Abstract: This study was carried out to describe physiological and morphological attributes that differentiate 12 Tunisian olive varieties cultivated in the collection of Chott Mariem (arid semi climate) in period of severe drought. Our results show that there were significant genetic differences for stomatal density, number and diameter of the trichomes peltates, length and the relative surface of stomatal pore, stomatal conductance, transpiration rate, elasticity of the petiole and for water losses from detached leaves. The classification of the various varieties according to their favorable behavior to water deficit show that 'Zarrazi' appears the most adapted to drought among the 12 studied varieties. In addition 'R'khami' appears the most sensitive variety to drought.
INTRODUCTION
Olive cultivation is spread in many regions around the world with temperate and subtropical climate, constituting an economically important crop for most Mediterranean countries. Olive, an alternate bearing evergreen tree, is of special interest, since its fruits and leaves have been demonstrated to be important organs for the synthesis of several biological compounds (Rugini and Fedeli, 1990).
Drought is one of the major causes for olive yield reductions and this is particularly true in the Mediterranean Basin where the climate is typically characterized by high potential evaporation and low rainfall during the growing season. Improvements in olive drought tolerance are therefore sought through plant breeding cultivars with better ability to access soil water and with improved water use efficiency could increase yields in an economically and environmentally sustainable way. According to Fernández et al. (1997) the knowledge of the mechanisms implied in drought resistance can help to optimize the water supply in olive orchards.
Olive leaves tolerate extremely low water potential (Rhizopoulou et al., 1991), characterizing them between sclerophyllous desert shrubs and mesophyte conifers (Bongi et al., 1987). Under water deficit, the olive tree shows a low growth rate (Giorio et al., 1999).
The olive-tree is a famous species for its rusticity and its adaptation to drought. Several aspects concerning its capacity to resist to the arid environments were studied, of which most recent are interested in the effect of water or saline stress on the photosynthetic assimilation and the anatomy of the leaves of several olive varieties carried out by Chartzoulakis et al. (1999, 2002); Gregoriou (1999), Bosabalidis and Kofidis (2002) and Loreto et al. (2003). Other studies showed that in addition to the structural aspects for resistance to water deficit, olive tree has active mechanisms which allow it a certain degree of control of the water losses (Fernández et al., 1997). The olive-tree (Olea europaea L.) has been considered as one of the hypostomatous species best adapted to the semiarid Mediterranean environment and is traditionally grown under drought conditions (Gimenez et al., 1997). The great capacity for absorption of the radicular system as well as the maintenance of a positive turgidity under the extreme conditions of water deficit was suggested as a key of a special adaptation to drought (Fernández et al., 1991; Dichio et al., 1997).
The development of olive tree in Tunisia is due to its capacity of resistance to drought. Indeed, this species increased the value of the arid and semi arid zones from the center and the south of the country. Nowadays, olive growing sector knows a difficult period due to the degradation of the traditional farming system and it is characterized by a mode of control based on an optimal exploitation of rainfed conditions. Thus the effect of drought, periodic phenomenon in Tunisia, is marked more and more on the trees.
As olive is a tree with a low growth rate, physiological rather than morphological adjustments are predominant adaptation mechanisms to water deficit in the short term (Lakso, 1985). Other studies are necessary to establish the degree by which the structural aspects and the active mechanisms play a role in the rationalization of water use. In this context the present research is a comparative study of some morphological and physiological aspects related to water losses of some Tunisian olive varieties in condition of drought, with the aim of understanding the strategies adopted by this species to resist to water deficit.
MATERIALS AND METHODS
Field conditions and plant material: This study was carried out in the experimental Station of Chott-Mariem in Sousse (Tunisia). This experimental station established on an area of 0.5 ha contains a collection of local and foreign varieties of olive-trees planted in 1991, in square with the spacing 7x7 m with the density of 200 tree/ha. The experiment coincided with one dry year. The two following years are also already dry. Indeed, the annual average rainfed recorded during the year of the study (2002-2003) and the two previous years which preceding it (2000-2001); (2001-2002), are respectively 250.6 mm against an annual average for the area calculated over 25 years of 350 mm.
For our study, we were interested in 12 local varieties. Such varieties were selected according to their origin in order to have plants from different regions. These varieties are: Zarrazi (south), Meski (north), Neb jemel (Sahel), Fouji (Gafsa), Gerboui (Téboursouk), Chetoui (north), Tounsi (Gafsa), R' khami (Cap bon), Dressi (Téboursouk), Dhemia (Nabeul), Fougi vert (Cap bon) and Dhabia (Korbous).
Evaluation parameters: Peltate trichomes were removed from the lower surface of the leaves using an adhesive tape before the measurement of the stomatal density for each cultivar because the lower leaf surface is densely covered by stellate hairs, it was impossible to get impressions unless the hair had been removed. Observations were conducted using the Windias software and a microscope type Leitz DIALUX 22 EB with the enlargement 250 times.
The relative surface of the stomatal pore was calculated starting from the following formula: RS = (a.b)/4.Ds.Π.10-4 RS is the relative surface of the pores in %, a and b are respectively the length and the width (which is taken arbitrarily equalizes with 6 μm) of the stomatal opening in μm, Ds the number of stomata per mm2, (Meinder and Mansfield, 1968).
To quantify the resistance of the petiole to bending (elasticity) a standard weight of 0.723 g was suspended from the test leaf by a 2 cm long thread held by a pin positioned exactly 4 cm from the point of attachment of the leaf to the stem (Schwabe and Lionakis, 1996).
Stomatal conductance and transpiration rate were evaluated by a portable porometer (Steady State Porometer model LI 1600, Neb U.S.A). The measurements were made on eight replicate leaves for each variety between 10:00 and 11:00 h.
The loss of water from detached leaves was measured by repeated weighing of individual leaves at 15 min intervals. The leaves were arranged with the lower surfaces in full daylight but not exposed to direct radiation from the sun. Water loss was expressed as percentage of the original fresh weight, immediately after leaf detachment.
Statistics: Statistical data analysis was performed by analysis of variance (ANOVA). Ducan tests were carried out to test significance of differences between treatment means using the SPSS for Windows, release 10.0.
RESULTS AND DISCUSSION
The stomata of olive-tree are present only on the abaxial surface of the leaves (hypostomata). Stomatal density oscillates between 312 and 470 mm-2 of leaf area depending on cultivars (Table 1). The extreme values of this parameter correspond to Meski (312 stomata mm-2) and to Tounsi varieties (470 stomata mm-2) (Table 1).
| Table 1: | Leaves morphological characteristics of the 12 studied olive tree varieties |
| * The numbers followed by the same letter(s) are not significantly different at 0.05% | |
Bongi et al. (1987) reported lower stomatal density in a cultivar originated from a warm area, however, Bosabalidis and Kofidis (2002) found an increase in stomatal number, under water stress, for Koroneiki. Hagidimitriou and Pontikis (2005) note values of stomatal density ranging between 399 and 500 stomata mm-2 on Greek olive-tree. Length of the stomatal pore varies from 12.14 to 16.89 μm depending on cultivars. These values are rather weak compared to those reported in other specie.
The relative surface of the stomatal pore ranged between 1.96 and 2.89% (Fig. 1). These values were relatively high compared to those reported for other specie. Mougou (1984), note values ranging between 0.62 and 1.35 % for this parameter in tomato varieties while Al Farraji (1983), found that this criterion varies between 1.8 and 3.4% for the six tree species studied under field conditions. Peltate trichomes are present on both faces of the leaf, but their number is about eight times greater on the abaxial than on the adaxial surface (Table 1). The diameter of these structures in the 12 studied cultivars ranged from 139 to 191 μm. the most significant values of this parameter are noted for the varieties Meski (184 μm) and Chetoui (191 μm) and the weakest ones in Zarrazi (139 μm) and Fougi (148 μm). Meski had the lowest trichomes number, 100 mm-2 and Tounsi the highest, one 200 mm-2 (Table 1).
Bongi and Pallliotti (1994) observed that trichomes are a barrier to the diffusion of CO2 and H2O, lowering the boundary layer conductance in the air surrounding the stomata. After removing trichomes from leaves of 'Manzanilla' olive, they found that the total boundary layer resistance was reduced more than 5 fold. Schwabe and lionakis (1996), however, questioned the efficiency of trichomes in reducing water loss, apart from increasing the reflection of radiation.
| Fig. 1: | Relative surface of the stomatal pore of the 12 studied olive tree varieties. Each value is the average of 30 measurements |
Gerboui variety, showed the highest elasticity of leaves (32.6°) whereas the lowest value of this parameter (13°) was recorded in Fougi vert and R' khami. The high elasticity of leaves increased the capacity of olive- to change the orientation of leaves relative to stem; this behavior reduces the amount of intercepted radiations in period of drought and influenced water consumption. The change of leaves orientation as quantified by the leaf/stem angle with the water status of the plant was reported by certain authors. According to Schwabe and Lionakis (1996), this angle is quantitatively related to the water content and water potential of the leaves and other characteristics such as the stomatal aperture, the rates of water loss and total desiccation of leaves.
Gerboui variety shows the highest level of transpiration rate (19.15 mg m-2 sec-1) (Table 2). The low level of this parameter is recorded in Fougi vert variety (9 mg m-2 sec-1) (Table 2). Boujnah (1996), found a mean values ranging between 12.5 and 15.5 mg m-2 sec-1 in various Tunisian olive cultivars. According to Kramer (1983), the intensity of transpiration is the result of a complex interaction between the factors of the environment, of which most significant are radiation, vapor pressure deficit, temperature, soil water availability and other factors related to the plant such as the leaf area, epidermal hairs, the stomatal density and root characteristics. According to this author, the change of one of these factors does not produce necessarily a proportional change in the rate of transpiration, because this parameter is controlled by more than one of these factors.
There were significant differences in stomatal conductance between the 12 cultivars (Table 2). The highest value of this parameter is recorded for the variety Dhabia (1.13 cm sec-1). Indeed, Gerboui and R'khami express a relatively low stomatal conductance estimated to 0.34 and 0.36 cm sec-1 respectively; these values are similar to those reported in the literature for olive trees grown under a various conditions, when measured during the hottest hours of a summer day Giorio et al. (1999); Proietti and Famiani, (2002) and Loreto et al. (2003).
| Table 2: | Transpiration (mg m-2 sec-1) and stomatal conductance (cm sec-1) measured from leaves of 12 studied cultivars. Each value is the average of 12 measurements |
| *: the Numbers followed by the same letters are not significantly different at 0.05% | |
Moriana et al. (2002) reported that leaf stomatal conductance, in olive trees, was high during autumn and low during summer when vapor pressure deficit was high. Studies on the effect of drought on olive leaf gas exchange showed that stomata play an important role in controlling photosynthesis (Angelopoulos et al., 1996; Fernandez et al., 1997). In many cases, good positive relationships were generally found between stomatal conductance, leaf water potential and soil moisture. This indicated that both hydraulic feedback and feed-forward mechanisms could be invoked in the response of stomata to soil drying (Giorio et al., 1999). Soil or root water status affecting directly stomata have been recognized in many plants when submitted either to split-root or to root pressurization experiments, but root to shoot chemical signaling has also been invoked to explain the independence of gs from shoot water status (Zhang and Davies, 1990).
The data of water loss from detached leaves are presented in Fig. 2-4. The curves of leaves desiccation follow a similar pattern, although the two varieties (Meski and Gerboui) close their stomata more faster, that is why their water losses are stabilized more quickly (from the first 17 min) in comparison with the other varieties which maintain their stomata opened for a longer time. In addition, the varieties (Dressi, Chetoui, Fougi vert and Tounsi) close their stomata only belatedly after 60 th min.
| Fig. 2: | Cumulative water loss from detached leaves of Neb jemel, Meski, Gerboui and Fougi varieties on lower surfaces during drying |
For the other varieties (Fougi, Zarrazi, Dhemia, R' khami) the water losses are stabilized with in 45th min. They seem to be best adapted to drought and can have the ability of maintaining a certain biological activity in spite of water deficit.
| Fig. 3: | Cumulative water loss from detached leaves of Dressi, Dhabia, Chetoui and Fougi vert varieties on lower surfaces during drying |
| Fig. 4: | Cumulative water loss from detached leaves of Tounsi, Zarrazi, Dhemia and R’khami varieties on lower surfaces during drying |
| Table 3: | Classification of the 12 varieties of olive-tree compared to their tolerance to drought |
The 12 studied cultivars seem to have different capacities of stomatal control. Similarly, Schwabe and Lionakis (1996), found a clear difference in stomatal behavior between irrigated and stressed trees of koroneiki variety.
CONCLUSIONS
Our results show that Olive leaves are well-adapted to avoid excessive water loss under the highly demanding conditions of the areas where the tree usually grows. They show not only several sclerophyllous characteristics, but also active mechanisms controlling water loss.
Many significant differences were found between the 12 studied varieties. These variations seem to be related to their genetic background. To have ideas on the aptitude of each cultivar to support the conditions of summer drought when this study is carried out we will try to establish a classification of the 12 varieties considering their aptitude to avoid, limit or support drought. We will give to each variety an order for each studied character. The first position is given to the variety which seems to be more tolerant to drought. The final score will be established by making the sum of all the filings for each variety (Al Farraji, 1983). So, the following statements are considered:
| • | A higher number of stomata is considered as a good criterion since it allows cooling of leaves, so the first position is given to variety with higher number of stomata. In addition a weak relative surface of stomatal pore is also a good criterion of adaptation because it allows to leaves to avoid intense water losses. |
| • | The trichomes act as a barrier to the diffusion of H2O and CO2, lowering the boundary layer conductance in the air surrounding the stomata, a high number of these structures are being effective in limiting water loss. |
| • | The aptitude of the leaves to change orientation (elasticity of the petiole) is a good criterion that reduces the exposure of the stomata to higher temperatures. |
| • | Stomatal closure is an active mechanism for preventing excessive water stress under conditions of high atmospheric demand, so species with higher transpiration rate are more adapted to drought |
| • | The water losses on detached leaves shows a better behavior for the varieties which are able to maintain their stomata open during one longer period in spite of water deficit. |
According to the results mentioned in Table 3 we then could classify the 12 varieties studied according to their tolerance to drought. Zarrazi variety appears the most adapted to drought and occupies the first position. In addition R'khami variety appears most sensitive to drought and occupies the last position. This selection will be continued in a future work by other anatomical and biochemical criteria.
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance provided in conducting the experiments by the technicians of the Institute of Olive Tree at Sousse Station.