Chemicals, media and instruments
Iprodione (purity ≥96%), N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine (purity ≥96%) were purchased from Toronto Research Chemicals Inc.(TRC). N-[[(3,5-dichlorophenyl) amino]carbonyl]glycine (purity ≥96%) was synthesized by Shanghai Nafu Biotechnology Co.,Ltd. Acetonitrile, acetone and n-hexane (GC grade) were provided by Fisher Scientific International Inc. Sodium chloride (AR grade) was provided by Chron Chemicals. Luria-Bertani (LB) broth consisted of the following components (g/L): 10.0 tryptone, 5.0 yeast extract, and 10.0 NaCl. Mineral salts medium (MSM) consisted of the following components (g/L): 1.0 NH4NO3, 1.0 NaCl, 1.5 K2HPO4, 0.5 KH2PO4, 0.2 MgSO4·7H2O, pH 7.0. Yeast morphology agar (YMA) consisted of the following components (g/L): 14.0 mannitol, 4.5 yeast meal, 0.1 MgSO4·7H2O, 0.4 K2HPO4, 0.3 NaCl, 0.01 CaCl2, pH7.0±0.2. Gas chromatograph (6890N, ECD with HP-5 Capillary column) was provided by Agilent Technologies. Gas chromatography-tandem mass spectrometer (450GC-320MS, EI with DB-5MS Capillary column) was provided by Bruker. Electronic Balance (JA2003N) was provided by Jinghua instruments. Ultraviolet visible photometer (TU-1901) was provided by Beijing Purkinje General Instrument Co.,Ltd. Ultrapure water preparation system (Milli-Q) was provided by Millipore.
Isolation of iprodione-degrading strain
Iprodione-degrading bacteria were isolated using enrichment culture technique. The samples were collected from the soil for vegetable growing in greenhouses at Lhasa, Tibet (29°66′84.4″N, 90°94′27.6″E, Altitude: 3667 m). A 5.0 g amount of soil sample was added into a 250 mL flask with 100 mL of sterile MSM containing 100 mg/L iprodione and was incubated on a rotary shaker (180 rpm) at 25℃ for 5 d. The suspension (5 mL) was successively transferred to fresh MSM containing 200 mg/L, 300 mg/L, 400 mg/L iprodione and incubated for another 5 d, respectively. After 4 rounds of enrichment, the culture was diluted and spread onto solid MSM plates containing 100 mg/L iprodione and incubated at 25°C for 7 d. A bacterial strain named as A1-3 that formed transparent halos around its colonies was purified for further study.
Phenotypic characterization and 16S rRNA gene analysis
The phenotypic characteristics of strain A1-3 were tested on yeast mannitol agar (YMA) in parallel. Cell morphology of strain A1-3 cultured at 25°C for 3 days were observed and photographed by light microscopy (CX31, Olympus). The temperature for optimal growth was tested at 5-40°C (5, 10, 15, 20, 25, 30, 37 and 40°C). The pH range for growth was measured from pH 4.0 to pH 12.0, with an interval of 1.0 units. The salt tolerance was determined with various NaCl concentrations (0, 1, 2, 3, 4, 5 and 6%, w/v). Other biochemical characteristics were carried out according to Ferreira et al (2020).
Genomic DNA was extracted from Strain A1-3 after growth in Luria-Broth for 48 h, using MiniBEST Bacterial Genomic DNA Extraction Kit Version 2.0 (TaKaRa Biotechnology Co., Tokyo, Japan). Amplification of 16S rRNA gene was performed under the following conditions: 95℃ for 10 min, followed by 94℃ for 45 s, 56℃ for 45 s, and 72℃ for 90 s for 30 cycles with a final 10 min extension at 72℃, the PCR products were detected by agarose gel electrophoresis and then sent to GENEWIZ.lnc for sequencing. Primers used for amplification and sequencing of 16S rRNA was as described by Pan et al (2021). 16S rRNA gene was aligned in EzBioCloud (https://eztaxon-e.ezbiocloud.net/). Maximum-likelihood (ML) tree was constructed using MEGA7.0 software with bootstrap values of 1000 replicates (Kumar et al. 2016).
Genome sequencing and comparative genomic analysis
The genomic DNA of strain A1-3 was sequenced with Illumina and Nanopore platform in MAGIGENE. The genomic sequence information of A1-3 had been submitted to the National Centre for Biotechnology Information (NCBI) database under the accession number JAMSLU000000000. Draft genome assemblies were prepared from the ONT reads using Apades v3.11.0, gene prediction using Glimmer 3.02 software. The predicted coding sequences were translated and used as queries to search the COG database.
The digital DNA-DNA hybridization (dDDH) values and confidence intervals were calculated using the recommended settings of Genome-to-Genome Distance Calculator (GGDC) (Meier-Kolthof et al. 2013). The average nucleotide identity (ANI) was determined between strains A1-3 and closely related strains of the genus Azospirillum by OrthANIu (Yoon et al. 2017). The whole-genome orthologous clusters were compared and analyzed by OrthoVenn2 (Xu et al. 2019). The whole-genome evolution trees were constructed using Type (Strain) Genome Server (Meier-Kolthoff et al. 2022).
Mensuration of iprodione and the metabolites
Cells of strain A1-3 were cultured in liquid LB medium for 24 h at 25°C and then collected by centrifugation at 8000 rpm for 5 min. The cell pellets were washed twice with sterilized MSM, adjusted to an optical density at 600 nm (OD600) of approximately 1.5, and used as the inoculant. An aliquot of the cells (5%, vol/vol) was inoculated into a 100-mL Erlenmeyer flask containing 30 mL of MSM supplemented with 50 mg/L iprodione as the sole source of carbon. The flasks were then incubated at 25°C with shaking (180 rpm). At each sampling point, six flasks were sacrificed for various measurements, three flasks were used to measure the iprodione concentration or for identification of metabolites by GC-ECD or GC-MS/MS, while three flasks were used to determine the values of OD600 of strain A1-3. Each treatment was performed in triplicate, and control experiments (medium without inoculum) were carried out under the same conditions.
Sample preparation of fermentation broth: 20.0 g sample were placed in 150 mL beaker, then 40 mL acetonitrile and 5-6 g NaCl were added, vibration at 180 rpm for 10 min, after 30 minutes of stratification, 10 mL of supernatant were rotatably evaporated to nearly dry, 5.0 mL acetone with n-hexane (1:9) was used as constant volume for GC-ECD or GC-MS/MS analysis (Celeiro et al. 2020).
The test conditions by Gas chromatography are as follows: HP-5 capillary column (30 m×0.25 mm×0.45 μm), carrier gas (N2, 99.999% purity), flow rate (3.0 mL/min), flow mode (10:1), sample volume (1 μL), inlet temperature (280℃), heating process: 150℃ for 0 min, 15℃/min to 210℃ and 10℃/min to 260℃, 20℃/min to 300℃ for 6 min, electron capture detector temperature (230℃).
The test conditions by Gas chromatography-triple tandem quadrupole mass spectrometer are as follows: DB-5MS capillary column (30 m×0.25 mm×0.25 μm), carrier gas (N2, 99.999% purity), flow rate (1.0 mL/min), no-flow mode, sample volume (1 μL), inlet temperature (230℃), heating process: 60℃ for 1 min, 15℃/min to 150℃ for 2 min, 10℃/min to 290℃ for 4 min. EI mode, electron bombardment energy (70ev), transmission line temperature (280℃), ion source temperature (230℃). Scan mode was used for qualitative analysis of each component. The scanning quality range was 50-500 amu (Özdoğan et al. 2018; Dai et al. 2022).