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The study of the rhizosphere by scanning electron microscopy
So11 BIOI Btochrln
Vol 5, pp. 747-752.
Pcrgamon
Press 1973. PrInted m Great Britain
THE STUDY SCANNING
OF THE RHIZOSPHERE BY ELECTRON MICROSCOPY
R. CAMPBELL and A. D. ROVIRA* Department of Botany, The University. (Accepted
Bristol BS8 1UG
15 Mav 1973)
Summary-Several
methods of preparing root and soil samples have been tested in order to produce specimens for the scanning electron microscope with the minimum of distortion and disturbance of the rhizosphere. Critical point drying and low temperature freeze-drying proved most satisfactory. The scanning electron microscope is useful for studying the roottsoil interface, the microorganisms on the root surface and the structure of the rhizosphere. INTRODUCTION THE APPLICATION of the scanning
electron microscope (SEM) to investigations of the rhizosphere and rhizoplane has been limited to three studies (Gray, 1967; Marchant, 1970; Dart, 1971). This limited use is surprising for the SEM should have advantages, including minimal preparation, and considerable depth of focus over a wide range of magnifications, over the transmission electron microscope and the light microscope for the study of spatial relationships between roots, soil and microorganisms in an undisturbed state. Preliminary studies by the authors indicated that the limited application of the SEM to rhizosphere studies could be due to difficulties in preventing distortion during specimen preparation and to difficulties in interpretation in natural soils. Dart (1971) in his studies on the colonization by Rhizobium of clover roots in agar culture concluded that the best preservation was obtained by infiltrating roots with glycol methacrylate (GMA). A disadvantage of this method is that the specimen must be passed through various solutions with the possibility of loss or disturbance of the microorganisms and soil in the rhizosphere. The GMA residues may obscure the true nature of the root surface, and furthermore, being difficult to remove from the smaller interstices of the soil, would limit the use of the SEM in studies of the distribution of the natural soil components in relation to the root surface. Marchant (1970) prepared his material by freeze-drying between -20 and -40°C. He attributed the irregularity of the surfaces he observed to cell debris and the break-down of epidermal cell walls. The purposes of this study were to develop methods by which roots, together with their rhizosphere microflora and soil, could be examined by the SEM with the minimum distortion of the specimens and secondly to assess the potential of the SEM for studying the rhizosphere. PRELIMINARY
The methods
of preparation
EXPERIMENTS
which failed are briefly listed below.
1. Roots dried in the air at room temperature. 2. Roots fixed in osmium vapour and dried as in (1). * Visiting Research Fellow. Permanent address: C.S.I.R.O.. Division of Soils, Adelaide, South Australia 747
748
R. CAMPBELL
AND A. D. ROVIRA
3. Roots fixed in glutaraldehyde/formaldehyde and dried as in (1). 4. Roots treated as in (3) but mercuric chloride was added to the fixative. Mercury salts have been reported (Boyde and Wood, 1969) to harden tissues and prevent collapse.
These four methods all resulted in extensive distortion and were abandoned. 5. Roots fixed in glutaraldehyde/formaldehyde, dehydrated in alcohol and impregnated with “Epon’” (Taab, Reading, England). Before polymerization the Epon was washed off the surface by brief immersion in propylene oxide. 6. Roots freeze-dried, with or without prior fixation in glutaraldehyde~formaldehyde. Fixation had no beneficial effect. The results varied with the method of freezing, the temperature at which the roots were dried and the rate of drying. Experimentation with the above variables led to the successful methods given below. MATERIALS
AND METHODS
Clover (~~foZi~~ rqens L. ) and rye grass ~Lol~~~ perenne L.) were grown in pots in a glasshouse, with soil from a natural grassland at Long Ashton near Bristol. This soil was pH 4.8, with 6.2 per cent organic matter (wet digestion) and a mechanical analysis of: Fraction Fine gravel Coarse sand Medium sand Fine sand Coarse silt Medium silt Fine silt Clay
Size (mm) >2 0.662 0*2-0.6 0.06-0.2 002-0.06 ~~~,02 0GO2-0@06 < 0.002
Per cent content 2.0 1-7 6.3 17.0 29.0 10-O 7-o 27.0
The roots were shaken to remove surplus soil, some were processed directly and others were gently washed to remove most of the soil. All roots were processed by one of the following methods: 1. Fresh roots were plunged into either isopentane at its freezing point or into liquid nitrogen. Pieces of the root were then rapidly transferred to the pre-cooled stage of an Edwards-Pearce Tissue Drier [EPTD Model 2, Edwards High Vacuum (Plant), Crawley, England]. The specimens were freeze-dried at -60°C and 0.01 Torr for 16 h, after which the stage was allowed to warm to room temperature while the vacuum was maintained. 2. Fresh roots were fixed for 1 h at room temperature in a buffered mixture of glutaraldehyde and formaldehyde (final concentration in mixture 2% v/v and 1% w/v respectively) and washed gently in water. The material was dehydrated in a graded series of ethanol/water (up to pure ethanol), followed by isopropanol (absolute) and then dried by the critical point method of Boyde and Wood (1969). When necessary the fixed material was stored at the 70% ethanol stage. *Material from both methods was stored over desiccants until coated with gold: palladium alloy (60:40, Johnson Mathey, London, England) and observed in a scanning electron microscope (Stereoscan model S4, Cambridge Instrument Co., Cambridge, England) at accelerating voltages of 6-10 kV.
SCANNING
ELECTRON RESULTS
MICROSCOPY AND
OF THE RHIZOSPHERE
749
DISCUSSION
The preliminary experiments with various preparation methods demonstrated the problems of preparing highly vacuolated root cells for the SEM. By the critical point drying and the Epon embedding methods, the surface contours of the root (Figs 14) were probably unaltered from the fresh state in the zone between 1 and 3 cm behind the apex, but the critical point drying method did not prevent the root hairs from collapsing. In most material prepared by critical point drying some of the epidermal cells collapsed slightly in the root beyond 3 cm from the apex but not to the extent of other methods. Another advantage of critical point drying was that the surface texture of the cells was preserved (Figs 2 and 3). Although Epon embedding prevented cell collapse, the root surface was obscured (Fig. 4) unless the Epon was removed carefully; if too much was removed the cells collapsed. The difficulties associated with GMA embedding (see above) also apply to Epon. Comparison of roots prepared by critical point drying and the freeze-drying method which was adopted finally showed little difference in the appearance of the roots; both methods produced some apparently undistorted root tissue although there was a tendency for the surface cell walls to collapse inward leaving the radial cell walls as ridges (Fig. 5). A disadvantage of the critical point drying method is that the roots need to be fixed and dehydrated in various solutions which may well disturb the orientation of the components of the rhizosphere and dissolve away materials soluble in water and alcohol. By contrast, freezing in isopentane and freeze-drying at -60°C should not change the relative positions of the various components of the rhizosphere and hence the freeze-drying was the preferred method for more extensive studies. The most obvious advantages of the SEM were its wide magnification range and depth of focus. Large areas of the root could be observed together with the surrounding mineral particles (Figs 5 and 8). Increased magnification allowed the microorganisms to be seen in relation to one another and to the root (Figs 5-9) while maintaining a depth of focus very much greater than that obtainable with the light microscope at comparable magnifications. It was possible to observe the shape, size and distribution of mineral grains, clay platelets and aggregates in relation to the root surface and the microflora (Figs 8-11). Fine sand grains in the clover rhizosphere were angular with mostly smooth flat faces (Fig. 10); colonization by bacteria occurred in the crevices and surface irregularities, presumably even such small surface features created slightly more favourable microenvironments. Thus the SEM could be a useful instrument for investigations in microecology, such as the distribution of the microflora in relation to the surfaces of different types of mineral particles. It is probable that because of their chemical nature and surface characteristics different minerals could harbour different populations. This preliminary study on the development of preparative techniques has given indications of differences between clover and rye grass in creating different microenvironments. Mineral grains adhering to clover roots generally had clean surfaces (Fig. 10); by contrast, on rye grass roots the mineral grains were frequently covered with a material in which bacteria and clay particles were embedded (Figs 11 and 14). This observation is being pursued as it may be linked with the improvement of soil structure under grasses. Microorganisms were visible on the true root surface of both clover and rye grass (Figs 3,9, 12 and 14) only where this was not covered by mucilaginous material. This material probably corresponds to the “mucigel” observed by Jenny and Grossenbacher (1963) and Greaves and Darbyshire (1972) by transmission electron microscopy of sections of roots and rhizoplane. Usually the roots were covered completely with this mucilaginous mater-
750
R. CAMPBELL
AND
A. D. ROVIRA
Abbreviations used: A, actinomycete; B, bacteria; C, clay or silt; F, fungus; M, mineral grain; Mu. mucilaginous material; R, root surface; S, sand grains. The bar in the lower left hand corner represents the number of pm indicated. FIG. 1. Root of rye grass (grown in quartz sand) prepared by critical point drying. The epidermal cells have convex surfaces, this is considered to be the natural condition, undistorted by preparation. The root hairs (arrows) are however collapsed. FIG. 2. Clover root surface prepared by critical point drying. The surface has a rough texture and has on it clay platelets and possibly bacteria. FIG. 3. Clover root prepared by critical point drying. There has been some collapse of the epidermal cell walls during preparation (cf Fig. 1). The surface texture of this root has been retained and there are individual bacterial cells, not covered by mucilage. FIG. 4. Clover root impregnated with Epon. The soil particles, bacteria and the root surface itself are coated with Epon, despite washing in propylene oxide. No surface texture is visible on the root. FIGS. 5, 6 and 7. Micrographs of increasing magnification of a clover root frozen in isopentane and freeze dried (cf. Figs. l-4 for preparation method). Freeze drying has caused some distortion of the root surface and the radial walls of the epidernial cells appear as ridges. The mucilage has dried to a thin sheet and peeled away from the root-surface (Lower left of Fig. 5). A fungal hypha can be seen in Fig, 5. There are clay platelets (Fig.7) on the root surface and bacteria on the surface of both mineral grains and the root (Figs. 6 and 7). These figures illustrate problems of distinguishing between clay aggregates and bacteria covered with mucilage. FIGS. 8 and 9. A clover root prepared by critical point drying illustrating mineral grain/root interfaces and microorganisms. There are fungal hyphae, actinomycetes and bacteria on the true root surface. The mucilage has peeled off and the edge of it is in the top right of these photographs. In close contact with the root surface there is a lamellated mineral crystal (Fig. 8) and many clay aggregates or particles oforganic matter. The rod shaped particle (arrow Fig. 9) may be a bacterium. although generally bacteria have rounded ends (Fig. 3). FIG. 10. A sand grain in the rhizosphere of clover, frozen in isopentane and freeze dried. The surface of the sand grain has no mucilage covering (cf Fig. 1 l), and the faces are smooth and flat. In the irregularities between the faces of the sand grain there appear to be clay particles and bacterial cells. FIG. 1 I. A sand grain in the rhizosphere of rye grass, frozen in isopentane and freeze dried. The sand grain and clay platelets and aggregates are coated in mucilaginous material which obscures bacterial cells. FIG. 12. A rye grass root frozen in isopentane and freeze-dried. The mucilage forms a sheet over the root surface. The surface is only visible where a part of the mucilage has flaked away. Bacteria are visible on this root surface. There are clay aggregates and possibly bacteria within mucilage. FIG. 13. A cross fracture of a freeze-dried clover root. The cortical picture) are covered with mucilage which has dried to a “honeycomb” bacteria occur within the mucilage.
cells (at the bottom of the structure. Clay particles and
FIG. 14. A rye grass root, freeze-dried. This illustrates the complexity of the root surface and its rhizosphere. There are mineralgrainscoated with mucilage and uncoated mineral grains supporting bacteria on their surfaces. On the root there are fungal hyphae, bacteria and clay aggregates. The oval structures with granules on their surfaces (inset) are probably fungal spores.
Frcs. I-f.
FIGS. 8 and 9.
FIGS. 10 and 11.
FIGS. 12-14.
SCANNING
ELECTRON
MICROSCOPY
OF THE
RHIZOSPHERE
751
ial which cracked and peeled away from the root, probably due to contraction during drying, to expose the true root surface (Fig. 12). The coating on mineral particles in the rhizosphere of rye grass may be an extension of this mucilaginous layer but only by examining surfaces of axenic roots as well as roots growing in natural soil could the contribution made by microorganisms to this coating be established. The outline of bacteria embedded in the mucilage can be seen in Fig. 12. Many of the bacteria on the root were irregular in outline, possibly due to their own mucilaginous,material, rather than being distinct rods, cocci, etc., as seen under the light microscope. This irregular appearance is also seen in pure cultures of bacteria when observed with the SEM (Passmore and Haggett, 1973). In natural soils the irregular shapes of bacteria in the rhizosphere made it difficult to distinguish bacteria amongst clay particles and organic debris or within the mucilage. The intimate association between bacteria and the mucilage was seen when broken ends of the roots were examined (Fig. 13). The depth of the mucilage appears to range from 5 to 20 pm for clover where the mucilage dried down to a “honeycomb” structure. As the material is a highly hydrated polysaccharide in its natural state (Northcote and Pickett-Heaps, 1966) it generally contracted to a thin film during drying (Fig. 12). The complex nature of the rhizosphere in which there are mineral grains of both angular and flat nature, clay platelets and aggregates, fungal hyphae and bacteria is clearly demonstrated in Fig. 14. There is a problem in SEM work of identifying structures of probable biological origin, e.g. the oval objects with granular protrusions on the surface (inset Fig. 14) could not be identified with certainty, although we believe that they may be fungal spores. Several genera have spores of this general size and shape but the genus Conoplea seem to agree most closely with the objects in Fig. 14 (Dr. J. H. Warcup, personal communication). A reference collection of photographs of known microorganisms and types of mineral particles on the root surface is needed to facilitate interpretation. Although these studies were made at 6610 kV, better resolution could be obtained by increasing the accelerating voltage to 20 kV. At these higher voltages any soil on the roots charged sufficiently to seriously limit the quality of the picture. Mounting the roots in colloidal silver and coating more heavily with the gold:palladium alloy helped to overcome this problem. CONCLUSIONS
This study has indicated both the problems and potential of the SEM in rhizosphere investigations. The initial problem of specimen preparation has been largely overcome by using either critical point drying or low temperature freeze drying; the latter method being well suited to studying the spatial relationships between soil and the root. The major difficulties of the SEM for this work lies in the interpretation. An aggregate of clay particles coated with mucilage can be indistinguishable from a micro-colony of bacteria. The complete cover of the roots by mucilage, under and on which organisms occur, places another limitation on the SEM for studies of the microbiology of the rhizosphere. On the other hand the SEM is most useful in presenting the three dimensional representation of the root-soil interface which will be valuable in forming concepts of the chemistry, mineralogy’ and microbiology of this zone. For example further SEM studies of the rhizosphere will help develop concepts of the flow paths of plant nutrient towards roots and organic exudates away from roots. To formulate such concepts will require the accumulation of experience and collections of photographs on which to base the interpretation of the spatial relationships of the various components in the Soil.
152
R. CAMPBELL
AND A. D. ROVIRA
Acknowlrdgernrnts~C. G. OGDEN and R. H. HARRIS of the British Museum (Natural History) and ELIZABETH PARSONS of Long Ashton Research Station, University of Bristol, are thanked for the use of their critical point drying and freeze-drying equipment. C. HASSETT. Botany Department% University of Bristol. is thanked for his skill and patience in operating the SEM. This study was supported by grants to the Botany Department, The University, Bristol (N.E.R.C. GR3j1731 and S.R.C. B/ RGl408).
REFERENCES BOYDE, A. and WOOD, C. (1969). Preparation of animal tissues for surface scanning electron microscopy. J. Microse. !Xl,221-249. DART, P. J. (1971). Scanning electron microscopy of plant roots. J. apt. Bat. 22, 163-168. GRAY, T. R. G. (1967). Stereoscan electron microscopy of microorganisms. Science, N.Y. 155, 1668-1670. GREAVES, M. P. and DARBYSHIRE, J. F. (1972). The ultrastructure of the mucilaginous layer of plant roots. Soil Biol. Biochem. 4,443-449. JENNY, H. and GROSSENBACHER,K. (1963). Root-soil boundary zones as seen in the electron microscope. Proc. Soil Sci. Sot. Am. 27, 273-217. MARCHANT, R. (1970). The root surface of Ammophila arenaria as a substrate for microorganisms. Trans. Br. Mycol. Sot. 54,479-482. NORTHCOTE, D. H. and PICKETT-HEAPS, J. D. (1966). A function of the Golgi apparatus in polysaccharide synthesis and transport in root-cap cells of wheat. Biochem. J. 98, 159-167. PASSMORE,S. M. and HAGGETT, B. (1973). The use of scanning electron microscopy to show confluent growth of Succharomyces sp. and Leuconostoc sp. J. appl. Bact. 36,89%92.
Vol 5, pp. 747-752.
Pcrgamon
Press 1973. PrInted m Great Britain
THE STUDY SCANNING
OF THE RHIZOSPHERE BY ELECTRON MICROSCOPY
R. CAMPBELL and A. D. ROVIRA* Department of Botany, The University. (Accepted
Bristol BS8 1UG
15 Mav 1973)
Summary-Several
methods of preparing root and soil samples have been tested in order to produce specimens for the scanning electron microscope with the minimum of distortion and disturbance of the rhizosphere. Critical point drying and low temperature freeze-drying proved most satisfactory. The scanning electron microscope is useful for studying the roottsoil interface, the microorganisms on the root surface and the structure of the rhizosphere. INTRODUCTION THE APPLICATION of the scanning
electron microscope (SEM) to investigations of the rhizosphere and rhizoplane has been limited to three studies (Gray, 1967; Marchant, 1970; Dart, 1971). This limited use is surprising for the SEM should have advantages, including minimal preparation, and considerable depth of focus over a wide range of magnifications, over the transmission electron microscope and the light microscope for the study of spatial relationships between roots, soil and microorganisms in an undisturbed state. Preliminary studies by the authors indicated that the limited application of the SEM to rhizosphere studies could be due to difficulties in preventing distortion during specimen preparation and to difficulties in interpretation in natural soils. Dart (1971) in his studies on the colonization by Rhizobium of clover roots in agar culture concluded that the best preservation was obtained by infiltrating roots with glycol methacrylate (GMA). A disadvantage of this method is that the specimen must be passed through various solutions with the possibility of loss or disturbance of the microorganisms and soil in the rhizosphere. The GMA residues may obscure the true nature of the root surface, and furthermore, being difficult to remove from the smaller interstices of the soil, would limit the use of the SEM in studies of the distribution of the natural soil components in relation to the root surface. Marchant (1970) prepared his material by freeze-drying between -20 and -40°C. He attributed the irregularity of the surfaces he observed to cell debris and the break-down of epidermal cell walls. The purposes of this study were to develop methods by which roots, together with their rhizosphere microflora and soil, could be examined by the SEM with the minimum distortion of the specimens and secondly to assess the potential of the SEM for studying the rhizosphere. PRELIMINARY
The methods
of preparation
EXPERIMENTS
which failed are briefly listed below.
1. Roots dried in the air at room temperature. 2. Roots fixed in osmium vapour and dried as in (1). * Visiting Research Fellow. Permanent address: C.S.I.R.O.. Division of Soils, Adelaide, South Australia 747
748
R. CAMPBELL
AND A. D. ROVIRA
3. Roots fixed in glutaraldehyde/formaldehyde and dried as in (1). 4. Roots treated as in (3) but mercuric chloride was added to the fixative. Mercury salts have been reported (Boyde and Wood, 1969) to harden tissues and prevent collapse.
These four methods all resulted in extensive distortion and were abandoned. 5. Roots fixed in glutaraldehyde/formaldehyde, dehydrated in alcohol and impregnated with “Epon’” (Taab, Reading, England). Before polymerization the Epon was washed off the surface by brief immersion in propylene oxide. 6. Roots freeze-dried, with or without prior fixation in glutaraldehyde~formaldehyde. Fixation had no beneficial effect. The results varied with the method of freezing, the temperature at which the roots were dried and the rate of drying. Experimentation with the above variables led to the successful methods given below. MATERIALS
AND METHODS
Clover (~~foZi~~ rqens L. ) and rye grass ~Lol~~~ perenne L.) were grown in pots in a glasshouse, with soil from a natural grassland at Long Ashton near Bristol. This soil was pH 4.8, with 6.2 per cent organic matter (wet digestion) and a mechanical analysis of: Fraction Fine gravel Coarse sand Medium sand Fine sand Coarse silt Medium silt Fine silt Clay
Size (mm) >2 0.662 0*2-0.6 0.06-0.2 002-0.06 ~~~,02 0GO2-0@06 < 0.002
Per cent content 2.0 1-7 6.3 17.0 29.0 10-O 7-o 27.0
The roots were shaken to remove surplus soil, some were processed directly and others were gently washed to remove most of the soil. All roots were processed by one of the following methods: 1. Fresh roots were plunged into either isopentane at its freezing point or into liquid nitrogen. Pieces of the root were then rapidly transferred to the pre-cooled stage of an Edwards-Pearce Tissue Drier [EPTD Model 2, Edwards High Vacuum (Plant), Crawley, England]. The specimens were freeze-dried at -60°C and 0.01 Torr for 16 h, after which the stage was allowed to warm to room temperature while the vacuum was maintained. 2. Fresh roots were fixed for 1 h at room temperature in a buffered mixture of glutaraldehyde and formaldehyde (final concentration in mixture 2% v/v and 1% w/v respectively) and washed gently in water. The material was dehydrated in a graded series of ethanol/water (up to pure ethanol), followed by isopropanol (absolute) and then dried by the critical point method of Boyde and Wood (1969). When necessary the fixed material was stored at the 70% ethanol stage. *Material from both methods was stored over desiccants until coated with gold: palladium alloy (60:40, Johnson Mathey, London, England) and observed in a scanning electron microscope (Stereoscan model S4, Cambridge Instrument Co., Cambridge, England) at accelerating voltages of 6-10 kV.
SCANNING
ELECTRON RESULTS
MICROSCOPY AND
OF THE RHIZOSPHERE
749
DISCUSSION
The preliminary experiments with various preparation methods demonstrated the problems of preparing highly vacuolated root cells for the SEM. By the critical point drying and the Epon embedding methods, the surface contours of the root (Figs 14) were probably unaltered from the fresh state in the zone between 1 and 3 cm behind the apex, but the critical point drying method did not prevent the root hairs from collapsing. In most material prepared by critical point drying some of the epidermal cells collapsed slightly in the root beyond 3 cm from the apex but not to the extent of other methods. Another advantage of critical point drying was that the surface texture of the cells was preserved (Figs 2 and 3). Although Epon embedding prevented cell collapse, the root surface was obscured (Fig. 4) unless the Epon was removed carefully; if too much was removed the cells collapsed. The difficulties associated with GMA embedding (see above) also apply to Epon. Comparison of roots prepared by critical point drying and the freeze-drying method which was adopted finally showed little difference in the appearance of the roots; both methods produced some apparently undistorted root tissue although there was a tendency for the surface cell walls to collapse inward leaving the radial cell walls as ridges (Fig. 5). A disadvantage of the critical point drying method is that the roots need to be fixed and dehydrated in various solutions which may well disturb the orientation of the components of the rhizosphere and dissolve away materials soluble in water and alcohol. By contrast, freezing in isopentane and freeze-drying at -60°C should not change the relative positions of the various components of the rhizosphere and hence the freeze-drying was the preferred method for more extensive studies. The most obvious advantages of the SEM were its wide magnification range and depth of focus. Large areas of the root could be observed together with the surrounding mineral particles (Figs 5 and 8). Increased magnification allowed the microorganisms to be seen in relation to one another and to the root (Figs 5-9) while maintaining a depth of focus very much greater than that obtainable with the light microscope at comparable magnifications. It was possible to observe the shape, size and distribution of mineral grains, clay platelets and aggregates in relation to the root surface and the microflora (Figs 8-11). Fine sand grains in the clover rhizosphere were angular with mostly smooth flat faces (Fig. 10); colonization by bacteria occurred in the crevices and surface irregularities, presumably even such small surface features created slightly more favourable microenvironments. Thus the SEM could be a useful instrument for investigations in microecology, such as the distribution of the microflora in relation to the surfaces of different types of mineral particles. It is probable that because of their chemical nature and surface characteristics different minerals could harbour different populations. This preliminary study on the development of preparative techniques has given indications of differences between clover and rye grass in creating different microenvironments. Mineral grains adhering to clover roots generally had clean surfaces (Fig. 10); by contrast, on rye grass roots the mineral grains were frequently covered with a material in which bacteria and clay particles were embedded (Figs 11 and 14). This observation is being pursued as it may be linked with the improvement of soil structure under grasses. Microorganisms were visible on the true root surface of both clover and rye grass (Figs 3,9, 12 and 14) only where this was not covered by mucilaginous material. This material probably corresponds to the “mucigel” observed by Jenny and Grossenbacher (1963) and Greaves and Darbyshire (1972) by transmission electron microscopy of sections of roots and rhizoplane. Usually the roots were covered completely with this mucilaginous mater-
750
R. CAMPBELL
AND
A. D. ROVIRA
Abbreviations used: A, actinomycete; B, bacteria; C, clay or silt; F, fungus; M, mineral grain; Mu. mucilaginous material; R, root surface; S, sand grains. The bar in the lower left hand corner represents the number of pm indicated. FIG. 1. Root of rye grass (grown in quartz sand) prepared by critical point drying. The epidermal cells have convex surfaces, this is considered to be the natural condition, undistorted by preparation. The root hairs (arrows) are however collapsed. FIG. 2. Clover root surface prepared by critical point drying. The surface has a rough texture and has on it clay platelets and possibly bacteria. FIG. 3. Clover root prepared by critical point drying. There has been some collapse of the epidermal cell walls during preparation (cf Fig. 1). The surface texture of this root has been retained and there are individual bacterial cells, not covered by mucilage. FIG. 4. Clover root impregnated with Epon. The soil particles, bacteria and the root surface itself are coated with Epon, despite washing in propylene oxide. No surface texture is visible on the root. FIGS. 5, 6 and 7. Micrographs of increasing magnification of a clover root frozen in isopentane and freeze dried (cf. Figs. l-4 for preparation method). Freeze drying has caused some distortion of the root surface and the radial walls of the epidernial cells appear as ridges. The mucilage has dried to a thin sheet and peeled away from the root-surface (Lower left of Fig. 5). A fungal hypha can be seen in Fig, 5. There are clay platelets (Fig.7) on the root surface and bacteria on the surface of both mineral grains and the root (Figs. 6 and 7). These figures illustrate problems of distinguishing between clay aggregates and bacteria covered with mucilage. FIGS. 8 and 9. A clover root prepared by critical point drying illustrating mineral grain/root interfaces and microorganisms. There are fungal hyphae, actinomycetes and bacteria on the true root surface. The mucilage has peeled off and the edge of it is in the top right of these photographs. In close contact with the root surface there is a lamellated mineral crystal (Fig. 8) and many clay aggregates or particles oforganic matter. The rod shaped particle (arrow Fig. 9) may be a bacterium. although generally bacteria have rounded ends (Fig. 3). FIG. 10. A sand grain in the rhizosphere of clover, frozen in isopentane and freeze dried. The surface of the sand grain has no mucilage covering (cf Fig. 1 l), and the faces are smooth and flat. In the irregularities between the faces of the sand grain there appear to be clay particles and bacterial cells. FIG. 1 I. A sand grain in the rhizosphere of rye grass, frozen in isopentane and freeze dried. The sand grain and clay platelets and aggregates are coated in mucilaginous material which obscures bacterial cells. FIG. 12. A rye grass root frozen in isopentane and freeze-dried. The mucilage forms a sheet over the root surface. The surface is only visible where a part of the mucilage has flaked away. Bacteria are visible on this root surface. There are clay aggregates and possibly bacteria within mucilage. FIG. 13. A cross fracture of a freeze-dried clover root. The cortical picture) are covered with mucilage which has dried to a “honeycomb” bacteria occur within the mucilage.
cells (at the bottom of the structure. Clay particles and
FIG. 14. A rye grass root, freeze-dried. This illustrates the complexity of the root surface and its rhizosphere. There are mineralgrainscoated with mucilage and uncoated mineral grains supporting bacteria on their surfaces. On the root there are fungal hyphae, bacteria and clay aggregates. The oval structures with granules on their surfaces (inset) are probably fungal spores.
Frcs. I-f.
FIGS. 8 and 9.
FIGS. 10 and 11.
FIGS. 12-14.
SCANNING
ELECTRON
MICROSCOPY
OF THE
RHIZOSPHERE
751
ial which cracked and peeled away from the root, probably due to contraction during drying, to expose the true root surface (Fig. 12). The coating on mineral particles in the rhizosphere of rye grass may be an extension of this mucilaginous layer but only by examining surfaces of axenic roots as well as roots growing in natural soil could the contribution made by microorganisms to this coating be established. The outline of bacteria embedded in the mucilage can be seen in Fig. 12. Many of the bacteria on the root were irregular in outline, possibly due to their own mucilaginous,material, rather than being distinct rods, cocci, etc., as seen under the light microscope. This irregular appearance is also seen in pure cultures of bacteria when observed with the SEM (Passmore and Haggett, 1973). In natural soils the irregular shapes of bacteria in the rhizosphere made it difficult to distinguish bacteria amongst clay particles and organic debris or within the mucilage. The intimate association between bacteria and the mucilage was seen when broken ends of the roots were examined (Fig. 13). The depth of the mucilage appears to range from 5 to 20 pm for clover where the mucilage dried down to a “honeycomb” structure. As the material is a highly hydrated polysaccharide in its natural state (Northcote and Pickett-Heaps, 1966) it generally contracted to a thin film during drying (Fig. 12). The complex nature of the rhizosphere in which there are mineral grains of both angular and flat nature, clay platelets and aggregates, fungal hyphae and bacteria is clearly demonstrated in Fig. 14. There is a problem in SEM work of identifying structures of probable biological origin, e.g. the oval objects with granular protrusions on the surface (inset Fig. 14) could not be identified with certainty, although we believe that they may be fungal spores. Several genera have spores of this general size and shape but the genus Conoplea seem to agree most closely with the objects in Fig. 14 (Dr. J. H. Warcup, personal communication). A reference collection of photographs of known microorganisms and types of mineral particles on the root surface is needed to facilitate interpretation. Although these studies were made at 6610 kV, better resolution could be obtained by increasing the accelerating voltage to 20 kV. At these higher voltages any soil on the roots charged sufficiently to seriously limit the quality of the picture. Mounting the roots in colloidal silver and coating more heavily with the gold:palladium alloy helped to overcome this problem. CONCLUSIONS
This study has indicated both the problems and potential of the SEM in rhizosphere investigations. The initial problem of specimen preparation has been largely overcome by using either critical point drying or low temperature freeze drying; the latter method being well suited to studying the spatial relationships between soil and the root. The major difficulties of the SEM for this work lies in the interpretation. An aggregate of clay particles coated with mucilage can be indistinguishable from a micro-colony of bacteria. The complete cover of the roots by mucilage, under and on which organisms occur, places another limitation on the SEM for studies of the microbiology of the rhizosphere. On the other hand the SEM is most useful in presenting the three dimensional representation of the root-soil interface which will be valuable in forming concepts of the chemistry, mineralogy’ and microbiology of this zone. For example further SEM studies of the rhizosphere will help develop concepts of the flow paths of plant nutrient towards roots and organic exudates away from roots. To formulate such concepts will require the accumulation of experience and collections of photographs on which to base the interpretation of the spatial relationships of the various components in the Soil.
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AND A. D. ROVIRA
Acknowlrdgernrnts~C. G. OGDEN and R. H. HARRIS of the British Museum (Natural History) and ELIZABETH PARSONS of Long Ashton Research Station, University of Bristol, are thanked for the use of their critical point drying and freeze-drying equipment. C. HASSETT. Botany Department% University of Bristol. is thanked for his skill and patience in operating the SEM. This study was supported by grants to the Botany Department, The University, Bristol (N.E.R.C. GR3j1731 and S.R.C. B/ RGl408).
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