Pollen Ole e 1 content variations in olive cultivars of different Portugal regions

Olive is recognized as a crop with great impact in agricultural, socioeconomic, environmental and public health sectors. The last is becoming more important during recent years as consequence of the increase of the pollen allergy in south Europe prompted by the widespread Olea pollen allergic reactions. The aim of the study was to quantify, for the first time, the variations of the Ole e 1 allergen amount in Olea pollen grains from four cultivars in three regions of Portugal. How weather parameters can affect the allergen production was also assessed. The study was conducted in three olive producer areas of Portugal from 2010 to 2013, Santarém (Central), Elvas (Southeast) and Mirandela (Trás-os-Montes region, Northeast). Mature pollen of four different cultivars (Cobrançosa, Arbequina, Picual and Verdeal) was collected during the olive flowering season. Ole e 1 was quantified using specific 2-site antibody ELISA. Pollen of the olive groves at the boundary Olea bioclimatic distribution in the Mirandela registered the higher allergen content for all varieties in each study year. Arbequina was the variety that showed the lower Ole e 1 allergen concentration, whereas the higher content was registered for Cobrançosa. The main meteorological parameters that influenced the allergen Ole e 1 concentration in the pollen grains were the rainfall and temperatures related variables. The knowledge of the allergenicity in different olive cultivars is an important tool in the selection of the most adequate for planting as ornamental crop and to adjust the pollen extracts used for diagnosis or even immunotherapy.

     

Screening of Ole e 1 polymorphism among olive cultivars by peptide mapping and N‐glycopeptide analysis

In the present paper, we have used 2‐DE coupled to MS analysis to examine the molecular variability of the Ole e 1 allergen in three olive cultivars (cvs). Our results confirmed that the predicted polymorphism of Ole e 1 at cDNA level is extended to the expressed protein. The profiles of both the Ole e 1 peptides and the N‐glycan variants significantly changed among cvs. We observed that Picual and Arbequina cvs presented the highest and lowest degree of Ole e 1 polymorphism, respectively. Some of these peptides and N‐glycans were distributed in a cv‐specific manner. The putative implications of this molecular polymorphism in the development of the allergy symptoms are discussed.     

Analysis of the pollen allergen content of twelve olive cultivars grown in Portugal

Olive pollen presents intercultivar variability as regards to its antigenic and allergenic composition. In this study, we report the presence of differences among the SDS-PAGE pollen protein profiles of twelve Portuguese olive cultivars. Though most soluble proteins from these extracts seemed similar, three bands of about 18, 20 and 22 kDa presented sharp differences in intensity among the cultivars analyzed. The dissimilarity of patient’s sera reactivity to these protein extracts and the presence of several allergens already characterized (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) in the extracts were also investigated. Epidemiological data indicated that 53.3 % out of the 428 patients analyzed with reactivity to pollen extracts, presented specific IgE levels to Olea europaea. A representative number of these sera were assayed in immunoblotting experiments. The cultivars ‘Galega’ and ‘Conserva de Elvas’ displayed low reactivity to the sera of atopic patients, whereas the extracts corresponding to the cultivars ‘Cobrançosa’, ‘Ascolana’ and ‘Verdeal de Serpa’ led to higher IgE reactivity. The use of antibodies to the allergens Ole e 1, Ole e 2, Ole e 5 and Ole e 9 in immunoblotting experiments also allowed cultivar discrimination. The cultivar ‘Verdeal de Serpa’ presented the highest Ole e 1, Ole e 5 and Ole e 9 allergen loads but the lowest Ole e 2. ‘Carrasquenha’ was the second cultivar in terms of the higher allergen content. Oppositely, the lowest allergen loads were those of the cultivars ‘Galega’ and ‘Conserva de Elvas’ coincidentally with their low IgE reactivity. These data may help interpret physiological differences in pollen performance for successful olive fertilization and, moreover, to better define future strategies for allergy diagnosis and treatment by specific immunotherapy.     

Identification of new polymorphic regions and differentiation of cultivated olives (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) through plastome sequence comparison

Background  

The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.     

Centennial olive trees as a reservoir of genetic diversity

Background and Aims

Genetic characterization and phylogenetic analysis of the oldest trees could be a powerful tool both for germplasm collection and for understanding the earliest origins of clonally propagated fruit crops. The olive tree (Olea europaea L.) is a suitable model to study the origin of cultivars due to its long lifespan, resulting in the existence of both centennial and millennial trees across the Mediterranean Basin.

Methods

The genetic identity and diversity as well as the phylogenetic relationships among the oldest wild and cultivated olives of southern Spain were evaluated by analysing simple sequence repeat markers. Samples from both the canopy and the roots of each tree were analysed to distinguish which trees were self-rooted and which were grafted. The ancient olives were also put into chronological order to infer the antiquity of traditional olive cultivars.

Key Results

Only 9·6 % out of 104 a priori cultivated ancient genotypes matched current olive cultivars. The percentage of unidentified genotypes was higher among the oldest olives, which could be because they belong to ancient unknown cultivars or because of possible intra-cultivar variability. Comparing the observed patterns of genetic variation made it possible to distinguish which trees were grafted onto putative wild olives.

Conclusions

This study of ancient olives has been fruitful both for germplasm collection and for enlarging our knowledge about olive domestication. The findings suggest that grafting pre-existing wild olives with olive cultivars was linked to the beginnings of olive growing. Additionally, the low number of genotypes identified in current cultivars points out that the ancient olives from southern Spain constitute a priceless reservoir of genetic diversity.     

High Genetic Diversity Detected in Olives beyond the Boundaries of the Mediterranean Sea

Background

Olive trees (Olea europaea subsp. europaea var. europaea) naturally grow in areas spanning the Mediterranean basin and towards the East, including the Middle East. In the Iranian plateau, the presence of olives has been documented since very ancient times, though the early history of the crop in this area is shrouded in uncertainty.

Methods

The varieties presently cultivated in Iran and trees of an unknown cultivation status, surviving under extreme climate and soil conditions, were sampled from different provinces and compared with a set of Mediterranean cultivars. All samples were analyzed using SSR and chloroplast markers to establish the relationships between Iranian olives and Mediterranean varieties, to shed light on the origins of Iranian olives and to verify their contribution to the development of the current global olive variation.

Results

Iranian cultivars and ecotypes, when analyzed using SSR markers, clustered separately from Mediterranean cultivars and showed a high number of private alleles, on the contrary, they shared the same single chlorotype with the most widespread varieties cultivated in the Mediterranean.

Conclusion

We hypothesized that Iranian and Mediterranean olive trees may have had a common origin from a unique center in the Near East region, possibly including the western Iranian area. The present pattern of variation may have derived from different environmental conditions, distinct levels and selection criteria, and divergent breeding opportunities found by Mediterranean and Iranian olives.These unexpected findings emphasize the importance of studying the Iranian olive germplasm as a promising but endangered source of variation.     

Computational annotation of genes differentially expressed along olive fruit development

Background  

Olea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.     

Development of a versatile tool for the simultaneous differential detection of <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pathovars by End Point and Real-Time PCR

Background  

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta.     

Detection of latent infections caused by Colletotrichum sp. in olive fruit

Aims

To set up a practical method to detect latent infections of Colletotrichum sp., the causal agent of olive anthracnose, on olives before the onset of disease symptoms.

Methods and Results

Freezing, sodium hydroxide (NaOH), ethanol and ethylene treatments were evaluated to detect latent infections on inoculated and naturally infected olive fruit by Colletotrichum sp. as non‐hazardous alternatives to paraquat. Treatments were conducted using fruit of cultivars Arbequina and Hojiblanca. The disease incidence and T₅₀ were calculated. Dipping in NaOH 0·05% solution and the paraquat method were the most effective treatments on both inoculated and naturally infected fruit, although the value of T₅₀ was lower for the NaOH method than for the paraquat method in one of the experiments. Subsequently, the dipping time in NaOH 0·05% was evaluated. Longer dipping times in NaOH 0·05% were better than shorter ones in cultivar Arbequina, with 72 h being the most effective in cultivar Hojiblanca.

Conclusions

NaOH solution is a practical method to detect latent infections of Colletotrichum sp. on immature olive fruit.

Significance and Impact of the Study

This study is relevant because we set up a viable, non‐hazardous alternative to paraquat to detect latent infections of Colletotrichum sp. using NaOH. The use of NaOH is a simple and eco‐friendly tool that allows the determination of the level of latent infections by Colletotrichum in olives. Therefore, our method will be useful in decision‐making processes for disease management before the appearance of the first visible symptoms.     

Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1

Background  

Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection.     

Development of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> on Tolerant and Susceptible <Emphasis Type="Italic">Olea europaea</Emphasis> L. cultivars: A Microscopic Analysis

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192 h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.     

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Background  

Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum.     

Apnea induction for invasive lung function testing in infant olive baboons: Comparison of intravenous propofol versus hyperventilation

Background

In various types of pulmonary research, pulmonary function testing (PFT) is performed to quantify the severity of lung disease. Induction of apnea and positive pressure ventilation are required for accurate PFT measurements in non‐cooperative subjects. We compared two methods of apnea induction in infant olive baboons (Papio anubis).

Methods

Pulmonary function testing results were compared during apnea induced by hyperventilation (CO₂ washout) vs. intravenous propofol (1 dose 10 mg/kg). PFT was evaluated using a hot‐wire pneumotachometer incorporated within an Avea ventilator in nine 1‐month‐old baboons.

Results

Propofol induced apnea faster and more reliably. In both groups, PFT values passed the statistical equivalence test and were not significantly different (Student's t‐test). There was a trend toward less data variability after propofol administration.

Conclusions

Intravenous propofol was non‐inferior to CO₂ washout for apnea induction in infant olive baboons. Propofol induced apnea faster and more reliably and yielded less variable PFT results.     

Immunocytochemical localization of allergenic protein (Ole e I) in the endoplasmic reticulum of the developing pollen grain of olive (Olea europaea L.)

The monoclonal antibody OL-1 and transmission electron microscopy were used to locate immunologically the major allergen of olive pollen. Ole e I, during pollen grain development. Within the pollen grain, allergenic proteins are located in the cisternae of the rough endoplasmic reticulum. Our findings indicate that the synthesis of these proteins starts in the vegetative cytoplasm of olive pollen during the early maturation stage.Dedicated to Professor Andreas Sievers on the occasion of his retirement     

Genetic and environmental control of the <Emphasis Type="Italic">Verticillium</Emphasis> syndrome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Verticillium spp. are major pathogens of dicotyledonous plants such as cotton, tomato, olive or oilseed rape. Verticillium symptoms are often ambiguous and influenced by development and environment. The aim of the present study was to define disease and resistance traits of the complex Verticillium longisporum syndrome in Arabidopsis thaliana (L.) Heynh. A genetic approach was used to determine genetic, developmental and environmental factors controlling specific disease and resistance traits and to study their interrelations.     

Cellular localization of ROS and NO in olive reproductive tissues during flower development

Background  

Recent studies have shown that reactive oxygen species (ROS) and nitric oxide (NO) are involved in the signalling processes taking place during the interactions pollen-pistil in several plants. The olive tree (Olea europaea L.) is an important crop in Mediterranean countries. It is a dicotyledonous species, with a certain level of self-incompatibility, fertilisation preferentially allogamous, and with an incompatibility system of the gametophytic type not well determined yet. The purpose of the present study was to determine whether relevant ROS and NO are present in the stigmatic surface and other reproductive tissues in the olive over different key developmental stages of the reproductive process. This is a first approach to find out the putative function of these signalling molecules in the regulation of the interaction pollen-stigma.     

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH₂-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH₂-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X₃-Cys-X₃-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.     

Plant water uptake by hard red winter wheat (Triticum aestivum L.) genotypes at 2°C and low light intensity

Background  

Hard red winter wheat (HRWW; Triticum aestivm L.) plants from genotypes selected in the Northern Great Plains of the U.S. have less tissue water after exposure to cool autumn temperatures than plants from the Southern Great Plains. It is generally assumed that the reduced tissue water content of northern compared to southern cultivars is due to an impedance to water uptake by northern plants as a result of the low autumn temperatures. We hypothesize that if low temperature impedes water uptake then less soil water would be removed by northern than by southern-selected cultivars. This hypothesis was tested by comparing plant water uptake of a northern (FR) and a southern (FS) cultivar in relation to their foliage water content at 2°C.     

Cytoplasmic male sterility in the olive (Olea europaea L.)

The olive tree is usually hermaphrodite but self-incompatible. In the Western Mediterranean some cultivars are totally male-sterile. Three different male-sterile phenotypes have been recognised. To infer the genetic basis of male sterility we studied its inheritance and cytoplasmic diversity in wild (oleaster) and cultivated Mediterranean olive. In the cross Olivière×Arbequina, the male-sterile trait was maternally inherited and affected all progenies. We also checked that both chloroplast and mitochondrial DNAs are maternally inherited. RFLP studies on chloroplast and mitochondrial DNAs revealed several cytotypes: two chlorotypes and four mitotypes in cultivars and oleaster (wild or feral Mediterranean olive). Furthermore, a total linkage desequilibrium between the CCK chlorotype and the MCK mitotype in cultivars and oleaster from different regions supports the fact that paternal leakage of organelles was not observed. The male sterility (ms 2) displayed by Olivière, plus six other cultivars and three oleaster was strictly associated with the CCK chlorotype and the MCK mitotype. These facts suggest that Olivière carries cytoplasmic male sterility. Male-fertile and male-sterile oleasters carrying this cytotype showed the presence of restorer alleles. This CMS might be due to a distant cross between olive taxa. The two other male-sterile phenotypes displayed by Lucques (ms 1) and Tanche (ms 3) were associated with the ME1 mitotype but we have not demonstrated CMS. Received: 26 July 1999 / Accepted: 27 August 1999