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Original article
Abstract
Changes in the soil microbial community structure and cellulolytic activity may reflect the effects of different amendment or
management strategies. In this study, cellulolytic activity dynamics and microbial community structure in an agricultural soil
undergoing treatment-induced cellulose decomposition in response to two commercial biofertilizers (G and Y) were investigated
under laboratory conditions. The rate of weight loss among filter paper strips buried in G-treated soil was significantly higher
than in untreated control soil (R), while that in Y-treated soil lower. A significant shift in the PCR-temperature gradient gel
electrophoresis (PCR-TGGE) fingerprints of fungal community members was observed during the process, while no dramatic
changes were observed in the bacterial community structure. The ITS3–4 sequence of one predominant TGGE band in a sample
from G-amended soil during the peak of cellulose decomposition was most similar to that of a wood-decaying species Meruli-
poria incrassata. Fungal species composition of the same sample was analyzed by clone library profiling and was found to differ
significantly from that of its parallel control sample. Several operational taxonomic units (OTUs) in a G-amended soil sample,
including the species represented by the predominant TGGE band, were suggested to be cellulose decomposing fungal species.
The data of this study demonstrate that structural shifts in the soil fungal community for cellulose degradation represent a
meaningful ecological indicator of the consequences of soil amendments with biofertilizers.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Cellulolytic activity; Temperature gradient gel electrophoresis (TGGE); Clone library profiling; Microbial community; Biofertilizer
Variation within a cellulolytic microbial community samples were then divided into four quarters of approxi-
reflects changes in microbial activity, and thus is an impor- mately 6 kg each. One quarter was mixed with 500 ml biof-
tant indicator of cellulose decomposition. Knowledge of ertilizer G diluted 200-fold in sterile water (the recom-
microbial succession on cellulose is particularly key to under- mended field dose) (G). One quarter was treated with 500 ml
standing the microbial aspects of residue decomposition [11]. biofertilizer Y diluted 300-fold in sterile water (the recom-
A few studies have focused on the microbial dynamics of mended field dose) (Y). The third quarter of the sample was
cellulose decomposition [11,28]. Though most soil microor- used as an absolute control (A) and was treated by autoclave
ganisms are not cultured as such [2], these studies used mostly at 121 °C for 20 min, then mixed with 500 ml sterile water.
conventional culture-dependent methods. The last quarter of the sample was used as a relative control
Molecular techniques based on DNA analysis are expected (R) and was mixed with 500 ml sterile water.
to circumvent the problems associated with culture-dependent Two brands of commercial biofertilizer frequently used in
methods, and are gaining popularity for elucidating micro- farms around Shanghai were selected for this study. Biofer-
bial population structures and dynamics in environmental tilizer G was produced by Chendu Nengsheng Bioengineer-
samples [2,24]. Molecular fingerprinting techniques such as ing Co., Ltd. (Sichuan Province, China) and was character-
temperature and denaturing gradient gel electrophoreses ized as being a yellow–brown liquid at pH 3.5. Biofertilizer
(TGGE and DGGE) are powerful tools for investigating Y was produced by Yiyijiu Bioengineering Co., Ltd. (Shang-
microbial diversity in a wide range of samples [9,21,23,25,44]. hai City, China) and was characterized as a light-yellow liq-
Cloning and sequencing may allow us to analyze phyloge- uid at pH 2.5. Both biofertilizers contained living microbial
netic community member types in various environments organisms beneficial to crop growth according to their prod-
[13,24,34]. For example, Weber et al. [40] used molecular uct labels. Analyses of the temperature gradient gel electro-
methods to examine bacterial populations that colonized and phoresis (TGGE) performed on the V3 region and of DNA
degraded rice straw. sequencing of the 16S rRNA gene indicated that biofertilizer
Biofertilizers may contain several microbial species that G contained five bacterial species related to Pseudomonas
benefit soil health and quality, plant growth and suppress soil- sp., Lactobacillus sp., Enterococcus sp., Streptococcus sp. and
borne plant pathogens [43]. Thus, biofertilizers are expected Bacillus mucilaginosus (sequence accession number in Gen-
to be ideal supplements to standard chemical fertilizers [27]. Bank: AY944300-AY944304), and Biofertilizer Y contained
Currently, many biofertilizer products are marketed in China. two bacterial species related to Lactobacillus sp. and Bacil-
However, the use of biofertilizers may alter soil microbial lus sp. (sequence accession number in GenBank: AY944305,
community structure and function, as well as microbial activ- AY944306). No fungal organisms were identified in both biof-
ity [10], thus necessitating an in-depth examination of biof- ertilizers by PCR amplification of ITS3–4 regions of fungi.
ertilizer impact. DNA sequencing analysis was performed with BLAST
In the present study, the composition and dynamics of cel- (Entrez, NIH).
lulolytic microbial communities were analyzed using DNA- A sterile dried filter paper strip (3 cm × 7 cm in size) was
based approaches, and cellulolytic activity was measured by weighed and then buried in 100 g treated soil loaded onto a
the weight loss of buried filter paper strips. The study objec- sterile plate (diameter 9 cm). Sixty replicates were prepared
tives were (1) to document the effects of two biofertilizers (G for each treated soil sample. A total of 240 plates were incu-
and Y) on the rate of cellulose decomposition and microbial bated at 20 °C in the dark. The soil moisture content was
community structure; (2) to examine the relationship between adjusted to 80% of field capacity before loading onto the plates
the rate of cellulose decomposition and the variation in micro- and was maintained by daily addition of sterile deionized
bial community structures; (3) to detail microbial commu- water. During the incubation period, six replicate plates of
nity composition in treated soils when the rate of cellulose each treated soil sample were removed from the incubator
decomposition is highest. every 5 days for cellulolytic activity and microbial commu-
nity analysis.
2.1. Soil sampling and treatments Cellulolytic activity was represented by the cellulose
decomposition rate, which is the percentage of dry weight
The original soil used in this study was sampled from the loss of the buried filter paper strips after each incubation
top layer (0–10 cm) in a field plot located at Shanghai Jiao period. For each treatment, the mean weight loss was calcu-
Tong University Experimental Farm (Shanghai; 121°24′E, lated for the six replicates sampled at each time point. T-tests
31°0′N), was characterized as silty-loam (WHC 45%, pH 5.8) were performed using Microsoft Excel 2000 software (Mi-
and was never exposed to biofertilizer. Freshly collected soil crosoft Corporation, Washington) to estimate the significant
samples were pooled and gently crushed to a size that would differences among the four treatment regimes on soil cellu-
passage through a 2 mm sieve. The sieved samples were then lolytic activity. Statistical significance was determined at the
stored for up to 48 h at room temperature until use. The soil 0.05 level (P < 0.05).
Y. Zhao et al. / European Journal of Soil Biology 41 (2005) 21–29 23
2.3. Extraction and purification of DNA from soil samples 2.5. TGGE analysis
The soil samples were removed from the six replicate fil- Before TGGE analysis, each PCR product was recondi-
ter paper strips and were pooled and sub-sampled. A total tioned for five cycles to reduce single-stranded and heterodu-
DNA sample was then obtained as follows: 500 mg of sub- plex DNA [33]. A 3 µl reconditioned sample (approximately
sampled soil was added to a 10 ml polypropylene tube with 50 ng of DNA) was mixed with 5 × gel loading dye and loaded
350 mg sterile glass beads (diameter 1 mm) and 2 ml DNA on an 8% (w/v) polyacrylamide, 7 M urea, 20% formamide
extraction buffer (100 mM Tris; 100 mM EDTA; 200 mM gel and subjected to electrophoresis using Biometra TGGE
NaCl; 2% PVPP; 3% CTAB; pH 9.0). The tube was vortexed Maxi for 3.5 h at a constant voltage of 200 V in 1 × TAE
for 10 min, after adding 500 µl lysozyme (100 mg ml−3). The running buffer. Electrophoresis was carried out over a tem-
tube was vortexed again for 5 min and then incubated at 37 °C perature range of 38–54 °C for bacteria and 35–48 °C for
for 30 min. After the incubation period, the tube was vor- fungi. The gel was then subjected to silver staining [4]. The
texed for 5 min and 2 ml SDS buffer (100 mM Tris; 200 mM stained gels were immediately photographed using a digital
NaCl; 3% SDS; pH 9.0) was added. The tube contents were camera DSC-F717 (SONY, The Japanese). The digital finger-
mixed by inverting five times, and then incubated at 65 °C printing images were analyzed using the UVIBAND/MAP
for 30 min. The supernatant was collected after centrifuga- V.99 software (UVItec Limited, UK). The Dice similarity
tion at 6000 × g for 10 min at room temperature and trans- coefficients among the TGGE patterns were calculated
ferred into a new 10 ml centrifuge tube. The supernatants according to Sneath and Sokal [31]. Cluster analysis of data
were extracted with an equal volume of chloroform/ and generation of dendrogram were performed using the Clus-
isoamyl:iso-amyl alcohol (24:1, v/v). The aqueous phase was ter program (software developed by UVItec Limited, UK).
recovered by centrifugation at 16,000 × g for 20 min and pre- Bands, marking the different structural characteristics
cipitated with 0.6 vol of isopropanol and 0.1 vol of 3 M NaAc between the treatments in TGGE fingerprinting, were excised,
at room temperature for 1 h. The pellet of crude nucleic acids purified and re-amplified for further sequencing analysis with
was obtained by centrifugation at 16,000 × g for 20 min at a clone library approach in which five clones were selected
room temperature, washed with cold 70% ethanol, and resus- to sequence for each band.
pended in sterile deionized water, to yield a final volume of
500 µl. 2.6. Clone libraries and sequence analysis
Crude DNA extracts were purified with 1 vol of phenol,
then passed through a Biocolor 3S DNA purification column, Clone libraries consolidating the amplified ITS3–4 prod-
according to the manufacturer’s instructions (Shanghai Bio- ucts representing significant fungal structural shifts at time
color Biotechnology Co., Ltd., Shanghai, China), and stored points with the highest cellulose degradation were con-
at –20 °C until used. structed for the samples collected at 20 days from G treat-
ment (G20) and R control (R20). The PCR products were
2.4. Polymerase chain reaction ligated into pGEM-T Easy Vector according to the manufac-
For TGGE analysis, a primer pair of P2 (ATTACCGCG- turer’s instructions (Promega, Madison, WI) and were then
GCTGCTGG) and P3-GC (CGCCCGCCGCGCGCGGC- transformed into E. coli DH5a. The resulting clones were
GGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGG- screened by insert length. Positive clones were sequenced with
CAGCAG) was used to amplify variable region three (V3) of an automatic sequencer (ABI PRISM 377 DNA Sequencer;
the 16S rRNA bacterial genes [22]. A primer pair of ITS3 PE Biosystems, Foster City, USA) by Shanghai BioAsia Bio-
(GCATCGATGAAGAACGCAGC) and ITS4-GC (CGCC- technology Co., Ltd.
CGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGG- The sequences with a similarity of greater than 99% were
GGGTCCTCCGCTTATTGATATGC) was used to amply regarded as being of the same OTU. The closest matches of
ITS3–4 regions of fungi [41]. Each 25 µl PCR reaction con- single-sequenced OTUs were determined in the EBI/NCBI
tained a final concentration of the following reagents: 10 ng database by FASTA3 (European Bioinformatics Institute,
of purified genomic DNA, 100 pM of each of the primers, UK).
200 µM dNTPs, 1 × Taq Reaction Buffer, 1.3 mM MgCl2, The clone library coverage was calculated using the fol-
and 1 unit Taq Polymerase (Promega Corporation, USA). A lowing equation:[30]:
Hybaid PCR Express thermal cycler (Hybaid Limited, Ash- C= 关 1 − 共 n/N 兲 兴 ×100%
ford, UK) was used in the PCR amplifications. For V3 region,
the amplification was carried out following a ‘touchdown where n is the number of unique sequences and N is the total
PCR’ process [22]. For ITS3–4 regions, amplified program number of sequences.
was as follows: one cycle of 94 °C for 3 min; 94 °C for 45 s,
51 °C for 45 s, and 72 °C for 1 min (30 cycles); and a final 2.7. Nucleotide sequence accession numbers
extension at 72 °C for 6 min.
For the clone libraries to analyze fungal community, the The nucleotide sequences were submitted to the Gen-
PCR conditions were the same as those described above but Bank database through NCBI under the following accession
using primer pairs without the GC-clamp ITS3 and ITS4. numbers: AY704731–AY704761.
24 Y. Zhao et al. / European Journal of Soil Biology 41 (2005) 21–29
Fig. 2. TGGE profiles of amplified V3 region of 16S rDNA fragments representing the bacterial community in different amended soil samples
(A) and cluster analysis result (B). G5, Y5, R5 represent G, Y, R amended for 5 days, respectively; G10, Y10, R10 represent G, Y, R amended for
10 days, respectively; G30,Y30, R30 represent G,Y, R amended for 30 days, respectively; G50,Y50, R50 represent G,Y, R amended for 50 days,
respectively.
Y. Zhao et al. / European Journal of Soil Biology 41 (2005) 21–29 25
Fig. 3. TGGE profiles of amplified ITS3–4 region fragments representing the fungal community in different amended soil samples (A) and
cluster analysis result (B). G5, Y5, R5 represent G, Y, R amended for 5 days, respectively; G10, Y10, R10 represent G, Y, R amended for 10 days,
respectively; G20, Y20, R20 represent G, Y, R amended for 20 days, respectively; G30, Y30, R30 represent G, Y, R amended for 30 days,
respectively.
No intense bands were detected in the TGGE profile of from clone libraries G20 and R20, respectively. In total, 30 dif-
the three samples collected after the cellulose filter paper strips ferent OTUs, denoted OTU-ZY01 to OTU-ZY30, were iden-
were buried for 5 days, however, an intense band (Band 1) tified with a similarity cutoff of 99%. The screening cover-
appeared in the soil treated withY biofertilizer after the paper age for the G20 and R20 libraries reached to 70% and 68.75%,
strips were buried for 10 days (Fig. 3A). Another intense band respectively.
(Band 2) appeared in G-treated soil after incubation for The relative abundance of each OTU in the two libraries
20 days, and several intense bands (Bands A–C) appeared in is shown in Fig. 4. OTU-ZY01 to ZY04 were common to
all three of the treated soil samples (indicated with arrows in both G20 and R20 libraries. OTU-ZY05 to ZY19 were spe-
Fig. 3A). After incubation for 20 days, some of the faint bands cific to the G20 library, and OTU-ZY20 to ZY30 were spe-
disappeared. In addition, specific intense bands appeared for cific to the R20 clone library. Most of the OTUs only appeared
G,Y and R treated for 30 days (Bands D–F, respectively, indi- in single clones. However, sequences of OTU-ZY01 to
cated with arrows in Fig. 3A). ZY08 and OTU-ZY20 to ZY21 were found in more than one
clone, marking the largest structural shift between the two
3.3. Phylogenetic analysis of fungal species represented by fungal communities.
Band1 and Band2 Table 1 presents the fungal species that were the most
closely matched to the 30 sequenced OTUs from libraries
Bands 1 and 2 (Fig. 3A) were excised from the TGGE gel, G20 and R20. Among the most highly matched fungal spe-
re-amplified and cloned. The amplified inserts of five selected cies, with the exception of several unknown fungi (OTU-
white clones from each band-clone library maintained their ZY19, ZY21, ZY30), most were soil-borne fungi (Table 1).
original TGGE patterns. The sequences of the five selected
clones were identical. According to the closest sequence
homology, the fungus represented by Band1 (AY704761)
matched to a plant pathogen fungus, Peniophora aurantiaca,
with a similarity of 66%, while Band 2 (AY704735) corre-
sponded to a wood-decaying fungus species, Merulipora
incrassata, with a similarity of 63%.
Table 1
Species of fungi with ITS3–4 sequences most similar to 30 OTUs from G20 and R20 clone libraries a
OTU numbers Fragment size Accession numbersb Fungal species with most similar sequence Similarity
(bp) (%)
Ascomycota
ZY10 338 AY704740 Scytalidium thermophilum (AB085927) 93
ZY14 350 AY704744 Leaf litter ascomycete strain its267 (AF502791) 96
ZY24 331 AY704754 Tricladium splendens (AY204636) 97
ZY25 340 AY704755 Salal root associated fungus UBCTRA1522.5 (AF284133) 87
Orbiliomycetes
ZY02 372 AY704732 Arthrobotrys amerospora (AF106533) 92
Pezizomycetes
ZY06 341 AY704736 Ascobolus immersus (AJ271628) 91
Sordariomycetes
ZY04 339 AY704734 Chaetoimium globosum (AY429056) 96
ZY07 393 AY704737 Cordyceps gunnii (AJ536551) 74
ZY11 353 AY704741 Bionectria ochroleuca (AJ509863) 98
ZY12 369 AY704742 Verticillium dahliae (AF104926) 91
ZY20 435 AY704750 Cf.Verticillium sp. 254/HP3 (AY172097) 96
ZY22 337 AY704752 Fusarium oxysporum (X78259) 100
ZY28 349 AY704758 Mycoleptodiscus terrestris (U97332) 99
Dothideomycetes
ZY08 346 AY704738 Alternaria mali (AY154683) 100
ZY15 346 AY704745 Cladosporium elatum (AF393699) 89
ZY16 344 AY704746 Didymella cucurbitacearum (AY293804) 99
ZY26 342 AY704756 Didymella cucurbitacearum (AY293804) 98
Eurotiomycetes
ZY13 334 AY704743 Geomyces sp. T489/9b (AY345348) 100
ZY27 353 AY704757 Penicillium brocae (AF484397) 97
Leotiomycetes
ZY23 333 AY704753 Chalara sp. LL-16.3 (AY188359) 92
Basidiomycota
ZY19 378 AY704749 Basidiomycete isolate wb436 (AF461413) 67
ZY21 400 AY704751 Basidiomycete from a bamboo (U65614) 63
ZY30 377 AY704760 Basidiomycete isolate wb436 (AF461413) 66
Basidiomycetes
ZY01 394 AY704731 Uncultured basidiomycete clone d484.32 (AY254866) 95
Hymenomycetes
ZY03 413 AY704733 Ceratobasidium sp. AGO (AF354094) 99
ZY05 402 AY704735 Meruliporia incrassata (AJ419913) 63
ZY09 397 AY704739 Meruliporia incrassata (AJ419913) 63
ZY17 407 AY704747 Inocybe nitidiuscula (AJ534934) 83
ZY18 415 AY704748 Ceratobasidium sp. CAG4 (AF354081) 99
ZY29 388 AY704759 Salal root associated fungus UBCTRA1041.2 (AF284135) 88
a
Sequences were compared to those in EBI and NCBI database.
b
Sequence were submitted to NCBI database.
Many species (matching OTU-ZY02, ZY03, ZY08, ZY12, ZY06 matched with 91% similarity to Ascobolus immerses,
ZY16, ZY18, ZY20, ZY22, ZY26 in identity) were plant- a species that may utilize fibrous residues in the feces of her-
root pathogen or soil nematophagous fungi; several (OTU- bivorous animals. OTU-ZY10 was found to be 93% similar
ZY01, ZY25, ZY29) were function-unknown soil fungi; some to a xylanase-producing thermophilic fungus isolated from
(OTU-ZY04, ZY05, ZY06, ZY09, ZY10, ZY14 and ZY17) Japanese soil, Scytalidium thermophilum. OTU-ZY14
may be related to cellulose decomposition. OTU- matched to a leaf litter ascomycete strain, with a similarity of
ZY04 matched to a xylanase-producing fungus Chaetoi- 96%. OTU-ZY17 corresponded to the cellulose- and xylan-
mium globosum, with a similarity of 96%; OTU-ZY05 and utilizing species Inocybe nitidiuscula, with a similarity of
ZY09 both correlated to a wood-decaying fungus, Merulipo- 83%. The sequence of OTU-ZY05 in library G20 was con-
ria incrassate, with the same similarity of 63%. OTU- sistent with that of Band2 in sample G20.
Y. Zhao et al. / European Journal of Soil Biology 41 (2005) 21–29 27
polymorphisms and observed that the sequence similarity [5] Y.C. Chen, J.D. Eisner, M.M. Kattar, S.L. Rassoulian-Barrett,
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