The Pennsylvania State University The Graduate School College of Agricultural Sciences A COMPARISON OF ANATOMICAL TRAITS RELATED TO THE DEVELOPMENT OF BRITTLE BUD UNIONS IN APPLE A Thesis in Horticulture by Michael R. Basedow Copyright 2015 Michael R. Basedow Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2015 The thesis of Michael R. Basedow was reviewed and approved* by the following: Robert M. Crassweller Professor of Horticulture Thesis Advisor Richard P. Marini Professor of Horticulture Head of the Department of Plant Science Kathleen M. Brown Professor of Plant Stress Biology Nicole R. Brown Associate Professor of Wood Chemistry *Signatures are on file in the Graduate School. ii ABSTRACT Anatomical and histological traits related to the development of mechanically weak unions were evaluated between relatively weak (‘Honeycrisp’/‘M.26 EMLA’, ‘Cripps Pink’ cv.Maslin/‘Geneva® 41’, ‘Scilate’ (Envy™)/‘Geneva® 41’) and strong (‘Honeycrisp’/‘M.7 EMLA’, ‘Zestar!’/‘M.26 EMLA’, ‘Zestar!’/‘M.7 EMLA’, ‘Cripps Pink’ cv.Maslin/‘M.9 NAKB T337’, ‘Scilate’ (Envy™)/‘M.9 NIC29’) scion/rootstock combinations of apple. The objectives of these studies were to identify differences between these groups to determine the cause of the weak unions, and to determine if these methods would allow for the rapid screening of future potentially weak scion/rootstock combinations. Discolored sapwood was observed in the rootstock tissues of all of the combinations, and was investigated because of its association with wood decay. Trees were cut longitudinally and the discolored wood was photographed and quantified as a percentage of the total wood area using ImageJ image analysis software. Discolored sapwood did not differ significantly between weak and strong union combinations, but there was significantly more present in the most vigorous rootstocks compared to the dwarfing rootstocks. The thickness of the fiber cell walls was measured between the combinations. Tissues were hand sectioned from three areas of the tree (below, at, and above the union) and were stained with toluidine blue for microscopic study at 400x magnification. Cell wall thickness was measured using photomicrographs and Olympus® CellSens software. Fiber cell wall thickness varied between some of the weak and strong combinations below and at the union. Few differences were observed between the ‘Honeycrisp’ and ‘Zestar!’ combinations in the scions, but the ‘Cripps Pink’ combinations varied there and both combinations of ‘Scilate’ were very thin. Xylem cells were divided into three categories of tissue based on their function within the wood. These categories included parenchymatous, fibrous, and conductive tissues. Xylem tissues from the most recent growth ring were sectioned and stained using toluidine blue. They were then examined at 200x magnification. The percentages of the three cell types were calculated between the combinations using ImageJ. Weak iii combinations contained more parenchymatous tissue and slightly less fibrous tissue than some of the strong combinations, and may be a sign of localized incompatibility between the scions and rootstocks. Laser Ablation Tomography (LAT) and an iodine starch indicating test were evaluated for their potential use for observing localized incompatibility at the union. LAT was performed by sectioning union samples and ablating the wood material. Images were simultaneously taken, and images were combined using Avizo imaging software to virtually reconstruct the sample into a three dimensional model. Using LAT we were able to distinguish differences in the tissues of weak and strong scion/rootstock combinations, including large areas of swirling xylem tissue in the weak combination ‘Honeycrisp’/‘M.26 EMLA’. However, abnormalities were also observed in some sections of the strong combinations. Iodine starch tests were conducted by cutting unions into longitudinal sections and then staining them with a 5% solution of iodine-potassium iodide. The iodine starch test was inconclusive, as the amount of tissues that stained varied greatly within each combination. Our results suggest anatomical differences can be determined between weak and strong scion/rootstock combinations, but these methods may not allow for the prediction of future weak unions due to their variability. iv Table of Contents List of Tables ................................................................................................................ vi List of Figures ............................................................................................................... vii Acknowledgements ........................................................................................................ ix Chapter I. Introduction ................................................................................................. 1 The role of the dwarfing rootstock in the modern orchard .................................. 1 Graft union failure of clonal rootstocks .............................................................. 2 Wood decaying fungi in apple ............................................................................ 3 The anatomical properties of dwarfing ............................................................... 4 The anatomical properties of brittle wood .......................................................... 6 Graft failure related to localized incompatibility ................................................ 7 Other symptoms commonly associated with incompatibility .............................. 9 Potential biochemical factors of incompatibility ................................................. 10 Viral infection and incompatibility ..................................................................... 14 Microscopic examination of graft unions ............................................................ 15 Other potential screening methods for incompatibility ....................................... 16 Hypotheses and objectives .................................................................................. 18 Literature Cited ................................................................................................... 21 Chapter II. The presence of discolored wood in the xylem tissue of strong and brittle scion/rootstock combinations of apple .......................................................................... 26 Introduction ......................................................................................................... 26 Materials and Methods ........................................................................................ 32 Results and Discussion ........................................................................................ 35 Literature Cited ................................................................................................... 41 Chapter III. Anatomical differences between the xylem of strong and brittle scion/rootstock combinations of apple .......................................................................... 44 Introduction ......................................................................................................... 44 Materials and Methods ........................................................................................ 50 Results and Discussion ........................................................................................ 52 Literature Cited ................................................................................................... 63 Chapter IV. Potential methods for the rapid determination of localized incompatibility within the xylem of strong and brittle scion/rootstock combinations of apple ............. 66 Introduction ......................................................................................................... 66 Materials and Methods ........................................................................................ 73 Results and Discussion ........................................................................................ 75 Literature Cited ................................................................................................... 84 Chapter V. Closing thoughts ......................................................................................... 86 Literature Cited ................................................................................................... 89 Appendix ........................................................................................................................ 91 v List of Tables Table 1.1. Combinations of scions and rootstocks used in this study, their relative strength, and their abbreviations used in this paper ...................................................... 20 Table 2.1. Percent of discolored wood in the rootstock tissues of eight scion/rootstock combinations of apple .................................................................................................... 35 Table 2.2. Percent of discolored wood in the rootstock tissues of eight scion/rootstock combinations by rootstock cultivar ............................................................................... 37 Table 3.1. Mean fiber cell wall thicknesses (µm) 7.0cm below, at, and 3.0cm above the unions of eight scion/rootstock combinations of apple ................................................. 53 Table 3.2. Percentages of wood tissues by combination in the unions of four scion/rootstock combinations of the apple scion cultivars ‘Honeycrisp’ and ‘Zestar!’