Mechanisms of Lolium rigidum multiple resistance to ALS- and ACCase-inhibiting herbicides and their impact on plant fitness.

Three putative resistant (R1, R2, R3) and one susceptible (S) Lolium rigidum populations originating from Greece were studied for resistance to ALS and ACCase inhibiting herbicides, using whole plant, sequencing of als and accase gene, and in vitro ALS activity assays. The S and two R (R1, R2) populations were also evaluated for fitness in competition with wheat. The whole plant assay indicated unsatisfactory control of the R populations with mesosulfuron-methyl + iodosulfuron-methyl or pinoxaden application, whereas sequencing of the als gene revealed that all ALS-resistant individuals had a Pro-197 substitution by Leu, Glu, Ser, Ala, Thr, or Gln. In addition, the accase gene of all pinoxaden resistant individuals had an Ile-2041 substitution by Asn or Thr. Furthermore, sequencing of the individuals surviving mesosulfuron-methyl + iodosulfuron-methyl or pinoxaden treatment revealed co-existence of point mutations in the accase or als genes, respectively, demonstrating multiple resistance. The in vitro activity of the ALS enzyme confirmed that resistance to mesosulfuron-methyl + iodosulfuron-methyl was due altered target-site. The recorded higher vigor and greater competitive ability of S population against wheat as compared with that of the R populations suggests an associated fitness cost with multiple resistance.

HRM analysis as a tool to facilitate identification of bacteria from mussels during storage at 4 °C.

High-resolution melting (HRM) analysis followed by sequencing was applied for determination of bacteria grown on plates isolated from farmed mussels (Mytilus galloprovincialis) during their storage at 4 °C. The V3-V4 region of the 16S rRNA gene from the isolates was amplified using 16S universal primers. Melting curves (peaks) and high resolution melting curves (shape) of the amplicons and sequencing analysis were used for differentiation and identification of the isolated bacteria, respectively. The majority of the isolates (a sum of 101 colonies, from five time intervals: day 0, 2, 4, 6 and 8) from non-selective solid medium plates were classified in four bacterial groups based on the melting curves (peaks) and HRM curves (shape) of the amplicons, while three isolates presented distinct HRM curve profiles (single). Afterwards, sequencing analysis showed that the isolates with a) the same melting peak temperature and b) HRM curves that were >95% similar grouped into the same bacterial species. Therefore, based on this methodology, the cultivable microbial population of chill-stored mussels was initially dominated by Psychrobacter alimentarius against others, such as Psychrobacter pulmonis, Psychrobacter celer and Klebsiella pneumoniae. P. alimentarius was also the dominant microorganism at the time of the sensory rejection (day 8). Concluding, HRM analysis could be used as a useful tool for the rapid differentiation of the bacteria isolated from mussels during storage, at species level, and then identification is feasible by the sequencing of one only representative of each bacterial species, thus reducing the cost of required sequencing.

Resistance of Rapistrum rugosum to tribenuron and imazamox due to Trp574 or Pro197 substitution in the acetolactate synthase.

Ten putative resistant and two susceptible Rapistrum rugosum populations originating from Greece were studied for resistance to acetolactate synthase (ALS)-inhibiting herbicides, using dose-response assays, sequencing of als gene and in vitro ALS activity assays. The dose-response assays showed that one (P1) out of ten putative resistant populations was cross-resistant to tribenuron and imazamox, while another population (P4) was resistant to tribenuron only. All populations were susceptible to MCPA at the recommended rate. Gene sequencing of als revealed that the P4 population had a point mutation at Pro197 by His providing resistance to tribenuron, whereas the P1 had a Trp574 by Leu point mutation conferring cross-resistance to tribenuron and imazamox. The in vitro activity of the ALS enzyme indicated I50 values (tribenuron concentration required for 50% reduction of the ALS activity) ranging from 66.68 to 137.01 µM, whereas the respective value for the S populations ranged from 0.29 to 0.54 µM. These results strongly support that two R. rugosum populations evolved resistance to ALS-inhibiting herbicides due different point mutations in the als gene.

Trp574 substitution in the acetolactate synthase of Sinapis arvensis confers cross-resistance to tribenuron and imazamox.

Rate-response experiments with nine putative resistant wild mustard (Sinapis arvensis) populations from Greece showed cross-resistance to tribenuron and imazamox. The calculated GR50 values [herbicide rate (gaiha-1) required for 50% reduction of fresh weight] of the nine resistant (R) populations ranged from 51.8 to 555.6gaitribenuronha-1 and from 66.3 to 900.4gaiimazamoxha-1. Regarding the susceptible population, GR50 value was not estimated for tribenuron as its lower treatment reduced fresh weight by >95%, whereas the respective value for imazamox was 0.5gaiha-1. Gene sequencing of als revealed that a point mutation at Trp574 position, leading to amino acid substitution by Leu in the ALS enzyme was present and the likely cause of resistance. The in vitro activity of the ALS enzyme indicated I50 values (herbicide concentration required for 50% reduction of the ALS activity) ranging from 19.11 to 217.45µM for tribenuron, whereas the respective value for the S population was 1.17µM. All populations were susceptible to MCPA at the recommended rate. These results strongly support that cross-resistance of 9 S. arvensis populations was due a point mutation of the als gene, which resulted in a less sensitive ALS enzyme.

Greek PDO saffron authentication studies using species specific molecular markers.

Saffron, the spice produced from the red stigmas of the flower of Crocus sativus L. is a frequent target of fraud and mislabeling practices that cannot be fully traced using the ISO 3632 trade standard specifications and test methods. A molecular approach is proposed herein as a promising branding strategy for the authentication of highly esteemed saffron brands such as the Greek Protected Designation of Origin (PDO) "Krokos Kozanis". Specific ISSR (inter-simple sequence repeat) markers were used to assess for the first time, the within species variability of several populations of C. sativus L. from the cultivation area of "Krokos Kozanis" as well as the potential differences with the band pattern produced by other Crocus species. Then, species-specific markers were developed taking advantage of an advanced molecular technique such as the HRM analysis coupled with universal DNA barcoding regions (trnL) (Bar-HRM) and applied to saffron admixtures with some of the most common plant adulterants (Calendula officinalis, Carthamus tinctorius, Gardenia jasminoides, Zea mays and Curcuma longa). The sensitivity of the procedure was tested for turmeric as a case study whereas HPLC-fluorescence determination of secondary metabolites was also employed for comparison. The overall results indicated that the Bar-HRM approach is quite effective in terms of specificity and sensitivity. Its effectiveness regarding the detection of turmeric was comparable to that of a conventional HPLC method (0.5% vs 1.0%, w/w). Yet, the proposed DNA-based method is much faster, cost-effective and can be used even by non-geneticists, in any laboratory having access to an HRM-capable real-time PCR instrumentation. It can be, thus, regarded as a strong analytical tool in saffron authentication studies.

Identification of Uvaria sp by barcoding coupled with high-resolution melting analysis (Bar-HRM).

DNA barcoding, which was developed about a decade ago, relies on short, standardized regions of the genome to identify plant and animal species. This method can be used to not only identify known species but also to discover novel ones. Numerous sequences are stored in online databases worldwide. One of the ways to save cost and time (by omitting the sequencing step) in species identification is to use available barcode data to design optimized primers for further analysis, such as high-resolution melting analysis (HRM). This study aimed to determine the effectiveness of the hybrid method Bar-HRM (DNA barcoding combined with HRM) to identify species that share similar external morphological features, rather than conduct traditional taxonomic identification that require major parts (leaf, flower, fruit) of the specimens. The specimens used for testing were those, which could not be identified at the species level and could either be Uvaria longipes or Uvaria wrayias, indicated by morphological identification. Primer pairs derived from chloroplast regions (matK, psbA-trnH, rbcL, and trnL) were used in the Bar-HRM. The results obtained from psbA-trnH primers were good enough to help in identifying the specimen while the rest were not. Bar-HRM analysis was proven to be a fast and cost-effective method for plant species identification.

Rapid discrimination between four seagrass species using hybrid analysis.

Biological species are traditionally identified based on their morphological features and the correct identification of species is critical in biological studies. However, some plant types, such as seagrass, are taxonomically problematic and difficult to identify. Furthermore, closely related seagrass species, such as Halophila spp, form a taxonomically unresolved complex. Although some seagrass taxa are easy to recognize, most species are difficult to identify without skilled taxonomic or molecular techniques. Barcoding coupled with High Resolution Melting analysis (BAR-HRM) offers a potentially reliable, rapid, and cost-effective method to confirm species. Here, DNA information of two chloroplast loci was used in combination with HRM analysis to discriminate four species of seagrass collected off the southern coast of Thailand. A distinct melting curve presenting one inflection point was generated for each species using rbcL primers. While the melting profiles of Cymodocea rotundata and Cymodocea serrulata were not statistically different, analysis of the normalized HRM curves produced with the rpoC primers allowed for their discrimination. The Bar-HRM technique showed promise in discriminating seagrass species and with further adaptations and improvements, could make for an effective and power tool for confirming seagrass species.

Genetic diversity and structure of natural Dactylis glomerata L. populations revealed by morphological and microsatellite-based (SSR/ISSR) markers.

Dactylis glomerata L. is an important forage species in the Mediterranean region, and in other regions with a similar climate. Genetic material from 3 locations in north, central, and south Greece was studied, using morphological traits, SSR, and ISSR molecular markers. Morphological analysis revealed differences among the geographic locations studied for all morphological traits, except the number of reproductive tillers. Moreover, the highest phenotypic variation was observed on the accessions from south, while the lowest was observed on the accessions from the north. Although the results of the molecular marker analysis are indicative, a high level of genetic diversity at the species level was revealed by ISSRs (GST=0.291) and SSRs (FST=0.186). Analysis of molecular variance showed that a high level of genetic diversity existed for ISSRs and SSRs within populations (62 and 83%, respectively), rather than among populations (38 and 17%, respectively). Cluster analysis divided the 3 populations in 2 groups, with the population originating from the island of Crete forming 1 group, while the populations from north Greece (Taxiarchis) and central Greece (Pertouli) were clustered in a 2nd group. In general, the results indicate that SSRs are more informative compared to ISSRs about the genetic variation within a population, whereas the ISSRs were more informative about the genetic diversity among populations However, a similar trend in diversity (genotypic and phenotypic) was observed in the morphological traits and microsatellite-based (SSR/ISSR) markers at the locations studied.

Barcoding the major Mediterranean leguminous crops by combining universal chloroplast and nuclear DNA sequence targets.

The ability to discriminate all species is the ultimate target in barcoding. The Mediterranean basin is a center of origin for legumes and thus they have played a key role in feeding the Mediterranean population. It is also a region with important protected designation of origin and protected geographical indication legumes that provide income in rural areas. We evaluated the use of two chloroplast regions, trnL and rpoC1, and a nuclear internal transcriber region, ITS2, for their efficiency to barcode the main Mediterranean leguminous crops. Twenty-five legume species were studied. Plant material of pasture and legumes was obtained from the Greek GenBank and the Fodder Crops and Pastures Institute (National Agricultural Research Foundation). DNA was extracted with the Qiagen DNeasy plant mini-kit and PCR amplification was performed using the Kapa Taq DNA polymerase using primers amplifying the chloroplast trnL and rpoC1 regions or the nuclear region ITS2. PCR products were sequenced and the sequences were aligned using CLUSTAL W. Species identification based on the sequence similarity approach was performed using the GenBank database. In order to evaluate intraspecific and interspecific divergence in legumes we used Molecular Evolutionary Genetics Analysis 5 and for pairwise Kimura 2-parameter distance calculations for all 3 DNA regions (2 chloroplast regions, trnL and rpoC1, and the nuclear region ITS2). Four tree-based methods (neighbor joining and maximum parsimony, maximum likelihood, and Bayesian inference analyses) were used to exhibit the molecular identification results to represent differences as an uprooted dendrogram. Additionally, the sequence character-based method was used with DnaSP and the information from each site was treated as a character to distinguish the species from one another. The DNA regions trnL and ITS2 successfully (100%) discriminated the Mediterranean crop legume species used, while rpoC1 identified only 72% of them. Furthermore, the use of the trnL region enabled the discrimination of even very closely related species, like Phaseolus lunatus and P. coccineus or Vicia faba subsp major with V. faba subsp minor, which are so closely related that even in NCBI they were both referred as Phaseolus vulgaris and V. faba, respectively. We conclude that trnL and ITS2 are efficient DNA barcoding target regions in order to discriminate Mediterranean leguminous crops and provide a reliable and efficient tool for the scientific, agricultural and industrial community.

A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera.

Hepatitis C virus (HCV) is a major disease agent affecting approximately 3% of the world's population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa precore protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome binding sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on Western blots providing a first step towards developing a chloroplast-based HCV diagnostic method.