, Global Ex Situ Conservation and Current Sustainable Exploitation Challenges.

From an ornamental viewpoint, tulips are famous clonally propagated crops. This research focuses on 15 wild-growing Greek tulip species including 11 range-restricted species, i.e., six Greek endemics and five Balkan or Aegean endemics and subendemics, among which seven are currently threatened with extinction (two Critically Endangered, three Endangered and two Vulnerable). An overview of the global electronic trade over the internet is presented herein for these valuable phytogenetic resources in an attempt to define the extent of their commercialization (25 nurseries in three countries, mainly bulb trade at various prices) with concomitant conservation implications. In the frame of the repatriation initiatives launched, their global ex situ conservation is overviewed according to the PlantSearch facility of the Botanic Gardens Conservation International (materials from 15 species stored in 41 botanic gardens of 14 countries). The results of this study on the Greek tulips showed that there are both well-established value chains and gaps in the market regarding the "botanical tulips"; revealed the compromised effectiveness of ex situ conservation for the majority of them; raised conservation concerns related to authorized access to these wild phytogenetic resources; and indicated that their future utilization should comply with the provision of national and international legislation. All these are envisaged and discussed within the framework of the newly launched research project TULIPS.GR which aims to be the pilot establishment of a national collection regarding all Greek tulips (currently holding 38 accessions of 13 species, including almost all of the threatened ones). The project's scope is to enable the creation of a sustainable value chain for the Greek tulips with authorized collections, sustainable conservation schemes, production of DNA barcoded propagation material, species-specific propagation and cultivation protocols, mycorrhizal investigations, field studies, applying innovative precise soil/foliar fertigation, and investigation of the postharvest treatment of fresh cut flowers, promoting networking and synergies with producers and associations in Greece and abroad.

Medicinal-Cosmetic Potential of the Local Endemic Plants of Crete (Greece), Northern Morocco and Tunisia: Priorities for Conservation and Sustainable Exploitation of Neglected and Underutilized Phytogenetic Resources.

Medicinal-aromatic plants (MAPs) are important sources for the development of new valuable products of interest to human and animal health, and are also used as ornamentals for the horticulture industry. However, the increased global demand and the uncontrolled exploitation of these plants constitute a threat to their sustainability. To date, few scientific investigations have focused on MAPs valorization and their domestication. The purpose of this study was to evaluate for the first time the medicinal-cosmetic potential of 399 local endemic Mediterranean plants confined to Crete (223 taxa), the Mediterranean coast-Rif of Morocco (94), and Tunisia (82). The new methodological scheme was developed by experts through three multidisciplinary co-creative workshops and was adjusted by end-users to point-scoring of nine attributes evaluating the potential of the targeted neglected and underutilized plants (NUPs) in the medicinal-cosmetic sector. The results were demonstrated as percentage of the maximum possible score. These assessments were further linked and discussed with respect to feasibility and readiness timescale evaluations for sustainable exploitation of the focal NUPs. A great diversity of local endemic NUPs (30 taxa, 11 families) were associated with interesting medicinal-cosmetic properties (>35% up to 94.44%). Among them, 8 taxa showed the highest medicinal-cosmetic potential (>55% of maximum possible score), half of which are threatened with extinction. Although ex-situ conservation efforts and applied research work are needed to safeguard and unlock the full potential of the local endemic NUPs evaluated herein, the proposed multifaceted evaluation scheme revealed that some local endemic NUPs of the studied regions can be sustainably exploited in short- or medium-term, following successful examples of Cretan NUPs e.g., Origanum dictramnus. The sustainable exploitation of high scored taxa of the studied regions can be fastened through targeted species-specific research bridging extant research gaps and facilitating conservation and stakeholder attraction.

GIS-Facilitated Seed Germination and Multifaceted Evaluation of the Endangered Abies marocana Trab. (Pinaceae) Enabling Conservation and Sustainable Exploitation.

In the frame of the sustainable use of neglected and underutilized phytogenetic resources, and along with numerous studies in Abies spp. due to the innate conservation value of fir forests, this research focused on the Moroccan endemic fir, Abies marocana. The aim was triple-fold: to assess its potential and dynamics in economic sectors for sustainable exploitation; to determine the ecological conditions in which the species naturally thrives; and to find the appropriate requirements for its successful seed germination. We sourced multifaceted evaluations for three economic sectors performed in three levels, using 48 attributes and eight criteria from previous studies of our own, and the relevant species-specific assessments are overviewed herein in detail. The species' ecological profile was constructed using Geographical Information Systems (GIS) and open access data (Worldclim). Seed germination trials were performed to examine the effect of cold stratification (non-stratified, one- and two-months stratified seeds), the influence of four temperatures (10 °C, 15 °C, 20 °C, and 25 °C), and interactions thereof in relation to germination percentage (GP) and mean germination time (MGT). The experiments showed that the interaction of cold stratification and germination temperature has a strong effect on the GP and MGT of A. marocana seeds. A detailed GIS-derived ecological profile of the focal species was created in terms of precipitation and temperature natural regimes, enabling the interpretation of the seed germination results. The multifaceted evaluations reveal an interesting potential of the Moroccan fir in different economic sectors, which is mainly compromised due to extant research gaps, unfavorable conditions, and low stakeholder attraction. The findings of this study fill in extant research gaps, contribute to in situ and ex situ conservation strategies, and can facilitate the sustainable exploitation of this emblematic local endemic plant of northern Morocco.

GIS-Facilitated Effective Propagation Protocols of the Endangered Local Endemic of Crete Carlina diae (Rech. f.) Meusel and A. Kástner (Asteraceae): Serving Ex Situ Conservation Needs and Its Future Sustainable Utilization as an Ornamental.

Conservation and sustainable exploitation of threatened endemic plants with medicinal and/or horticultural/ornamental value can be achieved through the development of effective propagation protocols. After unveiling the bioclimatic preferences of Carlina diae (Asteraceae) with geographic information systems (GIS), four propagation trials were conducted using seeds of this endangered local Cretan endemic for in vivo and in vitro germination, as well as seasonal vegetative propagation trials (softwood cuttings) and micropropagation (nodal explants). Seed germination was accomplished at a level of 77-90% in vivo (30 days) and 96% in vitro (10 days) using an MS medium with 2.9 µM gibberellic acid (GA3). The optimum treatments for cuttings' rooting were 1000 and 2000 ppm indole-3-butyric acid (IBA) (11-16 roots, 2-3 cm long, 100% rooting) within 40 days in mist. In vitro shoot propagation exhibited a 2.8 proliferation rate after six successive subcultures on an MS medium with 2.9 µM GA3. Both ex vitro rooting and acclimatization were successful in 40 days, with 96% microshoot rooting and an equal survival rate. The GIS-facilitated effective species-specific propagation protocols developed in this study can consolidate the perspective of successful re-introduction of ex situ-raised material of C. diae into wild habitats and may serve its sustainable exploitation for high-added value ornamental products.

Adaptor long-range PCR procedure for clone-specific characterization and chromosomal localization.

An efficient adaptor long-range PCR (ALR-PCR) procedure was developed to detect genomic rearrangements in high-plasticity genomic regions between closely related strains of bacteria. The method was precisely optimized using a combination of high-speed experimental steps for the chromosomal localization and elucidation of deletions, inversions, duplications, or inserted sequences within a clone-specific flanking region. The advantages of this strategy are: (i) ready-to-use polymerase mixtures and Master mix (ready-to-use reaction mixtures with polymerase MasterAmp and buffer 2x Premix 4); (ii) a 5-min ligation procedure; (iii) rapid purification of DNA digests; (iv) optimized DNA template concentration protocol to avoid nonspecific amplification and high backgrounds; (v) long-range PCR protocol to obtain at least 9.6 kb single PCR products; (vi) two-step PCR cycling with the same annealing and extension temperature at 68 degrees C; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. The application of this method for a tester-specific suppressive subtractive hybridization (SSH) clone of Brucella melitensis 16M revealed an 837-bp deletion and a 7255-bp DNA transfer from one chromosomal location to another for Brucella abortus 2308 used as a driver.

Molecular characterization of Brucella abortus chromosome II recombination.

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.