Interference from plant roots in the estimation of soil microbial ATP, C, N and P

Soil Biol. Biochem. Vol. 17, No. 3, pp. 275-278, 1985 Printed in Great Britain. All rights reserved

0038-0717/85$3.00+ 0.00 Copyright 0 1985Pergamon Press Ltd

INTERFERENCE FROM PLANT ROOTS IN THE ESTIMATION OF SOIL MICROBIAL ATP, C, N AND P G. P. SPARLING, A. W. WEST and KARINA N. WHALE N.Z. Soil Bureau, D.S.I.R., Private Bag, Lower Hutt, New Zealand (Accepfed 30 December

1984)

Summary-Excised, solution-grown roots of maize or ryegrass added to two pasture soils at the rate of 6.0 mg gg’ and 13.8 mg g-l, respectively, increased the flush (fumigated minus control values) of CO,-C by up to I .89-fold, KC1 extractable N by up to 1.88-fold, and NaHCO, extractable P by 3.28-fold. The ATP content of the soil was increased by up to 1.42-fold. Because of high variability the effect of the roots on the C and N flushes was not significant at P < 0.05. Incubation of the root-amended soils for 7 days at 25°C prior to fumigation much decreased the contribution from the roots to the C and N flush, and to the ATP content. There was, however, still a large significant effect of the roots on the P-flush, this being up to 3 times greater than the equivalent soil without roots. In soil samples with a high viable root density (>6 mg g-‘) such as may occur in dense pastures, greenhouse pot experiments or rhizosphere soil samples, it is recommended that they be incubated for 7 days prior to fumigation and analyses. Without such prior incubation there is the risk that root material may be included in the “microbial” biomass estimations.

INTRODUCTION

is a comparatively labile fraction of soil organic matter and represents an important reservoir of potentially available plant nutrients (Jenkinson and Ladd, 1981). The chloroform-fumigation technique has proved useful in quantifying the amounts of C, N and P in the microbial biomass (Jenkinson and Powlson, 1976; Ayanaba et al., 1976; Ladd et al., 1981; Brookes et al., 1982; Hedley and Stewart, 1982). Soils are generally sieved prior to biomass measurements, and because sieving removes larger roots, and an incubation before fumigation (preincubation) might be expected to allow decomposition of the smaller ones, the contribution of plant roots to the flushes of C, N and P after fumigation is generally considered to be small (Jenkinson and Powlson, 1976; Brookes et al., 1982; Hedley and Stewart, 1982). Lynch and Panting (1981) suggested that intact cores of soil are preferable to sieved soil; the roots remaining in the cores did not appear to contribute significantly to the C-flush after fumigation (Lynch and Panting, 1980). However, we have been examining soils under permanent pastures where root densities are high. It seemed possible that sieving and pre-incubation may not adequately diminish the contribution of fine roots to biomass measurements. The further incubation of both control and fumigated soils for 10 days during the assay may also reduce the contribution of roots to the C and N-flush, but the biomass-P method (Brookes et al., 1982) uses no such incubation, and interference from roots seemed likely. The presence of roots can also contribute substantially to the soil ATP content (Verstraete et al., 1983), and if this content were used to calculate microbial biomass C (Tate and Jenkinson, 1982) then this could result in overestimation. The soil microbial

biomass

We have therefore attempted to quantify in two grassland soils the contribution from roots to the content of ATP, and to the C, N and P released by fumigation. The experiments were performed by adding excised, solution-grown roots to soil and analyzing the soil immediately, or after a 7-day incubation. MATERIALS AND

METHODS

The experiments were done in two phases (i) the contribution of maize roots to C and N flushes and (ii) the contribution of ryegrass roots to the soil ATP content and P flush. Roots and soils

Maize, Zea mays var. Fantastic Hybrid or ryegrass, Lolium perenne were germinated on damp filter paper and grown for 28 or 76 days respectively in aerated solution culture (Darbyshire and Greaves, 1970). The plants (30) were grown in 601 tanks of solution and the solutions renewed every 14 days. Roots were harvested by cutting from the stem, and were washed in running tapwater, blotted dry, chopped with scissors into l-2 cm lengths, weighed and added to the soil within 30min of harvesting. The two soils, both under sheep-grazed permanent pasture, were Pomare, a Typic Dystrochrept and Waikanae, a Typic Udifluvent and were of low and high P status respectively. Brief analyses are: Pomare pH 5.56, 5.5 XC, 0.36%N, and Olsen P 6 pg gg’; Waikanae pH 5.59, 3.9 XC, 0.36%N and Olsen P 72 pg gg’. Soil from the top 75 mm was used for this study. To minimize the contribution from indigenous roots of the pasture grasses, the soils were sieved (< 2 mm) while moist and stored at 25°C for 41 days before the addition of ryegrass roots for P and ATP measurements, and for 110 days before the addition

G. P. SPARLING et al.

216

of maize roots for the C and N measurements. It was assumed that after these periods the contribution from the indigenous roots remaining after sieving would be negligible. The original moisture content of the soils (39-42% w/w) was maintained throughout the storage period and all subsequent experiments. Treatments and analyses Root-amended soils received 0.1 g maize roots gg ’ soil (5.99 mg dry wt) for C and N measurements or 0.2 g ryegrass roots gg’ soil (13.76 mgdry wt) for ATP and P measurements. Measurements of CO&, NH,-N and NO,-N were made on roots, soils and root-amended soils, with and without CHCl, fumigation (Jenkinson and Powlson, 1976), after a 10 day incubation at 25°C. Inorganic (Pi) and total (P,) levels in 0.5 M NaHCO, extracts were also measured with and without CHCl, treatment. Further samples of roots, soils and root-amended soils were pre-incubated at 25°C for 7 days and then analyzed as described above. Replication was in triplicate for all C, N and P measurements and quadruplicate for all ATP determinations. The methods of measuring C, N, P and ATP have all been previously described. Briefly, C02-C was measured by gas chromatography (Orchard and Cook, 1983), the incubation of control and fumigated soils (5 g dry wt) being in 1.8 1 preserving jars fitted with septa for gas sampling (Sparling, 1981). Soluble N was measured as NH,-N and NO,-N in 2 M KC1 extracts (Williams and Sparling, 1984). P, and P, were extracted in 0.5 M NaHCO, immediately following 2 h CHCl,-lysis of 5 g (dry wt) soil with 5 ml of liquid CHCl, (Hedley and Stewart, 1982; Williams and Sparling, 1984). Extractant-to-soil ratio was 20: 1, and the soils were extracted for 2 h on an end-overend shaker operated at 60 rev min-‘. Concentrations of P in the extracts were determined calorimetrically 1962) before and after (Murphy and Riley, perchloric acid digestion (Brookes and Powlson, 1981) to obtain Pi and P, concentrations. ATP in trichloracetic acid-phosphate-paraquat extracts was measured using luciferase-luciferin bioluminescence as described by Tate and Jenkinson (1982) except that measurements were made using an LKB 1251 Luminometer, Tris buffer (0.5 M, pH 7.2) instead of sodium arsenate, and measuring light output over a 5 s integration period, 10 s after enzyme addition. RESULTS

Carbon The flushes Table

(fumigated

minus

control

values)

of

CO,-C were generally greater in the presence of roots but, because of higher variability, the increases were not significantly greater than the soils without roots (Table 1). The C-flush from the root-amended soils without pre-incubation was a summation of the separate soil and root components, but after preincubation the effect was variable. The total amounts of CO,-C respired from both the control and fumigated soils were much higher in the presence of roots, the amounts being greater than the sum of the soil and root components. All values declined markedly with pre-incubation, the decline being greater in the presence of roots, so that the contribution of roots to CO2 levels after preincubation was accordingly reduced (Table 1). Use of the C-flush values to estimate the soil microbial biomass would result in values for the root amended soils being 26 and 89% greater than the non-amended Pomare and Waikane soils respectively. After pre-incubation the biomass-C estimate for the Pomare soil with roots was 41% lower than the soil without roots, whereas for Waikanae soil the biomass-C was 30% greater than the soil without roots. Nitrogen The effect of roots on the N-flush was not consistent because the prolonged storage before adding the excised roots resulted in the accumulation of high NH: and NO; concentrations, particularly in the Waikanae soil (Table 2). Roots decreased the flush of NO,-N, but had a variable effect on NH,-N, consequently most of the effects of roots on the flush were not significant. Generally the total amounts of NH,-N in the root-amended soil were a summation of the soil and root components, but the NO,-N concentration in the root-amended soils was increased even though the roots contained little extractable NO,-N (Table 2). After pre-incubation many of the flush values were negative indicating net immobilization of N. There was little change in the NO,-N concentrations of the control and fumigated soils, but NH,-N concentrations were increased by prior incubation. However, the increases in NH,-N were greater in the controls than the fumigated samples, resulting in lower overall N-flushes. Phosphorus The P-flush was increased by up to 3.28-fold by the addition of roots to the soils (Table 3). The roots contained a substantial amount of NaHCO,extractable P, the bulk of which was inorganic (P,). Fumigation increased the amounts of P, and total P,

1.E&t of maize roots (5.99 mg) and prior incubation for 7 days on the amounts of CO, (PgC g-‘) respired from soils after CHCI,-fumigation Not pre-incubated Treatment

Pre-incubated

Control

Fumigated

Flush

747

859

112

225

320

95

Pomare soil Pomare soil with root

335 1370

785 1931

450a 567a

293 599

679 825

38ba 22bb’

Waikanae soil Waikanae soil with root

227 1388

431 1773

204a 385a

lb3 335

316

153a 200a

Root alone

Control

Fumigated

535

Flush

‘For each soil, figures for the flush (fumigated minus control) which are followed by the same letter, do not differ significantly at P < 0.05

Roots and microbial biomass

277

Table 2. EtTect of maize roots (5.99 mg) and pre-incubation for 7 days on the amounts (PgN g-l) of NH,-N and NO,-N extracted from soils following CHCI,-fumigation Not pre-incubated Treatment Root alone Pomare soil Pomare soil with root Waikanae soil Waikanae soil with root >

Control

Fumirrated

Preincubated Flush

Control

Fun&&d

96 2.9

120 2.8

24
Flush

NH& NO,-N

177 2.6

168 2.9


NH,-N NO,-N NH,-N NO,-N

57 43 273 86

165 32 355 69

108a
164 39 383 82

185 34 381 70

21b’
NH,-N NO,-N NH,-N NO,-N

49 254 217 280

66 228 249 257

17a to 32a
62 242 292 294

75 235 296 255

13a
0.3

‘For each soil, figures for the N-flush (fumigated minus control) which are followed by the same letter do not differ significantly at P < 0.05. The flush was assumed to be zero (i.e. net immobilization) if the value for the control treatment was greater than the equivalent fumigated treatment. Table 3. Effect of ryegrass roots (13.76 mg} and pre-incubation for 7 days on the amounts (PgP g’) (P,) and total (P,) phosphorus extracted from soils following CHCl,-fumigation -Control

Treatment

Not pre-incubated --......_~ __ Fumigated Flush

Root alone

PI PI

14.9 17.2

16.5 73.0

61.6 55.8

Pomare soil

PI P,

6.5 30.3

15.4 44.5

Pomare soil > with root

Pi P,

15.9 43.9

Wdikanae soil

PI P,

89.0

P, P,

Waikanae soil with root >

-.._. Control

of inorganic

Prc-incubated Fumigated

Flush

99.3 115.7

107.7 127.3

8.4 11.6

8.9a 14.2x

5.9 45.2

12.6 59.4

6.7a 14.2x

41.3 72.7

25.4b’ 28.8~~

22.6 69.1

43.1 102.4

20Sb’ 33.3y

103.6

99.4 109.0

10.4a 5.4x

82.0 128.7

88.4 143.4

6.4a 14.7x

100.2 114.3

134.3 133.0

34.lb’ 18.7x

118.3 179.3

138.3 197.2

20.0b’ 17.9x

-

‘For each soil and for each form of P, figures for the flush (fumigated minus control) followed by the same letter do not differ significantly at P < 0.05.

extracted from the root by 5.1 and 4.2 times respectively. However, the extra Pi and P, in the flush from the root-amended soil was not a summation of the soil and root components. After pre-incubation the P-flush from the roots was much decreased, the extractable P concentration of the root being much increased, and fumigation causing only a small (8%) additional release. However, the Pi-flush from the amended soil remained much higher than the soil without roots, being some 3-fold greater, even after pre-incubation. The P,-flush of the preincubated soils was also greater in the presence of roots, although for Waikanae soil the increase (22%) was small and not significant. ATP

The roots contained substantial amounts of ATP and addition of roots to soil increased the ATP Table 4. Contribution from ryegrass roots (13.76 mg) to the ATP content (pgg-‘) of Pomare and Waikanae soil, before and after 7 day pre-incubation at 25°C Not pre-incubated

Pre-incubated

Root alone

2.94

0.53

Pomare soil Pomare soil with root

4.9fa 4.82a

3.77b’ 3.93b

Waikanae soil Waikanae soil with root

3.42a 5.85b

2.56c 2.62~

Treatment

‘For each soil, figures followed by the same letter do not differ significantly at P c 0.05. All results corrected for ATP recovery.

content of the Waikanae, but not of the Pomare soil. Root ATP content declined markedly during prior incubation, and after 7 days, roots had no significant effect on the ATP content of either soil, with all values showing an overall significant decline (Table 4). The anomaly of the root addition having little effect on the ATP content of Pomare soil is partly explained by the extraction efficiency of ATP in the presence of roots being apparently greater than 100%. At present we have no satisfactory explanation of this effect. DISCUSSION

The use of freshly-grown and excised roots in our experiments probably means that the contribution of such roots to the flushes of C, N and P after fumigation, and to the ATP content of the soils, was much greater than can be expected from field-grown roots. The weight of root used by us (6 to 13.8 mgg-‘) was comparable with the amounts present in grasslands (Sparling and Tinker, 1978), but a considerable proportion of pasture root mass is probably dead, of low nutrient content and not readily decomposed (Troughton, 1957). Consequently, the nutrients and COz released by fumigation of field-grown roots are likely to be less than in a laboratory study. In arable soils with a much lower root density than pastures the contribution from the roots to the flushes will be accordingly lower.

218

G. P. SPARLINC et al.

Our results therefore represent a “worst possible” case of root interference in the estimation of the microbial biomass by the fumigation method. However, in greenhouse pot experiments, or where rootto-soil ratios are high, such as in selectively-sampled rhizosphere soils, there is a clear risk of a substantial contribution from the root material to the “microbial” biomass. Interference from roots was greatest in the estimation of the microbial biomass P, interference was less in the biomass C and N determinations, probably because the latter two measurements involve incubation of the samples for 10 days. Our findings differ from those of Lynch and Panting (1981) who, using similar techniques and amounts of root, found no significant effect of roots on the C-flush. We found a large effect of roots on the flushes, although for C the increases were not significant because of the high variability. The physiological condition of the roots in the soil may be critical in determining the level of interference. Pre-incubation for 7 days was effective in reducing the interference from roots in the measurement of “microbial” ATP and for the flushes of C and N, but not the P-flush. We had anticipated that during the pre-incubation the microbial biomass and ATP content would increase as the organisms grew and colonized the decomposing root. However, subsequent experiments with axenical~y-grown, excised maize roots showed that the initial, high ATP level of 309 pg gg’ root decreased to 49 pg g’ during sterile incubation for 7 days, and to 55 pgg-’ when the roots were inoculated with a mixed culture of soil microorganisms at the onset of this incubation. This suggests that after root decomposition for 7 days, the microbial component of the extracted ATP was small (1 lx), and that there had been little synthesis of microbial ATP during incubation of the roots. This finding would accord with the absence of any detectable increase in the biomass C and P levels. However, it is recognized that small increases in microbial C, N and P occurring during the preincubation may well have been masked by the large effect of roots on the flushes. Unfortunately, because the flushes from the root-amended soils were not a summation of the soil and root components, there appears to be no ready means of allowing for the root contribution to the flush, even if the amount of root is known. We therefore recommend that for the estimation of microbial biomass C, N, P and ATP on soils with a high (> 5 mg g-l) viable root component, they should be sieved and incubated for 7 days at 25°C prior to analyses. Small increases in the biomass are likely during the pre-incubation but this error appears preferable to the potentially greater error of estimating root material as microbial biomass, which is the risk if the analyses are carried out immediately after sampling. The risk appears greatest in the estimation of the soil biomass P. Results obtained after a pre-incubation should still be valid for comparative purposes provided all the samples are pre-treated in a similar way.

W. West is an N.Z. National Research Advisory Council Junior Research Fellow. We

~ek~owledgement.~-A.

thank Janine Cowling for NH: and NO; Dr J. Reynolds for advice on statistics.

analyses, and

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