Zhang et al. AMB Expr (2017) 7:178 DOI 10.1186/s13568-017-0475-1 ORIGINAL ARTICLE Open Access Legacy efects of continuous chloropicrin‑fumigation for 3‑years on soil microbial community composition and metabolic activity Shuting Zhang1, Xiaojiao Liu1,2, Qipeng Jiang1, Guihua Shen1 and Wei Ding1* Abstract Chloropicrin is widely used to control ginger wilt in China, which have an enormous impact on soil microbial diversity. However, little is known on the possible legacy efects on soil microbial community composition with continuous fumigation over diferent years. In this report, we used high throughput Illumina sequencing and Biolog ECO micro- plates to determine the bacterial community and microbial metabolic activity in ginger harvest felds of non-fumiga- tion (NF), chloropicrin-fumigation for 1 year (F_1) and continuous chloropicrin-fumigation for 3 years (F_3). The results showed that microbial richness and diversity in F_3 were the lowest, while the metabolic activity had no signifcant diference. With the increase of fumigation years, the incidence of bacterial wilt was decreased, the relative abun- dance of Actinobacteria and Saccharibacteria were gradually increased. Using LEfSe analyses, we found that Saccha- ribacteria was the most prominent biomarker in F_3. Eight genera associated with antibiotic production in F_3 were screened out, of which seven belonged to Actinobacteria, and one belonged to Bacteroidetes. The study indicated that with the increase of fumigation years, soil antibacterial capacity may be increased (possible reason for reduced the incidence of bacterial wilt), and Saccharibacteria played a potential role in evaluating the biological efects of continu- ous fumigation. Keywords: Continuous fumigation, Bacterial community structure, Actinobacteria, Saccharibacteria Introduction soil fumigation has become an important global agricul- Soil fumigants are used extensively to control soil-borne tural practice (Ajwa et al. 2010). Methyl bromide (MB) pests including nematodes, pathogens, and weeds, so is mainly used for pre-planting fumigation of vegetables that increasing the yields of many crops (Mao et al. 2013; to manage many kinds of phytopathogenic organisms Wang et al. 2014; Ibekwe 2004). For instance, bacterial including R. solanacearum (Paret et al. 2010). However, wilt on ginger, caused by the soil-borne bacterial patho- MB had been phased out in developing countries owing gen Ralstonia solanacearum, is a major disease responsi- to its detrimental efects on the stratospheric ozone layer ble for enormous yield losses (Yang et al. 2012). Without (Bell et al. 2000). Chloropicrin is a potential replacement soil fumigation, it can cause an 80% crop failure in gin- for MB, which has been widely used to control ginger ger (Li et al. 2014). Terefore, in order to ensure a bet- bacterial wilt in China (Mao et al. 2013). ter crop yield and provide greater benefts to farmers, Soil bacteria have a high proportion of microorganisms, and they are responsible for many benefcially biological functions in biogeochemical and nutrient cycles, disease *Correspondence: [email protected] suppression, organic matter formation and decomposi- 1 Laboratory of Natural Products Pesticides, College of Plant Protection, tion, soil structure and plant growth promotion (Chap- Southwest University, No.2 Tiansheng Road, Beibei, Chongqing 400715, China arro et al. 2012; Kennedy 1999). In addition, soil bacteria Full list of author information is available at the end of the article play an important role in soil-borne disease suppression © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zhang et al. AMB Expr (2017) 7:178 Page 2 of 11 (She et al. 2016). Rhizosphere bacteria are antagonistic Te results will deepen our understanding of the possible to soil-borne pathogens by preventing their infection on bacterial mechanisms underlying continuous fumigation hosts (Latz et al. 2012). It is possible to bring back the soil to control bacterial wilt. suppressive status in a way changing the composition of rhizosphere bacteria (van Agtmaal et al. 2015). Materials and methods Fumigation can greatly change the bacterial com- Soil sampling position and decrease the abundance and diversity of Te samples were collected to study diferent fumiga- bacterial community (Chen et al. 2016). However, the tion years on ginger wilt soil microbiota in Weifang Actinomycetes and gram-positive bacteria have been city, Shandong Province, China. Te samples were col- reported to recover preferentially after 90 days of fumiga- lected from soils that non-fumigated (NF), fumigated tion (Klose et al. 2006). Liu et al. demonstrated that 1,3-D for 1 year (F_1), and continuously fumigated for 3 years had a rapid but short-lived impact on the indigenous soil (F_3). Te F_1 was fumigated in November 2014, and the bacteria community (Liu et al. 2015). Chloropicrin sig- F_3 was fumigated every November from 2012 to 2014. nifcantly altered bacterial population within 4 weeks of Te ginger’s growth period was from March to October application, and the efect on bacterial population struc- every year. Te details relevant to these trials are given in ture became non-signifcant 4 months later after treat- Fig. 1. Te fumigant was chloropicrin (Dalian Dyestufs ment (Wei et al. 2016). So recent studies showed that the & Chemicals Co., China), a commercial liquid prod- decrease of microbiome after fumigation could be mostly uct containing 99.5% chloropicrin. Chloropicrin liquid short-term, while the reconstructed system can exist for was injected into the soil at a 15 cm depth via a manual a long period (Yao et al. 2006). Terefore, it’s necessary to injection machine at a rate of 50 g/m2. After fumigating, compare the legacy efects of fumigation at a single time the soil was mulched by 0.04 mm impermeable flm for point, especially after crops planted. In addition, it is not 15 days. We uniformly collected samples in October 2015 clear what happens to the soil bacterial community after during the early ginger harvest. Te distance between several years of continuous fumigation. each sampling point was within 100 m, and the ecological According to our survey, we found that the incidence environment was consistent. Each treatment had three of ginger wilt was approximately 5% in continuous fumi- replicates, of which was collected and mixed to a com- gation for 3 years by chloropicrin; however, in non-fumi- posite sample from fve randomly point. Te soil cores gated felds, the value was approximately 40%. Terefore, were sampled at the 5–20 cm soil depth. After removal we hypothesized that the dominant bacteria in the soil of the plant residues, the samples were transported to the can be screened out by continuous fumigation, which laboratory in an ice chest (4 °C). Te basic physical and may give a positive efect on ginger bacterial wilt. We chemical properties of each soil sample is in Additional analyzed the changes of soil bacteria structure diversity fle 1: Table S1. of non-fumigation (NF), chloropicrin-fumigation for 1 year (F_1) and continuous chloropicrin-fumigation for DNA extraction and sequencing library construction 3 years (F_3). Specifcally, this study aimed to evaluate Total genomic DNA was extracted from 0.4 g of soil the biological legacy efects of continuous fumigation. using an Omega Biotek Soil DNA Kit (Omega Biotek, Fig. 1 The sample processing information. The green represents ginger growth period. The blue represents fallow period. The red represents fumi- gated treatment. The yellow represents sample collection period. M month, Y year, NF non-fumigation, F_1 chloropicrin-fumigation for 1 year, F_3 continuous chloropicrin-fumigation for 3 years Zhang et al. AMB Expr (2017) 7:178 Page 3 of 11 USA), following the standard protocol. DNA concentra- Statistical analysis tion and purity was checked on 1% (w/v) agarose gels. Raw Illumina fastq fles were demultiplexed, quality fl- PCR amplifcations were conducted with primers 338 tered, and analyzed using QIIME v1.7.0 (Quantitative forward (5′-ACTCCTACGGGAGGCAGCAG-3′) and Insights Into Microbial Ecology) (Caporaso et al. 2010). 806 reverse (5′-GGACTACHVGGGTWTCTAAT-3′), Operational taxonomic units (OTUs) were picked with which amplifed the V3–V4 region of the 16S rDNA gene a threshold of 97% pairwise identity. High-confdence (Xu et al. 2016). OTUs were identifed using the following criteria: any Polymerase chain reaction amplifcation consisted sample had a collective abundance of greater than 20 of an initial denaturation at 95 °C for 3 min, 28 cycles reads. Subsampling by the minimum number of sample of denaturation at 95 °C for 30 s, annealing at 55 °C for sequences. A comparison of overall microbial distribu- 30 s, elongation at 72 °C for 45 s, and a fnal extension at tion in diferent fumigation years was conducted on the 72 °C for 10 min. PCR reactions were performed in tripli- relative abundances of phyla, which were calculated using cate using 20 µL reactions with 2.0 µL 10× bufer, 2.0 µL OTUs based on taxonomy, using Origin 9.0 software for dNTP (2.5 mM), 0.2 µL rTaq Polymerase (Takara), 0.2 µL histogram analysis. BSA, 0.8 µL each of forward and reverse primers, and We calculated the Chao and Shannon indexes as meas- 10 ng of template. Amplifed products were run on 2% ures of α-diversity (within-sample species richness). agarose gel for identifcation; samples with bright main For β-diversity analysis, dissimilarity of bacterial com- strips between 400 and 450 bp were chosen for further munities was determined using cluster analysis based experiments. Amplicons were combined at roughly equal on Bray–Curtis dissimilarity and principal coordinate amplifcation intensity ratios, purifed using an AxyPrep analysis (PCoA) on unweighted and weighted Uni- DNA Gel Recovery Kit (AXYGEN) and submitted to the Frac distances among all samples.