Mobilization of nitrogen in the olive bearing shoots after foliar application of urea

Mature ‘on’ and ‘off’ ‘Manzanillo’ olives trees with three healthy branches of 10–12 cm in diameter were selected to determine changes in nitrogen levels in the bearing shoots after foliar application of urea. Each selected branch of each tree received one of the following treatments: (i) control without urea application; (ii) foliar application of urea to all the current-season leaves; and (iii) foliar application of urea to all the one-year-old leaves. Urea was applied in May, four days after full bloom in the ‘on’ year trees. Each treated leaf was immersed in a test tube containing a 4% urea solution and 0.1% Tween 20 for 5 s. Bearing shoots, composed of both one-year stems and leaves and current-season stems and leaves, were collected at intervals from the beginning of the experiment until 64 days after urea application. Nitrogen was determined in stems and leaves from each part of the bearing shoot, and in fruits during the ‘on’ year. Nitrogen uptake from the leaves was rapidly mobilized from the older to the current-season leaves of the bearing shoot, and thereafter to other storage organs of the tree or to the fruit, which is the largest nitrogen sink in the bearing shoot. No translocation of nitrogen from the current-season leaves to older leaves was observed. The rapid translocation of nitrogen from the younger leaves to other storage organs of the tree could explain the insensitivity of leaf analysis to detect excess nitrogen, since mature leaves from current-season shoots must be sampled to determine the nutritional status of the tree. The failure of leaf analysis to detect excess nitrogen may be a cause of nitrogen over-fertilization in olive orchards.     

Inflorescence architecture of olive

The influence of flower position on the inflorescence on opening day, gender, and petal persistence was studied in three olive cultivars: Manzanillo, Mission, and Frantoio. In each cultivar, 45 inflorescences were checked every morning from flower opening to petal fall. Perfect flowers opened mainly in the beginning of the flower opening period, and staminate flowers opened later. Flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than the flowers located on the secondary branches. Flower position had also a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. Among the primary branches, the branch arising immediately next to the terminal flower had the latest flowers to open and the lowest percent of perfect flowers. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal half of 120 inflorescences, on which the flowers tend to be perfect, in three trees of Manzanillo were removed about 1 month before full bloom. There was a highly significant effect on the percent of perfect flowers that opened on the proximal half. Flower competition may be a reason for pistil abortion in flowers located on secondary branches.     

Anatomy of the olive inflorescence axis at flowering and fruiting

In spite of the importance of the peduncle and pedicel in supporting and connecting flowers and fruits to the mother plant, these structures are little known in the olive, and more detailed information can be instrumental in studies of fruit development, ripening and abscission. The structure of pedicel and peduncle was studied at flowering and at fruit maturation for the Spanish olive cultivar “Picual”. Both organs have a stem-like structure. At flowering the pedicel is less developed as the transport requirements to individual flowers are minimal compared to that of the complete inflorescence, and the structure has high chances of being shed in the heavy flower abscission in this species. The peduncle is thicker, due to a ridged cortex, and has larger xylem vessel size. By fruit maturation in November, the overall diameter of both pedicel and peduncle increases, due to both conducting tissues and support tissues: in the pedicel the xylem increases 6-fold, in the peduncle 3-fold, while the phloem undergoes smaller increases. An important developmental feature of pedicel and peduncle is the increase in their capacity to mechanically support the growing fruit. To this end both organs, in addition to the increased thickness of the conducting tissues, produce a lignified sclerenchyma ring. Unlike the stem, however, which produces numerous sclereids, the sclerenchymatous ring only consists of fibres, which offer greater flexibility and make the structure less prone to breaking.     

Impacts of water stress on gas exchange,water relations,chlorophyll content and leaf structure in the two main Tunisian olive (Olea europaea L.) cultivars

Leaf structural adaptations for the reduction of water loss were examined in two olive (Olea europaea L.) cultivars (Chemlali and Chétoui) growing under water stress conditions. Leaf measurements included leaf tissue thickness, stomatal density, trichome density, specific leaf area, leaf density, water relations, and gas exchange. We found considerable genotypic differences between the two cultivars. Chemlali exhibited more tolerance to water stress, with a thicker palisade parenchyma, and a higher stomatal and trichome density. Chemlali leaves also revealed lower specific leaf area and had higher density of foliar tissue and lower reduction in net CO₂ assimilation rate. The mechanisms employed by these two cultivars to cope with water deficit are discussed at the morpho-structural level. The morphological and structural characteristics of the leaves are in accordance with physiological observations and contribute to the interpretation of why the olive cv. Chemlali is more drought-tolerant than cv. Chetoui. Furthermore, from the behaviour of Chemlali plants we consider this cultivar very promising for cultivation in semi-arid areas.     

Genetic diversity in Tunisian olive accessions and their relatedness with other Mediterranean olive genotypes

Eighty-four olive accessions obtained from the National Conservatory of Boughrara-Sfax (Tunisia), previously evaluated for morphological traits, were analysed with 47 random amplified polymorphic DNA (RAPD) markers. They were compared with other olive genotypes originated from Eastern or Western Mediterranean. The highest and lowest similarities between genotypes, estimated by simple matching algorithm, were 0.98 and 0.40, respectively. A dendrogram based on Ward's method and a factorial correspondence analysis (FCA) showed that most of Tunisian accessions are closely related to olive genotypes originating from the Eastern Mediterranean and some are clustering with genotypes originated from the Western Mediterranean. These findings suggested multiple and complex origin of Tunisian olive. A comparative study between a previous morphological analysis and current RAPD assay was carried out and discussed.     

DNA fingerprinting of olive varieties in Istria (Croatia) by microsatellite markers

The Istria region, where olives have been cultivated for many centuries, is characterized by a considerable variety of microclimates. The study of varieties traditionally cultivated in Croatian Istria and their relationships with varieties in historically and geographically connected regions is very important in order to identify native olive germplasm, well adapted to local conditions, and to characterize the oil of regional origin. Twelve olive microsatellite markers were used for identification and differentiation of a set of 27 olive accessions grown in Istria (Croatia). Among the 27 accessions, 18 different SSR profiles were discriminated. All 12 microsatellite markers analysed were polymorphic, revealing a total of 81 alleles. The number of alleles per locus ranged from four to nine. This is the first molecular characterization of olive germplasm in Croatian Istria. The analysis clarified the genetic relationships of varieties native to Croatian Istria with introduced olive varieties, as well as with varieties in the neighbouring Slovene Istria region. Numerous varieties in neighbouring regions showed high similarity and a few cases of synonymy (‘Bilica’-‘Bjankera’; ‘Buga’-‘?rna’) and one Croatian-Slovenian homonymy (‘Bu?a’-‘Buga’) were observed. The results provide useful information for a native germplasm survey and can be used for the construction of a unique database comprising all olive varieties in the Istrian region of Croatia and Slovenia.     

SSR markers reveal the uniqueness of olive cultivars from the Italian region of Liguria

Twenty-three important Ligurian olive accessions corresponding to 16 cultivars were studied using 12 SSR markers and 40 Mediterranean cultivars were included in the study in order to investigate the relationships between Ligurian and Mediterranean germplasm. All SSRs produced polymorphic amplifications. One hundred and forty-nine alleles were found in the 63 accessions analysed. Twenty-two alleles were specific to germplasm from Liguria and of these 12 were unique to single cultivars. Heterozygosity and discriminating power calculated in this regional germplasm were high on average (0.70 and 0.74) and not so much lower than the values in the total sample that includes cultivars from different Mediterranean countries (0.77 and 0.88 respectively). No cases of genetic identities were found between Ligurian and Mediterranean accessions. Several cases of homonyms and synonyms within the Ligurian germplasm were explained. Cluster analysis generally revealed a clear discrimination of the profiles from Liguria and Italy with respect to the cultivars from other Mediterranean countries. Only one Ligurian cultivar, “Negrea”, appeared to have a different origin, grouping with the Mediterranean cultivars.     

The effect of waste water irrigation on the extra virgin olive oil quality from the Tunisian cultivar Chemlali

The availability of fresh water is very scarce in the Middle East and North Africa. Yet consequently, brackish water or recycled wastewater can be used for irrigation, minimising the exploitation of natural water resources. The aim of this work was to study how irrigation with treated wastewater (TWW) over 4 years affects some parameters of “Chemlali” olive oil quality. Two treatments were considered: trees irrigated with treated wastewater and others cultivated in rain-fed conditions. Results showed that irrigation with TWW over 4 years did not affect free acidity, and specific ultraviolet absorbance at K₂₃₂ and K₂₇₀. For both the crop seasons the mean values of these parameters were lower than the upper limits established for “extra virgin” olive oil. However, irrigation with TWW cumulatively increased palmitic, palmitoleic, linoleic and linolenic acid contents and decreased oleic acid rates. A significant decrease of total phenol contents was also observed at the end of experimental period.     

Effect of alleviating products with different mode of action on physiology and yield of olive under drought

Two years old self-rooted Koroneiki olive trees (Olea europaea L.) were subjected to two irrigation regimes, i.e. the fully irrigated and the severely water stressed trees, while they were treated with three alleviating products of different mode of action. The products used were the osmolyte glycine betaine, the antioxidant Ambiol and the heat and irradiance reflecting kaolin clay particles. The effects of product application and water regime on leaf characteristics, shoot and root growth, photosynthesis, leaf compatible solids (carbohydrates) concentration and yield were evaluated. All products applied, exhibited significant alleviating action, based on the relative alleviation index. Irrigated trees exhibited greater growth than drought stressed ones, while the ameliorating products maintained the water content of the leaves under drought conditions and resulted in lower leaf tissue density. On the other hand carbon assimilation rate, stomatal conductance and intrinsic water use efficiency were significantly reduced under drought stress, while the opposite stood for intercellular CO₂. Drought stress resulted in elevated sucrose leaf concentration, while the application of Ambiol increased stachyose concentration and that of glycine betaine did the same with the mannitol concentration. Among the alleviating products tested in this experiment Ambiol and glycine betaine had a significant positive effect on leaf water content, photosynthesis and yield under both drought and well irrigated conditions.     

Calcium increases sodium exclusion in olive plants

‘Picual’ olive cuttings were grown in a greenhouse under saline conditions in 2 L plastic pots containing perlite. Plants were irrigated with a nutrient solution plus 75 mM NaCl and 0, 2.5, 10 or 40 mM CaCl₂. Vegetative growth, leaf and root Na⁺ and Ca²⁺ concentrations were measured. Na⁺ toxicity symptoms were observed in plants non-treated with Ca²⁺. Shoot length was higher in Ca²⁺ treated plants, although shoot growth was reduced at 40 mM CaCl₂, probably due to the high total ion concentration reached in the external solution. Ca²⁺ supply linearly increased leaf and root Ca²⁺ concentration and decreased leaf Na⁺ concentration. However, there were no differences in root Na⁺ concentration. Results indicate Ca²⁺ may take part in the Na⁺ exclusion mechanism, mainly preventing Na⁺ transport to the shoot, that may be an important ability for survival under saline conditions.     

Identifying the location of olive fruit abscission

For high quality oil, table olive characteristics, and reduction of alternate bearing tendencies, it is often desirable to harvest olive fruits prior to full physiological maturity, that is, before the natural separation zone has differentiated. Olive fruit abscission can occur at different positions, mainly fruit-pedicel, pedicel rachis and peduncle-branch, apparently varying according to cultivar, fruit weight, and fruit maturation. Precisely identifying the location of separation is critical for studying the differentiation of the abscission zone or zones and testing the effectiveness of harvest procedures, including the mode of action of different fruit-loosening chemicals. We determined the olive fruit abscission zone position under natural conditions, throughout the complete fruit maturation period, for two cultivars differing in fruit size. The major separation zone for the cultivars studied was between the fruit and pedicel. For cv. Picual, abscission in zones other that fruit-pedicel occurred in less than 15% of the fruit at all dates throughout the maturation period, whereas in cv. Hojiblanca, the percentages of fruits detached at peduncle-shoot (27%) and pedicel-rachis (19%) were initially substantial, and then decreased progressively during fruit maturation. Our data clearly indicate both varietal and temporal differences which should be taken into account not only when testing fruit-loosening compounds but in extrapolating test results to different varieties or dates. Fruit weight did not appear affect the abscission location. The method we used, of counting the different detached units, is simple and effective for determining the position of fruit abscission.     

Encapsulation of in vitro-derived explants of olive (Olea europaea L. cv. Moraiolo): II. Effects of storage on capsule and derived shoots performance

The experiments described in this paper were designed to examine the effect of storage at different temperatures for various periods on regrowth and proliferation ability of encapsulated olive microcuttings (capsules). The proliferation and rooting ability of the shoots derived from the capsules were compared with those maintained in normal proliferation by 45 days period of subculturing. A simple method of encapsulated microcuttings storage in plastic cuvettes was used. Interesting feature was the ability of the capsules to retain their viability and regrowth after storage at room temperature as well as at 4 °C for 15 and 30 days and both axillary buds on the nodes of almost all capsules sprouted successfully. Improvement in proliferation and rooting abilities were found in subsequent culturing of the shoots developed from the capsules. The study indicates bright prospects of commercial application of alginate nutrient capsule technology in various situations like supply of certified plant material from both official or private organizations to the plant nurseries and/or exchange of germplasm at international level without refrigerated containers just in plastic cuvettes.     

Effects of irrigation with treated wastewater on olive tree growth,yield and leaf mineral elements at short term

In arid regions, such as Tunisia, the reuse of treated wastewater (TWW) in agriculture can be a sustainable solution for water scarcity. A two-year field experiment was conducted in order to investigate the short-term effects of TWW on olive growth, yield and concentration of total nitrogen (N_t), potassium (K), phosphorous (P), and heavy metals (i.e. Zn, Mn, Pb and Cd) in olive leaves. Olive trees were subjected to the following irrigation treatments: (i) trees irrigated with well water (WW) and (ii) trees irrigated with treated wastewater (TWW). For both treatments, the TWW and WW were applied at a rate of 4.5 m³ day⁻¹ tree⁻¹ (5000 m³ ha⁻¹ year⁻¹). After two years, non-significant injuries caused by salts and/or heavy metals were observed on shoot growth of trees irrigated with TWW. The application of TWW significantly increased concentration of N_t, P and K in the leaves, whereas heavy metals (Zn and Mn) showed a significant increase only after the second year of irrigation.     

Cultivar-based fruit size in olive depends on different tissue and cellular processes throughout growth

In drupe fruits, in addition to fruit size, the proportions of mesocarp and endocarp tissues are critical objectives for fruit quality, crop production and management. The olive fruit is a typical drupe, with cultivars which show a wide range in both fruit size and the proportions of mesocarp and endocarp. Characterizing the roles of tissue and cellular processes in producing genetically based fruit size variability is necessary for crop improvement, as well as deepening our understanding of fruit developmental physiology. This study used microscope image analysis to evaluate cell number and size, the growth of mesocarp and endocarp tissues, and their developmental timing in producing fruit size among six olive cultivars with a large range of fruit size. We found that cultivar mesocarp and endocarp size increased linearly with fruit size, with larger sizes favoring an increasingly greater mesocarp/endocarp ratio. Within the mesocarp, cultivar-based fruit size related directly to cell number and was established soon after bloom by cell division rate. In spite of different cell division rates, all cultivars showed similar timing of cell division activity, with the majority of cells produced in the two months after bloom but, surprisingly, a substantial number of cells formed during the following 6 months. Cell expansion was high throughout fruit growth and an important factor in achieving final fruit size, but cell size did not differ among cultivars at any time. We can conclude that fruit size differences among olive cultivars are due at the tissue level to both mesocarp and endocarp sizes and at the cellular level to cell division throughout fruit growth. Furthermore, since cell size is consistent among cultivars in spite of variable cell division, it is likely that cultivar differences in cell expansion accompany those in cell division.     

Microsatellite markers based assessment of genetic diversity in Iranian olive (Olea europaea L.) collections

To study the genetic variation in Iranian olive collections and some foreign olive cultivars, 47 accessions of 18 local cultivars from 6 olive collections of Iran (Roudbar, Zanjan, Ahvaz, Dezful, Kazeroon and Shiraz), were analyzed along with 30 imported cultivars using 16 microsatellite primer pairs. All the used microsatellite loci revealed polymorphism in the studied genotypes, except GAPU14 and GAPU113 markers. Fourteen microsatellite primers amplified 126 polymorphic alleles in the 87 selected olive accessions. The average number of alleles per locus was 9, ranging from 3 to 14. Polymorphic information content (PIC) was 0.85. The genetic similarity based on Jaccard coefficient ranged from 0.15 to 1. The genetic relationships among accessions were investigated using cluster analysis and principal component analysis (PCA). Most of the accessions with the same name were grouped together; some exceptions were also observed. As expected, close relationship was observed among accessions within same cultivar. Most of the Iranian olive accessions were clustered to a main distinct group. Two-dimensional scatter plot of principal component analysis revealed a clear separation of most of the Iranian olives from Syrian and other introduced cultivars. These suggest that Iranian cultivars have different origin related to West Mediterranean basin cultivars and have evolved independently from the others. Between and within Iranian and foreign cultivars (cultivars including three or more accessions) genetic diversity was analyzed using analysis of molecular variance (AMOVA). AMOVA revealed higher within cultivar genetic variation (62.76%) as compare to that between cultivar variations (37.24%). The intra- and inter-cultivar variance tested by permutation test showed significant genetic variation at both levels. The high level within cultivar genetic variance could be due to mislabeling and presence of homonyms in cultivars produced by vegetative propagation from original plants.     

Early growth habit and vigour parameters in olive seedlings

Fruit tree growth habit and vigour are important traits for orchard management and production, yet are difficult to select for in breeding programs because the parameters for their evaluation are complex, and their expression often requires sufficient plant size and development, entailing valuable time. Thus, in olive (Olea europaea L.) breeding, as for other fruit crops, it is critical to define growth habit and vigour parameters with selection potential, and to determine the earliest age at which they can be measured. Furthermore seedling growth habit traits are important in themselves, particularly in their relation to juvenile period length and management. To explore these issues we evaluated a series of new and standard growth habit parameters during the first year of growth in a population of unpruned olive seedlings originating from six different crosses. The influence of plant age on growth habit traits was determined by comparing measurements at two or three different times. Both parent genotype and plant age significantly affected the vigour and growth habit of the olive seedlings, and 9 months was the most appropriate age for evaluating seedling growth parameters. ‘Picual’, ‘Arbosana’ and ‘Sikitita’ were shown to be promising cultivars for use as genitors, because of their tendency to produce offspring with desirable growth habit traits such as high vigour, weeping habit and low lateral shoot number. From among the 17 parameters studied, based on parent influence and the absence of correlation among parameters, five were identified which best described olive seedling growth habit: Primary Shoot Top Diameter, Primary Shoot Conicity, Secondary Shoot Number, Secondary Shoot Insertion Angle and Longest Secondary Shoot Internode Length.     

Fruit set is inversely related to flower and fruit weight in olive (Olea europaea L.)

Fruit weight is strongly correlated to ovary weight in olive as big-fruited cultivars have larger ovaries and flowers at bloom. We tested the hypothesis that larger ovaries imply stronger sinks and therefore reduce fruit set, expressed as number of fruits. Flower/fruit load per centimeter of shoot was assessed every month from flowering to harvest, on sample shoots of many different olive cultivars differing in fruit size. Data were taken for two years: 2007 and 2008. Additionally, in 2008, a fruit thinning experiment was carried out by leaving a single fruit per shoot, to assess the maximum potential fruit weight achievable by each cultivar. Results indicated that fruit size was mostly genetically determined as thinned fruits of small-fruited cultivars, although bigger than control fruits, never got as big as fruits from large-fruited cultivars, confirming that large ovaries are a prerequisite for large fruits. Among all cultivars, the number of flowers per inflorescence, the number of inflorescences, and the number of flowers per centimeter of shoot were not correlated to average flower weight while total flower mass per centimeter of shoot was positively correlated to average flower weight (i.e. large flowered/fruited cultivars had greater total flower mass). However, one month after bloom and thereafter, all parameters were negatively and exponentially correlated with fruit average weight across cultivars, except fruit mass per centimeter of shoot which was generally not correlated to fruit average weight, except in one year, at harvest, when fruit mass per centimeter of shoot was positively correlated to fruit average weight. These results suggest that fruit set is a consequence of, and inversely proportional to flower/fruit size in olive.     

Pistil abortion is related to ovary mass in olive (Olea europaea L.)

Pistil abortion is considered to be an evolutionary adaptation to save resources in andromonoecious species, balancing the number of pistils with the resources available. It follows that large-fruited species/cultivars should have greater levels of pistil abortion. Working in olive, we found that pistil abortion, expressed as percentage of staminate flowers, was positively correlated to the average ovary mass at bloom, across cultivars with different ovary/fruit mass. Since ovary mass correlated with fruit mass, pistil abortion also correlated with fruit mass. The absolute number of perfect flowers per inflorescence was negatively correlated with both ovary mass and pistil abortion, while the number of staminate flowers per inflorescence increased with both parameters. The total number of flowers (i.e. staminate + perfect) was not correlated to ovary mass or pistil abortion, suggesting that male function was not affected. The results support the hypothesis that pistil abortion is related to competition for resources among ovaries, and suggest that genetic differences in pistil abortion among olive cultivars may be explained by their different pistil mass and sink strength.     

Intra-cultivar variability of three major olive cultivars grown in different areas of central-southern Italy and studied using microsatellite markers

A collection of 70 olive samples, originating from diverse areas in central-southern Italy (Abruzzo, Apulia, Calabria, and Umbria) and corresponding to 3 major cultivars denominations (‘Carolea’, ‘Coratina’ and ‘Frantoio’), was genotyped at 10 microsatellite loci. In total, 44 alleles with a mean number of 4.4 alleles per locus were detected. The molecular analysis, allowed the study to show a clear genetic diversity between the three cultivars ‘Carolea’, ‘Coratina’ and ‘Frantoio’ and to state that ‘Carolea’ is a polyclonal cultivar, while ‘Coratina’ and ‘Frantoio’, are probably monoclonal ones. The analysis of intra-varietal polymorphism, through the SSR analysis, proved to be very useful both for varietal identification and for intra-varietal ones. Our work shows that the current designations of olive cultivars fall short of describing the genetic variability among economically important plant material. A thorough investigation of the existing variability will prove of major importance for both management and economic production of olive trees.