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.
Following the growth of plants of Podocarpus chinensis (P. macrophyllus)* in nitrogen free culture, Nobbe and Hiltner (1899) and Hiltner (1903) concluded that a true endotrophic mycorrhiza gave the plants the ability to use the free nitrogen of the atmosphere. Shibata (1902) made a detailed cytological examination of Podocarpus nodules, observing "digestion" of the fungal
endophyte and demonstrating the presence of a proteolytic enzyme. He stated that he had concluded, as had Magnus with Orchids and Prank with various plants, that there was an analogy with the absorption of materials from insects by certain plants. He noted, however, since there was only a slight connection between the fungal hyphae and the
exterior of the root, that there was scarcely any basis for assuming the uptake of nutrient to be significant.
Hiltner (1903) likewise believed that, while "digestion" took place^ it was not significant in the nutrition of the plant, and Petri (1903) also observed "digestion" in Podocarpus nodules and demonstrated the presence of proteolytic enzymes.
* I am much indebted to the Chief Botanist, N.S.W. National Herbarium, for checking all the names of conifers mentioned in this thesis. Where previous authors are quoted the names are given as in the original and followed, in brackets, by the correct name, if this differs. 5
During the next 30 years a number of workers claimed that the endophyte was not a fungus but a bacterium, identical with, or at least closely related to the
Rhizobium of legume root-nodules» Bottomley (1913), for instance, states that in 1906 he showed that the root- nodules of Podocarpus species contained nitrogen-fixing bacteria similar to those found in the nodules of
leguminous plants. Spratt (1912) continued this inves
tigation, recording nodules on all members of the Podocar- paceae except Pherosphaera (Microstrobus) and Acmopyle.
She concluded the nodules were lateral roots, the growth
of which was arrested by the entry of Pseudomonas
radicicola (Rhizobium sp.), and that the bacterium was
undoubtedly beneficial as it was able to assimilate
atmospheric nitrogen in culture. Although she concluded
that most of the hypha-like threads present in the cortical
cells were zoogleal threads, she also saw true hyphae in
the outer cells of Podocarpus nodules and suggested that
they may have been of mycorrhizal nature.
Sahni (1920) recorded nodules on Acmopyle paneheri
apparently agreeing with Spratt as to their nature and
endophyte, and McLuckie (1923) came to the same conclusion 6 for two species of Podocarpus in New South Wales. The latter also observed fungal hyphae to be present in the outer layers. Phillips (1932), like Spratt and McLuckie, observed bacteria in the nodules of Podocarpus spp. and, in a few instances fungal mycelium in the outer layers as well.
He stated that seedlings lacked nodules and were unthrifty when grown in sterilized soil, but developed nodules and recovered when either a suspension of bacteria from a crushed nodule or from a culture grown from such a nodule was added. During the period 1912 - 1932, however, other workers continued to support the earlier investigations. Yeates (1924), who recorded nodules on members of the Araucariaceae, Cupressaceae and Podocarpaceae in New
Zealand, considered the nodules to be lateral roots containing a non-septate, fungal endophyte and inferred
that mycorrhizal development was responsible for checking the growth of these lateral roots. He suggested that the
nodules functioned both in the storage of water and the absorption of organic matter resulting from digestion of the endophyte. Saxton (1930 a,b) who observed nodules on
both Podocarpus and Pherosphaera (Microstrobus), could 7 find no bacteria and concluded that mycorrhiza was the sole cause of nodulation. In view of the statements of
Spratt and McLuckie, however/ he suggested that in soils where Rhizobium was present it might obtain a footing and perhaps supplant the fungus. Kondo (1931) also reported the nodules of Podocarous to be mycorrhizal but, following comparative studies of the nitrogen status of nodules and roots of various ages and intensities of infection/ could draw no conclusions concerning their function. Since 1932 there have been no further reports of the presence of Rhizobium. Schaede (1943), Ferreira dos Santos (1947)/ Baylis et al.(1963), Bergersen and Costin (1964)/ Furman (1964), Becking (1965), Bond (1967), and Morrison and English (1967) have all observed or depicted a fungal endophyte in conifer nodules. The fungal nature of the endophyte in nodules of Podocarous nagi was established beyond doubt by the electron micrographs of
Becking (1965). This, together with the presence of vesicles and arbuscules, indicates a close relationship between the Podocarous endophyte and the vesicular - arbuscular infections occurring widely amongst the angiosperms, non-nodulated gymnosperms, pteridophytes and 8 bryophytes. Uemura (1964), on the other hand, isolated actinomy cètes from the nodules of Podocarpus and Sciadopitvs, and
Morrison and English (1967) reported that, as well as the fungus the nodules of Aqathis australis contained an actinomycète-like organism. As regards the cause of nodulation, Schaede (1943) observed that infection took place only at an advanced stage of Podocarpus nodule development; some nodules remain ing free from any extraneous organism, and thus concluded that the nodules did not arise in consequence of fungal infection. Shibata (1902), Baylis et al.(1963), Becking (1965), Bond (1967), and Morrison and English (1967) have also reported the presence of endophyte-free nodules. None of these, however, observed the development of nodules under completely aseptic conditions, although Baylis et al. (1963) reported nodulation in seedlings of Podocarpus dacrydioides in fungus - but not bacteria - free culture.
They concluded that nodulation under natural conditions was spontaneous, although the possibility remained that bacteria outside the root might play some part.
Unlike other recent authors, Furman (1964) apparently succeeded in isolating the fungal endophyte and reported 9 that, in a Koch's postulate test, nodules were formed on aseptic seedlings of Podocarpus rospigliosii. Uamura (1964), however, concluded that the actinomycetes he isolated from various plants could not produce nodules on host plants by themselves but presumed they might play an important part in nodule formation. Not only have there continued to be conflicting reports in recent times as regards the nature of the endophyte and the cause of nodulation, but the function of the nodules has also continued to be the subject of a certain amount of dissention, despite the application of modern biochemical techniques. Schaede (1943) concluded that, since the endophytic mycelium was in only very slight contact with the soil, there was no question of its activity as a channel of nutrient supply to the host. He regarded it as an harmless parasite, the absorption of which by the plant was a defence mechanism. Bond(1959, 1967) repeated the growth experiment of Nobbe and Hiltner (1899) using two species of Podocarpus in which the endophyte was present in a proportion of the nodules. He was unable to support their conclusion that nitrogen fixation had taken place and in
15N fixation tests with seven species no evidence of 10
fixation was obtained. the In experiments of Baylis et al.(1963), seedlings of Podocarpus with mycorrhizal nodules developed much better than similar but non-mycorrhizal seedlings in a soil highly deficient in phosphorus and calcium, and they concluded, without making any mention of nitrogen, that podocarps are dependant in poor soil upon the activities of an endotrophic mycorrhizal fungus or fungi. Furman (1964), like Bond (1959, 1967), also obtained a negative result with an 15N test using Podocarpus rospigliosii but Bergersen and Costin (1964), using a more sensitive method than that of Bond, recorded a low but significant fixation in short term experiments with nodulated roots of Podocarpus lawrencei. Becking (1965) recorded a somewhat larger fixation for P. rospigliosii and following tests for the presence of free-living, nitrogen-fixing organisms, concluded that the seat of fixation was the nodule containing the symbiont. In growth experiments similar to those of
Nobbe and Hiltner (1899) and Bond (1959, 1967), but in water culture, the plants became unthrifty and nitrogen deficient but had not died after 3 - 4 years whereas an 11 unnodulated alder had died after a few months. While the result of this experiment was somewhat at variance with that of the nitrogen-fixation test, he concluded that some fixation had probably occurred, although as Bond (1967) pointed out, the Podocarpus seeds would probably have contained much more nitrogen initially
than that of alder. Morrison and English (1967) suggested that they were fairly sure the presence of the mycorrhizal fungus did not account for the low fixation they obtained in
Aqathis australis, since it occurred in the roots of both mycorrhizal and non-mycorrhizal seedlings. Although the seedlings described as non-mycorrhizal were apparently not completely free of the fungal endophyte, they suggested that an actinomycete-like organism , which they observed to be present also, could have been responsible for the fixation. They stated that, while the low fixation obtained in three of the kauri samples could have been due to some transient member of the rhizosphere,
it would not have accounted for an enrichment of 0.048
atoms % in one sample nor the 2.6 atoms % obtained with nodules from Podocarpus alpinus (P. lawrencei).
Morrison and English also demonstrated enhanced 12
phosphorus uptake by the mycorrhizal nodules as compared with the non-mycorrhizal and similar but less striking effect with non-nodulated short roots. Thus it can be seen from the above account that, in spite of a considerable literature on the subject, as yet no entirely satisfactory conclusions can be drawn as to the nature and function of conifer nodules. No fully comparative studies have been made to define the nature of the nodule and its difference from the short root, nor has any systematic survey been made of the occurrence of nodules amongst conifers. While it seems probable that the nodules arise spontaneously as a normal feature of the root systems of those conifers which bear them, this has not yet been conclusively demonstrated in aseptic culture, nor has the identity of the endophyte or endophytes been fully clarified. As regards the function of the nodules, there is little evidence that it differs markedly from that of ordinary roots, or from the roots of other plants for that matter, since comparative studies are rare or lacking. That the fungal endophyte is beneficial seems evident from the studies of Baylis et al.(1963) and Morrison and English (1967), but there is no evidence to suggest that its presence is in any way 13
associated with the nitrogen fixation recorded by the authors mentioned above, nor that any such fixation as might occur is of any benefit to the plant concerned.
It was in an attempt to elucidate some of these problems that the present study was undertaken. 14
CHAPTER IT
CAUSE OF NODULATION
1. INTRODUCTION 2. ATTEMPTS TO PRODUCE PLANTS IN ASEPTIC
CULTURE
A. FROM CUTTINGS B. FROM SEED
C. FROM EXCISED EMBRYOS 3. CONCLUSIONS 15
1 . INTRODUCTION
Since the literature concerning the anatomy and development of podocarp nodules is so concerned with the presence and "behavior of an endophyte or endophytes, it seemed that the subject could he approached more satis factorily were it to he shown conclusively that the nodules developed as a normal feature of the root system and not under the influence of any microorganism.
The first authors to suggest any causal relationship were Nohhe and Hiltner (1899) who considered that an endo- trophic mycorrhizal fungus grew into developing lateral roots arresting their development. This view was supported hy Saxton (1930 a) and later hy Furman (196I4.), who reported that in Koch*s postulate test with a fungus from Podocarpus f nodular structures developed on aseptic seedlings of P. rospigliosii. Shihata (1902), however, reported that newly formed nodules at times reached their full growth in spite of the fact that no fungal infection could he discerned.
However, he stated that the complete development of the cell wall and the production of thickening only occurred where there had heen a successful fungal infection. Schaede (1943) ?
Baylis et al. (1963) and Becking (1965)* all observed fungus-free nodules under unsterile conditions and concluded, that the nodules did not arise as a result of fungal infection. 16
Following the development of nodules on Podocarous dacrvdioides in fungus - not bacteria-free culture, Baylis et al. (1963) concluded that nodulation under natural conditions was spontaneous, as did Becking (1965), although the possibility remains that bacteria outside the root may play some part in nodulation. Spratt (1912) and McLuckie (1923), on the other hand, r were of the opinion that the nodules developed following invasion of the developing lateral roots by a bacterium
identical with or closely related to Pseudomonas radicicola (Rhizobium so. ). Phillips (1932) claimed to have proved this by inoculation experiments. Schaede (1943), Baylis et al. (1963), Bergersen and Costin (1964) and Becking (1965), îiow-
ever? could find no bacteria and it now seems unlikely that endophytic bacteria are involved. There remain, however, the reports of the presence of actinomycètes by Uemura (1964) and Morrison and English (1967).
Uemura (1964) isolated actinomycètes from nodules of varierais non-leguminous plants including Podocarpus macrophyllus and Sciado-oitys verticil lata. In experiments with Alnus and Casuarina he failed to induce nodule forma
tion with the actinomycètes isolated, and concluded that they could not produce nodules on host plants by themselves , 17
"but presumed that they may play an important part in nodule formation,
Morrison and English (1967), working with the nodules of Agathis australis (Araucariaceae), observed that, as well as a fungal endophyte, there was present in some cortical cells a second endophyte which resembled an actinomy- cete in structure. They did not, however, suggest any causal relationship.
Although from the work of Baylis et al.(1963), it seems unlikely that any microorganism is involved in the develop ment of nodules, the following experiments were set up in an attempt to prove this beyond doubt,
2. ATTEMPTS TO PRODUCE PLANTS IN ASEPTIC CULTURE A. PROM CUTTINGS: Shoot cuttings of Podocarpus elatus, P. falcatus, P. lawrencei, and P. spinulosus were treated and planted in the manner described by Khan (1968b; Appendix 1V. 3)« Cuttings of all species gave rise to nodulated adventitious roots within four to five months of planting, but the cuttings of
P, lawrencei produced adventitious roots and nodules more prolifically (plate 2.1.) and earlier than those of other
species. //hen the soil was tested bacteriologically at the end 18 of the experiment, however, it was found that various bacteria and a Penicillium sp. were present. Hence it cannot he claimed that the nodulation was not influenced by the presence of microorganisms. However, microscopic examination of sections of the nodules did not reveal the presence of any endophyte, and no bacteria or fungi developed after three weeks at 20°C on malt-marmite agar slopes inoculated with crushed, surface-sterilized nodules of P. lawrencei in the manner described in Appendix 11.
These results are consistent with those of Baylis et al. (1963). B. FROM SEED Seeds of P. falcatus, surface sterilized in the manner described for cuttings (Appendix IV. 3)> were sown in April, 1966, in sterile pots containing mixture of sand and peat (equal parts by volume) which had been autoclaved.
The pots were covered and watered in the manner described for cuttings (Appendix IV. 3 ). (termination took place in August, 1966, and in September the seedlings were transferred, after surface
sterilization to a mineral agar medium (Appendix Til. 2)
in 9 x 1 inch rimless test tubes plugged with non-absorbent
Eotton wool 19
The roots of these seedlings did not become nodulated until June, 1967, 10 months after germination (Plate 2.2). This also agrees with the observations of Baylis et al.(1963) that 9-12 months elapsed before the roots of P. dacrydioides became nodulated. Although the cultures of P. falcatus appeared sterile it was still not possible to assume that the nodules had appeared spontaneously since the peat-sand mixture was found not to be sterile at the time the seedlings were transplanted. However, the nodules produced in agar cultures were tested in the manner described for those produced by cuttings and no microorganisms were detected. C. FROM EXCISED EMBRYOS Since considerable difficulties were experienced in germinating seeds and in maintaining cultures in a sterile condition for periods long enough to allow the development of nodules, an attempt was made to hasten the process using excised embryos of P. falcatus.
The embryos were excised under aspetic conditions and placed on a tissue culture medium and kept in the light as described by Khan (1968 a; Appendix IV. 2). The embryos were either placed on the surface of the agar, completely immersed, placed with the radicle immersed and the cotyledonary end free in the air, or with the 20 cotyledons immersed and the radicle free in the air. Fully differentiated embryos were approximately
2.5 mm in length (Plate 2.3), and those which were less than one third of this size failed to develop into seedlings (Plate 2.3). Fully developed embryos also failed to grow when completely immersed in the medium rather than placed in other ways (Plate 2.3). Those embryos planted with the cotyledons immersed in agar showed more rapid root growth initially than those planted with the radicle immersed (Plate 2.3), and no embryos could be induced to develop on Raghavan and Torrey's (1963) medium (Appendix III. 1) from which niacine, pyridoxin hydrochloride and thiamine were omitted (Plate 2.3). Once embryos had produced roots and shoots, however, the seedlings continued to develop normally when transferred to a medium without added vitamins.
Such seedlings growing under aseptic conditions on the surface of agar slopes became nodulated when they were one year old (Plate 2.4) (Khan 1967; Appendix IV. 1), and microscopic examinations and plating tests failed to reveal the presence of any microorganism.
A morphological and anatomical examination of the nodules produced under these conditions showed their structure to be fundamentally identical with that of those 21 developed under natural conditions (see Chapter tit),
3* CONCLUSIONS«
A seed dormancy of five or six months, the nature of which is not understood, and the difficulty of maintaining plants under sterile conditions for periods of at least 12 months have greatly hampered studies attempting to clarify the cause of nodulation . However, the production of nodules hy seedlings developing under aseptic conditions from excised embryos has shown conclusively that, unless nodulation is induced by some undetected endophyte trans mitted through the embryo, nodules are a normal feature of the root system of Podocarous falcatus and that they can develop without the influence of any microorganism.
The experiments reported above have also provided further information about the behavior of P. falcatus. It was observed, for instance, that the roots produced by cuttings became nodulated sooner than those of seedlings growing under similar conditions and, in all experiments, it was observed that the faster the roots developed the sooner nodules appeared In an effort to find a reliable method for the rapid production of nodulated roots for anatomical and physio- logical studio , an was set up to examine 22
the effects of temperature and growth substances on root and shoot production by cuttings from P. lawrencei (Khan 1968b Appendix IV, 3.) This experiment showed clearly that the formation of nodules was related to the rate of root develop ment. Also, although it did not provide an easy method for obtaining completely sterile nodulated roots, a simple method was devised for obtaining completely non-mycorrhizal nodules.
While seedlings have previously been grown from the excised embryos of conifers (Narayanaswami and Norstog, 1964), no pre vious record has been found of the culture of Podocarpus embryos. The failure of embryos to develop when excised before they had become one third or more of their length at maturity parallels the results of Dietrich (1924) and LaRue (1936) with other plants. Likewise the behavior of embryos planted with their cotyledons in the medium was similar to that reported by Brown and Gifford (1957) for Pinus lambertiana, in which sucrose entering the cotyledons greatly promoted root elongation. The behavior of Podocarpus embryos on an agar meduim lacking vitamins indicated that, under the conditions of the experiment, an external supply of growth-promoting substances was necessary for their further development. In an effort to find a suitable meduim for the excised embryos of Podocarpusf experiments were carried out to observe the effects of added growth substances on those of P. falcatus and P. spinulosus 23
(Khan, 1968a? Appendix IV. 2). The beneficial effect of vitamins and gi-bberellic acid was demonstrated. EXPLANATION OF PLATES EXPIRATION OF PLATES PLATE 2.1
Shoot cutting of Podocarous lawrencei producing
nodulated adventitious roots in fungus free culture. (X 0.2). PLATE 2.2
Seedlings of P. falcatus grown in aseptic
cultures. Note that the roots did not become
nodulated until the seedlings were 10 months old. (X 0.2).
PLATE 2.3
Excised embryos of P. falcatus grown on
agar slopes. Top left: A fully differentiated embryo. (X 10). Top second from left: An immature embryo, less than one third of its final length. (X 10). Top, second from left: Fully differentiated embyro completely immersed in the medium. (X 10). Top right: A fully differentiated embryo grown on a medium lacking vitamins. Note the absence of any growth and decolouration. (X 10). Bottom left: A seedling
developing from an excised embryo. Note the
immersed tips of cotyledons and the well developed
root. (X 8). Bottom right: A seedling developed from the excised embryo, the radicle of which was immersed in the medium. Note the poor root development, (X 8).
PLATE 2,k
Sterile seedling of P. falcatus. Note the root nodules (N). (X 2). months months months months PL.2 .3 PL.2.4 24
CHAPTER 111
MORPHOLOGY AND ANATOMY OF THE ROOT
SYSTEM OF PODOCARPS.
1. INTRODUCTION
2 . GENERAL MORPHOLOGY OF THE ROOT SYSTEM
3 . ANATOMY OF THE ROOTS
A. APICAL ORGANIZATION
B. PRIMARY STRUCTURE
C. SECONDARY STRUCTURE
4. DEVELOPMENTAL FEATURES OF LATERALS
A . SHORT ROOTS
B . NODULES
5. EPISODIC GROWTH
A . OF SHORT ROOTS
B . OF NODULES
6 . ANATOMY OF ENDOPHYTE-FREE ROOTS
A . STERILE ROOTS
B. ENDOPHYTE-FREE BUT UNSTERILE ROOTS
(1) LIGHT MICROSCOPE OBSERVATIONS
(2) ELECTRON MICROSCOPE OBSERVATIONS
7. CONCLUSIONS 24a
1. INTRODUCTION Once it became known for certain that the nodules of Podocarous could arise spontaneously without the intervention of any microorganism, it was decided that a careful examination of all parts of the root-system might enable a clearer picture of the true nature of the nodules to be constructed. In order to provide a simple background for such a study the appropriate literature is reviewed below, omitting any reference to the endophyte or endophytes. Ever since Hooker (1854) observed that the roots of Podocarous dacrvdioides were studded at intervals with 1__ 1 _ spherical bodies, 40 th to 60 th inch in diameter, attached by a short pedicel, sessile or sometimes sunk into the bark of the root, podocarp roots have received attention from time to time on account of this characteristic feature. According to McKee (1962), van Tieghem (1870) described the root nodules of P. nerlfolius as small hemispherical warts arranged in two opposite rows along the roots and placed so closely as almost to touch one another. Such an appearance, resulting from the origin of the nodules from the pericycle of the parent root opposite the protoxylem groups, is certainly characteristic of all 25
species of Podocarpus examined since. All authors who have come to any conclusion concerning the nature of the nodules consider them to be modified lateral roots or lateral roots of arrested growth (Hooker, 1854; van Tieghem, 1870; Janse, 1897; Nobbe and Hiltner, 1899; Shibata, 1902; Hiltner, 1903; Spratt, 1912; Sahni, 1920; McLuckie, 1923; Yeates, 1924; Baylis et al#, 1963; Becking, 1965; Morrison and English, 1967). Hooker (1854) apparently gave a detailed description of the anatomy of podocarp nodules, although his paper is reported only in abstract. He observed the nodules to be composed of a mass of spongy cellular tissue aggregated round a central vascular axis which extends from the wood of the root to the centre of the sphere. Such a structure has also been reported by subsequent workers and the presence of root hairs has been observed by several authors (von Tubeuf, 1896, according to Nobbe and Hiltner, 1899; Spratt, 1912; McLuckie, 1923$ Baylis et al,^ 1963; Becking, 1965).
Janse (1897), for example, observing the origin of the nodules in the pericycle of the parent root, concluded that the nodules are simply rootlets that arrest their growth
after having pierced the coi-tex of the mother root, their 26
structure being the same as that of the rootlets. Although Janse mentioned that the epidermis of the nodule was covered with several layers of dead cells/ analogous to the root-cap of ordinary roots, it is not clear from his drawings that such an analogy existed, although what is obviously a root-cap is figured for the rootlets. Baylis et al, (1963) stated that no distinct root cap could be recognised and seem to be the only other authors to have mentioned its presence or absence. Spratt (1912), however, figured epidermal hairs occupying the position where one would expect to have found a root cap had it existed.
The only person to draw attention to the absence of a meristematic zone in the mature nodule seems to have been Spratt (1912). Though the illustrations of Janse (1897), KcLuckie (1923), Baylis et al. (1963), Bergersen and Gostin (1964) and Becking (1965) show the nodule to be a fully differentiated structure with an endodermis completely enclosing the stele, Spratt (1912) and Shibata (1902) are the only authors to make special mention in the text of an endodermis overarching the apex of the stele. Spratt (1912), Baylis et al, (1963) and Becking (1965) noted that the endodermis lacked Casparian strips but was 27 well defined by a thickening of its outer walls and by brown tannin deposits. Janse (1897) seems to have been the only person to compare the anatomy of roots and nodules and figures a longitudinal section of a rootlet which has temporarily ceased growth and in which the endodermis is shown as both surrounding and overarching the stele (Plate Vlll fig. 16). According to Nobbe and Hiltner (1899), von Tubeuf (1896) reported that after a year the cortex began to die, since a cork ring formed beneath it. Janse (1897), however, appears to have been the first person to note that the nodules were perennial, a new one forming at the apex of the first, a process that might be repeated several times. He also observed that the ordinary short roots made repeated growth in a similar fashion and assumed a beaded appearance. This phenomenon was also noted in the short roots by Yeates (1924) and figured without comment for P. dacrvdioides by Baylis et al, (1963).
Since Janse's (1897) description, regeneration of the
nodules has been reported by various authors (shibata, 1902?
Spratt, 1912? Sahni, 1920? McLuckie, 1923? Yeates, 1924?
Baylis et al#> 1963? Bergersen and Costin, 1963? Becking, 1965) and, according to circumstances and species , the 28
new nodule may or may not break out of the shell of old tissue surrounding it* Baylis et al. (1963) reported that this annual extension is occassionally accompanied by branching so that two series of up to four nodules may surmount a single parent nodule. Spratt (1912) reported a similar occurrence in Saxeqothaea conspicua. Shibata (1902), Spratt (1912), McLuckie (1923), Baylis et alv (1963) and Becking (1965) all describe the origin of the new nodule from meristematic tissue which appears inside the endodermis at the tip of the vascular strand of the old nodule. According to Spratt (1912), a rapid formation of new tissue ensued, rupturing the endodermis and crushing the old cortical tissue, and when the rapid cell division ceased, a new endodermis was differentiated around the apex of the stele. Like other workers, she observed that this could happen year after year and that in many nodules several annual additions could be observed.
Sahni (1920) reported that the roots of Acmopyle pancheri bore tubercles and that several of them had proliferated, having grown into normal rootlets. He took this to support the view that the tubercles were modified rootlets. There are, however, no similar reports for other conifers. 29
Bar-like thickenings have frequently been reported on the walls of the cortical cells of nodules, particularly as they become older (von Tubeuf * 1896 , according to
Nobbe and Hiltner, 1899; Shibata, 1902; Spratt, 1912; McLuckie, 1923; Yeates, 1924; Baylis et alfJ 1963;
Becking, 1965), but only Becking (1965) seems to have recognised these thickenings as being like those found in the cortical cells of many gymnosperms. Baylis et al#
(1963) stated that in autumn the cortex lignified, the thickening being evenly deposited in the outer layers but in most of the tissue occurring in the form of bars, which gave the cells a striated appearance. Like earlier workers, they noted the disappearance of cytoplasm and nuclei.
Although there have been many detailed studies of the nodules, there seem to have been no comparative studies of rootlets and nodules, apart from the one observation of Janse (1897). Nevertheless most authors seem to have been content to assume that the nodules are modified rootlets or lateral roots of arrested growth.
It was to see how far this assumption is true that the following studies were made.
Except where otherwise mentioned, the roots used for anatomical studies were either fixed, processed, and 30
stained as described in Appendix 1,1, or prepared using a freezing microtome as described in Appendix 1.2.
The plates of whole roots showing development and
regeneration of nodules and short roots were made from roots kept in Sartory's Clearing Agent (Goldacre, 1959) for three to seven days.
bxcept where indicated, the roots examined were grown in natural soil and contained a well-developed fungal endophyte in the cortical cells.
2. GENERAL MORPHOLOGY OF THE ROOT SYSTEM
Seedlings of Podocard u s falcatus were selected as exhibiting a root-system characteristic of all species examined (see Chapter Vll). A two-year-old seedling of
P. falcatus has a slow growing tap-root from which arise a few large lateral roots which grow comparatively rapidly
(Plate 3.1). The tap-root and laterals are equivalent to
the "long-roots'1 of the "heterorhizic" state so character
istic of most trees (von Alten, 1909? Noelle, 1910? ftilcox, 1954, 1964). The long roots bear roughly
spherical nodules in two rows, as described by earlier
authors, "short-roots", which become only a small size
before ceasing growth. Both the nodules and short roots
are capable of renewed growth at intervals. This gives 31 the older nodules a characteristic appearance in section and causes the short roots to assume a beaded appearance, a constriction marking the position of each cessation in growth (see section 5 of this chapter),
3, ANATOMY OF THE LONG ROOTS A . APICAL ORGANISATION.
A longitudinal section of the root apex of Podocarpus falcatus is shown in Plate 3.2. The cells marked 1 are the apical initials which contribute cells to the stele/ root-cap, and cortex. Divisions in the derivatives of these initials give rise to the different regions of the root in the manner described by Schopf (1943) for Larix, Allen (1947) for Pseudotsuqa, and Wilcox (1954) for Abies procera.
The apical initials are in a single more or less saucer-shaped layer (Plate 3.2). The cell divisions in this zone are by partition walls transverse to the root- axis, so that cell production is directed both forwards and backwards. The internal (backward) derivatives of apical initials form the stelar-mother-cell zone (Allen, 1947), which gives rise to the root-stele. The external (forward) derivative form the column-mother-cell zone and cortical- mother-cell zone (Allen, 1947), which give rise to the 32
root-cap and the root-cortex respectively.
Over the surface of the root there is no continuous, uniseriate cell layer, derived from separate initial cells, and becoming free of the overlying root-cap cells. Hence there is no epidermis in the strict sense in the roots of
P. falcatus. These observations support von Guttenberg
(1941), who reviewed the numerous investigations that have revealed that the gymnosperms have no epidermis. Root- hairs are formed from the outermost cortical cells, and this outer layer is usually called the exodermis or hypodermis.
B. PRIMARY STRUCTURE.
Transverse sections of the tap root of P. falcatus showed the following characteristics(Plate 3.3).
(j) EPIDERMIS. The studies mentioned above show that in P. falcatus, as in gymnosperms in general, there is no epidermis. The outermost, closely packed cortical cells function as an epidermis and often bear root hairs,
(jj) CORTEX. The remainder of the root-cortex is composed of 5-7 layers of isodiametric cortical parenchyma cells and an endodermis, which lacks casparian strips but is well defined by a thickening of its outer walls and by reddish-brown tannin deposits, an observation in 33 agreement with those of earlier authors for the endodermis of the nodule (Spratt, 1912; Baylis et al„ 1963; Becking, 1965). The deposition of tannin, an endodermal character istic detectable histochemically, occurs during the meristematic activity at the boundary of vascular cylinder (Plate 3.2). The walls of the cortical layer outside the endodermis are characterised by bar-like thickenings which are laid down exactly opposite one another in adjacent cells (Plate 3.3). Similar observations were recorded by Russow (1875, pp. 72-73) for the roots of members of the Pomaceae and certain other families of dicotyledons and gymnosperms. He referred to this layer as the "exodermis" and noted that, in sections through the walls of adjoining cells, the thickenings resembled the Greek letter phi. The development of such thickenings was reviewed in detail by van Tieghem (1888). Guttenberg (1940, pp. 121-22) calls this layer the "inner cortical sheath", whereas Boureau (1939) has designated this layer in Libocedrus decurrens as "endodermis I" and the endodermis with Casparian strips as "endodermis II".
Wilcox (1962) recorded the presence of similar heavy thickenings on the walls of the layer of cells immediately outside the endodermis of L. decurrens and called this layer the "phi layer". He stated that the phi thickenings 34 gave reactions for lignin only, whereas the Gasparian strip of the endodermis was doth lignified and suherized.
He recorded that at a later stage, similar, hut less developed, thickenings were formed throughout the cortex.
The situation in P. falcatus is similar to that recorded hy Wilcox (1962) for L. decurrens. since in older roots and nodules the thickenings occur throughout the cortical parenchyma except for the outer layer or layers, in which they are usually sparse or absent. It seems probable that, with regard to the phi thickenings there is nothing unique about the layer immediately outside the endodermis, apart from the fact that such thickenings appear first in this layer. (ill) VASCULAR CYLINDER. The central part of the root consists of the vascular cylinder. The vascular system is surrounded by a multiseriate zone, the pericycle. The lateral roots and nodules arise
in this zone and the phellogen originates here. The ce^ls of the pericycle contain starch grains and show a deposition
of tannin. The vascular system consists of the xylem and phloem. The phloem occurs in the form of two strands distributed near the periphery of the vascular cylinder, beneath the
pericycle. The xylem forms a diarch strand,the pith being 35 absent. A few xylem elements at each pole of the xylem- plate represent the protoxylem. As seen in transection (Plate 3.3), the xylem elements at the poles are narrow and become wider towards the centre. The characteristic resin ducts of the primary vascular region of the root of many conifers (Guttenberg, 1943) is lacking in Podocarpus roots. Generally such ducts are found in the Araucariaceae, and Pinaceae, whereas they are absent from the primary vascular cylinder of the Taxaceae, Taxodiaceae and Cupressaceae.
C. SECONDARY STRUCTURE
Observations of transections of older roots of P. falcatus showed the following characteristics. The vascular cambium is initiated by divisions of the procambial cells remaining undifferentiated between the primary phloem and primary xylem. As shown in Plate 3.4, the cambium first appears as two curved strips on the inner sides of the phloem strands* This cambium, after producing some secondary xylem elements, becomes united into a continuous ring between the xylem and the phloem by the meristematic activity of the pericyclic cells located outside the protoxylem poles (Plate 3.4). The bottom photograph of Plate 3.4 is of a section showing 36
considerable secondary growth. The initiation of secondary vascular growth is followed by the formation of the periderm, pericyclic cells undergoing periclinal and anticlinal divisions. During this increase in the circumference of the stele through combial activity and proliferation of the pericycle, the cortex is forced outwards and finally sloughs off.
4. DEVELOPMENTAL FEATURES OF LATERALS A. SHORT ROOTS
In gymnosperms, as in angiosperms, lateral roots are initiated endogenously in the pericycle of the parent root, some distance from the apical meristem, and subsequently grow through the cortex of the latter (Guttenberg, 1940, 1941). The formation of lateral roots, which is similar for both long and short roots, is described below for P. falcatus, in which the distance from the root apex to the first lateral is much smaller in slow growing than in fast growing roots. This phenomenon also seems to be related to the rate of elongation as Wilcox (1954) recognized for Abies orocera.
A group of pericycle cells just beneath the endoderrais and opposite the protoxylem pole of the parent root becomes meristematic and the cells undergo anticlinal 37 divisions (Plate 3.5). Succeeding divisions result in the formation of a protrusion# the lateral root primordium. At first the endodermis streches over above the primordium (Plate 3.5) and latter ruptures and the lateral root pushes through the cortex# carrying the tannin-filled endodermal cells out at its apex (Plate
3.5). These cells eventually die and are shed. A definite growing point with its initial cells and root- cap is formed in the lateral root (Plate 3.6) and can be
discerned before the lateral has emerged from the cortex
of the mother root. Differentiation within the lateral root primordium occurs acropetally. The vascular system of the lateral becomes connected with that of the mother root through the intervening pericyclic derivatives. The cortical cells of lateral roots# as they become older# undergo similar structural changes to those occurring tn the tap-root# bar-like thickenings developing
on their walls.
B. NODULES.
The nodules also arise in the pericycle at some distance from the apical meristem# opposite the protoxylem groups of the diarch root# as first noted by Janse (1897). 38
Like the short roots, the distance from the root apex to the first nodule is much smaller in slov/-growing than in
fast growing roots, as can he seen in Plate 3.7. Again the pericycle cells divide per-iclinally and
anticlinally to produce a globular mass of cells. This
nodule primordium ruptures the endodermis of the parent root and crushes the overlying cortex (Plate 3.8.). The
globular shape of the primordium becomes more and more pronounced as it gradually pushes its way through the mother root cortex. There is no differentiation of an apical meristem and root cap but, during this period, a plerome becomes differentiated in the centrally placed cells of the protuberance and it develops into the vascular strand of the nodule (Plate 3«9.). Before the differentiation of plerome cells, a new endodermis differentiates overarching the plerome (Plate 3.8. and The cells of this en
dodermis are slightly thickened on the outside, although there are no evident Casparian strips, and as in the root,
they contain a deposit of tannin. The connection between the vascular systems of the
main root and the nodule is established through the intervening cells which are derivatives of the pericycle (Plate 3.9.). Some vascular elements mature in the nodule 39 before the connection with the vascular system of the main root is established basipetally. Unlike the lateral root, the mature nodule is a fully differentiated structure exhibiting the following tissues (Plate 3.10): in An outer cortical layer behaving as an epidermis, the cells of which, when young, have the ability to develop non-septate, hair-like outgrowths. (Plate 3.10). The occurrence of root-hairs on Podocarpus nodules has been noted by several authors, as mentioned in the introduction
to this chapter, and they are reported on nodules of representatives of all the genera of the Podocarpaceae studied by Spratt (1912). Under local conditions^the
development of root hairs appear3to be related to growth cycles and they persist for varying periods of a time, attaining a length about equal to or a little longer than that of the nodule. In older nodules the walls of the outer layer of cells become uniformly thickened, as noted by
Baylis et al. (1963).
• * in) Two to six layers of cortical parenchyma, all cells of which are similar and somewhat compressed. In older nodules
these parenchyma cells undergo degenerative process«similar
to those observed for roots and bar-like thickenings are
laid down which give the cells a striated appearance. As
in the roots these thickenings are often sparse in the ko outermost layer or layers or absent from them. (III) A definate endodermis like that of the parent root hut both surrounding and overarching the stele, completely enclosing it.
(IV) A stele, with three or four layers of pericycle cells containing tannin and starch grains, poorly developed phloem, and typically diarch xylem. In all species of Podocar^us and other genera with large nodules, the vascular tissue is quite well developed, whereas genera with smaller steles, the vascular strand, as observed by
Spratt (1912) and Baylis et al.(1963), is frequently very rudimentary.
3. Kir'ioODIC GROWTH A. OP SHORT ROOTS
"Short-roots" are produced by "long roots" of all orders and seem to be capable of only limited growth. The short roots, however, do exhibit renewed elongation from time to time. This episodic growth seems first to have been recorded by Janse (1897), who observed that, in P. cupressinus (P. imbricatus) growth of the nodules resumes following a short interuption only to stop again after forming a second spherical tubercle at the summit of the first, and that the rootlets often behave in an 41 analogous manner, although the cessations of’ growth are more distantly spaced, the successive articulations attaining a more considerable size.
Prat/ (1926) gave a particularly clear account of
the structure, mode of growth, and infection of the root system of Taxus baccata. in which he noted that the apices grew intermittently for more than one season, resulting in the formation of beaded rootlets.
The histological and chemical changes taking place in dormant root tips were summarized under the term
"Metakutisierung" by Muller (1906). This term was later translated as "metacutization" by Wilcox (1954). ,1918 Plaut (1909,1910/) described the dormant root tips of conifers, dividing them into four groups on the basis of the manner in which metacutization occurs in the root- cap (Figure 3.1). These types may be briefly described as follows.
TYPE I:- The outer layers of the root-cap metacutize and become continuous with the suberized exodermis. This
is the common type in cycads and some Pinus spp.
TYPE II:- The suberized exodermis is absent.
Metacutized layers form in the cap, but not necessarily on the surface. By means of a bridge across the cortex
these become continuous with the suberized cells of FIGURE :.3*1 /
Types of metacutization. Reproduced
from Romberger, 1963* Type I
Type IEL Type HZ
F igure 7.— Types of metacutized root tips. Cross hatching represents exten sive suberization. Stippling represents endodermis. See text for discussion. (Schematic after Plaut 1918.) 42
endodermis. The cortex and any cap cells outside the metacutized layers turn brown, collapse, and die,
Podocarpus totara, Aqathis robusta and Pseudolarix kaemferi are examples exhibiting this type.
TYPE Tils- A suberized exodermis is present and the metacutized layers of the cap become continuous with
it as in Type T. In addition the metacutized cell layers
in the cap are linked with the endodermis as in Type II.
Examples are Ginkgo biloba, Taxus baccata, Athrotaxus
selaqinoides, Secuoia qioantea, Crvptomeria laponica,
and Junioerus orostrata.
TYPE IVs- A suberized exodermis is present but the
suberization does not extend to the root tip and thus the
exodermis does not participate in the final phase of
metac'Jtization, which proceeds as in Type II. An example
is Araucaria excelsa.
Plaut's concepts were found substantially correct
and applicable to all the conifers studied by subsequent
investigators (Romberger, 1963).
In the present study, beaded rootlets were observed
in all the conifers investigated (see Chapter VII). The
banner in which metacutization occurs in the roots of
Podocarous falcatus was studied in detail. The sequence
of changes as a root ceases to elongate was as follows 43
(Plate 3.11);
(1 ) The dirf erent^ation of the endodermis down to a region close to the apical meristem.
(11) Metacutized (suberized) layers are formed in the cap, usually a few cells in from the surface.
(111) A bridge is formed across the cortex by
suberization of cells. This bridge connects the metacutized root-cap layers with the suberized cells of the endodermis. These changes result in the complete isolation of the apical meristem (including apical initials and their immediate derivatives) by the metacutized layers. The cortex and cap cells outside the metacutized layers turn brown, collapse and die. The tip of this dormant root appears dark brown (Plate 3.7). The metacutization process in P. falcalus thus con forms to Piaut’s Type IT. In addition to the events described above, other changes follow the process of metacutization. As occurs v/ith the endodermis^ vascular tissue differentiation in a dormant root occurs much closer to the apical initials than in an actively growing root (Plate 3.11). This indicates that maturation continues after the cessation of activity in the root initials. Also the tracheary elements are progressively shorter towards the apex of the dormant root. This suggests that elongation ceases or is much reduced after the cessation of cell divisions in the root-apex.
> s r Prat (1926) and Wilcox (1954) also found/Taxus baccata and Abies procers. respectively, that, in a dormant root-tip, differentiation had proceeded right up to the apical meristem.
The dormant roots resume their growth after a short pause. The apical initials start dividing again
(Plate 3*11) giving/to a new growth which breaks through the metacutized root-cap, leaving a transverse constriction in the cortex (Plate 3*12). This isolation of young extension-growths from the cortex of their parent root by dead cells, results in the beaded appearance of the rootlets (Plate 3.12). As many as eight ''beads’' could be seen on a single season's growth.
This suggests that the intermittent growth of Podocarpus roots, and perhaps other conifers, is not seasonal as suggested by Prat (1926) for Taxus but is cyclic, as noted by Wilcox (1954) for Abies.
•> These beaded rootlets are termed "pearl-necklace” rootlets by Kelley (1950), and according to him, they 45 are widly found among conifers (see also Chapter Vll) and also angiosperms such as Casuarina, Liquidambar,
Acer and Celtis.
B. OF NODULES
Despite the fact that no apical meristem is present in the conifer nodules, they, like the short roots, exhibit an episodic growth pattern and are perennial.
In P. falcatus some of the pericycle cells immediately below the endodermis, at the apex of' the nodular vascular strand, become meristematic (Plate 3*13)* The formation of new tissue beneath the endodermis causes the rupture of the latter, but later, when the rapid cell division ceases, a new endodermis is differentiated which
joins the older one and overarches the vasculat strand (Plate 3.13). The new cortex pushes its way through the old one and the vascular strand extends (Plate 3 • 3) • This renewal process may be repeated several times resulting into a series of nodules surmounting a single parent nodule (Plate 3 .1 4 ). In some cases the new cortex is completely ensheathed by the old, especially where the stele is relatively small. The new nodule thus arises within the old, rather than at its apex, the remains of previous nodules surrounding the most recent one. Still in other cases, the new nodule grows out from the remains of its immediate predecessor, hut the remnants all can he recognized still attached to its base (Plate 3.15).
Nothing comparable to the processes of metacutization exhibited by lateral short roots was found in the nodules. Each pause in the growth of an individual nodule was accompanied by complete differentiation including the formation of an endodermis overarching the stele. The resumption of activity results in the rupture of this endodermis, the remnant appearing in cleared roots, as a dark collar against a light background, marking the position of the renewal of growth (Plate 3*1U)« The cyclic growth behaviour of the podocarp roots and nodules was very marked under all conditions investigated, whether in the field, glasshouse, or test tube.
6. ANATOMY OF ENDQPHYTE-FREE ROOTS
A. STERILE ROOTS:-
Sterile nodulated roots from P. falcatus plants raised from excised embryos (Chapter It. 2.C) were sectioned and examined as before. Both roots and nodules exhibited a structure apparently indentical to that of plants under natural conditions, differing only in the absence of the endophyte (Plate 3.16), although of course, the endophyte kl must have a profound effect upon the cytoplasm of the invaded cells.
The nodules formed under completely aspetic conditions showed the same type of growth renewal described earlier and Plate 3*16 shows a meristematic zone in the pericycle immediately below the endodermis at the apex of the vascular strand.
B. ENDOPHYTE-FREE BUT UNSTERILE ROOTS
(1) Light Microscope Observations
The adventitious roots from the base of cuttings of Podocarpus species and the roots of seedlings of P. falcatus produced in earlier experiments (Chapter II. 2 . A,B) also bore typical nodules comparable in size and number with those of field-grown plants. Again the anatomical structrue of the roots and nodules appeared the same as in field grown plants, although no endophyte could be seen (Plate
3.17). The phenomenen of renewal of growth was again observed and in mature nodules of P. lawrencei it was observed that in the cortical cells a disorganization of the cytoplasm occurs and bars of thickenings are deposited on the walls
(Plate 3.17). (2) Electron Microscope Observations:- Preparations of' adventitious roots produced by cuttings of* P. lawrencei were made as described in Appendix 1.3* Election micrographs of ultra-thin sections, prepared from permanganate-fixed segments of non mycor- rhizal nodules, showed the presence in the younger cortical cells of peripheral cytoplasm (PC), cytoplasmic strands (CS), nuclei (N), plastids (P), endoplasmic reticulum (ER), mitochondria (M), golgi bodies (g ), vacuoles (V), and various other structures (Plates 3.18 to 3.24). No endophyte or endophytes were detected in any section. Details of the more conspicuous structures are given below. (a) The nucleus. Each cortical cell contains a single nucleus which is surrounded by the usual perforated double membrane (NM), which is continuous with portions of the endoplasmic reticulum (ER) (Plate 3.18). (b) The endoplasmic reticulum. There is an extensive endoplasmic reticulum (ER) in the cells, composed of long tubules and round or oblong vesicles (Plates 3*19 and 3. 20). The connection between the nuclear membrane and the endoplasmic reticulum in Plate 3*16 indicates that the space between the two components of the nuclear membrane are continuous with the membrane-enclosed spaces of the endoplasmic reticulum, suggesting as has been frequently k9 observed elsewhere, a possible.function of the endoplasmic reticulum in serving as a route of transport of materials from the nucleus to other parts of the cell.
(c) The Golgi bodies. These consist of a system of membrane-delimited, saucer-shaped discs or cisternae arranged approximately parallel to each other (Plates
3.19 and 3.23).' (d) Mitochondria. These are typical double walled structures, the outer membrane being smooth and the inner with many inwardly directed folds or cristae (Plates 3.19,
3.20, 3.21, and 3 .23). (e) Plastids. These are present in the cortical cells at all stages of development and are colourless, each usually containing two or more starch grains (Plate 3.19). The plastids are surrounded by a double membrane, and internally they may show various structures other than starch grains. Sometimes a few tubules or lamellae bounded by double membranes are also seen (Plate 3*19 * 3.20, and 3.21). (f) Vacuoles. Young cortical cells have three to four large vacuoles separated by thin layers of cytoplasm
(Plate 3.18) and, as a result, the bulk of the cytoplasm is present as a relatively thin layer aroung the periphery 50 of the cell. The structure of the vacuolar membrane or tonoplast could not be resolved in the sections used and the cell sap appears as a precipitate resulting from reactions with the permanganate fixdtive. (q) The cell wall. All cells are, of course, surrounded by conspicuous walls (W)5and a middle lamella (ML) can clearly be seen between the walls of adjacent cells (Plate 3.21). The structural changes taking place in cortical cells as they mature have already been noted, the nuclei and cytoplasm eventually disappearing and bar-like thickenings being deposited on the cell walls. These changes were further studied with the help of the electron microscope. The organization of the cytoplasm in older cortical cells (Plate 3.22) is quite different from that in younger ones (Plate 3.18). The cytoplasm is aggregated into ridges, rich in organelles against the cell wall, in a manner similar to that described by Hapler and Newcomb (1963) in parenchyma cells of Coleus which were redifferentiating into tracheary xylem elements. The golgi bodies, consisting of vesicles associated with the cisternae and presumably produced from their edges, near the primary wall (Plate 3.23). These vesicles were seen in close contact with the plasmalemma, as shown by means 51 of the arrows in Plate 3.23. It is well known (e.g. Iiollenhauer and Whaley, 1962?
Whaley et al#, 196 2? Mollenhauer and Whaley, 1963) that the development of the cell-plate during cell division in root meristems is associated with the Golgi bodies.
Their vesicles line-up in the plane of the future cell- wall and fuse laterally to form a sheet. The membrane of fused Golgi vesicles becomes the cell membrane on either side of the new cell wall. In the light of these observations, it seems possible that the golgi bodies in the cortical cells of conifer roots and nodules are concerned with the deposition of wall material. The close proximity of small vesicles, presumably produced from the edges of the Golgi cisternae, suggests the possibility of secretion of Golgi-produced material between the plasma membrane and the primary wall, resulting in the development of a thickening at that particular part of the cell wall (Plate 3.24). That Golgi-bodies play a similar role in developing xylem elements is suggested by Cronshaw and Bouck (1965) and Wooding and
Northcote (1964).
The endoplasmic reticulum is a further possible site of synthesis of precursors of wall polysaccharides or the enzyme system capable of producing them. In plate 3.23 52 the tubules of the ER are in close proximity to the plasmalemma. Similar observations were recorded by
Porter and Machado (1960) and Whaley et al,(1960) for the cell plates of root meristems. As the cortical cells become older, the wall bands or bars increase in size (Plate 3.24). The identity of the inclusions observed close to and appressed to these bars is not clear (Plate 3.24). It is impossible to decide whether all of these bodies are disintegrating plastids and mitochondria and whether they play any intimate role in the gro\rth of the bars. In common with the conducting elements of xylem, which develop secondary wall thickenings, these cortical cells lose their protoplasts as they mature, and are dead cells, although they may be functional in water storage or conduction.
7. CQIÏCHJSIOHS
It is evident from the above account that, while nodules develop laterally as normal structures on the long roots, there are, in P. falcatus and P. lawrencei, some marked developmental and histological differences between nodules and lateral roots. These differences have been observed to occur consistently in species of 53
Podocarnus and certain other coniferous genera (see
Chapter VII). While both lateral roots and nodules arise endogenously in the pericycle, they differ in their cellular configurations. The nodule primordium, in several Podocarpus species at least, is more or less spherical, while the lateral root primordium is usually conical. The developing lateral root, both before anlafter emergence from the cortex of the parent root, shows an obvious root cap and apical meristem, behind which differentiation of the tissues takes place, the stele being surrounded by an endodermis which remains open-ended. No example of the endodermis overarching the stele in a lateral root, similar to that figured by Janse (1897) for P. cupressinus (P. imbricatus) has ever been seen.
In the nodule, however, oneecell division has ceased, all cells become differentiated and, as reported by Shibate (1902) and Spratt (1912), the stele becomes completely enclosed by the endodermis and, as Spratt (1912) noted, there is no apical meristem. No evidence of any root-cap could be found nor any layers of dead cells analogous to it, such as described by Janse (1897), and this is in agreement with the observations of Baylis 54 et al. (1963)• The nodule ceases growth by complete differentiation of the meristematic cells but the lateral root, on the other hand, ceases growth after the cessation of divisions in the apical meristem followed by the process of metacutiztion. The lateral root resumes growth after a resumption of activity in the apical meristem and subsequent rupture of the metacutized layers, whereas in the nodule a new primordium appears in the pericycle during the subsequent development of which the endodermis and overlying cortical layers are ruptured.
All the differences between nodules and lateral roots mentioned above are present even when the nodules are elongated, rather than spherical, and superficially much resemble short roots (Plate 3.25). While it is very difficult to be certain, no evidence was obtained that nodules could grow on into normal lateral roots, as claimed by Sahni (1920) for Acmopyle pancheri, nor that regrov/th of a short root could lead to the development of a terminal nodule.
The loss of contents of the older cortical cells of the nodules and the deposition of bar-like thickenings on their walls, as reported by numerous authors, does not appear to be peculiar to the nodules, but rather a 55 characteristic of the cortical cells of the whole root- system. As Becking (1965) recognised, the thickenings are like those occuring in the cortical cells of many gymnosperms. In the view of their unique structure and behavior it is suggested that, nodules should not be regarded merely as modified lateral roots or lateral roots of arrested growth but as distinct features of the root system. EXPLANATION OF PLATES KEY TO LETTERING IN PLATES 3.1 - 3.17
Bar-like thickenings Cortex Parent root cortex Cortices of successively developing nodules or short toots. Cambium Cortex of nodule Dormant root-tip Endodermis New endodermis Root hair Root opical initials Unemerged lateral root primordium Meristematic region Metacutization layers Nodule Unemerged nodule primordium Pericycle Piero me Phi thickenings Remnants of previous nodules Root cap Root tip Stele Mother root stele Secondary xylem Xylem KEY TO LETTERING IN PLATES 3.13 - 3.2L
C Cristae of mitochondria
CS Cytoplasmic strands
ER Endoplasmic reticulum
G Golgi bodies
L Lamellae of plastids
M Mitochondria
ML Middle lamella
N Nucleus NM Nuclear membrane P Plastids PC Peripheral cytoplasm S Starch grains T Tonoplast V Vacuole VS Vesicles W Cell-wall Plate 3.1
Root system of two-year old seedling of Podocarpus
falcatus showing heterorhizic pattern. (X£).
Plate 3.2
Top: Median longitudinal section of the root tip of P. falcatus (X 100). Bottom: Root apical initials forming a single saucer-shaped layer. (X 250).
Plate 3.3
Top: T. S. of long root of P. falcatus showing
diarch stele (X 100). Bottom: Cortical cells immediately outside the
endodermis showing 0 (Phi) thickenings (X 250).
Plate 3.L
Transverse sections of long roots of P. falcatus showing different stages during the secondary growth. Top left: Formation of two cambial strips between xylem and phloem (X 100).
Top right: Formation of a continuous ring of cambium giving rise to secondary xylem towards inside (X 100). Bottom: A root showing
considerable formation of secondary tissue
(X 100). Plate 3.5
Development of a lateral root in P, falcatus. Top
left: Initiation of meristematic divisions in the
pericyclic cells opposite protoxylem group (X 150).
Top right: Advanced stage in the formation of
lateral root primordium (X 150). Bottom left: and
right: More advanced stages. Notice the stretching
and break down of the parent root endodermis and
the conical appearance of the primordium. (X 100).
Plate 3.6
Top: L.S. of a lateral root showing the character
istic root apical meristem with open-ended
endodermis and a root cap (X 100). Bottom: Line
drawing of the above showing root apex.
Plate 3.7
Cleared roots of P. falcatus. Left: Nodules which
have arisen a short distance from the root apex of
a slow growing root (X 8). Right: Nodules devel
oping at a greater distance from the root apex
of a fast-growing root (X 8).
Plate 3.8
Photomicrographs showing the development of a
nodule in P. falcatus. Top left: Formation of a globular nodule primordium opposite the protoxylem
group. Mote break down of the parent root
endodermis (X 100). Top right: An undifferentiated
nodule primordium embedded in the cortex of the
parent root. Note the ruptured endodermis of the
parent root at the summit of the emerging nodule.
(X 100). Bottom left: Differentiation of an
endodermis, plerome and cortex in the unemerged
nodule primordium. (X 100). Bottom right: Deposit
ion of tannin in the endodermal cells of the nodule
prior to vascular differentiation. Note the
overarching endodermis. (X 100).
Plate 3.9
L.S. of nodules of P. falcatus showing the differ
entiation of nodular stele and its connection with
that of the parent root. Top: Complete differ
entiation of overarching endodermis before the
emergence of the nodule from the parent cortex (X100)
Bottom left: Differentiation of the xylem elements
in the plerome. (X 100). Bottom right: Connection
of nodule stele with that of the parent root through
differentiation of the intervening cells which are the derivaties of pericycle. (X 100). Plate 3.10 Top left: Surface veiw of a nodule of P. falcatus showing"root-hairs”. (X 8). Top right; Line
drawing of an unemerged nodule. Note the over arching endodermis and absence of root cap or root
apical meritem. Bottom: T.S. of a root through a fully matured nodule. (X 100).
Plate 8.11 Photomicrographs of longitudinal sections through short root apices of P. falcatus showing the pro
cesses of metacutization. Top left: Root apex just before metacutization. (X 100). Top right: Root apex showing the formation of metacutization layers in the root cap. (X 100). Bottom left: L.S. through a dormant root tip. Note the differentiation of endodermis and vascular tissue near the ai)ical meristem which is enclosed by the metacutization
layers. (x 100). Bottom right: Root apex showing the beginning of regeneration. Note the resumption
of activity in the meristem and the rupture of the
suberized root tip. (X 1o0). Plate 3.12 Top: T.S. of a long root through a lateral short
root which has regenerated once. (X 100). Bottom: Cleared root showing the headed appearance of the short root. Note the dark "brown suherized root
tip and dark collars against a lighter back ground at the junctions between the ’'beads',' each marking the position of the beginning of a growth cycle. (X 8).
Plate 3."13 L.S. of nodules of P. falcatus showing regeneration in various stages of advancements. Top left: Initiation of meristematic activity in the pericyclic cells at the tip of the vascular strand underneath the endodermis. (X 250). Top right: An advanced stage. Note the stretching of the endodermis of the parent nodule. (X 250). Bottom left: Differentiation of the new endodermis (X 250). Bottom right: A new nodule which has developed at the apex of an old one. (X 100).
Plate 5.1U Cleared root and nodules of P. falcatus showing regeneration of the nodules, once (top) and twice (■bottom). Note the dark collars against a lighter background at the junction of' successive
growths. (X 8). Plate 3.15
Longisections of nodules /Microstrohos
fitgeraldii, which have regenerated several times, Note the remnants of previous nodules still
attached (left and right), and initiation of meristematic activity at the tip of the vascular
strand and stretching of the endodermis (right). (X 100). Plate 3.16 Sections through nodules of P. falcatus grown under completely aseptic conditions. Top; Section of the outer cortex only. Notice the absence of any endophyte in the cortical cells. (X 250). Bottom: L.S. of a nodule showing a structure similar to that of nodule produced by plants growing in the field.
Note the regeneration of the nodule commencing at the tip of the vascular strand. (X 250).
Plate 3*17 Top; T.S. of a root through a nodule from a seed ling of P. falcatus growing in sterile culture.
Note the absence of any endophyte. (X 100). Electronmicrographs of sections of nodules from P. lawrencei
cuttings grown in fungus-free culture.
PLATE 3.1 g
Top: A young cortical cell. Note the cyto plasmic strands and peripheral cyoplasm rich in organelles. (X 6,000). Bottom: Portion of
a cortical cell showing the nucleus, perforated nuclear membrane and the connection between the
nuclear membrane and the endoplasmic reticulum. (X 10,000).
PLATE 3.19
Portion of a cell showing the perpheral cyto plasm and its inclusions. (X 12,000). PLATE 3.20 Electronnicrograph showing the double-membrane •h bounded plastids and miochondria. Note the
presence of starch grains and lamellae in the plastids and cristae in the mitochondria. (X 18,000).
PLATE 3.21 Enlargement showing the cell wall, middle lamella, plastids containing starch grains and lamellae, mitochondria with cristae, g'olgi bodies with their
vesicles and the endoplasmic reticulum. (X 24,000).
PLATE 3.22 Section of an older cortical cell showing the uneven deposition of cytoplasm around the
periphery and absence of cytoplasmic strands. Note the large central vacuole. (X 6,000).
PLATE 3.23
Enlargements showing the abundance of endoplasmic reticulum and golgi bodies, with their vesicles, next to the cell wall. Note the position of the endoplasmic reticulum parallel to the cell wall in
all three micrographs. Arrows indicate the
apparent incorporation of vesicles in the cell wall giving an eneven appearance to the latter
(X 24,000).
PLriTK 3.24
Electronmicrographs showing the development of
bar-like thickenings on the cell walls of cortical
cells. Top left: Deposition of thickenings
opposite to one another in adjacent cells. Top
centre: Well developed phi thickenings. Top
right and bottom: Successive stages in the
development of bar-like thickenings. (X 18,00). PLATE 3,25
Top: L. S. elongated nodule showing a
structure essentially the same as that
of a spherical nodule, in spite of its
superficial resemblance to a short root. (X 100).
Bottom: Cleared preparation showing an elongated nodule which has regenerated twice. (X 10). PL. 3.1
PL. 3.3 PL.3.4 PL. 3.5 PL.3.6 PL. 3 .7 PL.3.8
P L . 3.10 P L. 3.11
P L . 3.13
Ü J *
P L . 3.15
PL. 3.18 P L . 3.19
PL. 3.21 ri * 4 * PL.3.22 mm PL. 3 .2 3 .2 3 PL. PL. 3.24 56
CHAPTER IV
MORPHOLOGY AND ID E N TITY OF THE ENDOPHYTE
1 . IN T RODU CT I ON
2. DESCRIPTION OF THE ENDOPHYTE IN THE PODOCüRP
ROOT SYSTEM
3. ATTEMPTS TO ISOLATE THE ENDOPHYTE
4. EXTRACTION OF ENDOGONE - TYPE SPORES FROM
SOIL
5. INOCULATION TESTS
A . WITH RED CLOVER
B. VilTH Podocarous lawrencei
6. CONCLUSION 57
INTRODUCTION
As regards the identity of the endophyte of podocarp nodules by far the majority of authors have described or figured a fungus of the vesicular-arbuscular type (Von
Tubeuf, 1896, according to Nobbe and Hiltner, 1899;
Janse, 1897; Nobbe and Hiltner, 1899; Shibata, 1902;
Hiltner, 1903; Petri, 1903; Yeates, 1924; Saxton, 1930a,b; Kondo, 1931; Shaede, 1943; Ferreira dos Santos, 1947;
Bond, 1959, 1967; Baylis et al., 1963; Bergersen and Costin, 1964; Becking, 1965; Morrison and English, 1967).
There have been similar reports for the nodules of conifers other than members of /the Podocarpaceae (Janse, 1897; Yeates, 1924; Bieleski, 1959; Baylis et al., 1963; Morrison and English, 1967).
However there have been a number of claims for bacteria (Spratt, 1912; Bottomley, 1913; McLuckie, 1923; Phillips, 1932) but, in most cases where such claims were made fungal hyphae were seen as well in the outer layers (Spratt, 1912; McLuckie, 1923; Phillips, 1932). None of the most recent workers has detected any bacteria, although Uemura (1964) claims to have isolated actinomycetes from the nodules of Podocarpus macrophyllus and Sciadooitvs verticillata and, -ed. Morrison and English (1967) record/that, in addition to a 58 fungal symbiont an actinomycete - like endophyte was present in the cortical cells of the nodules of Aqathis australis. Shihata (1902) reported a Streptothrix - like organism inside the fungal cells in Podocarpus where they had formed pseudoparenchyma, and suggested that it might he responsible for this deformation of the fungus.
The^e can he no douht, however, that, at least under natural conditions, the nodules are mycorrhizal and contain an endophyte of the resicular - arbuscular type. The fungal nature of this type of endophyte was established beyond doubt by the electron - micrographs of Becking (1965) and, as noted by Janse (1897), Nobbe and Hiltner
(1899), Baylis et al., (1963) and Morrison and English (1967), both the roots and nodules are invaded. In short, nodulated conifers form a mycorrhizal association of the type widely recorded in angiosperms, gymnosperms, pteridophytes and bryophytes, the literature concerning which has been re viewed by Kelley (1950), Harley (1959, 1965), Baylis (1962), Mosse (1963) and Nicolson (1967). There has been considerable controversy regarding the identity of fungi causing vesicular - arbuscular mycorrhizas the main claims being for the genera Endogone. Rhizophagus 59 and Pythium. lluch of the controversy has resulted from the failure of most workers to isolate the endophyte in pure culture, but the majority of later workers have demonstrated associations with the zygomycete genus Endoqone. It has become clear that inoculation with Endoqone stoores extracted from soil will lead to mycorrhizal development, although Mosse (1962) appears to have been the only person to achieve this under aseptic conditions.
It is also clear that several species of Endoqone are involved and it may well be further substantiated that species of other genera can enter into vesicular - arbuscular associations. The name Rhizoohagus has been applied to fungi for which no spores have been seen and it may well be that the fungi concerned are really species of Endoqone and, perhaps, Pythium. Barrett (1947, 1953, 1961) reported having isolated pure cultures of Rhizoohagus from various plants and to have synthesized the mycorrhiza. All cultures were isolated from extra - radical mycelium except one, which was isolated from thick-walled hyphae and vesicles that had developed in dead roots on growing sweet-potato plants in sand. His studies indicated that many forms of
Rhizoohagus exist, perhaps even distinct species. The
results of cross-inoculation tests showed that host specificity
did not exist and that the type of vesicular - arbuscular 60 mycorrhiza formed was governed by the host plant and not due to a specific form of the endophyte.
G-erdemann (1955) and Mosse (1956,1962) obtained infections in several plant species using the one type of Bndo^one spores, a result also indicating no clearly defined host specificity. G-erdemann and Nicolson (1963) obtained infections with Several different types of Endogone spores and also observed no host specificity. In addition Gerdemann (1965), showed, like Barrett (1958, 19 6 1) for Rhizaphagus. that the appearance of the mycorrhiza was governed by the host plant. Identification of the fungal endophyte in Podocarpus nodules has been hampered by the same difficulties encountered by those studying vesicular - arbuscular endophytes in other plants. Prom the appearance of the vesicles and arbuscules, which they considered to be reproductive bodies, and because the hyphae were non- septate, Nobbe and Hiltner (1899), Shibata (1902) and
Schaede (1943) considered it to be a member of the Peronosporales. Shibata (1902) reported, however, that he obtained a reaction for chitin and stated that, according to C. van Wissellingh, this should not occur in this group of fungi. 61
Petri (1903) attempted to isolate the fungus, hut was unahle to obtain any growth from sections of surface- sterilized, young mycorrhizal nodules. Prom Torula-like spores in the older roots, however, he obtained a fungus, close to Thielaviopsis, which developed, in culture, structures similar to the "prosporoidi" he had seen in the nodules. This led him to suggest that, in the case of Podocarpus, this was the endophyte. Saxton (1930 a) isolated a fungus apparently identical with that seen in sections of the root-nodules but gives no description or identification, nor did he report any inoculation experiments. Phillips (1932) isolated a non-septate fungus similar to that seen in the outer layers of nodules but reports that it produced no spores in culture. Furman (19614) reported that, in a Koch’s postulate test with a fungus, for which/identity was given, nodular structures were formed on aseptic seedlings of
Podocarpus. Baylis et al.,(1963) and Becking (1965) observed restricted hyphal growth into culture media, but reported that the organism was unable to survive
separation from the host tissue, an experience similar 62 to that of other workers with vesicular - arbuscular endophytes (Neill, 1944? Nosse, 1959 a? Greenall,
1963). As regards the identity of the endophyte in nodule bearing conifers other than Podocarous spp. there are few reports. Young (1940), although making no mention of nodules, claims that, in both pure culture and box experiments with Araucaria cunninqhamii, seedlings developed endotrophic mycorrhizae when §rown in association with Boletus granulosus. Morrison and English (1967) reported nodules of Aqathis australis to be infected with a fungal symbiont of the type regarded by Mosse as Endoqone, although no isolation or inoculation attempts are reported. The present situation with regard to the identity of the fungal endophyte in Podocarous and other nodule bearing conifers, then, appears to be similar to that pertaining to vesicular - arbuscular mycorrhizae in general. There are, however, no reported observations
of hyphal connections with Endoqone spores nor any
recorded inoculation experiments using Endoqone spores.
2. DESCRIPTION OF THE ENDOPHYTE IN THE PODOCARP
ROOT SYSTEM 63
Since, as mentioned earlier, the fungal endophyte in Podocarous has been described repeatedly, little detail will be given here since the findings in this study confirm those of earlier workers* No bacteria were seen in any sections, even those stained specially with appropriate stains, but a non- septate fungus was regularly observed growing inter - and intra - cellularly in the cortex of the nodules. The hyphae were constricted where they passed through the host cell walls (Plate 4.1) and loose coils of hyphae, 3 - 5 j a , in diameter, were observed in the outermost layers (Plate 4.2). Arbuscules at various stages of development and degeneration were present in the cells of inner layers (Plate 4.2). The arbuscules consisted of dichotomously branched, intracellular hyphae, the distal branches of which appeared to disintegrate soon after they formed. Becking (1965) described the fine structure of arbuscules in
Podocarous naqi in what appears to be the only election microscope study, so far, of a vesicular - arbuscular mycorrhiza. He reported that the extremities of the hyphae forming an arbuscule had much thinner walls than normal hyphae, and that the final distorted and com pressed appearance of the vesicular structures 64 suggested that their cytoplasmic contents were resorbed by the host cytoplasm. Thick-walled vesicles (Plate 4.1 and 4.3), approximately 35 - 45 a in diameter, were mostly terminal or, very rarely, intercalary. These vesicles were usually abundant in tissues which had been infected for some time but usually rare in newly infected tissues.
In Dacrvdium frank1inii, vesicles were abundant in roots in which phellogen formation had commenced. They appeared to be still alive in old tissues which were sloughing off (Plate 4.3). As observed by various authors (Janse, 1897;
Nobbe and Hiltner, 1899; Baylis et al«> 1963)/ the fungus was present in the cortex of the roots as well as that of the nodules. This was found to be the case for all the podocarps examined in this study (See Chapter VII).
Shibata, (1902) noted that the vascular elements were never invaded and in this study no sign of infection was seen in either the vascular or meristematic tissues. The endophyte does not penetrate into or beyond the endodermis, which appears to form a "tannin barrier" similar to that noted by Clowes (1951), in the mycorrhizal roots of Faqus svlvatica. Hyphae were also observed on the surface of the 65 roots and nodules. ¿-.xtramatrical chlamydospores, of the type described by Gerdemann (1961), were present on hyphae attached to the roots and some of the hyphae were observed entering roots and nodules through root hairs (Plate 4.4).
No extension of fungal hyphae from the cortex of the parent root into developing nodules was seen. This is in accord with the observations of Baylis et al., (1963), who stated that the external hyphae appeared to be the source of infection for the original cortex of a newly formed nodule.
3»ATTbiuPTS TO ISOLATE THE ENDOPHYTE
Plating surface sterilized nodules onto malt-marmite agar, potato-dextrose agar and potato-carrot agar gave rise either to no cultures or to colonies of common soil fungi. No species of Pythium were obtained nor any other non-septate fungi.
A further attempt was made using Barrett's (1947, 1961) baiting technique, substituting white millet seeds for hemp. Small pieces of mycorrhizal nodulated roots of
Podocarpus falcatus were either washed with distilled water, immersed for two minutes in 0.2% mercuric chloride and rinsed in several changes of sterile distilled water, treated for five minutes with 10% sodium hypochlorite 66
and rinsed in several changes of sterile, distilled water,
or washed and shaken with sterile water and sterile sand. Pieces of boiled millet embryo were placed in contact with the treated root pieces in sterile water in sterile
petri-dishes. For each type of root treatment 25 dishes were set up and kept at 19-20°C . The water was removed every two days and replaced with fresh, sterile water, and
the cultures were maintained for four weeks. Various fungi developed on the embryo pieces including an aspetate mycelium. Attempts to obtain this in pure culture failed. While this aseptate mycelium developed quite well in mixed cultures, a similar aseptate mycelium, which grew apparently free from contaminants from some of the surface-sterilised root pieces, developed only poorly and could not be cultured further.. While it is not known whether the fungus developing
in these cultures was the endophyte, a stimulation of the growth of phycomycetous endophytes by the presence of other
microorganisms has been recorded by Neill (1944), Mosse (1959a), and Greenall (1963).
4.S/CTiU\CTI0N OF KNDQGQKL SPORES FROM SOIL
Soils from around Podocarpus elatus, P. lawrencei 67
and P. soinulosus, groining naturally in New South Wales, were examined for the presence of Endogone spores by the wet-sieving and decanting method of Gerdemann (1955). The soil suspension in each case was passed through a sieve with pores 0.7 mm. in diameter to remove the larger particles of organic debris and then through sieves with pore diameters of 140 and 65fls. The residues retained on these sieves were each transferred to beakers of water. After the heavier particles had settled, the water, along with the lighter particles, was poured through a Whatman No. 1 filter paper. The filter paper along with its contents was then spread in a shallow petri dish and examined with a stereo-- scopic microscope. Bndoqone - type spores were picked out individually with a sharpened matchstick fixed in a metal holder, since it was found easier to pick up the spores this way than with a metal needle. The spores seperated in this manner resembled the
Endoqone - type spores extracted in a similar fashion from
soils elswhere, but could not, with confidence, be placed
in any of the categories described by Gerdemann and
Nicolson (1963). The spores extracted were spherical to
elliptical, double walled, and 90-110 JU, in diameter (Plate
4.5). In colour they ranged from pale yellow to dark broim 63
and it is possible that more than one species may have been present. In size and general appearance these spores resembled the chlamydospores of Endoqone fasiculata described and figured by Gerdemann (1965) and also appeared very similar to the vesicles seen in degenerating roots and nodules of Podocarpus and in the decaying roots of various other plants. They were, in fact, very similar in size and appearance to the vesicles figured by Mosse (1959 b, PI. 27, fig 6) in decaying apple rootlets. Dowding (1959) reported that Endoctone grew out from root fragments into the soil and produced chlamydospores indistinguishable from the vesicles of the same fungus found in the root cortex. Amongst the spores examined, one or two were found which showed a bulbous swelling at the base similar to that exhibited by Gerdemann's (1955) B-spores. It has been suggested (Nie, olson, 1967) that these spores are zygospores, the swollen attachment being the larger suspensor of a heterogamous pair. No spores as large as those described by Gerdemann
(1955), Mosse (1956, 1959 a)?Dowding (1959) and Gerdemann and Nicolson (1963) were found, nor were any sporocarps recovered. 5» INOCUA,1TION TESTS A. WITH RED CLOVER: 69
Spores extracted from soil from around the roots of Podocarpus species were washed in several changes of sterile water and used to inoculate red clover seedlings using the method of Gerdemann (1955), Small funnels were made by pressing a sheet of aluminium foil around a glass funnel. A cotton - wool plug was placed in the end of each aluminium funnel and the neck was filled with washed, autoclaved sand. About 100 Endogone - type spores were then poured over the surface of the sand and the funnel filled with sand and planted with red clover seeds (Plate 4,6). A control series was also set up to which no spores were added. The funnels were placed in a glasshouse and watered from above with sterile water until the seeds germinated, after which each funnel was stood in an Erlenmeyer flask of water, the water being replenished as the need arose.. After each four - week period, 4 ml. of Knops solution was added to each funnel and after 85 days the plants were removed and examined. Plants from the inoculated funnels showed external hyphae, apparently arising from the spores used as inoculum, and inter
- and intracellular hyphae in the root cortex, with arbuscules in the cells of the inner cortex. No fungi could be detected in the roots of plants from the control series.
Thussalthough the spores used were not sterile, the 70 findings are similar to those of earlier workers (Gerdemann,
1955, 1961, 1964, 1965; Mosse, 1956; Gerdemann and Nicolson, 1963) and it seems probable that they were responsible for establishing the vesicular - arbuscular infection.
B. WITH Podocarous lawrencei:
Endophyte - free plants of P. lawrencei were produced from cuttings in sand and peat as described in Chapter II.
Approximately 100 Endogone - type spores were added to the soil around each rooted cutting and the roots were examined after four and eight weeks. After four weeks no endophyte could be seen but after eight weeks a typical vesicular - arbuscular infection was observed in both roots and nodules. No endophtic fungus was detected in a control series. Around the roots and nodules of the inoculated plants there was a tangle of hyphae (Plate 4.7). This external mycelium consisted of coarse yellow - brown hyphae with fine, colourless branches(Plate 4.&). The coarse hyphae were 10-15 in diameter, had thick walls and were non - septate. The colourless branches were 3-6 in diameter, thin-walled, and septate. This mycelium was similar to that observed abundantly on Podocarous roots growing under natural conditions and 71
arbuscular to the extra - martical mycelium of vesicular/endophytes described by various authors including Gerdemann (1955), i.osse (1959 b), Nicolson (1959) and Greenall (1963). apparently nndogone - type spores were seen/giving rise to
coarse hyphae (Plates 4.7 and 4.8) and these spores contained oil globules (Plate 4.8). Penetration did not occur at the root
tips and some of the hyphae were seen entering the roots and nodules through hairs (Plate 4.9). The cortex of young roots and nodules was invaded at very early stages in their development, sometimes before the full differentiation of the vascular tissue (Plate 4.9). Coils were formed in the outer cortical cells (Plate 4.10) and arbuscules were abundant in the cells of the inner layers (Plate 4.10). The arbuscules appeared to persist for only a short time and were most often seen in their degenerative phases, giving a granular appearance to the host - cell contents (Plate 4.10). The infection appeared to be confined to the younger cells, no hyphae being seen in the cortex of older parts of the root system. 6. cone fusion Since attempts to obtain the fungal endophyte in pure culture were unsuccessful its identity must remain in doubt. However, the presence of Sndogone - type spores in 72
the soil around the roots of Podocarpus species, together with the establishment of vesicular - arbuscular infections
in both red - clover and P.ilawrencei using these spores,
albeit unsterile, as inoculum, does indicate that this vesicular - arbuscular endophyte is similar to those
occuring in other plants in other parts of the world. It
seems likely that the spores extracted from soils are the
infective propagules and that the endophyte or endophytes are species of Endogone♦ Also, since inoculation with the ht Endogone - type spores broug/about infection in both red
clover and P. lawrencei, it seems likely that, in line with observations elsewhere, there is little or no host-specificity.
The possibility that species of other genera may also form vesicular - arbuscular association with Podocarpus. of
course, remains. EXPLANATION OF P U T E S KEY TO LETTERING OF PLATES
A Arbuscle
C Cortex
CH Extramatrical chlamydospore
E Endodermis
EM External mycelium
H Fungal hyphae
N Nodule
0 Oil globule
P Pericycle
R Root
RH Root hair
S Endopone-type spore
V Vesicles
X Vascular strand Plate 4.1
Top: Cleared root of P. falcatus showing
the root cortex occupied by fungal
hyphae and vesicles* (x250).
Bottom: Intracellular fungal hypha
showing a constriction where it
passes through the host cell
wall* (xl500).
Plate 4.2
Top: L.S. Nodule of P. falcatus from
the field. Note the infection restricted
to the cortex only* (xl50).
Bottom left: Outer cortical cells of
a nodule of P.falcatus showing
loose hyphal coils in the outermost
layer. (x900). Bottom right:
Inner cortical cell of a nodule of
P* falcatus showing an arbuscule.
(x2000). Plate 4.3
Top left: Cortical cells of a root of
P. falcatus showing inter-and
intracellular fungal hyphae bearing
a terminal vesicle. (xlOO).
Top right: Enlargement of a vesicle.
(x500). Bottom: Old tissue of
a nodule of Dacrydium franklinii
showing many vesicles. (xlOO).
P late 4.4
Top: Extramatrical chlamydospore
on a fungal hypha attached to the
roots of P. falcatus. (x250). Bottom:
Fungal hypha coiling around and
entering a root-hair on a nodule of
P. falcatus. (x250).
Plate 4.5
Double-walled, spherical to elliptical
Endogone-type spore extracted by
wet-seiving and decanting method
from the soil around Podocarpus growing
naturally in New South Wales. (x800) Plate 4.6
Twelve-week-old red-clover seedlings grown in aluminium foil funnels.
(A) Control and (B)inoculated with Endogone-type spores.
Plate 4.7
Top and Bottom! Roots of P . lawrencei
from cuttings planted in autoclaved sand and peat and inoculated
with Endopone-type spores. Note the
tangle of hyphae around root-system and spores giving rise to these hyphae. (xlOO).
Plate 4.8
Top: External mycelium produced by
Endogone-spores. Note the oil-globule emerging from the broken spore. (xl50). Bottom: Enlargement of the external mycelium showing coarse and fine
hyphae. Note the septa in the fine hypha. (x250). Plate 4.9 Top: L.S. of a root of P . lawrencei
showing fungal hyphae and vesicles. (xlOO). Bottom left: T.S.
of a young root of P . lawrencei
showing the cortical cells
already infected by the endophyte
while the vascular tissues are still differentiating. (xlOO). Bottom right: A hypha entering the root of P . lawrencei through a root hair.
(x650).
Plate 4.10 Cortical cells of nodules of P. lawrencei showing coiled hyphae (top), arbuscules in the process of degeneration (centre),
and degenerated arbuscules,
which give a granular appearance to the host cell
contents (bottom). (x650). PL. 4.1
PL.4.3
PL.4 .6 PL. 4.7 PL. 4 • 8
*
73
CHAPTER V
CYTOLOGIC.iL EFFECTS OF INVASION BY THE FUNGAL
ENDOPHYTE
1. INTRODUCTION
2. CYTOLOGICAL OBSERVATIONS
3. CONCLUSIONS 74
INTRODUCTION.
As mentioned in Chapter III, no obvious structural differences could be seen between invaded and endophyte -
free nodules and roots. Neither were there any colour
differences of the type recorded by Jones (1924) for
peas and Gerdemann (1961) for maize. It is obvious,
however, that the extensive inter - and intracellular
development of the fungus must have a profound effect on
the tissues concerned.
In Podocarpus Janse (1897) observed that in invaded
cells the nucleus and protoplasmic strands were intact
beside the "pelotes1' of the endophyte. Shibata (1902), however, reported that the penetrating fungus evoked a
striking reaction from the host cell, the cytoplasm
"increasing" and the nucleus enlarging, becoming irregular
and dividing amitotically. He recorded that there were as many as eight nuclei in a single invaded cell. He
described various other changes and was of the opinion
that the increase in nuclei was directly related to
fungal infection, as in uninfected nodules all such cells
exhibited only one nucleus. Shibata (1902) also stated
that the complete development of the cell wall and
production of thickening only occurred where there had 75 been successful fungal invasion. Spratt (1912) and McLuckie (1923) described a similar amitotic nuclear increase in cells invaded by
what they considered to be a bacterium. Schaede (1943), however, reported that, while the
nuclei of cells invaded by the fungus were somewhat enlarged, the plurinuclear condition was common to both
infected and fungus-free cells, being a sequal to mitosis
without subsequent cell division and apparently occurring prior to invasion. He also observed that fungal invasion did not affect the size of the root nodule cells and only slightly increased their cytoplasm contents. Mosse (1963) briefly reviewed literature concerning the sequence of events in cells of other plants in which arbuscules develop and reported that a much enlarged nucleus with prominant nucleolus was usually attached to the centre of the arbuscule. She gave a table showing increase in nuclear diameter in such cells in onion, strawberry and apple roots and quoted Lihnell (1939) as having observed a similar enlargement in arbuscule - containing cells of juniper. It is clear from the electron micrographs of Becking (1965) that, in the case of Podocarpus naqi at least, even the much branched extremitities of the arbuscules are clearly delimited 76
from the cytoplasm of the host cell by the cell membrane,
Mosse (1963) also referred to the literature describing similar nuclear changes which occur during the “digestion" of pelotons in orchids. No mention was made of increase in nuclear number in cells with arbuscules but, in the case of orchids, for which similar nuclear enlargement has been reported during the “digestion" of pelotons, she reported Burgeff (1909) as having found several nucleoli in the enlarged nucleus of Platanthera chlorantha, which sometimes became "fragmented", giving rise to several nuclei. Nuclear enlargement and increase in nuclear numbers have been associated by most authors with increased activity in the invaded cells resulting in the "digestion" or elimination of the invading hyphae. The reported happenings in Podocarpus, however, are somewhat at variance with one another and a brief examination was made in an attempt to clarify the position.
2. CYTQLOGICAL OBSERVATIONS.
During the examination of the characteristics of the fungal endophyte in the host, as described in Chapter IV, it was observed that the infected cortical cells of
Podocarpus fa1catus were usually multinucleate, cells with 77 up to five nuclei being common (Plate 5.1), their nuclei being characterised by the presence within them of irregular, localised, deeply-stained areas. No mitotic figures were observed and it is not known whether the increased number of nuclei resulted from fragmentation or some form of equational division. Examination of the cortical cells of uninvaded roots and nodules of P. falcátus,produced as described in Chapter II, showed that they contained either one or two nuclei (Plate 5.1). There were no obvious differences
between root and nodule cells. The nuclei of uninvaded cells were not obviously smaller than those of cells containing the endophyte though they were more regular in shape and absorbed stains more uniformly. The invaded cells, however, frequently had more than two nuclei and it would ap »ear that this was brought about by the presence of the endophyte.
Examination of non-mycorrhizal roots of P. lawrencei also showed that cortical cells of the roots and nodules frequently contained two nuclei.
Invasion by the endophytic fungus did not affect the size of the nodules or of their cortical cells. The cytoplasmic contents, of course, appeared denser, largely due to the plurinuclear condition and the presence of the 73
endophyte. No differences in the degenerative processes described so well by Spratt (1912) and McLuckie (1923) were observed between mycorrhizal and endophyte-free nodules. The cytoplasm and nuclei of the cortical cells eventually disappeared and pronounced thickenings were laid down on the walls, as described in Chapter III, giving them a striated appearance.
3. CONCLUSION. *
Cortical cells of both roots and nodules of P.falcatus and P.lawrencei frequently have two nuclei and invaded cells of P.falcatus have been observed to have up to five per cell. Thus it appears that, while many cells had more than one nucleus prior to invasion, the number often increased following it. Whether this was caused by fragmentation or some form of nuclear replication is
not known, and clarification of this happening must await a detailed cytological study.
These observations are only partly in accord with those of bchaede (1943), who reported that the nuclear increase in P. chinensis (P. macroohyllus) and P. nubiqenus
took place prior to invasion. He reported, however, that invasion by the endophytic fungus did not affect the size 79 of the nodules or their cortical cells and this situation appears to hold for the species examined here. Neither were any colour differences seen of the type recorded by Jones (1924) for peas and Gerdemann (1961) for maize. The laying down of the thickenings did not appear to be influenced by the presence of the endophyte and in this, for P. falcatus and P. lawrencei at least, the situation is different from that described by Shibata (1902) for P. chinensis (P. macrophyllus), and P. naqeia (P. naqi). EXPLANATION OF PLATE Plate 5.1
Top: Infected cortical cells of a
nodule of P. falcatus showing
multinucleate condition. A cell
with upto 5 distinct nuclei (n) can be seen. (X 250). Bottom: A cotical cell of a
non-mycorrhizal nodule of
£. falcatus showing two nuclei, (n ), (X 360). PL. 5.1 80
CHAPTER VI
FUNCTION OF NODULES
1. INTRODUCTION.
2. UPTAKE OF PHOSPHORUS BY NODULES OF PODOCARPUS LAWRENCEI.
3. GROWTH EXPERIMENTS WITH SEEDLINGS OF P. FALCATUS.
¿1. NITROGEN FIXATION TESTS WITH P. LAWRENCE I.
5. TESTS FOR THE PRESENCE OF HAEMOGLOBIN IN NODULES OF P. LAWRENCEI.
6. CONCLUSIONS. 81
1. INTRODUCTION
Since Podocarpus nodules are now known to be a normal feature of the root system and since both the nodules and roots are usually invaded by vesicular-arbuscular endophytes a review of the influence of vesicular-arbuscular mycorrhiza on plant growth seems appropriate. Such a task, however, has been rendered largely unnecessary by the review of Mosse (1963) and the recent paper of Nicolson (19&7), who has dealt with this vexed subject with considerable clarity.
As he pointed out, the endophytes have not been obtained in pure culture by the investigators of mycorrhizal function and experiments have not been conducted under aseptic conditions. Nevertheless, in spite of possible criticism, a considerable body of evidence has been amassed indicating that under certain conditions the mycorrhiza is responsible for considerable growth enhancement, this being most marked under low nutrient conditions and with high infection levels This view has been further substantiated by the work of Baylis (1967). Increased phosphorus uptake is thought to exert the greatest influence of this growth.enhancement and increased phosphorus uptake by mycorrhizal roots has been reported by 82 a number of workers, some of whom have used 32 P. There is also evidence that mycorrhizal roots showed a marked increase in uptake from poorly available sources such as bone meal and rock phosphate. Differences in the absorption of other ions has been recorded as well (Baylis,1967), although as yet there are apparently no recorded cases of growth stimulation as a result of this.
A review of the literature concerning the function of conifer nodules, however, reveals a somewhat more complex state of affairs than might be expected from the literature concerning vesicular-arbuscular mycorrhiza in other plants. This is perhaps because an interest has been taken in conifer nodules by two different groups of research workers whose interests have not otherwise tended to overlap, those whose primary interest is mycorrhiza on the one hand and those who have devoted their attentions to the nodules of legumes and non-leguminous angiosperms on the other,
Hooker (1854) regarded the special function of conifer nodules as obscure, but supposed that they were”subservient of the office of the selection of nutrifiient ", a statement which recent researches suggest may well be true.
Nobbe and Hiltner (1899) found the growth of nodulated 83
Podocarpus chinensis (P. macrophyllus) plants in nitrogen- f'ree sand culture to be as rapid as controls of the same age planted in humus-containing garden soil, whereas an un- nodulated alder had exhibited symtoms of nitrogen deficiency after three to four weeks of similar sand culture. They noted, too, that the fungus was more abundant in the plants growing in sand culture than in rich soil, and concluded that a true endotrophic mycorrhiza gave the plants the ability to use the free nitrogen of the atmosphere. Hiltner (1903) reported that the plants grew on until for a further two years when they died, perhaps due to drying out, having main tained themselves in a nitrogen-free medium for eight years. Because of this and experience with legumes, alder and Blaeagnus, he concluded that, although, because of their failure to obtain nodule-free plants for comparison and inoculation experiments, the fixation could not be said to be proved in the widest sense, it must have taken place. After examining the endotrophic mycorrhiza of
G-inkgo and other conifers, he felt justified in assuming that an analogous situation existed in these and stated that more than ever he v/as convinced that the real
significance of endotrophic mycorrhizae lay in their ability to fix nitrogen. Shibata (1902) regarded Podocarpus nodules as specially arranged organs for the growth and consumption of fungi, demonstrating the presence of a proteolytic enzyme in the host cells, as did Petri (1903). Shibata (1902) decided that there was an analogy between the
"digestion" of the endophyte and the absorption of materials from insects by insectivorous plants. He did point out, however, that the slight connection between the fungal hyphae and the exterior gave scarcely any basis for assuming the uptake of significant nutriment.
Hiltner (1903) suggested that, while "digestion" of the respective endophytes found in nodules of Podocarpus. legumes, Alnus and Elaeaqnus undoubtedly took place, it was not significant in the nutrition of the plants. He believed that a balance was set up in which the endophytes protected themselves from digestion by fixing nitrogen and giving off a protein-rich material which was absorbed by the host. He considered the endophytes to be parasites which would damage the plants if they did not assimilate them or subject them to their service.
Yeates (1924), however, suggested, once more, that
*> the fungal endophyte of nodulated conifers was“digested and that thus organic matter was transferred to the host, though he gave no evidence for this. Schaede (1943), 85 however, "believed that, since the endophytic mycelium was in only very slight contact with the soil, there was no question of its activity as a channel of nutrient supply to the host. He regarded it as an innocuous parasite, the absorption of which by the plant was a form of defence mechanism.
Several authors (Spratt, 1912; McLuckie, 1923; Yeates, 1921+) reported that the older cortical cells of the nodules functioned in water storage. While this may be so, there is no evidence that they differ in this regard from the cortical cells of ordinary roots.
A number of authors (Spratt, 1912; Bottomley, 1913; McLuckie, 1923; Phillips, 1932) believed the endophyte to be a nitrogen-fixing bacterium, apparently functioning as in legume nodules. More recent workers, however, have not been able to substantiate this. Nevertheless the subject of nitrogen-fixation in conifer nodules has continued to claim the attention of investigators who considered, or consider, them to be mycorrhizal. For instance, Kondo (1931) made comparative studies of the nitrogen status of young Podocarpus nodules with abundant fungal endophyte, old nodules with little endophyte, and young roots. Although on the basis of dry v.eight, the nodules contained more nitrogen 86 than the old ones or the young roots, he pointed out that no conclusions could be drawn. Growth experiments similar to that of Nobbe and Hiltner (1899) were carried out by Bond (1959, 1967) and Becking (1965). Becking found that nodulated plants of three Podocamus species placed in nitrogen- free water-cultures, inoculated with crushed nodules of P. rospiqliosii and with fresh nodule material of p. nerifolius and P. blumei obtained from New Guinea, exhibited nitrogen-deficiency compared to plants supplied with nitrogen. The plants, however, were still alive after periods ranging from 2 - 4 years, whereas uninoculated alder plants died in a few months. Thus, like Nobbe and Hiltner (1899), he concluded that some nitrogen fixation probably occurred. The growth experiments of Nobbe and Hiltner (1899) and Becking (1965), have been critically discussed by Bond (1967), who repeated, as nearly as possible, Nobbe and Hiltner's
(1899) experiment and observed that over a period of 18 months, the plants, in which a fungal endophyte was observed to be present in a proportion of the nodules, continued to grow but the nitrogen level fell and they showed increasing nitrogen deficiency. He thus attributed the new growth largely or wholly to the nitrogen in the plants 87
initially. Since, in the case of Nobbe and Hiltner’s (1899) and Backing’s (1965) plants it is not known whether there was any increase in total nitrogen over the periods of their experiment, it seems wisest, as pointed out "by Bond (1967), to treat their claims with some reserve.
In 15jr tests with nodulated roots of seven species of Podocarpus, Bond (1959> 1967) obtained no evidence of fixation, though it was known that some nodules were
infected. A negative result was also obtained by Furman ( 1962+ ) with nodules of P. rospigliosii. The paper is reported only in an abstract wich gives no details. He reported a fungal endophyte and suggested that the mycorrhiza might be important in forestry practice, though not as a source of nitrogen. Bond (1967) pointed out that his tests were less sensitive than those of others who have subsequently recorded fixation of 15^* Bargersen and Costin (1962+) recorded fixation at a low rate in nodules of r. lawrencei, suggesting that this was probably due to the small proportion of nodules containing well-developed mycelium. Becking (1965) recorded a fixation, in rospiqliosii of 2+.2+ /ig nitrogen per gram nodule fresh weight in 22+ hours, compared with about 0.25 ,;wg in 4 hours by Bergersen and Costin (1964) for P .lawrencei. No enrichment was recorded by Becking in the roots themselves during the 24 hour period.
Morrison and English (1967) stated that they were fairly sure that the presence of the mycorrhizal fungus did not account for 4-5^ fixation they obtained in nodules of
Agathis australis. since it occured in nodules of both mycorrhizal and non-mycorrhizal seedlings. Although the latter plants were not endophyte-free, being described as having not more than 10$ of the root and nodule cells infected with the typical endophyte, compared with at least 50$ in the mycorrhizal plants, they suggested that an actinomycete-like organism, which they observed to be present also, could have been responsible. They statdd that, while the low enrichment (0.011 , 0.014 and 0.016 atoms $) obtained in three of the samples could have been due to some transient member of the rhizosphere, it would not have accounted for an enrichment of 0.04$ atoms % in
one sample nor the 2.6 atoms $ obtained with nodules from
P. alpinus (P. lawrencei).
In all the experiment with 15N there have.t then, been
no comparisons with completely endophyte-free nodules, with mycorrhizal and non-mycorrhizal short roots, 89
with sterile roots and nodules, or with mycorrhizal and non mycorrhizal roots of other plants. Hence there is no evidence that the fixations recorded are the result of any special property of the conifer nodules or that they are in any v/ay related to the presence or absence of the fungal endophyte. Nor is there any evidence that such fixations could he of benefit to the plants concerned. The results of Becking (1965) indicate little or no transfer from the nodules to the root during a 2ij.-hour period and the growth experiments of Nobbe and Hiltner (1899), Bond (1959, 1967) and Becking (1965) have provided no evidence that nodulated
Podocarpus plants are partially or wholly independent of external sources of combined nitrogen.
On the other hand, Baylis et al. (1963) in experiment with P. totara and P. dacrydioides, carried out along the lines of earlier experiments with G-riselinia (Baylis, 1959), showed that mycorrhizal seedlings developed very much better than non-mycorrhizal in a soil highly deficient in phosphorus and calcium, but which had been found to contain sufficient nitrogen for the growth of other plant species. They concluded that podocarps are dependant in poor soils upon the activities of a mycorrhizal fungus or fungi. Further evidence along these lines has been provided by Baylis
(1967) for Podocarpus totara and other plants. 90
Morrison and English (1967), using 32p, demonstrated a greater phosphorus uptake by mycorrhizal nodules of ^thia • .qnkqtralis than by non-mycorrhizal and a similar, but less striking, effect with the non-nodulated short roots. Their results are in line with those obtained using vesicular-arbuscular mycorrhizae of other plants if
(Nicolson, 1967). Work with ectotrophic mycorrhizae indicates that, in beech (Harley, 1959) and pine (Morrison, 1954,1962), they show similar enhanced absorption of phosphate compared with non-mycorrhizal roots. In the case of
Pinus there is some evidence,reviewed by 3ond(l967), that nitrogen fixation occurs. Ag with _ ibdo carpus. however, further work with 15^ and using adequate controls is necessary to clarify the position. The above review indicates that there is surprisingly little reliable evidence concerning the differences in behavior of the roots and nodules of nodule-bearing conifers when free from and invaded by vesicular-arbuscular and perhaps other endophytes. Hence the following studies were undertaken in an attempt to add a little to our knowledge. 2. UPTAKE OF PHOSPHORUS BY EXCISED NODULES OF
P0D0CARPUS LAWRENCEI 91
The nodules used in this experiment were obtained from endophyte-free roots from cuttings grown as described by Khan, 1868 b, (Appendix IV.3) and from mycorrhizal roots produced by cuttings grown in soil collected from the native habitat of the species.
The root samples were harvested immediately prior to the experiment, washed clean, and blotted dry.
Short segments with attached nodules were placed in
25 ml conical flasks, each containing 10 ml of 0.3mM 32 Na P0 (pH 5.0) with sufficient H PO to give an 2 k 3 k activity of 1 & Ci/ml. The solution containing the root-material was shaken in a water bath at 25°G. After uptake periods ranging from 5-120 minutes, the roots were washed in rapidly running water, for few minutes to remove surface radio-activity and dried in an oven at 80°C. for 2k hours. The nodules were detached from the dried roots by shaking them with a pair of fine-forceps and weigh ed. Samples of dried nodules were then placed on planchets and their activity estimated on a GeiSer - Muller type counter (EKCO Electronics, Type N620A) connected to a scaler
(EKCO Electronics, Type N529C). For each treatment counts were made with five 2 mg samples. The results recorded are thus the mean of FIGURE 6.1
Top: Specific activity of mycorrhizal and non-inycorrhizal nodules of
Podocarpus lawrencei immersed
in Na H2^2P O4 solution at 25^C. Bottom: Rate of specific activity of mycorrhizal and non-mycorrhizal
nodules.
92
five counts and are expressed as counts /100 sec/ mg dry nodules. The results are recorded in Pig. 6.1 and indicate a more rapid uptake "by the mycorrhizal nodules than the non-mycorrhizal and that adsorption was more rapid during the first 10 minutes of the exposure than during the rest of the period.
3. GROWTH EXPERIMENTS .7ITH SEEDLINGS OF P. FALCx/PUS. Seedlings were produced as described in Chapter II,
out instead of transferring germinated seeds to a mineral agar medium in tubes, seeds germinated in May, 196?, were transferred to pots of autoclaved, washed sand, four seedlings per pot, each pot being enclosed in a new polythene bag to aviod contamination. The pots were then divided into two series and to each of the pots
in one of these 100 Endogone-type spores, obtained as described in Chapter IV, were added. Each series was
then divided into five and subjected to the following
nutrient regimes: (i) low phosphorus, (ii) high phosphorus, (iii) low phosphorus + combined nitrogen, (iv) high phosphorus + combined nitrogen, (v) control. 93
Sterile nutrient solutions (Appendix III. ¿4.) were added aseptically at the rate of luO ml/pot at the corn lencement of the experiment and thereafter after every four weeks. Sterile distilled water was used for the control. Three replicates (3 pots, i.e. 12 seedlings) were set up for each treatment.
A third series was set up separately in which 100 ml. of fungus-free filtrate, from the Podocarpus soil from which the Endogone-spores were extracted (Appendix V), was added to each pot. Owing to a shortage of seedlings, however, there were only enough plants for two treatments, low phosphorus + combined nitrogen and high phosphorus + combined nitrogen. All pots were kept in a glasshouse and harvested in
July, lyb8 , when the seedlings were 13 months old and abundantly nodulated. The root-system were washed made clean and an examination./ for mycorrhizal infection.
Only those plants which had been inoculated with wndogone- type spores contained a vesicular-arbuscular endophyte and this was present conspicuously in about 5 0 /o of the nodules examined from all treatments, although infection appeared less dense in seedlings grown at the higher levels of fertility.
The seedlings were then dried in an oven at 100°q FIGURE 6.2 Mean dry weights and percentage
compositions of mycorrhizal
and non-mycorrhizal Qseedlings
of Podocarpus falcatus supplied
with different levels of nutrients.
CON.= controls LP= low phosphosrus;
HP= high phosphorus: LPN= low phosphorus
+ combined nitrogen: HPN= high phosphorus* combined nitrogen.
RESILTS OF SEEDLING GROfcTH EXPER 1.RENTS
TABLE 6. I
TREATMENT MM ORRIIIZAL NON MMORRIIIZAL
Dr \ we i y h 1 /s~eed 1 i ng t CAI» 0 .2 0 2 0 .1 5 7 • P < .O I »
C o n f r o 1 % Phosphorus 0 .7 9 0 . 6 9
% \ i t r o g e n 3 .4 4 3 .7 1
Dr\ w eighl/seedl i ng tG/MI 0 . IHM 0 . 170 • P< .O I » Low % Pnosphorus 0.76 0 .7 1 P h o s p h o ru s % N i t r o g e n 3 . 3 0 3 .H 2
Dr\ weighl/seedliny MAO O. 182 0 .1 7 9 « P > . 1 »
H ig h % Phosphorus 0 .7 7 0 .7 4 P h o s p h o ru s % N i 1r o g e n 3 .9 1 4 .0 2
Dr\ weighl/seedling MAO O. 177 0.187 i p * . 0 5 t Low % Phosphorus 0 .7 1 0 .7 7 Phosphorus f N i t r o g e n 3 . HO 4 .5 5 Combined Nitrogen %
Dr> w eighl/seedl i ng (CAO O . 176 0 .2 0 4 iP ^ .H S l H ig h % Phosphorus 0 .6 6 O.HI Phosphorus + Combined Nitrogen / Ni 1rogen 3 .^ 5 5 .2 5
TABLE 6.2
TREATMENT ) FILTRATE Non FILTRATE
Dr» weight /seedling MAO 0 .2 4 6 0 . 19B U w t P < . 0 S t Phosphorus + /« Phosphorus 0 .9 1 4 0 .6 B 9 Combined Nitrogen % N i t r o g e n 3.74 5 . 6 0
Dr> weighl/seedli ng IGMt H igh • P < .0 f» t 0 .3 6 2 0 .2 2 2 Phosphorus + P h o s p h o ru s 1 .1 5 0 0 .7 9 0 Combined Nitrogen N i l r o g e n 3 .5 4 0 .10 3k
for ¡48 hours and their dry weights determined. Also the percentage of nitrogen and phosphorus in hulked samples of
all seedlings from each treatment was assesed hy the methods described in Appendix VI. All these results are recorded
in table 6.1 and 6.2 and in figure 6.2 and 6.3. As can be seen in table 6.1 and figure 6.2, the mycorrhizal seedlings grew better and had a higher % phosphorus and lower % nitrogen when grown under a low nutrient regime (control, low P). The lower % nitrogen presumably resulted from the greater growth of the mycorrhizal seedlings and, by comparing dry weights and nitrogen contents, it can be seen that there is no evidence
of increased total nitrogen in the mycorrhizal series. This result is similar to that of Baylis (196?) for Podocarpus totara and other plants. In the high phosphorus treatment there was no obvious difference between the mycorrhizal and non-mycorrhizal
seedlings but, when combined nitrogen was added in addition
to phosphorus (low P+N, high P+N), the non-mycorrhizal better seedlings grew/than the mycorrhizal although they had a lower % phosphorous and a higher % nitrogen. In the seperate experiment in which fungus-free soil FIGURE 6.3 Mean dry weight and -percentage
composition of Podocarous falcatus
seedlings grown with If and without
the addition of soil filtrate. LPN= low phosphorus + combined nitrogen; HPN= high phosphorus + combined nitrogen. LPN HPN DRY WT.(gm)
8.0 95 filtrate was added, with the intention of' introducing microorganisms other than fungi, a similar effect occurred as was observed with the mycorrhizal seedlings at low nutrient levels. The plants to which the filtrate was added grew better and had a higher % phosphorus and lower % nitrogen (table 6.2, fig 6.5 )• Thus it cannot be assumed that the increased growth and phosphate absorption observed in the earlier experiment resulted from the activities of the mycorrhizal fungus, since the spores used for inoculation were not sterile and came from the same soil as t&e one from which the filtrate was prepared,
4. NITROGEN - FIXATION TEST ,/ITIl P. LA WHENCE I. Nitrogen - fixation tests were carried out with the following samples obtained from plants grown in a glass house. 1) Fungus - free, nodulated roots produced by cuttings s planted two years previously according to the method of
Khan (1968 b; Appendix IV. 3)« 2) Nodulated roots produced as in (1), but inoculated after six months with Endogone - type spores extracted from the soil of its native habitat as described in Chapter IV.
About half the nodules examined at the time of the test contained the endophyte. 96
3) Nodulated roots produced from cuttings as in (1) but planted in soil from the native habitat of P. lawrencei.
The fungal endophyte was present to approximately the same extent as in (2)•
The root samples were removed from the pots immediately 15 prior to the experiment and exposed to as described by Bergersen and Costin (1964). The roots were washed clean and blotted dry. Short segments bearing nodules were excised and placed in 25 ml conical flasks which were evacuated and flushed three times with argon before being filled with the gas mixture. The flasks were then incubated in a water bath at 25°C for four hours. The soluble non-protein nitrogen of each sample was extracted by grinding and digesting in 3N HCl and leaving
the mixture overnight at 4°C. The precipitated protein was removed by centrifugation at 22,000 x g and the supernatent treated by the Kjeldahl method. Duplicate
samples of Kjeldahl distillates, each containing 15 approximately 0.1 mg.N# was analysed for N using an
Atlas M £6 mass spectrometer. The results are given in
table 6.3. 97
TABLE 6.3
Fixation of nitrogen By detached roots of
P. lawrencei
Atoms 1^N2 Fresh Non- excess % Fixed Root Sample Weight Protein 15N m (fl#g) N. (mg) Fraction
1 Non-mycorrhizal 2.79 0.220 0.008 17.6
2 Mycorrhizal 1.31+ 0.200 0.016 32.0
3 Mycorrhizal 1.68 0.2 83 0.005 iu.o
o 1R (Roots incubated 1+ hr at 25 C, JN 2 values corrected to read as if gas phase contained 100 atoms % N; gas phase 1 R (total pressure 700 mm Hg):-0. 11+ atm. JN 2 (73 atoms %)
0.19 atm. 0^. 0.67 atm. A). 98
As can be seen there was an extremely low and doubt fully significant enrichment in the P. lawrencei roots whethef they were mycorrhizal or not. As with all negative results little can be concluded until further experiments are carried out.
The root and nodule surfaces were not sterile and, although no other microorganisms were recognised in any of the material examined, no special search was made and the possibility of their presence cannot be ruled out.
The results obtained, however, provide no evidence that the vesicular-arbuscular endophyte or endophytes are involved in nitrogen fixation.
5. TESTS FOR THE PRESENCE OF HAEMOGLOBIN IN
NODULES OF P_. lawrence i.
Although the haemoglobin in legume nodules has been claimed to be involved in their fixation of nitrogen (Keilin and Smith, 1947), it has now been established (Bergersen and Turner, 1967) that the bacterioids are the Nevertheless haemoglobin is present active componants and the haemoglobin is not involved/in effective legume nodules and it was decided that it might be of interest to examine both mycorrhizal and endophyte-free nodules of P. lawrencei for its presence,
The presence of haemin has been recorded in the nodules 99
and root-cortex o f P . neriifolius “by E g le and M ining
(195'l)> using a spectroscope.
The method described hy Davenport (i960) was followed but instead of the spectroscope, which he used, a Schimadzu spectrophotometer was used.
Nodulated roots were removed from the pots immediately prior to the experiments and were packed into crushed-ice after washing. The tests were initiated within 30 minutes of taking the samples.
The nodulated roots were macerated in a mortar with 0 . 2M t r i s (2-amino-2-hydroxymethylpropane-1 *.3 diol) buffer, pH 7 .hj containing 0 . 02M sodium ethylenediamine tetra-acetate (EDTA). Portion of the macerated nodules was centrifuged at 7700 x g for 20 minutes to remove large particles. Both the crude macerated nodules and the relatively clear supernatant fraction from the centrifuge tubes were examined fo r the presence of haemoglobin. Reduction (exclusion of dissolved oxygen) was carried out by adding sufficient sodium dithionite
to the tris-buffer medium. Oxidation of the preparations was carried out with potassium ferricyanide.
The samples, reduced or oxidized, were placed in
1 cm cuvettes and the difference spectra in the visible
light region were determin^-u^ing a Shimadzu recording
CO V" 3D | GOj r4RY 100
spectrophotometer (Model MPS 50L).
Intact-nodules, placed in 2 - 4- mm wedges of the spectrophotometer, were also tested for the presence of haemoglobin by direct spectrophotometric examination of dithionite reduced samples.
All the tests with macerated or intact nodules gave negative results. No absorption peaks were obtained at 544, 572 or 562 mq,, wavelengths at which absorption peaks would have been expected had haemoglobin been present (Davenport, i960).
6. CONCLUCIONS.
Unfortunately it was not found possible to produce sterile nodulated roots in sufficient quantities to examine their uptake of phosphorus and nitrogen, nor was it possible to produce otherwise sterile two membered cultures of Podocarpus and endophyte with which to compare them. Hence the results obtained here are subject to the same criticism that can be levelled at most of the previous work with vesicular-arbuscular mycorrhizae.
Although the mycorrhizal and non-mycorrhizal nodules of P. lavvrencei were not grown under identical conditions^ the mycorrhizal nodules showed a more rapid and greater 101
absorption of 32. than the non-myeorrhizal, a result similar to that obtained by Morrisom and English (1967) with nodules and roots of Agathis australis. Similar results have been reported by others who have compared roots of Ooher plants with ana without vesicular- arbuscular enuophytes (Micolson, 1967).
In the growth experiments with P. falcatus„the infection was less dense in the inoculated seedlings grown at the higher nutrient levels and this is in line with the observations of Baylis (1967) for
Coprosma robusta and Nicolson (1967) for tobacco and tomato. Also, the better growth of the mycorrhizal seedlings when grown under low nutrient regimes is a result similar to that reported by earlier workers
(Baylis et al?> 19¿3 ; Baylis, 1967), and the poorer growth with a high level of nutrients is similar to the result of Baylis (1967) with Coprosma robusta at the highest level of xjhosphorus used. However, the results obtained during the present study cannot be interpreted as they have been by earlier workers’ such as G-erdemann
(1964), since the addition of a fungus-free soil filtrate to plants grown at the higher nutrient levels produced
a similar effect to inoculation of plants grown at the lower nutrient levels v/ith Endo.gone-1ype spores. 102
It is known that phosphate absorption can he markedly
influenced by microorganisms external to roots (Nicholas,
1965; Barber, 1966; Bowen and Rovira, 1966) and it is clear that a fuller and more carefully designed experiment
is needed before any reliable conclusions can be drawn.
Nevertheless the recording of lower leveles of nitrogen in mycorrhizal as compared with non-mycorrhizal plants grown under nutrient regimes without added nitrogen suggests that, if nitrogen fixation had been taking place in the nodules, such a fixation was not associated with the presence of the mycorrhizal endophyte. The result of
Baylis (1967) for Podocarpus totara, Griselinia littoralis.
Coprosma robusta, Pittossorum tenuifolium and Myrsine australis could be interpreted similarly and indicate that a nodulated conifer behaved no differently from several angiosperms which do not bear nodules.
Likewise the result of the 15^ fixation tests give no cause for assuming that significant nitrogen fixation had taken place. Perhaps under different conditions, or with a different endophyte or endophytes, the results of such an experiment may have been more in line with those of other workers who have recorded significant fixation in conifer nodules. The fixations obtained here, however, although extremely small, are at least similar to the result of Morrison and English (1967) in that they suggest 103 that the vesicular - arbuscualr endophyte is not involved. As mentioned earlier, they suggested that an actinomycete - like organism, which they observed to he present also, could have been responsible for the fixation, No such organism was seen in the roots of P. lawrencei used here, but no exhaustive research was made and no doubt its presence, at least in an amount such as might be responsible for the minute fixations recorded, could have passed unnoticed. Somewhat surprisingly, no record has been found in the literature of 13^ fixation test with vesicular - arbuscular mycorrhizai from plants other than nodulated conifers. The unpublished results of Dandie (1966), however, show no fixation for mycorrhizai and non - mycorrhizai roots of ryegrass and tomato tested at the same time and under the same conditions as the Podouarpus roots examined in this
Study. The spectrophotometer examination of both mycorrhizai and non - mycorrhizai nodules of P. lawrencei did not
reveal the presence of haemoglobin. Should it be present
in amounts too small to be detected by the method used, it seems extremely unlikely that such minute amounts could be of any physiological significance.
The studies undertaken here, have unfortunately done 104 little to improve the knowledge concerning the function of mycorrhizal roots and nodules of conifers. They do, however, support the conclusions of earlier workers in that phosphate absorption is enhanced by the presence of the vesicular - arbuscular endophyte. The results obtained however, indicate that the vesicular - arbuscular endophyte present was not involved in nitrogen fixation. 105
CHAPTER VII
OCCURRENCE OF NODULES IN THE GINKGOALES,
TAXALES, AND CONIFERALES 106
Records of the occurrence of root-nodules amongst the conifers have been tabulated by Allen and Allen (1965) and a slightly fuller listing is given in table 7.1, Owing to difficulties with synonymy, it sometimes appears that an author has recorded one species under two different names.
Very probably different species were observed but, with only the names and no specimens to go by, it is impossible to be certain. Apart from the records for Podocarpus spp. c by numerous authors, for Dacrydium frank 1 inii, Microfachrys tetragona, Phyllocladus trichoma no ides and Saxegothaea conspicua by Spratt (1912), for Pherosphaera hookeriana
(Microstrobos nipho^hilus) and P. f itzgeraldii (M. fitzger- aldii) by Saxton (1930 a,b), and for species of Agathis,
Dacrydium, and Phyllocladus by Baylis et al. (1963), there is no evidence reported to indicate that the structures reported as nodules differ from short roots.
Sahni (1920) reported that the roots of Acmopyle pancheri bore tubercles but gave no detail of their structure, and his only illustration was of a longitudinal section of what he claimed to be a tubercle regenerating and becoming a root. This illustration shows no evidence of the structure peculiar to the nodules of other genera of the
Podocarpaceae.
Allen and Allen (1965) could find no nodules on TABLE 7.1 RECORDS OF THE PRESENCE OF NODULES AMONGST THE C0NIFERALE3
FAMILY, GENUS & SPECIES LITERATURE CITATIONS
ARAUC ARIAC EAE Agathis australis Cockayne, 1921; Yeates, 1924; Bieleski, 1959; Baylis et al., 1963; Morrison & English, 1967. A. roliusta Janse, 1897, (as Dammara robusta). X. vitiensi3 Allen & Allen, 1965. Araucaria spp. Hooker, 18 54. X. angustifolia Daugherty, 1963. X. heterophylla Janse, 1897; Yeates, 1924 (both as A. excelsa).
CUPPRESSACEAE Cupressu3 sp. Hooker, 1854. C . sempervirens Janse, 1897 (as C. fastigiata). Libocedru3 bidwillii Yeates, 1924. Sabina chinensis Janse, 1897 (as Junioerus chinensis) Thuja sp. (Plat.ycladus?) Hooker, 18 54.
PODOCARPACEAE Acmopyle pancheri Sahni (1920) Dacrydiuia sp. Hooker (1854) D. bidwillii Yeates, ex Allen & Allen, 1965. D. bifonne Yeates, 1924; Baylis et al., 1963. 5« colensoi Yeates, 1924. D. cupres3inum Yeates, ex Allen & Allen, 1965; Baylis et al., 1963. D. intermedium Yeates, ex Allen & Allen, 1965; Baylis et al., 1963. D. franklinii Spratt, 1912. D. kirkii Yeates, ex Allen & Allen, 1965. D. laxifoliiun Yeates, ex Allen & Allen, 1965. Microcachrys tetragona Spratt, 1912
Microstrobos fitzgeraldii Saxtcn, 1930 a,b (as P'herosphaera fitzgeraldii) M. niphophilus Saxton, 1930 a,b (as Pherosphaera hookeriana)
Phyllocladus sp. Hooker, 18 54• P. alpinu9 Baylis et al., 1963. P. glaucus Yeates, ex Allen & Allen, 1965 P. trichomanoides Spratt, 1912.
Podocarpus 9pp. Hooker, 1854; von Tubeuf, 1896 according to Nobbe & Hiltner, 1 Bond, 1959. P. acutifoliu3 Yeates, ex Allen & Allen, 1965. P. blumei Becking, 1965. P. dacrydioides Hooker, 1854 ; Petri, 1903; Yeates, 1924; Baylis et al., 1963. P . elatus Petri, 1903; McLuckie, 1923. elongatus Petri, 1903; Spratt, 1912; Phillips, 1932, (both as P. elongatus and P. thunbergii var. angustirolla) P. falcatus Phillips, 19*32. T. ferrugineus Yeates, 1924; Baylis et al., 1963. P. gracilior Parker, 1932. P. hallii Yeates, 1924; Baylis et al., 1963. T, henkelii Phillips. 1932. E* i^ricatua Janse, 1897 (as P. cuppressinus) F. latifolius Saxton, 1930 (as~P.~~tnunbergii ) : Phillips, 1932; B o n d ‘d 19 6 7 7 P. raacrophyllus Nobbe & Hiltner, 1899; Shibata, 1902; Petri, 1903 ; Schaede, 1943 (all a3 P. chinensis) Petri, 1903» P. macrophyllus var. maki Becking, 19&5. T. nagi von Tubeuf, 1896, according to Nobbe & Hiltner, 1899; Shibata, 1902 (both as P. nageia); Becking, 1965. P. neriifolius van Tieghem, 1870, according to Becking, 19o 5; von Tubeuf, 1896, according to Shibata, 1902; Egle & Munding, 1951; Becking, 1965. P. nivalis Yeates, ex Allen & Allen, 1965; Bond, 1967 P. nubigenus Schaede, 1943. F. 11 prostralo" Yeates, ex Allen & Allen, 1965. (no such name known) P. roapiglioaii Furman, 1964; Becking, 1965. F. aaligiU3 Spratt, 1912; Bottonley, 1913» (Both as P. chilina). P. SpjCatUS Yeatea, 1*924; Faylis et al., 1963. F. apinulosus MoLuckie, 1923. F. “botara Spratt, 1912; Yeatea, 1924; Baylis et al., 1963. P. "variegatus" Ferreira do8 Santos, 1947. "(a oultivar?) P. wallichianus Petri, 1903 (as P. latifolia Wall.)
Saxegothaea conspicua Spratt, 1912.
TAXODIACEAE Cunninghamia ap. Hooker, 1854•
Taxodium sp. Hooker, 1854• According to Hooker the elder de Candolle had earlier noted exostoses on T. diBtichum (Théorie Elémentaire, î a .T ,- pT 156).
SCIADOPITYACEAE Sciadopitvs verticillata Uemura, 1964 107
G-inkgo biloba t though they wrongly attributed Hiltner (1903) as having remarked that "this species bore nodules analogous to those on Podocarpus spp. They also wrongly report Yeates (1924) as having recorded them on Araucaria cunninghamii.
Usmura (1964) appears to be the only person to have reported nodules on Sciadopitys verticillata t stating that they closly resembled those of Podocarpus macrophyllus but were smaller and appeared as narrow ellipsoides. He gave no detail of their structure and reported that the nodules of S. verticillata were reported to be mycorrhizal by Noelle (1910) and Laing (1923)« An examination of these papers, however, has shown that, while these authors recorded the presence of endotrophic mycorrhizae on this
plant, they made no mention of nodules. Daugherty (1963) saw beaded roots on the fossil Araucariorhiza .joae and suggested that the bulb-like expansions at the tips might possibly be inicipient nodules,
though the cells showed no evidence of invasion by bacteria, fungi or blue-green algae. Such an occurrence, however, has not been observed in living conifers and it seems probable
that he was observing beaded roots at the commencement of a
new growth cycle. In view of the uncertainly of the occurrence of
structures analogous to the root-nodules of Podocarpus 108
species in other members of the Coniferales and Ginkgoales, a survey was carried out, the results of which are given in table 7.2.
All the species examined of genera of the Podocarpaceae bore on their roots numerous nodules, usually in two opposite rows (Plate 7.1). Their size was fairly regular in any one species but varied considerably between species.
The smallest nodules observed were those of Microstrobos fitzgeraldii (0*3 - 0«5mm diameter), those of Phyllocladus hypophyllus^ P. trichomanoides and Dacrydium franklinii were medium sized (0«5 - 0*9mm) and those of Podocarpus spp. the largest (0.8 - l*5mm). As observed by Spratt (1912) and Baylis et al. (1963), the vascular strand in species with small nodules is very rudimentary but in Podocarpus it is sufficiently developed to show a diarch structure• Unfortunately material of Microcachrys and Acmopyle was not obtained for examination. It would be particularly interesting to obtain material of the latter genus, since the only recorded evidence of the presence of nodules in this genus is the unconvincing report for A. pancheri
(Sahni, 1920). The roots of all species of Apathis and Araucaria examined bore nodules analogous to those occurring in TABLE 7.2
A record of nodules, mycorrhizae and beaded rootlets observed on species of the Ginkgoales, Taxales and Coniferales. ( + = present, - = absent, V - vesicular-arbuscular, E = cctotrophic)
Beaded Order, family, genus, species Nodules rootlets Mycorrhiza
Ginkgoales Ginkgoaceae Ginkgo biloba L. +V
Taxales Taxaceae Taxus baccata L. +V
Coniferales Araucariaceae
Agathis australis Salisbury + + +v A. dammara (!Lambe'rt) L.C.Richard + + +v X. moorei (Lindley) Masters + + +v A. robusta (C.Moore) F.M. Bailey + + +v A. vitiensis (Seemann) Drake + + +v
Araucaria araucana (Molina)K.Koch + + +v A. cunninghamii Aitón ex D. Don + + +v X. heteromylla (Sail sburv) Franco + + +v X. columnaris (Forster f. j Hooker + + +v Cupressaceae Austrocedrus chilensis (D.Don) ---- FI .orin et Eoutelje Callitris muelleri (Pari.) F.Mueller +v C. columellaris F. Mueller - - +v rhomboidea R.Br. ex A. et -— +v L.Ó. Richard Chamaecyparis obtusa (Siée, et — + +V Zuce.) Endl.
Cupressus arizonica Greene _ + +V G. funebri s Endl. — + + v ô. glabra Sudworth - + + v C. sempervirens L. - + + v Ô. torulosa I). Don - + +v
Fokienia hodginsii (Dunn) _ + + v Henry et Thomas
Juniperus communis L. - + + v
Libocedrus plumosa (D.Don) + + v Sargent
Platvcladus orientalis (L.) Franco - - + v
Thu.iopsis dolabrata (L. f.) . , Sieb, et Zucc.
Tetraclinis articulata (Vahl) +v Masters
Wid dringt onia wh.vtei Rendle - - + v
Pinaceae Abies nordmanniana (Steven) Spach - + +E
Cedrus deodara Loudon - - +E
Keteleeria davidiana (Eertrand) + +E Beissner
Larix kaempferi (Lambert) Carriere - + +E
Picea abies (L.) Karsten - + +E
Pinus radiata D. Don + +E P. wallichiana A.B. Jackson - + +E
Pseudotsuga menziesii (Kirbel) Franco - - +E
Tsuga canadensis (L.) Carriere - - +E Podocarpaceae Da crydiurn franklinii Hooker f. + + +v Microstroboa fitzgeraldii + + +v (F.Mueller) Garden et Johnson Phyllocladus hypophyllus Hooker f. + + +v P. trichomanoid.es D. Don + + +v Podocarpus Irassii Pilger + + +v P. compactus V/asscher + + +v P. elatus ITTBr. ex Endl. + + +v falcatus (Thunterg) R.Br. + + +v P. la dei fTl.:. Bailey + + +v F. latifolius (Thunberg) R.Br. + + +v F. lowrencei Hooker f. + + +v P. macrophyllus (Thunterg) D.Don + + +v F. spinul0 sus (3m.) R.Br. ex Mirtei + + +v Taxodiaceae Crypt omeri a .japonic a (L.f.) D.Don - + +v Cunninghamia lanceolata (Lambert) - + +v Hooker Glyptoatrotus pensilis (Staunton - - +v ex b.Don) K. Koch
Metasequoia glyptostroboides - - +v Hu et CÌieng Sequoia sempervirens (Lamb.) Endl. - - +v Se guoiadendron gi ganteum (Lindi.) - - +v Hucholz Taxodium diatichum (L.) L.C. Richard +v T. mucronatum Tenore mm +v Sciadopityaceae Sciadopitys verticillata (Thunterg) + + +v Si et. et ¿¡ucc. 109
Podocarpus. They were usually more elongated but structurally analogous, being fully differentiated,lacking a root cap and apical meristem, and having a vascular strand completely surrounded and overarched by the endodex*mis (Plates 7.2,
7.3). They also showed regeneration from cells of the pericycle. It seems reasonable to suppose that they are a constant morphological feature in the Araucariaceae.
The only other conifer in which such nodules were found was Sciadopitys verticillata (Plate 7.4), the single representative of the genus and familySciadopityaceae.
The regular and characteristic arrangement of the nodules as seen in members of the Podocarpaceae was not as pronounced in members of the Araucariaceae or in
Sciadopitys. In fact the roots of these plants appeared little different from those of members of the Taxaceae,
Cephalotaxaceae, Cupressaceae and Taxodiaceae, with their numerous short roots. Only in Araucaria araucana was any thing seen approaching the regular arrangement occurring in Podocarpus (Plate 7.5).
This lack of similarity may, however, have resulted from conditions under which the plants were grown, since Janse
(1897) considered the root systems of Araucar ia excelsa
(A. heterophylla) and Dammara (Agathis) robusta to bear a great resemblance to those of Todocarpus, although the 110
"tubercles" were a little more elongated in D. (A.) robusta.
He did, however, note that what he considered to be "tubercles" in Cupress» s fastigiata (C. sempervirens) and Juniperus (Sabina) chinensis were less numerous and more elongated.
No evidence of nodules could be found on roots of species of the Cephalotaxaceae, Cupressaceae, Pinaceae, and Taxodiaceae, or on those of Ginkgo biloba and Taxus baccata.
The long roots all bore small, blunt, lateral protuberances, but an anatomical study revealed them to be lateral roots, each with recognised root caps, apical meristem and open - ended endodermise (plate 7.6). Nearly all the plants examined were mycorrhizal and had beaded rootlets resulting from the episodic growth of short roots (table 7.2). The members of the Pinaceae examined all had ectotrophic mycorrhizae and all the others that were mycorrhizal contained vesicular - arbuscular endophytes. r No doubt the species found to be non - mycorrhizal in this survey would be found to be mycorrhizal under other conditions, just as it seems probable that those species, for which no beaded - rootlets were observed, might be found to form them were a more extensive search made. Hence there appears to be a clear pattern with regard to the occurrence of nodules and mycorrhizal amongst the
Ginkgoales, Taxales and Coniferales. Nodules of the 111
Podocarpus-type have been found only in the Araucariaceae, mycorrhizae Podocarpaceae and Sciadop ityaceae, and endotrophic/seem to be of general occurrence in all families except the
Pinaceae, the members of which form the ectotrophic type.
These observations concerning the mycorrhizae support the observations of earlier authors (Noelle, 1910; Laing, 1923).
The taxonomic significance of the occurrence of nodules has been mentioned by Spratt (1912), who discusses the affinities of the Podocarpaceae and Araucariaceae, noting that they have much the same geographical distrib ution. Both she and Saxton (1930 b) regard the universal occurrence of nodries to be a factor lending weight to the present grouping of genera in the Podocarpaceae. The occurrence of nodules in Sciadooitvs is also a factor strengthening the separation of this monotypic genus from the Taxodiaceae and its placement in a family of its own.
It is interesting that this Japanese plant occurs within the distribution of Podocarpus and that, according to
Dallimore and Jackson (1966), Greguss places it in the
Podocarpaceae, on the basis of similarities in wood structure. EXPLANATION OF PUTES KEY__TO LETTERING OF PLATES
C Cortex
E Endodermis
LR Lateral root
N Nodule
RC Root cap
RH Root-hairs
RM Root apical meristem
X Vascular strand Plate 7*1 Root nodules of podocarps. Top: Phvllocladus trichomanoides. (X 30). Centre: Microstrobos fitzgeraldiia(X 50). Bottom: Dacrydium franklinii.(X 30)
Plate 7.2 Top: Root nodules of Araucaria heterophylla. (X 40). Bottom:
L. S. of a root and a
nodule. Note the overarching
endodermis and absence of root-cap and apical meristem. (X 150). Plate 7*3» Top left: Root nodules of Agathis australis. (X 20). Top right: Cluster of root-nodules. (X 20) Bottom left and right: L. 5. of nodule showing anatomical features. Note the cortex invaded by the endophyte. (X 100).
Plate 7 .h Top: Root-nodules of Sciadopitys verticillata. (X 20 ). Bottom: L. S. of a typical nodule with endophyte. (X 150).
Plate 7.5. Root nodules of Araucaria araucana.Note the arrangement
in two opposite rows. (X 2 ) Plate 7.6
Top: A lateral root of Cupressus
sempervirens. Note the superficial
resemblance to a spherical nodule. (X 20). Bottom: L. S. of the
above showing a structure
essentially the same as that of
a lateral root# Note the root-cap and apical meristem. (X 100). PL. 7.1
PL. 7.3 PL. 7. 4 PL. 7.5
112
CHAPTER V ili
D I SCUSSION 113
Unless nodulation is induced by some undetected endophyte transmitted through the embryo, it can now be considered proved, as was indicated by the work of Baylis et al. (1963) and suggested by the observations of Shibata (1902)?
Schaede (1943), and Becking (1965), that nodules develop as normal features of the root-system of Podocarous species, and presumably of other conifers as well, and that their formation is not induced by any microbiological factor.
A comparative study has shown that the nodules of P. falcatus are distinct morphological features of the root system and should not be regared as modified lateral roots or lateral roots of arrested growth. Although both nodules and short roots arise in pericycle of the parent root, dif ferences can be detected very early in their development and long before they emerge from the cortex of the parent root. The nodule primordia appear spherical and those of short roots conical. Similar differences have been noted between short and long-root meristems in Pinus resinosa by Wilcox (1968). The nodule primordium rapidly develops into a fully differentiated structure without a root-cap and apical meristem. A vasular strand differentiates, becoming connected to that of the parent root, and this becomes 114 completely surrounded and overarched by the endodermis. Regeneration of the nodule follows the appearance or a new nodule primordium beneath the endodermis at the tip of the vascular strand. The short root, on the other hand, had an open-ended endoderrrnLs,and apical meristem and a well developed root-
cap. The short root exhibits an episodic growth pattern, not unlike that of the nodule but resulting from the resumption of activity of the apical meristem, rather than from the
appearance of a new meristem in the pcricycle. Under natural conditions both the young roots and nodules become invaded by a vesicular-arbuscula.r endophyte. Infections have been produced in endophyte-free plants inoculated with Bndoqone-type spores extracted from podocarp soil and, in view of similar observations with other plants, it seems reasonable to assume that the endophyte or endophytes are members of this genus. ITo evidence was obtained that infection noticeably stimulates, arrests or otherwise alters the nodules or roots which it invades. The situation is not very different from that pertaining to short roots of beech and pine and
thedr ectotrophic mycorrhizal fungi (Harley, 1959? Wilcox 1963). It is not known whether the presence of the 115
vesicular-arbuscular endophyte increases the longevity of
the cortex of nodules and short roots in Podocarpus spp. as
was observed for the ectotrophic mycorrhizae of Pinus
res inosa by Wilcox (1968).
With the differences between nodules and short roots
clearly established in Podocarpus. a survey was carried out
to determine the extent of the occurrence of such nodules
amongst the Ginkgoales, Taxales and Coniferales. Nodules were observed on all members of the Araucariaceae,
Podocarpaceae and Sciadopityaceae examined, but not on members of the Ginkgoaceae, Taxaceae, Cephalotaxaceae,
Cupressaceae, Pinaceae or Taxodiaceae. In all instances
the nodules observed were analagous in development, structure
and mode of regeneration to those occurring on Podocarpus
falcatust although in some genera they were elongated
and resembled short roots in shape.
It is clear that conifer root-nodules are markedly
different from those of legumes and non-leguminous angiosperms on which they develop only in response to
infection by the appropriate endophyte. The development
and structure of legume nodules is described by Allen and
Allen (1958) and that of non- leguminous angiosperm nodules
by Bond (1963), and it is clear that, while they have some
features in common with conifer nodules, they differ in 116
their origin, the presence of apical meristems, and the enlargement of the cells containing the endophyte. In the present study no such enlargement was observed in conifer nodules.
Spratt (1912) commented on the differences between the nodules of the Podocarpaceae, and those of legumes, noil-leguminous angiosperms, and cycads. The nodules of cycads, which are probably better termed apogeotropic roots, also have apical meristems, but there is no evidence that infection with the blue-green algal endo phyte is necessary for the initiation of their development. Conifer nodules are unique in having no persistent meristematic zone and in their regeneration from pericyclic cells which become meristematic. They also differ from the other nodules mentioned in that the endophyte is present in other parts of the root system as well.
Baylis et al. (19 6 3 ) suggested the ability of nodules of podocarp and araucarian roots to regenerate to be an adaptation for retaining the symbiont after the long roots have shed their cortex, and regarded them to be an equivalent development to the dichotomous short roots of pines, the outstanding example of root morphology modified to promote symbiosis with ectotropic 117
mycorrhizal fungi.
The repeated regeneration of conifer nodules, however, doe^s not seem to be more specialized system of retaining an abundance of young cortical cells than the episodic growth of short roots. Many conifers and other plants, such as Acer rubrum (Lyford and Wilson,
196A), appear to achieve the same end by the latter method.
Unless conifer nodules are shown to have inherent abilities differing from those of short roots, or it is found that a sebond endophyte, such as had been reported by Morrison and English (1967) in Agthis australis. has a particular affinity for them, it seems probable that they do not have any function markedly different from other parts of the root system, They might, then, be regarded as no more than morphological features characteristic of the root systems of members of the
Araucariaceae, Podocarpaceae, and Sciadopityaceae and their function,when invaded by a vesicular-arbuscular endophyte, to be similar to that of vesicular-arbuscular mycorrhizae in general.
Because of their appearance they have been called
T,nodules?T and compared with the nodules of other plants in which nitrgen fixation is known to take place. 118
Apart from the negative results of Dandie (1968), using those of tomato and ryegrass, the only vesicular- arbuscular mycorrhizae which have heen tested for nitrogen fixation are the nodules of Agathis and Podocarpus species. of While there are positive records for the incorporation fl 5-^
(Bergersen and Costin, 1961+; Becking, 1965; Morrison and English, 1967), there is as yet no evidence that such fixations are of Benefit to the plant, and what little evidence there is suggests that nitrogen fixation is not associated with the vesicular arhuscular endophyte. As
Bond (1967) has pointed out, there is no evidence from growth experiments that nodulated Podocarpus plants containing the endophyte are partially or wholly inde pendent of external sources of combined nitrogen. The fact that Both cycads and many legumes with effective nodules commonly contain well developed vesicular- arBuscular endophytes might/ also Be interpreted as suggesting that s^ch mycorrhizae are of little or no significance in nitrogen fixation.
In spite of the claims of Bergersen and Costin
(I96I4.) for P. lawrencei, there is also no evidence that nodule-Bearing conifers are Better able to colonise poor areas than many other plants without nodules, such an opinion Being supported By the results of experiments 119 conducted by Baylis (1967) with P. totara and certain angiosperms• Thus, while it can now reasonably be assumed that, as in the roots of other slants, the presence of a vesicular-arbuscular endophyte enhances the ability of the nodules and roots of conifers to take up phosphorus in soils in which it is low or poorly available, the cuestión of nitrogen fixation must, as it does with the ectotronhic mycorrhizae of Pinus, remain open. On present evidence, however, it seems unlikely that the nodules of conifers are the site of a symbiotic nitrogen-fixation of similar significance to that occurring in the nodules of legumes, non-leguminous angiosperms, and cycads. Experiments are needed involving both nodulated conifers and other plants, alone and in combination with different forms of fungal and perhaps other endophytes before this problem can be finally resolved. Until the as yet unsurmounted difficulties of obtaining both the plants and endophytes in pure culture are overcome, progress will probably continue to be slow. 120
APPENDICES
I METHODS FOR ANATOMICAL INVESTIGATIONS
I I METHOD FOR BACTERIOLOGICAL TESTS
I I I RECIPES FOR CULTURE MEDIA
IV PUBLISHED PAPERS
V PREPARATION OF SOIL FILTRATE
V I METHODS FOR NITROGEN AND PHOSPHORUS ESTIMATIONS 121
APPENDIX 1,
METHODS FOR ANATOMICAL INVESTIGATIONS
1. Paraffin - processing method, 2. Freezing - Microtome method.
3. Electron Microscopy. 122
(1) PARAFFIN - PROCESSING METHOD.
KILLING :JsID FIXING:-
Two killing and fixing agents were used. 1. F.A.A. being six parts commercial formalin, 3 parts glacial acetic acid
and 91 parts 70% ethyl alcohol by volumes.
2. Worcester's Fluid being 86 parts
saturated aquous mercuric chloride, 4 parts formalin and 10 parts 10%
aquous glacial acetic acid. DEHYDRATION AND INFILTBRATIQN:-
This was done using the tertiary butyl alcohol method (See Johansen, 1940)
EMBEDDING:-
Paraffin wax with a melting point of 56 °C was used exclusively. Materials were embedded using the paper boat method. (See Johansen, 1940). SECTIONING
6-12 microne thick sections were cut on a
rotary microtome; mainly longitudinally, but transverse sections were also cut. 123
ADHESIVE*—
Haupt's adhesive (See Johansen, 1940) was used
and a 3 to 4% aqueous solution of formald ehyde was used for floating sections• STAINING*- The staining procedures followed are described
by Johansen, (1940), The following combinations of stains were used.
1. safranin, light green; 2. safranin, fast green? 3. safranin, crystal violet? 4. safranin, Delafield's haematoxylin? 5. safranin, crystal violet, orange G? 6. safranin, tannic acid, ferric chloride?
7. safranin, crystal violet (Grams method for bacteris)? 8. carbol fuchsin?
9. gentian violet, Lugol's iodine, safranin. 10. Harris's haematoxylin.
MOUNTING Preparations were mounted in either Canada balsam or Euparal. 124 l2) _ _ „ FREEZING MICROTOME METHOD ARRANGEMENT OF MATERIAL:- Small pieces of roots preserved in F.A.A. or Worcestor's Fluid were
washed with water and oriented on the
freezing stage of Reichert's microtome.
The material was frozen quickly with
bursts from the carbon dioxide cylin
der. Water was used before the carbon dioxide to encase the material. CUTTING:- Sections ranging 15-40 microns in
thickness were cut with a wedge-shaped no.I knife. STAINING:- Staining was carried out on the slides
or in watch glasses, as described in
section ± or with 0.2% aqueous safranin. MOUNTING:- Sections were mounted either in 10%
glycerine in water or in glycerine jelly (Johansen, 1940). 125
(3) ELECTRON MICROSCOPY
FIXATIONS- Small pieces (0.5mm) of nodular tissue were fixed in
either 2% aqueous KMnO^ for 2 hours or 1% 0s04 in Kellenberger1s buffer at pH 6.0 (Ryter, et al#71958)
for 16 hours.*
STAINING After fixing, the pieces were placed in 2% aquous
uranyl acetate for 2 hours.
DEHYDRATION Ethyl alcohol and acetone were used in the procedure
described by Kay, (1965). EMBEDDING Using Kay*s (1965) procedure the pieces were embed
ded in either (a) Vestopal Vi (Martin Jaeger, Geneva, Switzerland); or (b) Araldite (CIBA), using either resin M'CY212,
N CY213 or MY740.
SECTIONING:- Ultrathin sections (700-1500 A) were cut on an
1KB microtome with glass khive§« MOUNTING:-
The sections were mounted on 100 and 200 mesh grids
coated with nitrocellulose and carboned for two seconds. 126
ELECTRON MICROSCOPE:-
The grids were examined with a Siemen*s Elmiskop 1 electron microscope. 127
APPENDIX XT.
METHOD FOR BACTERIOLOGICAL TESTS 128
METHOD FOR BACTERIOLOGICAL TESTS
Both the medium in which shoot-cuttings and seedlings of podocarps were growing (autoclaved Send and peat) and
the roots themselves were tested for sterility.
The nodules, produced on the adventitious roots arising
from the bases of shoot-cuttings and those from the seed
lings, were washed, surface sterilized for two minutes in
10% solution of sodium hypochlorite, washed six times with
autoclaved distilled water, and crushed with sterile inst
ruments.
The nodules produced on seedlings from excised embryos were crushed with sterile instruments without surface steri
lizing.
The crushed nodules and the medium (e.g. sand and peat) were transfered to malt-marmite agar slopes and the cultures o were incubated for three weeks at 20 C. 129
APPENDIX III
RECIPES FOR CULTURE MEDIA
1. Raghavan and Torrey*s Tissue Culture Medium
2. White*s Macronutrients
3* Heller*s Micronutrients
4. Nutrient solutions used in growth experiments with seedlings
(i) Low phosphorus
(ii) High phosphorus
(iii) Low phosphorus and combined nitrogen
(iv) High phosphorus and combined nitrogen
5 . Malt-Marmite Agar 130
RECIPES FOR CULTURE MEDIA
1. Raghavan and Torre.y^ Tissue Culture Medium
Macronutrients Mg/litre of water
Ca(N03 )2¿jH20 1+80.0
Mg So^ 7H20 63.0
K NO, 63.0 j K C1 1+2.0
k h 2 p o 4 60.0
Micronutrients
h 3b o 3 O.56 MnCl2 Í4H20 O.36
ZnCl2 0 .1+2
CuCl2 2H20 0.27
(n h 4 )6 Mo ?02í+ 4H20 1.55 Pe (C6H506) 3H20 3.08
Vitamina
Thiamine hydrochloride 0.1
Pyridoirin hydrochloride 0.1
Niacine 0.5 131
Sucrose 20,000.0
Difco-hacto Agar 9,000.0
White’s Macronutrients In 1000 ml water mg
k n o 3 80.0 Ca(N03 )2 H20 260.0
NaH2P0^ 2H20 16.5 KC1 65.0 MgSO. 7H20 360.0
Na2 Soi+ 200.0
Heller’s Micronutrients In 1000 ml water mg Pe Cl3 6H20 1 .0 Zn So 7H90 1 .0 k ¿ H, B0, 1 .0 MnSO^ ¿+H20 0.1 Cu so^ 5h 2o 0.03 132
A1C1, 0.03 j NiCl2 6H20 0.03 KI 0.01 Add 1ml/litre of macronutrients
4. Nutrient Solutions Used In Growth Experiments With Seedlings (i) Low phosphorus
I^HPO^ 2H2o 5mg/litre
(ii> High phosphorus K2HP0^ 2H20 20mg/litre
(iii) Low phosphorus and combined nitrogen K2HP0^ 2H20 5mg/litre Ca(N03 )2 UHgO 80mg/litre
n h u n o 3 236mg/litre (iv) High phosphorus andL combined nitrogen KgHPu. 2H20 20rag/litre Ca(H03 )2 UH20 80mg/litre
n h 4n o 3 236mg/litre 133
5. Malt-Marmite Agar
20 gms malt extract (Cornvell’s)
1 scant teaspoon Vegemite
15 gms Difco-bacto agar
1 litre water
The ingredients were "brought to the "boil to
dissolve the agar. The culture medium was then tubed
into rimless 6" x 1" iyrex test tubes with a tubing
syring to give accurate amounts (20ml/tube). The
tubes were plugged with non-absorbent cotton wool
and autoclaved for 15 minutes at 151b/sq. inch pressure. 134
APPENDIX IV
PUBLISHED PAPEPS 135
APPENDIX IV
p u b l i s h e d p a p e r s
1. Khan, A. Gr. (1967). Podocarpus root nodules in
sterile culture. Nature. (London). 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. Mirh.
^Trown in pure culture from excised emhroyos.
Aust. J. Sci, 3 0 , 372-3.
3. Khan, A.G-. (1968 h) Effect of temperature,
gihherellic acid, and Indolylacetic acid on
root and snoot growth of cuttings of Podocarpus
lawrencei Hook. f. Aust. J. hiol. Sci. 21. 573-7 ted from The Australian Journal of Science. Volume 30, Number 9, March, 1968, page 372
of Added Growth Substances on Seedlings stage II, making nine subsets in all. In addition, locarpus falcatus R.Br. and P. spinulosus two surplus seedlings growing on each of media (a), (6) and (c) of stage I were left untouched. R.Br. ex Mirb. Grown in Pure Culture from At the end of stage I, on basal medium alone, Excised Embryos there was no increase in embryo size, but chlorophyll production of nodules in pure culture by developed in the cotyledons. When B-vitamins rpus falcatus R.Br. has already been reported were present, {b), the embryos showed good shoot 1967). The study of nodule production in growth but poor root growth (Figure 1). When of Podocarpus has, however, been handicapped gibberellic acid was present, (c), the embryos showed dormancy of the seeds, which, in soil, very greater growth of shoot and of root (Figure 2). do not germinate for five to six months, e of this dormancy, surface-sterilized seeds not be induced to germinate in pure culture xperiments were commenced using excised >s. Seedlings have been grown from the L embryos of certain conifers (see Nara- rami and Norstog, 1964) but no record of the i of Podocarpus embryos could be found, he original study (Khan, 1967), seedlings of •atus were established from excised embryos on Raghavan and Torrey (1963) culture n, but embryos could not be induced to develop same medium from which niacin, pyridoxin hloride and thiamine were omitted. Once )S had produced roots and shoots, however, idlings continued to develop normally when rred to a medium without added vitamins, study of root development in pure culture of species of Podocarpus has also been hampered dormancy of the seeds and similar difficulties been experienced with P. spinulosus (Sm.) )x Mirb. jr-grown green seeds of P. spinulosus were id at Pearl Beach, N.S.W., in February 1967, sparated from the fleshy arils. These seeds surface-sterilized by immersion for ten is in a saturated solution of sodium hypo- 3 and washed six times with sterile water, rhich the embryos were excised aseptically and singly on nutrient agar slopes. At the tune ision the embryos were fully differentiated ire approximately 2*5 mm. in length, be’s macronutrients (White, 1943) plus Heller’s iitrients (reported by Morel, 1964), with ent. sucrose and 0-9 per cent. Difco bacto-agar as the basal medium, the pH being adjusted before autoclaving. experiment was carried out in two stages, each cli lasted for four weeks, fetage I, the following three media were used : al medium alone, (b) basal medium with ;./litre pyridoxin hydrochloride and niacin, ) basal medium with 10“ 7 mg./litre gibberellic Figure 1 (top) Embryos were placed in 20 tubes of each Figure 2 (bottom left) a. and kept in a shady glasshouse (photoperiod Figure 3 (bottom right) K 11 hr. to 13 hr. of light per day at the Lng and end of the experiment respectively ; The embryos, transferred from basal medium (a) r C. temperature range). of stage I to any of the three media of stage II, did stage II, media (a) and (c) were repeated, but not develop further. third medium (b'), thiamine at O-lmg./litre The seedlings, transferred from medium (b) of id the original two vitamins of medium (b). stage I to medium (a) or (c) of stage II, showed no end of stage I, each set of seedlings {a, b and c) further growth. The seedlings, transferred from tided into subsets of six and each subset was medium {b) of stage I (without thiamine), to medium rred to the fresh media (a), (b') and (c) of (//) (containing thiamine) in stage 11, showed added oot growth (Figure 3). The two surplus seedlings external supply of this B-vitamin satisfies a r< eft on medium (b) (without thiamine), still showed ment for root growth in these excised Podo to root growth. embryos. The observation that thiamine parti« The seedlings transferred from medium (c) of increased root growth is in agreement with tin tage I to any of the three media in stage I I showed of Lammerts (1942) on Prunus embryos and wi LO major changes in the distribution of growth, results of root culture experiments (Street and Ithough the growth rate of root and shoot was about 1963). ¡5 per cent, of that in stage I. This, however, Thus, while the nature of the dormancy of en »arallels the low growth rate observed following of Podocarpus species is still poorly underst« ;ermination of P. spinulosis seeds in soil. simple method has been devised for obtaining see The behaviour of embryos in stage I indicated that, in sterile culture. mder the conditions of the experiment, an external I wish to express my thanks for the encourageme upply of growth-promoting substances was necessary advice given to me by Dr. I. V. Newman throughout thi or further development of excised embryos. However, and to Professor S. Smith-White for the facilities pr mbryos maintained on the basal medium for four I would also like to thank Mr. B. Lester for his help vneks before exposure to thiamine or gibberellic preparation of photographic negatives for the illustration nid showed no response, although remaining healthy A. G. K i n appearance. It is assumed that during the four School of Biological Sciences, peeks on the basal medium they had entered a Botany Building, lormancy that could not be reversed, at least under University of Sydney, he prevailing conditions, by thiamine or gibberellic Sydney, N.S.W. nid at the concentrations used. 20 November 1967. The observations that gibberellic acid enhanced growth of shoot and root are similar to the effect References eported for excised barley embryos by Schooler B o n n e r , J. (1938) : Plant Physiol., 13, 865. Ch a t t e r j i, U. N., and Sa n k h l a , N. (1965): In Tissue < 1960) and for Merremia embryos by Chatterji and Edited by C. V. Ramakrishnan (Dr. W. Junk Put lankhla (1965). The Hague), p. 389. The need for vitamins in embryo-culture media is K h a n , A. G. (1967): Nature, 215, 1170. L a m m e r t s , W . E. (1942) : Amer. J. Bot., 29, 166. pell known. Bonner (1938), for instance, found that M o r e l , G. (1964) : Ann. Soc. Nat. Hort. France, No. i uacin accelerated the growth of the shoot of coty- 733. edonless embryos of Pea. In the present study, the N arayanaswami, S., and N o rsto g , K . (1964) : Boi esults of stage I show that, in these podocarp 30, 587. R a g h a v a n , V., and T o r r e y , J. G. (1963) : Amer. J. B »mbryos, pyridoxin hydrochloride and niacin 540. medium b) promoted vigorous leaf production on a Sc h o o l e r , A. B. (1960) : Agron. J., 52, 411. ihort stem but minimal root growth (see Figure 1). St r e e t , H. E., and J o n e s , O. P. (1963) : In Plant Tisi Seedlings from stage I medium (b) were stimulated Organs Cultures— A Symposium (International Soc. o Morphologists, Delhi). ;o strong root growth on transfer to stage II medium W hite, P. R. (1943) : In A Handbook of Plant Tissue b') containing thiamine. This indicates that an (Jacques Cattell Press, Lancaster, Pa., U.S.A.).
AUSTRALASIAN MEDICAL PUBLISHING CO. LTD. SEAMER & ARUNDEL STS., GLEBE, SYDNEY, 2037 Short Communication reprinted from the AUSTRALIAN JOURNAL OF BIOLOGICAL SCIENCES
EFFECT OF TEMPERATURE, GIBBERELLIC ACID, AND INDOLYLACETIC ACID ON ROOT AND SHOOT GROWTH | OF CUTTINGS FROM PODOCARPUS LAW REN C El HOOK f.*
By A. G. K h a n | .
It has been shown (Khan 1967) that roots of Podocarpus produce nodules r sterile conditions, thus proving that nodule production is a normal feature e root system and is not induced by the presence of any microoorganism. )uring this study it was observed that the roots produced by cuttings became ted sooner than roots of seedlings grown under the same conditions. Many fiative buds expanded on the shoot cuttings but root production and growth poor when daily air and soil temperatures were high, whereas the converse ihe case during a following period when temperatures were low7er. In an effort to find a reliable method for the rapid production of nodulated for anatomical and physiological studies, an experiment was set up to study fects of temperature and growth substances on root and shoot production by Igs. ials and Methods Vegetative material from the field was collected from Mt. Ginini, A.C.T. (the •om which material was obtained by Bergersen and Costin 1964) in February Shoot cuttings 6-7 cm long were surface-sterilized for 10 min with a 10% on of sodium hypochlorite, washed six times with autoclaved distilled water, ed of lowrer leaves, and planted in pots (five cuttings per pot) of autoclaved and peat (equal parts). The pots wrere completely enclosed in new polythene and were kept at “ low” temperatures (day 21°C, night 15°C) or “ high” sratures (day 28°C, night 21 °C) in Sherer environmental growth chambers with it photoperiod. In these chambers the source of light was VHO Cool White rtubes supplemented with Phillips 40-W incandescent lamps, giving a light sity of 4000 f.c. at the level of the plant bed as measured with a selenium cell light-meter. The light intensity, however, was reduced to 1000 f.c. by lg the pots with white paper. The experiment lasted for 9 weeks. Cuttings were removed under aseptic tions every 3 weeks for observation and recording. The pots were watered cally every 3 weeks with autoclaved distilled water.
* Manuscript received November 17, 1967. ■f School of Biological Sciences, University of Sydney, N.S.W. 2006.
Aust. J. biol. Sci., 1968, 21, 573-7 574 SHORT COMMUNICATIONS
[For explanation of Figures 1-12, see opposite SHORT COMMUNICATIONS 575
rvations and Results Effect of Temperature: Location Unchanged.—Two pots of cuttings were kept e low-temperature cabinet for the whole 9-week period without treatment with growth-promoting substances. The cuttings developed many roots (nodule-free), no shoot growth occurred (Fig. 1). By contrast, the untreated cuttings of two kept in the high-temperature cabinet for the whole 9-week period developed ¡r adventitious roots (nodule-free), and good shoot growth occurred with many s expanding (Fig. 2). Effect of Temperature: Location Interchanged.—The untreated cuttings of two i which were kept in the low-temperature cabinet for the first 3 weeks developed adventitious root primordia at their bases and no shoot growth occurred (Fig. 3). 6e cuttings were then transferred to the high-temperature cabinet where two to b vegetative buds per cutting expanded within the next 3 weeks (Fig. 4). At the of the experiment the cuttings showed good shoot growth and poor (nodule-free) growth (Fig. 5). The untreated cuttings of two pots, kept in the high-temperature net for the first 3 weeks, had two to three vegetative buds per cutting expanded . 6). These cuttings were then transferred to the low-temperature cabinet, where B was an increase in root development and a reduction in the rate of shoot msion during the next 3 weeks (Fig. 7). At the end of the experiment the roots 5 nodulated and much more extensive than those roots produced by cuttings , at the low temperature for the whole period (Fig. 8). Effect of Application of Gibberellic Acid to Tops of Cuttings.—Cuttings in two i in the low-temperature cabinet were treated weekly all over with a solution jbberellic acid (100 /¿g/1) in the form of a fine spray. Vigorous root and shoot db occurred and the roots were nodulated (Figs. 9 and 10). Shoot growth occurred at the temperature range of 15-21°C at which vegetative buds did not expand ntreated cuttings. Effect of Application of Indolylacetic Acid to Bases of Cuttings.—Cuttings in two in the high-temperature cabinet were treated, once before planting, with 1% ylacetic acid in the form of lanoline paste over the surface of the basal denuded on. Observations similar to those for cuttings sprayed with gibberellic acid in low-temperature cabinet were recorded, i.e. both root and shoot showed good dh (Figs. 11 and 12). Root growth here occurred at the temperature range of 8°C at which root development was very poor on untreated cuttings.
1-12.—Cuttings and root systems of P . Icavrencei showing the effects of low and high jeratures and growth substances on root and shoot growth. Cuttings illustrated are “average” lies from their treatment programme. Figs. 1 and 2, untreated cuttings after 9 weeks at the pig. 1) and the high (Fig. 2) temperature. xO-45. Figs. 3-5, untreated cuttings after 3 s at the low temperature (Fig. 3) and after transfer to the high temperature for 3 (Fig. 4) (Fig. 5) weeks. xO-9. Figs. 6-8, untreated cuttings after 3 weeks at the high temperature 6) and after transfer to the low temperature for 3 (Fig. 7) and 6 (Fig. 8) weeks. X 0 • 9. 9 and 10, whole cutting (Fig. 9) and central portion of nodulated root system (Fig. 10) after }ks at the low temperature. Cutting sprayed weekly with gibberellic acid solution. X 0 • 45 1-75 respectively. Figs. 11 and 12, whole cutting (Fig. 11) and central portion of nodulated pystem (Fig. 12) after 9 weeks at high temperature. Cuttings treated once before planting with indolylacetic acid-lanoline paste (arrow). xO-45 and 3-75 respectively. 576 SHORT COMMUNICATIONS
Conclusion
While the factors influencing root and shoot development are undoubted complex and imperfectly understood, it is generally considered that specmc substanci are concerned with the initiation of adventitious root primordia and that the maj< sources of these substances are the leaves, from which the substances are translocate to the bases of the cuttings (Sinnott 1960). The observations presented here sho that, in spite of vigorous shoot growth at the high temperature, root growth wj poor; but that when cuttings devoid of root primordia at the end of the first 3 wee] at high temperature (Fig. 6) were transferred to the low-temperature cabinet, spite of little further shoot growth, there was vigorous development of nodulate roots. Possible explanations for this behaviour of P. lawrencei cuttings under tl higher temperatures are either that an adequate supply of the root-inducing substam or substances was not formed in the leaves, or that the substance or substances we: destroyed or inhibited. The production of roots at higher temperatures by cuttinj treated with indolylacetic acid-lanoline paste could be interpreted as evidence support of these suggestions. Indolylacetic acid here extends upward the temperatu: range for root development, perhaps by replacing the root-inducing substance < substances inhibited in production or destroyed at higher temperatures. The present study also shows that P. lawrencei buds, dormant at lo temperatures, expanded at high temperatures. However, the buds were induced 1 expand at the low temperature by spraying them with a solution of gibberellic aci which here extended downward the temperature range for bud-break. The subsequei increase in root development might have resulted from some effect of gibberellic ac on the growth of the plant as a whole, possibly as a result of increased productio of the root-inducing substance or substances by the resulting larger shoot. Thej results are consistent with the findings of earlier workers such as Morgan and Mei (1956, 1958), Wittwer and Bukovac (1957), Leben, Alder, and Chichunk (1959), ar Biddiscombe, Arnold, and Scurfield (1962), who showed that gibberellic acid sprf induced some plants to grow at temperatures lower than those usually required f< the growth of untreated plants. The appearance of nodules under the conditions for good root growth (Figs.| and 11) is in harmony with the conclusion of the earlier study (Khan 1967) that nodul( are a normal morphological feature of the podocarp root system. The present stuc suggests that root development depends on the accumulation of sufficient roo inducing substance or substances, and that the time required varies with tl conditions. This is borne out by the fact that on removal from the chambers, at t] end of 4 months, all cuttings bore nodulated roots, even those kept under conditio] apparently unfavourable for root development. It is intended to study this proble further from this point of view. I am much indebted to several colleagues and especially to Dr. I. V. Newma Dr. P. G. Valder, Professor M. G. Pitman, and Mr. P. Martin, all of this Departmer and Dr. J. H. Palmer, School of Biological Sciences, University of New South Wale for helpful discussion and advice, and to Professor S. Smith-White for the faciliti provided. I would also like to thank Mr. B. Lester for his assistance in the preparatio of photographic negatives for the illustrations used. SHORT COMMUNICATIONS 677
'erences eigersen, F. J., and Co s t in , A. B. (1964).— Aust. J. biol. Sci. 17, 44-8. >d is c o m b e , E. F., A r n o l d , G. W ., and Sc u r f ie l d , G. (1962).— Aust. J. agric. Res. 13, 400-13. a n , A. G. (1967).— Nature, Lond. 215, 1170. b e n , C., A l d e r , E. F., and Ch ic h u n k , A. (1959).— Agron. J. 51, 116-17. r o a n , D. G., and M e s s , G. C. (1956).— Nature, Lond. 178, 1356-7. r g a n , D. G., and M e s s , G. C. (1958).— J. agric. Sci. 50, 49-59. in o t t , E. W. (1960).— In “ Plant Morphogenesis” , p. 391. (McGraw-Hill Book Co.: New York.) It t w e r , S. H., and B u k o v a c , M. J. (1957).— Q. Bull. Mich. St. Univ. agric. Exp. Stn 39, 682-6. 136
APPENDIX V
PREPARATION OP SOIL FILTRATE 127
PREPARATION OF SOIL FILTRATE About 250 gms. of soil from around the roots of Podocarpus falcatus was suspended in 1 litre of water. After the heavy particles had settled, the solution was poured through a kitchen sieve and then through a sieve with pore diameter of 65 to remove most of the Endogone type spores (as described in Chapter IV). The remaining liquid was then filtered through a Whatman Filter Paper No.l. This filtrate was tested for the presence of fungi Yv\ by plating it on malt-mar^Lte agar. The plates were incubated for two weeks at 20°C, after which colonies of bacteria were observed. Since there was no evidence of any fungal growth from the filtrate, it seems unlikely that any fungi, even those unable to develop on malt- marmite agar, were present in the filtrate. 138
APPENDIX VT
METHODS FOR NITROGEN AND
PHOSPHORUS ESTIMATIONS 139
METHODS FOR NITROGEN AND rHOSrHORUS ESTIMATIONS Plant material was digested in 100 ml Kjeldahl flasks on an open flame for 1 - 1 hr, 10 ml digestion mixture
(30 gms of Selenium dioxide powder dissolved in 2 L of 90% sulphuric acid; 20 mis of concentrated perchloric acid a<,ded to the mixture) "being added to 0.3 - 1.0 gm dried plant material. The cooled acid digest was diluted with distilled water to a volume of 250 ml and filtered.
Three samples from each digest were analysed using a Technicon Auto-Analyser, nitrogen and phosphorus being determined photocolorimetrically in a 15 mm tubular flow cuvette. The sample cups of the auto-analyser were filled with the solutions and submitted to the further treatments necessary for the determination o£ nitrogen and phosphorus. Nitrogen was determined by the automatic procedure of O ’Brien and Fiore (1962), as modified by the C.S.I.R.O. Plant Physiology Unit,Sydney. Absorption v/as measured at o25 mji using a sampling rate of ¿+0 per hr. Phosphorus was determined by an automatic procedure of Varley (1965), also modified by the C.S.I.R.O. plant
Physiology Unit, Sydney, Absorption was measured at kkO using a sampling rate of UO pei hr. A. standrad curve for each treatment was included at the FIGURE 9.1 Calibration curves for nitrogen
and Phosphorus. RECORDER CHART READING NITROGEN CONCENTRATION n o i t a r b i l a c
s e v r u c PHOSPHORUS 140 commencement and completion of each batch. Typical
standard curves are shown in Fig. 9 * 1 * 141
BIBLIOGRAPHY 142
-.lien, E • K. , and Allen, O.N. (1953). Biological aspects of symbiotic nitrogen fixation. In Lncyclopedia < Plant Physiolooy, 3,48-118, ed. Ruhland”, ’A, Springer-Verlay. Berlin, 1958.
______(1965). Nonleguminous plant symbiosis. In Microbiology and Soil Fertility, 77-106. Proc. 25th a . Biol. Colloq. Ore.st. Coll., ed. C. K. Gilmour and O. N. ¿».lien., Oregon State University Press, Corvallis.
-»lien G. S. (1947). Embryogeny and development of the apical meristem of Pseudotsuga. III. Development of the apical meristems. A m . J. Dot., 34, 204-11.
Barber, D.A. (1966). Effect of microorganisms on nutrient absorption by plants. Nature (bond.), 212, 638-40.
Barrett, J. T. (^1947). Observations on the root endophyte Rhizoygus in culture. Phytopathology., 37, 359-60.
____ (1958). Synthesis of mycorrhiza with pure cultures of Rhizophagus. Phytopathology, 48, 391.
_ — (1961) Isolation, culture, and host relation of the phycomycetoid vesicular-arbuscular mycorrhizal endophyte Rjiizophagus. Recent advances in Botany, 2 . 1725-7. University of Toronto Press.
-’.v.is, G. T. S. (1959). Effect of vesicu lar-arbuscular mycorrhiza on growth of Griselinia littoralis, (Cornaceae). New Phvtol.. 58, 274-3ÔT
— — j1?62- Rhizophacus. The catholic symbiont. •>ust. J. Sei. . 25, 195-200. 143
Baylis, G. T. S, (1967). Experiments on the ecological Significance of Phycomycetous mycorrhizas. New Phytol., 66,231-43.
______, McNabb R. F. R ., and M orrison, T. M. (1963). The Mycorrhizal nodules of Podocarpus. Trans. Br. myco 1, S o c., 4&, 378-84.
Becking, J. H. (1963). Nitrogen fixation and mycorrhiza in Podocarpus root nodules. PI. Soil, 23, 213-26.
Bergersen, F. J, and Cost in, A. B. (1964). Root nodules of Podocarpus lawrencei and their ecological signif icance. Aust. J. biol. Sci., 17, 44-8.
______and Turner, G. L, (1967). Nitrogen fix a tio n by the bacteroid fraction of breis of soybean root- nodules. Biochim. Biophys. Acta., 14, 507-15.
Bieleski, R. L. (1959). Factors affecting growth and distribution of the kauri (Agathis australis Salisb.). Aust. J. Bat., 2, 252-94*
Bond, G. (1959). The incidence and importance o f b io lo g ic a l fixation of nitrogen. Advan. Sci., 15, 382-6.
(1963). The root nodules of non-leguminous angiosperms. Symp. Soc. gen. Microbiol., 13, 72-91.
(1967). Fixation of nitrogen by higher plants other than legumes. A. Rev. PI. Physiol., 107-26.
Bottomley, W. B. (1913). The root nodules of the Podocarpeae. Rep. Br. Ass. Àdvmt. Sci., 82, 679. Boureau, E. (1939). As cited by Wilcox, 1962.
Bowen, G. D. and Rovira, A. D. (1966). Microbial factor in short term phosphate uptake studies with plant roots. Nature, (Lond.), 211„ 665-6.
Brown, G. L. and Gifford, E. M., Jr. (195$)• The relation of the cotyledons to root development of pine embryos grown in vitro. PI. Physiol.. 33. 57-64.
B u r g e f f , H. (1909). As cited by Mosse, 1963.
Clov/es, F. A. L. (1951). The structure of mycorrhizal roots of Fagus sylvatica.New Phytol.. 50. 1-16.
Coc k a y n e , L. (1921). The vegetation of New Zealand. Leipzig.
Cronshaw, J. and Bouck, G. B. (1965). The fine structure of differentiating xylem elements. J. Cell Biol.. 24. 415-31.
D a l l i m o r e , W. and Jackson, A. B. (1966). A Handbook of Coniferae and Ginkgoaceae. 4th Edition Revised by Harrison, S. G., Edward Arnold (Publishers) Ltd. London. 1966.
Dandie, A. K. (1968). M. Sc. Thesis, University of Sydney.
Daugherty, L. H. (1963). Triassic roots from the Petrified Forest National Park. Am. J. Bot., ¿0, 802-5« 145
Davenport, H. E- (i960). Haemoglobin in the root nodules of Casuarina cunninghamiana. Nature, (Lond.), 186,653-4. r Dietrich, K. (1924). Uber die Kulture von Embryonen ausserhalb des Samens. F lora , 117, 95-7.
Dowding, E. S. (1959). Ecology of Endogone. Trans. Br. mycol. Soc., 42, 449-57.
tl M M Egle, K. and Munding, H. (1951). Uber den gehalt an Haminkopen in den Uurzelknoliehen von Nicht-Leguminosen. Natnrwissenchaften. , 38, 548-9.
Ferreira Dos Santos. (1947). As cited by Allen and Allen, 1965.
Furman, T. E. (1964). Mycorrhizal nodules of Podocarpus. Abstract in Bull, ecol. Soc. Am., 45, 151.
Gerdemann, J. 7. (1955). R elation o f a large s o il-b o r n spore to phycomycetous mycorrhizal infections. Mycologia, 47, 619-32.
______( l 9 6 l ) . A sp ecies o f Endogone from com causing vesicular-arbuscular mycorrhiza. Mycologia, 53, 254-61.
______(1964). The effect of mycorrhiza on the growth of maize. Mycologia, 56, 342-9.
______(1965). Vesicular-arbuscular mycorrhizae formed on maize and tu lip tre e by Endogone fa s c ic u la ta . Mycologia, 57, 562-75. (Gerdemann, J . W . , and Nicolson, T. H. (1963). Spores of mycorrhizal Endogone species extracted from soil by wet-sieving and decanting. Trans. Br. mycol. Soc. 46, 235-44.
(Goldacre, P.L. (1959). Potentiation of lateral root induction by root initial in isolated flax roots. Mist. J. biol. Sci.. 12. 3S8-94*
(Grefinall, G. M. (1963). The mycorrhizal endophytes of Griselinia littoralls (Cornaceae). N. Z. J. 3ot.« 1 , 3Ö9-400.
(Guttenberg, H. von. (1940). Der primäre Bau der Angiospermenwurzel. In Handbuch der Pflanzenanatomie. Samenpflanzen Bd. VIII, Lief, 39. ed. Linsbauer, K., Gebrüder . Borntraeger, Berlin. ______(1941). Der primäre Bau der Gymnospermen wurzel. In Handbuch der Pflanzenanatomie. Samenpflanzen Bd. VIII, Lief, 41. ed. Linbauer, K., Gebrüder Borntraeger, Berlin. ______(1943). Die physiologischen scheiden. In Handbuch der pflanzenanatomie. Samenpflanzen Bd. V. Lief, 42. ed. Linsbauer, K., Gebrüder Borntraeger, Berlin.
Hapler, P. K. and Newcomb, E. H. (1963). The fine structure of tracheary xylem elements arising by redifferentiatiation of parenchyma in wounded Coleus stem. J* exp. Bot.. 1 4 > 496-503* 147
Harley, . L. (1959). The Biology of Mycorrhiza. A Plant Science Monograph. Leonard Hill (Books) Ltd., London.
___ (1965). Mycorrhiza. In Ecology of So il-Borne Plant Pathogens p. 218-30. ed. Baker, K. F., and Snyder, W. C., University of California Press, Berkeley.
H iltn er, L. (1903). Beitrage zur Mykorhizafrage. I. Über, die biologische und physiologische Bedeuntung den endotrophen Mykorhiza. Naturw. 2. Land-U-Forstw., 1, 9-25.
Hooker, J . D. (1854). On some remarkable spherical exostoses developed on the roots of various species of Coniferae. Proc. Linn. 6oc., 2, 335-6.
Janse, J. M. (1897). Les endophytes radicaux de quelques plantes javanaises. Annls. Jard. bot. Buitenz., 14, 53-201.
.Johansen, D. A. (1940). Plant Microtechnique. McGraw-Hill Co., New York.
.Jones, F. R. (1924). A mycorrhizal fungus in the roots of legumes and some other plants. J. argric. Res., 29. 459-70.
Kay, D. H . (1965). Techniques for Electron Microscopy« Blackwell Scientific Publications. Oxford.
Khan, A. G. (1967). Podocarpus root nodules in sterile culture. Nature. (Lond.), 215, 1170. Khan, A. Gr. (1968a). Effects 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, 372-3. ~
__ (1968b). Effect of tempaerature, gibberellic acid and indolylacetic acid on root and shoot growth of cuttings from Podocarpus lawrencei Hook f. Aust. J. biol. Sci. , 21_, 573-7*
Keilin, D. and Smith, J. D. (1947). Haemoglobin and nitrogen fixation in the root nodules of leguminous plants. Nature, (Lond.)., 159* 692-2+.
Kelley, A. P. (1950). Mycotrophy in plants. Waltham: Chronica Botanica.
Kondo, T. (1$3"0* Zur kenntniss des N-gehaltes des mykorrhiza- knollchens von Podocarpus macrophylla D. Don. Bot. Mag., ¿ 5 , 495-501.
Laing, E. V. (1923). Tree-Roots: Their action and develop ment. Trans. R. Scott. Arboric. Soc., 2 Z, 6-2 1 .
Lange, R. T, (19 6 6 ). Bacterial symbiosis with plants. Symbiosis. 1_, Chapter 3 . Academic Press Inc. N.x.
LaRue Carl, D, (1936), The growth of plant embryos in culture. Bull. Torrey Bot. Club,6 3 , 365-8 2.
Lihnel, D. (1939). As cit§d by Mosse, 19 6 3 .
Lyford, W. H. and Wilson, B. F. (1 962+). Development of the root system of Acer rubrum L. Harvard Forest Paper JJo. 1 0 . 149
McKee, H. S. (1962). Nitrogen Metabolism in Plants. Clarendon Press. Oxford.
McLuckie, J. (1923). Studies in symbiosis. III. Contribution to the morphology and physiology of the root^nodules of Podocarpus spinulosa and P. elata. Proc. Ijinn. Soc. N. S. W ., 4cJ, 82-93.
Heyen, J. (1829). Uber das Herauswachsen parasitischer Gewächse aus den Wurzeln anderer Pflanzen. Flora, (Jena), 12./ 49-64.
Mollenhauer,. H. H. and Whaley, W. G. (1962). A secretory function of the Golgi apparatus in certain plant cells. In Fifth International Congress for electron Microscopy. New York Academic Press, Inc., 2, YY-3.
______(1963). An observation on the functioning of the Golgi apparatus. J. cell Biol., H7, 222-5.~
Morrison, T. M. (1954). Uptake of phosphorus-32 by mycorrhizal plants. Nature, (Lond.), 174, 606.
_____ (1962). Absorption of phosphorus from soil by mycorrhizal plants. New Phyto1., 61, 10-20.
_____ and English, D. A. (1967). The significance of mycorrhizal nodules of Agathis australis. New Phytol., 66, 245-50.
iM-osse, B. (1956). Fructifications of an Bndogone species causing endotrophic mycorrhizas in fruit plants. Ann. Qot., Lond. N.S., 20, 349-62. 150
‘Mosse, B. (1959a). The regular germination of resting spores and some observations on the growth requirements of an Endogone sp. causing vesicular-arbuscular mycorrhiza. Trans. Br. mvcol. Soc.. 42. 273-236« ______(1959b). Observations on extramatrical mycelium of a vesicular-arbuscular endophyte. Trans. Br. mycol. Soc.. 1 2 . 439-43*
.(1962). The establishment of vesicular-arbuscular mycorrhiza under aseptic conditions. J. gen. Microbiol. 27. 509-20.
.(1963). Vesicular-arbuscular mycorrhiza: an extreme form of fungal adaptation. In Svmp. Soc. gen. Microbiol 11, 146-70
IMuller, H. (1906). As cited by Wilcox, 1962.
'Narayanaswami, S. and Norstog, K. (1964). Plant embryo culture. Bot. Rev.. 30, 537-023.
'Neill, J. C. (1944). Rhizophagus in Citrus. N. Z. J. Sei. Tech « A., 21, 191-201.
¡Nicholas, D. J. D. (1965) • Influence of the rhizosphere on the mineral nutrition of the plant. In Ecology of Soil- Borne Plant Pathogens, p. 210-17. ed Baker, K. F., and Snyder, W. C., University of California Press, Berkeley.
INicolson T. M. (1959 ). Mycorrhiza in the Gramineae. 1. Vesicular-arbuscular endophytes with special reference to the external phase. Trans. Br. mvcol. Soc.. 41, 421-33. 151
Nicolson, T. H. (1967). Vesicular-arbuscular mycorr' iza - -a universal plant symbiosis. Sci. Prog. Oxf.. 55. 561- 81.
Nobbe, F.. and Hiltner, L. (1899). Die endotrophe Mykorrhiza von Podocarpus und ihre physiologische Bedeutung, Landwirts. Versuchs. Stationen.. 51. 241-45.
Noelle, W. (1910). Studien zur vergleichenden Anatomie und morphologie der Koniferenwurzeln mit Rucksichtauf die Systematik. Bot. Ztg.. 68, 169-266
O ’Brien, J. E. and Fiore, J. (1962). Ammonia determination by automatic analysis. Wastes Engng..July. 1962.
Parker, R.N. (1932). Casuarina root nodules. Indian Foresten ¿8, 362-4.
Petri, L. (1903). Ricerche sul significato morfolofico e fisiologico dei prosporidi (sporangi oli di Janse) nelle micorize endotrophiche. Nuovo G. bot. ital.. 10. 541- 62. —
Phillips, J. (1932). Root nodules of Podocarpus. Ecology. 13. 189-95.
Plaut, M. (1909). As cited by Romberger, 1963.
______. (1910). As cited by Romberger, 1963.
______. (1918). As cited by Romberger, 1963.
Porter, K. P. and Machado, R. D. (1960). Studies on the endoplasmic reticulum. IV, Its fern and distribution during mitosis in cells of onion root tip. J. Biophys. Biochem. Cytol.. 2« 167-80 152
Prat, H. (1926). Etude des mycorrhizas du Taxus baccata. .vnnls. Sei. nat. Bat., 8, 141-62.
RaghavGu, V. and Torrey, J. G. (1963). Growth and morphogenesis of globular and older embryos of Capsella in culture. Am. J. Bot., 50, 540-51.
Aomberger, J. A. (1963). Meristems, growth, and development in woody plants. U.S. Dept, of agriculture. Tech. Bull. No. 1293.
II II Russow, E* (1375). Betrachtuqen tlber das Leitbundel-und Grundqewebe aus vergleichend morphologischen und phylogenetischen Gesichtspunkt. Schnakenburg's Anstalt, Dorpat.
Sahni, B. (1920). On the structure and affinities of /icmooyle oancheri Pilger. Phil. Trans. R. Soc., 210, 253-310.
.Saxton, '7. T. (1930a). The root nodules of Podocarpaceae. S. Af r . J. Sei., 27, 323-5.
_____ (1930b). Notes on Conifers. VII. Pherosohaera hookeriana archer. Ann. Bot. Lond. K.S., 44, 957-63.
Schaede, R. (1943). Die Symbiose in den Uurzelknollchen der Podocarpeen. Planta (Berl.), 33, 703-20. iSchopf, J. M. (1943). The embryology of Larix. Illinois Biol. Monoqr., 19, 1-97.
¿Shibata, K. (1902). Cytologische Studien über die endotrophen Mykorrhizen.. Jb. wiss.. Bot., 37, 643-84. 153
Spratt, E. R. (1912). The formation and physiological significance of root nodules in the Podocarpineae. Ann. Bot. Lond. , N..S. , 26, 801-14.
Uemura, S. (1964). Isolation and properties of microorganisms from root nodules of non-leguminous plants, review with extensive hihliography. Bull. Govt. Forestry Expt. Sta. (Japan)., 167, 59-91. van Tieghem, P. (1870). As cited hy Becking, 1965.
(1888). As cited hy Sahni, 1920.
Varley, J. A. (1965). Automatic methods for the determination of nitrogen, phosphorus, and potassium in plant, material. Analyst., 91 » 119-26. von Alten, H. (1909). As cited hy Wilcox, 1954. von Tuheuf, C. (1896). As cited hy Nobbe and Hiltner, 1899.
Whaley, W. G., Mollenhauer, H. H., and Kephart, J. E. (1962). Developmental changes in cytoplasmic organelles. In Fifth International Congress for Electron Microscopy. New York, Academic Press, Inc., 2, W-12.
______, and Leech, J. H. (i960). The ultrastructure of the meristematic cells. Am. J. Bot., 47» 401-50.
White, P. R. (1943). A Handbook of Plant Tissue Culture. Jacque Cattell Press: Lancaster, Pa.
Wilcox, H. E. (1954). Primary organization of active and 154
dormant root of noble fir, Abies procera. Am. J. Bot. t 41 . £12-21 .
Wilcox, H. E. (1962). Growth studies of the root of incense cedar, Libocedrus decurrens. I. The origin and development of primary tissues. Am. J. Bot.. 49. 221- 3 6 .
(1964). Xylem in roots of Pinus resinosa Ait. in relation to heterorhizy and growth activity. In The Formation of Wooc^in Forest trees. ed. Zimmermann, M. H., Academic Press, New York.
(1968). Morphological studies of the root of red pine, Pinus resinosa. II. Fungal colonization of roots and the development of mycorrhizae. Am. J. Bot. 25, 6&6-700.
Wooding, F. B. P. and Northcote, D. H. (1964). The devlopment of the secondary wall of the xylem in Acer pseudoolatanus. J. cell Biol.. 23. 327-37.
Yeates, J. S. (1924). The root nodules of New Zealand pines. N. Z. J1. Sci. Technol.. 2, 121-24.
Young, H. E. (1940). Mycorrhizae and growth of Pinus and Araucaria. The influence of different species of mycorrhiza-forming fungi on seedling growth. Aust. Inst. ap-ric. Sci., 6, 21-5