The role of temperature in the onset of the Olea europaea L. pollen season in southwestern Spain

Temperature is one of the main factors affecting the flowering of Mediterranean trees. In the case of Olea europaea L., a low-temperature period prior to bud development is essential to interrupt dormancy. After that, and once a base temperature is reached, the plant accumulates heat until flowering starts. Different methods of obtaining the best-forecast model for the onset date of the O. europaea pollen season, using temperature as the predictive parameter, are proposed in this paper. An 18-year pollen and climatic data series (1982–1999) from Cordoba (Spain) was used to perform the study. First a multiple-regression analysis using 15-day average temperatures from the period prior to flowering time was tested. Second, three heat-summation methods were used, determining the the quantities heat units (HU): accumulated daily mean temperature after deducting a threshold, growing degree-days (GDD): proposed by Snyder [J Agric Meteorol 35:353–358 (1985)] as a measure of physiological time, and accumulated maximum temperature. In the first two, the optimum base temperature selected for heat accumulation was 12.5°C. The multiple-regression equation for 1999 gives a 7-day delay from the observed date. The most accurate results were obtained with the GDD method, with a difference of only 4.7 days between predicted and observed dates. The average heat accumulation expressed as GDD was 209.9°C days. The HU method also gives good results, with no significant statistical differences between predictions and observations. Received: 18 April 2000 / Revised: 14 September 2000 / Accepted: 19 September 2000     

Behavior of storage lipids during development and germination of olive ( Olea europaea L.) pollen

Summary.  The presence of abundant oil bodies in the mature olive pollen grain has led us to focus on the behavior of these lipid bodies during pollen development and in vitro pollen germination. The appearance, increase, and accumulation of lipid bodies have been determined by following the sequential development of the pollen grain. Semithin slices of anthers and pollen grains were stained with Sudan Black B in order to identify neutral lipids. Ultrastructural studies were also carried out. Our results show a notable increase in lipid bodies between the young-pollen-grain stage and the mature-pollen-grain stage. Substantial polarization of lipid bodies was observed after 1 or 2 h of pollen incubation in germination medium. During pollen tube growth, the lipid bodies are located near the germinative aperture after 3 h of incubation, as well as inside the pollen tube, thus suggesting that the lipid bodies move from the pollen grain to the pollen tube. After 7 h of germination the presence of lipid bodies inside the pollen tube is no longer substantial. Our results support the idea that lipid bodies are involved in pollen germination, stigma penetration, and pollen tube growth. These results are discussed in connection with their implications for the pollen germination process. Received June 4, 2002; accepted October 29, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.     

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.)

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT)n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars. Received: 2 February 2001 / Accepted: 1 June 2001     

Heat requirement for the onset of the Olea europaea L. pollen season in several sites in Andalusia and the effect of the expected future climate change

Olives are one of the largest crops in the Mediterranean region, especially in Andalusia, in southern Spain. A thermal model has been developed for forecasting the start of the olive tree pollen season at five localities in Andalusia: Cordoba, Priego, Jaen, Granada and Malaga using airborne pollen and meteorological data from 1982 to 2001. Threshold temperatures varied between 5°C and 12.5°C depending on bio-geographical characteristics. The external validity of the results was tested using the data for the year 2002 as an independent variable and it confirmed the models accuracy with only a few days difference from predicted values. All the localities had increasingly earlier start dates during the study period. This could confirm that olive flower phenology can be considered as a sensitive indicator of the effects of climate fluctuations in the Mediterranean area. The theoretical impact of the predicted climatic warming on the olives flowering phenology at the end of the century is also proposed by applying Regional Climate Model data. A general advance, from 1 to 3 weeks could be expected, although this advance will be more pronounced in mid-altitude inland areas.     

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     

Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars

One hundred and two olive RAPD profiles were sampled from all around the Mediterranean Basin. Twenty four clusters of RAPD profiles were shown in the dendrogram based on the Ward’s minimum variance algorithm using chi-square distances. Factorial discriminant analyses showed that RAPD profiles were correlated with the use of the fruits and the country or region of origin of the cultivars. This suggests that cultivar selection has occurred in different genetic pools and in different areas. Mitochondrial DNA RFLP analyses were also performed. These mitotypes supported the conclusion also that multilocal olive selection has occurred. This prediction for the use of cultivars will help olive growers to choose new foreign cultivars for testing them before an eventual introduction if they are well adapted to local conditions. Received: 10 April 2000 / Accepted: 15 May 2000     

Production of total and stainable pollen grains in Olea europaea L

The production of the total and stainable number of pollen grains per flower and per inflorescence of 13 olive cultivars (Olea europaea L.) has been investigated. Grains stainability was evaluated by using the acetic carmine staining method whereas the production of pollen grains was calculated by using a Bürker haemocytometer. All pollen characteristics taken into account varied significantly among the cultivars. The number of flowers per inflorescence ranged from a maximum of 31.3 (‘Cipressino’) to a minimum of 13.0 (‘Leccino’), the number of pollen grains per anther ranged from 123?346 (‘Arbequina’) to 40?975 (‘Sant'Agostino’), and the pollen stainability from 97.6% (‘Cipressino’) to 48.0% (‘Cellina di Nardò’). The number of stainable pollen grains per inflorescence averaged 2?559?512, ranging from a maximum of 3?913?678 (‘Nociara’) to a minimum of 940?143 (‘Sant'Agostino’). All parameters were positively correlated, whereas a linear negative correlation was found between stainability and total pollen grains both per anther and per inflorescence.     

欧橄榄叶中的新裂环烯醚萜

从欧橄榄(Olea europaeaL.)干燥叶的乙酸乙酯部位分离得到5个裂环烯醚萜类化合物。通过波谱分析和理化常数对照等方法,上述化合物分别鉴定为6′-O-甲基橄榄苦苷(6′-O-methyloleuropein,1)、橄榄苦苷(oleu-ropein,2)、(1S)-methylelenolate(3)、3,4-二羟基苯乙基-4-甲酰基-3-甲酰甲基-4-己烯酯(4)和oleoside(5)。其中化合物1为新化合物。     

Airborne-pollen map for Olea europaea L. in eastern Andalusia (Spain) using GIS: Estimation models

Estimations based upon geostatistics and mapping have enabled the construction of a spatial model to predict the presence of biological particles in a particular region. This methodological proposal has been tested in a case study, at a regional scale, of airborne Olea pollen, using the data acquired from␣various sampling stations that are designed for the aerobiological monitoring of pollen levels. These sampling stations have been set up in cities throughout the region of Andalusia (southern Spain) at sites with very different characteristics in terms of biogeography, bioclimate, topography and vegetation. Pollen counts were made daily at all sites during 2003 using a volumetric spore-trap. Data were comparatively analysed in classical diagrams and by means of spatial-temporal maps. Space-time models were constructed using three coordinates, x, y (the UTM coordinates of each sampling station) and z, (the aerobiological data compiled for a specific period). The aerobiological data were interpolated by applying the traditional geostatistical method of Kriging. The introduction of the variable “space” into the model allowed us to predict pollen levels in different areas throughout the region. The interpolation method was used to make weekly estimations of Olea pollen values in areas where there was no aerobiological sampling station. In addition, the maps generated present a two-dimensional vision of the study area, showing that bioclimatic diversity of this region promotes a step-wise flowering of Olea.     

Microsatellite analysis of Latial Olea europaea L. cultivars

Olea europaea L. is one of the oldest domesticated tree species. O. europaea varieties cannot be confused because they are very different in morphology, genetics, and secondary metabolite content. It is important to study and establish the genetic structure of vegetal cultivars to better distinguish them, to solve past misclassification, to preserve plant biodiversity, and to increase their use, diffusion, selection, resistance to adversities, marketing, and scientific applications. Five simple sequence repeat loci (DCA-3, DCA-9, UDO99-9, UDO99-35, and EMO-3) were used to differentiate 39 individuals, representing 13 olive cultivars sampled in Latium (Central Italy). The markers showed a high discrimination power and were able to differentiate 39 alleles. Observed heterozygosity ranged from 0.538 at locus UDO99-9 to 1 at locus UDO99-35, with a mean value of 0.784. DCA loci were the most informative ones. Sample clustering, based on their genetic distance and similarity values, produced a phylogenetic network that has shown a unique major group of cultivars, composed of two sub-branches, and two independent taxa.     

Development of SCAR markers in olive (Olea europaea) by direct sequencing of RAPD products: applications in olive germplasm evaluation and mapping

RAPD markers generated by mixtures of two different 10-mer primers were developed for eight different olive cultivars used as parental lines in olive-breeding programs. Two RAPD bands were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step. The SCARs generated have maintained the original RAPD polymorphism among the cultivars and segregated according to Mendelian inheritance. Preliminary results suggest the use of the SCAR SCOeMS-2 for the marker-assisted selection of the high flesh/stone ratio. This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers with applications in olive mapping, paternal testing and germplasm characterization. The use of these markers in multiplexed PCRs, and the direct ethidium bromide detection of the PCR products in the test tube, facilitate their efficient and reliable breeding applications. Received: 1 November 2000 / Accepted: 24 November 2000     

Aerobiological and meteorological factors’ influence on olive (Olea europaea L.) crop yield in Castilla-La Mancha (Central Spain)

Knowledge of the main biological and climate factors influencing final harvest is becoming increasingly necessary in order to obtain reliable crop estimates and, thus, ensure optimised, effective private crop management. This knowledge is also of great value to public agricultural institutions for the planning of government subsidies. Castilla-La Mancha (Central Spain) is the second largest olive-oil-producing region in Spain, the highest olive-oil-producing country in the world. This study sought to identify the main factors influencing olive fruit production in this region, including atmospheric pollen as an index of flowering intensity, and meteorological data over the flowering and fruiting seasons in two main olive-producing provinces of the region: Ciudad Real and Toledo. Statistical analysis indicated that the annual pollen index (PI) was the variable influencing most the final olive crop in both provinces. The maximum temperature in March was the meteorological variable affecting most the annual olive crop. Also, the rainfall registered in October influences the final fruit production. The integration of aerobiological and meteorological data represents an important step forward in the development of future crop forecasting models in the region of Castilla-La Mancha.     

Influence of meteorological parameters on Olea flowering date and airborne pollen concentration in four regions of Portugal

For calculating the total annual Olea pollen concentration, the onset of the main pollen season and the peak pollen concentration dates, using data from 1998 to 2004, predictive models were developed using multiple regression analysis. Four Portuguese regions were studied: Reguengos de Monsaraz, Valença do Douro, Braga and Elvas. The effect of some meteorological parameters such as temperature and precipitation on Olea spatial and temporal airborne pollen distribution was studied. The best correlations were found when only the pre‐peak period was used, with thermal parameters (maximum temperature) showing the highest correlation with airborne pollen distribution. Independent variables, selected by regression analysis for the predictive models, with the greatest influence on the Olea main pollen season features were accumulated number of days with rain and rainfall in the previous autumn, and temperatures (average and minimum) from January through March. The models predict 59 to 99% of the total airborne pollen concentration recorded and the initial and peak concentration dates of the main Olea pollen season.     

Influence of wind direction on pollen concentration in the atmosphere

The daily pollen concentration in the atmosphere of Badajoz (SW Spain) was analysed over a 6-year period (1993–1998) using a volumetric aerobiological trap. The results for the main pollination period are compared with the number of hours of wind each day in the four quadrants: 1 (NE), 2 (SE), 3 (SW) and 4 (NW). The pollen source distribution allowed 16 pollen types to be analysed as a function of their distribution in the four quadrants with respect to the location of the trap. Four of them correspond to species growing in an irrigated farmland environment (Amaranthaceae-Chenopodiaceae, Plantago, Scirpus, and Typha), five to riparian and woodland species (Salix, Fraxinus, Alnus, Populus, and Eucalyptus), four to urban ornamentals (Ulmus, Arecaceae, Cupressaceae, and Casuarina), and three which include the most frequent pollen grains of widely distributed species (Poaceae, Quercus, and Olea). The results show that the distribution of the sources and the wind direction play a very major role in determining the pollen concentration in the atmosphere when these sources are located in certain quadrants, and that the widely distributed pollen sources show no relationship with wind direction. In some years the values of the correlations were not maintained, which leads one to presume that, in order to draw significant conclusions and establish clear patterns of the influence of wind direction, a continuous and more prolonged study will be required. Received: 6 May 1999 / Revised: 30 March 2000 / Accepted: 31 March 2000     

Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which differed in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to five distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, confirming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same specific pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones efficiently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas. Received: 25 November 1998 / Accepted: 19 December 1998     

Abnormalities induced by agricultural pesticides in the microsporogenesis of olive tree (Olea europaea L.) cultivars

The study evaluated the microsporogenesis of olive trees subjected to different agricultural pesticide applications during flowering. Inflorescences of cultivars Arbequina and MGS GRAP541 were subjected to agricultural pesticides: mineral oil, neem oil, dimethoate and deltamethrin. The floral buds were fixed in Carnoy for the microsporogenesis analysis and in Karnovsky for scanning electron microscopy. The slides were prepared by squash technique and staining with propionic carmine. The pollen viability was determined by Alexander’s stain and in vitro germination. Results show that the quantification of abnormalities in meiosis in the two cultivars caused significant effect among the treatments, being that all differed statistically from the control group. Both methods showed a higher percentage of viable pollens in the control treatment and lower percentage of viability with the agricultural pesticides. The method of pollen viability by staining presented the highest averages of viable pollens, but when compared together, both methods presented a strongly related positive linear correlation. It was concluded that the used chemical products increased the percentage of chromosomal abnormalities during microsporogenesis, which interfered in the pollen viability of the two analyzed cultivars. The product deltamethrin caused the strongest effect on meiosis and on pollen viability.