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Impact of liquid hog manure applications on antibiotic
resistance genes concentration in soil and drainage water in
field crops
Journal: Canadian Journal of Microbiology
Manuscript ID cjm-2019-0343.R2
Manuscript Type: Article
Date Submitted by the Author: 31-Mar-2020
Complete List of Authors: Larouche, Elodie; Research and
Development Institute for the Agri-EnvironmentGénéreux, Mylène;
Research and Development Institute for the
Agri-EnvironmentTremblay, Marie-Ève; Research and Development
Institute for the Agri-EnvironmentRhouma, Mohamed; University of
Montreal, Pathology and microbiologyGasser, Marc-Olivier; Research
and Development Institute for the Agri-EnvironmentQuessy, Sylvain;
University of Montreal, Veterinary medicineCôté, Caroline ;
Research and Development Institute for the Agri-Environment
Keyword: Hog manure, Soil, Drainage water, Tillage practices,
Antibiotic resistance genes
Is the invited manuscript for consideration in a Special
Issue? :Not applicable (regular submission)
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1 IMPACT OF LIQUID HOG MANURE APPLICATIONS ON ANTIBIOTIC
RESISTANCE
2 GENES CONCENTRATION IN SOIL AND DRAINAGE WATER IN FIELD
CROPS
3 Élodie Larouche, Mylène Généreux, Marie-Ève Tremblay, Mohamed
Rhouma, Marc-Olivier Gasser,
4 Sylvain Quessy, Caroline Côté.
5 Mylène Généreux. Research and Development Institute for the
Agri-environment (IRDA), 335
6 Vingt-Cinq East Road, Saint-Bruno-de-Montarville, Quebec,
Canada, J3V 0G7. Email:
7 [email protected]
8 Marie-Ève Tremblay. Research and Development Institute for the
Agri-environment, 2700
9 Einstein Street, Quebec, Canada, G1P 3W8. Email:
[email protected]
10 Mohamed Rhouma. Department of pathology and microbiology,
Faculty of veterinary
11 medicine, University of Montreal, 3200 Sicotte Street,
Saint-Hyacinthe, Quebec, Canada, J2S
12 2M2. Email: [email protected]
13 Marc-Olivier Gasser. Research and Development Institute for
the Agri-environment (IRDA),
14 2700 Einstein Street, Quebec, Canada, G1P 3W8. Email:
[email protected]
15 Sylvain Quessy. Department of pathology and microbiology,
Faculty of veterinary medicine,
16 University of Montreal, 3200 Sicotte Street, Saint-Hyacinthe,
Quebec, Canada, J2S 2M2. Email:
17 [email protected]
18 Caroline Côté. Research and Development Institute for the
Agri-environment (IRDA), 335
19 Vingt-Cinq East Road, Saint-Bruno-de-Montarville, Quebec,
Canada, J3V 0G7. Email:
20 [email protected]
21 Corresponding author: Élodie Larouche. Research and
Development Institute for the Agri-
22 environment (IRDA), 335 Vingt-Cinq East Road,
Saint-Bruno-de-Montarville, Quebec, Canada,
23 J3V 0G7. Phone number: 450-653-7368 extension 313. Email:
[email protected]
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24 Abstract
25 Agricultural practices such as manure applications could
contribute to the spread of antibiotic
26 resistance genes (ARGs) within the environment. The objective
was to assess the impact of some
27 fertilization methods (mineral or manure) and tillage
practices (reduced or conventional) on the
28 presence of ARGs and bacteria in soil and drainage water
under wheat and grain corn crops.
29 Targeted ARGs such as tet(T), sul1, and blaCTX-M-1 genes were
quantified by qPCR in liquid hog
30 manure, soil and water samples. The detection of mcr-1 and
mcr-2 was conducted using
31 conventional PCR. ARGs in control plots were detected despite
the absence of manure,
32 representing an environmental reservoir of resistant
microorganisms. The manure application rate
33 higher than 39m3/ha increased tet(T) and sul1 gene
concentrations in soil for more than 180 days.
34 Tillage practices had no impact on ARG concentrations in soil
and water samples. blaCTX-M-1 genes
35 were only detected in seven water samples in 2016, but no
link was established with the treatments.
36 The mcr-1 and mcr-2 genes were not detected in all tested
samples. This study demonstrated that
37 tet(T) and sul1 gene concentrations increased in soil after
liquid hog manure application as well as
38 in drainage water in the next weeks.
39
40
41
42
43
44 KEYWORDS: Hog manure, soil, drainage water, tillage
practices, antibiotic resistance genes.
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45 Introduction
46 Antibiotic resistance is now recognized globally as a major
public health issue and has raised
47 serious concern among physicians and veterinarians. The
decrease and sometime the loss of
48 antibiotics effectiveness for the treatment of bacterial
infections has led to a significant increases
49 in health care costs worldwide (O’Neill 2016). Antibiotics
are used in humans, animals, and crops
50 for the treatment as well as for the control and prevention
of bacterial infections. Some antibiotics
51 are also used as growth promoters to improve feed efficiency
in food-producing animals. Indeed,
52 tetracyclines, sulfonamides and β-lactams are among the most
common antibiotic families used in
53 pig production in Canada (Pakpour et al. 2012; Brown et al.
2017). Their use also exerted selective
54 pressure on microorganisms and led to the emergence of new
resistant strains (Looft et al., 2012).
55 The number and diversity of antibiotic resistant pathogenic
microorganisms have increased since
56 these compounds were adopted in medicine (Roberts 2005; World
Health Organization 2015).
57 Manure application on agricultural fields may introduce
antibiotic resistant microorganisms in soil
58 (Zhu et al. 2013). Indeed, hog manure contains resistant and
non-resistant microorganisms as well
59 as diverse antibiotics and their degradation products. There
may be genetic exchanges of ARGs
60 between bacteria and these genes can then be spread in the
environment (Frey et al. 2015). Tillage
61 practices are suspected to have an impact on propagation of
ARGs in the environment through
62 drainage water (Garder et al. 2014). However, the
contribution of the environment in the
63 dissemination of ARGs is still not well known (Zhang et al.
2015a).
64 Among ARGs that were found in soils fertilized with organic
fertilizers, there are genes conferring
65 resistance to sulfonamides (sul1, sul2, sul3, sulA),
tetracyclines (tet(A), tetA(P), tetB(P), tet(B),
66 tet(E), tet(G), tet(L), tet(M), tet(O), tet(T), tet(W),
tet(X)), beta-lactams (blaCTX-M-1, blaOXA-20,
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67 blaTEM-71), quinolones (qnr(A)), aminoglycosides (str(B)) and
macrolides lincosamides
68 streptogramins B (MLSB) (erm(A), erm(B), erm(F)) (Heuer and
Smalla 2007; Hartmann et al.
69 2012; Marti et al. 2013, 2014; Zhang et al. 2015a; Wang et
al. 2015). The occurrence of tetracycline
70 resistance genes coding for the NADPH-oxidoreductase leading
to ribosomal protection such as
71 tet(T) genes have been little studied (Marti et al. 2013).
Sulfonamide resistance was evaluated in
72 the environment by detecting sul1, sul2 and sul3 genes coding
for an enzyme dihydropteroate
73 synthase (Sköld 2000; Marti et al. 2013). The sul1 genes are
generally carried by a conjugative
74 plasmid which is included within a class I integron. It makes
it a gene of interest to assess the
75 impact of manure spreading (Gündoğdu et al. 2011; Bueno et
al. 2017; Razavi et al. 2017).
76 Resistance to β-lactams mediated by blaCTX-M-1 gene is the
most prevalent extended-spectrum β-
77 lactamase (ESBL) and is also widespread (Dohmen et al. 2015).
Colistin sulfate is a cationic
78 antibiotic peptide, which is approved for use in pigs in
several countries (Rhouma et al. 2016a,
79 2016c). However, colistin sulfate is not yet approved for use
in food animals in countries such as
80 Canada. This antibiotic was sometimes used under veterinarian
responsibility for the treatment of
81 post weaning diarrhea in pigs in Canada (Rhouma et al.
2016b). Since the first identification, in
82 2015, of a plasmid-mediated colistin resistance gene (mcr-1),
the environment has been
83 incriminated as a potential source of colistin resistance
spread (Schwarz and Johnson 2016).
84 Indeed, mcr-1 was found in Escherichia coli isolates from
animal production, meat, water and
85 vegetables. However, the role of hog manure applications in
the dissemination of colistin
86 resistance genes in agricultural land has not been
investigated yet.
87 Reducing transport of microbial contaminants, which may be
resistant to antibiotics, from the
88 surface soil to agricultural drains is therefore a major
challenge for improving water quality in
89 agricultural watersheds (Jamieson et al. 2002). We
hypothesized that repeated application of liquid
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90 hog manure increases E. coli and enterococci counts as well
as ARG concentrations in soil and
91 that environmental dissemination of these genes is influenced
by agricultural tillage practices. The
92 objective of the current study was: (1) to measure the effect
of fertilization and tillage practices on
93 microbiological quality of soil and drainage water in wheat
crop in 2016 and in grain-corn crop in
94 2017, and (2) to assess the effect of repeated hog manure
applications on the presence of ARGs in
95 soil and drainage water.
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96 Materials and methods
97 Field operations. The experimental site of the current study
is a long-term field set in 1978 at the
98 IRDA research farm located in Saint-Lambert-de-Lauzon,
Quebec, Canada. Crop rotation is grain
99 corn, wheat and canola since 2006, the site being dedicated
to grain corn production before this
100 period. Wheat and grain corn were grown respectively in 2016
and 2017. The 0-20 cm surface
101 texture of soil varies from a silty clay loam and loam,
through a silt loam and clay loam. Since
102 1998, a subsurface drain system was placed at 90 centimeters
below the ground surface, allowing
103 drainage water sampling for each plot individually. Since
spring 2011, half of plots were subjected
104 to reduced tillage, and the other half to conventional
tillage (Figure S1 in supplementary
105 materials). Reduced tillage consisted of superficially
incorporating manure (
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119 incorporation in post-application. Drainage water flow was
measured (Table S4), and weather data
120 were recorded every 10 minutes (air and soil temperature,
precipitation and air humidity).
121 Sample collection of hog manure, soil and drainage water.
Liquid hog manure was collected
122 three times during spreading to measure variability of its
properties. In 2016, soil was sampled in
123 each plot 10 days prior to hog manure application, a few
hours after application, and 116 days
124 after, corresponding to grain corn harvest day. Similarly,
in 2017, soil samples were taken 9 days
125 prior to hog manure application, as well as a few hours and
180 days after spreading. Soil was
126 sampled in each plot at a depth of 0-10 cm, 10-20 cm and
20-40 cm before manure application and
127 at the harvest day for a total of 84 samples per year. A
mixture of 5 sub-samples taken randomly
128 in each plot was made to be representative of the whole plot
area. Drainage water samples
129 associated to each plot were collected every time that water
flow at the end of the drain system
130 could be collected in a 500 ml bottle within a maximum of 2
hours. In 2016, six drainage water
131 sampling campaigns were done for a total of 72 samples. In
2017, four rain events leading to
132 drainage water occurrence were sampled as well as an event
with insufficient water flow in
133 October and another in spring 2018 during snow melt,
totalizing 65 water samples. Each sample
134 was collected aseptically to avoid cross contamination and
to maintain the integrity of the samples.
135 Isolation of E. coli and Enterococcus spp. To measure the
effect of fertilization and tillage
136 practices on microbiological quality of soil and drainage
water, the E. coli and enterococci bacteria
137 have been isolated since they are good indicators of fecal
contamination and frequently carry
138 ARGs. Bacteria were counted following the Quebec
government’s official protocols of CEAEQ
139 MA.700-Ec.BCIG 1.0 for the isolation of E.coli in water
samples and MA.700-Ent 1.0 for the
140 isolation of Enterococcus spp. Based on the previous two
procedures, a protocol was adapted for
141 the isolation of enterococci in soil and hog manure samples.
Fifty grams of hog manure and soil
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142 samples were weighed and diluted in a 0.0003M phosphate
buffer solution. Hog manure samples
143 were filtered with 1, 5 and 10 ml of a 1:1000 dilution. Soil
samples were filtered with 1, 5 and 25
144 ml of a 1:100 dilution. Water samples were not diluted. The
filtered volumes of water were
145 determined according to the turbidity of the sample and
varied between 10 and 150 ml. Enterococci
146 were confirmed with Enterolert* test kit based on IDEXX’s
patented Defined Substrate
147 Technology* (DST*). The volume allowing a count of bacteria
between 20 and 80 CFU per petri
148 dish was used to obtain the final bacterial count (CFU/g of
manure or soil and CFU/100ml of
149 drainage water). They were transformed with logarithm to the
base 10.
150 Genomic DNA extraction. Amounts of 500 mg of soil and 400 mg
of liquid hog manure were
151 weighed to carry out DNA extraction. Water samples were
filtered using a 0.45 μm pore membrane
152 to recover microorganisms and suspended matter. A maximum of
250 ml of water were filtered
153 per membrane. The resulting membranes from manure, soil and
water samples were then placed
154 in extraction kit microtubes. DNA extraction was done with
the Fast DNA Spin Kit for feces and
155 soil combined to the FastPrep® system from MP Biomedicals.
The same soil kit was used to isolate
156 genomic DNA from water samples. Concentration and purity of
DNA extracts were verified with
157 a Tecan Infinite F200 Pro spectrophotometer and 1% agarose
gel migration.
158 Quantification and detection of target genes. Quantification
of tet(T), sul1 and blaCTX-M-1
159 antibiotic resistance genes in DNA extracts was performed
using the Real-Time Polymerase Chain
160 Reaction (qPCR) method. The qPCRs were performed with the
CFX96 thermal cycler and Sso
161 Advanced™ Universal Inhibitor-Tolerant SYBR® Green Supermix
reagents from Bio-Rad.
162 Running of qPCRs was rigorously checked for each gene and
reaction conditions were adjusted as
163 needed. DNA amplification protocol of tet(T), sul1 and
blaCTX-M-1 genes is summarized in Table
164 1. To determine the number of gene copy in DNA extracts, a
standard curve was performed using
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165 known increasing concentrations of the target gene fragment
with an increment of dilutions 1:5.
166 These synthetic gene fragments and primers were obtained
from IDT company (Integrated DNA
167 Technologies, Inc., Iowa, USA) (Table S5). Regarding samples
in 2016, each reaction contained,
168 on average, 9.5 ± 1.1 ng of hog manure DNA, 6.4 ± 2.6 ng of
soil DNA or 7.8 ± 6.7 ng of drainage
169 water DNA extract. Regarding samples of 2017, each qPCR
contained an average number of 2.4
170 ± 0.6 ng hog manure DNA, 7.4 ± 2.7 ng soil DNA or 10.1 ± 8.0
ng of drainage water DNA extract.
171 Each sample was runned in three technical replicates to
confirm the precision of the target gene
172 quantification. A negative control without DNA was carried
out with deionized water. The total
173 volume of each reaction was 25 μl. The limit of detection
(LOD) of qPCR-targeted genes, define
174 as the minimum of gene copy numbers that can be detected
with the qPCR method, were 14 copies
175 per reaction for tet(T), 20 copies/reaction for sul1 and 39
copies/reaction for blaCTX-M-1. The limit
176 of quantification (LOQ), define as the gene copy numbers
that can be quantitatively determined
177 with accuracy and precision, were 357 copies/reaction for
tet(T), 98 copies/reaction for sul1 as
178 well as 195 copies/reaction for blaCTX-M-1. An internal
amplification control (IAC) was also added
179 to ensure that qPCR was not inhibited and that there were no
false negatives. Sequences of IAC
180 primers as well as those of phage lambda synthetic DNA
fragment used as IAC are summarized
181 in Supplementary material (Table S6). The purity and
specificity of amplicons were confirmed
182 with a melting curve as well as with a 3% agarose gel
migration for detection of target genes. It
183 was also possible to determine the number of samples
containing the targeted ARG and to calculate
184 gene prevalences of each sample type. All results were
reported as number of copies per gram of
185 hog manure or wet soil and per ml of filtered water and then
transformed using logarithm to the
186 base 10. In samples taken in 2016, mcr-1 and mcr-2 genes
were evaluated with conventional PCR
187 using a protocol previously described (Liu et al. 2016).
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188 Data analysis and statistical methods. Each factor has been
compared with a statistical analysis
189 for each year separately. Given the multiple variables and
different length of sampling time, the
190 results between the two year under study have not been
compared with a statistical analysis.
191 Association between each factor and prevalence of genes in
samples were verified using
192 contingency tables and Fisher exact test available with SAS
PROC FREQ (version 9.4). For counts
193 of bacteria and resistance genes, a general linear mixed
model with a logarithmic binding function
194 was fitted to count data in order to evaluate the effects of
factors and interactions (Littell et al.
195 2007). Binomial or negative Poisson distribution were
specified, and random effects and repeated
196 measures were considered in the model. Random part of the
model was simplified when
197 convergence was not respected. PROC GLIMMIX procedure of SAS
was used and threshold was
198 set at 0.05.
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199 Results
200 E. coli and enterococci enumeration in liquid hog manure and
soil. The average counts of E.
201 coli in hog manure were higher in 2016 than in 2017 with
respectively 5.0 ± 0.1 and 4.6 ± 0.1
202 Log10 CFU/g. The opposite was observed with the enterococci
counts which were more
203 concentrated in 2017 than in 2016 with respectively 4.8 ±
0.1 and 4.2 ± 0.1 CFU/g of hog manure.
204 The effect of tillage practice on bacterial and ARGs
transport was first analyzed at the soil surface
205 since tillage was done at a maximum depth of 10 cm. In 2016
and 2017, there were more E. coli
206 and enterococci in soil surface (depth of 0-10 cm) after
manure application than the two other
207 sampling dates (before application and harvest) (Figure 1A,
B, C and D). Also, tillage practice
208 did not affect the average bacterial counts in soil (results
presented only in supplementary material,
209 Table S7). There were more bacteria in the 2X manured soil
than in the 1X, and less in the MIN
210 than the other manure rates for the two years under study.
Bacterial counts were equivalent
211 between the MIN plots, although there was a slight increase
of average counts after manure
212 application in the spring. The E. coli and enterococci
counts after harvest in 2016 (116 days post-
213 application) (Figure 1A and C) and 2017 (180 days
post-application) (Figure 1B and D)
214 decreased to counts like those observed prior to
fertilization in the manure-receiving plots, except
215 in all plots in 2016 for enterococci counts and in the 1X
plots in 2017 for E. coli. Although not
216 significant, there was generally more E. coli and
enterococci in soil surface samples than in soil
217 sampled at a depth of 10-20 cm and 20-40 cm (results
presented only in supplementary material,
218 Table S7). Thus, there was no established link between soil
depth and bacteria counts in this study.
219 E. coli and enterococci populations in drainage water. E.
coli populations in drainage water
220 ranged between 0.0 and 3.2 Log10 CFU/100 ml in 2016, 0.0 and
2.3 Log10 CFU/100 ml in 2017,
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221 and between 0.0 and 1.3 Log10 CFU/100 ml in spring 2018. The
December 1, 2016 sampling date
222 was not included in the statistical analysis of E. coli
counts to obtain the convergence of the
223 statistical model. The populations of E. coli in 2016 were
influenced by the sampling date and the
224 fertilization method. Indeed, the average counts of E. coli
in drainage water at the outlet of the
225 drains was higher with 1X manure rate than with MIN in 2016
(Figure 2A). Also, the 2X manured
226 plots contained more E. coli counts in June 7 and June 13,
2016 than in 1X manured plots or MIN
227 control plots. E. coli counts in drainage water decreased
between June 7 and June 13, 2016,
228 corresponding to 19 and 25 days after hog manure
application. For the same sampling date, tillage
229 practice had no effect on bacterial counts in drainage water
regardless of the fertilization method
230 (results presented in supplementary material, Table S8). No
statistical analysis was done for E.
231 coli counts in drainage water in 2017 since average counts
were generally under the LOD of 1
232 CFU/100ml and did not vary between treatments. Counts were
higher on October 16, 2017 than
233 the other sampling dates in only three plots next to each
other. Given the proximity of the plots, it
234 is possible that the contamination was caused by an external
source such as wild animal feces.
235 Enterococci counts in drainage water ranged between 0.0 and
3.0 Log10 CFU/100 ml in 2016
236 (Figure 2B), 0.0 and 2.8 Log10 CFU/100 ml in 2017 (Figure
2C), and 0.7 and 1.9 Log10 CFU/100
237 ml in the spring of 2018. Enterococci counts in water taken
in 2016 and 2017 were influenced by
238 the sampling date. Enterococci counts decreased between June
7 and June 13, 2016. However,
239 there was an increase of enterococci counts in drainage
water on October 21, and a decrease on
240 November 4, 2016, corresponding to 169 days after hog manure
application. In 2017, regardless
241 of other treatments, enterococci counts were different
between all sampling dates. Despite an
242 increase in October 2016 and 2017, there was a decrease in
E. coli and enterococci counts in
243 drainage water samples during the agricultural season,
although enterococci persisted longer. The
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244 sampling date of October 16, 2017 has not been considered in
the statistical analysis as there were
245 seven missing data since some drains had an insufficient
flow to be sampled. Enterococci counts
246 at this date ranged from 2.1 to 2.4 Log10 CFU/100 ml. Counts
were also at their lowest prior to
247 manure application on May 24, 2017. They reached
approximately 2.6 Log10 CFU/100ml in
248 October (the first runoff event of drains after
fertilization in May 2017) and decreased to 1.7 Log10
249 CFU/100ml on November 6, corresponding to 166 days after
manure application. In 2017,
250 drainage water from plots with 1X manure rate and
conventional tillage contained more
251 enterococci (1.4 Log10 CFU/100ml) than those with reduced
tillage practice (1.0 Log10
252 CFU/100ml, p=0.0116) (Table S8). The same statistical
observation was made with the plots
253 fertilized with MIN (1.5 vs 0.9 Log10 CFU/100ml, p=0.0016).
There were more enterococci in
254 drainage water in 2017 from plots fertilized with 2X manure
rate and reduced tillage practice (1.4
255 Log10 CFU/100ml) than those receiving 1X (1.0 Log10
CFU/100ml) or MIN treatments (0.9 Log10
256 CFU/100ml, p=0.0088).
257 Concentration of ARGs in manure and soil. In 2016, average
concentrations of tetracycline
258 tet(T) and sulfonamide sul1 resistance genes in hog manure
were respectively 9.49 ± 2.97 and 8.35
259 ± 3.10 Log10 copies/g of wet soil. In 2017, the average
concentrations of tet(T) and sul1 genes in
260 hog manure were respectively 9.11 ± 3.24 and 8.44 ± 3.38
Log10 copies/g. Indeed, tet(T) and sul1
261 gene concentrations in 2016 and 2017 in manure were
similar.
262 The concentrations of tet(T) and sul1 genes in soil surface
in 2016 and 2017 varied depending on
263 fertilization method and sampling date. Prior to hog manure
application in both years,
264 concentrations of tet(T) genes were similar in plots
receiving 1X, 2X or MIN treatments (Figure
265 3A and B). Also, there were more tet(T) genes in soil
surface fertilized with 1X manure rate than
266 in those fertilized with MIN on May 15, 2017. The sul1 gene
concentrations increased as hog
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267 manure rate increased. Indeed, there was generally more sul1
genes in soil fertilized with 2X
268 manure rate than in those with 1X before manure application
for both years under study (Figure
269 3C and D). A few hours after hog manure application, there
were more tet(T) and sul1 genes in
270 plots receiving 1X or 2X manure rate than in those
fertilized with MIN in 2016 and 2017. In
271 addition, there were more tet(T) genes in plots with 2X
manure rate than in those with 1X in 2016.
272 At the harvest, plots receiving 2X manure rate had higher
tet(T) gene concentrations than the other
273 plots for both years. Even after 116 days in 2016 and 180
days following the manure application,
274 the concentration of tet(T) and sul1 genes were still higher
in 2X plots than MIN control plots. It
275 is possible that the delay between hog manure application
and harvest was not long enough to
276 allow a reduction of gene concentrations to comparable
levels observed in plots receiving MIN
277 (background levels). In 2017, tet(T) and sul1 gene
concentrations in MIN plots increased after hog
278 manure application. Also, at harvest, the average
concentrations returned to levels like those prior
279 to application. At the 2X manure rate, tet(T) gene
concentrations were higher after hog manure
280 application than before spreading or at harvest. Tillage
practice and soil depth did not affect the
281 average concentration of tet(T) and sul1 genes in both years
under study (results presented only in
282 supplementary material, Tables S9-10). There were generally
fewer tet(T) and sul1 genes in
283 deeper soil (10-20 cm and 20-40 cm) than in soil surface
(0-10 cm). Thus, there was no established
284 link between soil depth and ARGs in this study.
285 ARGs transport in drainage water. Since tet(T) gene
concentrations in drainage water in 2017
286 and 2018 were all under the LOQ, ranging from 0.00 to 300.00
copies/ml, no statistical analysis
287 was performed with these data. The sampling date had an
impact on concentration of tet(T) genes
288 in drainage water in 2016 and sul1 in 2017. Indeed, the
tet(T) gene concentrations in drainage
289 water were at its highest on 7 and June 13, 2016, at least
19 days after manure application (Figure
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290 4A). Since June 13, 2016, a significant decrease in
bacterial concentration was observed in
291 drainage water as well as a decrease in tet(T) and sul1
genes. The sul1 gene concentrations were
292 2.28 Log10 copies/ml in 2016 and 2.51 Log10 copies/ml in
2017 in drainage water in October.
293 Concentrations were similar and decreased in the next water
samples taken in November and
294 December as well as E. coli and enterococci counts in water
for both years. On June 13, 2016,
295 gene concentrations in water samples were generally higher
in conventionally-worked plots (2.36
296 for tet(T) and 2.21 Log10 copies/ml for sul1) than those in
reduced tillage (1.56 for tet(T) and 1.67
297 Log10 copies/ml for sul1) (results presented only in
supplementary material, Table S10). The sul1
298 gene concentrations in drainage water were influenced by the
fertilization method in 2016 and
299 2017 (Figure 4B and C). Indeed, there were more genes in
drainage water samples from plots
300 fertilized with 2X manure rate than those with 1X or MIN in
2016, and more genes in plots
301 fertilized with 2X and 1X than those with MIN in 2017. Mean
sul1 gene concentrations were
302 higher on June 7, 2016 and then decreased. There were fewer
sul1 genes in drainage water sampled
303 prior to hog manure application in May 2017 than in water
sampled in the fall of the same growing
304 season.
305 Prevalence of ARGs in environmental samples. The mcr-1 and
mcr-2 genes were below the
306 LOD of the conventional PCR and were not detected on the
agarose gel. The blaCTX-M genes were
307 lower in number than LOD of qPCR method, but beta-lactams
genes were still found on agarose
308 gel in some samples. In 2016, the prevalence of blaCTX-M
genes in drainage water was 9.7% and
309 100% in hog manure, but they were not detected in soil
samples (Table 2). The tet(T) and sul1
310 gene prevalences were mainly influenced by soil depth and
fertilization method. The prevalence
311 of sul1 and tet(T) genes after grain corn harvest in 2017
(respectively 88.9 and 100.0%) was higher
312 than after wheat harvest in 2016 (respectively 66.7 and
86.1%). Plots fertilized with MIN had
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313 slightly lower prevalence than other plots fertilized with
liquid hog manure. Interestingly, sul1
314 genes had high prevalences (75.0 to 100.0%) in all tested
soil samples. In 2016, the prevalence of
315 sul1 genes was higher in soil surface samples (100.0%) than
in the other deeper soil depth (87.5%
316 10-20 cm and 79.2% 20-40 cm). In addition, there was lower
prevalence of sul1 genes in plots
317 fertilized with MIN (75.0%) than with 1X (100.0%) or 2X
(96.4%) manure rates. The prevalence
318 of tet(T) genes in soil samples taken at 20-40 cm (45.8% in
2016 and 70.8% in 2017) were lower
319 than in the two other soil depth (79.2 to 100.0%). In 2016,
there was lower prevalence of tet(T)
320 genes in plots fertilized with MIN (78.6%) than with 1X
(96.4%) or 2X (100%) manure rates.
321 There were almost twice as many sul1 genes in water samples
as tet(T) genes for both agricultural
322 seasons under study. The unfertilized soil plots already
contained tet(T) and sul1 genes prior to
323 manure application. Indeed, the prevalence of sul1 has
increased by 8.3% in 2016 and 5.6% in
324 2017 after manure application, while those of tet(T)
increased by 22.2% in 2016 and 8.3% in 2017.
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325 Discussion
326 The objectives achieved in the current study were to measure
the effect of fertilization and tillage
327 practices on microbiological quality of soil and drainage
water and to assess the effect of repeated
328 hog manure applications on the presence of ARGs in samples.
This research was conducted in
329 2016 in wheat crop while in grain-corn crop in 2017. The
results combining the two years of the
330 study have been globally discussed but they could not be
statistically analysed together given the
331 multiple variables and length of sampling time. Both sul1
and tet(T) genes have been reported in
332 pathogenic bacteria isolated from humans and animals as well
as in the environment (water, soil,
333 plants, swine manure) (Clermont et al. 1997; Antunes et al.
2005; Roberts 2005; Marti et al. 2013).
334 Results of the current study showed that there was an
increase concentration of sul1 and tet(T)
335 genes in soil after the application of liquid hog manure for
the two years of the experiment.
336 Although less significant than in plots receiving manure,
there was also an increase in plots
337 receiving MIN, suggesting that concentrations of
tetracycline- and sulfonamide-resistant
338 microorganisms may have increased in soil at the beginning
of the growing seasons. This may
339 explain, in part, the increase of gene concentrations in
plots fertilized with hog manure. However,
340 at the high rate of hog manure (2X), the concentration of
tet(T) and sul1 genes remained higher
341 until wheat harvest in September 2016 as well as grain corn
in November 2017 than before manure
342 application. A delay of 116 days in 2016 and 180 days in
2017 (grain crop growing seasons in
343 Canada) after hog manure application was not enough to
reduce ARGs in soil at the background
344 levels observed in control plots. Thus, it would be better
to apply hog manure at an agronomic rate
345 (1X) to reduce the risk of spreading these genes across
crops. It is generally recommended to
346 harvest at least 90 to 120 days after manure application to
ensure that human pathogens, such as
347 E. coli and Enterococcus spp., reach undetectable levels
(Bernard et al. 2003; Marti et al. 2014).
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348 Several studies have reported high abundances of ARGs in
soils that have received hog manure
349 for more than one growing season (Knapp et al. 2010;
Hartmann et al. 2012; Garder et al. 2014;
350 Marti et al. 2014; Zhang et al. 2015a; Wang et al.
2015).
351 The presence of ARGs in soil after spreading is impacted by
various factors such as manure source,
352 spreading rate, application time, weather conditions and
soil type. It has been reported that fall
353 spreading tends to increase frequency of genes detection in
soil (Marti et al. 2014). In early spring,
354 the ARGs were less concentrated than at the end of autumn,
although more genes remained in plots
355 fertilized with organic fertilizers than in the control
plots receiving only MIN. Garder et al. (2014)
356 published results showing an increase in erm(B) and erm(F)
genes in silty soil after hog manure
357 application in France, but their abundance after 12 months
decreased to levels equivalent to those
358 observed in control plots that did not receive manure. They
also mentioned that detection of other
359 resistance genes could have led to other findings and
conclusions. Scott et al. (2018) reported an
360 increase in abundance of sul1, str(A), str(B), aad(A),
erm(B) and int1 genes after spreading a pig
361 manure compost on a clay loam soil of Brookston in Ontario
compared to unfertilized soil. This
362 increase in ARGs abundance was significant for at least 5
years after application, further
363 demonstrating long-term effects of organic fertilizer
applications on increased ARGs abundance.
364 In addition, heavy metals and antibiotic residues are added
to soil during manure application.
365 ARGs have been associated with heavy-metal resistance genes,
allowing co-selection of antibiotic
366 resistance (Zhu et al. 2013). In addition, subtherapeutic
levels of antibiotic substances in soil and
367 water exert a selection pressure for gene acquisition by
environmental microorganisms (Baquero
368 et al. 2008).
369 In the current study, prior to manure application in May
2016 and 2017, concentration of sul1 and
370 tet(T) genes was higher in plots fertilized with liquid hog
manure than in MIN-fertilized one. This
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371 higher concentration of genes in manure-fertilized plots
could be explained by the repeated
372 applications of previous years that led to an accumulation
of these genes in soil (Zhang et al.
373 2015a). Marti et al. (2014) reported that sul1 genes were
significantly higher in fertilized soils at
374 79 and 112m3/ha compared to unfertilized soils. Although 2X
rate was twice as low in this study
375 as in that of Marti et al. (2014), there were still more
genes for sulfonamide and tetracycline
376 resistance in these plots. It is normal to observe an
increase in the number of microorganisms and
377 resistance genes in soil and drainage water samples after
hog manure application. This increase in
378 genes concentration should not be attributed exclusively to
addition of ARGs contained in manure,
379 but also to the stimulation of soil microbial populations.
Manure applications as well as mineral
380 fertilizer provide nutrients useful for agricultural crops
to improve soil quality and crop yield, but
381 they also support growth of microorganisms already present
in soil (Larney and Angers 2012;
382 Meen et al. 2014).
383 The detection of genes in soil and water samples from plots
receiving only MIN has led to a better
384 knowledge of their amounts and prevalence when soil has not
been fertilized with organic fertilizer
385 for several years. Thus, this background concentration and
prevalence of ARGs was compared
386 with other plots receiving hog manure. It was found that all
soil samples, regardless of treatment,
387 contained tet(T) and sul1 genes after manure application.
The sul1 genes were found in different
388 soil types that were not always fertilized with organic
fertilizers demonstrating that indigenous soil
389 microorganisms may carry them (Heuer and Smalla 2007; Marti
et al. 2014; Zhang et al. 2015b;
390 Wang et al. 2015). Thus, there is already an established
reservoir of sul1 and tet(T) genes in soil,
391 but the prevalence and concentration of these genes has
increased after hog manure spreading
392 during at least one growing season. Researchers had detected
tet(T) genes in soil fertilized with
393 manure as well as in soil fertilized with organic fertilizer
(Marti et al. 2013). The same research
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394 team also reported that relative abundance of sul1 genes was
significantly higher in soils fertilized
395 with organic fertilizers than in soils fertilized with MIN
for at least one growing season (Marti et
396 al. 2014).
397 In the current study, blaCTX-M-1 genes were under the LOD of
qPCR in all samples. Prevalence of
398 extended-spectrum β-lactamase (ESBL) genes has been
associated with high use of antibiotics
399 such as penicillin and cephalosporin in animal breeding
(Dohmen et al. 2015). Results could have
400 been different depending on the use of antibiotics for pig
rearing. A study in France enabled
401 researchers to detect the blaCTX-M-1 or blaCTX-M-9 and
blaTEM-71 genes in soil of different agricultural
402 land receiving organic fertilizers. This study suggests that
beta-lactam resistance may be caused
403 by other resistance genes (Hartmann et al. 2012). The genes
of mcr-1 and mcr-2 were not detected
404 in liquid hog manure, soil and drainage water samples.
Indeed, these results seem to corroborate
405 the limited use of colistin on Canadian hog farms, primarily
for treatment of post-weaning diarrhea
406 in piglets (Rhouma et al. 2017). Guenther et al. (2017)
identified mcr-1 gene in swine manure in
407 Germany, which was associated with significant use of
colistin on these farms (Guenther et al.,
408 2017).
409 The results of the current study did not demonstrate that
conventional or reduced tillage practices
410 have impacted the bacterial counts and gene concentrations
in soil and in drainage water. Garder
411 et al. (2014) did not demonstrate that tillage practice had
an impact on presence of erythromycin
412 resistance genes in soil and on transport of these genes in
drainage water. The authors also
413 mentioned that other findings and conclusions could have
been observed if other genes have been
414 studied as part of their study. To date, our study is one of
the first to evaluate the impact of tillage
415 practices on transport of ARGs in drainage water. Further
studies will be needed to fully
416 understand relationship between tillage practice and
transport of ARGs in the environment through
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417 agricultural drains considering the characteristics of each
cultivated region (weather, moisture,
418 type of soil). It is known that tillage practice may impact
the movement of water through soil
419 macrospores (Jamieson et al. 2002). In addition, researchers
have indicated that diversity of
420 microorganisms in soil was greater in soil surface when
tillage was reduced (van Groenigen et al.
421 2010).
422 The current study has clearly demonstrated that hog manure
application on soil increased the
423 number of E. coli and enterococci as well as tet(T) and sul1
genes in drainage water. In October,
424 a decrease in bacterial concentration was observed in
drainage water as well as a decrease in tet(T)
425 in 2016 and sul1 genes in both years under study. The
results in soil and drainage water can be
426 explained by reduced number of sulfonamide resistant
microorganisms found in drainage water.
427 In 2016, May and June were relatively wet in
Saint-Lambert-de-Lauzon and precipitation reached
428 144mm in June. It is possible that antibiotic resistant
microorganisms and ARGs have left
429 agricultural soil via runoff rather than drains. However,
preferential transport of water in soil
430 would be particularly active in clay soil during floods
while soil profile is saturated and subjected
431 to surface runoff, thus promoting a relatively rapid flow to
drains by soil macropores (Jamieson et
432 al. 2002). It is know that bacterial counts and resistance
genes in water are influenced by weather
433 conditions (Sura et al. 2016).
434 Liquid hog manure contained high counts of E. coli and
enterococci, so this may explain the
435 increase in counts in soil and drainage water in both years
of culture. Enterococci generally survive
436 better than E. coli in the environment (Bernard et al. 2003;
Marti et al. 2014). Probabilities to
437 recover fecal microorganisms in drainage water depend on
their potential for survival and transport
438 through soil (Jamieson et al. 2002; Unc and Goss 2004).
Counts of indicator microorganisms and
439 fecal contamination such as E. coli generally follow an
exponential decay in soil and may persist
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440 for up to 100 days with initial high bacterial content
manure (Côté and Quessy 2005). This bacteria
441 survival is impacted by a multitude of factors such as
exposure to UV rays, as well as moisture,
442 temperature and microbiological activity of soil, which can
be influenced by tillage practices
443 (Licht and Al-Kaisi 2005). According to Jamieson et al.
(2002), the two most important parameters
444 influencing transport of microorganisms to drains are soil
moisture during application and
445 precipitation occurring within two or three weeks after
spreading. Many enteric microorganisms
446 are released in weeks following application. Also, changes
in soil microflora after organic fertilizer
447 application could have an impact on public health in longer
term, as well as the presence of
448 antibiotic resistant microorganisms in soil and water.
449 Results of this study are to be interpreted in the context
of agricultural lands in the Chaudière-
450 Appalaches region of Quebec (Canada), where rainy weather
prevailed in 2016 and 2017. Marti et
451 al. (2014) hypothesized that climatic conditions may impact
gene concentration. They reported
452 that warm and dry conditions reduce persistence of resistant
bacteria, while cool and wet
453 conditions promote growth of resistant bacteria. Wet weather
conditions at the experimental site
454 may have influenced transport of genes and bacteria in
drainage water, as well as their
455 concentration in soil. In the current study, tet(T) and sul1
gene concentrations increased in soil
456 after liquid hog manure application as well as in drainage
water in the next weeks. According to
457 various agricultural activities, soil could constitute a
reservoir of tetracycline and sulfonamides
458 resistance genes as well as potential resistant
bacteria.
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459 Acknowledgements
460 This study was founded by Ministry of Agriculture, Fisheries
and Food of Quebec (MAPAQ).
461 We thank K. Roseberry, A. Fortin, E. Latour and T. Raymond
for their important technical support
462 in the laboratory during this project. We also thank all the
farm workers and our farm cooperators.
463 We would like to thank Dr. Pascal Sanders and Prof. Dr.
Surbhi Malhorta-Kumar for providing
464 DNA from Escherichia coli harboring respectively mcr-1 and
mcr-2 genes.
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583 Rhouma, M., Fairbrother, J.M., Beaudry, F., and Letellier,
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584 pigs: risk factors and non-colistin-based control
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585 doi:10.1186/s13028-017-0299-7.
586 Roberts, M.C. 2005. Update on acquired tetracycline
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587 245(2): 195–203. doi:10.1016/j.femsle.2005.02.034.
588 Schwarz, S., and Johnson, A.P. 2016. Transferable resistance
to colistin: a new but old threat. J.
589 Antimicrob. Chemother. 71(8): 2066–2070.
doi:10.1093/jac/dkw274.
590 Scott, A., Tien, Y.-C., Drury, C.F., Reynolds, W.D., and
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591 resistance genes in soil receiving composts derived from
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592 food wastes, and evidence for multiyear persistence of swine
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593 Microbiol. 64(3): 201–208. doi:10.1139/cjm-2017-0642.
594 Sköld, O. 2000. Sulfonamide resistance: mechanisms and
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596 doi:10.1054/drup.2000.0146.
597 Sura, S., Degenhardt, D., Cessna, A.J., Larney, F.J., Olson,
A.F., and McAllister, T.A. 2016.
598 Transport of Three Antimicrobials in Runoff from Windrows of
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599 Manure. J. Environ. Qual. 45(2): 494–502.
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600 Unc, A., and Goss, M. 2004. Transport of bacteria from
manure and protection of water
601 resources. ResearchGate Applied Soil Ecology(25): 1–18.
602 doi:http://dx.doi.org/10.1016/j.apsoil.2003.08.007.
603 Wang, F.-H., Qiao, M., Chen, Z., Su, J.-Q., and Zhu, Y.-G.
2015. Antibiotic resistance genes in
604 manure-amended soil and vegetables at harvest. J. Hazard.
Mater. 299: 215–221.
605 doi:10.1016/j.jhazmat.2015.05.028.
606 World Health Organization. 2015, November. Antibiotic
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607 awareness survey. World Health Organization. Available
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609 Zhang, S., Gu, J., Wang, C., Wang, P., Jiao, S., He, Z.,
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610 Wang, P., Jiao, S., He, Z., and Han, B. 2015a.
Characterization of Antibiotics and
611 Antibiotic Resistance Genes on an Ecological Farm System,.
J. Chem. J. Chem. 2015.
612 doi:10.1155/2015/526143, 10.1155/2015/526143.
613 Zhang, X., Liu, D., Zhang, S., Wei, X., Song, J., Zhang, Y.,
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Stedtfeld, R.D., Hashsham, S.A., and
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doi:10.1073/pnas.1222743110.
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621 Tables & figures
622 Table 1 Protocol and primers selected for the quantitative
PCR
Name Sequence (5’ → 3’)Product
size (bp)
Primer
concentration
(nM)
Denaturation
temperature
(°C)
Annealing
temperature
(°C)
Primer
references
tet(T)
tet(T)-F
tet(T)-R
AAGGTTTATTATATAAAAGTG
AGGTGTATCTATGATATTTAC167 250 94 46
(Aminov et
al. 2002;
Marti et al.
2013)
sul1
sul1-F
sul1-R
GACTGCAGGCTGGTGGTTAT
GAAGAACCGCACAATCTCGT
105 200 98 64(Marti et al.
2014)
blaCTX-M-1CTX-M-F469
CTX-M-R532
CAGCTGGGAGACGAAACGTT
CCGGAATGGCGGTGTTTA64 400 98 60
(Hartmann
et al. 2012;
Marti et al.
2013)
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624 Table 2 Prevalence of beta-lactam, sulfonamide and
tetracycline resistance genes in drainage water, soil and hog
manure
Prevalence (%)
Year GeneDrainage water
Soil before
spreading
Soil after
spreadingHarvest Hog manure
blaCTX-Mb 9.7 0.0 0.0 0.0 100.0
sul1 94.4 91.7 100.0 86.1 100.02016
tet(T) 54.2 77.8 100.0 66.7 100.0
sul1 100.0 94.4 100.0 100.0 100.02017
tet(T) 67.3 91.7 100.0 88.9 100.0
sul1 100.0 NAc NA NA NA2018a
tet(T) 58.3 NA NA NA NA
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626 Figure 1 Bacterial counts in soil surface in 2016 and
2017
627
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633 Figure 2 Bacterial counts in drainage water in 2016 and
2017
634
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635 Figure 3 Tetracycline and sulfonamide resistance gene
concentrations in soil in 2016
636 and 2017
637
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638 Figure 4 Tetracycline and sulfonamide resistance gene
concentrations in drainage
639 water in 2016 and 2017
640
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642 Legends of figures and tables643 Table 2: aIn 2018, there
was only one sampling of drainage water in the spring, when the
snow
644 melted. bThe blaCTX-M gene was sought in 2016 but not in
2017 and 2018. cNA = Not Available
645 since there were no soil and hog manure samples in 2018.
646 Figure 1: A) E. coli counts in soil surface in 2016 B) E.
coli counts in soil surface in 2017 C)
647 Enterococci counts in soil surface in 2016 D) Enterococci
counts in soil surface in 2017. The data
648 presented are the average of 4 sampling replicates of
surface soil samples (0-10 cm). The error
649 bars represent the 95% confidence interval of the
statistical model. The LOD of the E. coli and
650 enterococci counts were 0.3 Log10 CFU/g of wet soil. Manure
rates are represented according to
651 recommended rate by CRAAQ 1X or 2X and mineral fertilizers
by MIN. The hog manure
652 application was made on May 19, 2016 (Julian day 140) and on
May 24, 2017 (Julian day 144).
653 The grain corn and wheat harvest were made respectively on
September 12, 2016 (Julian day 256)
654 and on November 20, 2017 (Julian day 324). *The average was
statistically different between
655 fertilization mode at the sampling date (p
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665 statistically different between fertilization mode at the
sampling date (p
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688 Log10 copies/ml of water while the LOQ was between 2.38 and
2.60 Log10 copies/ml of water. The
689 LOD of sul1 was between 1.12 and 1.34 Log10 copies/ml of
water while the LOQ was between
690 1.82 and 2.04 Log10 copies/ml of water. In 2016, May 9 was
10 days before hog manure application
691 (Julian day 130), May 19 was the date of application (Julian
day 140) (not shown on the graphs),
692 and wheat harvest was made on September 12 (Julian day 256).
In 2017, May 15 was 9 days before
693 hog manure application (Julian day 135), May 24 was the date
of application (Julian day 144), and
694 corn harvest was made on November 20 (Julian day 324). The
manure rates are represented
695 according to the recommended rate by CRAAQ 1X or 2X and
mineral fertilizers by MIN. *The
696 average was statistically different between fertilization
mode at the sampling date (p
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DraftJulian days 2016120 150 180 210 240 270
E.c
oli
(Log
10 C
FU
/g o
f w
et s
oil)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
MIN 1X 2X
Julian days 2017120 150 180 210 240 270 300 330
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Julian days 2016120 150 180 210 240 270
Ent
eroc
occi
(L
og10
CF
U/g
of
wet
soi
l)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Julian days 2017120 150 180 210 240 270 300 330
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
A) B)
C) D)
*
**
*
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Julian days 2016150 180 210 240 270 300 330
E. c
oli
(Log
10 C
FU
/100
ml)
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 MIN 1X 2X
Julian days 2016150 180 210 240 270 300 330 360
Ent
eroc
occi
(L
og10
CF
U/1
00m
l)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Julian days 201730 60 90 120 150 180 210 240 270 300 330
Ent
eroc
occi
(L
og10
CF
U/1
00m
l)
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
A)
B)
C)
†
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tet(
T)
(Log
10 c
opie
s/g
of w
et s
oil)
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5 MIN 1X 2X
Julian days 2016120 150 180 210 240 270
sul1
(L
og10
cop
ies/
g of
wet
soi
l)
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Julian days 2017120 150 180 210 240 270 300 330
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
Julian days 2017120 150 180 210 240 270 300 330
4.5
5.0
5.5
6.0
6.5
7.0
7.5
B)
C)
A)
D)
*
*
*
**
*
*
*
*
*
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Julian days 2016150 180 210 240 270 300 330 360
tet (
T)
(Log
10 c
opie
s/m
l)
-10.00-9.00-8.00-7.00
-2.50-2.00-1.50-1.00-0.500.000.501.001.502.002.503.003.504.00
MIN 1X 2X
Julian days 2016150 180 210 240 270 300 330 360
sul 1
(L
og10
cop
ies/
ml)
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Julian days 201730 60 90 120 150 180 210 240 270 300 330
1.00
1.50
2.00
2.50
3.00
3.50
sul 1
(L
og10
cop
ies/
ml)
A)
B)
C)
†
††
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