Stone pine (Pinus pinea L.) is a Mediterranean species characterized by great phenotypic plasticity, high tolerance to a wide range of environments and low genetic variability; it is also of great economic importance conferred by its edible seeds, the pine nuts, one of the most expensive and exclusive nuts worldwide. Advances in semi-intensive or intensive management for its cultivation as a fruit tree, however, have been limited, with fruits being harvested mostly from natural forests. This can be attributed to the long fruit development cycle (42 months), with one-, two- and three-year-old cones being present simultaneously on the tree, which makes nutritional and hydric management more challenging than in other fruit or forest trees, among other causes.
This thesis studied stone pine growth and fruiting along a climatic gradient in Chile; species variability through morphometric, chemical, Near Infrared Spectroscopy (NIRS) and molecular analyses (proteomic); species adaptation by assessing growth and fruiting response of European provenances in central Chile; and the effects of fertilization and irrigation on fruit production and growth.
Growth was examined in 4,094 trees located in 129 sites distributed across Coquimbo and Araucanía regions in Chile, via several climatic variables, whose relative contribution was assessed through regression trees and linear models. Three macrozones (MZs) showing marked growth differences were defined in the species distribution range: North, South and Dry Coast MZs. In the South MZ, growth rate was the highest for height (35 cm year-1) and DBH (1.50 cm year-1), whereas in the Dry Coast MZ, the species showed the lowest growth rate in height and DBH (23 and 0.87 cm year-1 respectively). Height growth was favored by winter thermal oscillation above 14°C, annual rainfall above 1,400 mm year-1, an annual average temperature below 14°C and a spring water deficit below 400 mm. DBH growth was also favored by an average annual temperature below 14°C and by a minimum average temperature below 6.6°C.
Fruiting was analyzed by recording cone number per tree (CN) on 3,464 trees distributed along a climatic gradient in Chile. Cone weight at harvest (CW) and in-shell pine nut number per cone (IS) were measured on 76 superior trees. Climatic and biometeorological
variables, defined based on 11 physio-phenological reproductive phases, were related to fruit production traits. Fruiting showed marked differences among MZs. The highest values of cone productivity (32 kg tree-1) and CN (62 cones tree-1) were recorded in the South. Stone pine cone production throughout Chile was favored in decreasing importance by autumn thermal oscillation below 12°C, spring hydric deficit lower than 345 mm, spring rainfall above 65 mm and annual thermal oscillation below 12°C. It was also favored by a minimum temperature above 7°C and a hydric deficit below 200 mm in the female flowering period, by a rainfall below 70 mm during male flowering and a thermal oscillation below 6°C during differentiation of reproductive shoots.
Cone weight was favored by an annual average temperature above 14°C, annual rainfall above 507 mm and an annual hydric deficit below 914 mm; at a maximum temperature higher than 23°C and a hydric deficit lower than 774 mm during embryo development also increased cone weight, as well as an accumulated rainfall higher than 14 mm during the 2 year-old conelet growth period.
IS was significantly increased by an annual hydric deficit below 914 mm and a maximum autumn temperature lower than 19°C, along with a maximum temperature above 24°C during 2 year-old conelet growth, as well as by accumulated rainfall during three key periods (values above 53 mm in induction of male primordia, 78 mm in 3 year-old cone growth and 133 mm during cone ripening). Therefore, water supply is recommended to mitigate the negative impact of water deficit on fruit productivity.
Species variability was studied based on morphological, chemical, NIRS and molecular characterization of stone pine fruits harvested from the three growth MZs. The morphometric analysis showed that stone pine selected trees from the different MZs differed in cone size and pine nut morphometry, and cone to kernel yield. Chemical composition assessed at the MZ level and for 38 selected trees was similar to values reported for the species in the main producing countries, (34.1-35.5% protein, 44.2-40.2% lipids and 4.7-4.4% ash respectively). Chemical composition of selected trees was homogeneous among MZs, with the exception of oleic acid, which was higher in the South MZ, and potassium, which was higher in the Dry Coast MZ. The analysis by macrozones showed that the North MZ had the highest ash content and the Dry Coast MZ the highest values of moisture, protein and lipids. Average phytosterol content reached 2,113 μg/g oil and tocopherols 113 μg/g oil. Stand-alone NIRS measurements were employed for identifying pine nut geographical origin by testing different sample presentations (in-shell and shelled pine nuts, and humid and dried flour). This analysis demonstrated the potential of this technology, since it is proved to be a rapid and accurate method for predicting the geographical origin of pine nuts in Chile; indeed, 87.8% of samples from the three MZs were correctly classified at the cross-validation stage, and the best model was obtained with shelled pine nuts. Proteomic analysis also allowed us to differentiate populations growing in the three macrozones of the species in Chile, and proved to be a useful tool to detect variability between populations of different environments, with seven proteins being identified as population markers. Proteins related principally to nutrient reserve, heat shock stress, glycolytic processes, oxidoreductase and peroxiredoxin activity, and in lower proportion, to carbon fixation and ATP binding, were identified, with 12% remaining unidentified. In summary, the present analysis of stone pine populations in Chile, involving different techniques, showed that the species variability is determined by geographical and climatic factors as a gene expression probably associated with adaptive strategies.
Growth and cone production of six European stone pine provenances established in central Chile were analyzed at the age of 19 years and survival during the first 7 years. The studied provenances significantly differed in cone number per tree and weight, and DBH, height and crown diameter growth. Provenances were separated into three groups, according to growth and productive variables: Italian and Slovenian; Andalucía and Sierra Morena (Spain); and Meseta Castellana (Spain), the latter showing the highest growth and productivity. Individual cone production was positively correlated with cone weight and growth variables.
Afterwards, individual and combined effects of fertilization and irrigation on growth and fruiting, and their correlation, were assessed in plantations located in central Chile. In a 30 year-old plantation (Cáhuil) treatments consisted of control, spring and autumn fertilization, spring and summer irrigation, and combined fertilization and irrigation; and in a 16 year-old plantation (Toconey), treatments included control and spring fertilization. In Cáhuil fertilization had a positive effect one year after application (23.5% superior height growth and 82.3% superior 1 year-old conelet production vs control); after two consecutive years of application, a significant influence on DBH (17.5% superior vs control), height (20.4% superior), and 1 year-old conelet production (36.4% superior vs control) was observed, independently of the hydric status of the trees. Irrigation enhanced fruiting but did not affect growth. The maximum 1 year-old conelet number was observed in fertilized and irrigated plots (60% superior). In Toconey, tree growth in fertilized plots was higher (41% superior height and 29% crown diameter), and bore 94% and 2.4 times more cones vs the control 3 and 4 years after fertilization. Fertilization also affected cone size, increasing the quantity of heavier cones during the two following harvests. Both cultural practices, applied either individually or combined, are useful tools to enhance fruit production in young and adult stone pine plantations, with a stronger response if applied at younger ages.
Finally, this thesis proposes an intensive management model for stone pine cropping for fruit production in Chile, including techniques commonly used in horticulture, such as fertilization, irrigation, weed control, grafting and different pruning types, including root pruning.
Overall, the scientific results obtained have confirmed the general hypothesis of this doctoral thesis, i.e., stone pine is well adapted to soil and climate of Chile within a wide range of environmental conditions, and has a production capacity that allows us to predict productive levels similar to or higher than those in its native habitat, which makes the species cultivation an important productive alternative for the country.
This thesis makes a valuable scientific contribution to the advancement of stone pine knowledge in several aspects relevant for its domestication, opening new research topics that should be addressed in the future.