G. Bond

Taxonomy and distribution of non-legume nitrogen-fixing systems

In this chapter more attention will be paid to plants bearing Alnus-type nodules than to other non-legume nitrogen-fixing systems, partly because it has been with the former that the author’s main research interest has lain, but also for the reason that of the various systems to be considered in the chapter, the nitrogen fixation associated with Alnus-type nodules seems to offer the best prospects for exploitation.

Effective and Ineffective Root Nodules in Myrica faya

In cross-inoculation trials, inocula containing the nodule endophytes of Myrica gale, M. cerifera, M. cordifolia and M. pilulifera respectively were applied to the roots of young plants of M. faya Ait. growing in nitrogen-free culture solution. All four inocula induced nodule formation, and except where the M. gale inoculum had been used the nodules were of effective type and enabled the plants bearing them to grow nearly as well as other M. faya plants associated with the normal endophyte. The nodules induced by the M. gale endophyte were very numerous, but remained small and fixed no significant amount of nitrogen, and were thus ineffective. Light and electron microscopy showed that in the effective nodules induced by the normal endophyte or by that of M. cordifolia, the endophyte was confined to a layer 1-2 cells deep near the middle of the nodule cortex, and that in respect of the width of the hyphae and their production of club-shaped internally subdivided vesicles, the endophytes resembled closely those in the nodules of the few other species of Myrica that have been studied by modern methods of microscopy. In ineffective nodules the disposition of the infected cells was unchanged, but within the cells only a sparse development of the endophyte was observed, and no vesicles were found. The finding that nodules lacking vesicles showed little or no fixation is consistent with other evidence that the vesicles normally produced by non-legume nodule endophytes are the main site of nitrogen fixation.

Effect of nitrate-nitrogen on the nodule symbioses of Coriaria and Hippophaë

Nodulated plants of Coriaria arborea and Hippophaë rhamnoides, one year old and grown previously in a rooting medium essentially free of combined nitrogen, were grown for a further 2½ months in the presence in the rooting medium of 0, 10 or 25 mg nitrate-nitrogen per litre of culture solution. The nitrate was labelled with 15N. Plant growth was promoted by the supplied nitrogen, especially in Hippophaë, but nodule growth and nitrogen fixation per plant were depressed, the latter, at the highest level of nitrate, being only 31% (in Coriaria) and 61% (in Hippophaë) of the fixation in plants at zero nitrate level. Although the 15N penetrated (probably indirectly) into the nodules, in both species and at both levels of nitrate the enrichment shown by the nodule tissues as a whole was only about one-fifth of that shown by the rest of the plant. This finding would be explained if four-fifths of the nodule nitrogen was in the endophyte and was wholly unlabelled nitrogen fixed from the atmosphere, while the remaining one-fifth was in the uninfected cells and these were in equilibrium with the tissues of the rest of the plant and carried the same 15N label. The implications of this hypothesis are considered.

Diurnal changes in nitrogen fixation in the root nodules of Casuarina

Measurements were made of the rate of nitrogen fixation and of the carbohydrate content of nodules detached at different times of day from plants of Casuarina cunninghamiana growing in water culture under semi-natural conditions in a greenhouse lit by daylight and subject to considerable diurnal variation in temperature also. The 15N method was used for the measurement of fixation. A distinction was made between ‘fixation potential’ - the rate shown by nodules incubated under 15N at a standard favourable temperature, and ‘actual fixation’, namely the rate shown by nodules incubated at the temperature prevailing in the greenhouse at the time. It was assumed that the values obtained for actual fixation, though known to be considerably below those prevailing in the nodules at the times of detachment, would be proportional to the latter. Actual fixation was low in the early morning, but soon rose and remained at a relatively high level for several hours centred about mid-afternoon, and then fell. A separate study showed that the rate of fixation increased steadily over the temperature range 10-36°C, and indicated that temperature fluctuations in the greenhouse were partly responsible for the diurnal changes in the rate of fixation. An additional factor, presumably light intensity acting through its effect on the supply of carbohydrates to the nodules, also appeared to be involved, but although evidence was obtained that during periods of low light or darkness the fixation potential was related to the level of nodular carbohydrates, over the part of the day when actual fixation was high the nodules showed a low carbohydrate content. The possible explanation of this situation is considered.

Some effects of combined nitrogen on the nodule symbioses of Casuarina and Ceanothus

Summary1. The effect of ammonium-nitrogen on the further growth and activity in fixation of nodules already present at the commencement has been studied inCasuarina cunninghamiana growing in water culture and inCeanothus velutinus var.laevigatus in Peralite culture.2. In Casuarina, at a low level of ammonium-nitrogen nodule growth remained similar to that in plants in nitrogen-free solution, but was stimulated in Ceanothus. In both genera nodule growth was strongly retarded at higher levels.3. Fixation of nitrogen fell continuously in Casuarina as the level of ammonium-nitrogen was increased, while that in Ceanothus was unaffected at a low level but markedly decreased at a higher one. These effects were compounded from the changes noted in 2 and a tendency for the efficiency of nodule tissues in fixation to fall in the presence of ammonium-nitrogen, though this was not always shown.4. In both genera but especially in Casuarina the growth of plants entirely dependent on nodule nitrogen was inferior to that of plants additionally supplied with ammonium-nitrogen. The reasons are discussed.5. The results indicate the effects likely to be produced in the field as the soil nitrogen level rises through the action of the nodules of these species.

Root-nodule formation in non-leguminous angiosperms

SummaryThe world-wide survey under the IBP of root-nodule formation in non-leguminous Angiosperms is progressing reasonably satisfactorily, and it is anticipated that when all the results have been collated a useful body of new data will be yielded.In recent studies, also forming part of the IBP, in the authors laboratory, the nodules of further species in the genera Alnus, Myrica, Ceanothus, Coriaria and Dryas have been examined for nitrogen-fixing properties, with positive results. Also the extent to which the nodule endophytes from species of Alnus and Myrica respectively are able to symbiose satisfactorily with other host species in the same genus has been investigated, and the conclusion reached that especially in Myrica there is very considerable specialisation among the endophytes.A marked diurnal variation in the rate of fixation of nitrogen in the nodules of non-legumes growing in a glasshouse lit by daylight has been found, with maximal rates being attained around midday. The implication is that this is the period of the maximal availability of carbohydrates in the nodules, but actual analyses have so far failed to reveal this.Analyses of the amino acid composition of the nodules in several genera have shown that except in Alnus, where citrulline is prominent, asparagine is in most cases the dominant amino acid.

IV.—The Effect of Malnutrition on Root Structure

1. Reference is made to experiments conducted by Flaskamper as a result of which he stated that the removal of the cotyledons at an early stage from seedlings of Phaseolus sp. and of Vicia Faba resulted in an elimination of the pith from the subsequently formed part of the root in both species, and further induced a reduction in the number of poles in the root of the second species. 2. The significance of such observations in a consideration of the theory of the Size Factor is pointed out. 3. The experiments have been repeated. The malnutrition resulting from the decotylation affected the varieties employed in the present work more adversely than with Flaskamper, and usually caused the death of the seedling after a shorter or longer period. 4. Sufficient evidence has been obtained, however, to throw very serious doubt on Flaskampers conclusions. The two reduction phenomena he described are now shown to occur in the normal development of the roots of these species, and there is no indication that their occurrence is affected by decotylation. 5. A teleological explanation, based on the theory of the Size Factor recently advanced by Bower, is made of this reduction in the number of protoxylem poles which occurs in the normal development of the roots of these plants. 6. The significance of the appearance of a pith in the basal parts only of these roots is discussed. In conclusion, I desire to express my indebtedness to Professor F. O. Bower, F.R.S., for his constant interest in this work, and to thank Dr S. Williams and other members of the Botany Department for much valuable help. Also my best thanks are due to the Executive Committee of the Carnegie Trust for a grant towards the expense of the illustrations both of the present work and those of a recent paper on “The Stem-Endodermis in the Genus Piper,” published in the Transactions of the Royal Society of Edinburgh .