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M818 3000 llllllllllllllllfIIIHHNIIHIHIlllllllllllllllllllllllllll 1293 02058 1959 This is to certify that the dissertation entitled QESfONgc 0‘: AWLG ewmmazs TO Dec L/CitH—x Mecmisfis mt ecgg‘rfl Ncs M9 The”: NON! mm NC} presentedby L50NA4¢00 Lo H 6.41301 N, has been accepted towards fulfillment of the requirements for ?l\ D . degree in Hoe-T1 QILZ‘U (2.9 Major professor Date “I/Z ({qu MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE moo «lemme-p.14 RESPONSE OF APPLE ROOTSTOCKS TO DROUGHT: MECHANISMS OF RESISTANCE AND THEIR MONITORH‘IG By Leonardo Lombardini A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1 999 ABSTRACT RESPONSE OF APPLE ROOTSTOCKS TO DROUGHT: MECHANISMS OF RESISTANCE AND THEIR MONITORING By Leonardo Lombardini The objectives of this dissertation were to characterize the adaptation of dwarfing apple (Malus domestica Borkh.) rootstocks (B.9, M.9, and Mark) to soil water deficit conditions. Three experiments were performed during the period 1997-1999. A first trial was designed to determine whether partial soil drying could influence transpiration, photosynthesis, and chlorophyll fluorescence in B.9, M.9, and Mark rootstocks, and, if so, to provide information on the mechanism of root-to-shoot-signaling. In particular, to determine whether a dry portion of the root system could improve the ability of other parts of the plant to withstand stress, roots subjected to uniform watering were compared with those receiving non-uniform watering. Results seemed to suggest an increase in tolerance to water deficit in plants with half of the root system growing in non-irrigated soil. Leaf water potential measured at predawn and midday indicated that B.9 experienced the biggest change in the water status, especially when equal water distribution was applied. Gas exchange analysis indicated that Mark had the greatest reduction in assimilation rate when only part of the roots were irrigated. Water use efficiency increased with time in stressed B.9, which also showed high stomatal conductance and transpiration rate. In Mark, assimilation rate and water use efficiency were frequently lower when half of the root system was not watered. The second experiment was conducted to test the use of infrared thermometry as a rapid, non- invasive tool to detect drought stress in potted plants of ‘TRECO Red Gala #42’ grafted on three different dwarfing apple rootstocks, B.9, M9 and Mark. The variation in leaf temperature induced by water deficit was studied together with the variation in assimilation rate, leaf expansion rate, and sap flow. Differences in canopy temperature could be detected as early as 3-4 days afier initiating the stress. Gas exchange appeared to be more affected by water deficit in ‘TRECO Red Gala #42’ grafted on B9 than on the other two rootstocks. Leaf expansion rate was reduced in ‘TRECO Red Gala #42’lMark within the first 2 days of stress. Heat-pulse sapflow sensors installed on ‘TRECO Red Gala #42’lMark indicated that the rate of the xylem sap flow decreased after 4 days of water deficit. In the third study, the stable isotope '3 C was used to evaluate the influence of soil water content on carbon partitioning among the various tree organs and to determine a possible correlation between carbon partitioning and root respiration. Canopies of 1-year-old rooted cuttings of 3.9 were pulsed with 13C02 and isotopic emissions were monitored for 16 days. The lower soil moisture had no effect on predawn leaf water potential or single leaf net assimilation rate but reduced the evolution rate of labeled l3C02 from the root. l3Carbon partitioning within the tree was not affected by water supply over the 16-day duration of the experiment, and evolution rate of 13 C from the roots was not related to the amount of labeled C allocated to the roots themselves. To my parents iv ACKNOWLEDGMENTS I would like to express my sincere thanks to my major professor and mentor Dr. Jim Flore for believing in me, more than four years ago, and giving me the opportunity to pursue a graduate degree here at MSU. His incredible enthusiasm, creativity, and above all, his friendship have been a great source of encouragement during these years. I am also grateful to the members of my guidance committee, Dr. Randy Beaudry, Dr. Ron Perry, Dr. Ken Poff, Dr. Joe Ritchie, and Dr. Irvin Widders for their suggestions with the dissertation projects. I would like to especially thank Dr. Riccardo Gucci for encouraging me to pursue a Ph.D. degree at Michigan State University. Appreciation is extended to my friends and colleagues, Dr. Moreno Toselli for sharing the experience of the ‘3C experiment, and Dr. Rita Giuliani, for the useful brainstorming relative to infrared thermometry. Thank you for your unique contribution in writing the relative manuscript. Specials thank to Sarah Breitkreutz for her constant help, friendship and support throughout these years. Valuable discussions regarding my research with the following scientists is acknowledged with great appreciation: Dr. John Everard, Dr. Abed Janoudi, Dr. Wayne Loescher, and Dr. Massimiliano Tattini. Thank you to Dr. Eugenio Magnanini for his help in the development of the MathCad program for the analysis of digital images. All the friends and colleagues that have worked in Dr. Flore’s lab: Ala Druta, Zafer Makaraci, Adriana Nikoloudi, and Costanza Zavalloni. All the students who, rain or shine, have worked hard in all these years helping with the data collection: Brie Genter, Beth Harkness, Allison Hopkins, Jon Luea, and Matt Reed. I am gratefirl for the fi'iendship, support, and comradery of Bruno Basso, Juan Hernandez, Priscilla Hockin, Mark Kehn, Rufino Perez, Erik Runkle, Silvanda Silva among others in the department. I also thank all the HOGS fellows, for the believing in our Student Organization in the Department of Horticulture. All the Italian friends that made the experience at MSU much easier and pleasant: Monica Accerbi, Bruno Basso, Barbara Casiraghi, Marilena Di Valentin, Laura Ghezzi, Valentina Maddalena, Eliana Messori, Andrea Pievaroli, Elda Toselli, Francesco Vianello. The support, enthusiasm and love from Sonia have helped me to go through these last years of graduate school. Thank you for being so understanding, especially in these last hard months. My sincere gratitude goes to my family. My parents have always believed in me and supported me and my career in the decision of staying away from home for such a long period. TABLE OF CONTENTS LIST OF TABLES - . ---.--VIII LIST OF FIGURES -- _- -- -- -- - - ....... -- -_ - - ------XI LITERATURE REVIEW - - -- - - u 2 1.1. Physiology of drought stress ........................................................................... 2 1.2. The importance of orchard irrigation ............................................................ 17 1.3. Apple rootstocks ........................................................................................... 19 1.3.1. History ......................................................................................................................... 20 1.3.2. Physiology of the dwarfing process ............................................................................. 22 Literature cited _ - - - - - - - -- _- -_ 27 CHAPTER 1. DROUGHT RESPONSES OF YOUNG APPLE ROOTSTOCKS GROWN WITH SPLIT-ROOT SYSTEMS - - 36 Abstract - — - - - 36 Introduction -_ - 37 Materials and methods -_ - _ _ - 42 Results - - - - 47 Discussion - -- 53 Literature cited ‘ - - - _ -- -- 59 CHAPTER 2. CANOPY TEMPERATURE AND WATER STRESS IN YOUNG APPLE TREES SUBJECTED TO WATER DEFICIT - - -_ 79 Abstract- - - - 79 Introduction.-." _ - .............................. _ -- - 80 Materials and methods -- -- _ - - - -- 84 Results - - - - - ...... - - - 93 Discussion - - - -- 100 Literature cited . __ 109 CHAPTER 3. LEAF ASSIMILATION, CARBON TRANSLOCATION AND ROOT RESPIRATION IN B.9 APPLE ROOTSTOCKS DURING DROUGHT STRESS- -133 Abstract- -- -- -- -- - - -- -- 133 Introduction ............. - --.-m---___-- - . 134 vi Materials and methods - Results - . Discussion Literature cited APPENDICES APPENDIX A APPENDIX B -- APPENDIX C -- - vii 136 141 144 148 159 160 164 - 165 LIST OF TABLES Chapter 1 Table 1.1. Total leaf area (cmz) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05 ........................................................................................................... 62 Table 1.2. Total leaf number measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. The absence of any letter within column means indicates that treatments are not significantly different at P S 0.05. ...................................................................... 63 Table 1.3. Total shoot length (sum of two shoots), in cm, measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. The absence of any letter within column means indicates that treatments are not significantly different at P s 0.05. ........................................ 64 Table 1.4. Root fresh weight (FW, g) and water content (WC, % FW) in B.9, M.9, and Mark apple rootstocks after 28 days of growth with the root systems split between two containers and subjected to root zone drought, equally and unequally distributed. The symbol "‘ indicates differences of treatment means between pots significant at P S 0.05. Treatments (smn of the two pots) within row means indicated by the same letter are not significant different P s 0.05. . 65 Table 1.5. Values of predawn leaf water potential (Ww, MPa) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05. ................................................. 66 Table 1.6. Values of midday leaf water potential (I/lw, MPa) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05. ................................................. 67 Table 1.7. Chlorophyll efficiency (Fv/Fm) measured on apple rootstocks. Six levels of root zone drought (either equally or unequally distributed) were imposed on Aug. 14 and released on Sep. 11 1997. Treatments within column means followed by the same letter are not significantly different at P s 0.10. ....................................... 68 viii Chapter 2 Table 2.1. Parameters derived from analysis of A-C, response curves performed on apple cultivar ‘Red Gala’ grafted on B.9, M9 and Mark rootstocks subjected to soil water deficit conditions. Data were collected on fully expanded apple leaves on day 0 and afler water was totally withheld for three days. ............................... 113 Table 2.2. Trend contrasts of net assimilation rate (A), intercellular C02 concentration (Ci), transpiration rate (E), stomatal conductance (g,), and leaf temperature (T.) with 7 increasing levels of temperature (10, 15, 20, 25, 30, 35, and 40 °C). 114 Table 2.3. Integrated values (over 1-day period) of sap flow and speed measured with heat-pulse technique for 8 days on one irrigated (F I) and one not-irrigated (NI) ‘Red Gala’lMark plant. ..................................................................................... 115 Table 2.4. Canopy temperature (Tc) and difference between Tc and air temperature (T,) measured daily between 1200 and 1300 HR EST using an infrared detector. Three sets of Tc and Tc — T. values were calculated, using the mean, median, and mode Tc values derived from pixel analysis of IR images. Treatments within column means followed by the same letter are not significantly different at P S 0.05 . .................................................................................................................. 1 16 Table 2.5. Results of the linear regression analysis performed between Tc and VPD, and between the difference Tc - T. and VPD (data plotted in Figure 2.12 and 2.13, respectively). The three values of TG (mean, median and mode) derived from digital infiared images were used. .................................................................... 117 Table 2.6. Summary table indicating the appearance of significant differences between F I and NI ‘Red Gala’lMark plant for most of the parameters investigated during the experiment. Tc data are based on the mean values derived form digital image analysis. Sapflow data are not based on statistical differences, but only on reduction of flow recorded in NI versus FI plants. ........................................... 118 Chapter 3 Table 3.1. Values of predawn water potentials measured on B.9 rootstocks during a moderate drought stress experiment. ................................................................ 150 Table 3.2. Variation of gas exchange in B.9 rootstocks in response to moderate drought stress conditions. A, net photosynthetic assimilation rate; gs, stomatal conductance: E, transpiration rate, Ci intercellular C02 concentration. ........... 151 Table 3.3. Effect of sampling day and water treatment on root evolution rate of 13C02 derived from supply and 13C enrichment in soil the headspace of potted B.9. 152 ix Table 3.4. Effect of sampling day and water treatment on partitioning of 13 C derived from supply among tree organs of B.9 rootstocks. ................................................... 153 Table 3.5. Fraction of ”C respired daily by root (percent of the total labeled C detected in root) of B.9 rootstocks. ..................................................................................... 154 Table 3.6. Levels of 13C measured in the water extracted from soil samples. Samples were collected at regular intervals after the 13C02 pulse. ................................. 155 Appendix A Table A. 1. Values of Fv/Fm derived from the preliminary experiment to determine the minimum dark adaptation time required by apple rootstocks. Lefl table, Fv/Fm was measured at 2-min intervals, and the interval necessary to maximize PV/Fm was determined. Right table, the found time interval was used to select the highest light level (expressed as percent of maximum light intensity obtained with PEA) necessary to saturate the leaves. Bold characters indicate time interval and percent light in correspondence of maximum Fv/Fm. ................... 160 Table A2. Variable chlorophyll fluorescence (F v) measured on apple rootstocks grown with the root systems split between two containers. Six levels of root zone drought (either equally or unequally distributed) were imposed on Aug. 14 and released on Sep. 11 1997. Treatments within column means followed by the same letter are not significantly different at P S 0.10. ..................................... 161 Table A3. Maximum chlorophyll fluorescence (F “1) measured on apple rootstocks grown with the root systems split between two containers. Six levels of root zone drought (either equally or unequally distributed) were imposed on Aug. 14 and released on Sep. 11 1997. Treatments within column means followed by the same letter are not significantly different at P S 0.10. ..................................... 162 Appendix C Table C. 1. Total carbon content (expressed in percent :1: SD) in the different organs of B.9 rootstocks during the drought experiment. Pots were isolated from the chamber atmosphere with plastic bags to prevent soil contamination with 13 C02. ........ 165 LIST OF FIGURES Chapter 1 Figure 1.1. Leaf expansion rate (LER) calculated on the first unrolled leaves for B.9 apple rootstocks grown with their root systems split between two containers and subjected to root zone drought, equally and unequally distributed. Leaf areas were measured at 2—day-interval during the first 10 days of the experiments. Treatments means indicated by the same letters are not significantly different at P .<. 0.05. ............................................................................................................. 69 Figure 1.2. Leaf expansion rate (LER) calculated on the first unrolled leaves for M9 apple rootstocks grown with their root systems split between two containers and subjected to root zone drought, equally and unequally distributed. Leaf areas were measured at 2-day-interval during the first 10 days of the experiments. Treatments means indicated by the same letters are not significantly different at P .<. 0.05. ............................................................................................................. 70 Figure 1.3. Leaf expansion rate (LER) calculated on the first unrolled leaves for Mark apple rootstocks grown with their root systems split between two containers and subjected to root zone drought, equally and unequally distributed. Leaf areas were measured at 2-day-interval during the first 10 days of the experiments. Treatments means indicated by the same letters are not significantly different at P S 0.05. ............................................................................................................. 71 Figure 1.4. Leaf, shoot and trunk water content (expressed as percent of fresh weight) calculated in the three apple rootstocks at the end of the drought stress experiment (day 28). Treatments means within the same plant organ followed by the same letter are not significantly different at P s 0.05 ................................... 72 Figure 1.5. Effect of 28 days of equal or unequal irrigation on dry matter partitioning (expressed as percent of total dry weight) in B.9, M9 and Mark apple rootstocks grown with their root systems split between two containers. Roots from the two pots (irrigated and non-irri gated) were kept separated during analysis .............. 73 Figure 1.6. Changes in single leaf net assimilation rate (A) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P s 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. ................................................................................ 74 xi Figure 1.7. Changes in stomatal conductance (g,) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non- significant differences ......................................................................................... 75 Figure 1.8. Changes in water use efficiency (WUE) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. ................................................................................ 76 Figure 1.9. Changes in leaf intercellular C02 concentration (Ci) calculated overtime in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. ................................................................................ 77 Chapter 2 Figure 2.1. Air temperature and soil water content (SWC) during the second period of measurements (August 23-30, 1998). A, air temperature measured at the site of the experiment. B, SWC calculated with a time-domain-reflectometer. .......... 119 Figure 2.2. Relative leaf expansion rate (RLER) during the short-term drought stress experiment. Values are means of four replicate plants. The symbol "' indicates means significantly different at the 5% level. .................................................. 120 Figure 2.3. Changes in single leaf net assimilation rate (A) in fully irrigated (F I) and non- irri gated (NI) apple trees during the short-term drought stress experiment. Symbols *,**,*** indicate means significantly different at P S 0.05, 0.01, or 0.001, respectively. ........................................................................................... 121 Figure 2.4. Changes in stomatal conductance (g,) in fully irrigated (F I) and non-irrigated (NT) apple trees during the short-term drought stress experiment. Symbols *,**,*** indicate means significantly different at P s 0.05, 0.01, or 0.001, respectively. ...................................................................................................... 122 xii Figure 2.5. Changes in leaf transpiration rate (E) in fully irrigated (FI) and non-irrigated (NI) apple trees during the short-term drought stress experiment. Symbols *,**,*** indicate means significantly different at P S 0.05, 0.01, or 0.001, respectively. ...................................................................................................... 123 Figure 2.6. Changes in leaf intercellular C02 concentration (C;) in fully irrigated (FI) and non-irri gated (NI) apple trees during the short-term drought stress experiment. Symbols *,**,*** indicate means significantly different at P s 0.05, 0.01, or 0.001, respectively. ........................................................................................... 124 Figure 2.7. Changes in leaf temperature (T1), measured with IRGA, in fully irrigated (F1) and non-irri gated (NI) apple trees during the short-term drought stress experiment. ....................................................................................................... 125 Figure 2.8. Variation of net assimilation rate in response to increasing leaf intercellular C02 concentrations (A-Ci curves). Data were collected on fully expanded apple leaves on day 0 and after water was totally withheld for three days. After photosynthetic parameters were calculated for each plant (Table 2.1), data from three plants per treatment were pooled, and curves were fitted by nonlinear regression models for illustrative purposes only. Symbols have the only purpose of facilitating the interpretation of the figure and do not correspond to actual measurements. .................................................................................................. 126 Figure 2.9. Variation of gas exchange in response of increasing temperature measured on ‘Red Gala’ plants grafted on B.9, M.9, and Mark rootstocks. A, net photosynthetic assimilation rate; gs, stomatal conductance: E, transpiration rate. Treatments means followed by the same letter are not significantly different at P s 0.05. ............................................................................................................... 127 Figure 2.10. Variation of leaf temperature (T1) and leaf intercellular C02 concentration (Ci) in response of increasing temperature measured on ‘Red Gala’ plants grafted on B.9, M.9, and Mark rootstocks. Treatments means followed by the same letter are not significantly different at P s 0.05. ..................................... 128 Figure 2.11. Sap flow (A) and sap speed (B) measured with heat-pulse technique for 8 days on one full irrigated (F I) and one not-irrigated (NI) ‘Red Gala’fMark apple plant. Air temperature (T.) measured during the days of measurements is also indicated. .......................................................................................................... 129 Figure 2.12. Relationship between canopy temperature (Tc) and vapor pressure deficit (VPD, graphs A, C, E) and soil water content (SWC, graphs B, D, F) measured daily on potted apple rootstocks, fully irrigated (O) or subjected to drought stress (0). Graphs A and B were obtained plotting mean Tc values obtained fiom pixel analysis of IR images. Graphs C and D with the median Tc, and graphs E and F with the mode Tc. Each symbol corresponds to a single data point. Values relative to the three different rootstocks were pooled. ............... 130 xiii Figure 2.13. Relationship between canopy temperature and air temperature (Tc — Ta) and vapor pressure deficit (VPD, graphs A, C, E) and soil water content (SWC, graphs B, D, F) measured daily on potted apple rootstocks, fully irrigated (O) or subjected to drought stress (0). Graphs A and B were obtained with using mean Tc values obtained from pixel analysis of IR images. Graphs C and D with the median Tc, and graphs E and F with the mode Tc. Each symbol corresponds to a single data point. Values relative to the three different rootstocks were pooled. .......................................................................................................................... 131 Chapter 3 Figure 3.1. Comparison of A-Ci curves performed on mature leaves of B.9 rootstocks in well watered conditions (solid line) and after four weeks of drought stress (dashed line). Each curve fitted data item three or four plants per treatment. Equations for the nonlinear regression models of the monomolecular asymptotic type were: control, y = 26.14 X (1.0 - 1.306 x exp(-0.004792 X x)), r'2 = 0.780; stress, y = 22.51 x (1.0 - 1.41 X exp(-0.006401 x x)), r2 = 0.673. .................... 156 Figure 3.2. Effect of sampling day on 13 C enrichment within the tree. Values indicate the means i SE. ...................................................................................................... 157 Figure 3.3. Effect of sampling day on ‘3 C partitioning within different tree organs. Values represent means t SE. ...................................................................................... 158 Appendix A Figure A. 1. Changes in leaf transpiration rate (E) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non- significant differences ....................................................................................... 163 Appendix C Figure C. 1. Diagram of the closed system for 13CO2 pulsing of potted apple trees. The components are lettered as follows: C) closed Mylar chamber; CD) cooler/dehumidifier; CG) l3C02 generator; F) fan; I) infrared gas analyzer; L) light source. ...................................................................................................... 166 xiv LIST OF SYMBOLS, UNITS AND ABBREVIATIONS Symbol V’w Amax Ci CSWI DFS DW gagagaa IRGA Parameter leaf water potential net C02 net assimilation rate abscisic acid maximum net C02 assimilation rate leaf intercellular C02 concentration crop stress water index (”C) derived from supply dry weight emissivity transpiration rate background or initial fluorescence fully irrigated maximum fluorescence variable fluorescence fresh weight stomatal conductance infrared infi'ared gas analyzer immersed weight carboxylation efficiency XV Units (MP a) (umol m”2 s") (umol rn'2 s'l) (umol mol") (2:) (mmol m'2 s") (relative units) (relative units) (relative units) (3) (mmol m"2 s") (g) (umol co; m'2 s" 111" L) LSD PAR RLER SD SE SWC VPD WC least significant differences non-irrigated photosynthetically active radiation relative humidity relative leaf expansion rate standard deviation standard error soil water content air temperature canopy temperature leaf temperature volume fraction of water vapor pressure deficit volume fraction of wood water content C02 compensation point Stefan-Boltzmann constant xvi (umol m'2 s") (%) (mm2 cm'2 d") (%) (°C) (°C) (°C) (%) (kPa) (%) (%) (umol co; moi") (5.670 x 10'8 w m’2 K“) LITERATURE REVIEW LITERATURE REVIEW 1.1. Physiology of drought stress Water deficit can have serious detrimental effects on the growth and development of plants. Among the many biochemical and developmental processes that are affected by water stress, decrease of photosynthesis (Fernandez et al., 1997b; Flore and Lakso, 1989), changes in water relations (Brough et al., 1986; Olien and Lakso, 1986), reduction of both cell division and expansion (Hsiao and Acevedo, 1974), ABA synthesis (Davies and Zhang, 1991; Zeevaart and Creehnan, 1988), and accumulation of sugars (Wang et al., 1995; Wang and Stutte, 1992) play a fundamental role. Stomatal and non-stomatal limitation could also be indicators of the upcoming plant stress conditions (Jones, 1985). Stomata are the ultimate regulators of both photosynthesis and transpiration responses (Jones, 1998). Stomata are regulated by internal components (leaf water and osmotic potentials, internal CO2 concentration, etc.), by environmental conditions (mainly net solar radiation and vapor pressure deficit), and by the interaction between transpiration ad photosynthetic activities (Jones and Corlett, 1992). Drought influences stomatal aperture, either directly, with the turgor loss of stomatal guard cells, or indirectly, with production of ABA or other inhibitory substances. Turgor is therefore critical to plant life since its loss induces decline in carbon assimilation, with physiological and morphological consequences in both the vegetative and reproductive growth and development. In the case of plants with commercial importance, the effects of water deficit also have negative repercussions on yield and link quality. It is now well established that depression of gas exchange is detectable at moderate leaf water deficits or even before leaf water status is influenced (Jones et al., 1985). Shoot and leaf growth seems to be firstly affected by lowering of leaf water potential (Flore and Lakso, 1989). When mild water stress was applied to peach trees, reductions in shoot and leaf expansion were detectable before assimilation rate and stomatal conductance were negatively affected (Andersen and Brodbeck, 1988). Plant water status is probably the best indicator of plant stress because it accounts for the effects of evaporative demands, the availability of water in the soil, and the hydraulic fluxes within the soil-plant-atmosphere continuum (Andrews et al., 1992). On the other hand, measurements of plant water status based on hydraulic parameters, 6. g. leaf water potential, can be extremely variable. Transpiration in plants is a fimction of plant water status and of the evaporative demand of the surrounding environment. This evaporative demand depends on the net radiation absorbed by leaves and on the drying power of the air (Nobel, 1991). Stomata represent the ultimate regulators of both photosynthesis and transpiration processes. Stomata are regulated by internal components (leaf water and osmotic potentials, internal C02 concentration, etc.), environmental conditions (vapor pressure deficit, light), and by the interaction between transpiration and photosynthesis (Jones and Corlett, 1992). When the plant is well watered, the transpiration rate is maximal and the leaf temperature (T1) is close to air temperature (T.). Vice versa, when water deficit increases, stomata are closed, transpiration is reduced and solar energy is no longer dissipated as latent heat for water evaporation, but rather converted into heat, which increases TI. This explains why, during hot summer days and when the soil water content becomes limiting, T. becomes higher than T.. Inversely, plants receiving an adequate amount of water through their roots have cooler leaves than those that are drought stressed. An alternative method for the evaluation of plant water status can be the measurement of leaf and canopy temperatures, and the difference between canopy and T. can be a tool to detect plant moisture stress (Jackson, 1982). The idea of using T] as a tool to detect plant water stress is not new. In 1963, Tanner (1963) was one of the first researchers to recognize the importance of monitoring T1 to predict the potential yield of a crop. However, it was not until the early 1980’s that the concept of measuring canopy temperature was developed and popularized. Nowadays, plant temperature is being measured not as much as a means of predicting crop yield, but rather as a means of quantifying plant water stress and then incorporating this parameter into crop water stress indices (CWSI). The concept of CWSI was first proposed by Idso (1981), who indicated an empirical approach to determine it on the basis of results obtained in the arid climate of Arizona. The CWSI can be also be calculated with the theoretical approach developed by Jackson (1981). In both methods, the index ranges from 0 to 1, with 0 indicating a plant transpiring at the maximum rate (no stress), and 1 a plant having no transpiration (stress). A good review of some of the CSWIs that have been proposed is presented by Andrews (1992). Temperature-based CSWI (Jackson et al., 1981) has been developed with an energy-balance approach, with surface temperature expressed as function of net radiation and VPD. Ehrler (1973) correlated the difference (T. — T.) with VPD. The result is a linear, inverse relationship. Idso (1981) and Jackson (1981) have used similar relationships. In other works the concept of stress-degree-day (SDD) has been used. SDD is defined by the accumulation of positive degrees derived from the difference between canopy temperature (T.) and T. measured near solar noon (Idso et al., 1977; Jackson et al., 1977). The concept of temperature-stress-day (T SD) has been defined as the difference in Tc between well- watered and water-stressed plots (Gardner et al., 1981). Canopy-temperature-variability is the range of canopy temperatures within a field (Clawson and Blad, 1982). In most cases, the Tc-related CSWI was calculated fiom data collected in the field on crops with uniform canopies exposed to VPD up to 5-6 kPa (Idso et al., 1981). Andrews (1992) evaluated Tc-based and infrared (IR) thermometer measurements of apple trees (Malus domestica Borkh. ‘Royal Gala’) grown in water deficit conditions. Measurements of Tc - T. were compared with point measurements of T1 — T., soil water storage and VPD in plots that were either irrigated or drought stressed. Values of Tc — T. and T1 — T. were significantly greater when water was withheld. Estimates of canopy temperature can be greatly affected by environmental conditions. Erroneous CSWI values can be estimated when windspeed is variable. High windspeed can cause low CSWI, with consequent underestimation of the stress condition and a delay in irrigation (O'Toole and Hatfield, 1983). Canopy temperature is affected also by change in radiation (i.e. cloud cover) (Jensen et al., 1990) and by VPD (Kirkham et al., 1983). In humid climates, the high degree of variability of VPD, net radiation, and evapotranspiration can lead to relevant errors in the estimation of CWSI (Campbell and Norman, 1990). That is why it is often difficult to estimate plant water status from canopy temperature. Andrews (1992) asserts that plants with canopies that have a heterogeneous shape, like fruit trees, are not very good predictors of plant water status, especially if grown in humid climates, where irrigation scheduling should be based more on the soil water content than on the T.. The gases present in the earth’s atmosphere (mainly water vapor) absorb radiated energy in the infrared except for two wavelength regions called the atmospheric windows. Both windows allow radiometric measurements with minimal losses. The longwave region (8-14 um) is exceptionally free of absorption except if very high atmospheric water content is present. The shortwave region (3-5 um) has relatively high transmission, but it usually requires compensation when high accuracy measurements are to be made. Modern thermal imaging radiometers are available with 8-14 um, 36 um, or 3-14 um (broadband) spectral response. Due to a higher thermal contrast at “earth” temperatures (-20 °C to 50 °C) in the longwave region, greater overall system performance can be achieved. Compared to traditional methods for temperature detection, IR thermometry represents a non-destructive, rapid, non-contact method of measuring temperature. A thermal image analyzer is able to sense the infrared emission from an object and convert the amount of energy into temperature, according to the Stefan-Boltzmann law: maximum radiant energy flux density = 07" where o is a constant (coefficient of proportionality, also known as the Stefan-Boltzmann constant, which equals 5.67 x 10'8 W m”2 K“) and T is the temperature of the object in kelvin. In the case where an object is not a perfectly efficient emitter (blackbody), the radiant energy flux density corresponds to eoT', where e is the emissivity of the object (Nobel, 1991). Emissivity has a maximum value of l for a blackbody and it is influenced by the surface material of the object. Emissivity for leaves and canopy varies between 0.97 and 0.98 (Jackson, 1982). Most common material surfaces have higher emissivities in the 8-14 pm waveband. The recent development of small portable infi'ared thermometers has made canopy temperature an easily measured parameter in the field. The energy is measured by a mercury-cadmium-telluride (HngTe) detector; the accuracy of such thermometers can be less or equal to 01°C at 30 °C. These portable infrared radiation detection units sense the amount of heat energy emitted from leaf surfaces and convert this energy to a digitally displayed temperature. Color infi'ared images are often called “false-color” image, because real colors do not correspond to the colors shown in IR images. Objects that are normally red appear green, green objects (except vegetation) appear blue, and “infrared” objects, which normally are not seen at all, appear red. For these reasons, in most cases, IR images are represented with gray-scale images, which replace the real colors with differences in gray intensities. The advantage of using digital images versus conventional ones is basically in the ability that computers have to distinguish 256 shades of gray, compared to 8-10 (optimistically) of them that a human interpreter can distinguish. Moreover, the analyst has control over the computer's presentation of the data and can group it any way he/she pleases, extract a portion of it, or display it in false color. Data sets can also be combined, compared, and contrasted with more ease and precision (not to mention speed) than if the task were left to humans alone. Human interpretations are highly subjective, hence, not perfectly repeatable. Conversely, results generated by computer (even when erroneous) are usually repeatable. Today, one of the primary uses of IR technology and photography is related to vegetation studies. This is because healthy green vegetation is a very strong emitter and reflector of infrared radiation and appears sharp and bright on infiared photographs. It seems that the method of measuring the increase in leaf temperature is particularly effective in arid, hot environments where leaves in a stressed plant can have a temperature of 10 °C or more higher than a well watered plant (Clarke, 1997). Even though quite a few studies have been done in this area, most of them focused on field detection with crop cover at 100%, or close to it. Only a few researchers have recognized the importance of monitoring plant temperature at much less than 100% cover (Alrneida and Slack, 1986; Heilrnan et al., 1981; Wanjura et al., 1984). An even smaller number of investigations have been conducted to; monitor the response of temperature on single plants or on foliage (Alkire and Simon, 1992; Ehrler, 1973). Among the reasons of the lack of studies, is the little success that followed either the empirical or the theoretical approaches to calculate the CWSI. Infrared thermometry has been used not only for water stress detection. Recently a few studies (Ceccardi et al., 1995; Wisniewski et al., 1997) showed the application of this technology to investigate the freezing process (ice formation is an exothermic event that visualized with an IR video camera). 0n stone fruits, infrared imaging has been used to visualize flower bud fi'eezing in peach (Ashworth, 1982). When remote sensing is used to determine plant temperature, only an average temperature of the canopy has to be considered. T. can vary with the position and the morphology of the leaf, the temperature of the air, the vapor pressure deficit, the intensity of the light, the angle at which it is incident on the leaf surface, etc. This is likely why, so far, the data collected with a remote sensing approach have not been precise enough to organize irrigation scheduling. As with all techniques, there are limitations in using IR thermometry. Problems arise mainly in the area of sampling and developing crop stress relationships for crops with limited ground cover and small leaves because of the interference of soil background. Furthermore, not much is known about the way leaf temperature is related to effective plant stress and to the other physiological and biochemical parameters. It is known from the above mentioned studies that leaf temperature is related to water deficit, however, to the best of our knowledge, the research is limited on the dynamics of this relationship with the development of a stress. With further studies, the approach could be refined and a better relationship between plant status and CWSI could be achieved, so that plant science (and, consequently, the growers) could benefit from this technique. Ahneida (1986) evaluated the correlation between measured and projected canopy temperature (calculated fiom composite measurements) under partial canopy situations, so the latter could be used for irrigation scheduling. While it was possible to estimate canOpy temperatures with reasonable accuracy under controlled conditions, their approach did not lend itself to field application. Loss of turgor induced by drought triggers physiological and biochemical adjustments that are important for turgor maintenance, and the likely importance of elastic and osmotic adjustments have been highlighted (Schulte and Henry, 1992). Elastic adjustment includes physical modifications in the cells, which make them more elastic, thereby facilitating tissue shrinkage during dehydration. Osmotic adjustment is the active accumulation of solutes inside the cell, with the consequent lowering of the osmotic potential and the maintaining of water absorption. These mechanisms help to maintain turgor even at low tissue water potentials and prevent mechanical damages to plasma membranes. The capacity for osmotic adjustment and maintenance of low osmotic potential may be useful criteria for selection and breeding of more drought-resistant species and cultivars. High solute concentrations can contribute to a greater capacity for turgor maintenance, but the contribution of electrolytes to osmotic adjustment is usually relatively low, if compared with other more compatible solutes. Inorganic ions can be toxic and disruptive to organelles, enzymes, and membrane-bound processes, whereas organic ions, usually referred to as osmolytes, may serve as more compatible solutes, being tolerated at high concentrations in the cytoplasm (Ahmad et al., 1979; Bieleski, 1982). Osmolytes are a group of low molecular weight compounds that are synthesized and accumulated in the cytosol. Many are the plants and bacteria that synthesize osmolytes in response to environmental stresses (Tarczynski et al., 1993). Osmolytes can also be referred to as compatible solutes or osmoprotectants. Examples of osmolytes are polyols (a.k.a. sugar alcohols), proline, and glycine-betaine. In many species belonging to the Rosaceae family, sorbitol is the primary product of photosynthesis and the form of carbohydrate that is most actively translocated (Bieleski, 1982). Although their function is still unknown, the primary function of sugar alcohols is likely to adjust the cytosolic osmotic potential to increase the cell tolerance against abiotic stresses (Tarczynski et al., 1993). The level of many osmolytes increases during the stress and declines when the stress is relieved. However, for many years it was uncertain whether this accumulation resulted from impaired metabolism or from storage during the stress (Wyn—Jones and Gorham, 1983). Considerable results have been obtained on experiments conducted on 10 transgenic tobacco plants where the gene for mannitol was inserted (Tarczynski et al., 1992; Tarczynski et al., 1993). Naturally, tobacco is not a mannitol-producer, but transformed plants produced mannitol (up to a maximum concentration of 100 mM) and seemed more tolerant to salinity stress. Transgenic plants were also able to produce new growth and new leaves, when exposed for 30 days to 250 mM NaCl. There are no assumptions on how accrunulation of intracellular mannitol may lead to new growth. The maintained production of roots and leaves, rather than a reallocation of resources, could explain the increased height and weight in transgenic plants (Tarczynski et al., 1993). In apple, mature leaves seem able to adjust osmotically, whereas young leaves and tips seem not to be able to do this. The opposite seems to occur in peach, where immature leaves indicate osmotic adjustment and mature leaves do not (Wang et al., 1995). Osmotic adj ustrrrent in drought-stressed roots was observed by Ranney et a1. (1991) for cherry trees. Sorbitol has been found to accumulate in apple leaves during drought stress. Perhaps this compatible solute plays a role in lowering leaf osmotic potential. However, not enough research has been conducted on young leaves, root and stems (Wang et al., 1995). Another important response to drought is the increase in the production of ABA. There is a considerable body of evidence that shows that leaves are not the only source of ABA and that ABA is also synthesized in roots (Cornish and Zeevaart, 1985; Davies and Zhang, 1991). The synthesis of ABA takes place in the apices as well in the non-growing regions, and in the cortex as well in the stele. Roots are able to detect the early stages of soil drying, and many of the shoot responses to soil drying occur before any detectable change in the leaf water status. In fact, it is now well established that the term “water 11 stress” does not refer only to situations where water relation parameters are altered. From several studies it has emerged that the soil water status, the leaf water relations and the plant responses are not always correlated (Davies and Zhang, 1991 and references therein). Among other parameters that can be involved in the perception of the incoming drought period (cytokinins, pH, ion concentrations, etc.), ABA is the most likely chemical involved in this signaling (Davies and Zhang, 1991). ABA has long been known to increase considerably in leaves of plants subj ected to drought. The marked increase is a consequence of the biosynthesis of this hormone, rather than of a release from storage forms present in the leaves (Dorffling, 1972). When roots “sense” the reduction of soil water potential, they start producing ABA, which is then sent to the leaves via xylem flow, thereby functioning as a long distance signaling molecule. It is now widely accepted that stomatal conductance is controlled by the soil water status via ABA. Although leaf water status is often not considered as influencing the response of stomata to ABA, recent works (Tardieu and Davies, 1993; Tardieu et al., 1992) report that leaf water potential might have an indirect role in the regulation of the stomatal conductance, via a modification of the stomatal sensitivity to ABA. An experiment conducted on the root system of sunflower seemed to confirm this hypothesis. Water potential was maintained high with the pressurization of the root system and a lower response of the conductance of ABA was observed (Schurr et al., 1992). A study conducted on sunflower (Gollan et al., 1986) has shown that, when leaf water potential and turgor do decline, stomatal conductance can be more closely related to the water status of the soil than to that of the leaves. In contrast to these assumptions, Munns and King (1988) removed ABA from the sap of water-stressed wheat, and found that the anti- 12 transpirant activity was still present. The authors concluded that ABA couldn’t be the only stress signal acting from roots to shoots. Any general condition of stress (water, temperature, osmotic, etc.) that has an effect on the photosynthetic machinery influences chlorophyll fluorescence. Therefore, chlorophyll fluorescence can be used to indicate how the electron transport through the photosystems is affected during the stress. When light shines on a plant, only less than 1% of the total absorbed solar irradiation is stored into photosynthetic products (Nobel, 1991). The rest of the energy is lost in a number of ways, including heat (latent heat, conduction/convection, and inflated radiation) or radiationless de—excitation and re- errrission as light (fluorescence). When a leaf is placed in the darkness or in dim light for several minutes and then is brightly illuminated, fluorescence rapidly rises to a peak and then it declines to reach a steady-state value. The variable component of chlorophyll fluorescence (F v) is the difference between the maximum fluorescence signal (P...) and the background level signal (F0). The ratio F vIF m is particularly important for physiological studies because it is proportional to the quantum yield of photochemistry (Butler and Kitajima, 1975). Measurements of chlorophyll fluorescence have the advantage of being non- destructive and non-invasive, and they are theoretically ideal for experiments that require repetitive samplings. Moreover, the measurements are very easy and quick to perform and the instrumentation is small and portable, therefore making the technique ideal for collecting large quantities of data in field conditions. Chlorophyll fluorescence data have been used as an indicator of the photosynthetic activity in several works (Bolhar- Nordenkampf and Oquist, 1993; Fernandez et al., 1997b; Massacci and Jones, 1990), 13 however, thus far, practical applications of fluorescence measurements have been limited, especially because of the difficulty in collecting reliable data and in their interpretation. Trees, including fruit trees, differ from annual plants because their life cycle spans through several seasons. This characteristic induces differences in the dynamics of carbon partitioning and biomass accumulation. The fate of new photosynthates and the way they are partitioned among sinks has been widely studied (Zarnski and Schaffer, 1996), but is still poorly understood, especially when roots are involved. On average, in annual plants, between 30 and 60% of the carbohydrates deriving from the photosynthetic process are allocated to the root systems. Out of this quantity, 16-76% is lost through respiratory processes, whereas 4-70% is released as organic carbon into the rhizosphere (Marschner, 1995). Despite the wide variability existing among the different species, it is evident that plants lose a great amount of photosynthate through the root system. The carbon costs of root growth and maintenance are strictly associated with plant growth, fi'uit quality and yield. Particularly, quantifying and understanding the function and the physiology of carbon in the root system during various environmental conditions could help to attain the optimum conditions to maximize growth and yield potential. The growth and dimension of the apple tree root system has been described by Atkinson (1983), but relatively little research has been conducted on root physiology, especially under drought conditions. Direct measurement of root respiration is very difficult because it is impossible to separate carbon dioxide fluxes deriving from root metabolism from other sources of respiration, such as rhizo-microbial respiration (Buwalda, 1993). As explained above, drought stress induces reduction of carbon assimilation. Consequently, a reduction in root growth would be expected when the soil 14 water potential declines. However, in many cases the rootzshoot ratio in plants growing in drying soil was higher than in well-watered controls (Buwalda and Lenz, 1992; Hsiao and J ing, 1987). The increase in rootzshoot ratio during the stress indicates that a greater proportion of photoassimilates is allocated to the roots, making a big impact on yield and fruit quality of perennial fruit crops. The use of heavy stable isotopes is a powerful approach to study C and nitrogen (N) interactions (Deleens et al., 1994), especially in the below-ground compartment. They are not as powerful as radioactive isotopes, because they are required in greater amounts and they are not as easy to detect in pulse/chase experiments. However, they do not present the disadvantages of radioactive isotopes and can be easily handled in the fields or in greenhouse environments. To our knowledge, more experiments have been conducted on crop species than on woody plants (Loreto et al., 1996; Vivin et al., 1995; Vivin et al., 1996). 13C and 15N are the most used stable isotopes of these important elements. They both can be easily and relatively inexpensively supplied to plants and used for labeling experiments. 13 C in form of 13CO2 as an input for the photosynthetic process (V ivin et al., 1995; Vivin et al., 1996), whereas 15N can be embodied within the nitrogen fertilizers, either as ammonium or as nitrate (Moing and Gaudillere, 1992; Toselli et al., 1999). In nature, there are two stable isotopes of carbon, 12C and 13C. ‘2C is the most abundant form, and it accounts for 98.9% of the total C. With the present level of technology, it is not possible to measure exactly the absolute abundance of a stable isotope. However, isotope-ratio mass spectrometers can measure minute differences between a sample and a standard. As a result, a differential notation ((5[3 C) has been 15 adopted to indicate the relative differences in stable carbon isotope ratios between samples and standards (Boutton, 1991a). The 6‘3 C value (expressed as %o) is calculated as follows: 5% = PM; 'R‘w‘d‘” :lx103 standard where R is the ratio between '3 C and 12C. The internationally accepted standard for '3 C was a limestone fossil of Belemnitella americana, known as PDB (PeeDee Belemnite), which has an atom percent abundance of 1.1100%. Today, the PDB standard is no longer available, but other standards have been calibrated against PDB, therefore allowing researchers to continue using PDB as the reference standard. The development of the use of the stable I3C isotope as a tracer for partitioning studies is relatively recent (Y oneyama et al., 1980). The overall abundance of 13 C relative to 12C is usually lower than the amounts expressed above. This occurs because plants discriminate for carbon isotope during the incorporation of CO2 into biomass (F arquhar et al., 1989). Discrimination against 13C occurs at the level of CO2 fixation by the enzime Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase), therefore resulting in relatively low 813C levels in C3 plants. C4 and CAM plants rely on the enzyme phosphoenolpyruvate (PEP) carboxylase as a primary acceptor of C02. PEP carboxylase does not discriminate against 13 C, and this explains why these types of plants have relatively high 513 C values (Boutton, 1991b). 16 1.2. The importance of orchard irrigation Irrigation and water availability have a fundamental role fi'orn the time of establishment of young trees in the orchard. The importance of the initial care of young trees for the successful performance of the orchard has been described in several works (Autio and Greene, 1991, and references therein). The selection of the proper rootstocks is also essential, especially for those areas where drought represents a potential danger for the establishment and the future performance of the orchard. In the productive years, water still represents one of the most important factors, because it has a great influence on plant growth and on size and yield of fruit crops. Water deficit conditions during fruit development can in fact induce a decrease in net productivity and affect fruit quality. There are several ways in which drought can affect fruit production and they can be related to alteration of a wide range of physiological and developmental processes. Among the many biochemical and developmental processes influenced by water deficit, reduction of both cell division and expansion, and decrease of the photosynthetic processes play a major role, together with a reduction of enzyme activity in general. Plants can experience drought not only when soil water content is limiting, but also when atmospheric water content declines or when both conditions are present. Among the physiological characteristics that confer certain species or varieties is their capacity to tolerate or avoid drought stress. In addition, flow resistances in the soil-plant pathway play an important role (Jones et al., 1985). Fruit trees are particularly sensitive to atmospheric conditions because they usually possess low hydraulic conductivity of the root system. Atkinson (1980) explains that low hydraulic conductivity of Malus and 17 Prunus species are correlated with the extremely low root densities that this genera tend to have, compared to the values reported for herbaceous species. Therefore, in Mt trees, a critical soil component of the hydraulic resistance can develop at higher soil til than for herbs. However, fruit trees possess big root systems that can explore great soil volumes and therefore maintain constant values of I]! and transpiration rate. For all the aforementioned reasons, irrigation is 'often economically profitable for the production of high quality fi'uit not only in arid and semiarid areas. Irrigation is usually suggested also in subtropical areas because of the irregular distribution of rainfall and the presence of soils with low water-holding capacity. Nonetheless, there are still many regions, mainly characterized by semi-humid climates, where tree fi'uits are grown without irrigation. That is the case for apple orchards in many regions of the US, like Ohio, where long-term studies (F erree and Schmid, 1990) have shown little benefit of irrigation under normal rainfall conditions. However, if growers are not equipped with an irrigation system they are unable to avoid serious consequences when severe drought conditions occur. The timing and frequency of irrigation are also important aspects to take into account to maintain good plant water status. In order to optimize plant water status and to reduce the costs related to irrigation, it is fundamental to develop knowledge of the natural processes that regulate plant water use. This information is necessary not only to develop a good method and schedule of irrigation, but also to formulate more accurate cultural techniques to achieve good control of plant water conditions, i.e. pruning, training systems, fertilization, chemical thinning, etc. Moreover, knowledge of plant physiology in relation to water consumption and use efficiency can undoubtedly help to 18 determine the most appropriate method of irrigation (sprinkler, trickle, over-tree mist, etc.). Even though, in recent years, drip irrigation has allowed control of this problem in a relatively economic way, irrigation is still responsible for a striking component of the farmer’s budget. 1.3. Apple rootstocks One of the most important requirements for apple rootstocks is the capacity to reduce tree vigor, increase precocity and yield efficiency, and allow high-density planting. Besides facilitating the harvesting and cultural processes, the use of intensive plantings of small trees allows the maximization of the interception of incident light, which is usually correlated with a higher dry matter production (Jackson, 1980). Smaller trees have in fact less internal shading, which corresponds to a higher photosynthetic efficiency per leaf area unit. Shaded leaves provide little contribution to the overall productivity of the plant, and can represent a negative term in the photosynthetic budget. However, differences in light interception do not account for all the variability that exists among rootstocks, and it is frequent for rootstocks that produce trees with similar size to have dissimilar yield efficiency (F erree and Morrison, 1975). Improved light interception and distribution also play a role in the partitioning of photosynthates to reproductive organs rather that to wood, therefore providing a further contribution to high yield efficiency. Among the fruit species, apple has the privilege of having a wide range of rootstocks, which is continuously extended with the selection of new rootstocks. l9 1.3.1. History The use of dwarf apple trees can be traced back to the third century BC However, it was only when grafting and budding techniques were developed (fifteenth century) that the use of dwarfing rootstocks became widespread. Rootstocks were imported to the United States from the early 19th century, but it was only in the 1920’s that they became objects of study and experimentation. The initial necessity for using rootstocks was driven by the necessity to obtain trees with size and/or weight different fi'om the ones deriving from seedlings. Behind this necessity, there were basic economic reasons for the growers to get earlier economic returns by planting trees on small precocious rootstocks. Trees produced by apple rootstocks have in effect size that can range from larger (e.g., M.25) to 15-20% the sizes that of seedling rootstocks (e.g., M.27) (F erree and Carlson, 1987). The advantage of using dwarfing rootstocks and therefore trees with small size has allowed intensive orchard management to become a standard practice for apple production in North America The early need to create smaller trees using rootstocks was rapidly flanked by the necessity to increase the productivity and the resistance to pests, diseases, climatic and soil extremes. For the investigations, an experiment was developed using three different apple rootstocks, MAC 9 (Mark), M.9, and Budagovski 9 (B.9). The former two rootstocks have been extensively studied, whereas B.9 is still relatively untested. However, all of them are known mainly for their dwarfing properties. M9 was selected in France in 1879 and is the worldwide standard rootstock for the production of small precocious trees (F erree and Carlson, 1987). When used for one- union trees, it produces trees 25-35% the size of the seedlings. For this reason, and 20 because of its high productivity and precocity, M9 is used in high-density plantings, especially in Europe. In North America, it is widely used to obtain small trees and where ‘you-pick’ plantings are desired. M9 is however susceptible to fire blight (caused by the bacterium Erwinia amylovora), crown rot (induced by Phytophtora cactorum), wooly aphids (Eriosoma lanigerum) and brittleness. Moreover, like many other dwarfing clones with thick rootstock bark, it seems very attractive to voles. The Mark rootstock was selected in 1959 by RF. Carlson fi'om an open pollinated seedling population of M9. It was patented in 1980 and released for commercial sale by Michigan State University in 1986 as a potential new apple rootstock (Carlson, 1980; Carlson and Perry, 1986; Perry and Carlson, 1986). The stature of trees grown on Mark is about 50% of the seedling stock. Since its release, Mark has been widely propagated and planted throughout the apple industry (Schupp, 1992), and provided a new candidate to compare to M9. The performance of Mark with the ‘Delicious’ cultivar has been extensively studied (Carlson, 1980; Carlson and Perry, 1986; Dennis, 1981; Ferree and Schmid, 1994). The Budagovski series was introduced by the College of Horticulture in Michurinsk (Russia) as rootstocks capable to withstand the severe climatic conditions of central Russia (-40 °C most every winter). B.9 (also known as ‘Bud.9’) originated from a cross of M8 with Red Standard and it has similar dwarfing potential and susceptibility to fire blight, wooly aphids and brittleness as M.9, but is hardier and more resistant to crown rot. It has been widely used in Poland as a dwarfing interstem, but it is still not very diffused in North America. Further investigation is being undertaken to verify the potential diffusion of this rootstock, because its capacity to adapt to North American soils 21 and climates is still uncertain. Mullins (1993) reported in fact that tree loss due to winter injury was higher (50% loss) when Starkspur Supreme Delicious trees were grafted on B.9 than when grafted on B.491, MAC 1, MAC 39, or Poland 1 (30, 20, 30 and 10% loss, respectively). 1.3.2. Physiology of the dwafling process Variations in size and in resistance to environmental factors (biotic or abiotic) represent the macroscopic results of a complex of physiological modifications that rootstocks induce to the scions. A lot of research has been conducted on rootstocks and on the way they affect growth and development of the scion (Hirst and Ferree, 1995; Lehman et al., 1990; Lockard and Schneider, 1981; Schechter et al., 1991b). Nevertheless, thus far, satisfactory explanations of the dwarfing mechanism in a rootstock or interstock have not been developed. Jones (1974) indicates the graft union as the site responsible for the growth potential of the scion. The author reports that the nutrient concentration in the xylem sap above the graft union is lower than below, especially for a dwarfing rootstock such as M.9. However, other works (see Lockard and Schneider, 1981, for a complete review) have disproved the hypothesis that the stocks could induce a reduction in the water and nutrient supply. No major differences were in fact detected between nutrient composition of the scion leaves when grafted to different rootstocks. Other theories have proposed feedback inhibition mechanisms or the synthesis of auxin inhibitors produced in the stocks. Plants are capable of regulating the balance between root and shoot and the growth of either part is controlled by the other. This capability is maintained in grafted 22 plants and the mechanism involved is what Wareing (1977) referred to as a growth substance interrelationship between root and shoot. The shoot-produced auxin has a major role on metabolite translocation and carbohydrate metabolism and therefore auxin is likely the growth regulator that mostly affects the mechanism for control of root growth by the shoot (Kramer and Kozlowski, 1979) and, therefore, between stock and scion (Lockard and Schneider, 1981). Back in 1935, Colby (1935) indicated that the more dwarfing apple rootstocks have higher starch content. It seems that auxin is the key compound to establish the communication between stock and scion (Lockard and Schneider, 1981). The level of auxin produced in the shoot and translocated to the roots is the main factor regulating their growth and metabolism. Auxin also plays a role in the metabolism of cytokinin, a group of hormones mainly synthesized in the roots and then translocated via xylem to the upper part of the plant. Cytokinins play a fundamental role in the regulation of apical dominance and senescence delay. Cytokinin synthesis is a process strictly controlled by the level of auxin produced by shoots and young leaves. Cytokinins seem also to enhance the sink effect of the tissue (Morris and Winfield, 1972), therefore facilitating the movement of nutrients to the growing regions. Once cytokinins arrive at the shoot tips, they control shoot growth and, consequently, auxin production. This delicate equilibrium between the two growth regulators is probably the key to the precise balance between shoot and root growth in plants. The balance between the two hormones is very important for carbon partitioning. Charles-Edwards (1979) emphasized that growth and development are more regulated by the control that growth regulators have on photosynthate distribution than by the photosynthetic activity. According to Lockard and Schneider (1981), when a scion is grafted on a dwarfing rootstock or 23 interstock, this equilibrium is somehow altered. The bark of the rootstock or of the interstock seems to be the part that has the stronger control over the passage of auxin from the scion to the stock. Thus, barks of different plants would have different levels of auxin that reach the roots and, consequently, changed levels of root growth, cytokinin synthesis and a smaller or larger shoot growth, depending on the type of stock. However, none of these theories have been fully proven. The reason being that, in most cases, different mechanisms act simultaneously in the induction of the dwarfing mechanism in plants. Yadava and Dayton (1972) found that the concentration of ABA is higher in some apple dwarfmg rootstocks, therefore suggesting a role of the hormone in reducing the scion vigor. Apple tree productivity is dramatically influenced by rootstock. Rootstocks have an effect on precocity and apical dominance, leaf size, rate and duration of growth (Tubbs, 197 3). The influence of rootstock on canopy net assimilation rate may be the main mechanism by which the rootstock exerts its effects on scion growth and productivity. Nevertheless, studies of the effects of the stock on the net assimilation rate (A) of the tree have produced a wide range of results. Photosynthetic rate was higher on seedling rootstocks than on Malling Merton (MM) 106 (F erree and Barden, 1971). Marro and Cereghini (1976) indicated that A in ‘Richared Delicious’ was higher on M.9 than on seedlings. Schechter et al. (1991a) reported that leaf net photosynthesis was lower in trees grafted on dwarfing rootstocks than on more vigorous rootstocks. Barden and Ferree (1979) reported no effect for a range of apple rootstocks on the levels of A and in transpiration of ‘Starking Delicious’. The performance of a certain scion-stock combination is usually quite specific and is not usually indicative of the performance of 24 another scion cultivar on the same stock or of the same cultivar on another stock (Tubbs, 1973). For example, Ferree et a1. (1980) reported that trees of the cultivar ‘Mollies Delicious’ were 28% smaller when grown on MM.106 than when grown on M.7. Viceversa, ‘Mutsu’ trees on MM.106 resulted 185% larger then those on M.7. There is still a lot of controversy regarding the capacity of rootstocks to confer drought resistance to scions. Landsberg and Jones (1981, and reference therein) indicated that dwarfing rootstocks, such as M.9, are usually more tolerant to water deficit than more vigorous rootstocks. However, Ferree and Carlson (1987) described the same rootstock, M.9, as drought sensitive. The influence of M9 EMLA and Mark rootstocks on drought tolerance of apple trees have been studied by Fernandez (1997a). Fernandez found an inverse relationship between the vigor of 5 rootstocks (M.9 EMLA, M.26, MM.106, MM.111, and Mark) and their root hydraulic conductance (Fernandez, 1992). The more dwarfing M.9 EMLA showed the greatest root hydraulic conductance while the vigorous MM.111 had the least. Mark showed intermediate values between M.9 EMLA and MM.111. When two levels of drought stress were imposed to ‘Imperial Gala’ apple on Mark, M.9 EMLA, and MM.111, Mark proved to be the most sensitive to the stress, and M9 EMLA the most drought resistant. Tree growth and whole plant photosynthesis were reduced by the stress to the greatest extent for trees grafted on Mark (F emandez et al., 1997a; Fernandez et al., 1997b). Today, many of the questions regarding the use and performance of the most common rootstocks have been answered, but there are continuously new clones and rootstocks that are selected and are being tested in different regions of the world. 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DROUGHT RESPONSES OF YOUNG APPLE ROOTSTOCKS GROWN WITH SPLIT-ROOT SYSTEMS Leonardo Lombardini Abstract Three apple rootstocks, B.9, M.9, and Mark, were grown with their root systems split between two containers to compare the effect of different soil water deficits applied to the root system either equally or unequally. Soil water content was maintained at 30, 25, and 20% (v/v) either in both containers or in only one, for a total of 6 irrigation treatments. Leaf expansion rate, total leaf area, predawn and midday leaf water potentials, single leaf gas exchange, and chlorophyll fluorescence were monitored at regular intervals during a 28-day experiment. Dry matter accumulation was also measured at the end of the experiment. Results seemed to suggest an increase in tolerance to water deficit in plants with half of the root system growing in non-irrigated soil. Total leaf area was reduced by water deficits in two of the three rootstocks investigated (B.9 and Mark), mainly when half of the root system was in dry soil. However, neither leaf number, nor shoot length were modified in any of the rootstocks. Relative leaf expansion rate was 36 mostly affected by drought for Mark rootstock, indistinctly whether irrigation was distributed equally or not to the root system. In most cases, non-uniform irrigation induced a reduction of fresh weight and water content in roots growing in the non- irrigated side. Leaf water potential measured at predawn and midday indicated that B.9 could tolerate conditions of non-uniform irrigation better than water deficit imposed equally to the entire root system. Gas exchange analysis indicated that Mark had the greatest reduction in assimilation rate and water use efficiency when treated with non- uniform irrigation. Water use efficiency increased with time in stressed B.9, which also showed high stomatal conductance and transpiration rate. Variable and maximal chlorophyll fluorescence were not as sensitive to stress as the other growth and physiological parameters investigated. More thorough research is needed to investigate the nature of the induction of stress tolerance by roots growing in water deficit conditions. Introduction Unlike most other cultural decisions, the choice of the correct rootstock has to be made before orchard establishment. The choice of the appropriate apple rootstock is likely the most important cultural decision that a grower can make because it affects tree size, growth habit, precocity, and productivity (Olien et al., 1995; Tubbs, 1973). Among the many requirements for apple rootstocks, predominant are the capacity to reduce tree vigor, increase precocity and yield efficiency, and allow high-density planting. The advantage of using dwarfing rootstocks, and therefore trees with small size, is that it 37 allows intensive orchard management facilitating the harvesting and cultural processes. For these reasons, the use of dwarfing rootstocks has become a standard practice for apple production in North America. Today, many of the questions regarding the use and performance of the most common rootstocks have been answered, but continuously there are new clones and rootstocks that are being tested in different regions of the world. Through the efforts of researchers, especially those associated with the USDA-CREES regional project NC-l40, the effects of different rootstocks on growth, productivity and performance of apple trees have been compared (Fernandez et al., 1997a; Fernandez et al., 1997b; NC-l40, 1996a; NC-140, 1996b; NC-140, 1996c; Schupp, 1992; Schupp, 1995). Water deficit can have serious detrimental effects on the growth and development of plants. Drought influences stomatal aperture, either directly, with turgor loss in stomatal guard cells, or indirectly, with the production of ABA or other inhibitory substances. However, loss of turgor induced by drought triggers physiological and biochemical adjustments that are important for turgor maintenance. The importance of elastic and osmotic adjustments has been highlighted by Schulte and Henry (1992). Shoot and leaf growth seem to be affected first by the lowering of leaf water potential (Flore and Lakso, 1989). When mild water stress was applied to peach trees, reductions in shoot elongation and leaf expansion were detectable before assimilation rate and stomatal conductance decreased (Andersen and Brodbeck, 1988). In the case of plants with commercial importance, like apple trees, the effects of water deficit also have negative repercussions on yield and fi'uit quality. 38 Despite the wealth of the lmowledge available on rootstock physiology, there is still considerable controversy regarding their capacity to confer drought resistance to scions. Landsberg and Jones (1981, and reference therein) indicated that dwarfing rootstocks, such as M.9, are usually more tolerant to water deficit than more vigorous rootstocks. Nevertheless, Ferree and Carlson (1987) described the same rootstock, M.9, as drought sensitive. Recently Fernandez et al. (1997a) described the influence of M9 EMLA and Mark rootstocks on drought tolerance of apple trees. Fernandez et al. found an inverse relationship between drought tolerance and the vigor of five rootstocks (M9 EMLA, M.26, MM.106, MM.111, and Mark). The root hydraulic conductance of the same five rootstocks has also been studied (Fernandez, 1992). The more dwarfing M.9 EMLA showed the greatest root hydraulic conductance while the vigorous MM.111 had the least. Mark showed intermediate values between M.9 EMLA and MM.111. When two levels of drought stress were imposed to ‘Imperial Gala’ apple on Mark, M.9 EMLA, and MM.111, Mark proved to be the most sensitive to the stress, and M9 EMLA the most drought resistant. Tree growth and whole plant photosynthesis were reduced by the stress to the greatest extent for trees grafted on Mark (Fernandez et al., 1997a; Fernandez et al., 1997b). Studies of the effects of the rootstock on net assimilation rate (A) of the shoot have produced a wide range of results. Photosynthetic rate was higher on seedling rootstocks than on MM.106 (F erree and Barden, 1971). Schechter et a1 (1991) reported that leaf net photosynthesis was lower in trees grafted on dwarfing rootstocks than on more vigorous rootstocks. Barden and Ferree (1979) indicated no effect for a range of apple rootstocks on the levels of A and in transpiration of ‘Starking Delicious’. 39 Conditions of non-uniform water availability (i.e. soil water potential) commonly affect field crops. This aspect is particularly important when the object of interest focuses on crops that require intensive irrigation. Water content within the soil usually varies with a gradient along the soil profile, due to the effect of evaporation fiom the soil surface, root distribution, and the presence of deep ground water. Soil water content also varies horizontally, which can be caused by soil heterogeneity, root distribution and non- uniform irrigation. Irrigation practices, like trickle irrigation, commonly induce a gradient of soil water content within and across the soil profile. Roots can therefore be exposed to different conditions even in a restricted volume of soil. Knowing the behavior of the rootstock in such conditions could help the grower in the choice of the most appropriate rootstock and method of irrigation. Split-root studies represent a way to simulate the heterogeneity in the field (Zhang and Kirkham, 1995), and have been widely used to examine the effects of differential irrigation in herbaceous plants (Williams et al., 1991, Zhang, 1995). Water uptake by one part of the root systems has been shown to be mostly dependent on the localized soil water potential. However, the relationship between soil water potential and water uptake may be affected by variations of the soil water status in other parts of the root system (Sirnonneau and Habib, 1994). Tan et a1. (1981) reported that tomato plants that had 25% of the root system watered, showed only 20% reduction in transpiration. The authors suggested that tomato roots could adjust their relative absorption capacity for water uptake in response to the transpirational demand. They did not speculate on the potential involvement of ABA as a signal to induce stomatal closure. Although circumstances of non-uniform water availability frequently occur in orchards, they have not been widely studied in terms the consequences on gas exchange and growth 40 in woody plants. Only a few studies have investigated the effects of non-uniform root zone stress on water relations and photosynthesis in hit trees (Gowing et al., 1990; Neri and Flore, 1990, Sirnonneau, 1994; Proebsting et al., 1989). Plant water status and water potential have been used as indicators of plant stress because they account for the effects of the availability of water in the soil, the evaporative demands of the surrounding environment, and the hydraulic fluxes within the soil-plant- atrnosphere continuum (Andrews et al., 1992). Chlorophyll fluorescence is also a useful tool to study photosynthetic activity, especially photosystem H (Karukstis, 1991). Any general condition of stress (water, temperature, osmotic, etc.) that has an effect on the photosynthetic machinery influences chlorophyll fluorescence. Therefore, chlorophyll fluorescence can be used to indicate how electron transport through the photosystem is affected during stress (Schreiber and Bilger, 1987). The present work was undertaken to determine whether partial soil drying would influence transpiration, photosynthesis, and chlorophyll fluorescence in rootstocks growing in different conditions of soil water content. In particular, by comparing plants with roots subjected to uniform and non- uniform watering, it should be possible to determine whether a dry portion of the root system could improve the ability of other parts of the plant to withstand stress. B.9, M.9, and Mark are known for their dwarfing properties. Mark and M9 have been extensively studied, whereas B.9 is still relatively untested and not widely used in North America. 41 Materials and methods Plant material and treatment application. Experiments were conducted in summer 1997 at Michigan State University on one-year old apple (Malus domestica Borkh.) rooted cuttings (trunk diameter approx. 1 cm) of three rootstocks, Budagovski 9 (B.9), Malling 9 NAKB T-337 (M.9), and MAC 9 (Mark). Plant material was purchased from Treco® Nursery (W oodbum, OR, USA). Twenty-four cuttings per rootstock were used. During potting, the root system of each cutting was pruned back to a length of approximately 15 cm. The stem of each cutting was positioned at the union of two coupled square pots (size 16.5 X 16.5 X 20.0 cm), with the root system split between two pots. Only plants that presented a natural separation of the root system were used for our investigation. The soil used was a 2:3 v/v mixture of Baccto (Michigan Peat Co., Houston, TX, USA) propagating mix (horticultural Sphagnum, perlite, vermiculite, lime, balanced nutrients, trace elements, wetting agent, pH = 5.9-6.2) and sandy loam soil (65% sand, 24% silt, and 11% clay). After budbreak, plants were pruned to two extension shoots per plant, regulme watered, and biweekly fertilized with a 560 ppm solution of Peters soluble 20N-20P-20K. Plants were grown outdoors at Michigan State University for ten weeks, before treatments were applied. One week prior to treatment, soil water content (SWC) during soil drying conditions was estimated on three plants per rootstock, fiom the same batch of nursery plants. Pots were covered with plastic fihn to reduce water loss through evaporation, and SWC was monitored using a time-domain reflectometer (TDR, Tektronix 1502, Tektronix Inc., Beaverton, OR, USA). For the measurements, a pair of steel TDR probes (length 170 mm, diameter 5 mm) was inserted 42 vertically in each pot to provide average soil water content within the pot soil profile. Plants were well watered and SWC was measured at field capacity. Plants were then left without irrigation to the wilting point. Wilting occurred in all plants 4 and 5 days after inigation was suspended. SWC was then measured again on the fifth day. Average SWC was 32% :I: 3 (v/v i SD) at field capacity, and 8% :1: 4 (v/v 1 SD) after 4-5 days without irrigation. For treatment application, three levels of SWC were chosen, firll capacity (30%), light water deficit (25%), and moderate water deficit (20%). Water was applied to the two halves of the root systems either equally or unequally. In equal irrigation (E), the three levels were maintained for both halves of the root system (treatments 30E, 25B, and 20B). In unequal irrigation (D), the three SWC levels were maintained on half of the root system, while the other half was left dry (treatments 30D, 25D, and 20D). SWC was measured daily in all pots and water was applied in the volumes necessary to achieve the desired SWC (d: 2%). Plants growing with the entire root system in field capacity conditions (30E) were considered as the control treatment. During the experiment, pots were covered with transparent polyethylene film to protect the soil fi'om rainfall events. The fihn was kept 3-4 cm above soil surface to allow air circulation. Experiment began on Aug. 14 and terminated on Sep. 11, for a total of 28 days of observations. Plant growth. The most recently unfolded leaves of each plant were marked every week with different colored yam. This allowed us to monitor shoot extension and to identify leaves of comparable age for measuring photosynthesis, chlorophyll fluorescence, and water potentials. Total leaf number, lamina length and width of each 43 leaf were measured on all plants every 7 days. Lamina length and width were measured using a caliper and total leaf area (LA) was estimated by multiplying their value by the factor 0.7 (Fernandez et al., 1997a). Shoot length was also measured at the same intervals, using a metric ruler. To evaluate whether the onset of water deficit condition had any effect on leaf expansion, the first unrolled leaf of the main growing shoot of each plant was tagged and LA was estimated as described above. LA of the tagged leaves was then monitored every other day for the first 10 days of the experiment. The obtained values of LA were then used to estimate the mean relative leaf expansion rate (RLER), which was calculated using the formula (Beadle, 1987): 1n(LA2)-1n(1-Al) tz—tl RLER= where t1 and t2 indicate the initial and final time, respectively, of a discrete time interval of measurements (two days in the present study), LA1 and LA2 are the values of the leaf area measured at t1 and t2, respectively, and In indicates the natural logarithm. At the end of the experiment (day 28), fresh weights of roots, trunk, shoots and leaves were measured for each single plant. Dry weights were measured after the tissues were oven-dried at 80°C for 48 hours (Roberts et al., 1987) and tissue water content (WC) was calculated as percent of fresh weight (FW). Roots from the two containers were kept distinct during the analysis. Leaf water potential. Predawn and midday leaf water potentials (M.,) were measured on one leaf per plant using a pressure bomb (Plant Moisture Stress Instrument 44 Co., Corvallis, OR, USA) on days 0, 3, 6, 12, and 24 from the beginning of the experiment. Leaves were excised and Ill“, was measured within 2 minutes, following the standard protocol described by Ritchie and Hinckley (1975) and modified for apple leaves by Davies and Lakso (1978). Measurements were made using the youngest fully developed leaves between 0400 and 0600 HR Eastern Standard Time (EST) for predawn yr“, and between 1200 and 1400 HR EST for midday law. Gas exchange. Single leaf gas exchange were measured on day 0, 7, 14, 21, and 28, between 1300 and 1400 HR EST using a CIRAS-l infrared gas analyzer (PP-Systems Inc., Haverhill, MA, USA). Gas exchange was measured on fully expanded, healthy leaves chosen between the yarns marking the leaves that had completed their expansion during the previous week. One leaf per plant was used. Each leaf was inserted into the leaf chamber (2.5 cmz), and one single measurement per leaf was collected, after waiting 2-3 minutes for the atmosphere in the chamber to equilibrate. Net assimilation rate (A), stomatal conductance (gs), transpiration rate (E), and leaf intercellular CO2 concentration (C;) were the parameters calculated at each measurement (see CIRAS-l user’s manual for reference on theory and calculations). The water use efficiency (WUE), also called transpiration ratio, was calculated by dividing the transpiration rate (expressed in umol H2O m'2 s") by the assimilation rate (umol CO2 m'2 s") Chlorophyll fluorescence. Chlorophyll fluorescence was determined with a plant efficiency analyzer (PEA, Hansatech Instruments Ltd., Norfolk, UK). A preliminary experimental procedure was performed to determine the required dark adaptation time 45 necessary to maximize the ratio between variable fluorescence (F v) and maximum fluorescence (F m). The protocol to calculate the dark adaptation time was described in the PEA instruction manual. For our plants, 16 minutes provided adequate dark adaptation. Once the time was estimated, a second preliminary procedure was performed to calculate the maximum light intensity level (0-100% of the maximum light intensity emitted by the PEA) to set the instrument in order to obtain the maximum Fv/Fm without overscaling (Appendix A, Table A.1). Leaves were fully saturated at 80% of the maximum light level, a value that was used therefore for the entire set of measurements. For data collection, leaves were covered with specific lightweight leafclips, at least 16 minutes before measurements to allow dark-adaptation. Measurements were collected between 1200 and 1300 HR EST on day 0, 6, 12, and 24. Experimental design and statistical analysis. The experimental design was a split- plot design, with the three rootstocks as the main plot, the six water treatments as the sub- plot and four single-tree replications per treatment. Plants were randomly assigned to the six treatments and blocked by the number of leaves to reduce the effect of the canopy size on the variability. Statistical analysis was performed by analysis of variance (ANOVA), and Least Significant Difference (LSD) test was employed for mean separation at P s 0.05 within each date of measurements. To compare data relative to roots growing in the two separate pots, t-test was also performed. Data analysis was completed using SAS software (SAS Institute Inc., Cary, NC, USA). 46 Results Plant growth. Variation of total LA throughout the experiment is reported in Table 1.1. In B.9, differences among the treatments were statistically significant on day 21 and 28. On day 21, the maximum value of total LA (880 cm2) was measured on control plants (30E), and the minimum (717 cm2) on 25D plants. On day 28, maximum LA was still recorded on 30E plants (1142 cmz), whereas 20D showed the mimimurn value (598 cmz). Treatments did not induce any significant change in total LA in M9. In Mark, maximum LA was recorded in 20D plants in both days 14 and 21, whereas minimum values were measured on 25D plants. On day 28, significant differences were detected between control plants (LA 1013 cm2) and 25D plants (587 cmz). Total leaf number and shoot length are reported in Table 1.2 and 1.3, respectively. Neither growth parameter was affected by irrigation and no significant differences were observed among the treatments. Results of the analysis of RLER in the first 10 days of application of treatments are reported in Figures 1.1-1.3. All rootstocks showed maximum RLER between day 2 and 4. A general decrease then followed, and relative leaf expansion was almost complete by day 10, regardless of rootstock used and water regimen applied. B.9 did not show differences among the six treatments (Figure 1.1) between days 0 and 2, and between days 6 and 10. Plants with the root system growing in soil with 20% WC (treatments 20B and 20D) had reduced RLER in days 2-4, when compared to 30E. Highest RLER was recorded in 30E during days 2-4, which represented the only time that differences between equal (30B) and unequal (30D) irrigation conditions were observed. Like in B.9, 47 RLER in M9 was not significantly different among the treatments during days 0-2 and 6- 8 (Figure 1.2). However, between days 2 and 6, 30E had higher RLER than 25D, 20E, and 20D. During the same period (days 2-6) and days 8-10, 25E always showed higher RLER than 25D. In the first four days of stress, the response of Mark to water deficit (Figure 1.3) was a lower RLER in plants with only half of the root system growing in field capacity conditions (30D) compared with plants with field conditions maintained around the entire root system (30E). Low values of RLER (less than 9 mm2 LA cm'2 (1") were observed in 20B and 20D throughout the entire period of measurements (Figure 1.3). Root FW and WC, measured separately for the two containers at the end of the experiment, are reported in Table 1.4. Significant differences in root FW and WC were not detected when the two halves of the root system were growing in soil containing the same amount of water. Conversely, when irrigation was applied unequally, the portion of the root system that did not received any water had usually lower F W and WC than the corresponding watered side. However, only in a few cases were these differences between the two parts of the root system significant. In B.9, a lower WC in roots growing in the non-irrigated side was observed only in 30D plants. In M.9 25D, FW in non- irrigated plants was 44.7% of the FW measured in the irrigated side. In Mark, root FW was lower in the non-irrigated side in 30D and 20D. Minimum value of root WC was measured in 20D Mark, where roots of the dry side had 58.5% water, compared to 69.6% of the irrigated side. When FW and WC data from the two containers were pooled and LSD test was used for treatment mean separation, results indicated a general reduction in 48 both parameters, parallel with the reduction of SWC (Table 1.4). FW in M9 was the only exception to this trend, with no differences observed among treatments. Values of tissue WC calculated for other plant organs at the end of the 28 days of treatments are shown in Figure 1.4. Leaf and shoot WC was not affected by water treatment in B.9. Instead, trunk WC was lower in 25E, 25D, and 20B than in control plants. In M.9, shoot and trunk WC was not affected by the water distribution. However, in the leaves of M.9, WC was lowest in 25E, 20B and 20D. Mark showed a significant reduction of leaf WC only when the same SWC was maintained in both pots (25B and 20B). Compared to control plants, reductions in the trunk WC were observed in 25E, 20B and 20D. Dry matter partitioning in the main plant organs (leaves, shoots, trunk, and roots) showed that, in B.9, leaf DW was 47.9% lower in 25E plants than in control plants (Figure 1.5). In M9 and Mark, the maximum reduction was observed between control plants and 20E (59.3% and 78.2%, respectively). As previously remarked, roots from the non-inigated containers had also reduced mass than the irrigated side (Table 1.4). The decreased growth was the main cause of the reduced dry matter allocation in roots in the dry side. Growth was instead stimulated on the watered side, therefore increasing the proportion of dry matter in roots growing in that side. Compared to the other two rootstocks, Mark had a lower percentage of dry matter accumulated in the trunk (between 42.2 and 59.6%) and a higher proportion of dry matter partitioned to the roots. Percent root dry matter was in fact 9.9-13.3% in B.9, 7.0-13.7% in M.9, and 166-19.7% of the total dry matter in Mark. In Mark, roots that grew in containers that never received water also showed a higher dry matter accumulation than the other two rootstocks. 49 Leaf water potential. In B.9, predawn Ill“, did not differ in any of the treatments during the first 6 days of experiment, with values between —0.18 and —0.42 MPa (Table 1.5). On day 12, w“, in control plants was —0.34 MPa, significantly higher than in 30D, 25D, 20B and 20D. However, on day 24, the lowest predawn I/lw was measured only in 25B and 20B treatments (-2.35 and -2.25 MPa, respectively), while in all the other treatments I/lw was similar to the control (Table 1.5). Like in B.9, in M9 day 6 was the first day in which differences in predawn M., were observed. 20D plants had the lowest w“, on both day 6 and 24 (—0.52 and —1.42 MPa, respectively). On day 24, predawn W in Mark was influenced by SWC rather than by water distribution between containers. Values of W.» in plants that had roots growing in soil with a specific SWC were in fact equivalent, regardless whether half of the root system was growing in drying soil or not. Like for predawn I/lw, midday VI“, in B.9 plants showed significant differences starting from day 12 (Table 1.6), when 20B and 20B had the lowest 1.1/w (—l.49 and —2.00 MPa, respectively). On day 24, W“, in 25E, 20B and 20D was significantly lower than in control plants. In both M9 and Mark, differences among treatments appeared from day 3. On day 24, WW was significantly lower in 25E, 20B and 20D than in control and 30D plants, just like it was observed for B.9. On day 24, midday w... showed a positive relationship with SWC (i.e., as SWC decreased, Ww decreased, as well) in all rootstocks. 30B and 30D plants had the highest I/lw, 20B and 20D had the lowest, and plants that were growing in soil containing 25% water had intermediate values of w... 50 —_—-,—-——— . -—————— ._ - . ..-..L-H 'M‘.‘ I”- — - - Gas exchange. The average A for control plants throughout the experiment was highest in B.9 (average of 10.6 umol m'2 s'l), followed by Mark (8.6 umol m'2 s") and by M9 (7.3 umol m”2 s") (Figure 1.6). Overall, B.9 responded better in maintaining a higher A rate during the stress than the other rootstocks, showing differences among the treatments on only two days out the four days of measurements. In B.9, A was significantly lower in 20E compared to 30E on day 28. In M.9, A had the lowest value in 20E on day 7 and 14, and the highest on day 21. In Mark, A seemed particularly affected by drought when this was imposed only on half of the root system. Plants with half of the root system non-irrigated had often significantly lower A than plants irrigated with the same SWC applied on both pots (Figure 1.6). On most days, stomatal conductance was not significantly affected by irrigation treatments and significant differences between equal and unequal irrigation were never observed (Figure 1.7). In M.9, no significant differences were detected between control and any of the other treatments throughout the entire experiment. In Mark, gs assumed the lowest value on day 14 (78.3 mmol m’2 s") in 20D plants, whereas the highest one (144.0 mmol rn’2 s") was measured in 25E (Figure 1.7). Like for g., E was not greatly affected by the water treatment (Appendix A, Figure AD. In Mark, E was significantly lower in 20D than in 25E on day 14. Among the rootstocks, Mark showed the greatest reduction in E, 7 days after the beginning of the stress. WUE slightly increased throughout the experiment in all the treatments applied to B.9, indicating that a higher amount of water was consumed by transpiration to allow CO2 assimilation (Figure 1.8). In M.9, WUE showed some fluctuations, but the trend was rather constant with time. In Mark, instead, WUE started decreasing after a maximum value was observed on day 7. In a few cases, Mark showed also significant differences of 51 WUE between equal and unequal water distribution. WUE was lower in B.9 on the day experiment started (Figure 1.8), in large part due to the higher A value that this rootstock showed compared to the other two rootstocks. In B.9 and M.9, WUE increased mainly because the stress induced a slight decrease of A (Figure 1.6) and increase of E over time. On day 7, leaf intercellular CO2 concentration calculated for B.9 was significantly lower at P s 0.05 in 25E than in control plants (Figure 1.9). Differences among the treatment means were not significant on days 14 and 21, but on day 28, 20B and 20D had higher C; than 25D. In M.9 differences were observed on days 7 and 21. On day 7, 20E had higher C, than 25E, but it was lower than 30E on day 21. In Mark, C; dropped after 7 days fi'om the application of the treatments in 20E, but then it increased again at later dates. On day 28, the lowest C. was detected in 30E, whereas 20E showed the highest Ci. Chlorophyll fluorescence. In B.9 and Mark, the ratio F.,/Fm did not indicate detectable differences (at 10% level) among the treatments until day 12, whereas, in M.9, differences were observed only on day 24 (Table 1.7). In B.9, the lowest level of FV/Fm was observed in 20E. However, in M9 the Fv/Fm value in control plants was significantly lower than what measured on 30D. On day 12, the highest values of F.,/Fm in Mark were detected in 25D and 20B, whereas the lowest FV/Fm was observed in 30D. On day 24, the situation was partially reversed with 30D having the highest F,,/Fm and 20D the lowest one. Results relative to FV and Fm are reported in Appendix A (Tables A2 and A3, respectively). 52 Discussion The rootstocks considered for the present study are known to have distinct morphological and physiological characteristics, and, consequently, different practical application (Perry, 1999, personal communication). For these reasons, a direct comparison of the three rootstocks could be sometimes misleading. The main objective of the present work was to compare plants growing in equal and unequal conditions of soil water availability in order to determine in which way the dry portion of the root system could affect the ability of the plant to withstand stress. Plants exposed to uniform soil conditions and in a container environment may behave differently from plants growing in the field. Soil water status varies horizontally and vertically, as water evaporates from the soil surface and plants take up water. Split-root studies provide a way to simulate the often non-uniform conditions of water availability existing in the field (Zhang and Kirkham, 1995). In the present study, conditions of non-uniform soil water availability were simplified by splitting the root system vertically and by imposing different soil water content on the two halves. Among the growth parameters investigated, a significant reduction of LA was measured during the last days of treatment application in two of the three rootstocks investigated (B.9 and Mark). Conversely, neither leaf number, nor shoot length were modified in any of the rootstocks during the 28 days of experiment. In terms of total LA and of RLER (estimated during the first 10 days of treatment), plants growing with half of the root system in drying soil conditions were more sensitive to water deficit. Leaf growth is very sensitive to reduction in water status (Hsiao and J ing, 1987) and leaf area reduction has been indicated as one of the first and evident symptoms of drought 53 stress in fruit crops (Flore and Lakso, 1989). Mark was the rootstock where RLER was mostly affected by drought, indistinctly whether irrigation was distributed equally or not to the root system. RLER was in fact much smaller in all plants growing in SWC lower than field capacity (Figure 1.3). If reduction in RLER can be considered an indicator of water deficit conditions, then these results would confirm what was found by‘ Fernandez et al. (1997a) who recently described Mark as the most sensitive among the three rootstocks investigated, M.9 EMLA, MM.111 and Mark. In some cases, non-uniform irrigation induced a significant reduction in the final F W and WC of roots growing in the non-irrigated side. These data confirm what was found by Sirnonneau and Habib (1994) who applied drought stress to one-half of the root system of a peach tree grown in nutrient solution and reported an increase in water uptake by the other half of the root system. Generally, root growth was stimulated as SWC was increased. It is known that root growth can be stimulated by lower SWC (Hsiao and J ing, 1987 ). However, in the current experiment, root growth was suppressed in non-inigated pots and high levels of root activity were maintained in irrigated pots. Thus plants favored root growth where water was present and avoided the utilization of energy and carbohydrate to stimulate root growth in soil portions where the presence of water was scarce (i.e., the dry pot). When considering WC in other plant organs, leaves in M9 had lower WC when SWC decreased equally in the two pots (Figure 1.4). The same happened for leaves and trunk WC in Mark. The high (less negative) predawn raw indicated that water deficit did not induce severe consequences to plant water status in the first 12 days of experiment (Table 1.5). The low predawn w“, measured in B.9 on day 24 in the 25B and 20B treatments (Table 54 1.5) indicated that this rootstock could tolerate conditions of non-uniform irrigation better than water deficit imposed equally to the entire root system. The same conclusion emerged from midday V’w, which again indicated that water status was better maintained when only half root system, rather than the whole, was exposed to SWC 25% or 20%. A possible explanation to these results could be related to the decrease in LA already observed when non-uniform irrigation was applied (Table 1.1). A lower LA would result in a lower net water loss via transpiration, therefore facilitating the maintenance of the water status. It could also be hypothesized that roots growing in the dry soil somehow stimulated stomatal closure. There is a considerable body of evidence that shows that ABA is synthesized in roots (Cornish and Zeevaart, 1985; Davies and Zhang, 1991) during the early stages of soil drying, and many of the shoot responses to soil drying occur before any detectable change in the leaf water status. ABA has long been known to increase considerably in leaves of plants subjected to drought. When roots “sense” the reduction of soil water potential, they start producing ABA, which is then sent to the leaves via xylem flow, thereby functioning as a long distance signaling molecule. It is now widely accepted that stomatal conductance is controlled by the soil water status via ABA. Although leaf water status is often not considered as influencing the response of stomata to ABA, recent works (Tardieu and Davies, 1993; Tardieu et al., 1992) report that leaf water potential might have an indirect role in the regulation of the stomatal conductance, via a modification of the stomatal sensitivity to ABA. Water deficit never induced total suppression of gas exchange and plants seemed able to maintain open stomata even when the most severe irrigation treatment was applied (Figure 1.7). However, a sensitive decrease in A and g3 was observed on day 14 55 in all rootstocks and treatments. The reason for this is quite certainly related to the high air temperature present on that day (26.7 °C, the highest value recorded in the 28 days, data not shown), accompanied by low relative humidity (53.9%, data not shown). This particular weather conditions likely induced the suppression in gas exchange observed in Figures 1.6 and 1.7. A previous study conducted on apple (Lakso et al., 1984) showed that moderate drought stress could inhibit the growth of immature tissue without affecting the photosynthetic rate of mature leaves. Results of A data indicated Mark as the rootstock that was mostly affected by non-uniform irrigation (Figure 1.6), particularly when SWC was 25%. M9 did not show any significant decrease in any of the treatments compared to control plants. The lower levels of internal CO2 concentration shown in Mark at the most severe drought conditions (Figure 1.9) suggest that the limitation to photosynthesis was almost entirely stomatal. The reduction in E observed in all the treatments on day 7 (Appendix A, Figure A. 1) was likely the result of the low daily air temperature values recorded during that day (daily mean temperature, 12.5 °C; maximum 17.2 °C, data not shown). This component, together with the several rainfall events that were observed during the first days during which the experiment was conducted, certainly reduced the evaporative demand of the environment, therefore reducing E. The analysis of WUE through time indicated that the amount of water lost via transpiration or evaporation to allow CO2 fixation intensified with time in all the treatments applied to B.9 rootstocks (Figure 1.8). Despite the fact that the loss of water increased during water deficit conditions, this behavior likely denotes an adaptation of B.9 to the unfavorable conditions. B.9 was the rootstock that had midday w“, unaffected 56 in the first 6 days of treatment (Table 1.6) together with the highest A, g., and E. The adaptation (possibly an osmotic adjustment) might have likely induced the capacity of maintaining stomata open (and, consequently, high gas exchange rates and, at the same time, high leaf W and even in conditions of water deficit. Our data do not provide enough information to prove whether B.9 was able to adjust osmotically or if it does not -— or does not need to — produce ABA, or any other signal, capable to induce stomatal closure. Therefore, it is not possible to speculate whether the ability of maintaining gas exchange was a consequence of osmotic adjustment, of reduced production of ABA, or if the opposite was true, i.e., B.9 had the capacity to photosynthesize even at low soil y/w, without increasing ABA synthesis to induce stomatal closure. Different were the responses of Mark to uniform and non-uni form water shortage. WUE decreased progressively in Mark as g. and C; rapidly decreased. There are some elements emerging from our data that suggest that the induction to close stomata might have been stronger in Mark rather than in B.9. Assimilation rate and WUE in 25D and 20D were in fact frequently lower than the correspondent uniform water treatments (Figures 1.6 and 1.8). Leaf water content was lower than control plants only in 25B and 20E (Figure 1.4), indicating a bigger water loss by plants that received uniform water treatments. M.9 showed intermediate characteristics between B.9 and Mark. The ratio Fv/Fm is particularly important for physiological studies because it is proportional to the quantum yield of photochemistry (Butler and Kitaj ima, 1975). The amount of energy emitted by chlorophyll through the fluorescence process has been used as an indicator for the stress condition of the plant. However, it seems that variable and maximal chlorophyll fluorescence and fluorescence quenching are not as sensitive to 57 stress as other physiological indicators. Fernandez (1997b) found a significant difference in leaf variable chlorophyll fluorescence in apple only 21 days after drought was imposed, whereas differences in assimilation rate, stomata conductance and transpiration were measured after 14 days. Our trials confirmed that chlorophyll fluorescence was not as sensitive as the other parameters investigated. Differences at 10% among treatments emerged only at day 12 in B.9 and Mark and even later (day 24) in M9. Overall, our data suggest that partial irrigation might induce a certain degree of tolerance to the adverse environmental condition. Whether the tolerance is more passive, (due to a reduction of leaf area and therefore of the amount of water loss by transpiration), caused by an active reaction to the stress (ABA production, osmotic adjustment, etc.), or by an interaction of both it is difficult to assess. Furthermore, the present work was conducted on ungrafted rootstocks. In applied horticulture, fi'uit trees are often grafted on rootstock that can induce specific characteristics to the grafted portion of the tree. Further study is certainly needed to examine whether the results of our investigation are confirmed when the water deficit is applied to grafted plants. In addition, more thorough studies are necessary to investigate the possible role that ABA plays in causing the different behavior among the water treatments and the different rootstocks. Split-root studies provide information on the responses of plants subjected to non-uniform drought conditions, however field studies are needed to investigate the long- term effects of non—uniform irrigation. 58 Literature cited Andersen, RC. and B.V. Brodbeck. 1988. Water relations and net CO2 assimilation of peach leaves of different ages. J. Am. Soc. Hortic. Sci. 113: 242-248. Andrews, P.K., D.J. Chahners, and M. Moremong. 1992. Canopy-air temperature differences and soil water as predictors of water stress of apple trees grown in a humid, temperate climate. J. Am. Soc. Hortic. Sci. 117: 453-458. Barden, J .A. and DC. F erree. 1979. Rootstock does not affect net photosynthesis, dark respiration, specific leaf weight, and transpiration of apple leaves. J. Am. Soc. Hortic. Sci. 104: 526-528. Beadle, CL. 1987. Plant growth analysis, p. 20-25. In: J. Coombs, D.O. Hall, S.P. Long and J .M.O. Scurlock (eds). Techniques in bioproductivity and photosynthesis, Pergamon Press, Oxford. Butler, W.L. and M. Kitajima. 1975. A tripartite model for chloroplast fluorescence. In: 3rd International Congress on Photosynthesis. M. Avron (ed), p. 13-24. Elsevier, Amsterdam. Cornish, K. and J .A.D. Zeevaart. 1985. Abscisic acid accumulation by roots of Xanthium strumarium L. and Lycopersicon esculentum Mill. in relation to water stress. Plant Physiol. 79: 653-658. Davies, F .S. and AN. Lakso. 1978. Water relations in apple seedlings: changes in water potential components, abscisic acid levels and stomatal conductances under irrigated and non-irrigated conditions. J. Am. Soc. Hortic. Sci. 103: 310-313. Davies, W.J. and J. Zhang. 1991. Root signals and the regulation of growth and development of plants in drying soil. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 42: 55-76. Fernandez, RT. 1992. Mechanisms of drought tolerance in apple as influenced by rootstock. Ph.D. Diss., Dept. of Horticulture, Michigan State Univ., East Lansing, MI. Fernandez, R.T., R.L. Perry, and J .A. Flore. 1997a. Drought response of young apple tree on three rootstocks: growth and development. J. Am. Soc. Hortic. Sci. 122: 14-19. Fernandez, R.T., R.L. Perry, and J.A. Flore. 1997b. Drought response of young apple trees on three rootstocks. H. Gas exchange, chlorophyll fluorescence, water relations and leaf abscisic acid. J. Am. Soc. Hortic. Sci. 122: 841-848. Ferree, DC. and RF. Carlson. 1987. Apple rootstocks, p. 107-143. In: R.C. Rom and RF. Carlson (eds). Rootstocks for Fruit Crops, John Wiley & Sons, New York. 59 Ferree, ME. and J.A. Barden. 1971. The influence of strains and rootstocks on photosynthesis, respiration, and morphology of "Delicious" apple trees. J. Amer. Soc. Hortic. Sci. 96: 453-457. Flore, J.A. and AN. Lakso. 1989. Environmental and physiological regulation of photosynthesis in fruit crops. Hortic. Rev. 11: 111-157. Gowing, D.J.G., W.J. Davies, and H.G. Jones. 1990. A Positive root-sourced signal as an indicator of soil drying in apple, Malus x domestica Borkh. J. Exp. Bot. 41: 1535- 1540. Hsiao, T.C and Jing J. Leaf and root expansive growth in response to water deficits, p.180-192. In: DJ. Cosgrove and DP. Knievel (eds). Physiology of cell expansion during plant growth, The American Society of Plant Physiologists. Karukstis, K.K. 1991. Chlorophyll fluorescence as a physiological probe of the photosynthetic apparatus, p. 769-795. In: H. Sheer (ed.). Chlorophylls, CRC, Boca raton, FL. Lakso, A.N., A.S. Geyer, and S.G. Carpenter. 1984. Seasonal osmotic relations in apple leaves of different ages. J. Am. Soc. Hortic. Sci. 109: 544-547. Landsberg, J .J. and H.G. Jones. 1981. Apple orchards, p. 419-469. In: T.T. Kozlowski (ed.). Water deficit and plant growth. Vol. 6, Academic Press, New York. NC-140. 1996a. Performance of the NC-140 cooperative apple rootstock planting H. A 10-year summary of TCA, yield and yield efficiency at 31 sites. Fruit Var. J. 50: 1 1-18. NC-140. 1996b. Performance of the NC-l40 cooperative apple rootstock planting. 1. Survival, tree size, yield and fruit size. Fruit Var. J. 50: 6-11. NC-140. 1996c. Rootstock and scion cultivar interact to affect apple tree performance: a five-year summary of the 1990 NC-140 cultivar/rootstock trial. Fruit Var. J. 50: 175-187. Neri, D. and J .A. Flore. 1990. Effects of soil water moisture stress and root pruning on ABA content, photosynthesis and root hydraulic conductivity of 'Cresthaven’ peach trees with split-root systems. Extd. Abstr. 23rd Intl. Hortic. Congr. 1: 273. (Abstr.) Olien, W.C., D.C. Ferree, B.L. Bishop, and WC. Bridges, Jr. 1995. Prediction of site index and apple rootstock performance from environmental variables. Fruit Var. J. 49: 179-189. Proebsting, E.L., P.H. Jerie, and J. Irvine. 1989. Water deficits and rooting volume modify peach tree growth and water relations. J. Am. Soc. Hortic. Sci. 114: 368- 372. 60 Ritchie, GA. and T.M. Hinckley. 1975. The pressure chamber as an instrument for ecological research. Adv. Ecol. Res. 9: 165-254. Roberts, M.J., S.P. Long, Tieszen L.L., C.L. Beadle. 1987. Measurements of plant biomass and net primary production, p. 1-19. In: J. Coombs, D.O. Hall, S.P. Long and J .M.O. Scurlock (eds). Techniques in bioproductivity and photosynthesis, Pergarnon Press, Oxford. Schechter, 1., DC. Elfving, and J .T.A. Proctor. 1991. Apple tree canopy development and photosynthesis as affected by rootstock. Can. J. Bot. 69: 295-300. Schreiber, U. and W. Bilger. 1987. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements, p. 27-53. In: J.D. Tenhunen (ed.). Plant response to stress. Vol. G15, Springer-Verlag, Berlin. Schulte, P.J. and LT. Henry. 1992. Pressure-volume analysis of tissue water relations parameters for individual fascicles of loblolly pine (Pinus taeda L.). Tree Physiol. 10: 381-389. Schupp, J .R. 1992. Early performance of four apple cultivars on Mark and other rootstocks in Maine. Fruit Var. J. 46: 67-71. Schupp, J .R. 1995. Growth and performance of four apple cultivars on M26 and mark rootstocks, with or without preplant mineral nutrients. Fruit Var. J. 49: 198-204. Simonneau, T. and R. Habib. 1994. Water uptake regulation in peach trees with split-root systems. Plant Cell Environ. 17: 379-388. Tan, C.S., A. Comelisse, and B.R. Buttery. 1981. Transpiration, stomatal conductance, and photosynthesis of tomato plants with various proportions of root system supplied with water Varieties. J. Am. Soc. Hortic. Sci. 106: 147-151. Tardieu, F. and W.J. Davies. 1993. Integration of hydraulic and chemical signalling in the control of stomatal conductance and water status of droughted plants. Plant Cell Environ. 16: 341-349. Tardieu, F., J. Zhang, and W.J. Davies. 1992. What information is conveyed by an ABA signal from maize roots in drying field soil? Plant Cell Environ. 15: 185-191. Tubbs, FR. 1973. Interactions of graftings and grafts on woody plants. Riv. Ortoflorofruttic. Ital. 57: 74-85. Williams, J .H.H., P.E.H. Minchin, and J .F . Farrar. 1991. Carbon partitioning in split root systems of barley: The effect of osmotica. J. Exp. Bot. 42: 453-460. Zhang, J. and MB. Kirkharn. 1995. Water relations of water-stressed, split-root C4 (Sorghum bicolor; Poaceae) and C3 (Helianthus annuus; Asteraceae) plants. Am. J. Bot. 82: 1220-1229. 61 Table 1.1. Total leaf area (cmz) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05. Day Rootstock Treatment 0 7 14 21 28 B-9 3013 463 540 710 880 a 1142 a 30D 365 431 586 717 ab 938 ab 25B 264 409 442 532 ab 733 ab 25D 361 440 590 717 b 916 ab 2013 364 335 517 608 ab 764 ab 20D 299 358 439 508 ab 598 b M9 305 166 222 289 357 435 301) 215 293 381 455 540 255 212 281 373 451 554 251) 225 263 333 393 486 205 223 267 331 384 460 201) 268 336 393 445 488 Mark 30E 437 510 654 ab 798 ab 1013 a 301) 424 579 553 ab 614 ab 695 ab 255 473 557 692 ab 806 ab 974 ab 251) 358 414 476 b 528 b 587 b 205 485 581 668 ab 742 ab 808 ab 200 477 604 727 a 830 a 930 ab 62 Table 1.2. Total leaf number measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. The absence of any letter within column means indicates that treatments are not significantly different at P S 0.05. Rootstock Treatment 0 7 14 21 28 B-9 3013 30 37 41 44 47 30D 27 35 38 40 43 255 23 29 33 37 42 25D 30 37 43 48 51 20B 25 29 34 38 43 20D 27 32 35 38 41 M9 305 21 29 34 39 46 301) 23 33 38 42 46 255 25 33 37 41 46 251) 28 33 37 41 46 205 24 32 35 39 42 201) 28 38 43 46 51 Mark 305 35 43 47 49 55 301) 33 40 43 45 48 255 35 45 50 54 57 251) 33 40 43 45 48 205 39 49 51 53 55 20D 37 47 51 53 56 63 Table 1.3. Total shoot length (sum of two shoots), in cm, measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. The absence of any letter within column means indicates that treatments are not significantly different at P S 0.05. Rootstock Treatment 0 7 14 21 28 B-9 3015 46.5 55.5 58.3 61.0 64.0 30D 45.0 51.5 54.8 57.8 59.8 2513 45.0 49.8 54.5 59.0 62.3 25D 40.8 46.0 49.8 53.8 56.3 2013 38.5 42.3 45.3 48.3 51.5 20D 42.8 47.3 50.5 52.5 53.5 M9 305 23.0 28.5 32.0 35.0 38.0 301) 30.8 37.8 41.3 43.5 45.0 255 29.5 36.3 39.8 43.3 46.0 250 36.5 42.0 45.3 47.5 48.8 205 34.0 39.3 43.0 45.8 48.0 201) 36.8 43.5 46.8 48.8 50.5 Mark 305 51.5 57.8 61.0 63.3 65.3 305 51.0 58.0 58.5 58.5 58.8 255 60.5 69.5 72.0 74.3 76.8 251) 50.3 56.5 57.8 56.0 59.0 205 60.8 69.3 71.3 72.3 73.0 201) 59.3 67.0 68.0 69.0 69.8 64 6 8 8 8 as s .. 3n 9% 36 4.8 Be 92 nee v.8 2: one 2:. was 5 e e... as as a 4 3.4. 8e 8.4 m2“ ed New e3. 8... . 2.... one :..m e? «a: e e e e as 4 Se 3e 3... 2e 2e 36 3e 3e 3e 3H «.2. 2:. a a a a a a 2: as a: m3 ._ as men 8.. and W2 ”3. 8a «.2 32 e e e as s s ”.8 Se ”.8 ”.8 3e 5% 3e 3e _. 35 a? a: N? e e at a... e 5 SN 3% :3” 9% Sn 9% as 8.... man was new man am See Amos and 6an Soc Sec fiancee o-o~ 8-8 In 2-2 Tom 8-2. 25: Eugene 02:22 23 gen 25 ca 3 ex. .038 «588 533 mom .36 w m «gum. “enemas 8: 8a 532 088 05 ,3 “.8885 «.508 38 £55 Eon e3. 05 .«e 853 aggafih .36 w m «a «SSE—um? Son :83qu «508 30.53... .3 mooeobg 838%.: ... 328% 2: 689955 33525 98 .533 43:96 2.3 88 9 38.33 28 303880 95 8233 5% mass... 82 ea as. area 5 95 3 see £8585 see as: as «.2 .3 a S... as .96 .858 ease as a .36 fire see as. .2 ease 65 Table 1.5. Values of predawn leaf water potential (w... MPa) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05. m Day Rootstock Treatment 0 3 6 12 24 B.9 30E -0.30 -0.42 -0.32 -0.34 a -0.92 a 30D -0.25 -0.38 -0.30 -0.51 be -1.01 a 25E -0.32 -0.37 -0.25 -0.45 ab -2.35 b 25D -0.28 -0.37 -0.30 -0.51 be -0.83 a 20E -0.30 -0.28 -0.18 -0.59 be -2.25 b 20D -0.30 -0.42 -0.38 -0.61 c -0.99 a M.9 30E -0.36 -0.33 -0.28 a -0.39 b -0.81 ab 30D -0.30 -0.43 -0.32 a -0.29 a -0.52 a 25E -0.29 -0.38 -0.30 a -0.50 e -1.29 c 25D -0.30 -0.52 -0.35 a -0.47 c -0.70 ab 20E -0.32 -0.35 -0.32 a -0.41 b -0.90 b 20D -0.33 -0.37 -0.52 b -0.41 b -1.42 c Mark 30E -0.34 -0.55 -0.28 -0.41 a -0.70 a 30D -0.33 -0.47 -0.32 -0.53 ab -O.76 a 25B -0.27 -0.50 -0.28 -0.63 b -l.50 b 25D -0.28 -0.57 -0.28 -0.44 a -1.38 b 20E -0.30 -0.62 -0.32 -0.65 b -2.13 c 20D -0.32 -0.50 -0.32 -0.54 ab -2.12 c 66 Table 1.6. Values of midday leaf water potential (al.,, MPa) measured on B.9, M.9, and Mark rootstocks grown with roots split between two pots and subjected to six watering regimens. Treatments within column means followed by the same letter are not significantly different at P S 0.05. M Day Rootstock Treatment 0 3 6 12 24 B.9 30E -0.30 -1.18 -1.08 -0.70 a -l.25 a 30D -0.28 -l.22 -l.20 -l.20 be -1.43 a 25E -0.33 -1.05 -1.15 -0.96 ab -3.00 b 25D -0.31 -0.97 -1.03 -0.80 ab -1.98 ab 20E -0.23 -1.27 -1.27 -l.49 c -2.88 b 20D -0.25 -l.10 -1.02 -2.00 d -2.85 b M.9 30E -0.32 -0.97 ab -0.97 ab -0.61 a -l.21 a 30D -0.35 -1.33 b -l.32 b -1.08 b -1.21 a 25E -0.30 -0.98 ab -0.88 ab -1.15 b -2.46 b 25D -0.31 -0.82 a -0.83 a -0.71 a -l.78 a 20E -0.28 -1.12 ab -1.13 ab -1.19 b -3.08 b 20D -0.26 -0.90 ab -1.00 ab -1.05 b -3.04 b Mark 30E -0.29 -l.18 ab -1.23 b -l.17 a -l.06 a 30D -0.30 -l.03 ab -0.92 a -0.97 a -l.18 8 25B -0.29 -l.22 b -1.23 b -1.02 a -2.10 be 25D -0.33 -0.98 a -0.95 a -1.38 ab -1.69 ab 20E -0.31 -1.18 ab -1.15 ab -1.80 be -2.70 c 20D -0.31 -1.00 ab -l.13 ab -l.98 e -2.03 be 67 Table 1.7. Chlorophyll efficiency (FJFm) measured on apple rootstocks. Six levels of root zone drought (either equally or unequally distributed) were imposed on Aug. 14 and released on Sep. 11 1997. Treatments within column means followed by the same letter are not significantly different at P S 0.10. Rootstock Treatment 0 6 12 24 B.9 30E 0.772 0.771 0.768 ab 0.785 ab 30D 0.776 0.780 0.783 a 0.808 a 25E 0.772 0.766 0.769 ab 0.774 ab 25D 0.756 0.781 0.768 ab 0.788 ab 20E 0.756 0.760 0.751 b 0.750 b 20D 0.777 0.784 0.773 a 0.798 8 M9 30E 0.766 0.773 0.759 0.791 b 30D 0.786 0.779 0.781 0.820 a 25E 0.808 0.787 0.765 0.808 ab 25D 0.778 0.769 0.755 0.795 ab 20E 0.769 0.770 0.749 0.803 ab 20D 0.769 0.767 0.749 0.791 ab Mark 30E 0.768 0.727 0.765 ab 0.786 ab 30D 0.750 0.737 0.741 b 0.812 a 25E 0.764 0.743 0.773 ab 0.797 ab 25D 0.784 0.751 0.781 a 0.792 ab 20E 0.787 0.743 0.783 a 0.802 ab 20D 0.759 0.759 0.759 ab 0.778 b .68 .36 w l ”a Bang magmas 8a 9a Ecto— oEum 05 .3 @8835 ESE £55qu Squaw—coax“. as he an“. 3 “Eu 06 wine itouflrnfirm 8 "8.5308 803 «33 ~34 €8.55va bis—eon: was 3138 45:8“. 2.3 82 8 @8833. was £055.50 95 5283 Emu «88m? 82 :05 55 E52» 30888.— oina arm he.“ agno— vo=2§ Spa 06 no @8233”. 95d 88 c2835 “no; .: Semi 95:55. 4 _ O - om . m - ON H ) m. 3 . a m... P. (r U 1 ov Qoml mom. mel mg- 1 an 988 BED 69 .86 w k «a 380% Language «on 03 Eute— 083 05 3 “333%.: 338 88:58; .fiuofitoaxo 05 we 95.. o— are 05 wet—6 igiiflrm “a 3:39: 0.53 «33 mg gafifimfi 3:538. v5 5:350 damage ones 82 8 @8833 98 ”58:8 23 5953 5% EPA.» 88 boa 5!» :38» 8.08302 can.“ «.2 he.“ 828— vo=8§ “we.“ 08 no Baas—8 $55 82 8.8.598 3 .NA 235 95:59 3 4 . o - S "d 8 H ) m. . m 1 on ~.L 5. (1 6 S 02... mom- Qmml mmml 1 on acmfl mcmD 70 .36 w m «a 380% bandage?“ «on one E032 088 06 .3 “383%.: 386 358329 .anofitonxo on. we gee S SE 06 macaw 32851.???“ 35308 803 82a use: 98333. Eggs. e5 E35» .3956 88 88 3 “388.33 can B03850 22 5053 5% Eu? 58 :05 as :38” £02308 03% x52 8m 3.32 3:85. “flu ofl ue moan—:23 Emu: 8a.. coin—axe 3 .mA 052m 95365:. Ne _ a ”n. O s E a a 1 o— ..s a . m 1 cm H ) L . m. 0 - a m... p. (1 M 3 Qoml mom- DmNB m3. 1 on comm womD 71 D305 111301) B.9 3° 1 I25E l25D ' a I 20E I 20D Water content (%F W) 8 8 .5 O Leaves Shoots Trunk M.9 70~ 50~ ' 'vvvvvv‘ .....I.... Water content (%FW) 8 40 Leaves Shoots Trunk Mark Water content (%FW) 8 Figure 1.4. Leaf, shoot and trunk water content (expressed as percent of fresh weight) calculated in the three apple rootstocks at the end of the drought stress experiment (day 28). Treatments means within the same plant organ followed by the same letter are not significantly different at P S 0.05. 72 Roots - Irrigated pot Z Roots - Non-irri ated t 1:1 Trunk 51 Shoots 8 p0 3'9 I Leaves Dry weight (% total) Dry weight (% total) Dry weight (% total) 3015 30D 25E 25D 20E 20D Irrigation treatment Figure 1.5. Effect of 28 days of equal or unequal irrigation on dry matter partitioning (expressed as percent of total dry weight) in B.9, M.9 and Mark apple rootstocks grown with their root systems split between two containers. Roots from the two pots (irrigated and non-irrigated) were kept separated during analysis. 73 B.9 Da A Trt. o 7 14 21 28 T.,, 30E NS b NS NS a ”a 301) NS ab NS NS ab _ 2515 NS ab NS NS ab E 25D NS ab NS NS ab < 20E NS a NS NS b 20D NS ab NS NS ab M.9 Da 7,. Trt. o 7 14 21 28 ...“ 3015 NS c abc c ab 5 30D NS a ab abc b g 2515 NS ab abc ab a v 25D NS c a be ab ‘1 20E NS c c a ab 20D NS bc bc ab ab Mark Da “7: Trt. o 7 14 21 28 ”a 3013 NS ab a a a "' 30D NS c ab ab a 3 25B NS a a ab ab : 25D NS c b c ab 20E NSababbcb 20D NSbc bbc a Time (days) Figure 1.6. Changes in single leaf net assimilation rate (A) calculated over time in apple rootstocks subjected to root zone dumght (either equally or unequally distribumd). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. 74 Trt. 30E 30D 25E 25D 20E 20D g, (mmol in2 s") Trt. 30E 30D 25E 25D 20E 20D g, (mol m'2 s") 3.9 Da 0 7 14 21 28 NS ab NS NS NS NS ab NS NS NS NS b NS NS NS NS b NS NS NS NS a NS NS NS NS ab NS NS NS Day 0 7142128 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS Trt. Mark Da 0 714 2128 30E 30D 25E 25D 20E 20D 3. (mmol in2 s") Time (days) NS NS ab NS NS NS NS ab NS NS NS NS a NS NS NS NS ab NS NS NS NS ab NS NS NS NS b NS NS Figure 1.7. Changes in stomatal conductance (g,) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with difi'erent amounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within cohimn means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. 75 B.9 Day r: Trt. o 7 14 21 28 8 30B NS a NS Ns NS 7.5 30D NS ab NS NS NS g E 255 NS b Ns NS NS :5. 25D NS b NS Ns NS 3 20B Ns ab NS NS NS 5 201) Ns ab NS NS NS M.9 Day 3 Trt. o 7 14 21 28 p 3013 NS ab b a NS '3 30D NS b b ab NS E g 2515 NS b b ab NS £1 25D NS b b b NS '5 20B NS a a b NS 5 201) NS ab ab ab Ns Mark Day 3 Trt. o 7 14 21 28 .9 3013 NS b b b b g 30D NS ab b ah ah E0 25E NS b b ab a :E' 251) Ns a a a ab 3 ZOE NS b b ab a v 20D NS ab b ab b Time (days) Figure 1.8. Changes in water use efficiency (WUE) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with difi'erent mounts of water. Tables on the right report significant differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly difi‘erent, whereas NS indicates non-significant difi‘erences. 76 B.9 Day Trt. 0 7 14 21 28 —.: 3013 NS a NS NS ab 5 30D NS ab Ns NS ab '5 255 Ns b NS Ns ab 3 25D NS ab NS NS b 5 20E NS ab NS NS a 20D NS ab NS Ns a M.9 Day ,.. Trt. o 7 14 21 28 "'— 30E NS ab Ns a NS E 301) NS ab NS ab NS 3 25E NS b NS ab NS V 250 NS ab NS ab NS ‘6 20B NS a NS b NS 20D NS ab NS ab NS Mark Day TA Trt. o 7 14 21 28 "g 3013 NS ab b NS b 3 30D NS a ab Ns ab 5 2515 NS ab ab NS ab :7 25D NS a a NS ab 0 20E Ns b b NS a 20D NS ab ab NS ab Time (days) Figure 1.9. Changes in leaf intercellular CO; concentration (0,) calculated over time in apple rootstocks subjected to root zone drought (either equally or unequally distributed). Plants were grown with root systems split between two containers, irrigated with different amounts of water. Tables on the right report signifith differences determined by LSD (P S 0.05). Treatments within column means followed by the same letter are not significantly different, whereas NS indicates non-significant differences. 77 Chapter 2 CANOPY TEMPERATURE AND WATER STRESS IN YOUNG APPLE TREES SUBJECTED TO WATER DEFICIT 78 CHAPTER 2. CANOPY TEMPERATURE AND WATER STRESS IN YOUNG APPLE TREES SUBJECTED TO WATER DEFICIT Leonardo Lombardini Abstract The recent development of small portable infrared thermometers makes it possible to estimate canopy temperature in the field. Our objective was to correlate a reduction of soil water content with foliage temperature and to compare it with other physiological parameters (assimilation rate, transpiration rate, stomatal conductance, relative leaf expansion rate, sap flow rate). During the summer of 1998 and spring of 1999, we evaluated the responses of young potted apple (Malus domestica Borkh.) trees to a rapid soil water deficit. The cultivar ‘TRECO Red Gala #42’, grafted on three dwarfing rootstocks, Budagovski 9 (B.9), Malling 9 (M.9) and MAC 9 (Mark), was used for the trial. Irrigation was withheld for 7 days, and the canopy surface temperature was measured once a day during the drought period, with a digital infrared radiometer. Canopy surface temperature, evaluated through mean, median, and mode statistical parameters, was usually higher than air temperature in non-irrigated plants. Conversely, 79 in irrigated plants, canopy temperature values were closer to or below air temperature. Differences between canopy temperature and air temperature could be detected as early as 3-4 days of initiating the stress. Net C02 assimilation rate seemed to be less reduced by water deficit in ‘Red Gala’IMark than in the other two rootstocks. At day 7, midday stomatal conductance was 38.0, 32.3, and 72.0 mmol m’2 s'1 in ‘Red Gala’lB.9, ‘Red Gala’lM.9, and ‘Red Gala’lMark, respectively (control values varied between 161.6 and 164.3 umol m'2 s" for all the rootstocks). Heat-pulse sapflow sensors installed on ‘Red Gala’/Mark indicated that the speed of the xylem sap decreased in non-irrigated plants after four days of water depletion (19—26 cm hr'1 for the controls vs. 15-21 cm hr'l for the stressed plants). Introduction Commercial fruit trees are composed of a cultivar, that possesses the desired fruit characteristics, grafted or budded onto a rootstock, thus resulting in a composite system of two different genomes. The choice of the appropriate rootstock offers the growers a means to increase tree performance against biotic and abiotic adversities and to increase orchard efficiency. Moreover, apple rootstocks are also chosen for their capacity to reduce tree size, therefore allowing high-density planting and facilitating the harvesting process (F erree and Carlson, 1987). Researchers are continuously challenged to find rootstocks that meet the demand of each fi'uit-producing region, and international committees, such as USDA-CREES regional project NC-l40, have been organized to test 80 new rootstocks over a wide range of climatic and soil conditions (NC-140, 1996a; NC- 140, 1996b; NC-140, 1996c). Water plays an essential role in plant growth and development, from orchard establishment (Autio and Greene, 1991) through full production at maturity. Where drought occurs, the selection of a proper rootstock, together with a good irrigation schedule, can prevent or reduce the negative effects of drought on the orchard’s performance. Growth, productivity, and fi'uit quality in apple are greatly affected by inigation or water deficit conditions (Erf and Proctor, 1989). The following biochemical and developmental processes have been shown to be affected in apple (Brough et al., 1986; Olien and Lakso, 1986): a reduction of both cell division and expansion (Hsiao and Acevedo, 1974), a decrease of photosynthesis (Fernandez et al., 1997b), ABA synthesis (Davies and Zhang, 1991; Zeevaart and Creehnan, 1988), and the accumulation of sugars (Wang et al., 1995; Wang and Stutte, 1992) play a fundamental role. Stomata are the ultimate regulators of both photosynthesis and transpiration responses. Stomata are regulated by internal components (leaf water and osmotic potentials, internal CO; concentration, etc.) (F arquhar and Sharkey, 1982; Jones, 1998), environmental conditions (mainly the net solar radiation and the vapor pressure deficit between leaf surface and air environment), and by the interaction between transpiration and photosynthetic activities (Jones and Corlett, 1992). Variation in stomatal and non-stomatal limitation could also be an indicator of the upcoming plant stress conditions (Jones, 1985). Plant water status has been used as an indicator of plant stress because it accounts for the effects of the availability of water in the soil, the evaporative demands of the air 81 environment, and the hydraulic fluxes within the soil-plant-atrnosphere continuum (Andrews et al., 1992). An alternative method for the evaluation of plant water status can be the measurement of leaf and canopy surface temperatures. When a plant is well watered, transpiration occurs at its maximum rate, and leaf temperature (T1) tends to be lower than air temperature (T.,) (Jackson et al., 1981). In most species, as soon as water deficit is perceived, stomata are induced to partially close, therefore causing the transpiration rate to reduce and leaf temperature to rise and to become closer to or higher than T... The increase in T1 is the consequence of the reduced energy dissipated as latent heat through water evaporation process (Nobel, 1991) and could be an additional signal of an incipient water stress occurrence. Thermocouples can only record point measurements and many are therefore needed to characterize a surface. Moreover, they are often invasive to the normal behavior of the sample because they are either attached to the tissue or they require that the tissue be pierced. In contrast, infrared (IR) thermometry represents a non- destructive, rapid, non-contact method of measuring plant canopy temperature. The gases present in the earth’s atmosphere (mainly water vapor) absorb radiated energy in the infrared except for two wavelength regions called the atmospheric windows. Both windows allow radiometric measurements with minimal losses. The longwave region (8- 14 um) is exceptionally free of absorption except if very high atmospheric water content is present. The shortwave region (3-5 pm) has relatively high transmission, but it usually requires compensation when high accuracy measurements are to be made. Modem thermal imaging radiometers are available with 8-14 pm, 3-5 pm, or 3-14 pm (broadband) spectral response. Due to a higher thermal contrast at “earth” temperatures 82 (-20 °C to 50 °C) in the longwave region, greater overall system performance can be achieved. Most common material surfaces have higher emissivities in the 8-14 pm waveband. Canopy temperature has been used to estimate plant water status during the early stages of drought in cotton (Ehrler, 1973) and wheat (Jackson et al., 1977). The development of IR thermometry greatly enhanced the potential for using the relationship between stress and canopy temperature to develop a crop water stress index (CSWI). This index is being used to correlate irrigation scheduling with water status on squash, soybean, and alfalfa crops (Idso et al., 1981). The present experiment was designed to test the use of infrared thermometry as a rapid, non-invasive tool to detect drought stress in potted apple plants. The cultivar ‘TRECO Red Gala #42’ grafted on three different dwarfing apple rootstocks, Budagovski 9 (B.9), Malling 9 (M.9) and MAC 9 (Mark) was used in the trial. The variation in leaf temperature induced by water deficit was studied together with the variation in assimilation rate, relative leaf expansion rate, and sap speed. The response of the three rootstocks were compared and the effect of timing of drought was detected to evaluate which of the monitored parameters results to be the most sensitive to the incipient drought stress condition. Because, during limited water availability, T. is expected to approximate or become higher than T., at whole-canopy level, the changes in the difference between Tc and T., should provide the indication of incipient plant water deficit. Successful results from the application of IR technology for detection of environmental stress should address orchard management and irrigation scheduling. 83 Materials and methods Plant material and growth conditions. The studies described here were carried out in summer 1998 and spring 1999 at Michigan State University, on ‘TRECO Red Gala #42’ (Red Gala) apple trees grafted on three rootstocks, M.9 NAKB T337 (M.9), Mark, and B.9. Plants were purchased from Treco® Nursery (W oodbum, OR, USA) and were about 1.5 cm in trunk diameter. Half of the plants were potted on May 11, 1998, whereas the other half was left in cold room until they were potted on May 25, 1998. Afier they were planted in 12-L pots, plants were transferred and grown outside on a gravel bed located on the MSU campus. The potting medium was 65% sand, 24% silt, and 11% clay (sandy loam). Pots were painted white and covered with transparent polyethylene, to prevent water from rainfall and soil evaporation. The polyethylene was supported by wood sticks to permit air movement under the cover and to prevent excessive heating of the soil. Two drought periods were imposed during summer 1998. The first one was applied on plants potted on May 11, it started on Aug. 11, and it lasted for 8 days. The second stress period was performed on plants potted on May 25, it began on Aug. 23, and it lasted 8 days, as well. Each session of the study lasted 8 days, after which non-irrigated plants reached the permanent wilting point. In the fall of 1998, all plants were stored in a cold room (temperature 4 °C, RH 70%). During Jan. 1999, the F1 plants used during the previous summer were taken out, transferred to a greenhouse and forced to grow. Average day/night temperature was 30/25 °C, RH 88-94%, and the photoperiod was extended to 14 hours using lOOO-W metal halide lamps (General Electric Lighting, 84 Cleveland, OH). Plants were regularly watered and fertilized with 20N-20P-20K. At the end of March, a stress period was imposed with the purpose of performing photosynthetic leaf response curves to air temperature and C02 concentration. Experimental design and statistical analysis. The trial was arranged with a split- plot experimental design, with rootstocks as the main plot and water treatments, firlly irrigated (FI) and non-irrigated (NI), as the sub-plot. Six plants per rootstocks were selected for uniformity of shoot length and leaf number and randomly divided in two groups to be assigned to the two treatments. Statistical analysis was performed by analysis of variance (AN OVA), and differences between means were determined by Least Significant Difference (LSD) at P S 0.05 within each date of measurements. Data analysis was completed using SAS software (SAS Institute Inc., Cary, NC, USA). Temperature response curves were analyzed with trend contrasts, to investigate whether the five parameters, such as A, E, g,, Ci, and TI change cubically, quadratically or linearly with the seven imposed levels of temperature. Weather conditions monitoring. Weather data relative the two drought periods were provided by the MSU Agricultural Weather Office web page (http://www.agweathergeo.msu.edu/Automated-Data/msuhor.hourly). Data were recorded by the sensors placed in the weather station located at the Horticulture Research Center (3 miles SW of MSU campus). In the second drought period, additional weather data were collected at the site of the experiment. Global solar radiation was measured with a LI—COR pyranometer sensor (Mod. LI-ZOOSA), whereas incoming 85 photosynthetically active radiation (PAR) was monitored with a LI-COR quantum sensor (Mod. LI-19OSA), placed horizontally, at height of 2.5 m, near the experimental plants. Dry- and wet-bulb air temperatures were measured using thermocouples, which were positioned in a shaded location at about 1 m from the ground near the plants. In particular, to measure wet bulb temperature, a cotton socket was placed around the thermocouple and inserted in a little reservoir filled with distilled water. A fan made it possible the continuous evaporation from the cotton socket. Before their use, the accuracy of both temperature thermocouples was tested by inserting them into boiling water and ice and by comparing the intermediate values with a mercury thermometer. All data were collected by a LI-COR (LI-COR, Inc. Lincoln, NE, USA) LI-lOOO datalogger, with 5- min logging period (switched to 1-min intervals during IR-thermometry measurements). Both relative humidity (RH) and vapor pressure deficit (VPD) were calculated from the measured dry- and wet-bulb air temperatures, using the equations reported in Appendix B.1. Soil moisture. Before starting the experiment, two steel probes (length 170 mm, diameter 5 mm) were inserted vertically in each pot 5-cm apart, to allow measurements of soil water content (SWC) by a time-domain reflectometer (TDR, Tektronix 1502, Tektronix Inc., Beaverton, OR, USA). In both drought periods, the SWC was estimated every other day in all the pots, immediately before irrigation of F1 plants. Relative leaf expansion rate. On day 0, the first unrolled leaves from each limb of each plant were tagged, and lamina length and width were measured at the widest point. 86 The same leaves were then measured every other day for the rest of the experiment. Area of each leaf was estimated by multiplying length and width by 0.7 (Fernandez et al., 2 1997a). Daily mean relative leaf expansion rate (RLER), expressed as cm cm'2 (1", was calculated over the two-day interval (t2 —t1) with the following formula (Beadle, 1987): 1n<[l—b>