Predicted microbial biomass in the rhizosphere of barley in the field

Plant and Soil 97, 303-314 (1987). �9 1987MartinusN~hoffPublishers, Dordrecht. Printed in the Netherlands.

Ms. 6694

Predicted microbial biomass in the rhizosphere of barley in the field

MARTIN WOOD Department of Soil Science, The University of Reading, London Road, Reading, RG1 5AQ, UK

Received 30 December 1985. Revised April 1986

Key words Barley C economy CO 2 fluxes Hordeum vulgare L. Microbial biomass Rhizosphere Roots

Summary Laboratory data for the loss of root material by barley and field data for the growth of barley plants in Syria and in England have been combined to predict the amount of material lost by barley roots during a season, and to predict the resulting microbial biomass in the rhizosphere. The predicted microbial biomass C in the rhizosphere ranged from 10-34% of the total plant biomass C depending mainly upon the value used for rate of loss of root material. Total loss of root material predicted during a season in England constituted 7.7~5.4 percent of C fixed by photosynthesis. The major assumptions made in these calculations are considered, and the predicted values discussed in relation to reported values for soil microbial biomass, CO, fluxes from soil and associative nitrogen fixation.

Introduction

The loss of organic material by plant roots is of major importance in determining the activity of soil microorganisms 3 and the soil adjacent to the surface of roots has long been recognised as a zone of intense microbial activity, defined as the rhizosphere 9. Furthermore, the loss of organic material and CO2 from plant roots forms an important component of the carbon economy of plants 5.

The nature and types of material lost from plant roots have been comprehensively reviewed 22'23 together with the response by soil microorganisms to this material 5'~3'~8 and the amount of C lost from plant roots has been quantified for cereals growing in a range of media in the laboratory 2'3't4'26'27. Estimates range from 14-23% of total net fixed C |4

to 1 7 - 4 0 ~ .

The C cycle within a soil-plant system is a complex network of pools and fluxes (Fig. 1) which are currently impossible to measure accurately under field conditions. Models, both conceptual 5 and mathematical ~8'25 are useful not only as a means of assimilating existing information, but also to indicate areas of ignorance. In the absence of field data on loss of root material by cereals, predictive data can be useful for formulating models and for assessing new techniqiues of field measurement. The aim of this paper is to use field data for growth of barley 4'6 and laboratory data for rates of loss of root material 2'27 to predict the amount of root

303

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/ ~. positive

' lm

~I~ORGANIC ~ / /

"]7

3 m

"1" CO 2

)CO 2 flux ~ Organic C flux

Fig. 1. Diagram showing the pools and fluxes of C in a soil-plant system (1~, respiration by the crop canopy; Pc, photosynthesis by the crop canopy; R,, respiration by the root; H, heterotrophic; A, autotrophic; R~, respiration by heterotrophic microorganisms; Rm, respiration by autotropic microorganisms).

material lost by barley stands during a season, and the resulting microbial biomass in the rhizosphere.

Assumptions The major assumptions made in these calculations are:

(1) The amount of material lost by a plant root depends upon (a) Root weight, (b) Rate of loss of material per unit root weight, (c) Time period of loss.

(2) The rate of loss of material per unit root weight remains constant throughout the season.

(3) The root material is utilized equally and readily by soil microorganisms (bacteria) for growth only (no maintenance).

(4) The yield of bacteria is considered as three consecutive batch cultures, each supplied with substrates by roots of different weights.

(5) Although the C cycle comprises both pools and fluxes (Fig. 1), only pools of root material (based on fluxes and pools measured in the laboratory) and microbial biomass are calculated. No recycling of respired CO2 or microbial C is therefore considered. The input of C to the soil from litter is also assumed to be negligible.

Root weight in the field Data were used for growth of barley in Syria 6 and in England 4. Data

for Syria are the mean of three varieties grown at Jindiress during the

MICROBIAL BIOMASS IN BARLEY RHIZOSPHERE 305

1982-1983 season which yielded 3.8 t ha- l grain. The data fell into three phases:

(i) Sowing to start of stem extension (0-18 weeks) (ii) Stem extension to anthesis (18-23 weeks)

(iii) Anthesis to maturity (23-27 weeks). Data for England are for Proctor barley grown at Nottingham during the 1972 season which yielded 4.9 t ha -~ grain. The data fell into three phases:

(i) Sowing to first measurement (0-5 weeeks) (ii) Measurements on root system of increasing weight (5-11 weeks)

(iii) Measurements on root system of decreasing weight (11-17 weeks). Anthesis occurred after 13 weeks and harvest after 22 weeks, therefore data for England was available for only 17 of the 22 weeks of the season. Data for Syria (Table l) show an increase in root dry weight throughout the season, in contrast to the data for England (Table 1) which show an increase to 11 weeks followed by a decrease in root weight from 11-17 weeks. Although it is recognised that these data are for net changes in root weight resulting from a balance between root growth and death, it was assumed that for the whole season in Syria and for the first 11 weeks of the season in England the root was growing and therefore all forms of active and passive loss of root material were operating. The laboratory data for loss of root material by growing roots were therefore applied to these periods, but not to the final period (11-17 weeks) of the season in England. During this particular phase a decrease in root dry weight occurred despite a continued increase in total plant biomass, and it was assumed that the loss of root material observed in the field (120 gm- : ) , was converted directly into heterotrophic microbial biomass. No additional contribution from root exudates was assumed, therefore the laboratory data were not applied to this phase.

The data presented in Table 1 are the cumulative root weights at different times throughout the season. In order to calculate the amount of root material lost over a period of time a constant root weight was assumed for that particular period; this was taken as the sum of the root

Table 1. Root weight (gin -2) for barley growing in Syria 6 and England 4

Site Time (weeks)

18 23 27

Syria 43 62 77

Time (weeks)

5 11 17

England 50 250 130

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weight at the start of the period plus half the increase in weight during that period. These values are shown in Table 3.

Loss of root material in the laboratory Reported rates for loss of root material by two varieties of barley

growing in a range of media in the laboratory are presented in Table 2. Rates (fluxes) have been calculated from the original data (pools) for plants growing for 16-21 days. The data for barley growing in soil and sand were obtained using ~4C-labelled CO2 and by measuring the amounts of CO2 and organic C lost from the roots. It is recognised that a component of this calculated C flux is due to root respiration, but in the absence of data for barley on the relative rates of CO2 flux due to root respiration and due to respiration by microorganisms utilizing material lost from the root in the rhizosphere it was assumed that root respiration was negligible compared to microbial respiration and therefore all of the C lost by the roots was in a form available to heterotrophic microorganisms. The data for barley grown in sterile solution culture are based on soluble carbohydrate measurements which may provide a more realistic estimate of the material available to microorganisms, but which may also underestimate the total amount of material lost from the root. The rate for non-sterile plants in solution culture was calculated from the measured microbial biomass, assuming 2.86 g equivalent root material g-~ bacteria. With the exception of Midas barley grown in sand (reported to be an extremely xeric treatment) the presence of microorganisms caused an increase in the rate of loss of material, hence the positive feedback loop shown in Fig. 1.

Predicted loss of material in the field Two of the reported rates of loss of root material for non-sterile barley

plants were used to predict the loss of root material by barley in the field.

Table 2. Rates of loss of root material (#g equivalent root material mg -1 root day -~) by barley varieties in the laboratory under sterile (ST) and non-sterile (NST) conditions

Variety Medium Rate of loss* Reference

Proctor Solution (ST) 9.5 3 Proctor Solution (NST) 46.0 3 Proctor Soil (ST) 22.4 2 Proctor Soil (NST) 35.2 2 Midas Sand (ST) 35.9 27 Midas Sand (NST) 26.0 27 Midas Soil (ST) 87.0 27 Midas Soil (NST) 117.0 27

t All rates based on total C lost except Ref. 3 based on soluble carbohydrate loss and the non-sterile value calculated from the measured microbial biomass.

M I C R O B I A L BIOMASS IN BARLEY R H I Z O S P H E R E 307

l17#g equivalent root material mg -1 root day -1 27 was used as the maximum rate, and 35/~g equivalent root material mg-~ root day-t was used as a more representative rate and also because it refers to Proctor barley to which the field data for England also applies. The amount of material lost was calculated as the product of rate of loss, root weight and time. Table 3 shows the predicted amounts of root material lost throughout a season by barley growing in Syria and in England using these two rates of loss of root material. The laboratory data were applied only to roots increasing in weight (Table 1) and not therefore to the final phase (11-17 weeks) of the season for barley growing in England for which the measured decrease in root weight in the field was used (120gm 2). The predicted data based on the lower rate of loss of root material (229-373gm 2) are similar to data estimated by a different method for wheat growing in South Australia of 227 g equivalent root material m -2 (100 g C m-2) j7.

Predicted microbial biomass in the rhizosphere in the field The microbial biomass in the rhizosphere of barley plants was cal-

culated from the estimated amounts of material lost by roots in the field using a conversion rate of 0.35g bacteria g-~ carbohydrate ~'3"8'27 (if bacteria contain 50% C and carbohydrates contain 40% C then this assumes a conversion efficiency of 44%). If it is assumed that all of the material lost by the roots is utilized equally and readily by the soil microorganisms (assumption 3) then glucose, carbohydrate and root material, which all contain 40% C, can be used interchangeably as

Table 3. Predicted loss of root material (g equivalent root material m-2) by barley growing in Syria 6 and England 4 assuming rates o f loss of (1) 117 #g equivalent root material rag- t root d a y - ~ :Tand (2) 35/~g equivalent root material mg -h root day -E~-

Time Root weight Root loss (gm -2) (weeks) (g m - 2 )

(l) (2)

Syr~ 6-18 22 317 95

18 23 53 215 65 23 27 70 228 69

Total NA 760 229

England 6-5 25 102 31 5 11 150 737 222

11 17 t 120 t 120 t

Total NA 959 373

NA not applicable. * observed root loss used.

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Table 4. Cumulative biomass C (g C m-2) as shoot and root (measured) and as rhizosphere bacteria (estimated) for barley growing in Syria 6 and England 4 assuming rates of loss of root material of (1) 117/ag equivalent root material mg - i root day - ~ 27 and (2) 35/~g equivalent root material m g - l root day- 12

Site C pool Time (weeks)

18 23 27

Syria Shoot 35 179 365 Root 17 25 31 Bacteria (1) 56 94 134 Bacteria (2) 17 29 41

Time (weeks)

5 11 17

England Shoot 11 196 436 Root 20 99 52 Bacteria (1) 18 147 168 Bacteria (2) 6 45 66

substrates in these calculations. Therefore: microbial biomass (gm -2) = 0.35 x loss of equivalent root material (gm-2).

Table 4 shows the predicted microbial biomass in the rhizosphere based on the estimated amounts of root material lost by barley plants growing in Syria and in England. The predicted microbial biomass based on the highest reported rate of loss of root material 27 is a factor of 3-4 larger than the final root biomass whereas the microbial biomass based on the lower rate of loss of root material 2 is approximately the same size as the final root biomass.

The predicted biomass C pools for barley growing in Syria and England in relation to the measured C pools are shown in Fig. 2 in which the size of the C pools is proportional to the area of the triangles. C pools were calculated by assuming bacteria contained 50% C and the root and shoot material 40% and 44% C respectively 4. The data shown for Syria (a dry environment with 417 mm annual rainfall) are based on the lower rate of loss of root material, similar to that reported for barley growing in dry sand 27 as is the data shown for England. Both total plant biomass C and microbial biomass C in the rhizosphere increases during the season. The microbial biomass C in the rhizosphere at harvest constitutes 34% of the total plant biomass C at both sites assuming the maximum rate of loss of root material, and constitutes 10 and 14% for barley in Syria and England respectively assuming the lower rate of loss of root material.

Measured microbial biomass and loss o f root material in the laboratory The rate of loss of material from roots is of critical importance in

predicting the microbial biomass in the rhizosphere and the values in


18 weeks 23 weeks 27 weeks

v v

5 weeks 11 weeks 17 weeks

. . . .

RL M ~, RL / ~ 1 1 / t ~ r S

Fig. 2. Diagram showing the predicted pools of C in the rhizosphere of barley growing in Syria and England during a season (area of triangles is proportional to C pool expressed as gm-2; S, shoot; R, root; G, grain; RL, root loss; M, microorganisms).

Table 2 vary widely. In one study 27 both the loss of root material and the number of rhizosphere microorganisms were measured for wheat and barley grown in sand and soil. If it is assumed that all of the material lost by the barley roots is available equally and readily to the rhizosphere bacteria (assumption 3) and that the conversion rate is 0.35 g bacteria g- equivalent root material, then the microbial biomass for barley in sand (mean cell weight 3.2 x 10-~2g, 6.2 x 107 bacteria mg -1 root) was 0.20mgmg -~ root. This would have required 0.57mg equivalent root material mg-l root compared to the measured value of 0.55 mg equivalent root material mg-x root. The same calculation applied to the data for barley growing in soil gives a requirement for root material based on the measured microbial biomass of 2.57mg equivalent root material mg -~ root compared to the measured value of 2.46mg equivalent root material mg-~ root. A quantity of fungal mycelia was observed in soil growing barley and this may place an additional (unknown) requirement on the supply of substrates by the roots. Also, the number of bacteria was counted on nutrient agar which probably underestimated the total number of bacteria.

These calculations demonstrate that observed rates of loss of root material of 117 #g mg-~ root day-~ (for barley growing in non-sterile soil)

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and 35#gmg -l day -1 (similar to the value for barley growing in sand) are in general agreement with the observed microbial biomass in the rhizosphere, and that the use of these values to predict the microbial biomass in the field is not unreasonable.

Predicted microbial biomass in relation to reported values The values for microbial biomass in the rhizosphere predicted here can

be compared with reported values for general soil microbial biomass measured by a range of techniques 1~ If the rhizosphere is the major zone of activity in the soil for microorganisms 3 then this comparison should be a valid one. Reported values for microbial biomass C in soils used to grow wheat range from 170-650 #g C g-~ soil ~~ equivalent to 43-162 g C m -2 (assuming 2,500 t ha-~ soil). The values predicted here for microbial biomass C in the rhizosphere of barley growing in the field range from 41-168gCm -2. These values agree well, even after taking into account a turnover time for the microbial biomass of 1.25-2.5 yr 1~ confirming that the rates of loss of root material used in these calculations are not unreasonable.

Predicted microbial biomass in relation to observed C02 fluxes In addition to data for weight of roots and shoots of barley growing

in England, data are also available for CO2 fluxes in the same barley stand". The flux of CO2 from the soil surface was measured and partitioned into the CO2 flux due to root respiration (calculated from the increase in root weight) and the flux due to respiration by soil microorganisms (by difference). The component of the total COz flux from the soil surface not due to root respiration was considered to be due to the decomposition of existing soil organic matter by microorganisms. How- ever, this component could also have been due to the utilization by soil microorganisms of material lost by the barley roots.

Values reported for the component of total CO2 flux from the soil surface not attributable to root respiration range from 0.05-0.14gCO2 m-2 h -14. This implies that the C lost from the soil surface as COz other than that due to root respiration during the period from 5-11 weeks was 50-141 g CO2 m -2 equivalent to 14-38 g C m -2. The predicted total C released from the roots during this period is 89-295 g C m -2 yielding a microbial biomass of 39-129 g m -2 (for barley growing in England). From assumption 3 the difference between the values for loss of C from roots and the values for microbial biomass C must equal the amount of C lost from the soil due to respiration by the rhizosphere microorganisms and the roots, which is 60-166gCm -2. This C flux is in excess of the measured values (14-38 g C m -2) and may reflect inaccuracies in the measurement of CO2 fluxes from the soil


surface in the field, incomplete decomposition of root material, or recycling o f C O 2 in the soil by autotrophic microorganisms (Fig. 1). These data suggest that the observed fluxes of CO2 from the soil surface attributable to soil microorganisms can be accounted for by the activity of microorganisms in the rhizosphere utilizing material lost from plant roots rather than existing soil organic matter, even after taking into account the fact that approximately 20 percent of the loss of C from the roots is due to production of CO2 by root respiration 24.

Based on an average rate of gross photosynthesis of 250gCO2 m -2 week -~4, the total photosynthetic CO2 flux during a season for barley growing in England can be estimated as 1160 g C m -2. The predicted loss of root material of 89-295 g C m -2 therefore constitutes 7.7-25.4 percent of the C fixed by photosynthesis during the season.

The potential for nitrogen fixation in the rh&osphere of barley It has been proposed that non-symbiotic nitrogen fixation by bacteria

associated with plant roots may be important in the N economy of plants 7. Long term measurements of nitrogen fixation in the rhizosphere of wheat using 15N isotope dilution have indicated, however, that under conditions found in the United Kingdom this contribution is of little significance ~2. A value of 10-15mgN fixed g-l sugar for free-living nitrogen fixing bacteria 21 has been applied to barley plants grown in solution culture 3 and can also be applied to barley grown in soil. It is assumed that all of the material lost by barley during a season is available equally and readily to free-living nitrogen fixing bacteria; therefore a mean conversion rate of 12.5 mg N fixed g-l equivalent root material was used.

The predicted loss of root material in a season by barley grown in Syria is 229-760 g equivalent root material m -z, which could support fixation of 2.9-9.5 g N m -2 (29-95 kg N ha-~). The corresponding loss of material by barley growing in England is 373-959g equivalent root material m -2 which could support fixation of 4 .7-12.0gNm 2 (47-120 kg N ha-l) . These are maximum values for the contribution of associative nitrogen fixing bacteria and they assume that all of the root material is utilized by nitrogen fixing bacteria, which is highly unlikely in the field. In practice the amount of N available to the plant would be less due to incomplete utilization of root material and unavailability of fixed N to the plants.

Limitations to these predictions Several major assumptions and simplifications have been made in

calculating the data presented here. These assumptions appear justified firstly in the absence of any improved data and secondly because in

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several cases the predictions do agree with reported data. One of the major limitations to the calculations is the lack of information on the loss of C by barley roots in the field, and on the compartmentalization of the C lost into that due to root respiration, microbial respiration utilizing root material, and other non-utilized root material. Values of 0.45-1.34 g C g-~ C incorporated into dry matter have been suggested for the C loss in respiration for growth including ion uptake by roots of a range of plant species (excluding barley) 1~ . If applied to the C increment from 5-11 weeks for barley growing in England (264 g m -E) this suggests a C flux from the soil surface of l19-354gCm -2 equivalent to 436-1298 g CO2 m -2. This is greatly in excess of the total measured CO2 flux from the soil surface in the barley stand of 241 g CO2 m - 2 which includes a component due to respiration by soil microorganisms 4.

The measurement and compartmentalization of CO2 fluxes for plants growing in non-sterile soil presents problems if fluxes are to be partitioned into those due to root respiration (maintenance and growth), uptake of CO2 by autotrophic microorganisms, respiration by heterotrophic microorganisms utilizing root-derived material and respiration by heterotrophic microorganisms utilizing existing soil organic matter (maintenance and growth). Assumption 5 was therefore a necessary simplification. In addition, despite recent advances in methods for measuring microbial biomass in soil ~~ techniques such as chloroform fumiga- tion may not be suitable for rhizosphere soil 16. For wheat growing in soil in a controlled environment chamber, 25 percent of the total CO2 formation in the root region was attributed to soil respiration and 75 percent to root respiration and root decomposition 24. Using a solution culture method the CO2 flux from wheat roots was partitioned into 80 percent due to root decomposition and 20 percent due to root respiration. These data suggest that the flux of CO 2 from decomposition of root material comprises the major CO2 flux in the root region of wheat plants. The same may therefore be true for barley. Although this root material may be heterogenous 22'z3 and not all equally and readily available to the soil microorganisms, ~4C data suggest that a large proportion is carbohydrate ~5, therefore assumption 3 may not be unreasonable.

There is a need to characterize and quantify more precisely the loss of organic material from plant roots growing in soil under field conditions; not only is the root weight changing continuously, but the rate of loss of root material per unit root weight is also likely to change throughout the season in response to both developmental changes within the plant e.g. at anthesis, and changes in environmental conditions e.g. moisture stress. Assumption 2 was a necessary simplification; the laboratory data used in these calculations gave only final root weights and total loss of root


material therefore the rates of loss of root material were calculated by:

total root loss rate of loss =

final root weight x time

For more precise estimates of root loss it may be necessary to consider the root as a series of small segments and to measure the change in rate of loss of material with time for each segment. The total amount of material lost per segment and, by summation, the total amount lost by the whole root could then be calculated (assumption 1).

A final limitation to thse estimates of microbial biomass is the con- sideration of the supply of substrates by the roots to the soil microorganisms as three consecutive batch cultures (assumption 4). The growth of microorganisms around plant roots may be more accurately considered as a fed batch culture system with kinetics of continuous culture 13'2~ and with inputs defined as above. A similar approach was incorporated in the mathematical model of Newman and Watson ~9 describing the changes in numbers of microorganisms in the rhizosphere over 10 or 40 days. This model assumed rates of loss of root exudates of 4-400 mg g-1 root dry weight increase, which, using the data for barley growing in England 4 is equivalent to 1.0-100 g root exudates m -2. This loss of root material is much lower than the predicted values used here of 373-959 g equivalent root material m -2 (Table 3). This illustrates the need for detailed information on the nature and rates of loss of material by plant roots in the field. Only when these data are available can more reliable estimates be made of the microbial biomass in the rhizosphere under field conditions.

References

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314 MICROBIAL BIOMASS IN BARLEY RHIZOSPHERE

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Predicted microbial biomass in the rhizosphere of barley in the field