Apeer-reviewed open-access journal

BioRisk 17: 31-43 (2022) ice :
doi: 10.3897/biorisk.17.76939 RESEARCH ARTICLE & RB Ke) R IS k

https://biorisk.pensoft.net

Differences in bacterial functional profiles from
loamy sand and clay loam textured soils
under fungicide Quadris® impact

Michaella Petkova', Anelia Kenarova?, Silvena Boteva?,
Stela Georgieva*?, Christo Chanev’, Galina Radeva'

| Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl.
21, 1113 Sofia, Bulgaria 2 Dept. of Ecology and Environmental Protection, Faculty of Biology, Sofia Uni-
versity “St. KL Ohridski’, 8 Dragan Tsankov Blvd, 1164 Sofia, Bulgaria 3 Dept. of Organic Chemistry and
Pharmacognosy, Faculty of Chemistry and Pharmacy, Sofia University “St. Kl. Ohridski’, 1 James Bourchier
Blud, 1164 Sofia, Bulgaria

Corresponding author: Silvena Boteva (sbboteva@biofac.uni-sofia.bg)

Academiceditor: Michaela Beltcheva | Received 23 October 2021 | Accepted 1 December 2021 | Published 21 April 2022

Citation: Petkova M, Kenarova A, Boteva S, Georgieva $, Chanev C, Radeva G (2022) Differences in bacterial
functional profiles from loamy sand and clay loam textured soils under fungicide Quadris® impact. In: Chankova S,
Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17:
31-43. https://doi.org/10.3897/biorisk.17.76939

Abstract

The non-target effect of the fungicide Quadris® on the bacterial community from grassland loamy sand
(LS) and cropland clay loam (CL) soils with unknown history of fungicide usage was investigated. Quad-
ris® was applied to soil mesocosms at 0.0 mg kg! (Az0), 2.90 mg kg" (Az1), 14.65 mg kg" (Az2) and
35.0 mg kg"! (Az3) calculated towards the active ingredient azoxystrobin (Az). Response of bacterial com-
munities to Quadris® was investigated during a 120-day incubation experiment, evaluating the shifts in
bacterial catabolic profiles by the community-level physiological profiling (CLPP) technique and Biolog
EcoPlates™ method. Quadris® decreased the overall catabolic activity (AWCD) of soil bacterial communi-
ties and the rate of decrease was independent of soil type and fungicide concentration. Fungicide affected
negatively the utilisation of amines and positively that of amino acids in both soil types, whereas the effects
on other carbon guilds (carbohydrates, carboxylic acids and polymers) corresponded closely to the respec-
tive soil type and fungicide concentration. Results indicated the presence of non-target effects of Quadris®

on bacterial functioning; hence, it is important to address the fungicide side-effects on soil health.

Keywords
Average well colour development, community-level physiological profiling, fungicide azoxystrobin,

Quadris®, soil bacterial communities

Copyright Michaella Petkova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

32 Michaella Petkova et al. / BioRisk 17: 31—43 (2022)

Introduction

Plant diseases are a common occurrence, often having a significant economic impact on
yield and quality; thus, managing diseases is an essential component of production for
most crops. For this reason, fungicides are used to kill fungi by damaging their cell mem-
branes, inactivating critical enzymes or proteins or by interfering with key processes, such
as energy production or respiration (McGrath 2004). One of the widely used classes of
fungicides is strobilusrins (Howell et al. 2014). The first patent for a strobilurin fungicide
(azoxystrobin) was introduced in 1996 (Bartlett et al. 2002) and, subsequently, a series
of strobilurin fungicides (pyraclostrobin, fluoxastrobin, kresoxim-methyl, trifloxystrobin,
picoxystrobin, mandestrobin and metominostrobin) were developed and marketed (Rod-
rigues et al. 2013). Strobilurin fungicides specifically bind to the quinol oxidation (Q )
site of cytochrome b and inhibit mitochondrial respiration (Bartlett et al. 2002). Stro-
bilurin fungicides control an unusually wide array of fungal diseases, including diseases
caused by water moulds, downy mildews, powdery mildews, leaf spotting and blighting
fungi, fruit rotters and rusts (Vincelli 2002). They are used on a wide variety of crops,
including cereals, field crops, fruits, tree nuts, vegetables, turf-grasses and ornamentals.

These pesticides are designed to manage fungal pathogens, although their broad-
spectrum mode of action also produces non-target impacts. Due to the unique mecha-
nism of action, strobilurins may directly affect soil fungi by inhibiting mitochondrial res-
piration, inducing a shift from fungal to bacterial dominance in soil activities (Ba¢maga
et al. 2015). Strobilurins may affect not only soil fungi, but also soil bacteria (Ba¢maga et
al. 2015), archaea (Howell et al. 2014) and invertebrates (Han et al. 2014). For instance,
Ba¢maga et al. (2015) reported negative effects of azoxystrobin not only on soil fungi, but
also on soil organotrophic bacteria and actinomycetes. Most of the earlier studies report-
ed low to negligible effects of Az alone or Az containing fungicides on bacterial diversity,
but the knowledge of fungicide effects on bacterial metabolic activity is still insufficient.

The aim of this study was to elucidate the effects of Quadris®, Az containing fun-
gicide, on soil bacterial metabolism. The study suggested that Quadris® can potentially
cause long-term adverse effects on soil nutrient turnover, affecting bacterial metabo-
lism, although bacteria are considered as fungicide non-target organisms.

Material and methods

Sampling site and preliminary soil preparation

In this study, two soils with different histories of management practice were used. Five sub-
samples were pooled randomly from the surface layers (0-20 cm) of grassland and crop-
land located near Gabra Village (Sofia Region, Bulgaria): 42°31'48.36"N, 23°37'28.20"E
(Fig. 1). The subsamples per soil type were sieved through a 2 mm mesh and mixed in
aliquots after determining the dry weights of 1 g sample at 105 °C in an oven for 24 hr.

Bacterial functional response under Quadris® impact 33
p p

Figure |. Map of Gabra Village territory (red line and red mark) and the sampling site (blue mark).
Mesocosm experimental design

Four sets of three replicated mesocosms (2 kg) were prepared for each soil type. ‘The fol-
lowing treatments were studied: control (Az0) and Quadris* amendments of 2.90 mg
kg! (Az1), 14.65 mg kg" (Az2) and 35.00 mg kg" (Az3), calculated towards the active
ingredient — Az. Soil water content was adjusted to 60% of the maximum water hold-
ing capacity and it was maintained with sterile distilled water during the experiment.
The mesocosms were incubated at 22 + 1 °C in dark to prevent physical degradation
of Az by light. Soil samples were collected randomly in triplicates from each mesocosm
on the 1* (D1), 30" (D30), 60" (D60), 90° (D90) and 120" (D120) day after fungi-

cide application.

Soil physico-chemical properties

Soil texture was defined and classified according to ISO 11277 (2009) and SSDS
(1993), respectively. Soil pH was measured potentiometrically (HANNA Instruments)
after mixing soil in 0.01 mol I! CaCl, solution and shaking for 30 min (1:5; weight:
volume). Soil nitrate (NO,-N) and ammonium (NH,-N) nitrogen and phosphates
(P,O,) were determined spectrophotometrically, according to the methods of Keeney
and Nelson (1982) and Olsen (1982), respectively.

Az residues in soil

The method of Az soil residues extraction and determination was explained in detail
in Aleksova (2020). Recovery rates of Az in each sample were satisfactory at 80.0%-—
85.0%. Modelling of Az dissipation in soils was conducted according to the recom-
mendations of the FOCUS group (2006), using an Excel file (FOCUS_DEGKIN V2)
provided online by the group. The same file was used to calculate the time of 50%
reduction in Az soil concentrations (DT50).

34 Michaella Petkova et al. / BioRisk 17: 31—43 (2022)

Community level catabolic activity and physiological profiling

EcoPlates (Biolog Inc., Hayward, CA, USA) were used to establish the changes in
CLPPs over time. The procedure of plates’ inoculation, cultivation and monitoring
(every 12 hr for 5 days) was described in detail in Kenarova et al. (2014). During the
initial data processing, the control OD was subtracted from the OD of each carbon
source (CS) well and the CSs with corrected OD < 0.25 were considered as non-
oxidised and their values were set to zero (Garland 1996). Biolog CSs were grouped
according to Weber and Legge (2009) into five carbon guilds (CGs) depending on
their chemical moieties: carbohydrates (CH; 10 CSs), polymers (Polym; 4 CSs),
carboxylic acids (CA; 9 CSs), amino acids (AA; 6 CSs) and amines/amides (Amin; 2
CSs). The Biolog-derived data were used to evaluate the bacterial metabolic activity

(AWCD) (Garland and Mills 1991) and the pattern of CLPP (Kenarova et al. 2014).

Data analysis

Each data point in the paper represented the mean value of the respective Az soil
amendment + standard deviation. One-way ANOVA, followed by Tukey’s test, was
performed to examine the differences in the means of soil (pH, NO,-N, NH,-N,
P,O,, Az) and bacterial (AWCD and CLPP) parameters. Principal component analysis
(PCA) was performed with soil abiotic data to assess the differences in soil physical
environments after Quadris® application. The differences in CLPPs between soil types
and amongst fungicide concentrations were assessed with the graph ‘one-to-one’ tech-
nique. The above statistics were performed with the package PAST (Hammer et al.

2001) at a level of significance p < 0.05.

Results

Soil environments

The soil textures of grassland and cropland were classified as loamy sand (LS; 2%
clay, 15% silt and 83% sand) and clay loam (CL; 27% clay, 37% silt and 36% sand),
respectively. Soils were well abundant in organic carbon (LS: 21.92 + 1.41 g kg! and
CL: 23.4 = 3.11 g kg") and Kjeldahl nitrogen (LS: 2.20 = 0.21 g kg! and CL: 2.64
+ 0.34 g kg"), both of them fluctuating insignificantly during the incubation time.
Soil pH was moderately acidic (5.63) at LS and neutral (6.99) at CL and, during the
incubation, it decreased significantly (LS: by Azl — 8%, Az2 — 12% and Az3 — 14%)
and insignificantly (CL: by Az1 — 0.8%, Az2 — 1.1% and Az3 — 1.3%) in fungicide
amended soil mesocosms. Quadris® application increased the overall soil NO,-N -
in LS by 10% (Az1), 13% (Az2) and 20% (Az3) and, in CL, by 70% (Az1), 34%
(Az2) and 19% (Az3). On the other hand, the overall soil NH,-N concentrations
decreased by 15% (LS) and 39% (CL). Soil concentrations of P,O, were much more

Bacterial functional response under Quadris® impact 35
12) p

stable than those of the inorganic nitrogen, decreasing during the incubation by
8.6% (LS) and 14.3% (CL).

Az soil residues decreased over time and the rate of decrease was higher for LS than
those for CL — DT50 ranged for LS from 36.5 + 7.1 (Az1) to 86.6 + 4.1 (Az3) days,
whereas those for CL ranged from 130.8 + 7.8 (Az1) to 212.1 + 3.2 (Az3) days.

PCA, based on soil physico-chemical properties and Az soil residues, was con-
ducted in order to elucidate the similarity amongst soil physical environments (Fig. 2)
and the analysis indicated: 1) the respective LS and CL mesocosms differed signifi-
cantly from each other, except Az1 where fungicide input approximated to the physi-
cal environments of LS and CL on D90; 2) significant differences within-soil physical
environments were detected, except those of Azl and Az2 at LS on D60; 3) temporal
fluctuations of CL physical environments were smaller than those of LS.

Bacterial metabolic activity

The AWCD of Az0 (CL) -1.69 OD was calculated to be around 35% higher than
that of AzO (LS) -1.25 OD. Quadris® application decreased the overall mean value of
AWCD (except Az1 at LS and Az2 at CL) and the changes were significant (Az3 at

PC 2 (0.12%)

PC 1 (20.2%)

Figure 2. Spatial projection of the first two principal components (PC 1 and PC 2) with an ordination
plot related to soil physical environments of Quadris® amended (Az1 — Az3) and un-amended (Az0)

loamy sand (LS) and clay loam (CL) soil mesocosms.

36 Michaella Petkova et al. / BioRisk 17: 31—43 (2022)

LS and Az1 and Az3 at CL) and insignificant (Az2 at LS). A stimulation effect was re-
corded for Az1 (LS) and Az2 (CL), being significant only for the second one. Temporal
Quadris® effects on bacterial metabolism were very similar, independent both on fun-
gicide concentration and soil type - AWCD profiles manifested a decrease in bacterial
metabolism for at least two months (D1 — D60), followed by recovery (D60 — D90)
and stimulation (D120). Different metabolic profiles over time were formed for Az2
and Az3 at CL — permanent stimulation (Az2, except on D1) and dramatic decrease
(Az3 after D60) after fungicide application. The values of Quadris® that influenced
AWCDs were much higher than that of Az0O on D120 (except Az3 at CL) and the
rates of stimulation were in reverse- (LS) and non- (CL) relationships with the applied
fungicide concentrations.

One-way ANOVA showed that the respective AWCD means of Az1 and Az3 did
not differ significantly between LS and CL (F < 2.05, p > 0.16), opposite to that of
Az2 (F = 18.5, p = 0.000).

Community level physiological profiling

It was obvious that bacterial metabolism was changed under Quadris* impact, but
AWCD was not sufficiently powerful to demonstrate the differences of these changes as
a dependence of the applied fungicide concentration and soil properties. Therefore, af-
ter grouping the EcoPlate carbon sources into carbon guilds (CG), the CLPP approach
and ‘one-to-one’ analysis were used to elucidate the intrinsic nature of AWCD changes
(Fig. 3). Between-soil analysis indicated that, after Quadris®, LS differed from CL by
the utilisation of: 1) CH and CA — Az], 2) all CGs, except CH — Az2, and 3) all CGs
— Az3. Within-soil ‘one-to-one’ analysis demonstrated the effects of increasing fungi-
cide concentrations on the utilisation of CGs in the respective soil type and they were
significant at: 1) LS — all fungicide concentrations influenced the utilisation of CH
(positively at Az1 and Az2 and negatively at Az3) and Amin (negatively at Az1 — Az3);
Az1 and Az2 stimulated the utilisation of AA; Az3 decreased the utilisation of Polym
and 2) CL - all fungicide concentrations influenced the utilization of-Amin (positively
at Az2 and negatively at Az1 and Az3); Az1 and Az2 stimulated the utilisation of AA
and CA (except Az1); Az3 decreased the utilisation of CH and Polym.

Metabolic diversity

In order to understand the insights of changes in CG utilisation rates under Quadris*,
the overall number of utilisable CSs were counted — metabolic richness, as well as the
index of carbon sources’ utilisation evenness per CG.

Most of the changes occurred in CGs related to shifts into the “evenness” rather than
the “richness” of utilisable CSs. For example, the richness changes under Quadris® were
detected only for the utilisation of CHs and CAs at CL - utilisation of carbohydrate D-
Xylose (Az1 — Az3) was inhibited, whereas that of carboxylic acids, y-Hydroxybutyric
acid (Az1) and 2-Hydroxy benzoic acid and a-Ketobutyric acid (Az2 and Az3), was

Bacterial functional response under Quadris® impact 37
p p

1.4 5 Ls

134 mae

1.2 4 14 -
oii QO oa-
o o Amin
- 09 4 3 0.8 4 CH in
2 08 5 é O74 Amin

CH

O07 ied 4
a an CA < 06 Polym
~ 06 05 3
a 0.5 0.7 0.9 1.1 ; 06 08 12

Az1 - Az3 of LS (OD) Az0 (OD)

149 CL

1.34 = AA

1.2 4 AA
Qo a al
oJ 144 He, heh HH Polym
ao 17 PS Polym
a og 4 Amin Polym
Ww 087
a CH

07 +4 +.

CA Amin
0.6
0.7 0.8 0.9 1 11 1.2
Az0 (OD)

Figure 3. ‘One-to-one’ comparison of CLPPs between loamy sand and clay loam (LS — CL) soil mesocosms
and between Quadris® un-amended (Az0) and fungicide-amended soil mesocosms (Az1 — Az3) per soil type.
Diamond symbols illustrate the mean (n = 3) utilisation rate of the respective carbon guild, bars illustrate the

standard deviations and colour denotes the fungicide concentration - Az1: green, Az2: blue and Az3: red.

stimulated. Much more changeable amongst soil mesocosms was the index of metabolic
evenness. The greatest between-soil type differences were detected at: 1) Azl, where
Quadris® increased the metabolic evenness of Polym, CA and Amin at LS and decreased
that at CL and 2) Az3, where Quadris® increased the metabolic evenness of CH, Polym
and CA at CL and decreased that at LS. The between-soil type similarity was found at
Az2, where Quadris® increased the metabolic evenness of CA, AA and Amin.

Discussion

Soil physical environment

Soil amendments with Quadris®, even by the lowest fungicide concentration, cre-
ated new physical environments referring mainly to changes in soil pH, nitrogen
pool and presence of allochthones substrates (Az and Quadris*’s adjuvants). Similar
soil acidification after Az application was also reported by earlier studies (Ghosh and
Singh 2009; Singh et al. 2010), explaining this fact by the formation of azoxystrobin
acid as the major product of fungicide degradation. In this study, the decrease in
pH was recorded immediately after fungicide application (D1), assuming that azox-
ystrobin acid was not the only determinant of soil acidification. Probably, some of

38 Michaella Petkova et al. / BioRisk 17: 31—43 (2022)

the Quadris® ingredients contributed also to soil acidification. We supposed that soil
acidification might influence directly and/or indirectly bacterial metabolism, shift-
ing community composition into growth of acidophiles and influencing nutrient
solubility (bioavailability).

Soil amendments with Quadris® influenced the soil nitrogen pool, changing bio-
available concentrations and forms of inorganic nitrogen which could be related to
the adjuvants presented in fungicide commercial formulations (Devare et al. 2007;
Mijangos et al. 2009), fungicide metabolisation (Cycon et al. 2011; Ba¢maga et al.
2017) and accumulation and degradation of proteins released from killed soil inhabit-
ants (Wu et al. 2014; Zhang et al. 2014). Since nitrification and mineralisation play
important roles in nutrient turnover (Edwards et al. 1995), it seems that shifts in soil
inorganic nitrogen could disrupt these processes and impact overall soil quality and
productivity. Additionally, proportions of soil NO,-N and NH,-N could also influ-
ence the utilisation rates of nitrogen containing CSs. We supposed that Az was more
bioavailable in LS compared to CL and it influenced its persistence, half-life and toxic-
ity to soil organisms. According to DT50, Az could be considered as a low to medium
persistent fungicide in LS and highly persistent in CL.

The ordination of soil physical environments demonstrated that Quadris® appli-
cation in increasing concentrations influenced soil properties, creating new physical
environments. We supposed that newly-created environments might influence soil
bacteria to adapt their metabolism.

Fungicide effects on soil bacteria

In this study, we evaluated the Quadris® effects on soil heterotrophic bacteria, which
display a substantial role in plant growth rates, mineralising dead organic matter and
detoxifying a range of exogenous substances. Bacteria are considered as Az non-target
organisms, due to the fungicide mode of action on mitochondrial respiration (Bartlett
et al. 2002). In fact, earlier reported data, referring to bacterial community composi-
tion (Howell et al. 2014) and functioning (Sutowicz et al. 2016; Wang et al. 2020)
under Az, were very contradictory. We hypothesised that fungicides influenced soil
bacterial metabolism indirectly by changes in soil biotic and abiotic properties. Two
main metabolic criteria were followed during the soil mesocosms’ incubation: 1) com-
munity metabolic activity expressed by AWCD and 2) community metabolic profiles
(CLPP) expressed by carbon guilds’ utilisation rates and metabolic diversity. In the case
of detected fungicide impacts, it was important to mention if there were any relation-
ships to soil type and, in particular, with some of the studied soil properties.

Community metabolic activity (AVWWCD)

Quadris*® application influenced bacterial metabolism for at least four months, decreas-
ing it in most of the soil mesocosms, except at Az1 (LS) and Az2 (CL). The most serious
negative effects were detected during the first two months after fungicide application,

Bacterial functional response under Quadris® impact by

followed by recovery and stimulation of bacterial metabolic activity (except Az3 at
CL). Some researchers reported inhibitory effects of fungicides on bacterial metabolic
activity (Zhang et al. 2014), although others advocated none or stimulation effects on
AWCD (Mufoz-Leoz et al. 2011). We suppose that these contradictions arise from
the fungicide chemical origin, applied concentrations and soil properties. In our study,
soil properties were of significant importance for the differentiation of Quadris* effects
on overall AWCD at Az2, but not at the other fungicide concentrations. Probably,
the delayed stimulation effects at Az1 decreased the differences in AWCD between LS
and CL, whereas the very high value of Az3 minimised the modulating effects of soil
peculiarities on fungicide mode of action.

Community level physiological profiles (CLPP)

Carbon guilds’ utilisation rates

Earlier studies (Bending et al. 2007) and our investigations (Aleksova et al. 2021)
reported that azoxystrobin did not affect bacterial community structure, suggesting
that the fungicide shifted bacterial metabolism via chemical toxicity and/or pheno-
typic bacterial adaptation to environmental changes. The dissimilarity between LS
and CL under Quadris®, applied at a field recommended concentration, was referred
towards CH and CA utilisation. These results confirmed the findings of some au-
thors (Kenarova et al. 2014; Yu et al. 2020) that the utilisation of carbohydrates and
carboxylic acids was sensitive to environmental disturbance and it could be used to
indicate the alterations that occurred in bacterial functional profiles under stress.
Additionally, in this study, we related that fungicide-affected soil physical environ-
ments. Probably, the differences in pH, clay content and organic matter concentra-
tion between the two soil types reflected differently on CH and CA bioavailability;
hence, on bacterial adaptations to changed soil nutrient pools. Great differences in
temporal profiles of CH and CA utilisation (not shown here) were detected in the
late fungicide exposure stage (D90—D120), when the utilisation of the two CGs
increased dramatically at LS (by 45%, on average) and stabilised (CH) or decreased
by"l5% (CA) at CL.

Higher fungicide concentrations (Az2 and Az3) widened the spectrum of impact-
ed CGs, but these effects could be related to soil chemical pollution, rather than to the
controlled use of Quadris® for plant protection.

Metabolic diversity

In both soil types, Quadris® application influenced metabolic evenness rather than
on metabolic richness, which might be explained by the intrinsic bacterial commu-
nity capacity to be metabolic resistant, resilient and redundant (Fenchel and Finlay
2004; Meyer et al. 2004). Some bacteria show a high degree of metabolic tolerance
(resistance) to changing environmental conditions (Meyer et al. 2004), whereas others

40 Michaella Petkova et al. / BioRisk 17: 31—43 (2022)

are capable to adapt quickly to the new nutrient inputs for rapid growth (resilience)
(Fenchel and Finlay 2004). Further, the extremely high abundance and diversity of
bacteria are arguments for their metabolic redundancy, ensuring ecosystem function-
ing (nutrient turnover), even in extreme conditions.

We assumed that Quadris® significantly changed soil nutrient pools and the
changes might occur due to soil accumulation of dead fungal biomass, induction of
detoxification agents (mainly proteins) - molecules that can later be metabolised by the
same microbiota (Degens et al. 2000), changes in soil pH and/or fungicide adjuvants’
inputs (Syngenta 2021). Changes in soil mesocosms affected in different ways bacte-
rial capacity to use CSs — ranging from inhibition to stimulation. Interesting was the
stimulation effects of Quadris® on the utilisation of y-Hydroxybutyric acid, 2-Hydroxy
benzoic acid and o-Ketobutyric acid at CL. Stimulated utilisation of 2-Hydroxy ben-
zoic acid and a-Ketobutyric acid under fungicide tetraconazole was observed earlier by
Sulowicz et al. (2016).

Conclusions

The study showed that soil properties (soil texture, pH and organic and inorganic
substances) were of significant importance for the fate of applied fungicide Quadris*,
as well as its effects on bacterial metabolism. ‘The fungicide decreased for at least four
months the overall bacterial activity (AWCD), shifted the metabolite profiles (CLPPs)
of bacterial communities and changed the preferred carbon sources and metabolic
diversity. Fungicide also affected the mode of environmental control on bacteria, in
accordance with soil peculiarities.

Acknowledgements

This study was financially supported by the National Research Fund of the Bulgarian
Ministry of Education and Science (grant DN 11/6 - Dec, 2017).

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