Comparative analysis of the effects of five soil fumigants on the abundance of denitrifying microbes and changes in bacterial community composition

Ecotoxicology and Environmental Safety 187 (2020) 109850

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Comparative analysis of the effects of five soil fumigants on the abundance of denitrifying microbes and changes in bacterial community composition

T

Wensheng Fanga, Xianli Wanga, Bin Huanga, Daqi Zhanga, Jie Liua, Jiahong Zhua, Dongdong Yana, Qiuxia Wanga, Aocheng Caoa,∗, Qingli Hanb a b

Institute of Plant Protection, Chinese Academy of Agricultural Sciences, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Beijing, 100193, China College of Biodiversity Conservation, Southwest Forestry University, Kunming, 650224, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Soil fumigation DMDS AITC Soil microorganism Denitrifying bacteria

Soil fumigation is currently the most effective method for controlling soil-borne pests and diseases in high-value crops. To better understand the effect of chloropicrin (CP), dazomet (DZ), dimethyl disulfide (DMDS), allyl isothiocyanate (AITC) and 1,3-dichloropropene (1,3-D) fumigants on soil microorganisms, this study monitored changes in the diversity and community composition of soil bacteria involved in denitrification using real-time PCR and high-throughput gene sequencing techniques. These five fumigants significantly decreased the bacterial population size in some phyla including Proteobacteria, Chloroflexi and Acidobacteria, and increased the bacterial population size in other phyla such as Firmicutes, Gemmatimonadetes, Actinobacteria, Verrucomicrobia, Saccharibacteria and Parcubacteria. Although bacterial diversity declined after CP fumigation, it was briefly stimulated by the other four fumigants. Meanwhile, all five fumigants temporarily decreased populations of denitrifying bacteria containing the napA, narG, nirS or nirK enzyme-encoding genes. Denitrifiers bearing the cnorB, qnorB or nosZ genes were relatively stable following DZ and DMDS fumigation. However, cnorB and nosZ decreased initially following CP, AITC and 1,3-D fumigation. Simultaneously, the abundance of qnorB significantly increased in AITC and 1,3-D fumigated soils. These results showed that soil fumigation significantly shifted the abundance and community structure of denitrifying bacteria. This study will help to predict the response of different phyla of denitrifying bacteria to soil fumigation.

1. Introduction

borne pathogens, they can also be detrimental to soil bacterial diversity and bacterial communities (Yan et al., 2015). Previous studies have reported that the fumigant 1,3-D significantly altered soil microbial diversity and caused a significant shift in the predominant microbial populations (Liu et al., 2015). CP fumigation significantly changed microorganism populations and their community structure (Li et al., 2017b). DZ fumigation triggered a reduction in both the bacterial and archaeal amoA-expressing populations and caused long-term effects for > 28 days (Feld et al., 2015). Functional soil microorganisms, particularly bacteria, play crucial roles in maintaining soil quality and crop yields by regulating nutrient transformation (Álvarez-Martín et al., 2016; Crouzet et al., 2016). Substantial research effort has shown that soil fumigation slows down microbial nitrification processes resulting in ammonium nitrogen accumulation (Yan et al., 2013). For example, the concentration of NH4+-N in soil increased ten-fold after CP, MITC or MeBr fumigation (Ebbels, 2008). In addition, 7 days fumigation with CP, 1,3-D or MITC significantly stimulated NH4+-N, while NO3−-N was reduced by 5%–9% after fumigation and maintained low concentrations

Fumigants have been used for decades to disinfest soil of soil-borne pathogenic bacteria, fungi, nematodes and insects in soil seeds (Abou Zeid and Noher, 2014). Methyl bromide (MeBr) was the most widely used fumigant in the past, but due to its stratospheric ozone depletion it has been banned from use as an agricultural soil fumigant (Martin, 2003). To date, the most commonly used alternatives to MeBr are 1,3dichloropropene (1,3-D), chloropicrin (CP), dazomet (DZ) and metham sodium (MS) (Cao and Wang, 2015). In moist soil, both DZ and MS rapidly release methyl isothiocyanate (MITC) which is effective against pathogenic fungi. The nematicide 1,3-D can control root knot nematodes as effectively as MeBr (Desaeger and Csinos, 2006). CP and DZ are reported to protect crops against a range of fungi including Ralstonia solanacearum, Ganoderma orbiforme, Phellinus noxious, Macrophomina phaseolina, Fusarium oxysporum and Phytophthora infestans (Mao et al., 2014). Although fumigants protect crop production by controlling soil-

∗

Corresponding author. E-mail address: [email protected] (A. Cao).

https://doi.org/10.1016/j.ecoenv.2019.109850 Received 21 May 2019; Received in revised form 28 September 2019; Accepted 20 October 2019 0147-6513/ © 2019 Published by Elsevier Inc.

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After the fumigants were added to the jars, they were closed immediately and incubated at 28 °C. The fumigation time was 10 d. On the tenth day, the jars were moved to a ventilation hood to vent the fumigant. Ten grams of soil were sampled from each treatment. The jars were returned to the incubator. Soil samples were extracted 10, 24, 38 and 59 d after the start of fumigation and stored at −80 °C. Deionized water was added to adjust the water content to 45% WFPS during incubation. At the same time, a continuous flow automated analyzer (Futura Continuous Flow Analytical System, Alliance Instruments, France) was used to analysis the mineral nitrogen (NH4+-N and NO3–N) in soil sample extracted with 2 M KCl.

for 14 days after fumigation (Zhang et al., 2011). Although we now understand that fumigation has negative effects on soil microorganisms, the response of denitrifying microbes to fumigation is still unclear. Denitrifying microbes are of great importance to nutrient cycle as they are involved in the conversion of NO3−→NO2−→NO→N2O→N2 (Ji et al., 2012). Microbial denitrification not only releases NO3−-N which pollutes groundwater but also emits gaseous nitrogen (as NO or N2O) which pollutes the atmosphere (Farmaha, 2014). However, agricultural operations such as fertilization, cultivation and fumigation, as well as soil geochemical conditions (pH and the availability of nitrogen and organic carbon), can also influence microbial denitrification (Braker and Conrad, 2011; Čuhel et al., 2010; Philippot et al., 2013). For example, both CP and DZ have been shown to significantly stimulate denitrification and enhance N2O emissions (Spokas et al., 2006; Yan et al., 2015). In addition, the abundance of denitrifier nirS and nosZ genes significantly increased following CP fumigation (Li et al., 2017b), while MS treatment significantly suppressed the expression of the functional genes narG, nirS and nosZ (Li et al., 2017a). The differential response of functional genes to fumigants encouraged us to compare the changes in soil denitrification microbes following different fumigant treatments. Recently, dimethyl disulfide (DMDS) and allyl isothiocyanate (AITC) have been reported to effectively control soil insects, pathogens, nematodes and weed seeds (Le Bechec et al., 2015; Ren et al., 2018). DMDS is a highly effective nematicide that induces mitochondrial dysfunction and activates K-ATP channels to kill pests (Dugravot et al., 2003; Gautier et al., 2008), while the insecticidal action of AITC is achieved through a carbamoylation reaction at nucleophilic sites (such as amines, hydroxyls and thiols) in the enzyme molecule (Lin et al., 2000). Both DMDS and AITC are believed to be good replacements for MeBr. However, to date, there have been few studies focusing on the response of soil microorganisms to DMDS and AITC fumigants. We carried out a 59-day experiment to assess the effects of multiple soil fumigants on soil microbes using real-time quantitative PCR (qPCR) and 16S rRNA gene amplicon sequencing. The aims of our study were to (i) assess the non-target effects of soil fumigation on the abundance, diversity and community structure of bacterial microorganisms; (ii) compare the different responses of denitrifying microbes to these five fumigants and their recovery after fumigation; and (iii) to pay particular attention to the response of soil bacteria and denitrifying bacteria following fumigation with DMDS and AITC. Our overall intention was to better understand the interaction of microorganisms in fumigated soil ecosystems.

2.2. Measure of residual fumigant in soil Residual fumigant concentrations in the soil samples were determined following procedures previously described by Wang et al. (2016). A soil sample (8.8 g dry weight) was weighed into a 21 mL headspace vial. Each fumigant was added achieve specific rates (Table S1). All vials were crimp-sealed with an aluminum cap and incubated at 28 °C. The residual fumigant concentrations were monitored daily for a total of 168 h of incubation. The residual fumigants were extracted with 8 g of anhydrous sodium sulfate and 8 mL ethyl acetate, vortexed for 30 s and then shaken for 60 min. The supernatants were filtered 0.22 mm nylon syringe filter for fumigant analysis by GC-MS. The concentrations of the five fumigants were determined using a GCMS-QP2010 SE Standard Gas Chromatograph-Mass Spectrometer (Shimadzu, Japan) set in selective ion mode (SIM) (Fang, 2019). The qualitative ions for 1,3-D were 112 and 110 m/z, and the quantitative ion was 112 m/z. Qualitative ions for MITC were 73, 72 and 45 m/z, and the quantitative ion was 73 m/z. Qualitative ions for CP were 121, 119 and 117 m/z, and the quantitative ion was 119 m/z. Qualitative ions for DMDS were 94, 79 and 45 m/z, and the quantitative ion was 94 m/z. Qualitative ions for AITC were 72 and 99 m/z, and the quantitative ion was 99 m/z. 2.3. Microbial DNA extraction and real-time quantitative PCR MoBio Powersoil® DNA Isolation Kit (MoBio Laboratories, USA) was used to extract the total genomic DNA from each soil sample. The DNA quality was determined by gel electrophoresis (1% agarose). The DNA concentration was determined by NanoDrop 1000 UV spectrophotometer (Thermo Scientific, USA). The total bacteria in the soil were quantified using 16S rRNA gene abundance. The abundance of bacteria capable of reducing nitrate, nitrite, nitric oxide and nitrous oxide was quantified by determining the number of napA and narG, nirS and nirK, qnorB and cnorB, and nosZ genes in the soil, respectively. SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and gene-specific primers (Tables S2–S3) were used to quantify these denitrification functional marker genes and 16S rRNA by CFX96 real-time PCR system (Bio-Rad, USA). All operations were followed the MIQE guidelines. Special amplification of target genes and eligible amplification efficiency were achieved (Table S4).

2. Materials and methods 2.1. Soil sample collection and experimental setup Soil samples were collected in May 2016 during the tomato harvest in Beijing MiYun (40°22′38.33″N, 116°50′35.41″E). Soil diseases were evident in this plot as fumigants had not been used for many years. The top 20 cm of soil samples were taken from 5 locations following a ‘W’ line. Soil samples were filtered through a 2 mm sieve and stored at 4 °C before any treatment. The soil had a pH of 8.2 which was typical for alkaline fluvo-aquic soil. Other soil physicochemical parameters were: 72.9% sand, 24.3% silt and 2.7% clay; 3.07% organic matter; 9.4 mg NH4+-N kg−1; 58.9 mg NO3–N kg−1; pH 8.2; and 899 us cm−1 electrical conductivity. The laboratory incubation tests were conducted using a 500 mL jar that contained 300 g of soil. The six treatments included five fumigants (chloropicrin, CP; dazomet, DZ; dimethyl disulfide, DMDS; 1,3-dichloropropene, 1,3-D; allyl isothiocyanate, AITC) and one control (CK) (Table S1). All treatments had three replicates. Fumigants application amounts refer to Table S1 here were added into the jars to simulate field fumigation (Mao, 2015; Spokas et al., 2005; Yan et al., 2013). The final moisture content of the soil was 45% water filled pore space (WFPS).

2.4. High-throughput sequencing and bioinformatics analysis The universal primers 338 F [5′-ACTCCTACGGGAGGCAGCAG-3′] and 806R [5′-GGACTA CHVGGGTWTCTAAT-3′] were used to amplify bacterial 16S rRNA genes in V3–V4 hypervariable regions. MiSeq sequencing was conducted on an Illumina® MiSeq sequencer (Illumina, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). A total of 2,681,891 high-quality 16S rRNA reads was obtained after quality-filtering and merging. All sequences of the 16S rRNA gene were clustered with Operational Taxonomic Units (OUT) at 97% similarity levels. The Silva (SSU123) 16S rRNA database was used to determine the taxonomy of each 16S rRNA gene sequence at a confidence threshold of 70%. All raw data were uploaded to the National Center for 2

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3.3. Changes in the abundance of 16S rRNA and denitrification functional marker genes Statistical analysis revealed that all five fumigants significantly lowered 16S rRNA gene copy numbers compared with the control groups on day 24 (Fig. 2). However, total bacterial abundance in DZ-, DMDS-, AITC- and 1,3-D-fumigated soils recovered to the unfumigated levels on days 38 or 59. Gene copy numbers of 16S rRNA were significantly higher following DMDS and AITC fumigation than in the control group on day 59. By contrast, fumigation with CP decreased the bacterial population throughout the incubation period. The abundance of bacteria capable of reducing nitrates, nitrites, nitric oxides and nitrous oxides during denitrification were quantified by determining the copy numbers of napA and narG, nirS and nirK, qnorB and cnorB, and nosZ genes in the soil, respectively (Fig. 2). Fumigation with CP caused a 59-day decrease in the abundance of napA, narG, qnorB, and nosZ, but only briefly decreased nirS, nirK and cnorB which recovered to control levels on day 38. The other four fumigants caused a transient decrease in the abundance of napA, narG, nirS and nirK, whereas qnorB significantly increased on day 10 following AITC and 1,3-D fumigation. The cnorB and nosZ genes were relatively stable in DZ- and DMDS-fumigated soils but significantly decreased compared with the control following AITC and 1,3-D fumigation. Furthermore, some fumigants were able to stimulate napA, nirS and nirK genes. The nirS and nirK genes were significantly more abundant on day 38 in DMDS and AITC-fumigated soils than the control.

Fig. 1. Degradation of five fumigants in Beijing soil held at 28 °C. CP: chloropicrin; MITC: dazomet; DMDS: dimethyl disulfide; AITC: allyl isothiocyanate; 1,3-D: 1,3-dichloropropene.

Biotechnology Information database (No. SRP124701).

2.5. Statistical analysis 3.4. Changes in soil bacterial diversity Duncan's new multiple-range test (SPSS statistical software package, V 18.0; IBM, USA) was used to compare the difference in abundance of functional genes as well as physiochemical parameters following the five fumigant treatments. Heatmap figures and correlation analyses were performed using vegan package and pheatmap package in R, respectively (Version 2.15.3). LEfSe with Kruskal–Wallis sum-rank test was used to detect changes in genera abundance between the five fumigant treatments and the control group.

Non-metric multi-dimensional scaling analysis (NMDS) showed CP treatments and the control groups were separated by NMDS1 and NMDS2, while most other treatments were grouped together (Fig. S2). This result suggested CP significantly dispersed the soil bacterial community whereas the other four fumigants had a weaker affect. The saturated rarefaction curves in the present study (Fig. S1) suggested that both sampling and the 16S rRNA gene sequence database were sufficient to estimate the diversity of bacterial communities. Good's coverage estimations (0.956–0.977) revealed that 95% ~ 97% of the species were present in all samples at 97% sequence identity (Table 2). After fumigation with CP, OTUs together with diversity indices Shannon, Chao1 and ACE significantly decreased (P < 0.05) over the whole incubation period, as well as significantly increased in Simpson, suggesting CP significantly decreased bacterial diversity. In samples collected 24 days after fumigation, ACE, Chao1 and Shannon diversity indices in the soils treated with DZ, DMDS, AITC or 1,3-D significantly increased (P < 0.05) relative to unfumigated soil. Although the values of the Simpson index were relative stable in DZ- and AITC-fumigated soil, DMDS and 1,3-D triggered a significant (P < 0.05) decrease on day 24. However, statistical analysis revealed no significant difference in the diversity indices between DZ-, DMDS-, AITC- or 1,3-D-fumigated soils and the control at later sampling times. These results demonstrated that DZ, DMDS, AITC and 1,3-D promoted an increase in soil microbial community diversity, but only in the short term. The stimulation affect was transient and the microbial diversity was restored to unfumigated levels by day 38. In addition, Venn diagrams showed that the first samples on day 10 had the most shared OTUs between the fumigant treatments and the control, whereas the second set of samples on day 24 shared the fewest OTUs. By day 59, the number of shared OTUs gradually increased until they approximated those found initially (Fig. S3).

3. Results 3.1. Determination of fumigants concentrations The degradation half-life values for the five fumigants in Beijing soil varied from 29 h to 86 h (Fig. 1 & Table S5). AITC, CP and MITC had half-life values of 29.3 h, 34.1 h and 35.2 h respectively. They were degraded faster than DMDS, cis 1,3-D and trans 1,3-D that had half-life values of 67.1 h, 86.6 h and 79.9 h respectively. AITC, CP and MITC concentrations were undetectable 168 h (7 d) after the start of fumigation.

3.2. Changes in inorganic nitrogen Fumigation with CP, DZ and AITC significantly increased the concentration of NH4+-N compared to the unfumigated samples (Table 1). The increase in NH4+-N was larger and lasted longer with CP than DZ and AITC. The NH4+-N concentration in CP-fumigated soil was 2.5-fold greater than that in unfumigated soil and it did not recover to control levels until day 59; DZ and AITC triggered a 0.5 and 1.5-fold increase in NH4+-N concentration, respectively; NH4+-N contents recovered to the control levels by day 38 and day 24, respectively. Concurrently, NO3−N significantly decreased on days 10 and 24 following CP and DZ fumigation, respectively. However, statistical analysis revealed that NH4+ and NO3− concentrations in the soil did not change significantly after fumigation with DMDS or 1,3-D.

3.5. Changes in soil bacterial community composition The phyla Proteobacteria, Chloroflexi, Acidobacteria, Firmicutes, Gemmatimonadetes and Bacteroidetes were predominant across all samples (Fig. S4). Further comparative analysis between fumigant 3

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Table 1 NH4+-N and NO3−-N concentrations in soil 0, 10, 24, 38 and 59 days after fumigation with chloropicrin (CP), dazomet (DZ), dimethyl disulfide (DMDS), allyl isothiocyanide (AITC), 1,3-dichloropropene (1,3-D) or left unfumigated (CK). Parameter

Treatment

Concentration (mg kg−1 soil) 0d

NH4+-N

NO3−-N

CK CP DZ DMDS AITC 1,3-D CK CP DZ DMDS AITC 1,3-D

7.76 7.46 7.73 7.88 7.22 7.47 47.2 45.2 46.5 48.6 46.2 49.2

10 d ± ± ± ± ± ± ± ± ± ± ± ±

0.54a 0.24a 0.35a 0.74a 0.51a 0.34a 1.62a 1.99a 1.09a 1.77a 1.69a 1.67a

7.21 25.1 12.4 9.23 18.1 16.1 49.2 41.2 43.1 50.3 45.2 51.4

24 d ± ± ± ± ± ± ± ± ± ± ± ±

0.44c 4.94a 2.51bc 1.23c 3.29ab 1.75bc 1.67ab 0.77c 1.03c 1.97a 1.62bc 0.71a

6.74 23.3 10.3 8.01 6.76 6.62 53.6 45.9 46.0 54.9 55.0 54.6

38 d ± ± ± ± ± ± ± ± ± ± ± ±

0.24c 1.26a 0.56b 0.36c 0.54c 0.26c 0.77a 2.73b 0.55b 0.51a 0.26a 0.99a

5.45 8.77 5.72 6.26 5.23 6.38 58.6 57.1 62.5 56.8 60.5 56.5

59 d ± ± ± ± ± ± ± ± ± ± ± ±

0.54b 1.97a 0.56ab 0.10ab 0.38b 0.94 ab 1.04a 2.05a 2.55a 0.76a 0.91a 2.05a

4.96 4.45 5.61 4.51 5.09 4.21 68.2 72.2 67.4 70.2 72.1 72.7

± ± ± ± ± ± ± ± ± ± ± ±

0.69a 0.73a 0.22a 0.36a 0.56a 0.38a 1.64a 0.48a 8.20a 1.42a 0.95a 1.08a

Means within columns followed by the same letter are not significantly different (P = 0.05) according to Duncan's new multiple-range test.

Pseudomonas and Devosia were increased in short-term, but this effect disappeared by day 38. Massilia significantly decreased throughout the incubation period following AITC fumigation, whereas Streptomyces significantly increased. Fumigation with 1,3-D caused a significant increase in Cellulosimicrobium and Nitrosospira during the 59-day incubation. However, Pseudomonas, Sphingomonas and Shinella significantly decreased on day 38–59. Notably, Paenibacillus and Paracoccus were relatively stable in CP-fumigated soil, but increased on day 38–59. Sphingobium initially increased in all fumigated groups but significantly decreased in CP- and DMDS-fumigated soils on day 38 and 59.

treatments and the control was conducted (Fig. 3). All fumigants caused a significant (P < 0.001) decrease in Proteobacteria by day 24, but the inhibition was transient and the levels of Proteobacteria returned to unfumigated levels on day 59. At the same time, fumigation with DMDS resulted in a significant (P < 0.001) increase in Proteobacteria by day 59, suggesting that DMDS promoted the growth of Proteobacteria. Fumigation with CP resulted in a 59 day decline in Chloroflexi and Planctomycetes. However, Firmicutes, Gemmatimonadetes, Actinobacteria and Verrucomicrobia were significantly (P < 0.01) elevated throughout the incubation period. Acidobacteria initially decreased and then recovered to normal levels at later sampling times. Following DZ fumigation, Acidobacteria and Gemmatimonadetes significantly decreased (P < 0.001), whereas Firmicutes, Verrucomicrobia and Saccharibacteria increased at by 10 and 24. Gemmatimonadetes significantly decreased (P < 0.001) by day 24 following DMDS fumigation, whereas Verrucomicrobia and Saccharibacteria increased at the same time points. Most phyla, such as Chloroflexi, Acidobacteria, Firmicutes, Gemmatimonadetes, Actinobacteria and Planctomycetes were relatively stable in AITC-fumigated soil, whereas Saccharibacteria and Parcubacteria significantly increased (P < 0.01) by day 24. Concurrently, Verrucomicrobia initially increased and then significantly decreased (P < 0.01) at the last two sampling times. Fumigation with 1,3-D resulted in a transitory decrease in Gemmatimonadetes, as well as an increase in Chloroflexi and Acidobacteria. However, Verrucomicrobia, Saccharibacteria and Parcubacteria first increased and then significantly decreased (P < 0.01) by day 38 and 59.

4. Discussion 4.1. Effects of fumigants on soil bacterial diversity We observed that bacterial diversity significantly decreased following CP fumigation, which was consistent with the results of previous studies (Li et al., 2017b; Rokunuzzaman et al., 2016). CP disrupts normal cell functions in bacteria through a facile metabolic dechlorination process that takes place when CP reacts with biological thiols, which in turn disrupts multiple targets within the cell (Hutchinson et al., 2000; Sparks et al., 1997). CP exhibits cellular activity in various microorganisms which eventually kills them and causes a decrease in biomass (Stromberger et al., 2005). In contrast, we observed that fumigation with DZ, DMDS, AITC or 1,3-D initially increased bacterial diversity (Table 2), but this increase was transitory and there was no significant difference between fumigated and unfumigated groups at the end of incubation. Liu et al. (2015) reported that 1,3-D-treated soils had a slightly higher bacterial diversity than untreated soil. However, total bacterial diversity was reported to decrease following DZ fumigation and then return to normal after 12 d (Feld et al., 2015). The stress of MS fumigation on soil bacterial community structure significantly decreased bacterial diversity in the long term (Li et al., 2017a). In moist soil, DZ and MS rapidly produce MITC which kills soil-borne diseases and pests (Spyrou et al., 2009). Inconsistencies in the effects of DZ on soil bacteria that we observed might therefore be due to the soil's different physicochemical properties. The soil used in our study was loamy sand with a pH of 8.2, but the pH in the above-mentioned soils were 6.4 and 6.3 respectively. Soil pH is known to significantly influence bacterial activity. In general, bacterial activity is higher under alkaline conditions (ŠImek and Cooper, 2002). Previous researchers have indicated that fumigants were able to create a temporary “biological vacuum” shortly after fumigation by initially reducing the bacterial population (Yakabe et al., 2010). Many microorganisms both maleficent bacteria and some probiotics could readily

3.6. Changes in denitrifying bacteria community composition All 16S rRNA gene sequences were selected to assess the changes in denitrifying microbes at finer resolution as they could be assigned to known functional genes involved in the denitrification process. Thirtynine “most wanted” genera of denitrifying bacteria from 18 orders were investigated in this section (Fig. S5 & Fig. S6). Across all samples, the orders Bacillales, Sphingomonadales, Gemmatimonadales, Rhizobiales, Pseudomonadales and Streptomycetales were predominant. LEFSe analysis showed the biomarkers in the genera between the fumigation and control groups at different time points (Figs. 4 and 5). CP fumigation caused a 59-day inhibition in Azoarcus, Cupriavidus, Massilia, Pseudomonas, Hyphomicrobium and Nitrosospira, while Gemmatimonas and Cellulosimicrobium were continuously stimulated. After fumigation, DZ triggered a decrease in Cupriavidus, Massilia, Pseudomonas, Sphingomonas and Shinella, as well as an increase in Anoxybacillus, Nitrosospira, Pantoea and Rhizobium on day 38 or 59. DMDS fumigation appeared to have only a temporary effect on denitrifying microbes. For example the abundance of Azoarcus, Bradyrhizobium, Mesorhizobium, 4

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Fig. 2. Gene copy number per gram dry soil over time for the 16S rRNA gene and seven key genes (napA, narG, nirK, nirS, cnorB, qnorB and nosZ) involved in the bacterial denitrification process in soil fumigated with chloropicrin (CP), dazomet (DZ), dimethyl disulfide (DMDS), allyl isothiocyanide (AITC), 1,3-dichloropropene (1,3-D) or unfumigated soil (CK). Error bars represent standard errors of the means (n = 3). Statistically significant differences at a specific time point are shown by different letters according to Duncan's new multiple-range test. Bars within a graph with the same letter are not significantly different at the P = 0.05 level. 5

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Table 2 Estimated Operational Taxonomic Units richness, diversity indices and estimated representation of phyla in soil samples at the 97% sequence identity level. Day

Treatment

0TU

10

CK CP DZ DMDS AITC 1,3-D CK CP DZ DMDS AITC 1,3-D CK CP DZ DMDS AITC 1,3-D CK CP DZ DMDS AITC 1,3-D

2850 2305 2888 2914 2575 3077 2452 2137 2939 2847 2906 3087 2813 1968 2837 2843 2586 2852 2740 2033 2571 2561 2616 2664

24

38

59

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

103a 42c 38a 95a 102b 91a 71b 89c 63a 108a 69a 93a 85a 89b 98a 93a 35a 94a 98a 74c 85 ab 28ab 39ab 58ab

Shannon

ACE

Chao1

6.62 6.07 6.73 6.68 6.49 6.76 6.58 6.09 6.74 6.78 6.71 6.79 6.81 5.96 6.70 6.73 6.69 6.74 6.76 5.87 6.60 6.57 6.57 6.62

3773 ± 164ab 3153 ± 54c 3770 ± 27 ab 3843 ± 92 ab 3490 ± 164bc 3917 ± 116a 3453 ± 112b 2908 ± 132c 3958 ± 29a 3928 ± 74a 3894 ± 106a 4059 ± 118a 3794 ± 41 ab 2775 ± 120c 3837 ± 93a 3838 ± 96a 3513 ± 48b 3684 ± 133 ab 3685 ± 125a 2788 ± 135c 3487 ± 56ab 3441 ± 60ab 3507 ± 8ab 3610 ± 87a

3744 3176 3765 3788 3491 3936 3474 2894 4010 3965 3916 4058 3769 2751 3892 3802 3503 3644 3663 2747 3462 3470 3489 3662

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08ab 0.02c 0.03a 0.08a 0.01b 0.04a 0.00b 0.03c 0.04a 0.00a 0.03a 0.04a 0.01a 0.03c 0.03 ab 0.04ab 0.02b 0.02ab 0.03a 0.04c 0.04 ab 0.00ab 0.00ab 0.02ab

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Simpson 156ab 51c 14ab 74ab 148bc 129a 103b 136c 63a 59a 94a 142a 80ab 142c 85a 105ab 45b 127ab 157a 107c 54ab 76ab 13ab 107a

0.00354 0.00869 0.00297 0.00399 0.00441 0.00287 0.00361 0.00593 0.00306 0.00271 0.00297 0.00293 0.00257 0.00743 0.00291 0.00334 0.00289 0.00273 0.00284 0.00942 0.00326 0.00351 0.00344 0.00330

Representation ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0003b 0.0000a 0.0001b 0.0010b 0.0000b 0.0001b 0.0001b 0.0002a 0.0001 ab 0.0000c 0.0001ab 0.0002c 0.0000b 0.0003a 0.0001b 0.0005b 0.0000b 0.0000b 0.0001b 0.0005a 0.0002b 0.0000b 0.0001b 0.0000b

0.971 0.972 0.969 0.968 0.969 0.974 0.959 0.977 0.966 0.960 0.968 0.969 0.961 0.975 0.967 0.965 0.964 0.973 0.975 0.979 0.968 0.970 0.970 0.968

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.003a 0.001a 0.002a 0.000a 0.002a 0.000a 0.002c 0.001a 0.001bc 0.004c 0.000bc 0.001 ab 0.003c 0.001a 0.002bc 0.002c 0.002c 0.000ab 0.000ab 0.000a 0.002c 0.000bc 0.002bc 0.000c

Means within columns followed by the same letter are not significantly different (P = 0.05) according to Duncan's new multiple-range test.

involvement in the decomposition of organic materials (Sykes and Skinner, 1973). Notably, CP produced a dramatic change in the population of soil bacteria, which is consistent with the observation that CP has the longest effect on nitrifying bacteria (Yan et al., 2013). Specifically, fumigation with DMDS and AITC resulted in 2.3-, 5.1- and 2.5fold and 0.9-, 3.9- and 2.9-fold increases in the abundance of Verrucomicrobia, Saccharibacteria and Parcubacteria (Tables S8–S11)., respectively. Previous reports have demonstrated that Verrucomicrobia consumes methane (Dunfield et al., 2007). Hence, Verrucomicrobia stimulation by DMDS or AITC could serve to act as a biofilter to reduce methane emissions into the atmosphere. Parcubacteria species are deemed to ectosymbionts or parasites of other organisms because they are lack biosynthetic and DNA repair capabilities (Nelson and Stegen, 2015). The enhanced abundance of Parcubacteria would inevitably cause an increase in its parasitism. Growing evidence suggests that additional nitrogen could alter the diversity and composition of soil microbial communities (Campbell et al., 2010; Ramirez et al., 2010; Zhang et al., 2014). In our study, we only observed higher NH4+-N contents following CP, DZ and AITC fumigation. However, recent studies indicated that DMDS and 1,3-D can increase the accumulation of NH4+-N in soil (Yan et al., 2013, 2015). The low concentrations of NH4+-N in the soil in our study might be due to longer DMDS or 1,3-D fumigation periods than used in these studies. In general, enhanced NH4+-N pool would increase the relative abundance of copiotrophic taxa, such as Proteobacteria and Firmicutes and reduce that of oligotrophic taxa, typically represented by Acidobacteria (Fierer et al., 2007). As a result, N enrichment has been proposed to cause a shift toward labile carbon decomposition (Ramirez et al., 2010).

colonize this empty niche (Liu et al., 2015), which might explain why DZ, DMDS, AITC or 1,3-D treatments had a higher bacterial diversity than the control. 4.2. Effects of fumigants on soil bacterial members The predominant and ubiquitous bacteria in our soil samples included the phyla Proteobacteria (20%–39%), Chloroflexi (6%–29%), Acidobacteria (5%–21%), Firmicutes (8%–23%), Gemmatimonadetes (6%–13%) and Actinobacteria (4%–19%). This result is consistent with previous research (Janssen, 2006; Spain et al., 2009). This is also in agreement with the observation that soil fumigation did not significantly change the predominant bacterial groups in the bacterial community (Li et al., 2017b; Liu et al., 2015). However, the abundance of some predominant bacterial members did change significantly following soil fumigation. For example, the most dominant bacterial group Proteobacteria was reduced by 33%, 40%, 31%, 40% and 46% after fumigation with CP, DZ, DMDS, AITC or 1,3-D, respectively (Tables S8–S11). Simultaneously, DMDS caused a 34% increase in Proteobacteria abundance toward the end of the incubation period. The phylum Proteobacteria is the most commonly reported phyla in soil libraries probably because of its predominant role in global carbon, nitrogen and sulfur cycling (Kersters et al., 2006). We observed significantly higher NH4+-N in fumigated soil than in unfumigated soil, which is consistent with reports that soil fumigation depresses microbial nitrification (Yan et al., 2015, 2017). Many nitrifier species belong to the phylum Proteobacteria, such as Nitrosospira, Nitrosomonas and Nitrosococcus. The reduction in nitrifier abundance could be contributed to the reduction in nitrification that was observed in our research. The phylum Chloroflexi is involved in organohalide respiration, such as sugar- and plant-derived-compound degradation (Hug et al., 2013). Acidobacteria have the ability to grow in nutrient-limited environments (Ward et al., 2009). Both Chloroflexi and Acidobacteria were relatively stable in DZ, DMDS, AITC or 1,3-D fumigated soils, but decreased 75% and 76%, respectively, after CP fumigation. In contrast, Firmicutes, Gemmatimonadetes and Actinobacteria increased in CPfumigated soil by 137%, 89% and 366%, respectively (Tables S8–S11). Firmicutes and Actinobacteria are believed to play important roles in organic matter turnover and carbon cycling because of their

4.3. Effects of fumigants on functional microorganisms involved in denitrification Although the abundance of napA, narG, nirS and nirK genes was significantly decreased at an early stage by all fumigants (Fig. 2), they ultimately led to an increase in the relative abundance of denitrifiers with napA- or narG-encoded enzymes, such as Streptomyces, Bordetella, Bacillus and Pantoea, as well as the nirS- or nirK-bearing denitrifiers Devosia and Bradyrhizobium. The nitrate-reducing membrane-bound 6

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Fig. 3. One-way ANOVA bar plot (95% confidence intervals) of the top 10 bacterial phyla in chloropicrin (CP)-, dazomet (DZ)-, dimethyl disulfide (DMDS)-, allyl isothiocyanide (AITC)- or 1,3-dichloropropene (1,3-D)-treated Beijing soil, compared to the control groups (CK) (*p < 0.05, ** < 0.01, *** < 0.001). Graphs (a), (b), (c) and (d) show the proportion of bacterial phyla recorded 10, 24, 38 and 59 days after fumigation, respectively.

was also observed, whereas CP fumigation resulted in an increase in the abundance of nirS and nirK genes (Li et al., 2017b). The difference in the performance of functional genes following CP fumigation may be because the soil-type encouraged nitrate- and nitrite-reducing bacteria. Furthermore, DZ, DMDS and AITC fumigation caused a remarkable increase in the abundance of nirS and nirK on day 38 (Fig. 2), indicating that these three fumigants have the ability to promote the growth of nirS and nirK–expressing bacteria. Cytochrome cd1 nitrite reductases (encoded by nirS) and Cu-containing nitrite reductases (encoded by nirK) are responsible for nitrite reduction (Kandeler et al., 2006). We observed greater inhibition of nirK than the nirS gene following fumigation, suggesting that the denitrifying microbes possessing copper

(Nar) reductase and periplasmic (Nap) nitrate reductase are involved in processes of denitrification, i.e., dissimilatory/assimilatory nitrate reduction to ammonium, which would be stimulated by enhanced napAand narG-bearing denitrifiers (Bru et al., 2007). The significant decrease in NO3−-N on the first two sampling days following CP and DZ fumigation (Table 2) further demonstrated that nitrate-related metabolism was occurring. Our observations were consistent with previous results that showed that soil fumigation stimulated microbial denitrification (Yan et al., 2015), which can be contribute to N2O emissions. However, Li et al. (2017b) found that there were no significant differences in the abundance of napA and narG genes between CP fumigation and the control. In addition, the inhibition of nirS and nirK by MS fumigation 7

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Fig. 4. Linear discriminant analysis (LDA) coupled with effect size measurements to identify the genera abundance in soils fumigated with chloropicrin (CP), dazomet (DZ), dimethyl disulfide (DMDS), allyl isothiocyanide (AITC) or 1,3-dichloropropene (1,3-D), compared to the control groups (CK), on days 10 (a) and 24 (b) after fumigation. Genera with an LDA score > 2 are displayed.

quinol-oxidizing single-subunit class (qnorB) (Braker and Tiedje, 2003). The poor stability of Nor, together with the cytotoxic effects of NO, has resulted in less research effort on Nor and NO. However, NO reduction is the most direct mechanism for NO consumption in soil. The increase in cnorB and qnorB-expressing denitrifier abundance would stimulate nitric oxide consumption, resulting in less cytotoxic nitric oxide gas emissions. Conversely, an increase in cnorB and qnorB gene abundance also promotes nitrous oxide production, which has been identified as an important greenhouse gas (Gvakharia et al., 2007). Furthermore, the

nitrite reductase are more sensitive to fumigants than those without copper nitrite reductase. Soil fumigation triggered a slight decline in the abundance of cnorB, qnorB and nosZ compared to napA, narG, nirS and nirK genes. Although the populations of cnorB-, qnorB- and nosZ-bearing denitrifiers were uninfluenced by DMDS fumigation, AITC and 1,3-D caused an 89% and 84% increase in the abundance of qnorB, respectively. Nitric oxide reductases (Nor) are responsible for catalyzing the reduction of NO to N2O, which are encoded by cytochrome bc-type complex (cnorB) or 8

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Fig. 5. Linear discriminant analysis (LDA) coupled with effect size measurements to identify the genera abundance in soils fumigated with chloropicrin (CP), dazomet (DZ), dimethyl disulfide (DMDS), allyl isothiocyanide (AITC) or 1,3-dichloropropene (1,3-D), compared to the control groups (CK), on days 38 (a) and 59 (b) after fumigation. Genera with an LDA score > 2 are displayed.

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of the N-cycling soil microbial community; and (ii) improves the general understanding of soil nutrient cycling based on the responses of soil microbes to soil fumigation. However, long-term field studies that apply various doses of fumigants to different types of soil will be needed to confirm the findings of this study and to further advance our understanding on the impact of soil fumigation on the N cycling microbial community.

decline we observed in the abundance of nosZ would theoretically result in the accumulation of large amounts of nitrous oxide. For example, several studies have reported that fumigation with CP and DZ results in higher N2O emissions (Spokas et al., 2006; Yan et al., 2015), and this could be linked to a decline in the abundance of nosZ. However, denitrifiers containing nosZ, such as the predominant genera Gemmatimonas, Bradyrhizobium, Sphingobium and Streptomyces, increased after fumigation.

Declaration of competing interest 4.4. Effects of fumigants on the recovery of functional microorganisms The authors declare no conflict of interest. The physical parameters that we monitored, as well as the functional gene abundance, recovered to unfumigated levels within 38 or 59 days of DZ, DMDS, AITC or 1,3-D fumigation. However, microbial activity did not recover until day 59 in CP-fumigated soil. The recovery of microorganisms is closely linked with the persistence of residual fumigant in the soil (Li et al., 2017a; Liu et al., 2015; Yan et al., 2017). We observed half-life values from 29 h to 86 h for these five fumigants indicating that they degraded rapidly in Beijing soil. Our observations were consistent with previous reports (Qin et al., 2016; Wang et al., 2016; Gan et al., 2000). Fumigants are usually small molecule compounds that can rapidly degrade in soil and have half-life values of several days (Qin et al., 2016). For example, 1,3-D has a half-life of approximately 2–6 d (Wang et al., 2016), CP 0.2–4.5 d (Gan et al., 2000), MITC 0.7–2.5 d (Fang et al., 2016) and DMDS 1.1–10.9 d (Han et al., 2017). We postulate that fumigant effects on the microbial community are transitory, which is confirmed by most of the results of our study. Regardless of the short-term impact on the soil's biochemical parameters and the inhibition of gene abundance, these effects disappeared shortly after fumigation. The recovery of denitrification genes also suggests that fumigants have no sustaining direct toxic impact on the overall microbial metabolic activity and biomass, which is consistent with the results of previous studies (Li et al., 2017a, 2017b; Liu et al., 2015). However, it remains unknown whether the soil microbial community would recover faster in the presence of crops or in conjunction with farming operations such as fertilization and irrigation. In addition, soil fumigation not only disturbs bacterial communities, but also produces effects on other microbes including fungi and nematodes, which are effects that need to be addressed in the future.

Acknowledgments This work was supported by the National Key Research and Development Program of China (2017YFD0201600) and Beijing Innovation Consortium of Agriculture Research System (BAIC01-2017). We thank Dr Tom Batchelor for providing comments on the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109850. References Abou Zeid, N., Noher, A., 2014. Efficacy of DMDS as methyl bromide alternative in controlling soil borne diseases, root-knot nematode and weeds on pepper, cucumber and tomato in Egypt. VIII International Symposium on Chemical and Non-Chemical Soil and Substrate Disinfestation 1044, 411–414. Álvarez-Martín, A., Hilton, S.L., Bending, G.D., Rodríguez-Cruz, M.S., Sánchez-Martín, M.J., 2016. Changes in activity and structure of the soil microbial community after application of azoxystrobin or pirimicarb and an organic amendment to an agricultural soil. Appl. Soil Ecol. 106, 47–57. Braker, G., Conrad, R., 2011. Diversity, structure, and size of n(2)o-producing microbial communities in soils-what matters for their functioning? Adv. Appl. Microbiol. 75, 33–70. Braker, G., Tiedje, J.M., 2003. Nitric oxide reductase (norB) genes from pure cultures and environmental samples. Appl. Environ. Microbiol. 69, 3476–3483. Bru, D., Sarr, A., Philippot, L., 2007. Relative abundances of proteobacterial membranebound and periplasmic nitrate reductases in selected environments. Appl. Environ. Microbiol. 73, 5971–5974. Campbell, B.J., Polson, S.W., Hanson, T.E., Mack, M.C., Schuur, E.A., 2010. The effect of nutrient deposition on bacterial communities in Arctic tundra soil. Environ. Microbiol. 12, 1842–1854. Cao, A., Wang, J., 2015. Principle and Application of Soil Disinfection. Science Press, Beijing. Crouzet, O., Poly, F., Bonnemoy, F., Bru, D., Batisson, I., Bohatier, J., Philippot, L., Mallet, C., 2016. Functional and structural responses of soil N-cycling microbial communities to the herbicide mesotrione: a dose-effect microcosm approach. Environ. Sci. Pollut. Res. 23, 4207–4217. Čuhel, J., Šimek, M., Laughlin, R.J., Bru, D., Chèneby, D., Watson, C.J., Philippot, L., 2010. Insights into the effect of soil pH on N2O and N2 emissions and denitrifier community size and activity. Appl. Environ. Microbiol. 76, 1870–1878. Desaeger, J.A., Csinos, A.S., 2006. Root-knot nematode management in double-cropped plasticulture vegetables. J. Nematol. 38, 59–67. Dugravot, S., Grolleau, F., Macherel, D., Rochetaing, A., Hue, B., Stankiewicz, M., Huignard, J., Lapied, B., 2003. Dimethyl disulfide exerts insecticidal neurotoxicity through mitochondrial dysfunction and activation of insect KATP channels. J. Neurophysiol. 90, 259–270. Dunfield, P.F., Yuryev, A., Senin, P., Smirnova, A.V., Stott, M.B., Hou, S., Ly, B., Saw, J.H., Zhou, Z., Ren, Y., 2007. Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature 450, 879. Ebbels, D.L., 2008. Effects of soil fumigation on soil nitrogen and on disease incidence in winter wheat. Ann. Appl. Biol. 67, 235–243. Fang, W., 2019. Effects and Mechanisms of Soil Fumigation on Nitrogen Cycling Microorganisms and N2O Production. Chinese Academy of Agricultural Sciences, Beijing. Fang, W., Wang, Q., Han, D., Liu, P., Huang, B., Yan, D., Ouyang, C., Li, Y., Cao, A., 2016. The effects and mode of action of biochar on the degradation of methyl isothiocyanate in soil. Sci. Total Environ. 565, 339–345. Farmaha, B.S., 2014. Evaluating Animo model for predicting nitrogen leaching in rice and wheat. Arid Land Res. Manag. 28, 25–35. Feld, L., Hjelmsø, M.H., Nielsen, M.S., Jacobsen, A.D., Rønn, R., Ekelund, F., Krogh, P.H., Strobel, B.W., Jacobsen, C.S., 2015. Pesticide side effects in an agricultural soil ecosystem as measured by amoA expression quantification and bacterial diversity changes. PLoS One 10, e0126080. Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classification of soil

5. Conclusion In this study, we investigated the effects of five fumigants on the diversity and community composition of soil bacteria, as well as the response of denitrifiers in fumigated soil. Soil fumigation appeared to not significantly change the compositional structure of the predominant bacterial groups. However, fumigants caused a significant decrease in phyla such as Proteobacteria, Chloroflexi and Acidobacteria; and increased Firmicutes, Gemmatimonadetes Actinobacteria, Verrucomicrobia, Saccharibacteria and Parcubacteria. In addition, bacterial diversity declined following CP fumigation. However, it was briefly stimulated by fumigation with DZ, DMDS, AITC or 1,3-D. Meanwhile, the abundance of denitrifying bacteria changed significantly in fumigated soil. All five fumigants briefly decreased populations of denitrifying bacteria carrying the napA, narG, nirS or nirK enzyme-encoding genes. However, denitrifying microbes expressing cnorB, qnorB or nosZ were relatively stable following DZ and DMDS fumigation; cnorB and nosZ initially decreased following CP, AITC or 1,3-D fumigation. Concurrently, the abundance of qnorB significantly increased in AITC- and 1,3-D-fumigated soils. These results demonstrated that soil fumigation results in a significant shift in the abundance of denitrifying bacteria. However, the activities of functional microorganisms recovered to the unfumigated levels in day 38 or 59. Our laboratory work that documents changes in the relative abundance of nitrogen transforming microbes provides (i) new mechanistic insights into the effect of soil fumigation on the structure and functioning 10

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