A Study of the Morphology, Anatomy, and Function Of
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A STUDY OF THE MORPHOLOGY, ANATOMY, AND FUNCTION OF CONIFER ROOT-NODULES, WITH PARTICULAR REFERENCE TO THOSE OF PODOCARPS. by Abdul Ghaffar Khan A Thesis presented to the University of Sydney in partial fulfilment of the requirements for the Degree of Doctor of Philosophy. September# 1968 CONTENTS PAGE PREFACE (i) SUMMARY (iv) CHAPTER I. INTRODUCTION 1 CHAPTER II. CAUSE OF NODULATION 14 1. Introduction 15 2. Attempts to produce plants 17 in aseptic culture A. From cuttings 17 B. From seed 18 C. From excised embryos 19 3* Conclusions 21 CHAPTER III. MORPHOLOGY AND ANATOMY OF THE ROOT 24 SYSTEM OF PODOCARPS 1. Introduction 24a 2. General morphology of the 30 root system 3. Anatomy of the roots 31 A. Apical organization 31 B. Primary structure 32 C. Secondary structure 35 4- Developmental features of laterals 36 A. Short roots 36 B. Nodules 37 PAGE 5. Episodic growth 40 A. Of short roots 40 B, Of nodules 45 6. Anatomy of endophyte-free roots 46 A. Sterile roots 46 B. Endophyte-free but 47 unsterile roots 7. Conclusions 52 CHAPTER IV MORPHOLOGY AND IDENTITY 56 OF THE ENDOPHYTE 1• Introduction 57 2. Description of the endophyte 62 in the podocarp root system 3. Attemps to isolate the endophyte 65 4. Extraction of Endogone - type 66 spores from soil 5. Inoculation tests 68 A. With red clover 68 B. With Podocarnus lawrencei 70 6. Conclusions 71 PAGE CHAPTER V CYTOLOGICAL EFFECTS OF INVASION 73 BY THE FUNGAL ENDOPHYTE 1. Introduction 74 2. Cytological observations 76 3. Conclusions 7& CHAPTER VI FUNCTIONOF NODULES 80 1. Introduction 81 2. Uptake of phosphorus by 90 nodules of Podocarpus lawrencei 3. Growth experiments with 92 seedlings of P. falcatus 4. Nitrogen fixation tests 95 with P. lawrencei 5. Tests for the presence of haemoglobin 98 in nodules of P. lawrencei 6. Conclusions 100 CHAPTER VII OCCURRENCE OF NODULES IN THE 105 GINKGOALES, TAXALES, AND CONIFERALES PAGE CHAPTER VIII DISCUSSION 112 APPENDICES 120 APPENDIX I METHODS FOR ANATOMICAL 121 INVESTIGATIONS APPENDIX II METHOD FOR BACTERIOLOGICAL TESTS 127 APPENDIX III RECIPES FOR CULTURE MEDIA 129 APPENDIX IV PUBLISHED PAPERS 132, APPENDIX V PREPARATION OF SOIL FILTRATE 136 APPENDIX VI METHODS FOR NITOGEN AND 13g PHOSPHORUS ESTIMATIONS BIBLIOGRAPHY 141 (i) P RE F A_C E The work described in this thesis was carried out in the School of Biological Sciences, University of Sydney, during the period April, 1964 to September, 1968. Except where specified the work described is my own and has not been presented previously for a degree at this or any other University. This Study was made under the supervision of Dr. I.V. Newman until his retirement in December, 1967, when Dr. P .G. Valder was appointd my supervisor for the re maining period. I am particularly indebted to them for their advice, encouragement, and assistance. The first part of the investigations was carried out with members of the Podocarpaceae only but, at the sug gestion of Dr. P. G. Valder, the observations were extend ed to all families of the Coniferae. Through his kind resources and those of the Director of the Royal Botanic Gardens, Sydney, and the Forestry Commission Nursery, Pennant Hills, N.S.W. roots of a considerable number of species were obtained. My thanks are also due to the staff of the School of Biological Sciences and the C.S.I.R.O. for numerous dis cussions and assis tance. For the help with investigations concerning the function of the nodules.I owe much to (ii) Dr. N. Scott, and Mr. J. Smydauk of C.S.I.R.O. Plant Physiology Unit, Sydney, and to Dr. F. J. Bergersen of C.S.I.R.O. DIVISION of Plant Industry, Canberra, A.C.T. The electron micrographs contained in this thesis were made at the Electron Microscope Unit, University of Sydney, and I wish to thank Dr. D. G. Drummond and his staff for their instruction and help. Part of the work reported in this thesis has already been published and the following reprints are enclosed as Appendix IV. (1) Khan, A. G. (1967). Podocarpus root-nodules in sterile culture. Nature, (Lond.), 215, 1170. (2) Khan, A. G. (1968 a). Effect of added growth substances on seedlings of Podocarpus falcatus R. Br. and _P. spinulosus (Sm.) R. Br. ex Mirb. grown in pure culture from excised embryos. Aust. J. Sci., 30 372-73. (3) Khan, A. G. (1968 b). Effect of temperature , gibberellic acid, and indolylacetic acid on root and shoot growth of cuttings from Podocarpus lawrencei Hook. f. Aust. J. biol. Sci., 21, 573-77 (iii) I am most grateful to the University of Sydney for employing me as a tutor during the period concerned and to Professors F. V. Mercer and S. Smith-White for making avail able to me the facilities of the School of Biological Sciences. Finally, I extend sincerest thanks to my wife for her patience and interest during these years, when she and my family must have found me especially trying. A. G. Khan. Department of Biological Sciences. The University of Sydney. Michaelmas Term, 1968. (iv) SUMMARY It has been shown that nodules develop in completely aseptic cultures of Podocarpus falcatus. and hence that they are a normal feature of the root system, developing in response to internal stimuli, A comparative study has shown that nodules differ markedly from short roots in their development, structure> and mode of regeneration. The nodules are fully differentiated structures with no root cap or apical meristem, and with an endodermis completely surrounding and overarching the vascular strand. A survey of members of the Ginkgoales, Taxales,and Coniferales has revealed such structures to be regularly present on species of the Podocarpaceae, Araucariaceae and Sciadopityaceae, but not on species of the Cephalotaxaceae, Cupressaceae, Ginkgoaceae, Pinaceae, Taxaceae, or Taxodiaceae. Vesicular-arbuscular endophytes were almost universally present in the roots and nodules of all species examined except members of the Pinaceae, in which ectotrophic mycorrhizae were of universal occurrence. Inoculation of Podocarpus lawrencei with Endogone-type spores, extracted from podocarp soil by the wet-sieving (v) and decanting method, resulted in the formation of vesicular-arbuscular mycorrhizae, the fungal infection being most pronounced in the nodules. No differences in the colour, morphology or anatomy of the nodules were observed following the fungal infection. Cortical cells of P. falcatus containing arbuscules were observed to contain up to five nuclei, whereas cortical cells from the roots and nodules of non-mycorrhizal plants of P. falcatus and P. lawrencei contained either one or two nuclei. Hence it appears that an increase in the number of nuclei was a consequence of fungal infection. In 15n fixation tests with P. lawrencei there was a very low and doubtfully significant fixation with both mycorrhizal and non-mycorrhizal nodulated roots. The results of this study and of growth experiments indicated that, if there had been any nitrogen fixation, the vesicular-arbuscular endophyte was not involved. With regard to phosphorus uptake, mycorrhizal nodules were shown to be more efficient accumulators of 32p than non-mycorrhizal but the results of growth experiments were inconclusive. No evidence was found that nodulated roots of Podocarnus spp. behaved any differently from the vesicular- (vi) arbuscular mycorrhizae of other plants, and the function of conifer nodules is discussed in the light of findings by other workers. 1 CHAPTER 1 INTRODUCTION 2 INTRODUCTION The literature concerning the structures which have been called "nodules'* in Podocarpus and other coniferous genera has been reviewed in recent years by Kelley (1950), McKee (1962), Baylis et al. (1963), Allen and Allen (1965), Lange (1966), and Bond (1967)* Since, however, the array of evidence and opinion on the subject is both confusing and conflicting, it has been considered appropriate to review it yet again. As reported by McKee (1962), root-nodules were figured without comment for Vicia faba by Fuchs in 1542 and described in 1587 by Dalechamps. In 1758, Du Hamel Du Monceau reported that such structures were of general occurrence on roots of legumes and, in 1829, Meyen described nodules on the roots of Alnus glutinosa (^etulaceae)• Hooker (1854) recorded the presence of "exostoses" on the roots of conifers and, to date, apart from more than 1,000 species of Leguminosae, nodules have been reported on 110 species of 13 genera within eight families of dicotyledons (Bond, 1967), 26 of the 85 to 90 species of cycads, and 49 species of 11 genera of conifers (Allen and Allen, 196 5). Following Hooker's (1854) observations of "exostoses" 3 on conifer roots, similar structures were reported for species of Podocarpus by Van Tieghem (1870), Von Tubeuf (in 1896 according to Nobbe and Hiltner, 1899), Janse (1897), Shibata (1902), Hiltner (1903), and Petri (1903). Janse (1897) recorded ‘'mamelons", as he called them, in other genera as well, including representatives of Aqathis and Araucaria, the two genera of the Araucariaceae, and all these workers, with the exception of Van Tieghem, described a fungal endophyte of what would now be classified as the vesicular arbuscular type. Hooker (1854), Von Tubeuf (1896), Janse(1897) and Nobbe and Hiltner (1899) gave description of the structure and development of the nodules. Hooker (1854) regarding them as transformed root fibrils and Van Tieghem (1870), Janse (1897) and Nobbe and Hiltner (1899), as lateral roots of arrested growth, the last named authors considering the fungal invasion to be responsible for the deformation. The other authors suggested no casual relationship and Janse (1897), for instance, is one of the many authors who have described a similar fungus in the roots of plants without nodules. Nobbe and Hiltner (1899), however, had observed the fungus in the cortex of the roots as well as that of the nodules, as had Janse (1897), and considered them to be endotrophic 4 mycorrhizas in the sense of Prank.