A COMPARISON OF ROOT ARCHITECTURE AND SHOOT MORPHOLOGY OF CERCIS SILIQUASTRUM L. BETWEEN TWO WATER REGIMES

A COMPARISON OF ROOT ARCHITECTURE AND SHOOT MORPHOLOGY OF CERCIS SILIQUASTRUM L. BETWEEN TWO WATER REGIMES Rahman M.S., Tsitsoni T.K. and Tsakaldimi M. N. Aristotle University of Thessaloniki, Department of Forestry and Natural Environment, Laboratory of Silviculture, P.O. Box 262, 54124, Thessaloniki, Greece e-mail: tsitsoni@for.auth.gr Summary The survival of seedlings in the field depends on their morphological and physiological characteristics and the environmental conditions of the planting site. This study was undertaken to identify seedlings morphological characteristics that ensure better field performance Cercis siliquastrum L. The purpose of this research specifically, was to answer to the following questions: (1) to what extent do the seedlings shoot morphology and root architectural attributes differ under water stress compared to well- watered conditions (2) do these variations effect, in the field. The first question will help to determine the drought responses to seedling morphology and the second question relates to seedling desired attributes that will enhance the survival and growth of transplanted C. siliquastrum seedlings in the field. The study was conducted at the Arboretum of the Laboratory of Silviculture, AUTh, Greece. The experiment was set in Randomized Complete Block Design with two treatments and three replications. Treatment 1 (Control): Regular spot irrigation was applied daily. Treatment 2 (Stressed): No additional water except rain water during the summer months (from June, July, August and September 2012). All seedlings were hand- planted on November 2011 with planting shovel and spade. In the field, measurements were taken after 6 months (before summer), after 8 months (midsummer) and after 10 months (after summer). In each harvest, 12 seedlings (4 seedlings 3 replications) per treatment were chosen randomly and excavated from the field for root morphology estimation. Although mean shoot height, collar diameter, number of branches and length of central root increased more in watered seedlings than stressed individuals over time, they did not differ significantly in a same growth stage. Length and number of first order later roots especially increased significantly in watered seedlings than stressed individuals in 8 months after transplantation (in July 2012) but their mean diameter was significantly similar in both water regimes. To reduce the mortality rate of C. siliquastrum seedlings in the field, first year irrigation may be recommended during summer months. Key words: water stress, well- watered conditions, seedlings survival, drought, mortality rate 1

Introduction The survival of seedlings in the field depends on their morphological and physiological characteristics and the environmental conditions of the planting site (Margolis and Brand 1990). Among shoot characteristics, height and diameter are widely used to determine seedling growth. However, height and diameter are not correlated in all cases (Jacobs et al 2005). To survive and to start growth quickly, a seedling mainly depends on how its root system performs after transplanting. For bareroot seedlings, sufficient root systems along with long first order lateral roots (FOLR) should be ensured (Thompson and Schultz 1995). When natural regeneration is inadequate and supplementary outplanting is needed. However, in Greece as in other Mediterranean countries, reforestations frequently failed due to low survival rates during the dry period (Tsitsoni, 1997). Generally in Greece, dry period begins in the middle of April and lasts until September but severe drought period is from June to September (Tsitsoni and Karagiannnakidou, 2000). In these conditions, transpiration of the seedlings occurs very rapidly but the seedlings do not get enough soil moisture. Thus, it is important to assess drought performance, i.e. growth and survival of seedlings under water stress compared to well-watered individuals. Although many studies were conducted in the field condition for above ground morphology or physiology (Wilson et al 2007), little studies were reported on morphology of the whole seedlings with root systems by conducting soil excavation in the field condition (Zida et al 2008; Tsakaldimi et al. 2009), especially on C. siliquastrum in relation to water stress. Thus, this study was undertaken to identify seedlings morphological characteristics that ensure better field performance Cercis siliquastrum L. The purpose of this research specifically, was to answer to the following questions: (1) to what extent do the seedlings shoot morphology and root architectural attributes differ under water stress compared to well- watered conditions (2) do these variations effect seedlings survival in the field. The first question will help to determine the drought responses to seedling morphology and the second question relates to seedling desired attributes that will enhance the survival and growth of transplanted C. siliquastrum seedlings in the field. Study area - Methods Three years old bareroot seedlings of C. siliquastrum L. (Family- Fabaceae) were collected from the Lagadas Nursery of the Forest Service Department of Thessaloniki, Northern Greece. After lifting the seedlings from the bareroot nursery, their root systems were undercut. 2

The study was conducted at the Arboretum of the Laboratory of Silviculture, AUTh, Greece. The experiment was set in Randomized Complete Block Design with two treatments and three replications. Treatment 1 (Control): Regular spot irrigation was applied daily. Treatment 2 (Stressed): No additional water except rain water during the summer months (from June, July, August and September 2012). Each replication of a treatment contained 20 seedlings. Seedling to seedling distance within a row was maintained 2.0 m and also same distance was maintained among rows. All seedlings were hand- planted on 26 th November 2011 with planting shovel and spade. Initially 30 seedlings were taken randomly (Zida et al. 2008) for the measurement of morphology. In the field, measurements were taken 6 months after planting (before summer), 8 months (midsummer) and 10 months (after summer). In each harvest, 12 seedlings (4 seedlings 3 replications) per treatment were chosen randomly and excavated from the field for root morphology estimation (Tsakaldimi, 2006). Seedlings were excavated and extracted by the total excavation method (Martínez-Sánchez et al 2003). First-order lateral roots (FOLR 1 mm) were counted and measured (Dey and Parker1997). After measuring the length and diameter of each FOLR, they were arranged into five different diameter classes D1 (1-2 mm), D2 (> 2-3 mm), D3 (> 3-4 mm), D4 (> 4-5 mm) and D5 (> 5 mm). Survival rate (%) of seedlings was taken from the initial time to 17 months after transplantation. After the 10 months in the field, the plants of both treatments were left in same natural environment without irrigation. The equality of means of the morphological variables was tested for water stress and control treatments with independent samples t-test. All statistical analyses were carried out using SPSS 19. RESULTS AND DISCUSSION Morphological characteristics of shoot and root Six months after planting, mean shoot height of C. siliquastrum seedlings was found significantly lower than the initial stage (Table1). Initially shoot die back was observed almost in all the seedlings in that field condition and the shoot height decreased 20.32%. Height of seedlings of both water regimes was increased slowly from six to eight months after transplantation (from June to July 2012); especially new leaf flashed in some new and old branches. At this stage, mean shoot height increased 12.53 % in control seedlings (water seedlings) while 9.46 % in stressed ones but they were statistically similar. This shoot height increment was not consistent further. As air 3

temperature was high, it was observed that mean shoot height decreased from midsummer to end of the summer (from 8 month to 10 months after transplantation). Partial shoot die back, followed by formation of lateral shoots is common after planting in dry sites. Our results were similar with many researchers (e.g. Ritchie 1984; Dey and Parker, 1997; Haase 2007) where they mentioned that total shoot height of outplanted seedlings often becomes less than the initial height. Root Collar diameter was not affected seriously by the drought (Table 1). Mean collar diameter was observed to increase over the time. Control seedlings (watered seedlings) showed better collar diameter growth than the stressed ones during the months of stressed condition. Collar diameter has been found to be a useful predictor of field survival and growth (Barnett 1984; Tsakaldimi et al. 2012). Initially number of living branches per plant was 7.67. After six months, it was recorded as 10.97 (43.02% increment) which was statistically higher. After 8 months, some top branches dried and the number of branches reduced in both water regimes. However, watered seedlings showed more branches than the stressed ones (Table1). In our study, we found seedlings showed more branches in the lower part of shoot due to shoot dieback. Table 1: Morphological characteristics of shoot and root of C. siliquastrum seedlings in different times at two water regimes in the field Πίνακας 1: Μορφολογικά χαρακτηριστικά του βλαστού και της ρίζας των φυταρίων του είδους C. siliquastrum σε υδατική καταπόνηση και σε υδατική επάρκεια μετά από 6, 8 και 10 μήνες από τη φύτευση στο πεδίο. Time after transplanting Treatments Shoot height (cm) Root Collar diameter(mm) Number of branches Length of central root 0-Month - 147.12a±5.9 16.34a±0.9 7.67b±0.5 30.12b±1.7 6- month - 117.22b±5.8 17.09a±0.6 10.97a±0.8 39.88a±4.2 8-Month Control 131.91a±9.3 19.91a±1.2 10.48a±0.8 41.0a±8.9 Stressed 128.31a±9.6 19.86a±1.1 8.34a±0.8 36.67a±1.9 10-Month Control 124.95a±9.7 21.90a±1.0 11.16a±1.0 41.75a±4.2 Stressed 117.42a±12 21.17a±1.9 8.37a±1.20 37.08a±3.7 Note: Significant differences (P < 0.05) between 0 and 6 months without treatment or between two treatments (at 8 and 10 months) are in column by different letters of lower case (a, b). Values are means ± S.E. During 10 months after transplanting mean central root length increased over the time from 30.12 cm to 41.75cm, for control seedlings and to 37.08 cm in stressed seedlings. 4

In 8 and 10 months after planting, mean central root length was greater in watered plants (control) than in stressed, but this difference was not statistically significant. Root architecture Six months after field planting, mean length of FOLR was 33.49 cm. After the water plant situation began to diversify and especially in the heart of the summer (8 months after planting), mean length of FOLR was significantly reduced from 36.20 cm in watered plants (control) to 29.13 cm in stressed plants. 10 months after planting (in September) stressed plants also had shorter mean length of FOLR (37.95 cm) than watered plants (40.38 cm) but this difference was found not statistically significant (Figure1). This expose the fact that after applying stressed treatment during the summer, some FOLR become dead due to shortage of water. Before the water plant situation began to diversify (6 months after planting), mean diameter of FOLR was 4.03 mm. Stress treatment did not affect the mean diameter of FOLR and in the heart of the summer (8 months after planting) the mean diameter of FOLR in both water regimes was statistically same as was at 6-month stage (Figure 2). 10 months after planting, the situation remained the same except for the fact that the mean diameter of FOLR of watered plants was found slightly thinner than that of stressed plants. This can be attributed to the fact that when the number of new FOLR increased in watered seedlings (Figure 4), they were of smaller diameter initially and this caused the apparent reduction. Figure 1: Mean length (cm) of FOLR of C. siliquastrum Σχήμα 1: Μέσο μήκος (cm) των ριζών πρώτης τάξης του C. siliquastrum Figure2: Mean diameter (mm) of FOLR of C. siliquastrum Σχήμα 2: Μέση διάμετρος των ριζών πρώτης τάξης του C. siliquastrum 5

Absolute maximum lengths of FOLR increased over time and remained significantly greater in control (watered) plants compared to stressed plants (Figure 3). Total number of FOLR (diameter 1mm) per plant increased from initial time of transplanting to the last measurement period i.e. after 10-month (Figure 4) in control plants and was significantly greater than that of stressed plants. The lack of water and the high air temperature caused the significant reduction of the number of FOLR in stressed plants. Figure 3: Absolute maximum lengths (cm) of FOLR over time. Σχήμα 3: Απόλυτο μέγιστο μήκος (cm) των ριζών πρώτης τάξης, σε 6, 8 και 10 μήνες μετά τη φύτευση. Figure 4: Total number of FOLR (diameter 1mm) per plant over time. Σχήμα 4: Συνολικός αριθμός ριζών πρώτης τάξης ανά φυτάριο σε 6, 8 και 10 μήνες μετά τη φύτευση. 6

Number of FOLR (diameter 1mm) per plant by diameter class is shown in Table 2. Control plants showed gtreater number of FOLR in each diameter class. In D1 and D2 classes the number of FOLR was found significantly higher in control plants compared to stressed ones (P 0.05). However, in higher diameter classes no significant differences were observed between treated plants. To use bareroot seedlings, sufficient root systems along with large first order lateral roots (FOLR) should be ensured (Thompson and Schultz 1995). Thompson (1991) also observed in some excavations that these FOLR remained present and new roots grow from them. Table 2: Number of FOLR per plant in different diameter classes and in two water regimes of C. siliquastrum in 8 and 10 months after planting. Πίνακας 2: Αριθμός των πρώτης τάξης ριζών του C. siliquastrum ανά κλάση διαμέτρου, σε υδατική καταπόνηση και σε υδατική επάρκεια μετά από 8 και 10 μήνες από τη φύτευση. Time period after planting 8 months 10 months Treatment Number of FOLR per plant in different diameter class D1 D2 D3 D4 D5 Control 3.08a±.67 2.17a±.4 0.92a±0.1 0.75a±0.2 2.75a±0.5 Stressed 2.7a±0.68 1.0b±0.4 0.6a±0.2 0.6a±0.2 1.7a±0.3 Control 5.75a±1.0 2.25a±0.4 1.75a±0.5 0.42a±0.1 2.92a±0.6 Stressed 2.25b±0.3 0.92b±0.2 1.08a±0.3 0.58a±0.2 1.83a±0.4 *Significant differences (P < 0.05) between control and stressed plants are in column by letters of lower case (a, b). Values are means ± S.E. Survival rate Survival rate of C. siliquastrum was monitored up to17 months after field transplanting (Figure 5). When stressed treatment was applied (in June 2012), the air temperature was increasing and the soil moisture was decreasing, seedling mortality increased rapidly in stressed plants. After withdrawal of irrigation from the plants, the first two dry hot months were very critical and the mortality rate of stressed plants was very high in that stage. After 8 months, trend of survival of stressed treatment was almost same. After the 10 months, when the plants of both treatments were left in same natural environment without irrigation, the survival of control (watered) plants decreased in a greater rate than of stressed plants. This observation indicates that the plants survived after long stress during the summer were better adapted to the field conditions. 7

Figure 5: Mean survival rate of C. siliquastrum Σχήμα 5: Μέσο ποσοστό επιβίωσης του C. siliquastrum Conclusions Shoot dieback was observed in all bareroot seedlings which decreased shoot height. This may be due to transplanting shock, slow root growth or other causes of the undercut seedlings. Although mean shoot height, collar diameter, number of branches and length of central root increased more in watered seedlings than stressed individuals over time, they did not differ significantly in the same growth stage in the field. Length and number of first order lateral roots significantly increased in watered seedlings than stressed individuals in 8 months after transplantation (in the heart of the summer July 2012), while their mean diameter was significantly similar in both water regimes. The withdrawal of irrigation from the plants during the dry hot months was very critical and the mortality rate of stressed plants was very high. The high survival rate of watered seedlings during the dry summer months can be attributed to the moisture availability and the higher number and length of first order lateral roots in relation to stressed plants. To reduce the mortality rate of C. siliquastrum seedlings in the field, first year irrigation may be recommended during summer months. 8

ΣΥΓΚΡΙΣΗ ΤΗΣ ΑΡΧΙΤΕΚΤΟΝΙΚΗΣ ΤΗΣ ΡΙΖΑΣ ΚΑΙ ΤΗΣ ΜΟΡΦΟΛΟΓΙΑΣ ΤΟΥ ΒΛΑΣΤΟΥ ΤΗΣ ΚΟΥΤΣΟΥΠΙΑΣ (CERCIS SILIQUASTRUM L.) ΣΕ ΠΟΤΙΖΟΜΕΝΑ ΚΑΙ ΜΗ ΦΥΤΑΡΙΑ Rahman M.S., Θ.Κ.Τσιτσώνη και Μ. Ν. Τσακαλδήμη Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης, Τμήμα Δασολογίας & Φυσικού Περιβάλλοντος, Εργαστήριο Δασοκομίας, Τ.Θ. 262, 54124, Θεσσαλονίκη e-mail: tsitsoni@for.auth.gr Περίληψη Η επιβίωση των φυταρίων στην ύπαιθρο εξαρτάται από τα μορφολογικά και φυσιολογικά χαρακτηριστικά τους και τις περιβαλλοντικές συνθήκες της περιοχής φύτευσης. Η μελέτη αυτή πραγματοποιήθηκε για τον εντοπισμό εκείνων των μορφολογικών χαρακτηριστικών των φυταρίων Cercis siliquastrum L., που εξασφαλίζουν καλύτερη επιβίωση στην ύπαιθρο. Ο σκοπός αυτής της έρευνας συγκεκριμένα, ήταν να απαντήσει στα ακόλουθα ερωτήματα: (1) σε ποιο βαθμό τα μορφολογικά χαρακτηριστικά του βλαστού και η αρχιτεκτονική της ρίζας διαφέρουν σε συνθήκες υδατικής καταπόνησης σε σύγκριση με συνθήκες όπου τα φυτάρια είναι καλά-ποτιζόμενα (2) επιδρούν οι διαφορές αυτές στην επιβίωση των φυταρίων στην ύπαιθρο? Η πρώτη ερώτηση καθορίζει την επίδραση της ξηρασίας στη μορφολογία των φυταρίων και η δεύτερη σχετίζεται με τις επιθυμητές ιδιότητες που θα ενισχύσουν την επιβίωση και την ανάπτυξη των μεταφυτευμένων φυταρίων C. siliquastrum στο ύπαιθρο. Η μελέτη διεξήχθη στο Arboretum του Εργαστηρίου Δασοκομίας, Α.Π.Θ. Το πείραμα σχεδιάστηκε σε πλήρη τυχαιοποιημένο σχέδιο με δύο χειρισμούς και τρεις επαναλήψεις. Χειρισμός 1 (Control): Κανονική άρδευση εφαρμόστηκε ημερησίως. Χειρισμός 2 (Stressed): Όχι επιπλέον νερό, εκτός από το νερό της βροχής κατά τους θερινούς μήνες (Ιούνιος, Ιούλιος, Αύγουστος και Σεπτέμβριος 2012). Όλα τα φυτάρια φυτεύτηκαν τον Νοέμβριο του 2011. Οι μετρήσεις ελήφθησαν μετά από 6 μήνες (πριν από το καλοκαίρι), μετά από 8 μήνες (μέσα καλοκαιριού) και μετά από 10 μήνες (μετά το καλοκαίρι). Σε κάθε μία από τις τρεις παραπάνω περιόδους, 12 φυτά (4 3 επαναλήψεις) ανά χειρισμό επιλέχθηκαν τυχαία και εξήχθησαν από το έδαφος για την εκτίμηση της μορφολογίας της ρίζας. Τα αποτελέσματα έδειξαν ότι αν και το μέσο ύψος του βλαστού, η διάμετρος στο ριζικό κόμβο, ο αριθμός των κλαδιών και το μήκος της κεντρικής ρίζας αυξήθηκαν περισσότερο στα φυτά που ποτίζονταν από αυτά που δεν ποτίζονταν, οι διαφορές δεν βρέθηκαν στατιστικά σημαντικές στο ίδιο στάδιο ανάπτυξης. Το μήκος και ο αριθμός ριζών πρώτης τάξης αυξήθηκε ιδιαίτερα σημαντικά 9

στα φυτά που ποτίζονταν στους 8 μήνες μετά τη μεταφύτευση (Ιούλιος 2012), αλλά η μέση διάμετρός τους ήταν παρόμοια και στους δύο χειρισμούς. Το ποσοστό θνησιμότητας, κατά τη διάρκεια των θερμών θερινών μηνών, για τα φυτάρια που δεν ποτιζόταν ήταν αρκετά υψηλό. Αντίθετα το ίδιο διάστημα, τα ποτιζόμενα φυτάρια έδειξαν αρκετά υψηλό ποσοστό επιβίωσης το οποίο μπορεί να αποδοθεί στη διαθέσιμη υγρασία και στη καλύτερη αρχιτεκτονική ρίζας των φυταρίων. Έτσι από την παρούσα προτείνεται ότι για να εξασφαλιστεί ένα αρκετά υψηλό ποσοστό επιβίωσης των φυταρίων C. siliquastrum στην ύπαιθρο, συνιστάται άρδευση κατά τους καλοκαιρινούς μήνες του πρώτο έτους. Λέξεις κλειδιά: υδατική καταπόνηση, υδατική επάρκεια, επιβίωση φυταρίων, ξηρασία, ποσοστό θνησιμότητας. References Burdett, A. N. (1979). New methods for measuring root growth capacity: their value in assessing lodgepole pine stock quality. Canadian Journal of Forest Research, 9(1), 63-67. Dey, D. C., & Parker, W. C. (1997). Morphological indicators of stock quality and field performance of red oak (Quercus rubra L.) seedlings under planted in a central Ontario shelterwood. New Forests, 14(2), 145-156. Haase, D. L. 2007. Morphological and Physiological Evaluations of Seedling Quality. USDA Forest Service Proceedings RMRS-P-50. Jacobs, D. F., Salifu, K. F., & Seifert, J. R. (2005). Relative contribution of initial root and shoot morphology in predicting field performance of hardwood seedlings. New Forests, 30(2-3), 235-251. Margolis, H. A., & Brand, D. G. (1990). An ecophysiological basis for understanding plantation establishment. Canadian Journal of Forest Research, 20(4), 375-390. Martínez-Sánchez, J. J., Ferrandis, P., Trabaud, L., Galindo, R., Franco, J. A., & Herranz, J. M. (2003). Comparative root system structure of post-fire Pinus halepensis Mill. and Cistus monspeliensis L saplings. Plant Ecology, 168(2), 309-320. Ritchie, G.A., 1984. Assessing seedling quality. In: Duryea, M.L., Landis, T.D. (Eds.), Forest Nursery Manual: Production of Bareroot Seedlings. Corvallis, OR, pp. 243 259. The Hague (The Netherlands): Martinus Nijhoff/Dr. W. Junk Publishers. 10

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