Bilal Et Al., 2011
Bilal Et Al., 2011
Bilal Et Al., 2011
Review/Revue
A R T I C L E I N F O A B S T R A C T
Keywords: This study was focused on investigating the role of the initial residue community, i.e.
Soil microorganisms and enzymes from the epiphytic and endophytic compartments, in soil
Maize residue decomposition processes. Aerial and underground parts (leaves and roots) of maize (Zea
C mineralization mays L.) plants were g-irradiated, surface-sterilized with sodium hypochlorite (NaOCl)/
Decomposition ethanol or non-sterilized (controls), while the outer surface morphology of maize leaves
Enzyme activity
and roots was examined by scanning electron microscopy (SEM). Non-sterilized and
Colonizing microorganisms
sterilized maize leaves and roots were incubated in soil to study carbon (C) mineralization
Sterilization
kinetics and enzyme dynamics (L-leucine aminopeptidase, CBH–1, xylanase, cellulase and
laccase). SEM results showed that initial microbial colonization was more pronounced on
non-sterilized leaf and root surfaces than on sterilized samples. The hypochlorite
treatment removed a part of the soluble components of leaves by washing and no specific
effect of any type of colonizing microorganisms was observed on C mineralization. In
contrast, g irradiation and hypochlorite treatments did not affect root chemical
characteristics and the quantitative effect of initial residue-colonizing microorganisms
on C mineralization was demonstrated. The variations in C mineralization and enzyme
dynamics between non-sterilized and sterilized roots suggested that activities of epiphytic
and endogenic microorganisms were of the same order of magnitude.
ß 2011 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
R É S U M É
Cette étude a été réalisée pour étudier le rôle des communautés microbiennes initialement
Mots clés :
Sol présentes sur les résidus végétaux, c’est-à-dire les micro-organismes et les enzymes
Résidus de maı̈s provenant des compartiments épiphytes et endophytes, sur les processus de décomposi-
Minéralisation du carbone tion dans les sols. Les parties aériennes et souterraines (feuilles et racines) du maı̈s (Zea
Décomposition mays L.) ont été soit g-irradiées, soit stérilisées en surface avec de l’hypochlorite de sodium
Activité enzymatique (NaOCl)/éthanol ou enfin non stérilisées (témoin), alors que la morphologie des surfaces
Micro-organismes colonisateurs externes des feuilles et des racines de maı̈s a été observée par microscopie électronique à
Stérilisation balayage (MEB). Les feuilles et les racines de maı̈s non stérilisées et stérilisées ont été
* Corresponding author.
E-mail address: [email protected] (I. Bertrand).
1631-0691/$ – see front matter ß 2011 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crvi.2011.07.004
B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836 825
incubées dans le sol pour étudier les cinétiques de minéralisation du carbone et les
dynamiques enzymatiques (L-leucine aminopeptidase, CBH–1, xylanase, cellulase et
laccase). Les observations au MEB ont montré que la colonisation microbienne initiale était
plus importante sur les surfaces des feuilles et des racines non stérilisées que sur les
échantillons stérilisés. Le traitement à l’hypochlorite a éliminé une partie des composés
solubles des feuilles par lavage et donc aucun effet spécifique de la colonisation
microbienne initiale sur la minéralisation du carbone n’a pu être constaté. En revanche, les
traitements irradiation g et hypochlorite n’ont pas affecté les caractéristiques chimiques
des racines et l’effet quantitatif des micro-organismes colonisateurs des résidus initiaux
sur la minéralisation du carbone a été démontré. Les variations de minéralisation du
carbone et des dynamiques enzymatiques entre les racines non stérilisées et stérilisées
suggèrent que les activités des micro-organismes épiphytes et endogènes sont du même
ordre de grandeur.
ß 2011 Académie des sciences. Publié par Elsevier Masson SAS. Tous droits réservés.
replicates of each treatment were incubated at 15 0.5 8C genic group alone (4-MUB or 7-AMC) as catalysis products.
for 43 days. Soils with no added residue were prepared and Equivalent 200 mM of substrates were initially pre-
incubated at the same time to serve as a control treatment. dissolved in 1 mL of methanol, then made up to 50 mL
Soil moisture was maintained by weighing weekly through- with deionized water and kept in brown bottles at 4 8C. All
out the incubation period and readjusting with deionized further dilutions were made in a 50 mM sodium acetate
water when necessary. Carbon mineralization (C-CO2) was buffer (pH 6). For each treatment of leaf or root extracts,
measured at regular intervals after the beginning of 20 mL aliquots were pipetted into well microplates
incubation, (after 3, 7, 14, 21, 28, 35 and 43 days) by placing followed by 25 mL of 200 mM substrate solution and
10 mL of 1 M NaOH in 250 mL glass jars to trap the CO2 80 mL of buffer to maintain the final volume at
released by soil samples (equivalent to 50 g dry soil). The CO2 125 mL well1.
concentrations trapped in the NaOH solutions were mea- The assay plates with 6 analytical replicates by
sured by continuous auto-analyzer (TRAACS 2000, Bran and treatment were incubated in the dark (20 8C) for up to
Luebbe). 3 h. The incubation times were based on preliminary
assays of the linearity of the reaction over time. Reactions
2.5. Enzyme extraction were terminated by adding 10 mL of 1 M NaOH to each well
to raise the pH to 9, which corresponds to the optimal pH
After 0, 8, 14, 21, 28, 35 and 43 days of incubation, the for fluorescence of these fluorogenics [37]. After excitation
maize residues and soil particles were manually separated at 365 nm, the fluorescence emission intensity was
to prepare leaf and root extracts. Day 0 corresponds to leaf measured at 460 nm by a computerised microplate
and root residues that were thoroughly mixed with their fluorimeter (Spectra Max Gemini, Molecular Devices)
relevant soils but removed after 10 min to prepare leaf and which was programmed to shake the microplate for 5 s
root extracts. Control extracts from non-sterilized residue, at 20 8C before reading, in order to homogenize the
sodium hypochlorite- or g-radiation-treated residues were reaction medium. Enzyme activities of all the samples
obtained from residues that had not been added to soil. The were determined in reference to a specific standard curve
maize leaves and roots were ground to 2 mm with a based on the appearance of fluorogenic products from the
grinder (Retsch, Cross Beater Mill) and sodium acetate was fluorogenic conjugates.
used to extract proteins from the plant residue [32]. For
each treatment, leaf and root extracts were prepared in 2.6.2. Colorimetric assay
50 mM sodium acetate buffer, pH 6 to give a plant residue- Xylanase standards were prepared using the method
to-buffer ratio of 1:125 [33]. After over-end shaking on a reported in Biely et al. [34]. One gram of RBB xylan
rotating shaker for 1 h, the suspensions were allowed to (Remazol Brilliant Blue R D-xylan, Fluka), which is a
settle for 1 h before filtration through 0.45 mm filters soluble chromogenic substrate for the assay of endo-1,4-b-
(PTFE). The resulting plant residue extracts were used for xylanases, was dissolved in 80 mL of hot water (90 8C). The
enzyme activity measurements. solution was then cooled at room temperature and the
volume was adjusted to 100 mL.
2.6. Enzyme assays For the cellulase assay, 2 g of powdered substrate (AZO-
CM-Cellulose, Megazyme) were added to 80 mL of boiling
The activities of five enzymes were determined using water on a hot plate and stirred vigorously using a
fluorometric enzyme assays for L-leucine aminopeptidase magnetic stirrer until apparent homogeneity (approx.
(LAP) and cellobiohydrolase-1 (CBH–1) [33] or colorimet- 20 min). After cooling, 5 mL of 2 M sodium acetate buffer
ric enzyme assays for cellulase, xylanase [34] and laccase (pH 4.5) were added to the solution then the pH and final
[35,36], according to previously reported methods. En- volume were adjusted to 4.5 and 100 mL, respectively. A
zyme assays were performed on the relevant leaf and root solution was prepared to precipitate high molecular
extracts to determine enzyme activities as equivalent fragments of substrate (AZO-CM-cellulose) by dissolving
absolute activity in enzyme units per gram dry matter 40 g of sodium acetate trihydrate and 4 g of zinc acetate in
(U g1 DM). Enzyme activities were expressed in Interna- 200 mL of deionized water and adjusting the pH to 5 with
tional Unit (U) which is defined as ‘‘the amount of enzyme 5 M HCl. The final volume of this precipitation solution was
activity which catalyzes the degradation of one micromole made up to 1 L with ethanol (95%) and stored at room
of the substrate per minute under standard conditions’’. temperature.
Laccase activity was determined as in the assays
2.6.1. Fluorimetric assay described by Buswell et al. [35] and Floch et al. [36].
The two substrates: 4-methylumbelliferyl-b-D-cello- One hundred milliliters of 0.55 mM of laccase substrate
bioside (4-MUB-cellobiose) for CBH–1 activity measure- (ABTS, 2,20 -azinobis-3-ethylbenzothiazoline-6-sulfononic
ment and 7-amino-4-methyl coumarin hydrochloride (7- acid diammonium salt, Sigma) were prepared by using
AMC-leucine) for LAP activity measurement, as well as sodium acetate buffer (50 mM, pH 4.5) followed by
their respective fluorogenic compounds (4-MUB and 7- vigorous shaking on a magnetic stirrer.
AMC) were obtained in crystalline form from Sigma- For each treatment, enzyme activities were measured
Aldrich. The fluorimetric assays were conducted in black in four replicates by adding 0.5 mL of residue extracts to
96-well microplates (NuncTm, FluoroNuncTm). A 200 mM 0.5 mL of the relevant substrate solution (RBB xylan/AZO-
stock solution was prepared either for the substrate (4- CM-cellulose) in the case of xylanase and cellulase, and
MUB or 7-AMC fluorogenic-conjugates) or for the fluoro- 1 mL of residue extract to 0.5 mL of ABTS substrate solution
828 B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836
in the case of laccase. All samples were incubated for 3 h at Chemical features related to non-decomposed leaves
room temperature (20 8C). The added high molecular and roots were expressed in relation to the initial dry
fragments of substrates (RBB xylan/AZO-CM-cellulose) matter (non-decomposed–dry matter, ND–DM) by taking
were precipitated by adding 2 mL of ethanol (96% v/v) with into account the loss of mass residue by decomposition.
vigorous shaking by vortex shaker (Heidolph, REAX 2000) This mass loss was calculated from the amount of C
while the low molecular weight fragments produced by mineralized.
enzymes remained in solution. Samples were then allowed A two-way repeated measures ANOVA (rmANOVA) was
to equilibrate at room temperature for 10 min before used to determine the effects of residue type (leaves and
shaking and were then centrifuged at 5000 revolutions per roots) treatments (g-irradiated, surface-sterilized and
minute for 5 min. The absorbance of the supernatant non-sterilized) across time (until 43 days) on C minerali-
solutions was measured at 590 nm for xylanase and zation kinetics and enzyme activities of L-leucine amino-
cellulase and at 414 nm for laccase, using a spectropho- peptidase, CBH–1, xylanase, cellulase and laccase. The
tometer (Helios g, Thermospectronic). rmANOVA examines the variation between type of residue
Xylanase and cellulase activities of all the samples were and treatments as well as the variation within time, where
determined by reference to a standard curve obtained with each factor is considered in combination with time to
purified endoxylanase from Thermobacillus xylanilyticus produce factorial interaction terms. Since the effect of type
and commercial cellulase (322 U mL1, endo-b-glucanase of residue was always significant (P < 0.05), this was
purified from Aspergillus niger, Megazyme) at concentra- dropped from the analyses to examine more closely the
tions in sodium acetate buffer (50 mM, pH 6) ranging from effect of treatments over time. Thus for each type of
0.01–0.1 U mL1 and 0.001–0.01 mU mL1, respectively. residue, one-way repeated measure ANOVA (rmANOVA)
Laccase activity was determined on the basis of ABTS were performed on dependent variables namely carbon
degradation by a purified laccase from Trametes versicolor and enzyme kinetics (LAP, xylanase, cellulase, CBH–1 and
(21.8 U mg1, Sigma) used at concentrations ranging from laccase) to interpret the effects of treatments interacting
0.001–0.1 mU mL1 in sodium acetate buffer (50 mM, pH with time. The data were log or square root transformed to
4.5). meet the assumptions of normality and homoscedasticity.
Data analyses were performed with software Statistica 6.1
2.7. Scanning electron microscopy (SEM) (Statsoft, Inc., 1984–2003).
Table 1
Total C, N and biochemical characteristics of non-sterilized and sterilized maize leaves (NSL, HSL, gSL) and roots (NSR, HSR, gSR) before soil decomposition
(ND).
Variables Total-C Total-N C to N ratio Cell wall fraction Total sugars Glucan Xylan Klason lignin
Data are means of 2 replicates and expressed as % of non-decomposed dry matter (%ND-DM). Means not sharing a common letter within the same column
are significantly different (P 0.05).
B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836 829
highest value for HSR. Due to their lower N contents, the C was residue type (F = 90.01, P < 0.001) (data not shown) in
to N ratios of the sterilized samples were higher than those which maize leaves (sterilized treatments included)
of non-sterilized samples, especially in roots. decomposed more rapidly than roots (Fig. 2a). Depending
The cell wall contents were higher in non-sterilized upon the type of sterilization treatment, the amount of
roots than in leaves. Cell wall contents did not vary after g- carbon mineralized did not vary significantly in case of
sterilization, whereas hypochlorite treatment resulted in maize leaves but was significantly different over incuba-
significant enrichment in both leaf and root. Total sugar tion time as indicated by the ‘between’ P-values from
contents were slightly higher in leaves than roots, and repeated measures ANOVA (F = 28.97, P < 0.001) (Table 3).
were not significantly modified by the sterilization The amount of carbon mineralized was highest for non-
treatments. Glucose and xylose were the main monomers sterilized treatments and lowest for gamma-sterilized
recovered after acid hydrolysis of samples, reflecting the treatments (Fig. 2a). Repeated measures ANOVA also
presence of cell wall glucan and xylan, respectively. Both indicated that the interaction of incubation time and
root and leaf contained approximately 35% glucan, and sterilization treatments (T Tr) had significant effects on
sterilization did not affect this level. The same was true for the amount of mineralized carbon from both maize leaf-
xylan content, but leaf xylan (close to 24% DM) was and root-amended soils.
significantly higher than root xylan (mean of 18% DM). In Non-sterilized and g-sterilized leaves (NSL and gSL)
contrast, Klason lignin contents were higher in roots than showed the same pattern of decomposition, in contrast to
in leaves. Klason lignin was only significantly increased hypochlorite-sterilized leaves (HSL). However, almost the
after hypochlorite treatment, in both leaf and root (Table same amount of leaf carbon was mineralized for all three
1). treatments up to the end of incubation with 39.1% 1.4,
41.5% 3.3 and 42.1% 2.8 of added C, respectively (Fig. 2a).
3.2. Modification of plant residue surface induced by In the case of HSL, an immediate effect of sterilization was
sterilization treatments, as observed by SEM observed which corresponded to a smaller amount of carbon
mineralized from 0–14 days of incubation (23.1% 0.3 of
The presence of microorganisms and the effect of added C), as compared to NSL and gSL (26.8% 0.9 and
sterilization treatments on the outer surface morphology 27.8% 1.9 of added C, respectively). After this lag phase and
of maize leaves, roots and the respective sterilized until the end of incubation, HSL samples were mineralized in
treatments, before any contact with soil (controls), were the same way as NSL and gSL (Fig. 2a). Roots gave rise to less
observed by scanning electron microscopy (SEM) (Fig. 1a– cumulated C mineralized than leaves. No effect of steriliza-
f). Compared to roots (Fig. 1b), microbial colonization was tion was observed over the 0–14-day interval for roots, but
more apparent on leaf surfaces where long and contrasting the total amount of carbon mineralized (% of added carbon)
fungi hyphae or actinobacteria were observed (Fig. 1a). In up to the end of incubation, differed significantly and in the
contrast, smaller particles and possibly bacteria were more following order: NSR (32.1% 0.6) < HSR (28.9% 0.8) < gSR
frequently found as speckles on the root surface (Fig. 1b). (25.4% 0.5) (Fig. 2a).
The epiphytic communities encountered on both leaf and The rates of CO2 production in control soil, leaf (NSL,
root surfaces were drastically reduced after hypochlorite HSL, gSL) and root (NSR, HSR, gSR) are shown in Fig. 2b. In
treatment (Fig. 1c, d). In addition to removing the the control soil, the initial low rate of CO2 production
microorganisms, this treatment concomitantly washed decreased rapidly at the beginning of incubation and then
off almost all the surface particles on leaf (Fig. 1c), whilst became constant over a function of time (Fig. 2b). In the
some were still visible on root (Fig. 1d). Distinct patterns of amended soils, the rates of CO2 release from leaf
apparent microbial colonization were observed after treatments were initially (8–14 days) much higher and
hypochlorite and g-sterilized treatments. The leaf and faster than from roots but lower at the later stages of
root surfaces subjected to g-radiation bombardment incubation, the cross-over point occurring during the 20–
displayed probably dead and speckled particles (possibly 30-day period. Maximum rates for both leaf and root were
bacteria), respectively (Fig. 1e, f) and were similar to the attained at day 8, except for the hypochlorite-sterilized
corresponding non-sterilized control samples in terms of samples which showed a delayed maximum at day 14 of
epiphytic communities. Therefore, the surface appearance incubation. In the case of leaves, a rapid and maximum
of the g-irradiated samples was rather different from the decrease of mineralization rate was observed during the
clean surfaces observed after hypochlorite treatment. In 14–28-day period, while in the case of root treatments, this
general, no visible epidermal damage of the plant tissues or rate decreased continuously until the end of the incubation
alteration of physical structure was apparent in the experiment (Fig. 2b).
pictures of sterilized samples as compared to non-
sterilized samples. For example, the leaf surfaces showed 3.4. Effect of sterilization treatments and time on enzyme
open stomata cells (Fig. 1a, c, e) whatever the treatment. kinetics
3.3. C mineralization of maize leaf and roots The effect of sterilization was examined by determining
the initial enzyme activities on maize leaves, roots and the
The amount of mineralized carbon in maize leaf- and corresponding sterilized residues, before incubation in soil
root-amended soils varied markedly as a function of time (Table 2). In general, significantly higher enzyme activities
(Fig. 2a, Table 3). In general, the main factor explaining the were found on leaf samples than on root samples except
variance in carbon mineralized (% of carbon mineralized) for L-leucine aminopeptidase (LAP). LAP activities did not
830 B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836
Fig. 1. Scanning electron micrographs of representative surface observations from non-sterilized and sterilized maize leaves and roots. (a) and (b) represent
non-sterilized maize leaf and root (NSL & NSR); (c) and (d) represent hypochlorite-sterilized maize leaf and root (HSL & HSR) and (e) and (f) represent g-
sterilized maize leaf and root (gSL & gSR), respectively.
change after g-sterilization of either residue whereas LAP 2). CBH–1 activities measured on leaves were significantly
activity decreased significantly by 44% after hypochlorite reduced after sterilization, especially after hypochlorite
treatment of roots (Table 2). g-sterilization had no treatment, but no effect of sterilization treatments was
significant effect on xylanase, cellulase and laccase observed on roots (Table 2).
activities whereas hypochlorite sterilization led to a In general, all enzyme activities differed significantly
significant and strong decrease of these activities (Table ‘between’ the sterilization treatments for both maize
B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836 831
40
35 with sterilization treatments and incubation time
30 (Fig. 3a–j).
25 L-leucine aminopeptidase activities, which mirror a
20 major protease family, differed significantly by steriliza-
15 tion treatments for maize leaves (F = 16.34, P < 0.01) and
10 roots (F = 16.26, P < 0.01) (Table 3) and were relatively low
5
in both leaf and root treatments (between 0.1–0.4 U g1
DM) at time 0 (Fig. 3a, b). When the respective residues
0
0 10 20 30 40 50
were added to soil, maximum L-leucine aminopeptidase
Days activities were obtained for NSL and NSR when 38% and
29% of C were mineralized, respectively. The measure-
50 ments of L-leucine aminopeptidase enzyme activities on
45
(b) NSL
mg C-CO2 kg-1 dry soilday -1
HSL all the sterilized samples indicated that the HSL, gSL, HSR
40 γSL and gSR values were lower than NSL and NSR and
35 NSR remained similar till the end of incubation (Fig. 3a, b).
30 HSR Xylanase activities which provide an indication of
25 γSR enzymatic degradation of the major cell wall heteroxylan,
20 Control soil varied significantly for sterilization treatments in case of
15 maize leaves (F = 75.44, P < 0.0001) and roots (F = 27.14,
10 P < 0.001) being greater on leaf treatments than root
5 treatments (Table 3; Fig. 3c, d). Xylanase activities were
0 similar in NSL and gSL from the start to end of incubation,
0 10 20 30 40 50 attaining a maximum when 28% of C was mineralized
Days (Fig. 3c). In contrast xylanase activities in HSL were 4 times
lower at the start but finally joined the enzyme activity
Fig. 2. Cumulative carbon mineralization kinetics over time expressed as
curve for NSL when 34% of C was mineralized (Fig. 3c). In
% added C (a) and carbon mineralization rates (b) measured in soil
without maize residue (control) and after addition of non-sterilized, case of roots, xylanase activities of NSR showed 2 to 5 times
hypochlorite-sterilized and g-sterilized maize leaves (NSL, HSL & gSL) more xylanase activity than gSR and HSR. Maxima were
and roots (NSR, HSR & gSR), respectively. Data are means of 4 incubation reached when 13% of C was mineralized, i.e. earlier than in
replicates (n = 4). leaves. After this maximum, the root treatments ranked as
NSR > HSR > gSR and followed the same trend until the
residues with an exception of laccase in case of maize end of incubation (Fig. 3d).
leaves (Table 3). At the same time, all enzyme activities Cellulase activities of maize leaves and roots were also
were found to be significantly different over incubation significantly affected by sterilization treatments (F = 11.38,
time as indicated by the ‘within’ P-values from repeated P < 0.01; F = 12.64, P < 0.01, respectively), being greater in
measures ANOVA (Table 3). Repeated measures ANOVA leaf treatments as compared to root treatments (Table 3;
also indicated that the interaction of incubation time and Fig. 3e, f). Cellulase activities of leaf extracts showed
sterilization treatments (T Tr) had significant effects on almost the same patterns as xylanase activities, with HSL
the enzyme activities except laccase in maize leaves and extracts showing 2.5 less cellulase activity than NSL and
xylanase and cellulase in case of maize roots (Table 3). gSL at time 0 (Fig. 3e). Cellulase activities in HSL remained
On the basis of these differences, enzyme activities lower until 33% of C was mineralized. Cellulase activities
measured on non-sterilized and sterilized leaf and root measured on hypochlorite-treated roots were approxi-
mately 2 times lower at time 0 than under other
Table 2 conditions, and reached their maximum earlier when only
Enzyme activities of L-leucine aminopeptidase, xylanase, cellulase, CBH– 8% of the C was mineralized. In contrast to leaves, the
1 and laccase of non-sterilized and sterilized maize leaves (NSL, HSL, gSL)
patterns of cellulase activity measured in NSR and gSR
and roots (NSR, HSR, gSR) before adding to soil.
differed from those of xylanase activity and the decrease
Variables LAP Xylanase Cellulase CBH–1 Laccase until the end of incubation was smaller (Fig. 3f).
NSL 0.15b 60.8d 54.9c 15.7c 0.0072d CBH–1 activities of maize leaves and roots varied
HSL 0.11ab 4.9c 6.9 ab 0.7ab 0.0059c significantly across the sterilization treatments
gSL 0.16b 60.3d 52.9c 13.2b 0.0070d (F = 291.88, P < 0.0001; F = 284.24, P < 0.0001, respective-
NSR 0.18b 2.5b 13.4b 0.5ab 0.0028b ly), being lower in root treatments as compared to leaf
HSR 0.10a 0.2a 2.4a 0.1a 0.0019a treatments (Table 3; Fig. 3g, h). CBH–1 activities were
gSR 0.15b 2.1ab 9.6ab 0.1ab 0.0023ab considerably decreased by sterilization treatments in both
LSD 0.03 2.01 8.02 0.66 0.0006 leaves and roots (Fig. 3g, h) and remained 10 times higher
Data are means of at least 4 replicates and expressed as enzyme units per
for leaves than for roots. At time 0, CBH–1 activities
gram of dry matter (U g1 DM). Means not sharing a common letter measured on NSL were 20 and 1.2 times higher than on HSL
within the same column are significantly different (P 0.05). and gSL, the extent of variation being much greater than
832 B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836
Table 3
Statistical summary of repeated measures analysis of variance (rmANOVA) representing effects of sterilization treatments on C mineralization and enzyme
kinetics.
Maize Between-subject
leaf Treatment (Tr) 2 0.50 0.63 16.34 0.004 75.44 < 0.0001 11.38 0.009 291.88 < 0.0001 0.37 0.706
Within-subject
Time (T) 6 827.73 < 0.0001 7.17 < 0.0001 181.05 < 0.0001 246.35 < 0.0001 757.50 < 0.0001 118.74 < 0.0001
Interaction 12 6.12 < 0.0001 2.78 0.009 9.29 < 0.0001 4.16 < 0.0001 22.93 < 0.0001 1.93 0.064
(T Tr)
Maize Between-subject
root Treatment (Tr) 2 28.97 < 0.001 16.26 0.004 27.14 0.001 12.64 0.007 284.24 < 0.0001 23.33 < 0.001
Within-subject
Time (T) 6 7470.15 < 0.0001 4.95 < 0.001 404.93 < 0.0001 188.34 < 0.0001 79.73 < 0.0001 15.04 < 0.0001
Interaction 12 15.72 < 0.0001 3.27 0.003 1.37 0.226 1.20 0.320 6.78 < 0.0001 4.72 < 0.0001
(T Tr)
Analyses were performed with the values of C mineralized and enzyme kinetics at different dates of incubation for each treatment. df: degrees of freedom;
F: value of the statistic; P: probability levels of the analysis. Significant effects at P < 0.05, 0.01 and 0.001 probability levels.
with cellulase (Fig. 3e). In leaves, CBH–1 activities reached residue, in agreement with a recent report [39]. The
their maximum more rapidly than cellulase and the validity of these conclusions is strengthened by the present
difference between treatments remained significant until study since higher levels of C mineralization and enzyme
35% C was mineralized. Almost no CBH–1 activity was activities were monitored in leaves than in roots,
observed in leaf extracts at the end of incubation (Fig. 3g). independently of the sterilization treatments. In the
CBH–1 activities on roots did not follow the cellulase present study, we focused on the impact of initial residue
patterns (compare Fig. 3h and Fig. 3f) and were comparable colonization in an attempt to separate the role of epiphytic
to those on leaves. At time 0, NSR showed 4.5 times more activities, i.e. those on the external surface of the residues,
CBH–1 activity than HSR and gSR (Fig. 3h). Maximum from endophytic activities which occur within the residue.
CBH–1 activities in NSR were obtained when 14% of C was Our hypothesis was that the impact of residue-colonizing
mineralized, in contrast to the activities in both HSR and microorganisms could be assessed by subtracting the
gSR for which maxima were reached when 7% of C was effect of g-irradiation from non-sterilized treatments,
mineralized (Fig. 3h). while insight into the specific contribution of endophytes
Sterilization treatments did not have significant effect might be obtained by subtracting the effect of hypochlorite
on laccase activities of maize leaves but did significantly treatment (i.e. surface sterilization) from non-sterilized
affect laccase activities of maize roots (F = 23.33, P < 0.001) treatments.
(Table 3), resulting in greater laccase activities in leaf
treatments than root ones (Fig. 3i, j). In contrast to the 4.1. Interactions between residue quality and sterilization
other enzyme activities tested, laccase activities in root treatments
and leaf extracts at time 0 were comparable, so no
significant variations were observed between treatments. Surface sterilization has frequently been applied to
After the start of incubation when 12% of C was eliminate surface-colonizing microbial populations of
mineralized, a transitory period of less activity was fungi and bacteria from plant residue [40,41]. SEM
determined for HSL, as compared to NSL and gSL, while comparisons of hypochlorite-sterilized treatments and
no difference between treatments was observed during the non-sterilized treatments, confirmed that the microorgan-
remaining period of incubation (Fig. 3i). For roots, lower isms which were precursors of enzyme production were
laccase activities were observed on sterilized samples completely removed from leaf and root surfaces without
when 6–8% of C was mineralized (Fig. 3j). After this stage, any visible physical damage to the residue surface (Fig. 1c,
gSR activities remained parallel as a function of mineral- d). On the contrary, g-sterilized leaf and root treatments
ized C while the NSR and HSR activities increased until the revealed the presence of dead microorganisms and debris
end of incubation (Fig. 3j). (e.g. fungal hyphae or actinobacteria) on the residue
surfaces which presented the same physical features as in
4. Discussion the non-sterilized treatment. Total N contents in both
residues were reduced after hypochlorite sterilization,
The effect of adding residues of contrasting quality (i.e. resulting in a slight increase in cell wall, C and Klason
roots versus leaves) on C mineralization kinetics and Lignin contents. These hypochlorite effects can possibly be
enzyme dynamics was discussed in a previous paper [38]. attributed to the removal of soluble compounds by a
In that study we demonstrated that the changes in ‘washing effect’ during dipping of the plant residue in the
activities of polysaccharide-degrading enzymes provided NaOCl/ethanol solution. This also suggests that residue-
an exact reflection of decomposition and that most of the colonizing microorganisms have assimilated a portion of N
relevant biological activity was concentrated on the which is removed with their elimination from the residue
B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836 833
0.2 0.2
0.1 0.1
0.0 0.0
0 10 20 30 40 50 0 5 10 15 20 25 30 35
120 14
(c) Xylanase 12
(d) Xylanase
100
10
U g-1 DM
80
8
60
6
40
4
20 2
0 0
0 10 20 30 40 50 0 5 10 15 20 25 30 35
300 70
(e) Cellulase 60
(f) Cellulase
250
50
200
U g-1 DM
40
150
30
100 20
50 10
0 0
0 10 20 30 40 50 0 5 10 15 20 25 30 35
30 0.8
25
(g) CBH-1 (h) CBH-1
0.6
U g-1 DM
20
15 0.4
10
0.2
5
0 0.0
0 10 20 30 40 50 0 5 10 15 20 25 30 35
0.14 0.06
0.12
(i) Laccase (j) Laccase
0.05
0.10
0.04
U g-1 DM
0.08
0.03
0.06
0.02
0.04
0.02 0.01
0.00 0.00
0 10 20 30 40 50 0 5 10 15 20 25 30 35
Mineralized carbon (% of C added) Mineralized carbon (% of C added)
Fig. 3. L-leucine aminopeptidase (a, b), xylanase (c, d), cellulase (e, f), CBH–1 (g, h) and laccase (i, j) activities of non-sterilized, hypochlorite-sterilized and g-
sterilized maize leaves (NSL, HSL & gSL) and roots (NSR, HSR & gSR) treatments represented as a function of mineralized carbon (% of C added), respectively.
Enzyme activities were expressed as enzyme units per gram dry matter (U g1 DM). Error bars represent the standard error of the mean of four replicates
(n = 4).
surface. Accordingly, it has been shown [13] that in the washing effect of hypochlorite treatment. Sodium
maize roots, microorganisms (bacteria and fungi) are rich hypochlorite/ethanol sterilization was considered to
in N, as compared to the cell wall. Nevertheless some N remove surface-colonizing microorganisms, however,
could also correspond to the soluble proteins removed by given the significant decrease of enzyme activities in
834 B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836
hypochlorite-treated leaves and roots (Table 2), we These enzymes work progressively either on the reducing
hypothesize that such a treatment would concomitantly end or the other end. Further, b-glucosidase hydrolyzes the
wash off enzymes and some soluble compounds in cellobiose units in glucose monomers. The enzyme
addition to removing the surface-colonizing microorgan- dynamics during the first step of decomposition suggest
isms (Fig. 1c, d; Table 1). that oligoglucosides were present in the soluble fraction of
In contrast, g-irradiation had no significant effect on the roots on which the epiphytic and endogenic microorgan-
chemical composition of either leaf or root residues, as g- isms were acting. Few studies have described the spectrum
irradiated residues were not immersed in any liquid during and activity of endophytes colonizing roots at the
the sterilization process. To ensure complete inactivation senescent stage. It has already been found [47] that
of the microorganisms, a 45 kGy dose of g-irradiation was endophytic microorganisms in sugar beet promote growth
used, as 25 kGy is sufficient to eliminate all fungi and and increase beet yield but no published data exist for
bacteria (except radioresistant bacteria) [42] and is also plants after harvesting. The variation in C mineralization
currently used in industrial food and medical sterilization and enzyme dynamics between non-sterilized and steril-
processes. The selected g-irradiation dose is considered to ized roots suggests that the activities of epiphytic and
kill all microorganisms while conserving extracellular endogen microorganisms would be of the same order of
enzyme activities and protein functions [23]. Accordingly, magnitude, assuming that g-irradiation killed all micro-
the treatment used did not significantly affect the enzyme organisms [22] but left enzymes active. It is possible that
activities measured in leaf and root extracts (Table 2). the entire residue structure would be slightly altered by
Nevertheless some proteins may be denatured more the treatments. Cellulose and hemicellulose have been
rapidly, depending on local water conditions [43,44], so shown to be less recalcitrant to enzymatic hydrolysis after
no generalization can be made. Structural proteins are high dose g-irradiation [48]. In our case after irradiation at
reported to be less solvated in g-irradiated samples which much lower dose (45 KGy), lower mineralization rates
may impact their function [45]. were observed with roots, and no variation could be
detected for leaves. Hence, the enzyme activities measured
4.2. Impact of colonizing microorganisms on enzyme in g-irradiation-treated roots were reduced throughout
activities and residue decomposition almost the entire incubation period (Fig. 3). This fact plus
the major differences between our treatment and those in
Non-sterilized and g-treated leaves reached the same the above-cited reports, i.e. g-irradiation dose and in vitro
level of cumulated C mineralization after 43 days of degradation of polymers, permit us to assume that the cell
incubation, suggesting that residue-colonizing microor- wall would be slightly modified without any strong impact
ganisms, whether endophytic or epiphytic had no effect. In on the overall mineralization dynamics and would thus
contrast, hypochlorite-treated leaves mineralized at a permit treatment comparisons.
slower rate during the first 7 days of incubation (Fig. 2), L-leucine aminopeptidase (LAP) activities in leaf and
which can be explained by the decrease in HSL soluble root samples were slightly affected by the sterilization
components (10% DM) compared to NSL and gSL, as treatments. These proteases did not vary much with
previously demonstrated [5,6,38,46]. The enzyme activity residue decomposition in soil (Fig. 3) as a function of
patterns (xylanase, cellulase, CBH–1), following incubation mineralized carbon, possibly due to the non-limiting soil
of residues in soil, indicated that leaf samples were mostly nitrogen conditions.
affected by hypochlorite treatment, in contrast to roots,
suggesting a greater presence of those enzymes on the leaf 5. Conclusions
surface and/or in the soluble fraction. Hypochlorite
treatment was followed by washing until no chorine Firstly, no effect attributable to any kind of colonizing
was detectable, so the weaker activities of xylanase, microorganisms, epi- or endophytic, on C mineralization
cellulase and CBH measured on leaves during their kinetics and enzyme dynamics was observed on leaves.
mineralization should not be due to a residual toxic However the side effect of hypochlorite highlighted the
component. In this respect, laccase activities displayed key role of soluble compounds during short-term decom-
similar patterns during the decomposition of non-steril- position in soil.
ized and sterilized leaves. Secondly, the significant role of colonizing microorgan-
The cumulated amount of C at day 43 for roots ranked isms and enzymes on C mineralization of roots was
as follows NSR > HSR > gSR. Hypochlorite treatment demonstrated. These underground tissues, as well as their
reduced the amount of soluble components to a much original locations, are more exposed to microorganisms
weaker extent in roots than in leaves, possibly due to the (rhizosphere). Overall, colonizing microorganisms whatev-
lower liquid permeability of roots. Therefore the observed er their location, will promote post-harvest decomposition.
decrease in C mineralization between non-sterilized and In addition, epiphytic and endophytic microorganisms of
hypochlorite-treated roots (about 10% of added C) would roots act in the same way during the soil decomposition
be mainly due to colonizing microorganisms. However, no process.
differences in the enzyme kinetics of sterilized roots were The major differences in C mineralization, observed in
recorded until 15% C was mineralized except for CBH–1 roots versus leaves, could be attributed in part to variations
(Fig. 3). CBH–1 is an accessory exo-cellulase which assists in the colonizing microorganism communities and enzyme
microbial assimilation by catalyzing the 2–4 terminal units pools initially present on the surface or within the residue
at one end of the polysaccharide chain releasing cellobiose. structures, while specific chemical characteristics explain
B.A. Zafar Amin et al. / C. R. Biologies 334 (2011) 824–836 835
the main differences. Nevertheless the influence of residue communities at different developmental stages of Medicago truncatula
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