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Apparatus to study the quantitative relationships between root exudates and microbial populations in the rhizosphere
Soil Bid. Biochem. Vol. 14, pp. 315 to 317. 1982 Printed in Great Britain. All rights reserved
0038-0717/82/030315-03$03.00/O Copyright 0 1982 Pergamon Press Ltd
SHORT
COMMUNICATION
Apparatus to study the quantitative relationships between root exudates and microbial populations in the rhizosphere R. MARTENS* Department
of Microbiology,
The Macaulay Institute for Soil Research, Aberdeen AB9 245, U.K.
Craigiebuckler,
(Accepted 20 September 198 1)
Assessments of the quantity of microorganisms in rhizosphere soil have been based on the plate count technique and have often been confined to bacteria. Such investigations have revealed that the number of microorganisms in the rhizosphere is S-10 times higher than in non-rhizosphere soil (Rovira and Davey, 1975; Brown, 1975). Disadvantages of the plate count technique are well known (Russell. 1961: Jensen, 1967: Grav et al., 1967) and new methods, specifically developed to- estimate microbial biomass in soils (Jenkinson and Powlson, 1976; Anderson and Domsch. 1978), are more likely to give a better insight into the dynamics of the microbial population near the plant root. Unfortunately, the very small volume of the rhizosphere soil limits the accuracy of the determinations with these methods and it is difficult to show the decrease of the biomass with increasing distance from a living root. Newman and Watson (1977) tried to overcome these experimental problems by developing a computer model to predict the microbial abundance in the rhizosphere. The ideas and results of this mathematical model were an encouragement to devise an apparatus that could be used to study the quantitative relationship between rhizosphere microbial populations and root exudates in soil. The most important part of the apparatus proposed here is the “artificial root” which simulates the region of a plant root where inorganic and organic materials are released into the soil. This artificial root should be round in section and have a surface that allows a slow uniform release of sterile synthetic exudate into the surrounding soil. The size of the artificial root should make it feasible to collect several distinct soil samples at different distances from the root surface. Also. the artificial root should be resistant to microbial decay or mechanical damage and should be sterilizable. After sterilization the pore size of the artificial root surface must prevent penetration of microorganisms from soil into the inner part of the artificial root as well as the contamination of the reservoir of the synthetic exudate. During an experiment it should also be possible to collect CO2 respired by soil organisms. Three different types of readily-available cylinders made from sintered ceramic or metallic materials with pore diameters between 0.5 ana 2pm were tested as artificial roots, but all became contaminated with soil bacteria. Unsatisfactory results were also obtained when PVC or polyamide membranes (pore sizes 0.1-0.2pm and normally used for bacterial filtration) were attached to the surfaces
* Present address: Institut fur Bodenbiologie, forschungsanstalt fur Landwirtschaft, Bundesallee Braunschweig, Federal Republic of Germany.
Bundes50, 3300
of the described cylinders. Eventually, filters called Immersible CX-Molecular Separators (Millipore Intertech. Inc., Bedford, Massachusetts) were found to be the most suitable artificial roots, because they satisfied all the requirements mentioned above. The filters (length 35 mm, o.d. 11 mm) consist of a Pellicon molecular filtration membrane (13 cm2) moulded onto a porous polyethylene core with a polystyrene cap. These plastic materials are heat labile (max. temp. 50°C) and can only be sterilized with chemical agents. Figure 1 shows the whole apparatus which was set up as follows: To exclude diffusion of exudate from the tip of the filter, this area was coated with several layers of a hexane solution of silicone rubber (Bathtub caulk, Dow Corning, Michigan). The filters were sterilized with formaldehyde (2% v/v) for 24 h. Each filter was attached to a sterile vacuum flask by a short length of silicone tubing with a T-Swagelok fitting and a septum inserted in it. Fifty ml of the formaldehyde solution were sucked through the membrane into the flask. After 24 h of incubation at 35°C. the filter was rinsed with distilled water (approx. 200 ml) until the chromotropic acid calorimetric test for formaldehyde (Bremanis, 1949/50) was negative. The distilled water remaining in the membrane body was then largely replaced by sucking 10 ml of the synthetic exudate into the system. The filter cylinder was then connected to a sterile 25 ml pipette by a 1.5 m long Tygon tube as shown in Fig. 1. The glass bulb (6 in Fig. 1) in the Tygon tube trapped any gas bubbles in the exudate before they reached the filter. The system was filled with 20 ml of synthetic exudate by attaching a vacuum line to the side arm of the pipette (at 2 in Fig. 1) and closing the upper end of the glass tubing inside the pipette. A PVC drainpipe (142 x 103 mm i.d.), closed by two cylindrical PVC stoppers at either end, was used as the incubation chamber (5 in Fig. 1) for rhizosphere studies. The filter was introduced into this chamber through a hole in the upper stopper and the tip of the filter was inserted into a central hole (11 mm dia) of a plastic disk (6 x 102 mm) on top of the lower stopper. With this arrangement, the filter was held in a fixed position while the chamber was filled with soil. To ensure a good and even contact between the soil and the membrane, the soil was tamped down and the pore volume was reduced to about 50%. The size of the soil cylinder around the membrane was determined by the height of the membrane (32 mm) and the inner diameter of the stopper (103 mm). With the formula IV = 0. V (4 = specific weight of the soil with the desired pore volume, V = volume-of the soil cylinder reduced by the volume of the membrane filter cylinder). the necessary amount of soil was calculated. This amount was divided into four portions and each portion was pressed with a suitable plastic disk to a quarter of the total height of 32 mm. During this procedure, it was 315
316
Short communications
CO*-free air -
vacuum pump -
Fig. 1. Diagram of the apparatus. 1. Reservoir for artificial exudate; Connection to vacuum pump; 3. Inner glass tube; 4. Magnetic valve; 5. Incubation chamber: 6. Tygon tube and glass bulb; 7. Molecular filter; 8. Soil; 9. Plastic disc; 10. Greased rubber rings; 11. Tube with water: 12. Trap to prevent return flow; 13. CO,-trap filled with ethanolamin and methanol or NaOH.
necessary to keep the filter exactly perpendicular. After the chamber was filled with soil, the upper stopper was screwed into position and the glassware shown in Fig. 1 was connected to the incubation chamber. The flow of the exudate through the membrane was caused by hydrostatic pressure dependent on the difference in height between the membrane and the lower end of the tube inside the pipette. Capillary action of the soil also encouraged the flow of the exudate. The daily supply of exudate to the soil was regulated by means of a time controlled magnetic valve (Model1 Hl, Schiitt, Gottingen, West-Germany), and could be measured on the graduated pipette. A flow of 1 ml day-’ was chosen for the preliminary experiments. After 10 days, the incubation chamber was opened and the artificial root was carefully replaced by a metallic cylinder (32 x 11 mm). Soil samples were collected at different distances from the membrane by means of cork borers. Various experiments demonstrated that the apparatus can be used to study quantitative aspects of the microbial rhizosphere population. As synthetic exudates, three different concentrations of a rSC-glucose solution (88, 440 and 2200 pg C ml ‘) with NHINO, and KH,PO, (C:N:P = 10:2:1; pH = 4.5) were used. The two lower concentrations of C supply were in the range of data given by Newman and Watson (1977) and calculated from plant experiments described in the literature. In two different acid soils the formation of microbial biomass after 10 days of exudation was investigated as described by Jenkinson and Powlson (1976). For this estimation, only the i4C02
evolved from the reinoculated fumigated samples and from the non-fumigated samples was used to calculate biomass i“C formed from the “C-labelled glucose. Table 1 shows the results of these estimations carried out in sections at increasing distance from the surface of the membrane. The values in Table 1 demonstrate the expected result that with increasing concentration of glucose in the exudate the diffusion of the C source extended further into the surrounding soil. However. the two lower concentrations showed only slight ditlerences. With both soils and concentrations, formation of new biomass occurred mainly in the first section O-2 mm. After the addition of 88OngC substrate, 90 and 84”, respectively. of total “C-1abelled biomass was formed in the volume of the first 2 mm; with 44OOpg C substrate 81 or 75”, of the new biomass were found in the first section. In the experiment with a carbon supply of 22,OOOpg. the formation of biomass occurred mainly in the first and second section (O-4 mm). In soil B. however, the glucose at this large concentration initiated a noticeable proliferation of microorganisms even in the area between 4 and 6 mm from the membrane. The appearance of labelled biomass in the outer sections was presumed to be due to autotrophic fixation of iJCOz, respired by the heterotrophic organisms, In experiments with lower concentrations of glucose the distribution of newly formed biomass corresponded to the distribution of total residue ‘%activity in soil, estimated by combustion of soil in a Sample Oxidizer 306 (Packard Inst. Comp. Inc., 2200 Warrenville, Illinois). Between 80 and 90”, of total residue activity was found in the first
Short communications Table added
317
1. Formation of microbial biomass (pg i4C) from i4C-labelled glucose to soils during a lo-day period in an apparatus to measure quantitative aspects of the rhizosphere Amount
Distance of soil from surface of membrane (mm)
A”
Bb
t%2 24 4-6 68 8-10 l&14 1419 19-52
54 1.5 0.4 0.6 0.2 0.3 0.3 3
81 7 0.6 0.5 0.4 0.5 0.7 6
60.3 479
6.9
Total in soil CG*-i4C (p(P) respired Biomass i4C as a percentage of 14C added to soil “Arable soil C 5.37;; pH ‘Arable soil 6”b; org. C
880
of “‘C-glucose (pg C 10 days-‘) added to soil 4400 22,000 A
B
A
B
226 34 3 1.1 1.0 1.3 1.5 13
262 49 6 1.5 1.9 2 3 24
382 271 30 5 8 10 12 89
325 327 126 26 6 8 13 96
96.7 529
280.7 2516
349.2 2455
807 14,366
927 13,860
11.0
6.4
7.9
3.9
4.3
from the north east of Scotland (Sand 74%. silt 190/L, clay 7:/i; org. 10 mM CaCl, 4.7). from the eastern parts of West-Germany (Sand 43%, silt 51”/,, clay 1.29;; pH 10 mM CaCl, 5.3).
section O-2 mm. After carbon had been supplied at 22,000 pg almost the same high proportion (about 75T0) of residue activity was found in the innermost section although the newly formed “‘C-1abelled biomass in the &2 mm section amounted only to 35 and 47:/, respectively. of the total new biomass, This contradiction can be explained by the formation of a thin layer of a white amorphous material at the soil-membrane interface. From the experiment with soil A, the material was isolated with forceps and freed from adhering soil particles by sonification in distilled water. After lyophilization, the white material was identified by liquid scintillation counting and i.r.-spectroscopy as i4C-labelled polysaccharides. The extensive accumulation of these polysaccharides suggests that 382 and 325 pg respectively, were the maximal amounts of C in biomass that could be formed in the 2.6 g soil (dry weight) of the &2mm section. Any excess organic carbon was transformed into polysaccharides or diffused further into the outer sections. A remarkable result of all experiments was the very low efficiency by which the microbial population used the available carbon source for its own proliferation. Between 54 and 65% of the added carbon was respired as CO2 and only 4 to 11’8; was transformed into microbial biomass.
Acknowledgements-This research was supported by a grant from the Deutsche Forschungsgemeinschaft. I thank Dr John Darbyshire for his support during the course of the work and critical review of the manuscript. I also thank J. D. Russel for i.r.-spectroscopy of polysaccharides.
REFERENCES ANDERSON J. P. E. and DOMSCH K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215-221. BREMANIS E. (1949/50) Die photometrische Bestimmung des Formaldehyds mit Chromotropsaure. Zeitschrift fir Analytische Chemie 130,4447. BROWN M. E. (1975) Rhizosphere microorganisms-opportunists, bandits or benefactors. In Soil Microbiology (N. Walker. Ed.), pp. 21-38. Butterworth. London. GRAY T. R. G., BAXBY P., HILL J. R. and GCI~DFELLOW M. (1967) Direct observation of bacteria in soil. In The Ecology of Soil Bacteria (T. R. G. Gray and D. Parkinson, Eds), pp. 171-197. Liverpool University Press. JENKINSON D. S. and POWL~~N D. S. (1976) The effects of biocidal treatments on metabolism in soil-V. A method for measuring soil biomass. Soil Biology & Biochemistry 8, 209-213. JENSEN V. (1967) The plate count technique. In The Ecology of Soil Bacteria (T. R. G. Gray and D. Parkinson, Eds). pp. 1588170. Liverpool University Press. NEWMAN E. J. and WATSON A. (1977) Microbial abundance in the rhizosphere: A computer model. Plant and Soil 48, 17-56. ROVIRA A. D. and DAVEY C. B. (1974) Biology of the rhizosphere. In The Plant Root and Its Encironment (E. W. Carson, Ed.), pp. 1533204. University Press of Virginia, Charlottesville. RUSSELL E. W. (1961) Soil Conditions and PIant Growth, pp. 146147. Longman, London.
0038-0717/82/030315-03$03.00/O Copyright 0 1982 Pergamon Press Ltd
SHORT
COMMUNICATION
Apparatus to study the quantitative relationships between root exudates and microbial populations in the rhizosphere R. MARTENS* Department
of Microbiology,
The Macaulay Institute for Soil Research, Aberdeen AB9 245, U.K.
Craigiebuckler,
(Accepted 20 September 198 1)
Assessments of the quantity of microorganisms in rhizosphere soil have been based on the plate count technique and have often been confined to bacteria. Such investigations have revealed that the number of microorganisms in the rhizosphere is S-10 times higher than in non-rhizosphere soil (Rovira and Davey, 1975; Brown, 1975). Disadvantages of the plate count technique are well known (Russell. 1961: Jensen, 1967: Grav et al., 1967) and new methods, specifically developed to- estimate microbial biomass in soils (Jenkinson and Powlson, 1976; Anderson and Domsch. 1978), are more likely to give a better insight into the dynamics of the microbial population near the plant root. Unfortunately, the very small volume of the rhizosphere soil limits the accuracy of the determinations with these methods and it is difficult to show the decrease of the biomass with increasing distance from a living root. Newman and Watson (1977) tried to overcome these experimental problems by developing a computer model to predict the microbial abundance in the rhizosphere. The ideas and results of this mathematical model were an encouragement to devise an apparatus that could be used to study the quantitative relationship between rhizosphere microbial populations and root exudates in soil. The most important part of the apparatus proposed here is the “artificial root” which simulates the region of a plant root where inorganic and organic materials are released into the soil. This artificial root should be round in section and have a surface that allows a slow uniform release of sterile synthetic exudate into the surrounding soil. The size of the artificial root should make it feasible to collect several distinct soil samples at different distances from the root surface. Also. the artificial root should be resistant to microbial decay or mechanical damage and should be sterilizable. After sterilization the pore size of the artificial root surface must prevent penetration of microorganisms from soil into the inner part of the artificial root as well as the contamination of the reservoir of the synthetic exudate. During an experiment it should also be possible to collect CO2 respired by soil organisms. Three different types of readily-available cylinders made from sintered ceramic or metallic materials with pore diameters between 0.5 ana 2pm were tested as artificial roots, but all became contaminated with soil bacteria. Unsatisfactory results were also obtained when PVC or polyamide membranes (pore sizes 0.1-0.2pm and normally used for bacterial filtration) were attached to the surfaces
* Present address: Institut fur Bodenbiologie, forschungsanstalt fur Landwirtschaft, Bundesallee Braunschweig, Federal Republic of Germany.
Bundes50, 3300
of the described cylinders. Eventually, filters called Immersible CX-Molecular Separators (Millipore Intertech. Inc., Bedford, Massachusetts) were found to be the most suitable artificial roots, because they satisfied all the requirements mentioned above. The filters (length 35 mm, o.d. 11 mm) consist of a Pellicon molecular filtration membrane (13 cm2) moulded onto a porous polyethylene core with a polystyrene cap. These plastic materials are heat labile (max. temp. 50°C) and can only be sterilized with chemical agents. Figure 1 shows the whole apparatus which was set up as follows: To exclude diffusion of exudate from the tip of the filter, this area was coated with several layers of a hexane solution of silicone rubber (Bathtub caulk, Dow Corning, Michigan). The filters were sterilized with formaldehyde (2% v/v) for 24 h. Each filter was attached to a sterile vacuum flask by a short length of silicone tubing with a T-Swagelok fitting and a septum inserted in it. Fifty ml of the formaldehyde solution were sucked through the membrane into the flask. After 24 h of incubation at 35°C. the filter was rinsed with distilled water (approx. 200 ml) until the chromotropic acid calorimetric test for formaldehyde (Bremanis, 1949/50) was negative. The distilled water remaining in the membrane body was then largely replaced by sucking 10 ml of the synthetic exudate into the system. The filter cylinder was then connected to a sterile 25 ml pipette by a 1.5 m long Tygon tube as shown in Fig. 1. The glass bulb (6 in Fig. 1) in the Tygon tube trapped any gas bubbles in the exudate before they reached the filter. The system was filled with 20 ml of synthetic exudate by attaching a vacuum line to the side arm of the pipette (at 2 in Fig. 1) and closing the upper end of the glass tubing inside the pipette. A PVC drainpipe (142 x 103 mm i.d.), closed by two cylindrical PVC stoppers at either end, was used as the incubation chamber (5 in Fig. 1) for rhizosphere studies. The filter was introduced into this chamber through a hole in the upper stopper and the tip of the filter was inserted into a central hole (11 mm dia) of a plastic disk (6 x 102 mm) on top of the lower stopper. With this arrangement, the filter was held in a fixed position while the chamber was filled with soil. To ensure a good and even contact between the soil and the membrane, the soil was tamped down and the pore volume was reduced to about 50%. The size of the soil cylinder around the membrane was determined by the height of the membrane (32 mm) and the inner diameter of the stopper (103 mm). With the formula IV = 0. V (4 = specific weight of the soil with the desired pore volume, V = volume-of the soil cylinder reduced by the volume of the membrane filter cylinder). the necessary amount of soil was calculated. This amount was divided into four portions and each portion was pressed with a suitable plastic disk to a quarter of the total height of 32 mm. During this procedure, it was 315
316
Short communications
CO*-free air -
vacuum pump -
Fig. 1. Diagram of the apparatus. 1. Reservoir for artificial exudate; Connection to vacuum pump; 3. Inner glass tube; 4. Magnetic valve; 5. Incubation chamber: 6. Tygon tube and glass bulb; 7. Molecular filter; 8. Soil; 9. Plastic disc; 10. Greased rubber rings; 11. Tube with water: 12. Trap to prevent return flow; 13. CO,-trap filled with ethanolamin and methanol or NaOH.
necessary to keep the filter exactly perpendicular. After the chamber was filled with soil, the upper stopper was screwed into position and the glassware shown in Fig. 1 was connected to the incubation chamber. The flow of the exudate through the membrane was caused by hydrostatic pressure dependent on the difference in height between the membrane and the lower end of the tube inside the pipette. Capillary action of the soil also encouraged the flow of the exudate. The daily supply of exudate to the soil was regulated by means of a time controlled magnetic valve (Model1 Hl, Schiitt, Gottingen, West-Germany), and could be measured on the graduated pipette. A flow of 1 ml day-’ was chosen for the preliminary experiments. After 10 days, the incubation chamber was opened and the artificial root was carefully replaced by a metallic cylinder (32 x 11 mm). Soil samples were collected at different distances from the membrane by means of cork borers. Various experiments demonstrated that the apparatus can be used to study quantitative aspects of the microbial rhizosphere population. As synthetic exudates, three different concentrations of a rSC-glucose solution (88, 440 and 2200 pg C ml ‘) with NHINO, and KH,PO, (C:N:P = 10:2:1; pH = 4.5) were used. The two lower concentrations of C supply were in the range of data given by Newman and Watson (1977) and calculated from plant experiments described in the literature. In two different acid soils the formation of microbial biomass after 10 days of exudation was investigated as described by Jenkinson and Powlson (1976). For this estimation, only the i4C02
evolved from the reinoculated fumigated samples and from the non-fumigated samples was used to calculate biomass i“C formed from the “C-labelled glucose. Table 1 shows the results of these estimations carried out in sections at increasing distance from the surface of the membrane. The values in Table 1 demonstrate the expected result that with increasing concentration of glucose in the exudate the diffusion of the C source extended further into the surrounding soil. However. the two lower concentrations showed only slight ditlerences. With both soils and concentrations, formation of new biomass occurred mainly in the first section O-2 mm. After the addition of 88OngC substrate, 90 and 84”, respectively. of total “C-1abelled biomass was formed in the volume of the first 2 mm; with 44OOpg C substrate 81 or 75”, of the new biomass were found in the first section. In the experiment with a carbon supply of 22,OOOpg. the formation of biomass occurred mainly in the first and second section (O-4 mm). In soil B. however, the glucose at this large concentration initiated a noticeable proliferation of microorganisms even in the area between 4 and 6 mm from the membrane. The appearance of labelled biomass in the outer sections was presumed to be due to autotrophic fixation of iJCOz, respired by the heterotrophic organisms, In experiments with lower concentrations of glucose the distribution of newly formed biomass corresponded to the distribution of total residue ‘%activity in soil, estimated by combustion of soil in a Sample Oxidizer 306 (Packard Inst. Comp. Inc., 2200 Warrenville, Illinois). Between 80 and 90”, of total residue activity was found in the first
Short communications Table added
317
1. Formation of microbial biomass (pg i4C) from i4C-labelled glucose to soils during a lo-day period in an apparatus to measure quantitative aspects of the rhizosphere Amount
Distance of soil from surface of membrane (mm)
A”
Bb
t%2 24 4-6 68 8-10 l&14 1419 19-52
54 1.5 0.4 0.6 0.2 0.3 0.3 3
81 7 0.6 0.5 0.4 0.5 0.7 6
60.3 479
6.9
Total in soil CG*-i4C (p(P) respired Biomass i4C as a percentage of 14C added to soil “Arable soil C 5.37;; pH ‘Arable soil 6”b; org. C
880
of “‘C-glucose (pg C 10 days-‘) added to soil 4400 22,000 A
B
A
B
226 34 3 1.1 1.0 1.3 1.5 13
262 49 6 1.5 1.9 2 3 24
382 271 30 5 8 10 12 89
325 327 126 26 6 8 13 96
96.7 529
280.7 2516
349.2 2455
807 14,366
927 13,860
11.0
6.4
7.9
3.9
4.3
from the north east of Scotland (Sand 74%. silt 190/L, clay 7:/i; org. 10 mM CaCl, 4.7). from the eastern parts of West-Germany (Sand 43%, silt 51”/,, clay 1.29;; pH 10 mM CaCl, 5.3).
section O-2 mm. After carbon had been supplied at 22,000 pg almost the same high proportion (about 75T0) of residue activity was found in the innermost section although the newly formed “‘C-1abelled biomass in the &2 mm section amounted only to 35 and 47:/, respectively. of the total new biomass, This contradiction can be explained by the formation of a thin layer of a white amorphous material at the soil-membrane interface. From the experiment with soil A, the material was isolated with forceps and freed from adhering soil particles by sonification in distilled water. After lyophilization, the white material was identified by liquid scintillation counting and i.r.-spectroscopy as i4C-labelled polysaccharides. The extensive accumulation of these polysaccharides suggests that 382 and 325 pg respectively, were the maximal amounts of C in biomass that could be formed in the 2.6 g soil (dry weight) of the &2mm section. Any excess organic carbon was transformed into polysaccharides or diffused further into the outer sections. A remarkable result of all experiments was the very low efficiency by which the microbial population used the available carbon source for its own proliferation. Between 54 and 65% of the added carbon was respired as CO2 and only 4 to 11’8; was transformed into microbial biomass.
Acknowledgements-This research was supported by a grant from the Deutsche Forschungsgemeinschaft. I thank Dr John Darbyshire for his support during the course of the work and critical review of the manuscript. I also thank J. D. Russel for i.r.-spectroscopy of polysaccharides.
REFERENCES ANDERSON J. P. E. and DOMSCH K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215-221. BREMANIS E. (1949/50) Die photometrische Bestimmung des Formaldehyds mit Chromotropsaure. Zeitschrift fir Analytische Chemie 130,4447. BROWN M. E. (1975) Rhizosphere microorganisms-opportunists, bandits or benefactors. In Soil Microbiology (N. Walker. Ed.), pp. 21-38. Butterworth. London. GRAY T. R. G., BAXBY P., HILL J. R. and GCI~DFELLOW M. (1967) Direct observation of bacteria in soil. In The Ecology of Soil Bacteria (T. R. G. Gray and D. Parkinson, Eds), pp. 171-197. Liverpool University Press. JENKINSON D. S. and POWL~~N D. S. (1976) The effects of biocidal treatments on metabolism in soil-V. A method for measuring soil biomass. Soil Biology & Biochemistry 8, 209-213. JENSEN V. (1967) The plate count technique. In The Ecology of Soil Bacteria (T. R. G. Gray and D. Parkinson, Eds). pp. 1588170. Liverpool University Press. NEWMAN E. J. and WATSON A. (1977) Microbial abundance in the rhizosphere: A computer model. Plant and Soil 48, 17-56. ROVIRA A. D. and DAVEY C. B. (1974) Biology of the rhizosphere. In The Plant Root and Its Encironment (E. W. Carson, Ed.), pp. 1533204. University Press of Virginia, Charlottesville. RUSSELL E. W. (1961) Soil Conditions and PIant Growth, pp. 146147. Longman, London.