Acute glyphosate exposure does not condition the response of microbial communities to a dry–rewetting disturbance in a soil with long history of glyphosate–based herbicides

Dry–rewetting perturbations are natural disturbances in the edaphic environment and particularly in dryland cultivation areas. The interaction of this disturbance with glyphosate–based herbicides (GBHs) deserves special attention in 10 the soil environment due to the intensification of agricultural practices and the acceleration of climate change with an intensified water cycle. The objective of this study was to assess the response of microbial communities in a soil with long history of GBHs to a secondary imposed perturbation (a single dry–rewetting event). A factorial microcosm study was conducted to evaluate the potential conditioning effect of an acute glyphosate exposure on the response to a following dry– rewetting event. A Respiratory Quotient (RQ) based on an ecologically relevant substrate (p–coumaric acid) and basal 15 respiration was used as physiological indicator. Similarly, DNA–based analyses were considered, including quantitative PCR (qPCR) of functional sensitive microbial groups linked to cycles of carbon (Actinobacteria) and nitrogen (ammonia– oxidizing microorganisms), qPCR of total bacteria and denaturing gradient gel electrophoresis (DGGE) of ammonia– oxidizing bacteria (AOB). Significant effects of Herbicide and of Dry–rewetting perturbations were observed in the RQ and in the copy number of amoA gene of AOB, respectively. However, no significant interaction was observed between them 20 when analyzing the physiological indicator and the copy number of the evaluated genes. PCR–DGGE results were not conclusive regarding a potential effect of Dry–rewetting × Herbicide interaction on AOB community structure, suggesting further analysis by deep sequencing of amoA gene. The results of this study indicate that the perturbation of an acute glyphosate exposure in a soil with long–history of this herbicide does not have a conditioning effect on the response to a subsequent dry–rewetting disturbance according to a physiological indicator or the quantified bacterial/archaeal genes. 25


Soil sampling and microcosm set up
Sampling was conducted in the same agricultural plot (ZAV H ) with long history (more than 20 years) of exposure to GBHs 60 that was described in a previous study (Allegrini et al., 2015). Fifteen subsamples were taken at a 0-10 cm depth, sieved (<5.6 mm) and pooled to obtain a composite sample. Soil was stored at 4°C and used within 6 days for the microcosm study.
Twelve microcosms (equivalent to 40 g of oven dry soil) were prepared in 100 ml sterile screw-cap polypropylene flasks, loosely capped to reduce water evaporation whilst leaving enough space for free passage of air. All flasks (60 % WHC) were pre-incubated in the dark at 25 °C (Ingelab I.501PF Incubator) for 1 week. Then, microcosms were randomly assigned to the 65 following treatments, in a 2×2 factorial design with 3 replicates per treatment: "Herbicide" (two levels: with GBH "CG" and control with distilled sterile water "SG") and "Dry-rewetting" (two levels: with desiccation "CD" and untreated control "SD"). First, microcosms received either the CG or SG treatments (day 0). The herbicide (Roundup Full II, Monsanto™, N-(phosphonomethyl)glycine potassium salt, 66.2 % w v -1 , additives not specified) was applied in a final volume of 0.2 ml (with distilled water) at a rate of 49 µg active ingredient g -1 soil similarly to other studies with silt loam soils (Haney et al., 70 2000;Ratcliff et al., 2006). This dose mimics the concentration of glyphosate found in soil after a 1× application rate in the field (0.84 kg ha -1 ) considering a 2 mm soil interaction penetration due to the high absorptivity and low leachability of glyphosate (Haney et al., 2000). Microcosms were initially incubated for 14 days under conditions described above for the pre-incubation step. The dry-rewetting disturbance was imposed at day 14 and microcosms were returned to incubation for
Decimal dilutions of a plasmid harboring one copy of 16S rRNA gene of Streptomyces albus DSM 40313 were used as 95 standards (serial 10 -1 dilutions to obtain between 4.97×10 6 and 4.97×10 2 copies). All amplifications were conducted in ABI 7500 Real Time System (Applied Biosystems, Foster City, CA).
The abundance values of these genes were used as surrogates of population sizes, although no attempt was made to convert copies into cell numbers to avoid introducing errors (e.g. errors related with an unknown number of operons per cell in mixed bacterial communities) (Zabaloy et al., 2017;Ouyang et al., 2016). The efficiencies of qPCR assays were 84.1% 100 (amoA AOB ), 78.57% (amoA AOA ), 91.07% (total bacteria 16S rRNA) and 93.67% (Actinobacteria 16S rRNA); and R 2 values were ≥ 0.99 in all assays. 5 stability of them (group separation assessment) was verified in GelCompar II using the statistical method Jackknife resampling with average similarities (GelCompar II™ v. 4.6, Software Manual).

Respiratory responses
The mean RQ values for the different treatments are indicated in Fig. 1. According to two-way ANOVA (Table 1), no interaction was observed between factors (P > 0.05). Thus, main effects were considered. No statistical significance was observed for the main effect of Dry-rewetting. Conversely, Herbicide showed a significant effect (P < 0.05) with a higher RQ value in CG microcosms relative to the untreated microcosms (SG). 125

Quantification of indicators genes
For all the indicators genes, the results of two-way ANOVA (Table 32) indicated no statistical significance of Herbicide main effect as well as no interaction, while a significant Dry-rewetting effect was detected only for AOB (P < 0.05). The equations obtained after linear regression of qPCR standard curves and the respective efficiencies are indicated in Table 2. 130 Mean copy numbers for each treatment and each gene are shown in Fig. 2 and Table 43. For all the indicators genes, the results of two-way ANOVA (Table 3) indicated no statistical significance of Herbicide main effect as well as no interaction, while a significant Dry-rewetting effect was detected only for AOB (P < 0.05). The abundance of amoA AOB (averaged for both levels of Herbicide factor) was 1.27 fold higher in microcosms with dry-rewetting dessication (CD) than in undisturbed (SD) microcosms (Table 43). 135

DGGE of ammonia-oxidizing bacteria
DGGE profiles showed few bands and high similarity values (Pearson coefficients) among replicates of the four treatments, with no separation in four treatment-clusters. Similarly, no obvious separation was observed between microcosms with (CD) and without (SD) dry-rewetting or between glyphosate-treated (CG) and untreated microcosms (SG). At 80 % similarity level (cut-off), a separation in two clusters was observed (Fig. 3, grey branches). In one of them, we observed two replicates 140 of CD/SG treatment. In the second cluster the three replicates of CD/CG treatment clustered together with microcosms in which no dry-rewetting was applied (SD).
In this study we evaluated whether an acute in vitro glyphosate application on a soil with long history of application of GBHs modulates the response of the microbial communities to the following dry-rewetting disturbance. 145 We hypothesized that if no PICT was observed in the studied soil after long exposure in the field (Allegrini et al., 2015), a single glyphosate application to microcosms would have no effect in the structure of the microbial community, as the probability to change to an alternative state is more likely in response to a press disturbance (chronic exposure) than to a pulse disturbance (Shade et al., 2012). These changes in microbial communities associated with greater tolerance to a pesticide might, at the same time, conceal a higher sensitivity in the response to other perturbations (a "cost of tolerance"; 150 Clements and Rohr, 2009). Thus, for the soil assessed in this study, we expected no conditioning effect in the the sensitivity to a secondary perturbation will not be conditioned by the presence/absence of a previous acute glyphosate exposure (Clements and Rohr, 2009). This hypothesis was confirmed by our results: no interaction was observed between Herbicide and Dry-rewetting in an acute exposure to both perturbations with a physiological indicator (Table 1) and with DNA-based methods (Table 2), supporting the absence of a PICT response. The non-significant interaction observed for Actinobacteria 155 (Table 2) indicates that one of the main characteristics of this microbial group, the high tolerance to desiccation (Evans and Wallestein, 2011), is not conditioned by the previous exposure to a single application of a GBH, even when negative effects of GBHs on this phylum have been reported (Barriuso et al., 2010). For amoA, the absence of interaction is also a relevant observation considering that AOB are particularly sensitive to pesticides and also to water availability (Franzluebbers et al., 19951994;Hastings et al., 2000;Gleeson et al., 2010). Thus, our results suggest that the sensitivity expected to each 160 perturbation alone does not necessarily results in a synergic effect when combined.
Ammonia-oxidizing archaea were more abundant than AOB for all treatments. Also, they clearly differentiated from AOB as no significant dry-rewetting effect was observed (Table 2). This observation is consistent with the results of Gleeson et al. (2010), who reported that AOB are more responsive to water availability than AOA. The statistical significance of dryrewetting main effect on the abundance of AOB indicates that the microbial community of the soil assessed in this study is 165 particularly sensitive to the perturbation. Conversely, the abundance of AOB seems to be less sensitive to GBH exposure (no significance detected for this factor), supporting previous results with the same soil and the same herbicide formulation in which no effects of repeated applications were detected on absolute abundance (up to three applications) (Allegrini et al., 2017). As indicated in Tables 3 2 and 43, the dry-rewetting perturbation enhanced the abundance of amoA AOB relative to the untreated microcosms (SD). Most gram negative bacteria are affected by a rapid rewetting after desiccation events and a 170 recover to the initial abundance values has been reported for AOB at 18 days after rewetting (Hastings et al., 2000). A t functional level (nitrification rate), Fierer and Schimel (2002) found a significant increase in the activity of autotrophic nitrifying communities after several dry-rewetting cycles, in agreement with the higher abundance that we observed for amoA AOB and with a correlation between amoA copy number and nitrification potential observed in different soils (Rudisill et al., 2016;Zabaloy et al., 2017).

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The low number of bands observed in the DGGE profiles of amoA AOB amplicons suggests a low richness of AOB in the studied soil. This result is in agreement with a previous biogeographic study which reported a low diversity of amoA sequences in soil AOB communities, with most of them in the Nitrosospira lineages (Fierer et al., 2009). More recently, a microcosm study with a loam sandy soil from Pampa region observed low diversity in AOB community with DGGE (Zabaloy et al., 2017). An obvious separation among DGGE profiles of microcosms with and without dry-rewetting was not 180 observed, indicating no effects of this perturbation on the community structure of AOB. Thus, even that though qPCR indicated an increase in the abundance of amoA AOB sequences, the profiling (fingerprinting) of the community structure did not show the same sensitivity to the dry-rewetting disturbance (Fig. 3).
The separation observed at 80 % similarity level (Fig. 3) between two replicates of CD/SG treatment and the three replicates CD/CG could be indicating an interaction as no comparable separation was detected between SD/SG and SD/CG. However, 185 more evidences are still necessary to determine whether or not there is a significant interaction effect on the structure of AOB. Amplicon sequencing of amoA AOB and beta diversity analysis could provide substantially more information in this regard.
In conclusion, our study demonstrates that acute exposure to a GBH does not have a conditioning effect on the response of microbial communities to a secondary disturbance (dry-rewetting) in a soil with chronic exposure to GBHs. To obtain more 190 evidences supporting our conclusion, future studies should assess the effects of several dry-rewetting cycles.

Point-by-point response to the reviewers' comments
Anonymous Referee #1 Received and published: 22 April 2020 General comments: In this manuscript, the authors evaluate the potential conditioning effect of an acute glyphosate 375 exposure (first imposed perturbation) on the response of soil microbial communities to a single dry-rewetting event (second imposed perturbation), in soils with a long history of exposure to glyphosate-based herbicides. The topic under study is relevant, the hypothesis is sound, and the experimental design is suitable for the aim of the study. In addition, the manuscript is concise, well written and organized; therefore I recommend its publication after some minor revisions. 380 Technical corrections: L27-28: The phrase "(e.g. pesticides)" is repeated in both sentences; maybe it's not necessary to mention it twice.
We agree with the comment. The phrase was removed in line 27. Table 2 We agree with the suggestion. Table 2 was removed and the information was inserted in the text in the same way as suggested by the reviewer. Changes in microbial communities associated with the development of a greater tolerance (PICT) to a pesticide in the field (chronic exposure) might, at the same time, conceal a higher sensitivity in the response to other perturbations (a "cost of tolerance" when adapting to an environmental stress; Clements and Rohr, 2009). Thus, if no PICT response was observed in 395 the studied soil after long exposure in the field (Allegrini et al., 2015), it could be expected that a single glyphosate application to microcosms (acute exposure) would have no effect at all in the structure of the microbial community and, consequently, no conditioning effect of this acute glyphosate exposure should be observed on the response to a secondary perturbation (dry-rewetting in this case). The absence of conditioning effect is consistently reflected in the non-significant interaction term of ANOVA. 400

L124-125 and
However, it is important to mention that even if a PICT response would have been observed in this soil, the higher tolerance could have associated costs in the response to only some environmental stresses (e.g., to stresses caused by other xenobiotics but no to a dry-rewetting stress). Thus, a non-significant interaction could be also observed for a soil in which a PICT has been detected. Based on this argument, we consider that the absence of interaction in our study is not a conclusive result supporting the absence of a PICT response. In other words, the result we observed in the microcosm assay (no conditioning effect of an acute glyphosate exposure to dry-rewetting response) is an expected result for a soil in which no PICT was observed (as explained above) but it cannot be considered a supporting evidence of the absence of a PICT response in this soil.
We have removed the phrase "supporting the absence of a PICT response" in line 146. Also we have introduced the concept of "cost of tolerance" after line 142, as explained before in response to the reviewer comment. 410 L147 and L155: Can you please check Table numbers? I believe it's Table 3.
As indicated by the reviewer it is Table 3 and not Table 2.

L152: "does not necessarily result"
Ok, the error was corrected.

L173: Maybe "even though" instead of "even that"?
We agree with the suggestion. All references were checked and the modifications were introduced as indicated by the reviewer.

2-As
Reviewer #1 suggests, the concept of PICT response and the absence of interaction could be explained with 435 more detail.
We agree with the need of clarification of this concept. Please see the response to the third comment of Reviewer 1 (L145-146). The change was introduced in the text as indicated by the reviewer.

List of all relevant changes
All relevant changes were introduced in response to the reviewers' comments and were indicated previously.
In addition to these changes, the following changes were also introduced: 445

Abstract (Lines 23 to 25 of the revised version of the manuscript)
The following lines were introduced in response to the Topical Editor comments: "The results of this study indicate that the perturbation of an acute glyphosate exposure in a soil with long-history of this herbicide does not have a conditioning effect on the response to a subsequent dry-rewetting disturbance according to a physiological indicator or the quantified bacterial/archaeal genes." 450

Data availability
A typing error was found in the name of the database and was corrected: "4TL Database" changed by "4TU Research.Data Database"