Soil Enzymes in Relation to Old Field Succession: Amylase, Cellulase, Invertase,
Dehydrogenase, and Urease1
SUNIL K. PANCHOLY AND ELROY L. RicE2
ABSTRACT
Amylase, cellulase, invertase, dehydrogenase, and urease
activities in soil were determined every other month for a year
in two old-field successional stages and a climax stand in each
of three vegetation types; tall grass prairie, post oak-blackjack
oak forest and oak-pine forest. The activities of amylase, cellu-
lase, and invertase were highest generally in the first succes-
sional stage, intermediate in the second stage, and lowest in
the climax stand. On the other hand, dehydrogenase and
urease activities were generally lowest in the first successional
stage, intermediate in the second stage, and highest in the
climax. These trends were observed in all three vegetation
types throughout the year. No correlation was found between
soil enzymatic activity and amount of organic matter or soil
pH. The type of vegetation and thus the type of organic
matter added to the soil during succession seem to be the chief
determiners of the activity gradients of the enzymes under
study.
Additional Index Words: carbohydrases, hydrolases, plant
succession.
OOTH (2) found four prominent successional stages in
B the revegetation of infertile abandoned fields in cen-
tral Oklahoma and southeastern Kansas: (i) a pioneer weed
stage which lasts 2-3 years; (ii) an annual grass stage which
persists for 9-13 years; (iii) a perennial bunchgrass stage
which lasts for 40 years or more; and (iv) the climax
prairie. Rice and colleagues have conducted extensive inves-
tigations to identify changes which occur during succession
in an effort to explain its causes (1, 3, 16, 18, 19, 23, 24,
25, 26, 27).
Rice, Penfound, and Rohrbaugh (23) found that the or-
der in which species of plants invade revegetating old-fields
is the same as the order based on increasing requirements
for nitrogen and phosphorus. Enzymes in the soil are obvi-
ously involved in the decomposition of organic matter and
many other chemical transformations in the soil. Neverthe-
less, we have found no reports in the literature correlating
activities of soil enzymes with stages of old-field succession.
Amylase, cellulase, and invertase are some of the impor-
tant enzymes in soil which are partially responsible for the
rate and course of decomposition of plant litter. Ross (28)
reported that the composition of pastures influences the ac-
tivities of carbohydrase enzymes in soil which suggests that
changes might occur due to changes in vegetation during
succession. Khan (14) found that invertase activity in plots
with a 5-year rotation of grains and legumes was almost
double that of a wheat-fallow sequence.
Urease activity in soil appears to correlate in general with
1 at 30C for 24 hours. After incubation the contents of the beaker
This project was done under Grant GB-22859 from the
National Science Foundation. Received July 19, 1972. Ap-
proved Sept. 5, 1972.
2
Post Doctoral Research Associate and Professor, respec-
tively, Dep. of Botany and Microbiology, University of Okla-
homa, Norman, Oklahoma 73069.
47
the number of microbes and is greatest in the rhizosphere
(9, 29). Dehydrogenase activity is a good measure of the
total microbiological activity of soil according to Stevenson
(30). Kozlov and Mikhaylova (15) found pronounced
changes in dehydrogenase activity in soil with changes in
the type of vegetation.
Because of the information cited above, we hypothesized
that the activities of amylase, cellulase, invertase, urease,
and dehydrogenase in the soil would change markedly dur-
ing old-field succession. Experiments were designed to test
the hypothesis in two old-field successional stages and a
climax stand in each of three vegetation types; the tall grass
prairie, the post oak-blackjack forest (Quercus stellata,
Quercus marilandica), and the oak-pine (Quercus, Pinux)
forest.
ENZYME ASSAY
Eight soil samples were taken every other month from the
0-15-cm depth in each plot and immediately brought to the
laboratory. Adjacent pairs of samples were combined, mixed
thoroughly, and then screened through an ASTM No. 10 sieve,
thus giving four samples per plot for separate analyses.
The methods used for determination of amylase and inver-
tase activities were those of Ross (28) except that glucose was
determined by Nelson's method (8). For determination of cel-
lulase activity, 5 g of soil were placed in a 50-ml flask. Five-
tenths of a milliliter of toluene were added; the soil was mixed
thoroughly; and after 15 min, 10 ml of acetate buffer (pH 5.9)
were added followed by 10 ml of 1% carboxy methyl cellulose.
The flask was then incubated for 24 hours at 30C. After incu-
bation, approximately 50 ml of distilled water were added, the
suspension was filtered through Whatman 30 filter paper, and
the volume of the filtrate was made up to 100 ml with distilled
water. The reducing sugar content of the filtrate was determined
by Nelson's method (8). Controls consisting of soil without
carboxy methyl cellulose and autoclaved soil with carboxy
methyl cellulose were run concurrently.
Dehydrogenase activity in soil was determined as described
by Stevenson (30) with modifications suggested by Casida et al.
(7). Five grams of fresh soil were put in a test tube and satu-
rated with 2.0 ml of a 1% solution of 2, 3, 5—triphenyl tetra-
zolium chloride. The contents were mixed thoroughly, the tubes
were tightly sealed with plastic caps and were then incubated at
32C for 24 hours. Following incubation, 25 ml of methanol
were added to each tube and the contents were stirred. The
resulting slurry was washed into a Buchner funnel (Whatman
no. 30 paper), extracted with successive aliquots of methanol,
and the filtrate was made up to 100 ml. The absorbance of tri-
phenylformazan in the filtrate was determined spectrophoto-
metrically at 485 nm, using methanol as the reference blank.
Concentrations were determined from a standard curve of tri-
phenylformazan in methanol. Results were recorded as milli-
grams of formazan formed per kilogram of soil.
To determine urease activity; 5 g of soil, 10 ml of phosphate
buffer (pH 6.7), 10 ml of a 10% urea solution, and 0.5 ml of
toluene were added to a 50-ml beaker. The soil was incubated
were filtered and 15 ml of IN KC1 were added to the soil in
the beaker. After 10 min, this solution was filtered and the total
filtrate was brought up to 100 ml with distilled water. The
amount of ammonia in a 10 ml sample was determined by
steam distillation as described by Bremner (5).
48 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973

Table 1 — Results of vegetational analyses of study plots. Data Table 2 — Characteristics of soils, 0-15 cm. Figures are
given only for the herbs in each plot which made up 3% averages of ten replicates
or more of the total herbaceous composition, but data Organic
given for all tree species sampled. Pi and P2 were Vegetation Sand Silt Clay pH carbonf
successional stages in old fields, whereas Ps % O7

was a climax stand in each vegetation type Tall grass prairie

Post oak-blackjack P,* 59.05 14.26 26.68 6.82 0.561
Tall grass prairie forest Oak-pine forest PI 65.29 17.94 16.75 6.49 0.589
Ps 78.16 7.17 14.66 6.81 0.885
Herb species P, P, P, P, P, P, P^ P, Pf
Post oak-blackjack
% Composition P, 79.40 8.48 13.24 6.23 0.484
Agrostls elllotlana 11.8 Pj 82.88 5.36 11.76 6.58 0.637
Ambrosia pslloa'ta'chya 3.1 12.4 P, 83.20 6.32 10.48 5.91 0.480
Andropogon gerardi 22.4 8.6 Oak-pine
A, scopariua 42.4 47.6 P! 75.06 12.30 12.64 5.52 1.151
A. Ylrglnlcua 13.9 8.1 71.1 P, 75.22 15.08 9.32 6.06 0.342
Antennaria planta- P, 65.52 20.00 14.48 5.42 1.096
glnlfolla 3. 2 * P! and P2 are successional stages in old fields, P3 is a climax stand.
Aristida oligantha 63.3 9.4
Coreopsis tlnctorla 63.7 6.8 f Walkley and Black value.
Cynodon dactylon 7.9 3.0
Cyperua ovularts
G. atrlgoaus
3.5 3.8
13.7
Table 3 — Amylase activity in soil. Figures are averages of four
Dlgltarla sangulnalla 12.9 23.6 replicates as nig glucose/g soil per 24 hours
Erlgeron canadensls 16.8 4.3 3.0 Tall grass prairie Post oak-blackjack Oak-pine
E. strlgosus 7.9 3.9 Date P! P, P, P, Pj P, Pj P2 P,
Gnaphaiium purpureum 5. 1 Apr. 71 0.800 0.668* 0.6561 0.484 0.316* 0.264ft 0.528 0.404 0.200ft
Haplopappus ctliatus 7.1 14.5 June 71 0.812 0.456* 0.380ft 0.464 0.360* 0.2841 0.460 0.368 0.232ft
Leptoloma cognatum 8.6 8.3 Aug. 71 0.840 0.580* 0.5201 0.660 0.480* 0.336ft 0.488 0.352* 0.332t
Lespedeza stlpulacea 19.9 16.7 Oct. 71 0.700 0.820 0.250ft 0.285 0.050* 0.0501 0.280 0.230 0.240
Medtcago satlva 15. 3 Dec. 71 0.680 0.800 0.300ft 0.365 0.175* 0. 150t 0.260 0.180 0.180
Panlcum llnearlfollum 4, 3 Feb. 72 0.700 0.750 0.470f 0.350 0.180* 0. 120t 0.250 0.200 0.200
P. scrlbnerlanum 3. 2 * Difference between Pj and P2 significant at 0.05 level or better,
P. vlrgatum 11.2 f Difference between Pj and Ps significant at 0. 05 level or better,
Paspalum pubescens 34. 1 4, 5 t Difference between P, and Pj significant at 0. 05 level or better.
Ptillmniom nuttallll 4. 1
and S. lutescens 4. 1
Sorghaatrum nutans 13.6 3.8
Sorghum halepense 5.3
Sporobolus asper 4.3 replicates as mg glucose/g soil per 24 hours
Twaao Importance Percentage
Tall grass prairie Post oak-blackjack Oak-pine
Carya >««n. 2 5 Date P, Pj Pj PI Pj P, P! Pj P,
C. toroentoaa 6
Ptnus echlnata 54 June 71 1.480 0.692* 0.330ft 1.064 0.510* 0.572ft 1.356 0.506* 0.228ft
Quercus falcata 2 Aug. 71 4.180 2.740* 1.330ft 1.012 0.532* 0.664ft 1.240 0.720* 0.416ft
t. marllandlca 64 5 Oct. 71 2.520 1.100* 0.795ft 0.245 0.180* 0.340ft 0.260 0.210* 0.190t
. stellata 26 23 Dec. 71 2.400 1.800* 0.9501 0.180 0.180 0.350 0.240 0.210 0.185
Q. yelutlna 6 Feb. 72 2.000 0.925* 0.7001 0.200 0.200 0.320t 0.250 0.200 0.200
Ulpma alata 7 * Difference between Pt and P9 significant at 0. 05 level or better.
* Sparse--not sampled,
t Difference between P! and P3 significant at 0.05 level or better.

RESULTS patches consisting of Andropogon gerardi, A. scoparius,

Vegetation of Study Plots
The first successional stage, (Pj) was in the 1st year
after abandonment from cultivation in the oak-pine and
tall grass prairie areas and in the 2nd year in the post oak-
blackjack area. All P! plots were in the pioneer weed stage
of succession, Stage 1 of Booth (2) (Table 1). The second
successional stage (P2) was in the 6th year after abandon-
ment in the tall grass prairie area and was dominated by
Aristida oligantha which represented Stage 2, the annual
grass stage, of Booth. (Nomenclature follows Fernald, 11).
The P2 plot in the post oak-blackjack area was in the 8th
year after abandonment and was dominated by Ambrosia
psilostachya, Andropogon virginicus, Aristida oligantha, and
Lespedeza stipulacea. The P2 plot in the oak-pine area was
abandoned from cultivation for 25 years and was dominated
by Andropogon virginicus; and few pine and oak seedlings
were present. The climax prairie (P3) was dominated by
Andropogon gerardi and A. scoparius with Panicum virga-
tum and Sorghastruin nutans as important secondary spe-
cies. The climax post oak-blackjack oak stand was domi-
nated by Quercus marilandica and Q. stellata with ground
cover primarily of Andropogon scoparius (Table 1). The
climax oak-pine stand was dominated by Pinus echinata and
Quercus stellata. This stand had virtually no herbaceous
ground cover. There were a few very small and sparse
Desmodium laevigatum, and Tephrosia virginiana.
General Soil Characteristics
All plots had a sandy loam soil except the Pt plot in the
tall grass prairie area which had a sandy clay loam soil (6)
(Table 2). Moreover, the average pH's were similar in all
three plots of each vegetational type (Table 2). The amount
of organic carbon often varied considerably in the different
plots with the only consistent trend in relation to succession
occurring in the tall grass prairie plots (Table 2).
The activities of amylase, cellulase, and invertase were
highest generally in the first successional stage, intermediate
in the second stage, and lowest in the climax stand under
all three vegetation types throughout the year (Tables 3, 4,
5). The chief exception to this trend was the somewhat low
cellulase activity in the second successional stage of the
post oak-blackjack area.
The highest amylase activity occurred in spring and sum-
mer, and the highest amylase activity consistently occurred
in the prairie area. Seasonal variations in amylase activity
were higher in the post oak-blackjack forest area and the
oak-pine forest area than in the tall grass prairie area (Table
3). Under all three vegetation types there were more vari-
ations in the second successional stage and the climax than
in the first successional stage. Amylase activity was usually
 PANCHOLY & RICE: SOIL ENZYMES 49
lowest in the months of October and December in all plots. Table 5—Invertase activity in soil. Figures are averages of
No positive correlation was found between amylase activity four replicates as mg glucose/g soil per 24 hours
and soil pH or amount of organic carbon. Tall grass prairie Post oak-blackjack Oak -pic le
Date PI Pj Pj PI PI P. P> P, Pj
Cellulase activity generally showed trends similar to amy- Apr. 71 3.70 3.32 5.64 1.48* 0. 970tt 5.48 1.04*
2. 88tt 0. 105tt
lase in all plots with highest activity in spring and summer June 71 4.60 3.78* 3.68} 4.66 1.66* 0. 574ft 4.96 1.31* 0.132tt
Aug. 71 7.90 6.68 5.60J 3.94 1.18* 0.690tt 6.42 1.56* 0. 988tt
and in the prairie area (Table 4). Again seasonal variations Oct. 71 5.34 2.64* 2.33J 6.65 8.05 3.00tt 5.45 1.50* 0.670tt
Dee. 71 4.35 2.50* 2. 10J 3.45 3.30 3.00 3.25 1.90* 0. 850J
were higher in the forest areas than in the prairie area, the Feb. 72 3.80 3.00 2.90 3.50 3.00 2.80 3.30. 2.00 1. 100J
lowest activities occurred in the fall and winter, and no pos- * Difference between P: and Pa significant at 0.05 level or better,
t Difference between P2 and P8 significant at 0.05 level or better.
itive correlations were found between cellulase activity and J Difference between Pt and P8 significant at 0.05 level or better.
soil pH or amounts of organic carbon.
Invertase activity generally showed trends similar to amy- Table 6—Urease activity in soil. Figures are averages of four
lase and cellulase and was consistently high throughout the replicates as mg of NHa liberated/g soil per 24 hours
spring, summer, and fall in most plots. The highest invertase Tall grass prairie Post oak-blackjack Oak-pine
activity occurred in the month of August in the tall grass
prairie area and the first successional stage in the oak-pine
area and in October in the post oak-blackjack oak forest
area. Strangely enough, the peak invertase activity occurred
in February in the second successional stage and the climax • Difference between P, and P2 Is significant at 0.05 level or better.
plot in the oak-pine area, with a secondary peak in August t Difference between Pa and Ps Is significant at 0.05 level or better.
J Difference between Px and Pa Is significant at 0.05 level or better.
(Table 5). Small seasonal variations were observed in most
plots. Again no correlation was found between invertase Unlike urease, dehydrogenase activity did not show any
activity and soil pH or amount of organic carbon. particular trend toward an increase or decrease. The high-
The urease activity of soil was generally lowest in the est dehydrogenase activity in the tall grass prairie occurred
first successional stage (Pj), intermediate in the second in the month of June in the first successional stage, in Au-
successional stage (P2), and the highest in the climax stand gust in the second stage, and in April in the climax. The
(P3). This trend was remarkably consistent throughout highest activities in all plots in the post oak-blackjack area
most sampling periods and all vegetation types (Table 6). occurred in June, and in the oak-pine they occurred in
Differences between Px and P2, P2 and P3, and P1 and P3 October (Table 7).
were found to be usually statistically significant. The chief Soil dehydrogenase activity in the tall grass prairie was
exception to this trend occurred in the month of October about 2 to 10 times higher than in the oak-pine or post
when one successional stage showed a higher urease activity oak-blackjack oak forest. Unlike urease activity, dehydro-
than the climax in the oak-pine and post oak-blackjack genase activity was generally similar in the oak-pine forest
areas. and post oak-blackjack oak forest.
Urease activity increased from April to August in the tall
grass prairie plots and from April to October in the post
DISCUSSION
oak-blackjack oak forest and oak-pine forest plots. A grad-
ual decline was subsequently noted in all plots (Table 6). There were striking and remarkably consistent changes in
Urease activity in the tall grass prairie soil was approxi- activity of the five soil enzymes under investigation during
mately thrice that in the post oak-blackjack forest and old-field succession in all three diverse vegetation types.
twice that in the oak-pine forest. This trend was consistent The carbohydrases had high activities in the first succes-
throughout the year. sional stage and decreased with succession toward the cli-
Dehydrogenase activity showed a trend similar to urease max. On the other hand, urease and dehydrogenase had low
activity under all three vegetation types, with the activity activities in the first stage of succession and increased as
being lowest in the first successional stage, intermediate in succession progressed. Apparently, these consistent gradi-
the second successional stage, and highest in the climax ents in activities of these soil enzymes during old-field suc-
(Table 7). The chief exceptions to this trend occurred again cession represent a widespread phenomenon because they
in October when the activity was higher in one successional occurred in all three diverse vegetational areas, even though
stage than in the climax in the post oak-blackjack and oak- these vegetational types occur on different soil orders and
pine areas. This was true also for the oak-pine in December. in different climatic conditions. The average annual precipi-
Differences between Pj and P2, P2 and P3, and Pl and P3 tation varies from 112 cm (44 inches) in the oak-pine to
were usually statistically significant. 97 cm (38 inches) in the post oak-blackjack area and 84
Table 7—Dehydrogenase activity in soil. Figures are averages of four replicates as mg formazan/kg soil per 24 hours
Tall grass prairie Post oak-blackjack Oak~plne
Date PI Pj Pj PI Pj Pj PI Pj Pj
April, 1971 2.50 5.00* 320.00ft 8.00 132.00* 188.00« 5.80 5.60 21. 60ft
June, 1971 92.00 122.00 156.00t» 102.00 158. DO- 198.00ft 9.00 10.20 17.60ft
August, 1971 42.50 205.00* 274.001 9.00 13. 80* 20.00f» 10.00 10.00 ll.lOf
October, 1971 41.20 62.50 137.00ft 12.50 75.00* 30.00f 66.20 26.20* 27.50J
December, 1971 30.00 58.00* 92.00tt 10.00 45.00* 90.00ft 65.00 30.00* 45.00J
February, 1972 30.00 6.00* 260. 00 t 9.00 86.00* 100.00ft 60.00 40.00 40.00
' Difference between Px and P2 la significant at 0.05 level or better.
t Difference between Pa and P( la significant at 0.05 level or better.
t Difference between Pt and P, Is significant at 0.05 level or better.
50 SOIL SCI. SOC. AMER. PROC., VOL. 37, 1973

cm (33 inches) in the tall grass prairie (12) and mean an-
nual evaporation varies from 147 cm (58 inches) in the
oak-pine to 178 cm (70 inches) in the tall grass prairie.
The activities of the enzymes did not correlate with
amounts of organic matter as previously reported by Khan
(14) nor did they correlate with pH as reported by McGar-
ity and Myres (17) for urease. It appears, therefore, that
the type of vegetation and thus the type of organic matter
added to the soil during succession are the chief determiners
of the activity gradients of the enzymes under study.
Although no specific data are available at present, it ap-
pears on the basis of anatomy, general morphology, and
texture that most pioneer weedy invaders of old-fields have
a considerably higher ratio of cellulose and other polysac-
charides to lignin than do the perennial grasses which in-
vade later in succession and which are important in the cli-
max prairie and post oak-blackjack forest. Certainly the
woody vegetation in the climax forests has a much lower
ratio of polysaccharides to lignin than do the herbaceous
species which invade early in succession. It would appear
logical therefore that organisms which decompose cellulose
would be more active in early stages of succession and thus
cellulase activity should be higher in the early stage. Fur-
ther experimentation is planned to elucidate this relation-
ship. One point is obvious and that is that the amount of
soil organic matter is not the important determiner of car-
bohydrase activity. As previously stated, Ross (28) suggested
that the composition of a pasture can influence carbohy-
drases, but no specific data were given to support the sug-
gestion. It is interesting to note that a decrease in the
invertase activity in soils has been associated with the accu-
mulation of bacteriostatic substances contained in the crop
roots and absorbed by the clay minerals. Also high inver-
tase activity was reported to be associated with low catalase
activity and vice versa (29).
Although available evidence indicates that the gradients
in activity of urease and dehydrogenase are related to the
type of vegetation and thus the type of organic matter
added to the soil, the possible reasons for this relationship
are not apparent to us at this time.