S. Afr. J. Bot., 1988,54(5): 491-495 491

SEM observations on cyanobacteria-infected cycad coralloid roots

D.C.N. Chang 1, N. Grobbelaar* and J. Coetzee2

'Department of Botany, University of Pretoria, Pretoria, 0002 Republic of South Africa and 2Electron Microscopy Unit, University of Pretoria, Pretoria, 0002 Republic of South Africa

1 Present address: Department of Horticulture, National Taiwan University, Taipei, Taiwan (10764), Republic of China

Accepted 17 May 1988

In Nostoc-infected coralloid roots of the cycads Encephalartos trans venosus Stapf & Burtt Davy and E pauciden­tatus Stapf & Burtt Davy the intercellular cyanobacteria are present in a narrow circular band between the outer and the inner cortex only. When the cyanobacterial density is very great, some intracellular infection is present. In uninfec­ted coralloid roots, cells with a high concentration of phenolic substances are evenly distributed throughout the outer and inner cortical layers, but in infected roots, such cells are restricted largely to the outer cortex. Cyanobionts newly isolated from cycad coralloid roots consist of cells arranged in filaments. Some large spherical structures, similar to those which each leads to the development of a new filament during the late heterocystous phase of the nostocacean life cycle, are found in the roots. On some cyanobacterial cells other micro-organisms are visible. These bacteria-like micro-organisms are probably the heterotrophic bacteria that usually occur in cultures of coralloid root endophytes.

In Nostoc-ge'infekteerde koraalwortels van Encephalartos trans venosus Stapf & Burtt Davy en E. paucidentatus Stapf & Burtt Davy is die intersellulere sianobakteriee beperk tot 'n area wat tussen die binneste en die buitenste korteks gelee is. Waar buitengewoon baie sianobakteriee teenwoordig is, mag intrasellulere infeksie voorkom. Selle wat ryk is aan fenoliese stowwe, is bykans eweredig deur die binneste en buitenste kortekslae van onge'infekteerde wortels versprei, maar is tot die buitenste kortekslae van ge'infekteerde wortels beperk. Onmiddellik na isolering uit die koraalwortels bestaan die sianobionte uit selle wat in filamente gerangskik is. Groot sferiese strukture, soortgelyk aan die wat tydens die laat-heterosist-stadium in die Nostoc-Iewensiklus elk aan 'n filament oorsprong gee, is in die wortels aanwesig. Mikro-organismes, wat moontlik die heterotrofiese bakteriee is wat gewoonlik in kulture van die endofiete van koraalwortels teenwoordig is, is op sommige filamente sigbaar.

Keywords: Coralloid roots, cyanobacteria, cycads, intracellular, phenolic substances

*To whom correspondence should be addressed

Introduction Although the structural changes that occur in cycad coral­loid roots after infection by cyanobacteria have been inten­sively studied (Lindblad et ai. 1985 and references cited by them), there are still some aspects that require additional investigation. Three of these that have received attention in this study concern (a) the intracellular occurrence of the cyanobiont, (b) the occurrence and distribution of root cells that are rich in phenolic compounds and ( c) the presence of endophytes other than cyanobacteria in the coralloid roots .

Cyanobacteria invade the coralloid roots of cycads, but they usually remain restricted to the intercellular spaces of the central aerenchymatous cortex (Wittmann et ai. 1965; Grilli Caiola 1974). Nathanielsz & Staff (1975) were how­ever the first authors to claim that the endophyte can also occur intracellularly. They worked on the coralloid roots of Macrozamia communis, using light microscopy. Obuko­wicz et al. (1981) published the first electron micrographs of the intracellular occurrence of cyanobacteria in cycad coralloid roots. In the coralloid roots of Encephalartos species, the endophyte has thus far apparently been found only intercellularly (Grilli Caiola 1975).

Tannins (Wittmann et al. 1965) or phenolic compounds generally (Obukowicz et al. 1981), were reported to occur in the coralloid roots of Macrozamia communis and Cycas revoluta respectively. Nothing appears to be known about the distribution of phenolic compounds in the coralloid roots of other cycads.

Early workers (Schneider 1894; Life 1901; Bottomley 1909; McLuckie 1922) claimed that in addition to cyano­bacteria, other bacteria also inhabit the coralloid roots of cycads. Grilli Caiola (1974, 1975) however specifically mentions that she did not observe endophytes other than cyanobacteria. Grobbelaar et al. (1987) found that all cyanobacterial isolates from surface-sterilized coralloid roots contain other bacteria, which suggests that apart from

the cyanobiont, other bacteria also commonly occur within the coralloid roots of cycads.

Due to the fact that only one scanning electron micro­scopic study of a cycad-Nostoc symbiosis (Lindblad et al. 1985) appears to have been carried out previously, this technique was applied in an attempt to gain a better under­standing of the structural aspects of cycad coralloid roots with special regard to the three aspects referred to above.

Methods Coralloid roots of Encephalartos trans venosus Stapf & Burtt Davy and E. paucidentatus Stapf & Burtt Davy were collected in March 1987 from plants growing in the gardens of the University of Pretoria. Samples were taken close to the stem of plants where the roots were readily detected as a coralloid mass protruding slightly above soil level. Uninfec­ted and infected roots were gathered from different root clusters of the same plant. In the case of the infected roots, the presence of the cyanobiont can clearly be seen with the unaided eye as a dark green ring in a transverse section of the root.

Root segments, approximately 10 mm long, were fixed for one week at room temperature in 2,5% glutaraldehyde in 0,1 mol dm-3 phosphate buffer at pH 7,4. This extended time in the aldehyde was found to be necessary to com­pletely stabilize the phenolic substances in the cells. After the initial fixation in glutaraldehyde, the segments were washed in the same buffer (3x, 10 min each) and cut into thin transverse sections by hand. These sections were then post-fixed in 0,5% aqueous OS04 for 2 h at room tem­perature (Coetzee & van der Merwe 1985), rinsed in water and dehydrated with acetone. Freon (1,1,2-trichlorotri­f1uroethane) was used as intermediate before critical point drying with CO2 ,

Cyanobacteria were isolated by sectioning fresh roots by hand and suspending the sections in autocIaved distilled

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water for about 30 min to permit some of the cyanobacteria to migrate out of the root tissue into the water. The result­ing cyanobacterial suspension was then filtered through a membrane filter with O,2-lLm pores (Sartorius type SM 11607) and the retained material fixed in 2,5% glutaralde­hyde for 1,5 h. In some cases the fresh sections were directly fixed in 2,5% glutaraldehyde. During the initial time in the fixative some cyanobacteria also emerged from the cut sur­face of the sections. The fixed material (sections or mem­brane filters) was post-fixed in 0,5% OS04 for 1 h, dehydra­ted in acetone and critical point dried.

Hand sections of fresh roots were embedded in glycol methacrylate (Feder & O'Brien 1968) to observe the dis­tribution of phenolic compounds in the coralloid roots . The cells containing phenolic compounds turned dark brown during this embedding procedure.

Special precautions were taken in order to remove the enveloping mucilage layer of the cyanobionts which severe­ly obscures details of their morphology. Fixing the cycad roots for 1 week or even longer proved very effective in that (1) this eliminates most ofthe mucilage and therefore yields much cleaner specimens, (2) it prevents the cyanobionts from migrating from the coralloid root samples into the sur­rounding fluid in large numbers , and (3) it makes root cells which are rich in phenolic compounds much more conspi­cuous at light and SEM levels.

Results and Discussion Low magnification views of transverse sections of non­infected (Figure 1) and infected roots (Figure 2) show that the infected roots have a larger diameter and that the cyanobacterial zone is found at the junction of what appears to be the outer and inner cortex (Figure 2). Wittmann et at. (1965), however, claims that the coralloid roots of Macrozamia communis have a persistent root cap and that the cyanobacterial zone occurs at the junction of the cortex and the tissue of this persistent root cap.

During prolonged fixation, the root cells which are rich in phenolic compounds develop a brownish colour which makes it easy to locate these idioblasts in a root section by light microscopy. In un infected roots, the tanniniferous cells are evenly distributed in the cortex, with some such cells also present in the stele (Figure 1). In infected roots, however, the number of cells containing phenolic com­pounds in the outer cortex (tissue exterior to the cyanobac­terial zone) is much higher than in the inner cortex (Figure 2) . During prolonged fixation, the contents of the cells that are rich in phenolic compounds also become solidified. The resultant slightly porous masses can easily be observed in the SEM (Figure 3). Phenolic compounds are widely distri­buted amongst plants. Many possible functions have been attributed to these ubiquitous compounds, but they are generally believed to be involved in static or dynamic de­fence mechanisms against pathogens (Swain 1977). The re­sults in Figures 1 & 2 suggest that some kind of relationship exists between the distribution of phenolic cells in coralloid roots and the presence of cyanobionts.

Crystalline and phenolic compounds (Figure 4) are found inside the root cells of both infected and non-infected coralloid roots. Starch grains, when present, are usually found in the uninfected coralloid roots only (Figure 5). The demands of the endophytic cyanobacteria for carbohy­drate, both for growth and for nitrogen fixation, probably explains the lack of starch grains in the infected coralloid roots.

Most of the cyanobacteria in the roots appear to exist as single cells or as short chains (Figures 6 & 7) . The cyano­biont appears to occur mainly intercellularly in a girdle (Figure 2) which is radially traversed by widely spaced

S.-Afr. Tydskr. Plantk., 1988,54(5)

elongated cortical cells. Root cells which adjoin the cyano­bacterial zone occasionally contain intracellular cyano­biont cells (Figures 8 & 9). Although the intracellular presence of cyanobacteria was rarely observed, there can be no question about the fact that cyanobacteria do occasionally occur intracellularly . The presence of structures that are most

1 1 mm

Figure 1 Light micrograph of a transverse section of an uninfected root of E. trans venosus. Cells containing phenolic compounds appear black.

• • . \ • .. •

2· t , . 1mm

Figure 2 Light micrograph of a transverse section of an infected root of E. transvenosus. CZ: cyanobacterial zone, P: periderm. Cells con­taining phenolic compounds appear black.

Figure 3 Phenolic cell contents (P) solidified by prolonged fixation . Cortical cells of E. transvenosus.

S. Afr. J . Bot., 1988, 54(5)

probably nuclei of cortical cells (Figures 10 & 11), or crys­talline inclusions (Figure 7) , in the same compartment as that in which the cells of the cyanobiont are found, must be considered as providing additional evidence for the intra­cellular location of at least some of the cyanobiont cells.

The outer surface of a cycad coralloid root, at some dis­tance away from the apex where periderm development has not yet commenced , is shown in Figure 12. The loosely packed surface cells are not typical of root epidermal cells

Figure 4 Transverse section of an infected coralloid root of E. trans­venosus . CZ: cyanobacteria-infected zone , P: solidified phenolic cell contents , C: crystalline inclusion.

Figure 5 Starch grains (S) in uninfected roots.

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and are rather suggestive of the arrangement of the super­ficial sloughing cells of the root cap (Milindasuta 1975). This observation supports the contention of Wittmann et at. (1965) that the root cap tissue of the coralloid roots of cycads is retained as the outermost layer of the root prior to periderm development.

The cyanobiont within E. transvenosus coralloid roots appears to consist of isolated cells or cells arranged in short filaments (Figures 6, 7 & 9). However, some of the

Figure 6 Intercellular cyanobacteria in roots of E. transvenosus.

Figure 7 Crystal (C) surrounded by cyanobacteria in the vicinity of the cyanobacterial zone in E. transvenosus.

Figures 8 & 9 Cortical root cells adjacent to the main cyanobacterial zone with intracellular cyanobacterial cells (arrowed) (E. transvenosus) .

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cyanobacteria isolated from E. paucidentatus consist of long filaments (Figure 13). Whether the length of the fila­ments is representative of different stages in the life cycle of the cyanobacterium, or is due to differences between sepa­rate Nostoc species or strains, remains to be investigated. Large spherical structures, often with a visible pore, are present (Figure 13) amongst the isolated cyanobacteria as well as in the cyanobacterial zone of the coralloid roots.

Figure 10 Intracellular nucleus-like structure (N) in a cyanobacteria­free cell of a root of E. trans venosus.

Figure 11 Nucleus-like structure (N) and cyanobacteria (CY) in the cyanobacterial zone of E. transvenosus.

Figure 12 Loosely packed outer cells of the root surface of E. trans­venosus.

S.-Afr. Tydskr. Plantk., 1988, 54(5)

Grilli Caiola & Pellegrini (1980) described structures found in the late stage of the heterocystous phase of the life cycle of Nostoc spp. which are very similar to the spherical bodies seen in Figure 13. It is not clear whether the cyanobacterial filaments that are in contact with the spherical bodies origi­nate from within these bodies (Figure 13).

It was commonly found that a few small, spherical bodies are attached to the outer surface of some of the cyanobac­terial cells (Figures 13-15). Occasionally a cluster of such small spherical bodies appears to be associated with the cyanobacteria (Figure 16). The size of these small (less than 1-fLm diameter) spherical bodies suggests that they might be heterotrophic bacteria associated with the cyanobac­teria. This could explain why cyanobacterial cultures, ob­tained from surface-sterilized infected coralloid roots, are habitually contaminated with heterotrophic bacteria (Grobbelaar et al. 1987).

Acknowledgements The financial assistance from the National Science Council of the Republic of China and the Foundation for Research Development of the Council for Scientific and Industrial Research of South Africa is gratefully acknowledged.

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Figure 13 Long filament of a Nostoc sp. with large spherical structure (S), isolated from E. paucidentatus roots.

Figure 14 Chains of Nostoc cells with terminal heterocyst (H), isola­ted from E. paucidentatus roots.

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Figures 15 & 16 Cyanobacterial cells with adhering small bodies, isolated from roots of E. transvenosus.

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