Role of wheat root exudates in associative nitrogen fixation

Soil Biol. Biochem. Vol. 15, No. I, pp. 33-38, Printed in Great Britain. All rights reserved

0038-0717/83/010033-06%03.00/O

1983 Copyright

0

1983 Pergamon

Press Ltd

ROLE OF WHEAT ROOT EXUDATES IN ASSOCIATIVE NITROGEN FIXATION S. M. Department

of Bacteriology

BECK and C. M. GILMOUR

and Biochemistry, (Accepted

University

of Idaho,

Moscow,

ID 83843,

U.S.A

15 July 1982)

Summary-Root exudates and related exudate diffusion gradients were studied using “C-radioisotope techniques. With inoculated wheat plants (Triticum aestivum CV. Nugaines), 3.7% of the “‘C-labeled photosynthate was released as soluble exudate whereas 3.0% was found with axenic plants. Root surface areas averaged 54 cm* plant ‘. The microbial cells produced were sufficient to colonize 7.4%, of the root surface with a cell monolayer. Gradient studies showed that with inoculated root systems, rapid utilization of soluble exudate markedly decreased the distance of exudate diffusion. Microbial colonization also was a function of the physiological features of the test culture. The relationship between root colonization, exudate production and potential for associative nitrogen fixation is discussed.

vitamin B,,, 0.05 g yeast extract, 0.34 g K,HPO, and 0.17 g KH,PO,.ll’ distilled water (pH 6.9 without adjustment). Single cultures were tested for N,ase activity by inoculating into 30 ml serum bottles containing 15 ml of semi-solid L&L medium, incubating for 3 days at 28°C closing with serum bottle stoppers, replacing 5% of the bottle atmosphere with C,H,, incubating again for 24 h and determining C2H, reduction by the method of Stewart et al. (1967). The cultures for seed inoculation were grown individually in glucose yeast nutrient (GYN) broth (5 g peptone, 3 g beef extract, 5 g glucose, 0.5 g yeast extract). 1-I distilled water, centrifuged, washed and resuspended in L&L broth. In the sand-plant studies of total exudate, selected seedlings were dipped into an equal mixture of the 5 organisms in L&L broth, each having an absorbance of 0.39 at 500nm, and blotted against the side of the tube. In the agar gel gradient experiments, a grass-root organism was culture I and a newly isolated Gram-negative rod was culture II. Surface-sterile germinated seeds were used in the axenic plant studies.

INTRODUCTION

The presence of free-living N,-fixing bacteria in the rhizosphere and microbial association with the rhizoplane have attracted considerable attention. Of special interest and speculation is the amount of root exudate C required to meet the energy needs for significant N,ase activity. Rovira and McDougall (1967) using hydroponic solutions and axenic wheat cultures found that O.llO.4% of the photosynthate was excreted. Subsequently Barber and Gunn (1974) showed that 59% of total plant weight was released as exudate. Barber and Martin (1976) reported that wheat or barley root exudates could comprise 7-I 3”/, of the total dry matter content of axenic plants growing in soil, and in nonsterile soil, root exudates increased to 19-25x of the plant dry matter produced. We have reassessed the magnitude of plant exudates and related secretion gradients in respect to root colonization by N,-fixing cultures. MATERIALS AND METHODS

Cultures and Inoculum

Seed Surface Sterilization

A mixed inoculum of five Gram-negative, motile, N,ase-positive bacterial isolates was used. The morphology, cultural characteristics and high N,ase activity of two isolates from native Idaho grasses suggest that they belong in the family Azotobacteraceae. Variable pigment formation and lower rate of C2H, reduction indicate that the two cultures from rice and one from wheat probably represent different taxonomic entities. All culturing was done aerobically. The cultures were isolated and maintained on a modified Line and Loutit (1971) medium (L&L) consisting of 5 g glucose, Noble agar (2.5 g for semisolid or 10 g for solid medium), 0.5 g MgS0,.7H,O, 17 mg FeCl,.6H,O, 3.8 mg MnS0,.4H,O, 5 mg ZnS0,,7H,O, 4 mg CuS04.5H,0, 25 pg CoSO,. 7H,O, 5 mg Na,MoO,.2H,O, 5 mgH,BO,, 92 mg CaCl,. 2H,O, 0.75 mg biotin, 1.4mg thiamine, 1.5 pg

Wheat seed (Triticum aestivum cv. Nugaines) were wetted in vacua, soaked in distilled water for 1 h, slowly shaken for 2.5 h in covered Petri dishes in a solution of NaOCl (1 mg ml-‘) which was changed after 5 min and after 30 min to compensate for Cl, depletion, rinsed twice with sterile distilled water, immersed in 0.1% (w/v) Na,S,O,. 5H,O for 5 min, and finally rinsed twice with sterile water. They were aseptically transferred to plates of GYN agar for germination and observation for contamination. Plant Growth The plant nutrient solution (pH 6.7) contained (1-I distilled water): [0.49 g MgSO,. 7H,O, 0.40 g KNO,], [I.18 g Ca(N0&.4H,O], [0.34 g K*HPO,, 0.17 g KH,PO,], [lo mg ferric citrate.(n-hydrate)], [2 mg MnSO,,H,O, 2mg ZnS0,.7H,O, 1 mg CuSO,. 33

FRR 1511 c

S.

34

M.

BECK and C. M. GILMOUK

5H,O, 25 pg CoS0,.7H,O, 1 mg Na,MoO,.2H,O and 3 mg H,BOJ. Compatible constituents [in brackets] were diluted in large portions of the total water, mixed with vigorous stirring, and filter sterilized. Narrow-mouth 125 ml Pyrex bottles containing 150 g washed 0.60--0.85 mm-standard Ottawa silica sand were weighed to f 10 mg and covered with black construction paper. Loosely fitting Pyrex towers (3.5 cm i.d x 19 cm height} stood on the shoulders of the bottles and were topped with inverted Pyrex dishes. The glass assemblies were autoclaved, 25 ml sterile plant nutrient solution added, and an axenic or inoculated sprouted seed placed into a 1.5 cm depression and covered with the sand. Twelve assemblies were placed in an 18 x 24 x 32 cm high rectangular Pyrex jar and sealed with a gasketed and clamped glass lid. The atmosphere was maintained at 0.330.60/;:, ‘%Oz by injecting 0.71 M Na,‘4C0, (sp. act. = 0.157 @CI mg-’ C) into an excess of 55% H,SO, contained in a 32 x 250 mm CO2 generator tube inside the plant growth chamber. To distribute and mix the ‘%ZOz, an external stainless steel vacuum-pressure pump withdrew atmosphere and delivered it back through the COz generator or through pin holes of a distribution tube around the inside base of the jar. Gas connections between pump and jar were through l-hole and 2-hole rubber stoppers in the plate glass lid. Day length was 16 h at temperatures of 25‘C (day) and 2OC (night). Light intensity was 40pE ss’ m ’ at mid-plant height. After 14 days, 25 ml distilled water was added to each bottle. Individual plants were removed, placed in 150 ml beakers, and the roots washed with distilled water. Washings and loose sand were returned to their respective bottles which were then weighed to 210 mg to determine total added liquid. Bottles were stoppered and shaken vigorously for 3 min. Ahquots of each supernatant liquid were immediately removed, weighed into tared combustion boats, made alkaline with a drop of 0.01 N NaOH. and dried at 40-50°C under a heat lamp. The “‘C-content of each dried exudate sample was determined by dry combustion in O2 and absorption of the 14C0, in a train containing 20% (v/v) monoethanolamine in methanol. Each train was diluted to 50 ml with methanol, 10ml aliquots were mixed with 10ml portions of 0.47; BBOT in toluene and ‘%I was counted on a Beckman LS-I 00 scintillation counter. The residual seed coat was discarded. Roots and shoots were placed into separate weighed combustion boats, air-dried and weighed. Organic matter was determined by dry combustion. Absorption train contents were diluted to 100ml with methanol, and 1 ml aliquots counted for 14C. Dry weights and ‘x ash of the shoots were directly determined; those for the roots were calculated using 47%C for the organic matter and 15*,’ ,” ash . Exudate

tubing (3.2 mm i.d. x 2.4 mm wall) arranged to leave a 2.0-2Scm hole at the top center of the chamber (Fig. 1). Small triangular hardwood blocks 7 mm thick were clamped between the two plates at each of the four corners to prevent compression during handling. Inside was a glass “filler” piece 3 x 146 x 95 mm high with the two bottom corners cut off to make it rest on the chamber bottom. The hole in the top was plugged with cotton, and a folded heavy aluminum foil cover protected the entire upper edge of the chamber. To facilitate exudate diffusion and spread of bacterial growth, a semi-solid L&L medium containing 0.60/;, Difco Noble agar was used. After autoclaving, the medium was aseptically pumped into the chamber to the top of the filler plate which decreased the agar slab thickness and enhanced planar diffusion of root exudate. Before it solidified, an axenic or inoculated seedling was placed aseptically on the agar surface with the three primary roots pointing into the medium. When the shoot was tall enough, its tip was aseptically led out of the hole at the top and the cotton plug replaced. Plants were grown for 5-8 days in a growth chamber using a day length of 16 h, temperatures of 25°C (day) and 20°C (night), and a light intensity of 56jiE s ’ m ’ at mid-plant height. After sufficient plant growth, four chambers (Fig. I) were placed in a 30.5 x 9.8 x 25.4cm high glass chromatography tank which was sealed with a gasketed and clamped glass lid. Remaining gas volume was about 4 1. At the end of an 8 h dark period, the plants received a singie pulse of 10 6rCi ‘“CO, which after 12-15 h. Gas circuwas 90-97’:; assimilated DOUBLE

PLATE

CHAMBER

\

\

)---AR

AREA

.ATES

“C

ROOT EXUDATES

Gradients and Root Colonization

Double plate growth chamber studies Four large spring paper clips clamped two 3 x 165 x 127 mm high plates of double strength window glass against a 49 cm section of gum rubber

Fig.

1. Growth

chamber for ‘“C-diffusion gradient studies.

Wheat

root exudates

and associative

lation, light, and temperatures were as in the total exudate studies. After pulsing, chambers were placed on a dark

background and a root map sketched on a clear plastic sheet. They were refrigerated at 5-10°C for 1S-20 min to harden the semisolid agar and then laid flat while clamps, top plate, and gasket were removed and water of syneresis was blotted off with filter paper. Selected bars of agar 3 cm long were cut perpendicular to the root axis with a gel cutter having parallel razor blades 1 cm apart. Each agar bar was placed on a large rubber stopper and sectioned parallel to the contained piece of root with another gel cutter having 1.5 mm spaces. Agar sections stood 30min in scintillation vials in 10ml absolute methanol; 10 ml 0.4’j/, (w/v) BBOT in toluene were added and ‘C determined. RESULTS

With inoculated wheat plants 3.7% of the Ylabeled photosynthate appeared as exudate whereas 3.0% was released from axenic plants (Table 1). Since respired 14C0, was not measured, these data do not include metabolized exudate C and must be regarded as minimal C release values. The 14C0, respiration data reported by Barber and Martin (1976) with non-sterile root systems showed that a highly significant proportion of the soluble exudate was oxidized to CO,. If their respiration values are used to adjust our mean percentage exudate data (Table l), our mean exudate values then become ca. 11% for the inoculated and ca. 4% for the axenic plants. The exudate data also are presented on the basis of total plant C. The mean values of 133.0 mg g ’ inoculated dry roots and 118.4 mg g-’ for the axenic roots are within the ranges reported by Barber and Martin (1976). The C and N compounds which are excreted by plant roots undoubtedly serve as nutrients for the rhizoplane and rhizosphere flora, but the site, nature Table

1. Distribution

of photosynthate

in inoculated

and amount of such exudates will determine the microbial colonization potential of the root complex (Bowen and Rovira, 1976). SEM photomicrographs obtained by Elliott et al. (1979) showed scattered cell aggregates of microcolonies on and adjacent to young and mature wheat roots and brought special focus on mucigel embedded colonies. Unfortunately, no accurate numerical estimate of root coverage can be obtained by the TEM or SEM approach. In our study the wheat seedlings had a mean root surface area of 54 cm2. Inclusion of microscopically estimated root hairs increased the total root surface area by a factor of 2-3. The 14Ctotal counts for exudate C were used to calculate percentage particulate matter. Membrane filtration of the water extract showed that ca. 30% was insoluble and 70”/, soluble in water. With the inoculated plants the insoluble particulate matter “C-counts were considered to represent microbial biomass C plus a small contribution from detached root cells. The insoluble portion of the axenic culture extracts contained only sloughed off root cells. The difference in “C-counts between the inoculated and axenic samples gave an approximation of microbial biomass C. The “C-specific activity of the plant organic matter was used to calculate a representative weight of biomass C. The air-dry weight of a single bacterial cell was assumed to be 0.25 pg and its total C content to be 52%. From these, the mean microbial population weight for the inoculated plant roots was calculated to be 51.3 pg and the related cell count 3.9 x lO*plantt’. This is sufficient to provide a cell monolayer covering approximately 7.4% of the root surface, and is in close agreement with the low microbial coverage of plant root systems reported by Christie et al. (1974) Rovira et al. (1974) and by model predictions of Newman and Watson (1977). Exudate

1 3 5 9 Mean + SD 4 6 10 12 Mean + SD F (1.6) P>F

lo5 dpm

’ plant - ’

root secretions

and axenic

that C and N compounds of are quickly used by rhizoplane flora.

Triticum aestiuum cv. Nugaines

wheat

seedlings

Distribution 14C photosynthate (%) C(mg) plant

’

69.2 75.0 67.8 71.6

41.2 42.8 40.1 42.8

70.9 k 3.2

41.7 f 1.3

Roots

Inoculated~ 67.3 66.9 70.4 71.5

28.5 29.6 25.8 25.3

4.1 3.4 3.9 3.2

143.0 115.1 148.9 124.9

27.3 + 2.1

3.7 * 0.4

133.0 * 15.7

23.3 25.6 26.5 26.1

3.7 3.5 2.3 2.5

157.3 135.0 85.1 96.0

25.4 & 1.4

3.0 + 0.7

118.4 + 33.6

2.32 0.179

2.52 0.164

NS NS

69.0 + 2.3

46.5 47.9 51.6 56.0

84.8 + 7.1

50.5 * 4.3 15.54 0.008

71.7 + 0.9 4.76 0.072

Exudate*

Exudate* (mg g ’ dry root)

Shoots

Non inoculated (axenic) 72.9 70.9 71.4 71.5

77.2 82.3 85.6 94.1

12.81 0.012

gradients

It is well recognized

Total photosynthate Plant No.

35

N,-fixation

*Exudate includes all dissolved and suspended material in growth medium. tInoculum was equal mixture of 5 N,-fixing bacterial isolates (absorbance = 0.39 at 500 nm). NS-not significant.

36

S. M.

BECK and

Table 2. Comparative 14Ccontent of root tips and adjacent root sections 10) dpm - ’ section

’

Adjacent sections Plant No.

Root tip*

1 1 2 2 3 3 4

20.4 28.9 43.8 93.4 14.2 39.0 90.0

4 Mean

1

2

3.8 9.0 19.2 5.2 10.6

ND 11.8 9.8 18.9 12.2 12.2

6.2

ND 2.3

21.3 ND

42.0

8.3

14.4

8.1

*I cm section includes root cap. The Newman and Watson model predicts that, with an initial microbial concentration of 2 pg cmm3, the final cell densities after 10 days growth would be 1509 pgcmm3 at 1.8 mm from the root boundary. The site of maximum microbial activity is the root surface or rhizoplane. Table 2 emphasizes differences in 14C-exudate excretion along the length of sample roots. Much higher counts were obtained with root cap sample sections and associated mucigel sheath areas than with older sections located above the root caps. Individual plants and even different roots of the same plant gave This may be a variable root section “C-counts. reflection of different root ages and activities at the time of VO, pulsing. Figure 2 summarizes exudate gradient patterns obtained with the agar gel growth chamber. Agar section%counts successively distant from each side of the root were plotted to illustrate the overall

350 -

;

t

300-

Culture

II

5 5 3

C. M.

exudate movement from various 1 cm root samples. Gradients with axenic roots were quite different from those with inoculated roots. Rapid consumption of water soluble exudate on or near the root surface by the introduced N,-fixing cultures effectively immobilized it and diminished diffusion of 14C except for lesser amounts of simple water soluble metabolic waste products of the microorganisms. Figure 3 provides a more uniform picture of gradient development in axenic and inoculated root systems. Points on these curves are mean values of two agar sections taken equidistant from opposite sides of the root. Root sections of similar ‘“C-content were selected to minimize variation due to total count. As with the two-sided gradients of Fig. 2, colonized roots showed “C-gradients extending much shorter distances from the roots than in the axenic plant roots. Figure 4 illustrates the influence of physiological features of the test cultures upon the exudate gradient. Culture II effectively utilized and immobilized the exudate in a dense, compact growth zone at the root surface resulting in a 14C-decrease nearly to background level at 1.5 mm from the root. Culture I produced a scant growth zone, and the exudate gradient extended nearly as far from the root as in the axenic plants. DISCUSSION

The observed diversion of plant photosynthate C to root exudates makes clear that water soluble exudate alone cannot supply sufficient substrate C for significant root N,ase activity. However, Warembourg (1975), Warembourg and Billes (1978), Coleman (1973) and others have shown that utilization of other more complex carbon moieties of the soil contribute to the soluble carbon pool, and this undoubtedly enhances root colonization. In this regard, cell growth and maintenance kinetics as described by Barber and Lynch (1977) provide additional insight into the dynamics of the root colonization process. Ecological aspects of root colonization also merit attention. Newman and Bowen (1974) called attention to the non-random distribution of rhizoplane bacteria. Micro and macro colonization were regarded as salient features of the cell distribution

250-

250

= % 5 I I a

GILMOUR

I

?

P 200 L t

\

Iz 2oo

“,I

\

l

Axenic

*

Culture

I[

150-

P d 2

loo-

50-

‘0 LflI11.5 DISTANCE

FROM

ROOT

(mm)

Fig. 2. Distribution of ‘%I in agar-gel sections from roots of inoculated and axenic wheat seedlings.

3.0

4.5

6.0 I

25 I

9.0 I

10.5 I 12.0 II

13.5 15.0 c 16.5 10

18.0

DISTANCE FROM ROOT (mm)

Fig. 3. Mean 14C-content of agar-gel sections from roots of inoculated and axenic wheat seedlings.

Wheat

root exudates

and associative

300.

7

250.

l

GROWTH

ZONE

l None

[Axenicl

o Slight

(Culture

5

0 Diffuse

F 0 lJl 200.

A Dense lculture

5 r: 7,

. \

\

* =

o\I’._. loo

4 ::

II III

0 150 _\

I

?

I)

ICulture

•~o,\o_o~., 50-

. o-0

O\o \

AlA \/;Az>:; Co

1.5

3.0

4.5

DISTANCE

6.0 FROM

25

9.0

ROOT

Fig. 4. Relationship between microbial and V-content of agar-gel

10.5

12.0

(mm)

colonization sections.

density

Such colony patterns were also demonstrated with mature and seedling wheat plants by Elliott et al. (1979). Our observation that the apical meristems showed the highest exudate concentrations fall in line with the data of Rovira and Davey (1974). It may be that active growth and colonization characterize the root cap area where they are initiated, and that both growing and resting cell aggregates comprise the remainder of the total rhizoplane population. This idea is not supported by Rovira (1956). One wonders whether proportional root coverage should be regarded as a realistic value since root section counts probably reflect variable cell numbers and physiological states along the root surface. It has been long been recognized that rhizosphere bacterial counts are influenced by proximity to the root boundaries. However, earlier studies did not emphasize root surface (rhizoplane) populations. Our gradient data with inoculated root systems supports the theoretical prediction of the Newman and Watson (1977) model that a sharp decline in bacterial numbers occurs at a relatively short distance from the root boundary. In our case, the significance of this observation lines in the definition of the ecological niche for associative nitrogen fixation. Obviously the primary seat of N,ase activity is on the root surface or perhaps in the decomposing cortex of the root. Our agar systems differ from soil systems in that they do not possess the complexities of discontinuity, barriers of impermeable minerals, presence of solute adsorption and exchange sites, and other heterogeneous features of the soil. For this reason, our exudate gradients extend a greater distance from the root. Nevertheless, they establish the existence of the gradient, its usage by the microflora of the rhizoplane, and the improbability of significant nitrogenase activity resulting from utilization of exudates as the main energy source. Table 1 shows an overall decrease of 17.5% in total plant C from 50.5 mg in the axenic to 41.7 mg in the inoculated seedlings. This approaches the 19-25%

pattern.

N,-fixation

37

diversion of photosynthate to production of root exudate and respired carbon in non-sterile soil obtained by Barber and Martin (1976). It is somewhat higher than the 11% obtained by correcting our total exudate of the inoculated plants with the Barber and Martin root respiration values. Table 1 also shows a slight tendency for inoculation to decrease the shoot size from 71.7 to 69.0% of the photosynthate with a corresponding increase in the root size from 25.4 to 27.3% of the photosynthate. Our recent work indicates that these results may vary considerably with both wheat variety and inoculum used. Data in Table 1 also may be used to estimate the potential for root exudate to serve as a substrate and support significant associative microbial nitrogen fixation on the rhizoplane of a total wheat crop. Assuming a 2:l straw:grain weight ratio (Dondlinger, 1908) and 1O:l shoot:root weight ratio at maturity (Russell, 1977), a yield of 4000 kg wheat ha-’ also would be accompanied by 8000 kg straw and 1200 kg roots for a total crop biomass of 13,200 kg ha-‘. If the 3.7% excretion of photosynthate as exudate by the inoculated plants in Table 1 is assumed to continue over the entire season, as approximation, or even an overestimate, of 488 kg of root exudate is obtained. Using an N-fixation efficiency of 10 mg N gg’ glucose (or exudate) utilized (Becking, 1974), the root exudate could support a fixation of 4.9 kg N ha- ’ if all of it were metabolized by N-fixing organisms with none being consumed by competitors. Since neither of the latter conditions exists in nature, much of the exudate would be consumed by organisms not fixing N. The use of less than 4.9 kg fertilizer N ha-’ would have such little effect, even when applied continuously on a favorable plant location such as the rhizoplane, that crop improvement by rhizoplane colonizing microorganisms is more likely to result from plant growth hormone production than from N fixation. Even when our inoculated plant exudate value of 3.7% is adjusted to 11% using Barber and Martin’s (1976) respiration data, the N-fixation potential is still only 11 x 3.7-l x 4.9 = 14.6 kg N haa’ yr’. These calculations reveal the limited amount of root exudate available as a substrate for microbial N-fixation in this wheat variety as compared to other substrates available from soil organic matter and from decaying crop residues (Staniforth, 1979), and suggest that attention might more profitably be directed toward other aspects of biological sources of plant N. Acknowledgements-This research was supported by grants from the United States Department of Agriculture (Science and Education Administration) and from the University of Idaho Agricultural Experiment Station. Published with approval of the Director of the Idaho Agricultural Experiment Station as Research Paper 8259.

REFERENCES

Barber D. A. and Gunn K. B. (1974) The effect of mechanical forces on the exudation of organic substances by the roots of cereal plants grown under sterile conditions. New Phylologist 73, 39-45. Barber D. A. and Lynch J. M. (1977) Microbial growth in the rhizosphere. Soil Biology & Biochemistry 9, 305-308.

38

S. M. BECK and C. M. GILMOUR

Barber D. A. and Martin J. K. (1976) The release of organic substances by cereal roots into the soil. New Phytologist 76, 69-80. Becking J. H. (1974) Gram-negative aerobic rods and cocci. Family II. Azotobacteraceae Pribram 1933,5. In Bergey’s Manual of Determinative Bacteriology (R. E. Buchanan and N. E. Gibbons, Eds), 8th edn, p. 253. Williams & Wilkins, Baltimore. Bowen G. D. and Rovira A. D. (1976) Microbial colonization of plant roots. Annual Review of Phyioparhology 14, 121-144. Christie P., Newman E. I. and Campbell R. (1974) Grassland species can influence the abundance of microbes on each others roots. Nature, London 250, 570-571. Coleman D. C. (1973) Compartmental analysis of “total soil respiration”: an exploratory study. Oikos 24, 361-366. Dondlinger P. T. (1908) The Book of Wheat, p. 24. Orange Judd, New York. Elliott L. F., Gilmour C. M., Cochran V. L., Coley C. and Bennett D. (1979) Influence of tillage and residues on wheat root microflora and root colonization by nitrogenfixing bacteria. In The Soil-Root Interface (J. G. Harley and R. S. Russell, Eds), pp. 2433258. Academic Press, New York. Line M. A. and Loutit M. W. (1971) Non-symbiotic N,-fixing organisms from some New Zealand tussockgrassland soils. Journal qf General Microbiology 66, 309-318. Newman E. I. and Bowen H. J. (I 974) Patterns of distribution of bacteria on root surfaces. Soil Biology & Biochemistry 6, 205-209. Newman E. I. and Watson A. (1977) Microbial abundancp

in the rhizosphere: A computer model. Plant and Soil 48, 17-56. Rovira A. D. (1956) A study of the development of the root surface microflora during the initial stages of plant growth. Journal ef Applied Bacteriology 19, 72-79. Rovira A. D. and Davey C. B. (1974) Biology of the rhizosphere. In The Plant Root und Its Environment (E. W. Carson, Ed.), pp. 1533204. University of Virginia Press, Charlottesville. Rovira A. D. and McDougall B. (1967) Microbiological and biochemical aspects of the rhizosphere. In Soil Biochemistry (A. D. McLaren and G. G. Petersen, Eds), Vol. I, pp. 417463. Marcel Dekker, New York. Rovira A. D., Newman E. I., Bowen H. J. and Campbell R. (1974) Quantitative assessment of the rhizoplane microflora by direct microscopy. Soil Biology & Biochemistry 6, 211-216. Russell R. S. (1977)Plant Root Systems: Their Function and Interaction With the Soil, p. II. McGraw-Hill, London. Staniforth A. R. (1979) Cereal Straw, p. 77. Oxford Univ. Press. Stewart W. D. P., Fitzgerald G. P. and Burris R. H. (I 967) In situ studies on nitrogen-fixation using the acetylene reduction technique. Proceedings of the Nationul Academy qf Science of the United States of America 58, 207 I-2078. Warembourg F. R. (1975) Le degagement de CO, dans la rhizosphere des plantes. Bulletin de lu Societe Botanique de France, Colloque la Rhizosphere 122, 77-87. Warembourg F. R. and Billes G. (1978) Estimating carbon transfers in the plant rhizosphere. In The Soil-Root Interface (J. L. Harley and R. S. Russell, Eds), pp. 181-196. Academic Press, New York.