Download Effect of Different Bacterial-Feeding Nematode Species on Soil

Pedosphere 24(1): 116–124, 2014
ISSN 1002-0160/CN 32-1315/P
c 2014 Soil Science Society of China
Published by Elsevier B.V. and Science Press
Effect of Different Bacterial-Feeding Nematode Species on Soil
Bacterial Numbers, Activity, and Community Composition∗1
XIAO Hai-Feng1,2 , LI Gen2 , LI Da-Ming2 , HU Feng2 and LI Hui-Xin2,∗2
1 Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303
(China)
2 College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095 (China)
(Received July 31, 2013; revised December 7, 2013)
ABSTRACT
The effects of bacterial-feeding nematodes on bacterial number, activity, and community composition were studied through
a microcosm experiment using sterilized soil inoculated with soil bacteria (soil suspension) and with bacteria and three species
of bacterial-feeding nematodes (Cephalobus persegnis, Protorhabditis filiformis, and Caenorhabditis elegans). Catalyzed reporter
deposition-fluorescence in situ hybridization, CO2 evolution, and denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA gene fragments were used to investigate bacterial numbers, activity, and community composition, respectively. Our
results showed that bacterial numbers and activity significantly increased in the presence of bacterial-feeding nematodes, which indicated that bacterial-feeding nematodes had a significant positive effect on soil bacteria. The different nematode species had different
effects on bacterial numbers and activity. C. persegnis and P. filiformis, isolated from native soil, increased the bacterial number and
activity more than C. elegans. The DGGE analysis results showed that dominant bacterial species significantly differed among the
treatments, which suggested that bacterial-feeding nematode species modified the bacterial community composition in soil. Further
gene sequence analysis results showed that the dominant bacterial species in this study were gram-negative bacteria. Given the completely same conditions except nematode species, the varied selective feeding behavior of different nematode species was the most
likely reason for the altered bacterial community composition. Overall, the alteration of bacterial numbers, activity and community
composition resulting from the bacterial-feeding nematodes may ultimately affect soil ecological functioning and processes.
Key Words:
CARD-FISH, CO2 evolution, DGGE, gene sequence, gram-negative bacteria
Citation: Xiao, H. F., Li, G., Li, D. M., Hu, F. and Li, H. X. 2014. Effect of different bacterial-feeding nematode species on soil
bacterial numbers, activity, and community composition. Pedosphere. 24(1): 116–124.
Bacterial community structure and activity are
central factors that influence terrestrial ecosystem
functions (Kennedy and Gewin, 1997) and are changed
by bacterivorous predators such as bacterial-feeding
nematodes (Griffiths et al., 1999; Rønn et al., 2002;
Djigal et al., 2004) that graze in soils. Bacterial-feeding
nematodes have been recognized as a main bacterivorous predator (Griffiths, 1994; Li and Hu, 2001) because of their greater abundance and consumption in
soils. For example, Bernard (1992) and Liang et al.
(2000) found that nematodes are the most abundant
metazoans in the soil, ranging from 7.6 × 105 m−2 in
deserts to 2.9 × 107 m−2 in mixed deciduous forests.
Furthermore, Venette and Ferris (1998) found that an
adult bacterial-feeding nematode consumes 1 × 106
cells daily. Bacterial-feeding nematodes have a huge
grazing potential on soil bacteria. However, this does
not mean that the number of bacteria will be substantially reduced. Bacteria respond variably to nematode
∗1 Supported
grazing and conclusions regarding the changes in bacterial numbers caused by nematode grazing have been
inconsistent. Researchers found that bacteria increase
when predators graze on them (Abrams and Mitchell,
1980; Traunspurger et al., 1997; Bardgett et al., 1998),
whereas others obtained the contrasting results (Coleman et al., 1977; Anderson et al., 1983).
Numerous studies have also reported bacterialfeeding nematodes that graze on bacteria, thereby influencing bacterial activity (usually by detecting soil
respiration and enzyme activity) (Anderson and Coleman, 1977; Trofymow et al., 1983; Djigal et al., 2004;
Fu et al., 2005). Most of these studies showed increased bacterial activity in the presence of nematodes. For example, Fu et al. (2005) found that microcosms with nematodes produced significantly more
CO2 than those without nematodes. Although the alterations of bacterial numbers and activity in the presence of bacterial-feeding nematodes were reported by
by the National Natural Science Foundation of China (Nos. 41271270 and 31200409).
author. E-mail: [email protected].
∗2 Corresponding
NEMATODE EFFECT ON SOIL BACTERIA
many previous studies, related reasons were deficiently
discussed and need to be explored more deeply.
Aside from affecting bacterial abundance and activity, bacterial-feeding nematodes that graze on bacteria even modify the bacterial community composition (Griffiths et al., 1999; Djigal et al., 2004). However, previous studies did not examine which bacterial species changed, and the possible mechanisms
have been rarely reported and need more investigation. Generally, nematodes are selective and display
specific preferences when grazing. In turn, different
bacterial resources may be appropriate for different nematode species and affect nematode growth and fecundity. Venette and Ferris (1998) found that six different
bacterial-feeding nematode species have different reproduction rates according to the ingested bacterium.
Selective feeding behavior may induce competition between microbes, thereby altering the community composition and distribution in soil (Fu et al., 2005; Blanc
et al., 2006).
Considering the complexity of environmental conditions and the limitation of research tools, observing the activity of microfauna and bacteria directly
in the soil is difficult. Recently, however, catalyzed
reporter deposition-fluorescent in situ hybridization
(CARD-FISH) with horseradish peroxidase (HRP)labeled oligonucleotide probes and tyramide signal amplification and DNA fingerprinting (such as denaturing
gradient gel electrophoresis, DGGE) have been used to
investigate the numbers and the community structure
of bacteria, respectively. CARD-FISH, instead of conventional monolabeled FISH (oligonucleotide probes
labeled with Cy3 fluorochrome), was suitable for soil
microorganism analysis (Pernthaler et al., 2002; Eickhorst and Tippkötter, 2008). Both the detection sensitivity and specificity of CARD-FISH were higher than
those of monolabeled FISH (Haugland, 2005; Eickhorst
and Tippkötter, 2008). Muyzer et al. (1993) and Pernthaler et al. (2002) successfully used CARD-FISH and
DGGE to investigate the bacterial distribution and
community structure.
Alterations in bacterial abundance, activity, and
community composition are related to soil ecological
functions because bacteria are the drivers of many biochemical reactions in soil. Considering that different
nematode species likely have different effects on bacterial community composition because of their selective
feeding behavior, we hypothesize that bacterial-feeding
nematodes modify the bacterial community composition because of their preference for different bacterial
species. Two native bacterial-feeding nematode species
117
isolated and identified from the soil used in this study,
namely, Cephalobus persegnis and Protorhabditis filiformis, and Caenorhabditis elegans were used to compare the effect of different bacterial-feeding nematode
species on the soil bacterial number, activity, and community composition. Altered bacterial species were also
examined in this study.
MATERIALS AND METHODS
Soil
The soil used in this study was a sandy soil (57.5%
sand, 26.6% silt, and 15.9% clay) collected from Nantong City, Jiangsu Province, China. The soil contained
6.03 g kg−1 organic C, 0.70 g kg−1 total N, and 0.68
g kg−1 total P, and the pH (H2 O) was 6.85. Before
use, the fresh soil was passed through a 2-mm mesh to
remove stones, macrofauna, and some broken roots. A
portion of the soil was used to isolate nematodes and
prepare bacterial suspensions, and the remainder was
sterilized by heating at 121 ◦ C (102.9 kPa) for 30 min
(Xiao et al., 2010a).
Nematode isolation and incubation
Two native nematode species were isolated from
the soil via a modified cotton wool filter method (Liang
et al., 2009) and identified as Cephalobus persegnis and
Protorhabditis filiformis, both of which are bacterialfeeding nematodes that belong to the family Rhabditidae. A single individual of each was allowed to multiply
on an agar plate with Tryptic soy broth culture containing mixed soil bacteria (soil bacterial suspension
being coated onto the plates) as the food source. C.
elegans maintained in long-term cultures in our laboratory was selected because it also belongs to the family
Rhabditidae and considered a model organism. All
three nematode species were reared on agar plates to
generate enough for inoculation. These three nematode
species had similar generation times of 4 to 5 d when
incubated at 28 ◦ C.
Soil preparation
To obtain nematode-free soil abundant with bacteria, the soil was sterilized and then treated with a
bacterial suspension to increase the bacterial count.
The bacterial suspension was filtered through two filter
membranes with 5 μm pores to eliminate nematodes.
Each pot contains 1 000 g of sterilized soil. Up to 40
mL of nematode-free inoculum of the mixed soil bacteria (about 2 × 106 g−1 dry soil) was inoculated into
each pot.
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Nematode inoculation
Each bacterial-feeding nematode species was inoculated into a pot at 20 individuals g−1 dry soil. Before the nematodes were inoculated into the soil, they
were surface disinfected for 20 min with a mixture of
1.0 g L−1 of streptomycin sulfate and 0.02 g L−1 of
cycloheximide and then centrifuged (3 000 × g) for 3
min, after which the supernatant was discarded. The
nematodes were then washed 5 to 6 times with sterile
water to minimize bacterial interference during their
transfer into the soils. The control pots were not inoculated with nematodes. Each nematode species and
the control treatment were performed with three replicates, with a total of 12 pots. Then, the soil moisture
content was adjusted to 25% using distilled water. Finally, all the pots were placed in an incubator at 28
◦
C in darkness for 28 d. Soils weighing 30, 0.5 and
20 g were sampled non-destructively weekly from the
beginning of experiment to determine nematode numbers, bacterial abundance, and soil respiration. At the
end of the incubation, DNA was extracted from 5 g of
soil to analyze the bacterial community structure.
CARD-FISH
The samples were prepared on glass slides according to the method in Eickhorst and Tippkötter
(2008). Briefly, 0.5 g of fresh soil was weighed and
transferred to 2 mL centrifuge tubes. Then, 320 μL
of 25% (w/v) particle-free paraformaldehyde solution
(4% (w/v) final concentration) was added, filled with
1 × phosphate-buffered saline (PBS) (137 mmol L−1
NaCl, 2.7 mmol L−1 KCl, 10 mmol L−1 Na2 HPO4 , and
2 mmol L−1 KH2 PO4 ), mixed, and stored at 4 ◦ C. After 6 h, the suspension was centrifuged at 10 000 × g
for 5 min at 4 ◦ C, washed twice with 1 × PBS, centrifuged again under the same condition, and stored in
PBS/ethanol (1:1, v:v) at 20 ◦ C for further processing.
Then, 100 μL of the fixed sample was diluted with 900
μL of PBS/ethanol. Up to 30 μL of the diluted sample,
60 μL of 1 × PBS and 10 μL of 0.01% (v/v) sodium
dodecyl sulfate (SDS) were placed on a glass slide. The
CARD-FISH procedure of Pernthaler et al. (2002) was
followed with slight modification. The samples on the
slides were hybridized with the probes EUB338 (5 GCT GCC TCC CGT AGG AGT-3 ) (Eickhorst and
Tippkötter, 2008). HRP-conjugated oligonucleotides
probes were purchased from TaKaRa, Japan. Cy3labeled tyramide was purchased from China Isotope
Corporation. Automated cell counting was performed
on 10 to 15 randomly selected visual fields, the selected
visual fields were photographed under 40 × magni-
H. F. XIAO et al.
fication (64 and 112 μm2 ), and the counts were extrapolated onto 1 g of soil (dry soil). To prevent the
weakening of the fluorochrome under excitation light,
the samples were treated with Citifluor AF1 (Citifluor
Ltd., London) at the surface. Excitation was performed
under blue light before UV light because high-energy
UV may destroy the weak fluorescence signals.
Soil respiration analysis
CO2 evolution from the soils was measured using
the alkali absorption method (Anderson, 1982). Briefly,
20 g of soil was weighed and transferred into 300-mL
jars, and small vials containing 5 mL of 0.05 mol L−1
NaOH were placed inside the jars. The jars were sealed
and left for 24 h. After incubation, the excess NaOH
was titrated using 0.025 mol L−1 of HCl with phenolphthalein as an indicator and soil respiration rate
was calculated.
Extraction and purification of DNA from soil samples
After 28 d of incubation, DNA was extracted from
5 g of the soil samples by mixing them with 13.5 mL
of DNA extraction buffer (100 mmol L−1 of Tris-HCl,
100 mol L−1 of EDTA-Na2 , 100 mmol L−1 of Na3 PO4 ,
1.5 mol L−1 of NaCl, and 10 g L−1 cetyltrimethylammonium bromide (CTAB, pH 8.0)). The samples were
then incubated for 30 min at 37 ◦ C in a horizontal
shaking bath at 225 r min−1 . After three rounds of
freezing in liquid nitrogen and thawing in a 65 ◦ C water bath, 1.5 mL of 200 g L−1 SDS was added and the
samples were further incubated for 2 h at 65 ◦ C with
agitation every 15 min. The samples were then centrifuged (6 000 × g) at room temperature for 10 min
to collect the supernatant. The supernatant was transferred into a 50-mL centrifuge tube, extracted with
phenol and then purified with chloroform-isoamyl alcohol (24:1, v:v). The aqueous phase was transferred
into 50-mL centrifuge tubes. Then, 0.6 volumes of isopropanol were added and the mixture was incubated
at room temperature for 1 h. The samples were then
centrifuged (6 000 × g) at room temperature for 20
min. After centrifugation, nucleic acid was collected
and washed with cold 70% (v/v) ethanol, dissolved in
300 μL of sterile ultrapure water, and the crude DNA
product was purified using an OMEGA purification kit
(Omega Bio-Tek, USA) (Xiao et al., 2010a).
PCR and DGGE
Universal bacterial primers that amplified a 194 bp
fragment of the 16S rRNA gene, including the third
variable (V3) region, were used in this experiment. A
40 bp GC clamp was added at the 5 end of the for-
NEMATODE EFFECT ON SOIL BACTERIA
ward primer. The PCR protocol included 5 min of initial denaturation at 94 ◦ C, 30 cycles at 94 ◦ C for 30
s, at 61 ◦ C for 30 s, and at 72 ◦ C for 30 s, followed
by a final extension at 72 ◦ C for 5 min. The reaction
mixtures (50 μL) contained 1 × PCR reaction buffer
(TaKaRa, Japan), 100 ng of DNA template, 10 pmol
L−1 of the forward and reverse primers, 200 μmol L−1
of deoxynucleotide (dNTP) mix, and 2.5 units of Ex
Taq DNA Polymerase (TaKaRa, Japan).
The PCR-DGGE was performed with a DCode
mutation detection system (Bio-Rad, USA). Polyacrylamide gels (8% (v/v) of a 37.5:1 acrylamidebisacrylamide mixture in 1 × Tris-acetate-EDTA
(TAE) buffer) with a gradient of 30% to 70% denaturant (100% denaturant containing 7 mol L−1 urea and
40% (v/v) formamide) (Muyzer et al., 1993). Approximately 200 ng of each PCR product was loaded, and
the gels were electrophoresed for 5 h at 200 V and 60
◦
C. The gels were silver stained and fixed for image
analysis using Quantity One gel analysis software version 4.62 (Bio-Rad, USA).
119
at 20 individuals g−1 dry soil and the growth of the
nematodes is shown in Fig. 1. The number of nematodes significantly increased over time (F = 1 443,
P < 0.01). C. persegnis and P. filiformis were significantly more than C. elegans after Day 14, but the number of C. persegnis was not significantly different from
that of P. filiformis. In addition, time significantly interacted with the nematode treatment (F = 279.448,
P < 0.01).
Cloning and sequencing
PCR amplifications without GC clamp were conducted using the OMEGA quick PCR purification
kit prior to cloning. Approximately 4 μL of purified products were ligated into the PMD-19T vector (TaKaRa Cloning Kit) (TaKaRa, Japan) and
then further transformed into Escherichia coli competent cells DH5α (TaKaRa, Japan). The white colonies
were randomly selected from each cloned sample, grew
overnight, and then sequentially reacted on an ABI
377 apparatus (BGI Company, China). The nucleotide
sequences were deposited in the GenBank database
and assigned with accession numbers FJ911506 to
FJ911520.
Statistical analysis
A repeated measures analysis of variance (ANOVA) was used to determine significant differences
among treatments at different dates. Differences among treatments were tested by a least significant difference (LSD) test (P < 0.05). For DGGE analysis,
Quantity One software was used to test the band intensity. All the bands were normalized and principal
component analysis (PCA) was performed using SPSS
16.0 software.
RESULTS
Growth of bacterial-feeding nematodes in soil
The soil was initially inoculated with nematodes
Fig. 1 Bacterial-feeding nematode numbers of the soils inoculated with three nematode species (C. persegnis, P. filiformis,
and C. elegans). Error bars represent standard deviations of
the means (n = 3). Different letters denoted statistically significant differences between the treatment groups according to least
significant difference (LSD) test (P < 0.05).
Soil bacterial numbers
The bacterial numbers significantly increased during the experiment (F = 92.605, P < 0.05), with
those in the nematode treatments about twice that
of the control (Fig. 2). The number of bacteria in the
C. persegnis and P. filiformis treatments were significantly higher than those with C. elegans on Day 14,
with no significant difference between the C. persegnis
and P. filiformis treatments. By Day 28, the number
of bacteria in the C. persegnis treatment was significantly higher than those in the P. filiformis and C.
elegans treatments.
Soil respiration
Soil respiration was significantly higher in the presence of bacterial-feeding nematodes than the control
(F = 344.675, P < 0.01) (Fig. 3). Soil respiration
showed no significant difference over time (F = 1.765,
P > 0.05), but time significantly interacted with the
treatments (F = 8.053, P < 0.05), which indicated
that the bacterial-feeding nematodes significantly increased the soil respiration. The nematode treatments
did not significantly differ except on Day 14, wherein
120
H. F. XIAO et al.
cated that the bacterial diversity increased in the presence of the bacterial-feeding nematodes. The intensity
of some bands (e.g., a, b, d, f, i, and j) increased; however, the intensity of band c decreased in the presence
of C. persegnis and P. filiformis. Additionally, bands g
and h were associated with C. persegnis. The intensity
of band k was sharply increased in the treatment with
P. filiformis and intensity of bands l and m increased
in the presence of C. elegans. The PCA of the DGGE
profile differentiated the four treatments, especially the
nematode treatments from the control (Fig. 5).
Fig. 2 Effect of bacterial-feeding nematodes on bacterial numbers of the control soil and the soils inoculated with three nematode species (C. persegnis, P. filiformis, and C. elegans). The
control consisted of sterilized soil only inoculated with soil bacteria (without the nematodes). Error bars represent standard
deviations of the means (n = 3). Different letters denote statistically significant differences between the treatment groups according to the least significant difference (LSD) test (P < 0.05).
Fig. 4 Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene sequences amplified from DNA extracted
from three replicate samples of the control soil and the soils inoculated with three nematode species (C. persegnis, P. filiformis,
and C. elegans).
Fig. 3 Effect of bacterial-feeding nematodes on soil respiration
of the control soil and the soils inoculated with three nematode
species (C. persegnis, P. filiformis, and C. elegans). The control consisted of sterilized soil only inoculated with soil bacteria
(without the nematodes). Error bars represent standard deviations of the means (n = 3). Different letters denote statistically
significant differences between treatment groups according to the
least significant difference (LSD) test (P < 0.05).
the soil respiration in the C. persegnis and P. filiformis
treatments was significantly higher than that in the C.
elegans treatment.
Soil bacterial community structure
Fig. 5 Principal component analysis of the denaturing gradient
gel electrophoresis (DGGE) band patterns affecting the bacteria
community structure. The error bars represent the least significant difference; hence, the coordinates are significantly different
(P < 0.05) when the error bars do not overlap with the mean
from another treatment.
The differences among the four treatments were
clearly discernible from the bands in the DGGE gel
(Fig. 4). The number of bands increased in the nematode treatments compared with the control, which indi-
To further analyze which bacterial species was altered by the different nematode species, we cloned and
sequenced all the labeled bands (Fig. 4). The sequences
were aligned with previously published sequences using
NEMATODE EFFECT ON SOIL BACTERIA
the Basic Local Alignment Search Tool (BLAST) in the
NCBI database, and the results are shown in Table I.
DISCUSSION
Effects of bacterial-feeding nematodes on bacterial
numbers and activity
The numbers of nematodes gradually increased
over time, peaked on Day 14, and gradually decreased
thereafter (Fig. 1). The bacterial generation time was
much shorter than that of the nematodes. Thus, the
bacterial growth rate was faster than the nematode
growth rate in soil. This is why the bacteria rapidly
increased and almost peaked on Day 7, whereas the
number of nematodes peaked on Day 14. With the increase in the number of bacteria, the number of nematodes increased gradually because of the increased bacterial food resource. However, with the consumption
of the substrate, both the bacteria and the nematodes
decreased after two weeks. Therefore, sufficient substrate for bacterial growth is an important factor for
stabilizing predator-prey fluctuation. In barren soil or
in closed experimental systems with limited resources,
the number of bacteria might decrease with the deficient substrate under the pressure of grazing bacterialfeeding nematodes. Additionally, the lag times associated with different nematode and bacterial generation
times were important factors that affect the numbers
of nematodes and bacteria (Neher, 2010). However,
the generation times of the three different nematode
species in this study were similar, around 4 to 5 d at
121
28 ◦ C. Therefore, we consider that the differences between the number of C. elegans and those of the two
native nematode species may have resulted from differences in their adaptability to soil.
Our results also showed that the number of soil bacteria significantly increased in the presence of bacterialfeeding nematodes (Fig. 2), which is consistent with
many previous studies (Traunspurger et al., 1997; Bardgett et al., 1998; Fu et al., 2005). There are two possible explanations for these results. First, according to
the nutritional dynamic hypothesis proposed by Carpenter et al. (1985), reproduction rates of one trophic level are maximal under moderate predation pressure by higher trophic level animals. Therefore, moderate nematode grazing may keep the bacterial number
growth rapidly. Fu et al. (2005) considered that the
bacteria-to-nematode ratio is a critical index for the
predator and prey relationship in soil, which determines bacteria and nematode growth. This surmise was
confirmed by our previous study (Xiao et al., 2010b),
which we designed with different nematode concentrations and found a significant “density regulating effect”
between the nematodes and the bacteria. Second, the
nematodes also excrete inorganic nitrogen and other
organic matter, which provide nutrition for bacteria
and stimulate their growth (Anderson et al., 1983; Ingham et al., 1985; Griffiths and Bardgett, 1997). Anderson et al. (1983) reported that nematodes excreted
significant amounts of amino acids into soil. Furthermore, most bacteria maintain activity during passage
through the nematode alimentary canal (Yeates, 1969;
Smerda et al., 1971; Ingham et al., 1985; Bird and Ry-
TABLE I
Alignment of 16S rRNA sequences of bacterial species from the control soil and the soils inoculated with three nematode species (C.
persegnis, P. filiformis, and C. elegans)
Banda)
a
b
c
d
e
f
g
h
1
2
1
2
i
j
k
l
m
a) See
Sequenceb)
Accession no.
FJ911506
FJ911507
FJ911508
FJ911509
FJ911510
FJ911511
FJ911512
FJ911513
FJ911514
FJ911515
FJ911516
FJ911517
FJ911518
FJ911519
FJ911520
Gram staining characteristic
Similarity
Nearest relative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
%
100
100
98
99
99
96
98
96
96
100
98
93
100
96
98
Sphingobacterium sp.
Uncultured Flavobacterium sp.
Uncultured Sphingobacterium sp.
Uncultured Pseudomonas sp.
Flavobacterium sp.
Chitinophaga sp.
Uncultured Bacteroides sp.
Uncultured Fluviicola sp.
Uncultured Fluviicola sp.
Sphingobacterium sp.
Sphingobacterium sp.
Uncultured Solitalea sp.
Uncultured Flavobacterium sp.
Uncultured Stenotrophomonas sp.
Sphingobacterium sp.
Fig. 4 for the band code.
some bands, two separate bands were obtained after rerunning the excised band.
b) From
122
der, 1993), and these bacteria possibly obtained hormones and nutrients to help them grow. Although some
studies indicated that nematodes negatively affect bacterial numbers (Coleman et al., 1977; Anderson et al.,
1983), these inconsistencies may be nematode speciesspecific (Ingham et al., 1985). Although all the nematode species may be capable of stimulating bacterial
growth, the additional production may be consumed
before a net increase in numbers can be observed (Anderson et al., 1983).
Although nematode grazing increased the number
of bacteria in our study, the different nematode species
affected the number of bacteria differently. As shown
in Fig. 2, the numbers of bacteria in the C. persegnis
and P. filiformis treatments were significantly higher
than that in the C. elegans treatment, except on Day
7, with no significant difference in the number of bacteria between the C. persegnis and P. filiformis treatments. This result indicated that the two native nematode species were more effective in increasing bacterial
number than C. elegans.
Respiration is an important index for bacterial
activity in soils (Coleman et al., 1983). Our study
showed that compared with the control, soil respiration was significantly increased by both the native
bacterial-feeding nematode species and C. elegans after
7 d (Fig. 3). This result indicated that nematode grazing increased bacterial activity (Woods et al., 1982;
Coleman et al., 1983; Djiagal et al., 2004; Fu et al.,
2005). Results of soil respiration also showed that the
CO2 evolution in all the treatments peaked on Day
7, which indicated that the bacteria exhibited higher
activity under the stimulation of the bacterial-feeding
nematodes when the bacteria were actively growing.
Similar to the bacterial numbers, the effects of native
nematode species on soil respiration were stronger than
that of C. elegans (Fig. 3).
Effect of bacterial-feeding nematodes on bacterial community composition
In the DGGE profile analysis, we assumed that the
number and intensity of the bands in the DGGE profile reflected bacterial diversity and abundance. The
number and intensity of the bands were significantly
different among the four treatments (Fig. 5), which indicated that the bacterial community was significantly
changed by the bacterial-feeding nematodes, consistent
with the previous studies by Griffiths et al. (1999), Djigal et al. (2004), and De Mesel et al. (2004). Djigal et
al. (2004) found that different nematode species influence the soil microbial community differently. For
example, Zeldia punctata clearly has less impact on
H. F. XIAO et al.
the microbial community structure than Acrobeloides
nanus andCephalobus pseudoparvus. A. nanus resulted
in the greatest change in the structure of the microbial
community. Size-selective feeding by nematodes may
alter the bacterial community composition because nematodes can utilize probolae to restrict the size of food
entering the buccal cavity (Lee and Atkinson, 1977). In
the current study, P. filiformis was about 0.4 to 0.5 mm
long, with a 16- to 18-μm long buccal cavity. C. persegnis was about 0.7 to 0.8 mm long, with a 10- to 12-μm
long buccal cavity (Wu, 1999). Adult C. elegans was
about 1 to 1.5 mm long (Wood, 1988), with a 20-μm
long buccal cavity. These different oral morphologies
probably preferred bacteria with different sizes. Therefore, the morphology and size differences of the nematodes may have led to differences in bacterial community composition.
In addition, bacterial-feeding nematodes may be
capable of distinguishing the structure of bacterial cell
walls and selecting the food edibility while rapidly excreting the unsuitable bacteria. Bacterial-feeding nematodes generally favor Gram-negative bacteria over
Gram-positive bacteria because their thinner cell walls
are easier to digest (Tortora et al., 2000), which is
supported by Salinas et al. (2005), who found that
Cephalobus brevicauda preferred Gram-negative bacteria. In this study, whether the different nematode
species preferred the dominant bacterial species in
each treatment was unclear. However, all the examined
dominant bacterial species were Gram-negative bacteria (Table I).
Moreover, nematodes can identify the smell of
different bacteria to distinguish their favorite foods
(Zhang et al., 2005). Newsham et al. (2004) also found
that different nematode species (Geomonhystera villosa, Plectus spp., and Teratocephalus spp.) have different preferences for microbes (two microalgae, three
microfungi, and six heterotrophic bacteria) in Antarctic soil. Other indirect factors affect the composition of
bacterial communities, such as nutrient and substrate
availability, may also be operating.
CONCLUSIONS
The presence of bacterial-feeding nematodes increased the bacterial numbers and activity and changes
the microbial community composition through a variety of selective feeding behavior. Native nematodes
had higher reproduction rates than C. elegans, with
the effect of the former stronger than that of the latter.
These results help us better understand the interaction
between microfauna and bacteria as well as provide
a basis for understanding the influence of microfauna
NEMATODE EFFECT ON SOIL BACTERIA
grazing on microbial communities and functioning in
soil ecology.
ACKNOWLEDGEMENTS
The manuscript was greatly improved by the insightful suggestions of the anonymous reviewers. We
also thank the Public Technology Service Center, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, for soil analyses.
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