Modern olive breeding points to a plant model characterized by low vigour, high productivity, and resistance to biotic and abiotic stresses, all traits required by the intensive and superhigh-density (SHD) systems of olive tree growing. The Italian Don Carlo and FS-17 Favolosa stand out among the new cultivars that are being tested. They were obtained not by breeding but by mass selection from two seedling populations of the Frantoio cultivar (maternal parent). Here, a multidisciplinary approach was used to determine the paternal parent of Don Carlo and FS-17, and then to investigate the inheritance of interesting traits such as fruit cell dimensions and oil content in these cultivars. Microsatellites were applied in phylogeny and kinship analyses, along with two functional markers previously developed on OeACP1 and OeACP2 genes. Ascolana Tenera cultivar was identified as the paternal parent of both new cultivars. This result was also supported by the analysis of the self-incompatibility group of the new cultivars and their most likely paternal parents. Light and electron microscopy [Cryo Scanning Electronic Microscopy (CRYO-SEM), Electronic Scanning Microscopy (E-SEM), and Transmission Electron Microscope (TEM)] techniques were used to analyze the fruit development concerning oil accumulation. Significant differences in cuticle thickness, size and shape of mesocarp and exocarp cells, and oil content were detected among cultivars. Our results suggested that the rearrangement of the traits studied led to an improved progeny compared to the parents. FS-17 exhibited an oil storage efficiency higher than Frantoio. Don Carlo showed fruit traits and oil content almost intermediate between the parents, making it a dual-purpose cultivar.

Bitter vetch (Vicia ervilia (L.) Willd.), one of the Near Eastern founder crops, is an annual cleistogamous legume domesticated during the Neolithic period. Originally used for human consumption, over time it was replaced by other pulses and downgraded to a fodder crop. When coupled with a small degree of cross hybridization, cleistogamy confers evolutive plasticity to the plant species. The aim of the present work consisted in setting up optimal conditions to overcome the existing cross hybridization barriers in V. ervilia. Genotypes of Turkish origin, characterized by an erect growth habit were crossed with Italian counterparts characterized by high seed production. A detailed cyto-histological analysis of flower development was undertaken to determine the optimal stage for emasculation and manual cross. Ninety-eight crosses were carried out and the hybrid nature of the putative F1 progenies was assessed by SSR (simple sequence repeat) DNA markers. Fifty-five seeds were obtained of which only five gave rise to hybrid plants. Among these, only three turned out to be fertile and two of which generated a consistent number of F2 seeds whose plants were assessed in greenhouse for seed production and plant growth habit. Most of the evaluated traits showed mean values of the F2 plants intermediate between the two initial parents, confirming that intraspecific hybridization is not only possible but also useful to exploit the diversity confined in different bitter vetch populations.

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

The ability to form biofilms of pathogenic microorganisms in a wide variety of environments, surfaces and conditions constitute an important risk, both for the food industry and for public health. The aim of this work was to evaluate and to compare the effects of the methodology applied and the culture medium used on the ability of a non-O157 verotoxigenic Escherichia coli strain and an enteropathogenic strain to form biofilm on polystyrene surface. Two methodological variants were tested in static culture and culture mediums with different composition were used. The results showed that both strains were able to form a greater biofilm under culture in LB supplemented with glucose, with medium replacement at 24 h and the quantification of the biofilm carried out at 48 h of incubation. These conditions could be used in future studies on biofilm formation. (C) 2018 Published by Elsevier Espana, S.L.U. on behalf of Asociacion Argentina de Microbiologia.

Light spectra influence growth, development, and quality of plants and seedlings, that is one of the main aspects engaging the interests of private and public researchers and nursery industries. Propagation of hazelnut (Corylus avellana L.), which in the past has been held in low consideration because of the widespread use of rooted suckers directly collected in the field, today is taking on increasing interest due to the strong expansion of hazelnut cultivation. In order to improve the quality of plants and seedlings in greenhouse acclimatization, the effects of light emitting diodes (LED) lights during the ex vitro growth of two hazelnut cultivars (Tonda di Giffoni and Tonda Gentile Romana) were investigated. Plantlets were maintained in a growth chamber and exposed to three different continuous spectrum LED systems as a primary source of illumination and to fluorescent lamps used as control. LEDs differed in the percentage of some wavelength ranges in the spectrum, being AP673L rich in green and red wavelengths, NS1 in blue and green light, G2 in red and far red wavelengths. After a 4-week experimental period, morphometric, biochemical, and histological analyses were carried out. Shoot and leaf growths were influenced by LEDs more than by fluorescent lamps in both cultivars. G2 positively affected biomass increment more than the other LEDs, by inducing not only cell elongation (increase in shoot length, new internodes length, leaf area) but also cell proliferation (increase in new node number). G2 exposure had negative effects on total chlorophyll content but positively affected synthesis of flavonoids in both varieties; therefore, plants grown under this LED showed the lowest nitrogen balance index. Leaf morpho-anatomical analyzed traits (thickness, palisade cell height, number of chloroplasts, number of palisade cells), were influenced especially by G2 and, to a less extent, by NS1 light. Significant differences in some parameters were observed between the two cultivars in response to a same light source. The results obtained underline the importance of light modulation for hazelnut, providing useful information for ex vitro growth of hazelnut plantlets.

Olive, representing one of the most important fruit crops of the Mediterranean area, is characterized by a general low fruit yield, due to numerous constraints, including alternate bearing, low flower viability, male-sterility, inter-incompatibility, and self-incompatibility (SI). Early efforts to clarify the genetic control of SI in olive gave conflicting results, and only recently, the genetic control of SI has been disclosed, revealing that olive possesses an unconventional homomorphic sporophytic diallelic system of SI, dissimilar from other described plants. This system, characterized by the presence of two SI groups, prevents self-fertilization and regulates inter-compatibility between cultivars, such that cultivars bearing the same incompatibility group are incompatible. Despite the presence of a functional SI, some varieties, in particular conditions, are able to set seeds following self-fertilization, a mechanism known as pseudo-self-compatibility (PSC), as widely reported in previous literature. Here, we summarize the results of previous works on SI in olive, particularly focusing on the occurrence of self-fertility, and offer a new perspective in view of the recent elucidation of the genetic architecture of the SI system in olive. Recent advances in research aimed at unraveling the molecular bases of SI and its breakdown in olive are also presented. The clarification of these mechanisms may have a huge impact on orchard management and will provide fundamental information for the future of olive breeding programs.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx(2a) alone or together with stx(2c) or, less frequent, with stx(2d), all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Shiga toxin-producing Escherichia coli (STEC) are pathogens of significant public health concern. Several studies have confirmed that cattle are the main reservoir of STEC in Argentina and other countries. Although Shiga toxins represent the primary virulence factors of STEC, the adherence and colonization of the gut are also important in the pathogenesis of the bacteria. The aim of this study was to analyze and to compare the presence of putative virulence factors codified in plasmid -katP, espP, subA, stcE- and adhesins involved in colonization of cattle -efa1, iha- in 255 native STEC strains isolated from different categories of cattle from different production systems. The most prevalent gene in all strains was espP, and the less prevalent was stcE. katP was highly detected in strains isolated from young and rearing calves (33.3%), while subA was predominant in those isolated from adults (71.21%). Strains from young calves showed the highest percentage of efa1 (72.46%), while iha showed a high distribution in strains from rearing calves and adults (87.04 and 98.48% respectively). It was observed that espP and iha were widely distributed throughout all strains, whereas katP, stcE, and efa1 were more associated with the presence of eae and subA with the eae-negative strains. A great proportion of eae-negative strains were isolated from adults -dairy and grazing farms- and from rearing calves -dairy and feedlot-, while mostly of the eae-positive strains were isolated from dairy young calves. Data exposed indicate a correlation between the category of the animal and the production systems with the presence or absence of several genes implicated in adherence and virulence of STEC.

Pigs are important reservoirs of Shiga toxin-producing Escherichia coli (STEC). The entrance of these strains into the food chain implies a risk to consumers because of the severity of hemolytic uremic syndrome. This study reports the prevalence and characterization of STEC throughout the pork production chain. From 764 samples, 31 (4.05%) were stx positive by PCR screening. At farms, 2.86% of samples were stx positive; at slaughter, 4.08% of carcasses were stx positive and at boning rooms, 6% of samples were stx positive. These percentages decreased in pork meat ready for sale at sales markets (4.59%). From positive samples, 50 isolates could be characterized. At farms 37.5% of the isolates carried stxl/stx2 genes, 37.5% possessed stx2e and 25%, carried only stx2. At slaughter we detected 50% of isolates positive for stx2, 33% for stx2e, and 16% for stxl/stx2. At boning rooms 59% of the isolates carried stxl/stx2, 14% stx2e, and 5% stx1/stx2/stx2e. At retail markets 66% of isolates were positive for stx2, 17% stx2e, and 17% stxl/stx2. For the other virulence factors, ehxA and saa were not detected and eae gene was detected in 12% of the isolates. Concerning putative adhesins, agn43 was detected in 72%, ehaA in 26%, aide in 8%, and iha in 6% of isolates. The strains were typed into 14 E. coli O groups (O1, O2, O8, O15, O20, O35, O69, O78, O91, O121, O138, O142, O157, O180) and 10 H groups (H9, H10, H16, H21, H26, H29, H30, H32, H45, H46). This study reports the prevalence and characterization of STEC strains through the chain pork suggesting the vertical transmission. STEC contamination originates in the farms and is transferred from pigs to carcasses in the slaughter process and increase in meat pork at boning rooms and sales markets. These results highlight the need to implement an integrated STEC control system based on good management practices on the farm and critical control point systems in the food chain.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Olea europaea subsp. cuspidata, belonging to the olive complex, is largely distributed in Asia and Africa. In this research, reproductive structures were studied at both structural and functional levels in Asian and African cuspidata accessions with the aim to broaden the knowledge about the floral biology of this subspecies. Morpho-structural observations showed that the cuspidata flower is very similar to the europaea one. Nevertheless, some biometric parameters (length of pollen, pistil, ovule, and embryo sac) significantly differed between the two subspecies. Furthermore, numerous and interesting differences were observed between Asian and African cuspidata accessions for the morphology of stigma papillate cells, degree of secondary cell wall thickenings of the endothecial cells, persistence of the tapetum, ovule and embryo sac length. At functional level, main differences were in the synchrony/asynchrony of the microsporogenesis, percentage of male meiotic abnormalities and sterile pollen, development of ovules. The influence of the growth environment on these features is discussed. (C) 2016 Elsevier GmbH. All rights reserved.

Adventitious shoot organogenesis and somatic embryogenesis are the basis for implementing new genetic variability and biotechnological approaches in woody species, particularly if mature tissues from valuable cultivars are used. To date, these technologies are only applied to few tree species due to the absence of efficient regeneration protocols. In hazelnut, adventitious shoot organogenesis and somatic embryogenesis have only been carried out with zygotic embryo tissues up to now. Here we report plant regeneration from explants of somatic origin by using in vitro rejuvenated mature tissues (leaves, petioles and stipules). A histological analysis carried out on calli grown on various media, showed significant evidence of shoot regeneration, as proved by the presence of vascular elements such as tracheids with annular or helical secondary wall thickening. Subsequently, the optimization of the regeneration protocol performed by pre-treating the explants with antibiotics (carbenicillin, vancomycin, and cefotaxime) as molecules with auxin-like effects enabled us to achieve shoot organogenesis in hazelnut. The organogenic competence strictly depended on the explants and antibiotics used in the experiments. Following an antibiotic pre-treatment of cv Tonda Gentile Romana explants in the proliferation stage, organogenic responses (frequencies of 40 %) were obtained. According to the results obtained, the best protocol for inducing shoot organogenesis in hazelnut should include the use of explants (leaves and petioles) conditioned in a double-liquid layer of cefotaxime 1000 mg L−1 added to the proliferation medium, cultured on the induction medium, consisting in solid MS medium supplemented with 3 % sucrose, 6-benzylaminopurine 1 mg L−1, indole-3-butyric acid 1 mg L−1 and kinetin 2 mg L−1, and then by sub-culturing the newly-formed calli to regeneration medium, consisting of half-strength solid MS medium with sucrose (30 g L−1) and 6-benzylaminopurine (0.5 mg L−1).

To enrich the source of germplasm of cultivated olive ( Olea europaea subsp. europaea L.), inter-subspecific hybrid plants have been produced by experimental crosses between several varieties of cultivated olive and Asian and African accessions of the wild related subspecies cuspidata . Germination of putative hybrid seeds was enhanced by using in vitro embryo culture. The genetic make-up of germinated seedlings was assayed with the aid of both AFLP and SSR molecular markers and their hybrid nature was proved by the presence of male-specific alleles in their molecular patterns. Most of the parent specific alleles showed segregation among F 1 progenies indicating high heterozygosity content of the parental lines. The majority of the hybrids derived from crosses in which an African accession of cuspidata was used as female parent. The overall morphological aspect of hybrids resembled that of the female parent. The production of inter-subspecific hybrid plants in Olea is discussed in relation to the genetic improvement of cultivated olive.

Apomixis, a clonal plant reproduction by seeds, is controlled in Paspalum spp. by a single locus which is blocked in terms of recombination. Partial sequence analysis of the apomixis locus revealed structural features of heterochromatin, namely the presence of repetitive elements, gene degeneration, and de-regulation. To test the epigenetic control of apomixis, a study on the distribution of cytosine methylation at the apomixis locus and the effect of artificial DNA demethylation on the mode of reproduction was undertaken in two apomictic Paspalum species. The 5-methylcytosine distribution in the apomixis-controlling genomic region was studied in P. simplex by methylation-sensitive restriction fragment length polymorphism (RFLP) analysis and in P. notatum by fluorescene in situ hybridization (FISH). The effect of DNA demethylation was studied on the mode of reproduction of P. simplex by progeny test analysis of apomictic plants treated with the demethylating agent 5'-azacytidine. A high level of cytosine methylation was detected at the apomixis-controlling genomic region in both species. By analysing a total of 374 open pollination progeny, it was found that artificial demethylation had little or no effect on apospory, whereas it induced a significant depression of parthenogenesis. The results suggested that factors controlling repression of parthenogenesis might be inactivated in apomictic Paspalum by DNA methylation.

Apomixis is an alternative route of plant reproduction that produces individuals genetically identical to the mother plant through seeds. Apomixis is desirable in agriculture, because it guarantees the perpetuation of superior genotypes (i.e. heterotic hybrid seeds) by self-seeding without loss of hybrid vigour. The Paspalum genus, an archetypal model system for mining apomixis gene(s), is composed of about 370 species that have extremely diverse reproductive systems, including self-incompatibility, self-fertility, full sexual reproduction, and facultative or obligate apomixis. Barriers to interspecific hybridization are relaxed in this genus, allowing the production of new hybrids from many different parental combinations. Paspalum is also tolerant to various parental genome contributions to the endosperm, allowing analyses of how sexually reproducing crop species might escape from dosage effects in the endosperm.In this article, the available literature characterizing apomixis in Paspalum spp. and its use in breeding is critically reviewed. In particular, a comparison is made across species of the structure and function of the genomic region controlling apomixis in order to identify a common core region shared by all apomictic Paspalum species and where apomixis genes are likely to be localized. Candidate genes are discussed, either as possible genetic determinants (including homologs to signal transduction and RNA methylation genes) or as downstream factors (such as cell-to-cell signalling and auxin response genes) depending, respectively, on their co-segregation with apomixis or less. Strategies to validate the role of candidate genes in apomictic process are also discussed, with special emphasis on plant transformation in natural apomictic species.

Self-incompatibility is one of the most important barriers adopted by many flowering plants to prevent inbreeding, thus maintaining diversity within species. The elucidation of this reproductive constraint is crucial in olive (Olea europaea L.) because it may help to improve breeding strategies and orchard management. The available literature reports that a gametophytic self-incompatibility (GSI) system might exist in olive, even though neither cytological nor molecular data were provided. A molecular approach failed to find S-RNase (S-locus ribonuclease) and SLF (S-locus F-box containing protein), the genes responsible for GSI. In this paper, cyto-histological observations and bio-molecular evidence is presented, supporting the presence in olive of a sporophytic self-incompatibility (SSI) system. The main genes known to play a crucial role in SSI are SRK (S-locus receptor kinase) and SLG (S-locus glycoprotein), the female determinants, and SCR (S-locus cysteine rich protein), the male determinant. OeSLG and OeSRK genes cloned by PCR with degenerated primers were shown to be differentially expressed in flower organs of self-compatible (cv Frantoio) and self-incompatible (cv Leccino) genotypes by means of Real-Time PCR analysis. Since the same approach failed to isolate SCR, a 454 pyrosequencing library derived from flowers at different developmental stages was screened. One of the sequences displaying a conserved amino acid structure in terms of cysteine residuals, named OeSCR-like, proved to be specifically expressed in anthers at blooming stages. The overall data so far collected support the existence of an SSI system in olive, at least in the tested genotypes.

Deploying whole and dissected nuclear genome of wild Triticeae species in the homoeologous wheat genetic background through inter-specific hybridization and introgression is a lower cost and effective option to prepare wheat germplasm with unexploited genes for disease resistance and enhanced grain yield and quality traits. The whole nuclear genomes of Dasypyrum villosum (Dv) and T. turgidum var durum have been combined, and an homoploid derivative of the original amphiploid displayed typical ‘farro’ spike morphology, tough rachis and the adaptive traits of Dv such as high resistance to diseases (caused by Tilletia tritici, Blumeria graminis f. sp. tritici, Puccinia triticina and P. graminis f. sp. tritici), heading earliness and fortified caryopses (high protein and micronutrient contents). The dissection of the Dv genome by either ‘Triticum aestivum cv Chinese Spring (CS) × hexaploid amphiploid’ or ‘(CS × Dv) × CS’ hybridization and backcrossing provided wheat introgression breeding lines (IBLs) expressing one or more of the Dv adaptive traits. Molecular analyses revealed that either cryptic or Genomic In-situ Hybridization (GISH) detectable Dv chromatin introgression occurred in those IBLs. The IBLs, after 2 years of low-input field tests and genetic analyses in Italy and Hungary, showed simple inheritance, dominance and stability of the adaptive and disease resistance traits.

Variations in the nuclear DNA, mainly as a result of quantitative modulations of DNA repeats belonging to different sequence families of satellite DNA and to the activity of transposable elements, have been assessed within several angiosperm species. These variations alter the amount and organization of the DNA and therefore the genotype, rather than the genome proper. They take place on an evolutionary time scale as the result of selection processes after the occurrence of uncontrolled events in the genome or may be due to direct responses of plant genomes to environmental stimuli that occur under plant-level control within a short developmental period of a single generation. These DNA changes are correlated to changes in the developmental dynamics and phenotypic characteristics of the plants, and the capability to carry out genotypic variation is an evolutionary trait that allows plant species to adapt to different environmental conditions, as well as to the variability of conditions in a given environment. The link between developmental and environmental stimuli and repetitive DNA that elicits the intraspecific diversity of plant genotypes may provide models of evolutionary change that extend beyond the conventional view of evolution by allelic substitution and take into account epigenetic effects of the genome structure.