APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2003, p. 2116–2125 Vol. 69, No.

4
0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.4.2116–2125.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Spatial and Temporal Analysis of the Microbial Community in Slow

Sand Filters Used for Treating Horticultural Irrigation Water
Leo A. Calvo-Bado,1 Tim R. Pettitt,1 Nick Parsons,2 Geoff M. Petch,1 J. Alun W. Morgan,1*
and John M. Whipps1
Plant Pathology and Microbiology Department1 and Biometrics Department,2 Horticulture Research
International, Wellesbourne, Warwickshire CV35 9EF, United Kingdom
Received 29 October 2002/Accepted 17 January 2003

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water
through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor
global changes in the microbial community, bacterial and fungal numbers were estimated on selective media,
direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S
rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the
bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active
microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and
diverse bacterial populations. The major changes in the microbial populations occurred during the first week
of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear
mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples.
According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were
represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column
indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as
Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an un-
characterized environmental clone. This study describes the characterization of the performance, and micro-
bial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of
naturally suppressive population of microorganisms either directly or by manipulation of the environment in
an SSF may provide a more reproducible control method for the future.

Fungal plant diseases are a major problem within the hor-
ticultural industry, resulting in reduced yields and occasionally
major crop damage. Contaminated irrigation water has long
been recognized as an important source of fungal plant patho-
gens and is an important factor in disease spread on commer-
cial horticultural nurseries (4). Water from many commonly
used sources, such as rainwater from glasshouse roofs and, in
particular, water stored in open reservoirs and ponds, can
often contain large numbers of infective propagules of fungal
plant pathogens, such as Pythium and Phytophthora spp. (1, 4,
34, 46, 48, 53). A particularly high risk of disease spread is
associated with the collection, recycling, and reuse of irrigation
water, often referred to as recirculation (45, 57, 58), which is
becoming more popular as attempts are increasingly being
made to conserve valuable water supplies. Rapid dispersal of
the pathogen in water is often achieved by asexual flagellate
zoospores, and a key element for pathogen control is the re-
moval of zoospores from water supplies. A wide range of
treatment techniques such as UV-radiation, ozonation, pas-
teurization, ultrafiltration, slow sand filtration, and dosing with
sterilant chemicals have been shown to be effective at control-
ling fungal plant pathogens in water supplies (23, 36, 50, 51, 53,
* Corresponding author. Mailing address: Department of Plant
Pathology and Microbiology, Horticulture Research International,
Wellesbourne, Warwickshire CV 35 9EF, United Kingdom. Phone: 44
(0) 1789 470382. Fax: 44 (0) 1789 470552. E-mail: alun.morgan@hri.ac
.uk.
55, 61, 70). Slow sand filters (SSFs), with their simple design
and operation, reliability, flexibility, and comparatively low
cost of installation and operation, have great appeal to horti-
cultural nurseries. The form of SSF currently used in horticul-
tural practice is based upon construction guidelines set out by
the World Health Organization (63) for the safe supply of
potable water. This type of filtration has a long history of
successful use in drinking water production (20) but has only
recently been introduced into horticultural production systems.
The action of SSFs against many microorganisms, including
some human pathogens and plant pathogens, is still not fully
understood but is considered to be a combination of physico-
chemical and biological processes which remove and consume
much of suspended particulate organic carbon from the water
passing through the sand column (24, 35, 65, 66). Using tradi-
tional microbial methods, the diversity and dynamics of bacte-
ria in SSF used in the water industry have been widely studied
(8, 13, 18). However, much of this work has relied upon con-
ventional plating and isolation techniques which do not study
the nonculturable and fastidious species generally thought to
dominate environmental samples (49). Direct methods to
study microbial populations overcome this limitation, and in
recent years, a genetic fingerprinting technique, denaturing
gradient gel electrophoresis (DGGE) (40, 41), has been widely
applied to ecological studies of microbial populations (39).
This method, like any other, is not without its problems: dif-
ferences in the efficiency of DNA extraction from different cell
types are likely to exist, 16S rRNA copy number per cell is

2116
VOL. 69, 2003
FIG. 1. Schematic representation of the SSF used in this study. The water supply system (storage tank), recirculation system (headwater and
sump), and filtration system are indicated. Arrows indicate the direction of the water flow.
, positions of pumps.
known to vary, and the kinetics of PCR amplification of the
different molecules present in samples containing a diversity of
bacteria may not be uniform (7, 64). However, PCR amplifi-
cation of DNA and DGGE analysis of the products has pro-
vided a useful means to directly characterize many bacterial
populations within samples.
This paper describes the characterization of experimental
SSF units shown to efficiently remove zoospores of Phytoph-
thora cryptogea. Both spatial and temporal information on the
bacterial community present within the SSF column were ob-
tained over the critical early stages of the filter maturation
process. For this, conventional plating, direct microscopic ob-
servation, and ATP analysis techniques were used in combina-
tion with PCR DGGE profiling of the bacterial population. To
our knowledge, the results presented here are the first where
DGGE has been applied to the study of the dynamic spatial-
temporal community in SSFs.
MATERIALS AND METHODS
Setup and operation of experimental SSF unit. Three replicate experimental
SSF rigs were constructed, each supplied from a common source of untreated
water (Fig. 1). The filters were made with 3 m of 160-mm-diameter Terrain
polyvinyl chloride pipe (Geberit, Aylesford, Kent, United Kingdom) mounted
vertically in a mild steel tubing frame. The bottom of each filter column was
sealed with an end cap fitting. To hold the sand in place and allow free drainage
of filtered water from the column, a drilled stainless steel plate (8-mm-diameter
holes arranged in a radial pattern at 15-mm spacing) was mounted inside the end
cap. A nylon mesh (150-␮m-pore-size mesh) was placed over the steel plate. The
end cap was drilled and fitted with a 40-mm-diameter polyvinyl chloride outlet
pipe. From this pipe ran the outlet and a manometer tube, fitted to allow
determinations of head loss (26) during filter runs. The water flow from each
filter was regulated using a 1/4-in. straight Wade-coupling needle valve inserted
MICROBIAL ANALYSIS OF SSFs 2117
Downloaded from http://aem.asm.org/ on March 5, 2020 by guest
into the end of the 40-mm-diameter pipe. The water flow rate for all experiments
was set at 0.15 m h⫺1 (height of water column passing per hour). To the valves,
8-mm-diameter flexible opaque gas tubing (RS Ltd., Corby, Northamptonshire,
United Kingdom) was used to drain the water, and the outflow water samples
from the SSF columns were collected from this tubing. Each column was con-
structed with a sand depth of 1 m and a 1.5-m-deep head of water above it. In this
system, water flow through the column was gravity assisted. Water was pumped
to the top of the column (headwater) from a tank containing the headwater (300
liters) at a continuous rate (1 liter min⫺1) which was supplied from a large
storage tank containing untreated water from a local reservoir (Wellesbourne).
This storage tank was refilled each week with water from the reservoir. An
overflow pipe was used to maintain the water level, and the water from this was
pumped back to the tank containing the headwater via an overflow sump (50
liter). Sand was loaded into the filter column through four inspection hatches
fitted to the front of each column. The sand used in all experiments was a finely
washed plasterer’s sand sourced in southwest Hampshire, United Kingdom (New
Milton Sand and Ballast Co., New Milton, Hampshire, United Kingdom). This
sand met the recommendation of Visscher et al. (63) for drinking water filter
sands and contained ⬍1% (by weight) calcium carbonate and had an effective
size of 0.30 mm and a uniformity coefficient of 1.87. Prior to use, the sand was
autoclaved at 120°C for 20 min and reautoclaved 48 h later and the pipework was
cleaned by using 1,000 ␮g of peroxyacetic acid (Jet 5; Hortichem Ltd., Amesbury,
Wiltshire, United Kingdom) liter⫺1. Sand and water samples were removed via
the inspection hatches or a series of ports mounted at 100-mm intervals down the
column. The SSF runs were carried out three times, each with three replicate
SSF columns, and run for a minimum of 4 weeks.
Zoospore removal by SSF. To test the efficacy of the SSF column, an inoculum
of P. cryptogea zoospores was applied to each filter column and the filtrate
(outflow water) was monitored. Zoospore suspensions (104 spores ml⫺1) were
prepared as described by Pettitt and Pegg (44) and a 20-ml inoculum was
introduced into the water immediately above the sand surface through a specially
designed port. Following inoculation, five 1-liter samples of filtered water were
collected from each of the SSF columns (20). Each 1-liter sample was assayed for
the presence of Phytophthora CFU by using the membrane filtration technique of
Ali-Shtayeh et al. (2). Filter pieces from this were placed in universal bottles
containing 5 ml of resuspension medium and placed on a rotary arm flask shaker
2118 CALVO-BADO ET AL.
at medium speed for 5 min. The resuspension medium, containing antibiotics
and fungicides at the same concentration as the modified BNPRA oomycete
selective medium (44), consisted of 0.09% (wt/vol) agar dissolved in sterile
distilled water and benlate (1 mg ml⫺1), pimaricin (1 mg ml⫺1), PCNB (2.5 mg
ml⫺1), rifamycin (1 mg ml⫺1), ampicillin (50 mg ml⫺1), and nystatin (2.5 mg
ml⫺1). One-milliliter aliquots of resuspension medium were pipetted and spread
onto modified BNPRA plates and incubated at 25°C for 24 to 36 h. Once visible
colonies had formed, CFU per plate were counted, and from these, the mean
numbers of Phytophthora (CFU liter⫺1) were calculated.
Sampling from SSF columns. Sand samples from different depths within the
SSF column (top, 1 cm; middle layer, 50 cm; bottom layer, 80 cm) were taken 1,
2, and 4 weeks after starting each SSF run. A sterile metal corer was used to take
samples of ca. 5 g (wet weight) of sand from the columns via sampling ports. For
water samples, 1 liter of water was taken from the storage tank, headwater tank,
and outflow water.
Viable bacterial plate counts. Routinely, 1 g (wet weight) of sand was placed
in 10 ml of sterile water and vortexed for 10 s every 2 to 3 min for 15 min. Water
samples were used directly. A tenfold dilution series of these samples was made
by using sterile water. The dilutions were plated in triplicate on appropriate
media. Bacteria were enumerated on 1/10-strength tryptic soy agar (0.1 TSA;
Oxoid Ltd., Basingstoke, Hampshire, United Kingdom); fungi were enumerated
on potato dextrose agar (Oxoid Ltd.) containing 10 mg of aureomycin ml⫺1; and
Pseudomonas species was enumerated on P1 agar (29). All plates were incubated
at 20°C for 7 days, and plates with between 30 and 200 colonies were counted.
Direct count of fungal spores. Sand samples (1 g [wet weight]) were taken 2
and 4 weeks after the start of two SSF runs from the top, middle, and bottom
layers of the SSF column and mixed with 1 ml of sterile water. The samples were
vortexed for 3 min and centrifuged at 4,000 ⫻ g for 5 min. Numbers of fungal
spores in dilutions of the supernatant were determined by hemocytometer counts
at a magnification of ⫻40.
ATP extraction and measurement. To three replicate 0.5-g (wet weight) sand
samples, 0.5 g of glass beads (0.1-mm diameter; BioSpect Products, Bartlesville,
Okla.) and 500 ␮l of ATP extraction buffer (0.1 M Tris-acetate [pH 7.75]) were
added. The cells within the sample were disrupted by bead beating for 3 min with
a mini-bead beater (BioSpect Products) at homogenizing speed. The samples
were cooled on ice and centrifuged at 5,000 ⫻ g for 5 min. ATP was measured
by using the Enliten assay system (Promega, Southampton, United Kingdom)
and a luminometer from LKB-Wallac (Uppsala, Sweden), model 1250. Relative
light units were converted into ATP concentrations (in micrograms gram⫺1) by
using an internal standard curve.
DNA isolation. DNA was extracted from 1 liter of water and 0.5-g (wet weight)
sand samples. The water samples were filtered (0.22-␮m-pore-size filters; GV-
Durapore Millipore Ltd., Watford, United Kingdom), and the cells were washed
off the filter surface with 5 ml of sterile water. Each sample was then centrifuged
at 13,000 ⫻ g for 20 min, and the pellet was saved. To the cell pellets or the sand
samples, 1 ml of extraction buffer (0.12 M K2HPO4, pH 8.0) and 1/3 of a volume
of 0.1-mm-diameter glass beads (BioSpect Products, Inc.) was added. Each
sample was shaken vigorously for 3 min in a mini-bead beater (BioSpect Prod-
ucts, Inc.). Immediately, 0.1% (wt/vol) sodium dodecyl sulfate was added and the
sample was homogenized and placed on ice for 10 min. One milliliter of phenol
(pH 8.0; Sigma, Cambridge, United Kingdom) was added, and each sample was
centrifuged at 5,000 ⫻ g for 15 min. The supernatants containing the DNA were
recovered, and 1 ml of chloroform-isoamyl alcohol (24:1) was added to the DNA
suspension. After mixing, each sample was centrifuged at 5,000 ⫻ g for 15 min
and the aqueous phase was saved. The samples were precipitated with a 0.6
volume of isopropanol and a 0.1 volume of 5 M NaCl and washed with 70%
(vol/vol) ethanol. The DNA was further purified by using a Geneclean spin kit
(Bio 101, Inc., Nottingham, United Kingdom) according to the manufacturer’s
recommendations. The DNA from each sample was finally eluted in 50 ␮l and
analyzed by agarose gel electrophoresis to estimate the yield. Appropriate dilu-
tions were made for PCR amplifications.
PCR amplification. The V3 region (39) of the 16S rRNA-borne gene between
positions 341 to 534 on the Escherichia coli numbering system was amplified by
PCR. Routinely, 10 to 50 ng of DNA template, 25 pmol of each primer, 20 mM
deoxynucleoside triphosphate mix, 1.25 U of thermostable DNA polymerase, 1⫻
reaction buffer, and 1.5 mM MgCl2 (Advance Biotechnologies, Surrey, United
Kingdom) was used in a final reaction volume of 100 ␮l. The following cycling
conditions were used: one cycle at 95°C for 1 min followed by 35 cycles of 95°C
for 45 s, 55°C for 30 s, and 72°C for 45 s, and a final extension cycle at 72°C for
10 min. PCR products were analyzed by agarose gel electrophoresis to determine
yield and purity.
DGGE. DGGE analysis was performed with a DCode mutation detection
system (Bio-Rad, Hemel Hempstead, Hertfordshire, United Kingdom). Gels of
APPL. ENVIRON. MICROBIOL.
8% acrylamide (37:1 acrylamide-bisacrylamide) were formed between 20 and
70% denaturant, with 100% denaturant defined as 7 M urea and 40% (vol/vol)
formamide (39). Linear denaturant gradients were made by using a gradient
maker (BDH, Lutterworth, Leicestershire, United Kingdom) in a 16-cm gel with
a 1-mm gel width. Normally, 300 to 500 ng of PCR product was loaded onto each
lane of the gel. Gels were run at 60 V for 16.5 h and maintained at a constant
temperature of 60°C in 7 liters of 0.5⫻ TAE buffer (40 mM Tris-acetate and 1
mM EDTA [pH 8.0]). A set of seven laboratory strains (Agrobacterium rhizo-
genes, Arthrobacter polychromogenes, Bacillus subtilis, Burkholderia phenazium,
Paenibacillus amyloyticus, Pseudomonas fluorescens, and Sphingomonas yanoiku-
yae) were used to construct a standard marker for DGGE analysis.
Sequencing of DGGE bands. Selected DGGE bands were excised from the gel
with a sterile blade. The PCR products were cloned into the pGEM-T easy vector
system I (Promega) following the manufacturer’s instructions, and plasmid DNA
was extracted from clones (⬎3) with the Qiagen 8 Ultra Plasmid kit (Qiagen,
West Sussex, United Kingdom). Sequencing reaction mixtures (20-␮l volume)
with the ABI PRISM BigDye terminator cycle sequence ready reaction kit
Downloaded from http://aem.asm.org/ on March 5, 2020 by guest
(Perkin Elmer-Applied Biosystems, Warrington, United Kingdom) and standard
PCR sequencing reaction conditions were used according to the manufacturer’s
recommendations. Products were analyzed on an ABI PRISM 377 DNA cycle
sequencer, and all sequences were edited and assembled by using the DNAstar
SeqMan II sequence analysis package (Lasergene, Inc., Madison, Wis.). Se-
quences were compared to those on the Ribosomal Database Project II (http:
//rdp.cme.msu.edu/html/) (32) by using sequence match and to EMBL DNA data-
base sequences by using FASTA 3.0 (http://www.ebi.ac.uk/embl/index.html) (56).
Statistical analysis. For bacterial and Pseudomonas fungal populations, CFU
data from all three depths for all three SSF runs and their replicate SSF columns
were used in an analysis of variance. For ATP contents and spore counts, data
from all three depths from only two SSF runs and their replicate SSF columns at
weeks 2 and 4 were analyzed. The significance of differences among treatment
means was determined by using Duncan’s multiple range tests and a P value of
0.05.
The DGGE banding patterns were scored manually, and a binary matrix was
made based on the presence (1) or absence (0) of the bands. The binary data
representing the banding patterns were used to generate a distance matrix by
using the Dice coefficient (42). The distance matrix was analyzed by using clas-
sical multidimensional scaling (11) and Sammon’s nonlinear mapping (52) and
used to construct a dendrogram by cluster analysis with the UPGMA (un-
weighted pair group mean average) linkage method (54). Multidimensional
scaling and Sammon’s nonlinear mapping gave representations of the interband
distance in a two-dimensional space, approximating the corresponding interband
distance in the original space. The maps were used to visualize and interpret
relative spatial and temporal changes in the bacterial community structure be-
tween groups. More-complex self-organized maps can also be used for interpret-
ing the data generated from rapid profiling techniques, such as DGGE, and are
particularly useful for large and variable data sets (15). However, for the visu-
alization of relatively small and structured data sets, such as those presented in
this paper, the more-conventional mapping and clustering techniques were pre-
ferred. The statistical significance of differences in structure between groups,
based on the original distance matrix, were assessed by comparing the mean
distances between groups to the within-group distance distribution. Between-
group distances were considered significant at the 5% level if they were greater
than the mean within-group distance plus t0.05[n] standard deviations of the
within-group distance, where t0.05[n] is the appropriate critical value of the t
distribution on n degrees of freedom.
Nucleotide sequence accession number. All sequences from excised DGGE
bands have been deposited in the EMBL database under accession no. AJ536093
to AJ536099.
RESULTS AND DISCUSSION
SSF efficacy. SSF efficacy was determined by testing for the
removal of P. cryptogea zoospores from the water passing
through the column. At the start of all three SSF runs, the
removal of zoospores was ⬍30%, but by week 3, all filters were
achieving 100% removal of zoospores (Table 1). In the second
run, filtration was continued for 64 days, during which time full
efficacy was maintained from day 18 onwards. In the first 4
weeks of SSF operation, little change in water head loss was
observed (maximum ⫽ 103 mm). There was no evidence of
VOL. 69, 2003 MICROBIAL ANALYSIS OF SSFs 2119

TABLE 1. Development of SSF efficacy as indicated by removal The sand had a low number of microorganisms as it was
rates of applied P. cryptogea zoospores loaded into the SSF columns (8.8 ⫻ 104 CFU of bacteria g⫺1,
Time of Zoospore count 233 CFU of pseudomonads g⫺1, and 123 CFU of fungi g⫺1). It
Run no. SSF run (CFU liter⫺1) ina: % Removal was rapidly colonized by microorganisms, by the end of the first
(days) Headwater Outflow water week, the sand appeared to be fully colonized, and these pop-
ulations remained relatively constant during the 4-week study
1 0 221 (31.8) 204 (25.5) 8
7 317 (22) 130 (14.6) 59 (Fig. 2). The bacterial population in the top of the filter was
21 289 (5.9) 0 100 significantly higher than that of the middle and bottom layers
at week 1 (Fig. 2A). This difference was small and not apparent
2 0 302 (12.7) 236 (45.1) 22
5 306 (28.4) 33 (10.3) 89 at week 2 or 4 and may simply reflect the natural variation seen
10 255 (33.3) 9 (7.0) 97 within the system. The number of colonies growing on Pseudo-
18 300 (15.7) 0 100 monas selective media was lower than that recorded on 1/10-
28 399 (42.0) 0 100 strength TSA and showed significant differences between lay-
40 380 (13.0) 0 100
64 359 (32.7) 0 100 ers at weeks 1 and 2 but not at week 4. Lower numbers of

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

3 0 430 (25.8) 370 (32.1)
7 368 (27.6) 46 (14.8)
28 394 (28.9) 0 100
a
Standard deviations are shown in parentheses; n ⫽ 3.
increased retention times for zoospores or cysts passing
through the columns, with the majority being isolated from the
first of the five 1-liter samples collected from each column at
each sampling time. The removal of zoospores by primed fil-
ters highlights the efficiency of SSF for the control of this plant
pathogen. As zoospores are key to the spread of disease in
water, SSF would result in disease suppression within this type
of growing system. Although these results indicate efficacy
against Phytophthora zoospores, the processes involved in re-
moval are not fully understood. The action of SSF is consid-
ered to rely on a combination of physical, physicochemical, and
biological processes (24, 62, 69). The results in Table 1 agree
with these findings and demonstrate that a clean SSF unit is
inefficient at removing zoospores. However, good zoospore
removal is coincident with the development of an active mi-
crobial population. SSF have been successfully used to remove
propagules of a wide range of plant pathogens from contami-
nated irrigation water, including Cylindrocladium spp., Fusar-
ium spp., Phytophthora spp., Pythium spp., Thielaviopsis spp.,
Verticillium dahliae, Xanthomonas spp., tobacco mosaic virus,
and pelargonium flower break virus (3, 61). Biological pro-
cesses are known to be central to the removal of plant patho-
genic Xanthomonas spp. (6), and the same is likely to be true
for the removal of fungal zoospores. The structure of the SSF
provides a large surface area that is readily colonized by mi-
croorganisms that may be responsible for zoospore removal.
Dynamics of the total microbial population. The sizes of the
bacterial populations of the storage tank and the outflow water
samples during the study were significantly different from each
other. The water from the storage tank showed 3 to 4 times
higher bacterial populations (900 CFU of pseudomonads ml⫺1
and 2.9 ⫻ 105 CFU of total bacteria ml⫺1) compared to the
outflow water (300 CFU of pseudomonads ml⫺1 and 7.2 ⫻ 104
CFU of total bacteria ml⫺1). A significant difference in the
total fungal population between the storage tank and outflow
water samples was also observed. Fungi were detected in the
water from the storage tank at a level of 3 ⫻ 103 CFU ml⫺1,
and there was a 30-fold reduction in this fungal population in
the outflow water to 100 CFU ml⫺1.
bacteria (with the same population pattern described previ-
14 ously for TSA) were observed by direct counting of bacteria
88
with acridine orange staining (data not shown). It is possible
that most of the microbial communities present within the SSF
are tightly attached to the sand grains within biofilms and that
lower numbers exist as free-living bacteria, and these are
counted. Previous studies, where scanning electron microscopy
was used to examine the sand grains in a SSF, showed the
existence of discrete colonies of bacteria on the sand grain
surface (6, 22).
The fungal population was significantly higher in the top of
the filter compared to the middle and bottom samples on all
weeks by plate counting (Fig. 2C). No CFU of fungi were
detected in the bottom layer during the first week in all three
SSF runs. After 4 weeks, the fungal population in the top layer
(1.4 ⫻ 105 CFU g⫺1 [dry weight]) of the filter was at least 40
times and 130 times higher than that observed at the middle
(3.6 ⫻ 103 CFU g⫺1 [dry weight]) and in the bottom (1 ⫻ 103
CFU g⫺1 [dry weight]), respectively. Similar results were ob-
served from direct counts of the fungal spores at different
depths of the SSF columns (Fig. 2D). The number of spores in
the middle (3.5 ⫻ 104 spores g⫺1 [dry weight]) and bottom
layers (2.1 ⫻ 104 spores g⫺1 [dry weight]) were significantly low-
er than those in the top layer (1.6 ⫻ 106 spores g⫺1 [dry weight]).
These results show that large microbial populations develop
in the top of the sand filter within 1 week of setting it up, and
Pseudomonas species seem to dominate the culturable bacte-
rial population. This higher population at the top of the filters
may be related to the higher concentration of particulate or-
ganic carbon known to accumulate in the upper layers of SSFs
(13).
ATP content. The ATP contents at different depths in the
SSFs were determined as an indicator of microbial activity.
Statistically significantly higher levels of ATP were detected at
the top of the filter bed (0.1296 mg g⫺1 [dry weight]) than in its
middle (0.0036 mg g⫺1 [dry weight]) or at the bottom (0.0038
mg g⫺1 [dry weight]) (Fig. 3). At each sampling location, no
significant differences in the ATP content with time was de-
tected between 2 and 4 weeks. Using these measurements, the
top of the sand column had almost 30 times greater ATP levels
than the other two layers. ATP has been used as a relevant
biochemical indicator for estimation of microbial biomass and
metabolic activity in soil for more than 2 decades (28). If total
bacterial and fungal counts on laboratory media are represen-
tative of the populations present, then these results would
2120 CALVO-BADO ET AL. APPL. ENVIRON. MICROBIOL.

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

FIG. 2. Dynamics of the microbial community on selective media from the top, middle, and bottom layers of the SSF over a 4-week period.
(A) Total bacterial population on 0.1 TSA medium; (B) Pseudomonas population on P1 medium; (C) total fungal population on potato dextrose
agar medium; (D) direct counts of fungal spores 4 weeks after loading. Bars represent treatment means (⫾ standard errors), and different letters
above the bars indicate that values differed significantly at a P value of ⬍0.05.

indicate that the cells in the top layer of the SSF have the
greatest activity.
Since the greatest retention of what little organic matter
there is within the reservoir water is likely to be in the top layer
of the sand column, it is not surprising that this area has the
greatest microbial activity. However, since nonculturable bac-
teria could dominate the top fraction to a greater extent, these
results may just indicate a larger total microbial biomass within
this area. Attempts have been made to evaluate microbial
populations of SSFs by using methods such as particulate or-
ganic carbon, bacterial counts, nitrogen content of volatile
solids, acriflavine direct cell count, growth on selective R2A
medium, and bacterial respiration levels (13, 19, 21), and our
results are in general agreement with these. However, in this
study, we have demonstrated that the majority of biological
activity occurred in the top layer of the sand column. Biochem-
ical analyses which used BIOLOG (sole-carbon-source utiliza-
tion pattern) and phospholipid fatty acid analysis of sand filters
have also shown that the microbial community present in the
top of the sand column utilized the biodegradable dissolved
organic carbon more quickly than the communities pres-
ent in the lower depths (37). Nevertheless, the phospholipid
fatty acid profiles showed that the communities were closely
grouped taxonomically, regardless of filter depths (38).
Bacterial community structure based on DGGE analysis.
Analysis of the V3 region of the 16S rRNA genes within the
microbial population by PCR DGGE showed changes in the
structure of the bacterial community. PCR amplification of
DNA from different extractions, and samples from different
replicate columns with different dilutions (1:10, 1:100, and
1:1,000), followed by DGGE separation showed a highly re-
producible profile, indicating the accuracy of the molecular
technique (data not shown). Overall, the replicate samples
showed a variation in the DGGE banding pattern between 2 to
4%. Major changes in the microbial structure were observed
during the first week of the experimental work (Fig. 4). During
the experiment, the number of bands within the DGGE pro-
files was relatively constant. In week 1, between 31 and 35
bands were detected in the three depths, and with time, the
bands tended to remain and increase slightly (faint DGGE
bands difficult to detect) but some became more prominent
within the profile. By week 4, 41 of the bands were from the
top, 33 bands were from the middle, and 31 bands were from
the bottom fraction of the sand column, of which only 24 bands
VOL. 69, 2003 MICROBIAL ANALYSIS OF SSFs 2121

represent the dominance of particular bacteria within the sys-

tem. Bruggemann et al. (7) support the contention that profiles
of bacterial communities generated by PCR-based methods
are a reasonable estimation of dominant in situ community
structure. However, this may not always be the case, and some
element of bias is likely (7, 64), as there will be with any
method applied to such diverse samples. Since the PCR
DGGE profile is obtained directly from the filter sample, it
provides one of the best direct measurements of the total
bacterial population. The sand used to fill the columns showed
a DGGE profile of 24 bands. Some of these bands do not
appear after 1 week of the SSF operation and may represent
bands derived from DNA remaining from the killed bacterial
population. The DGGE banding pattern within the sand itself

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

FIG. 3. ATP content at top, middle, and bottom layer of the SSF
at weeks 2 and 4 after loading. Bars represent treatment means
(⫾ standard errors), and different letters above the bars indicate that
values differed significantly at a P value of ⬍0.05.
were common within the sand column, with between 2 and 5
unique bands for each depth. With time, the top layer of the
sand column showed the greatest changes in band intensity and
diversity. If it can be assumed that the intensity of the DGGE
band represents the relative abundance of a particular species
in the population (17, 27), then the bands within the profile
(before filling the sand columns), the storage tank, and outflow
water samples were less complex and different from the total
sand column populations. A slightly higher number of bands (5
DGGE bands) was observed in the water from the storage tank
compared to the outflow water (Fig. 5). Similar results were
found during the recycling of nutrient solutions through a sand
filter in a glasshouse cropping situation (47). However, when
the bands from within the water from the storage tank and
original sand samples were compared by addition, bands rep-
resenting all those seen in the samples from the SSF columns
could be detected. This is not surprising since it would be
expected that colonization of the filter would be from bacteria

FIG. 4. Ethidium bromide-stained DGGE gel (negative image) showing the 194-bp PCR-amplified fragment of the 16S rRNA genes (V3
region) from a spatial and temporal analysis of the bacterial community from three different depths in the SSF over a 4-week period after loading.
Lanes: 1, original sand; 2, 5, and 8, top; 3, 6, and 9, middle; 4, 7, and 10, bottom; 11, storage tank; 12, headwater. Samples were taken in weeks
1 (lanes 2 to 4), 2 (lanes 5 to 7), and 4 (lanes 8 to 10). Lane M represents a bacterial marker containing P. fluorescens (a), S. yanoikuyae (b), B.
subtilis (c), B. phenazium (d), P. amyloyticus (e), A. rhizogenes (f), and A. polychromogenes (g). ‹ indicates the DGGE bands selected for cloning
and sequencing (SSF-1 to SSF-7). These bands were excised from the gel from the top (SSF-1 and SSF-2) or middle and bottom layers (SSF-3,
SSF-4, SSF-5, SSF-6, and SSF-7) of the SSF from samples at week 4.
2122 CALVO-BADO ET AL. APPL. ENVIRON. MICROBIOL.

20% of the DGGE bands were common between the direct

and culturable fractions from the SSF column. This indicates
that the majority of bacteria grow slowly on the media and do
not dominate the culturable biomass or that they may be un-
culturable on the media used. Four to six major bands were
observed from all the depths on 0.1 TSA or P1 medium. The
banding pattern on P1 medium was similar at all the depths
except for one band missing in the top layer. This indicates that
the culturable pseudomonad population from all areas of the
sand filter was essentially the same. Greater variation in the
DGGE patterns from 0.1 TSA media was observed with some
similarity in DGGE banding patterns from the middle and
bottom layers. These results confirm that PCR DGGE is a
powerful culturability-independent technique for monitoring

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

bacterial populations (25).
Classical multidimensional scaling and Sammon’s nonlinear
mapping were applied to the complex DGGE banding patterns
in order to provide two-dimensional visual representations of
overall changes (spatial and temporal) in the structure of the
microbial community. The map produced from classical mul-
tidimensional scaling proved to be a poor representation of the
original distance data (stress ⫽ 0.16), whereas Sammon’s non-
linear mapping (Fig. 7) proved to be a good representation of
the original data (stress ⫽ 0.07). In Fig. 7, replicate samples

FIG. 5. Ethidium bromide-stained DGGE gel (negative image)

showing the 194-bp PCR-amplified fragment of the 16S rRNA genes
(V3 region) from the storage tank and outflow water over a 4-week
period after loading. Lanes: 1 to 3, storage tank; 4 and 5, outflow water.
Samples were taken from the storage tank in weeks 1 (lane 1), 2 (lane
2), and 4 (lane 3) and from outflow water in weeks 2 (lane 4) and 4
(lane 5) after loading. Lane M represents a bacterial marker contain-
ing P. fluorescens (a), S. yanoikuyae (b), B. subtilis (c), B. phenazium
(d), P. amyloyticus (e), A. rhizogenes (f), and A. polychromogenes (g).

present within these samples. The results therefore indicate

that a diverse bacterial population develops within the sand
column. The banding pattern also indicates little change down
the sand column and, hence, relatively little stratification of the
populations with depth. This may be expected as continual
movement of water down the column will replenish nutrients
and oxygen and remove waste products from the system (24).
The DGGE profiles from the sand samples in all depths
were different from the profiles taken from bacteria represent-
ing the culturable fraction of the bacterial population. The
DGGE banding patterns obtained from community DNA from
different depths and from culturable bacteria on 0.1 TSA and
P1 agar media were compared with samples from the top,
middle, and bottom layers at week 4 (Fig. 6). The DGGE
analysis showed great variation in the number of bands de-
pending on the agar medium used for enrichment. Based on
the similarity of the denaturing migration distances of the FIG. 6. Ethidium bromide-stained DGGE gel (negative image)
showing the 194-bp PCR-amplified fragment of the 16S rRNA genes
DGGE bands in the gel, most of the bands observed in DGGE (V3 region) from total and culturable bacterial DNA extracted from
banding patterns from SSF were not observed in the DGGE three different depths in the SSF in week 4 after loading. Samples were
from the culturable bacteria on the media used. The DGGE taken from the top (lane 1), middle (lane 2), and bottom (lane 3) of the
bands that were strong in intensity from the culturable bacteria sand column, and bacterial colonies were grown on 0.1 TSA and P1
media, respectively, from the top (lanes 4 and 5), middle (lanes 6 and
showed a faint DGGE band in the SSF profiles, indicating that 7), and bottom (lanes 8 and 9). Lane M represents a bacterial marker
they represent a minority species within the bacterial popula- containing P. fluorescens (a), S. yanoikuyae (b), B. subtilis (c), B. phena-
tion in the sand column. It was estimated that less than 10 to zium (d), P. amyloyticus (e), A. rhizogenes (f), and A. polychromogenes (g).
VOL. 69, 2003 MICROBIAL ANALYSIS OF SSFs 2123

with phylogenetic-based analysis software. The sequences pre-

sented were obtained from clones of extracted bands as a
full-length double-stranded sequence could not be obtained
from the product, since ca. 50 bp from each end is lost in direct
sequencing in both directions, resulting in only 50% coverage
of a 200-bp product. To determine if each band represented
one product, a number of clones were investigated, and only
one sequence was obtained, which is presented. Sequencing of
the selected DGGE bands and a comparison to the database
indicated that each band had the most similarity to bacterial
16S rRNA genes from groups such as Bacillus megaterium,
Cytophaga, Desulfovibrio, Rhodococcus rhodochrous, Sphingo-
monas, and one environmental clone. Similar sequences to the
microorganisms described here have been found in water from

Downloaded from http://aem.asm.org/ on March 5, 2020 by guest

sea or lake and sediment environments. Cytophaga spp. are
bacteria common in soil and fresh seawater environments and
are well known for the production of cellulase, amylase, and
chitinase (9, 30, 60). Rhodococcus ruber is well known for the

FIG. 7. Two-dimensional Sammon map of the DGGE banding pat-

terns from all the SSF runs and their replicate SSF columns (a, b, c, d,
and e). Labels represent bacterial communities at the top (T), middle
(M), and bottom (B) layers of the SSF at 1, 2, 3, and 4 weeks after load-
ing and original sand (S), storage tank (ST), headwater tank (HW),
and outflow water (OFW).

within an experimental treatment are grouped together, em-

phasizing the fact that variation between replicate samples
within experimental treatment groups proved to be consider-
ably lower than variation between treatment groups. The sta-
tistical significance of differences in structure between treat-
ment groups, based on the original distance matrix, showed
that the only groups that did not differ at the 5% level were the
middle (M1) and bottom (B1) at week 1. The Sammon map
illustrates the proximity of the M1 and B1 groups and shows
the other treatment groups as distinct clusters. As a compar-
ative approach, a dendrogram (Fig. 8) was built from the UP-
GMA algorithm and showed similar clustering to the Sammon
map. With time, populations from the sand column changed,
indicating shifts within the microbial populations. Clearly, bac-
terial populations in the top, middle, and bottom of the col-
umns did develop in different groups even though there were
dominant bands common to all samples. To our knowledge,
this experimental study is the first where DGGE has been
applied to analyze spatial and temporal changes in the micro-
bial community in SSFs.
DGGE band characterization. Six dominant bands at differ-
ent gradient levels were present within the bacterial DGGE
profile from all the depths within the sand column at week 4.
These bands were selected for further studies (Fig. 4), as they
would provide information on the types of bacteria dominating
the sand column at all levels. A comparison of the sequence of FIG. 8. Dendrogram derived from a distance analysis of bands and
bands to those within databases revealed homology to charac- the UPGMA method of clustering to show relationships between
terized strains. The results are given in Table 2. Since only a DGGE patterns from all the SSF runs and their replicate SSF columns
(a, b, c, d, and e). Labels represent bacterial communities at the top
part of the 16S rRNA gene is sequenced (ca. 194 bp), the data (T), middle (M), and bottom (B) layers of the SSF at 1, 2, 3, and 4
are only used as an indicator of the types of bacteria present weeks after loading, and original sand (S), storage tank (ST), headwa-
rather than an accurate affiliation of the band to a species level ter tank (HW), and outflow water (OFW).
2124 CALVO-BADO ET AL.
TABLE 2. Sequence similarities of partial 16S rRNA clones from selected DGGE bands from the SSF
DGGE band
Group of bacteriaa
(EMBL accession no.)
SSF-1 (AJ536093) Cytophaga group I
SSF-2 (AJ536094) Cytophaga group I
SSF-3 (AJ536095) R. rhodochrous
SSF-4 (AJ536096) B. megaterium
SSF-5 (AJ536097) Sphingomonas
SSF-6 (AJ536098) Desulfovibrio
SSF-7 (AJ536099) Environmental clone S027
a
Sequences were matched to the closest relative from the Ribosomal Database Project (33).
b
Sequences were matched to the closest relative from the EMBL DNA database (63).
degradation of a wide range of organic pollutants and can
catalyze many useful biotransformations (5, 10, 12, 14). An
additional DGGE PCR band within the sand column had se-
quence similarity to Legionella cherrii. A detailed study on this
group of bacteria in the inflow water, outflow water, and sand
column was carried out and is reported separately. Some of
these microorganisms might exhibit antagonistic properties
against fungal plant pathogens by the production of fungal cell
wall degrading enzymes or extracellular compounds that influ-
ence the retention or immobilization of the fungal spores in
the sand bed (62). In addition, the enrichment of a pathogen
such as Legionella within an SSF system is undesirable, and
methods to induce a natural suppressive population without
encouraging the growth of this pathogen would be an advan-
tage. The introduction of suppressive bacteria into an SSF may
provide good fungal pathogen control and prevent coloniza-
tion by Legionella species. Already, several microbial antago-
nists have been tested as biological agents in horticulture crops
including species of Bacillus, Pseudomonas, and Trichoderma
(16, 31, 33, 43, 59, 68); however, these have had variable suc-
cess (67, 68). Bacteria isolated from an SSF, tested for sup-
pressive activity, and then reintroduced into the system may
provide the best option to this problem in the long term. Identi-
fying bacterial groups associated with PCR DGGE bands could
allow the development of selective media to isolate groups indi-
vidually from the filter. This information could also be used to
directly relate isolates back to the composition within the filter.
This study describes the characterization of the performance
and microbial composition of SSFs used for the treatment of
water for use in the horticultural industry. Although there is
still uncertainty concerning the mechanisms involved in SSF
functioning against fungal plant pathogens, the results pre-
sented here clearly show that fungal zoospores are efficiently
removed during passage of water through the SSF and that this
is concurrent with the development of a highly active microbial
population. This study has also shown that the groups of bac-
teria that colonize the SSF and are detected by PCR DGGE
are maintained after 1 week of operation. The bacteria de-
tected may be associated with the removal of zoospores. Cer-
tainly, increasing pressure has been placed on the horticulture
industry worldwide to minimize the use of chemicals to control
pathogens and to recycle water. Utilization of a naturally sup-
pressive population of microorganisms either directly or by
manipulation of the environment in an SSF may provide a
more reproducible control method. Further characterization
APPL. ENVIRON. MICROBIOL.
Most closely related bacterial sequence (EMBL accession no.)
(% similarityb)
Uncultured crater lake bacterium (AF316797) (90.5)
Cytophaga sp. (AF235126) (91.1)
R. ruber, R. rhodochrous, gram-positive bacterium B1G-1B (AF439261,
RR16SR6, AF323255) (100)
Bacillus infernus (BI20384) (99.5)
Porphyrobacter tepidarius, Blastomonas natatoria, Lutibacterium
anuloederans (AB033328, AB024288, AY026916) (100)
Uncultured eubacterium (UEA005994) (98.8)
Uncultured bacterium (AF245330) (99.4)
Downloaded from http://aem.asm.org/ on March 5, 2020 by guest
of the bacterial, fungal, and protozoan populations in many
different sand columns is desirable.
ACKNOWLEDGMENTS
This work was funded by the Department for Environment, Food
and Rural Affairs DEFRA (London, United Kingdom) projects
HH1751 and HH3207.
We thank the nursery and technical staff at HRI and Julie Jones for
statistical analysis of the data.
REFERENCES
1. Ali-Shtayeh, M. S., and J. D. MacDonald. 1991. Occurrence of Phytophthora
species in irrigation water in the Nablus area (West Bank of Jordan). Phy-
topathol. Mediterr. 30:143–150.
2. Ali-Shtayeh, M. S., J. D. MacDonald, and J. Kabashima. 1991. A method for
using commercial ELISA tests to detect zoospores of Phytophthora and
Pythium species in irrigation water. Plant Dis. 75:305–311.
3. Berkelmann, B., W. Wohanka, and G. Wolf. 1994. Characterisation of the
bacterial flora in recirculating nutrient solutions of a hydroponic system with
rockwool. Acta Hortic. 361:372–381.
4. Bewley, W. F., and W. Buddin. 1921. On the fungus flora of glasshouse water
supplies in relation to plant disease. Ann. Appl. Biol. 8:10–19.
5. Bock, C., R. M. Kroppenstedt, and H. Diekmann. 1996. Degradation and
bioconversion of aliphatic and aromatic hydrocarbons by Rhodococcus ruber
219. Appl. Microb. Biotechnol. 45:408–410.
6. Brand, T., and W. Wohanka. 2000. Importance and characterization of the
biological component in slow filters. Acta Hortic. 554:313–321.
7. Bruggemann, J., J. Stephen, Y.-J. Chang, S. J. Macnaughton, G. A. Kowal-
chuk, E. Kline, and D. C. White. 2000. Competitive PCR-DGGE analysis of
bacterial mixtures an internal standard and an appraisal of template enu-
meration accuracy. J. Microb. Methods 40:111–123.
8. Burman, N. P. 1978. Slow sand filtration. H2O 11:348–351.
9. Casamayor, E. O., H. Schäfer, L. Bañeras, C. Pedrós-Alió, and G. Muyzer.
2000. Identification of and spatio-temporal differences between microbial
assemblages from two neighboring sulfurous lakes: comparison by micros-
copy and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol.
66:499–508.
10. Chaineau, C. H., J. Morel, J. Dupont, E. Bury, and J. Oudot. 1999. Com-
parison of the fuel oil biodegradation potential of hydrocarbon-assimilating
microorganisms isolated from a temperate agricultural soil. Sci. Total Envi-
ron. 277:237–247.
11. Chatfield, C., and A. J. Collins. 1996. Introduction to multivariate analysis.
Chapman and Hall, London, United Kingdom.
12. Chauvaux, S., F. Chevalier, C. Le Dantec, F. Fayolle, I. Miras, F. Kunst, and
P. Beguin. 2001. Cloning of a genetically unstable cytochrome P-450 gene
cluster involved in degradation of the pollutant ethyl tert-butyl ether by
Rhodococcus ruber. J. Bacteriol. 183:6551–6557.
13. Collins, M. R., T. T. Eighmy, J. M. Fenstermacher, and S. K. Spanos. 1992.
Removing natural organic matter by conventional slow sand filtration. J. Am.
Water Works Assoc. 84:80–90.
14. Colquhoum, J. A., S. C. Heald, L. Li, J. Tamaoka, C. Kato, K. Horikoshi, and
A. T. Bull. 1998. Taxonomy and biotransformation activities of some deep-
sea actinomycetes. Extremophiles 2:269–277.
15. Dollhopf, S. L., S. A. Hashsham, and J. M. Tiedje. 2001. Interpreting 16S
rDNA T-RFLP data: application of self-organizing maps and principal com-
ponent analysis to describe community dynamics and convergence. Microb.
Ecol. 42:495–505.
16. Duijff, B. J., P. A. H. M. Bakker, and B. Schippers. 1994. Suppression of
VOL. 69, 2003
Fusarium-wilt of carnation by Pseudomonas putida WCS358 at different
levels of disease incidence and iron availability. Biocontrol Sci. Technol.
4:279–288.
17. Duineveld, B., A. S. Rosado, J. D. van Elsas, and J. A. van Veen. 1998.
Analysis of the dynamics of bacterial communities in the rhizosphere of the
chrysanthemum via denaturing gradient gel electrophoresis and substrate
utilization patterns. Appl. Environ. Microbiol. 64:4950–4957.
18. Duncan, A. 1988. The ecology of slow sand filters, p. 163–180. In H. J. D.
Graham (ed.), Slow sand filtration. Horwood, Chichester, United Kingdom.
19. Eighmy, T. T., M. R. Collins, S. K. Spanos, and J. Fenstermacher. 1992.
Microbial populations, activities and carbon metabolism in slow sand filters.
Water Res. 26:1319–1328.
20. Ellis, K. V. 1986. Slow sand filtration. Crit. Rev. Environ. Control 15:315–354.
21. Ellis, K. V., and M. E. Aydin. 1995. Penetration of solids and biological
activity into slow sand filters. Water Res. 29:1333–1341.
22. Esch, P., and A. Nehrkorn. 1988. Rasterelektronenmikroskopische untersu-
chung über die mikrobielle besiedlung von langsamsandfiltern. Zentbl. Bak-
teriol. Hyg. B 185:569–579.
23. Goldberg, N. P., M. E. Stanghellini, and S. L. Rasmussen. 1992. Filtration as
a method of controlling Pythium root rot of hydroponically grown cucum-
bers. Plant Dis. 76:777–779.
24. Haarhoff, J., and J. L. Cleasby. 1991. Biological and physical mechanisms in
slow sand filtration, p. 19–68. In G. S. Logsdon (ed.), Slow sand filtration, vol.
1. American Society of Civil Engineers, New York, N.Y.
25. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-
independent studies on the emerging phylogenetic view of bacterial diversity.
J. Bacteriol. 180:4765–4774.
26. Huisman, L., and W. E. Wood. 1974. Slow sand filtration. World Health
Organisation, Geneva, Switzerland.
27. Jackson, C. R., E. E. Roden, and P. F. Churchill. 1998. Changes in bacterial
species composition in enrichment cultures with various dilutions of inocu-
lum as monitored by denaturing gradient gel electrophoresis. Appl. Environ.
Microbiol. 64:5046–5048.
28. Karl, D. M. 1980. Cellular nucleotide measurements and applications in
microbial ecology. Microbiol. Rev. 44:739–796.
29. Katoh, K., and I. Kunio. 1983. New selective media for Pseudomonas strains
producing fluorescent pigment. Soil Sci. Plant Nutr. 29:525–532.
30. Laetitia, B., H. Schäfer, F. Joux, C. Courties, G. Muyzer, and P. Lebaron.
2000. Genetic diversity of total, active and culturable marine bacteria in
coastal seawater. Aquat. Microb. Ecol. 23:1–11.
31. Leeman, M., F. M. DenOuden, J. A. van Pelt, C. Cornelissen, A. Matamala-
Garros, P. A. H. M. Bakker, and B. Schippers. 1996. Suppression of Fusar-
ium wilt of radish by co-inoculation of fluorescent Pseudomonas spp. and
root-colonizing fungi. Eur. J. Plant Pathol. 102:21–31.
32. Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. Parker, Jr., P. R. Saxman, R. J.
Farri, G. M. Garrity, G. J. Olsen, T. M. Schmidt, and J. M. Tiedje. 2001. The
ribosomal database project (The RDP-II). Nucleic Acids Res. 29:173–174.
33. McCullagh, M., R. Utkhede, J. G. Menzies, Z. K. Punja, and T. C. Paulitz.
1996. Evaluation of plant growth-promoting rhizobacteria for biological con-
trol of Pythium root rot of cucumber grown in rockwool and effects on yield.
Eur. J. Plant Pathol. 102:747–755.
34. McIntosh, D. L. 1966. The occurrence of Phytophthora spp. in irrigation
systems in British Columbia. Can. J. Bot. 44:1591–1598.
35. McNair, R. D., R. C. Sims, D. L. Sorensen, and M. Hulbert. 1987.
Schmutzdecke characterization of clinoptilolite-amended slow sand filtra-
tion. J. Am. Water Works Assoc. 79:74–81.
36. McPherson, G. M., M. R. Harriman, and D. Pattison. 1995. The potential for
spread of root diseases in recirculating hydroponic systems and their control
with disinfection. Med. Fac. Landbouww. Univ. Gent 60/2b:371–379.
37. Moll, D. M., and R. S. Summers. 1996. Performance, biomass and commu-
nity structure profiles of biological rapid media filters, p. 159–166. In N.
Graham and R. Collins (ed.). Advances in slow sand and alternative biolog-
ical filtration. John Wiley and Sons, Chichester, United Kingdom.
38. Moll, D. M., R. S. Summers, and A. Breen. 1998. Microbial characterization
of biological filters used for drinking water treatment. Appl. Environ. Mi-
crobiol. 64:2755–2759.
39. Muyzer, G., E. C. De Waal, and A. G. Uitterlinden. 1993. Profiling of
complex microbial populations by denaturing gradient gel electrophoresis
analysis of polymerase chain reaction-amplified genes encoding for 16S
rRNA. Appl. Environ. Microbiol. 59:695–700.
40. Myers, R. M., S. G. Fisher, L. S. Lerman, and T. Maniatis. 1985. Nearly all
single base substitution in DNA fragments joined to a GC-clamp can be
detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13:
3131–3145.
41. Myers, R. M., T. Maniatis, and L. S. Lerman. 1987. Detection and localiza-
tion of single base changes by denaturing gradient gel electrophoresis. Meth-
ods Enzymol. 155:501–527.
42. Nei, M., and W. H. Li. 1979. Mathematical model for studying genetic
variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA
76:5269–5273.
43. Ongena, M., F. Daayf, P. Jacques, P. Thonart, N. Benhamou, T. C. Paulitz,
P. Cornélis, N. Koedam, and R. R. Bélanger. 1999. Protection of cucumber
MICROBIAL ANALYSIS OF SSFs 2125
against Pythium root rot by fluorescent pseudomonads: predominant role of
induced resistance over siderophores and antibiosis. Plant Pathol. 48:66–76.
44. Pettitt, T. R., and G. F. Pegg. 1991. The quantitative estimation of Phyto-
phthora cactorum in infected strawberry tissue. Mycol. Res. 95:233–238.
45. Pettitt, T. R., A. R. Finlay, M. A. Scott, and E. M. Davies. 1998. Development
of a system simulating commercial production conditions for assessing the
potential spread of Phytophthora cryptogea root rot of hardy nursery stock in
recirculating irrigation water. Ann. Appl. Biol. 132:61–75.
46. Pittis, J. E., and J. Colhoun. 1984. Isolation and identification of pythiaceous
fungi from irrigation water and their pathogenicity to Antirrhinum, tomato
and Chamaecyparis lawsoniana. Phytopathol. Z. 110:301–318.
47. Postma, J., E. A. van Os, and G. Kritzma. 1999. Prevention of root diseases
in closed soilless growing systems by microbial optimization. Med. Fac.
Landbouww. Univ. Gent 64:431–440.
48. Pottorff, L. P., and K. L. Panter. 1997. Survey of Pythium and Phytophthora
spp. in irrigation water used by Colorado commercial greenhouses. Hortic.
Technol. 7:153–155.
49. Roszak, D. B., and R. R. Colwell. 1987. Survival strategies of bacteria in the
natural environment. Microbiol. Rev. 51:365–379.
Downloaded from http://aem.asm.org/ on March 5, 2020 by guest
50. Runia, W. T. 1994. Elimination of root-infection pathogens in recirculation
water from closed cultivation systems by ultra-violet radiation. Acta Hortic.
361:361–371.
51. Runia, W. T. 1995. A review of possibilities for disinfection of recirculating
water from soilless culture. Acta Hortic. 382:221–229.
52. Sammon, J. W. 1969. A nonlinear mapping for data structure analysis. IEEE
Trans. Comput. C 18:401–409.
53. Shokes, F. M., and S. M. McCarter. 1979. Occurrence, dissemination, and
survival of plant pathogens in surface irrigation ponds in southern Georgia.
Phytopathology 69:510–516.
54. Sneath, P. H. A., and R. R. Sokal. 1973. Numerical taxonomy, p. 230–234.
W. H. Freeman and Company, San Francisco, Calif.
55. Stanghellini, M. E., L. J. Stowell, and M. L. Bates. 1984. Control of root rot
of spinach caused by Pythium aphanidermatum in a recirculating hydroponic
system by ultraviolet irradiation. Plant Dis. 68:1075–1076.
56. Stoesser, G., W. Barker, A. van den Broek, E. Camon, M. Garcia-Pastor, C.
Kanz, T. Kulikova, R. Leinonen, Q. Lin, V. Lombard, R. Lopez, N. Redaschi,
P. Stoehr, M. A. Tuli, K. Tzouvara, and R. Vaughan. 2002. The EMBL
nucleotide sequence database. Nucleic Acids Res. 30:21–26.
57. Strong, S. S., B. K. Behe, C. F. Deeke, K. L. Bowen, and G. J. Keever. 1997.
Cultivar and spacing effects on transmission of Phytophthora parasitica in an
ebb-and-flow subirrigation system. Plant Dis. 81:89–95.
58. Thomson, S. V., and R. M. Allen. 1974. Occurrence of Phytophthora species
and other potential plant pathogens in recycled irrigation water. Plant Dis.
Rep. 58:945–949.
59. Utkhede, R. S., C. A. Koch, and J. G. Menzies. 1999. Rhizobacterial growth
and yield promotion of cucumber plants inoculated with Pythium aphanider-
matum. Can. J. Plant Pathol. 21:265–271.
60. van Hannen, E., G. Zwart, M. P. van Agterveld, H. J. Gons, J. Ebert, and H.
Laanbroek. 1999. Changes in bacterial and eukaryotic community structure
after mass lysis of filamentous cyanobacteria associated with viruses. Appl.
Environ. Microbiol. 65:795–801.
61. van Os, E. A., J. J. Amsing, A. J. van Kuik, and H. Willers. 1999. Slow sand
filtration: a potential method for elimination of pathogens and nematodes in
recirculating nutrient solution from glasshouse-grown crops. Acta Hortic.
481:519–526.
62. van Os, E. A., M. A. Bruins, J. van Buuren, D. J. van der Veer, and H.
Willers. 1997. Physical and chemical measurements in slow sand filters to
disinfect recirculating nutrient solutions, p. 313–327. In Proceedings of the
Ninth International Congress on Soilless Culture, St. Helier, Jersey, Channel
Islands, 12 to 19 April 1996.
63. Visscher, J. T., R. Paramasivam, A. Raman, and H. A. Heijnen. 1987. Slow
sand filtration for community water supply. Planning, design, construction
and maintenance. Technical paper no. 24. International Reference Centre
for Community Water Supply and Sanitation, The Hague, The Netherlands.
64. Watanabe, K., Y. Kodama, and S. Harayama. 2001. Design and evaluation of
PCR primers to amplify bacterial 16S ribosomal DNA fragments used for
community fingerprinting. J. Microbiol. Methods 44:253–262.
65. Weber-Shirk, M. L., and R. Dick. 1997. Biological mechanisms in slow sand
filters. J. Am. Water Works Assoc. 89:72–83.
66. Weber-Shirk, M. L., and R. Dick. 1997. Physical-chemical mechanisms in
slow sand filters. J. Am. Water Works Assoc. 89:87–100.
67. Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizo-
sphere. J. Exp. Bot. 52:487–511.
68. Whipps, J. M., and K. G. Davies. 2000. Success in biological control of plant
pathogens and nematodes by microorganisms, p. 231–269. In G. Gurr and S.
Wratten (ed.), Biological control: measures of success. Klumer Academic
Publishers, The Netherlands.
69. Wohanka, W., and M. Helle. 1997. Suitability of various filter media for slow
filtration, p. 551–557. In Proceedings of the Ninth International Congress on
Soilless Culture, St. Helier, Jersey, Channel Islands, 12 to 19 April 1996.
70. Wohanka, W., H. Luebke, H. Ahlers, and M. Luebke. 1999. Disinfection of re-
circulating nutrient solutions by slow sand filtration. Acta Hortic. 481:539–544.