Proc. Indian Acad. Sci., VoL 85 El, No. 1, 1977, pp. 3_4-41

P r e l i m i n a r y s tudies o n the d e v e l o p m e n t a n d s t ruc tu re o f r o o t nodu l e s in Casuarina equisetifolia L.

SHRI KANT AND H. S. NARAYANA

Department of Botany, University of Rajasthan, daipur 302004

MS received 9 August 1976; in revised fo~m 30 September 1976

ABSTRACT

The structure and development of the root nodule of Casuarina equi- setifolia have been studied.

1. INTRODUCTION

THE root nodules of non-legumes are as important as those of legumes in the nitrogen economy of soil. The causal organism and the major events in the development and structure of leguminous root nodules are fairly wel 1 known. However, these aspects are not understood well in non-leguminous root nodules. Earlier investigations in this regard were due to Hawk~ and Fraymouth, 1 Fletcher 2 and Bond a with special referev_ce to morphology and significance of root nodules of Elaegnus, t-Iippophae, Alnus, Myrica anct Casuarina. The available information with regard to the mode of infection, the structure and branching of effective and ineffective nodule, the spread of infection and the degeneration of infected cells in non-leguminous root nodules in general and in root nodules of Casuarina in particular, is very scarce. The work of Aldrich-Blake ~ and Mowry 5 refers only to the nature of causal organism and the significance of root nodules in nitrogen nutrition of Casuarina. In this paper a preliminary account of the development and structure of root nodules of Casuarina equisetifolia is presented.

2. MATERIAL AND METHODS

Nodules of Casuarina equisetifolia of different ages were obtained from the plants raised in field soil, sand and test-tube cultures on agar deep inocu- lated by the suspension of crushed nodules of Casuarina equisetifolia in water (30 gm of nodules in 100 ml of water). They were fixed in Navaschin's

34

ROOT NODULES IN Casuarina equisetifolia L. 35

aqueous fixative for 5-15 days, washed in water and stored in 70~o alcohol. The nodules of different sizes were examined under microscope to ensure that they have many infected root hairs. The nodules were passed through alcohol and xylol and also tertiary butyl alcohol series, infiltrated and embedded in paraffin. Sections were cut in different planes at 8-10 microns thick and were stained by Heidenhain's haematoxylin and erythrosine, and safranin and fast-green combinations. The latter combination was found better.

3. OBSERVATIONS

In Casuarina equisetifolia the first nodule is observed on the primary root close to the hypocotyl region on the 26th day of the inoculation when the epicotyledonary shoot has five to six internodes. Subsequently more nodules are principally added on the primary root and secondarily on roots of other orders. To begin with these nodules are spherical. Later, they become flask-shaped with terminal growing point. They branch into a bilobed structure after 20 or 25 days of inoculation and become frequently branche6 into a spherical corolloid mass after 40 to 45 days of inoculation. The nodules are generally distributed on the roots of various orders to a depth of 3 to 4 inches close to the surface of the soil (plate I, figure 1).

The mode of entry of the causal organism is through the root hairs and is never directly through the surface layer. The root hairs which get infected may be long or short, branched (plate I, figure 3) or unbranched but coiled (plate I, figures 2, 4). The organism enters the root hair in the coiled region and forms infection threads. No definite cellulosic sheath is seen surrounding the infection thread. The infection threads are many and are interlaced in the root hair. They are rarely solitary. If the root hair is branched the infection threads extend even into the lobes of root hair. They generall'y grow towards the base of the root hair and penetrate the cortex subsequently (plate I, figure 5). The infected root hair, however, does not enlarge. Only its wall becomes thickened. As the nodule becomes old, the infected root hair gets detached and the hypodermal cells which contain the endophyte form the surface layer (plate 1, figure 6). When the infection threads penetrate the root cortex, the hypodermal cell may or may not divide and the infection thread passes through it into the deeper lying cortical tissue (plate I, figure 5). On the other hand, the sub-hypodermal cells of the root divide around the region "~ of infection. Their derivatives become thick-walled and them lumen become filled with tanniniferous substance as a defence reaction. Such a tanniniferous layer is formed all-round the nodule except in the region where the infection threads enter the cortex (plate I, figure 6).

B. 5--Jalt. 77

36 SHRI KANT AND H. S. NARAYANA

Text Figures 1--4 (d, drus~; end, endodermis; hyl, hypodermal layer; lrt I, lateral root of first order; lrt 11;

lateral root of second order; lrt co, latei al root cortex; nco, nodule cortex; n m, nodule meristem ; p, periderrn; rtst, root stele: sec xy, secondary xylem; secph, secondary lrhloem; shy 1, sub- hypodermal layer ; tc, tannin-filled ~ell; vb ; vascular bundle)

Figure 1. V.S. nodule showing different regions (semi-diagremmatic). Figure 2. Invasion of cells of nodule cortex by the infection threads. Figure 3. Infected and uninfected cells of the nodule cortex, note the uninfected cells with

tannin and druses. Figure 4. Vascular bundle at the base of the nodule showing secondary growth.

Shri Kant and H. S. Narayana Plate I

Proc. bufian Acad. Sci., Vol. 85 B, No. 1, 1977, pp. 34-41

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Figures 1-7 ( facing page 36 )

Shri Kant and H. S. Narayana Plate H

Proc. hMian Acad. Sci., I1"ol. 85 B, No. 1, 1977, pp. 34--4l

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8 -12 (facing page 37)

ROOT NODULES IN Casuarina equisetifolia L. 37

The loci of infection leading to nodule initiation are generally more than one through the root hairs. It may be confined to one side of the root and the root stele is excentric (plate I, figure 6), or the infection may be all-round and the root stele is centric.

The cells of the cortex through which the infection threads traverse are generally large. The penetration of the cortex by the threads is intracellular and the depth of penetration is up to the endodermis which is four to five cells deep. The endophyte induces the adjacent cells of the cortex to divide and enlarge and subsequently infects some of them initiating the nodule formation.

The infection thread is not smooth along its side. It is not enclosed by a cellulosic sheath either in the root hair or in the root cortex. The thread shows affinity with the nucleus of the host cell and moves towards it, often enclosing the latter without causing any damage (text-figure 2). The host nucleus, on the other hand, becomes amoeboidal and darkly-stained. In most cases the infection thread either remains uniform (text-figure 2; plate 2, figure 10) or forms slight swelling close to the cell wall through which the endophyte passes into the adjacent cell. The infection threads may be small or large in size. Both the types may be presertt nearby in the same nodule.

The first formed nodule on the roots of Casuarina equisetifolia is spherical consisting of only the infected cortex representing the endophytic zone. The central vascular cylinder of the root itself functions as the nodule vascula s bundle and the cortex of root functions as the cortex of nodule along with the endophyte. There is no nodule meristem. This spherical nodule gives rise to a branch which is flask-shaped or cylindrical (text-figure 1) to begin with irregularly lobed (plate I, figure I) subsequently. The origin of this nodule branch in reality is the origin of a lateral root in the nodule region of the parertt root (plate I, figure 7) and is endogenous and pericyclic. The meristem of the lateral root very soon functions as the meristem of the emerged nodule (nodule branch) and produces not only the lateral root at the centre but also adds cells to the nodule cortex (plate II, figures 8, 9) which is the cortex of the parent root enclosing the lateral root (" nodule root "). Thus, the emerged nodule (nodule branch) consists of three regions: the nodule meristem which is really the meristem of the lateral root, the nodule cortex which is the infected cortex of the parent root and the central vascular cylinder which is the growing young lateral root possessing its own central vascular cylinder and a small cortex (text-figure 1, plate II, figure 8).

The nodule meristem is prominent at the distal end of the emerged nodule. It consists of a layer of small thin-walled cells each with a conspi-

38 SHRI KANT AND H. S. NARAYANA

cuous nucleus embedded in dense cytoplasm. Just behind the region of meristem there is the region of maturation where the cells produced by the nodule meristem enlarge and mature into components of nodule cortex at~d central vascular cylinder of the nodule (plate II, figure 9). Some of the newly added cells of the nodule cortex are invaded by the infection threads and the endophyte is released into them. In this way, the infection threads continue to move towards the distal end of the nodule as new cells are added to the nodule cortex by the nodule meristem.

The emerged nodule frequently branches. Each branch bears an endo- genously producedmeristem of lateral rootlet. This subsequently functions as the nodule meristem and causes the growth of the nodule branch. The branches of nodule are short and stumpy (plate I, figure 1) resembling the corolloid mass of roots of Cycas or mycorrhizal roots of Fagus. In this way the presence or absence of the nodule meristem and its activity determine the shape of the nodule.

The nodule cortex consists of infected parenchyma intermixed with cells filled with tannin or druses (text-figure 3). The extent of tannin- or dluses-filled cells to the infected parenchymatous cells is variable. Some nodules have high percentage of uniseriate tannin-filled in relation to the infectedcells. The uniseriate tannin-filled cells and the alternating infected parenchymatous zones are predetermined by the nodule meristem. The subhypodermal tanviniferous tissue all-round the nodule functions as barrier protecting the underlying cells of the nodule from the invasion of extraneous micro-organisms. In the large old nodules the hypodermal parenchymatous cells contribute to the phellogen which forms a few-layered phellem and phelloderm. The organization of this periderm may be discontinuous in regions where the surface layer of the nodule is ruptured or may be conti- nuous. The periderm, however, is not formed in young nodules.

The vascular cylinder of the nodule is di- to pentarch, exarch and radial. The secondary growth of the vascular cylinder is seen only at the base of the main nodule (text-figure 4) and not in other regions irrespective of the age of the nodule. The xylem archs may be united with the absence of pith or may be free with a few parenchymatous cells in between them representing the pith. The phloem patches are alternating with the protoxylem points. The tannin-filled cells may be associated with the phloem and the pith. The pericycle is parenchymatous and one-ceil thick. It is rarely two cells thick at certain legions. The endodermis is unilayered and tannin-filled. The structure of the vascular cylinder of mmdul¢ is similar to that of the mair~ root on which it is borne.

Root NODULES IN Casuarina equisetifolia L. 39

The divisions of cells iv. infected tissue of the nodule cortex is not obvious. The spread of endophyte in the host tissue is caused by the continuous inva- sion of new cells formed by the nodule meristem by the endophyte. The infection of the emerged nodule is ov.ly brought about by the spread of endo- phyte from the nodule of the parent root.

The infected cells enlarge more than their normal size. The nucleus slightly increases in size. It is spherical to begin with and becomes amoe- boidal and darkly stained subsequently. The uninfected cells are small and have comparatively thin cytoplasm and small nuclei. Some of the uninfected cells get filled with tannin and divide the infected zone into compartments. Others get filled with druses.

The process of degeneration of the endophytic zone starts from the base. The endophyte clumps iv. the beginning (plate II, figure 11) and subsequently disappears leaving absorption spots or spaces.

The ineffective nodules (plate II, figure 12) are very few in comparison to the effective nodules. The mode of infection in them is through the root hairs (plate 2, figure 12). The delimitation of nodular tissues conforms to that of effective nodule. The nodule meristem is distinct. It forms the nodule cortex and central vascular cylinder. At the proximal end the cells of the nodule cortex contain the endophyte released into the host cell while in the lemaining region they contain only the infection threads of the organism fiequently branching and invading the new cells formed by the nodule meristem. The infection threads are devoid of cellulosic sheath. The cells which are filled with; tannin and druses are very meagre. Even the causal organism is very little in quantity.

4. DrseussloN

Bond 3 has observed nodulation in Casuarina equisetifolia and C. cunning- hamiana between 26 and 35 days after inoculation. Similar result is obtained by the authors not only in the sand but also in the test-tube cultures on agar inoculated with the suspension of crushed nodules. Bond 3 has also reported pronounced deformation and branching of root hairs reminiscent of those in legume roots. However, he has not made any study of the infection process. Fletcher ~ considers the infection in Myriea gale to be through the root hair on the basis of similarity of the organism present in the root hair and the inner lying host cells. On the other hand, the present authors have not only observed deformation and branching of root hairs under the influ- ence of the inoculum but have also seen the presence of distinct infection threads iv. the coiled roo t hairs and their penetration into the root cortex causing nodule formation,

40 SHRI KANT AND H. S. NARAYANA

The nodule emergence in Myrica gale 2 is associated with great distur- bance in the cortical cells of the parent root unlike the lateral root which has a digestive cap and which consequently causes little disturbance. Further, in the early stages of the nodule in Myrica there is no protective cap of the nodule meristem and the origin of vascular strand from the meristem is not distinct. The meristematic cells are filled with tannin which renders the detection of endophyte difficult. However, in Casuarina equisetifolia the authors have observed no disturbance in the cortical cells of the parent root during the emergence of branches of nodule. The meristem of nodule is not tannin-filled and there is a clear zone of vascular strand originating from the meristem.

Secondary growth of the vascular cylinder is also seen at the base of the nodule in Casuarina equisetifolia. This observation is on similar lines reported in the perennial nodules of Alnus and Elaeagnus. 6

ACKNOWLEDGEMENTS

Our thanks are due to Prof. B. Tiagi, Drs. D. Singh and N. Chandra for encouragement and facilities. We are grateful to ICAR for financial support of the scheme.

S UMMARY

The structure and development of the root nodule of Cas..:arina equiseti- folia have been studied.

Both effective and ineffective nodules are produced although the latter are,very few. The former are pink, cylindrical or irregularly lobed and are present on roots of all orders, while the latter are small, spherical present on the roots of secondary ::nd subsequent orders and are not pink.

The causal organism e r e r s through the deformed root hairs and form many infection threads. The infection thread is rugged at the sides. No cellulosic sheath is seen either in the root hair or in. the cortical cells. The thread penetrates up to the evdodermis irrespective of the size of the cortex in the primary, secondary or tertiary roots. The spread of the organism is by the invasion of newly formed cells.

The nodule is the frequently branched infected root. The meristems of branched roots of different orders show restricted activity in nodule formation for a period. The nodule consists of three regions: the nodule meristem, the nodule cortex and the central vascular cylinder. The nodule meristem is the meristem of lateral root. The nodule cortex is the infected

ROOT NODULES IN Casuarina equisetiJblia L . 41

cor tex o f pa r en t roo t a n d the centra l vascu la r cy l inder is the growing young lateral root . The g rowth o f the nodule is b rough t a b o u t by the dis ta l mer i - s tem. The b ranch ing o f the nodule is no t by division o f me r i s t em in to segments but by endogenous deve lopmen t o f mer i s t em o f la te ra l root let . This mer i s t em funct ions as nodule me r i s t em a n d causes g rowth o f the nodu le branch. The nodule cor tex consists o f infected p a r e n c h y m a inter- mixed with tann in- or druses-filled cells. The infected cells a re enlarged and the nucleus o f the infected cells becomes a m o e b o i d a l and darkly-s ta ined. Some o f the uninfec ted cells a re filled wi th t ann in a n d divide the infected zone into c o m p a r t m e n t s . The others a re filled with druses.

The degenera t ion o f the infected cells is g radua l f r o m the vase upwards . The ineffective nodules a re smal le r and parasi t ic .

REFERENCES

J. Hawker, L. E. and Fraymouth, J. J., Gen. Microbiol. 5 369 t1951). 2. Fletcher, W. W., Ann. Bot. London 19 501 (1955). 3. Bond, O., Ann. Bot. London 21 373 (1957). 4. Aldrich-Blake, R. N., Oxford Forestry Memoirs 14 20 (1932). 5. Mowry, H., Soil Sci. 36 409 (1933).

6. Spratt, E., Ann. Bot. London 26 119 (1912).

EXPLANATION OF PLATES

Plate I

Figures 1-7. 1. Root bearing nodules. 2. Young nodule with coiled and infected root htirs. 3. Dzformed and branched root hairs. 4. Coiled root hair with many infection threads. 5. Penetration of root cortex by the infection threads from root hai~, see the three infection tlueads in the root hair. 6 T.S. spherical nodule with many loci of infection. 7. T.S. spherical nodule showing the emergence of nodule branch.

Plate I I

Figures 8-12. 8. R.l.s. mature nodule. 9. L.s. distal end of nodule. 10. Magnified view of infected cells, see the uniform size of the infection threads close to the cell wall. 11. Degenerating infected tissue. 12. T.s ineffective spherical nodule.