Screening and identification of pyroxasulfone degrading bacteria

Introduction

Herbicide play an important role in agricultural production, and their use is currently essential for ensuring and enhancing crop yields. (Wang et al., 2023) [3] . Herbicides remain the most effective, efficient and economical way to control weeds; and its market continues to grow even with the plethora of generic products. With the development of herbicidetolerant crops, use of herbicides is increasing around the world that has resulted in severe contamination of the environment. The strategies are now being developed to clean these substances in an economical and eco-friendly manner (Singh and Singh, 2016) [21] . Microbial biodegradation is environment-friendly treatment technique for detoxification of persistent organic pollutants, compared with conventional methods. Some bio-technological applications such as biodegradation of organic pollutants could be given as examples of biodegradation. Bacteria and fungi are both responsible for biodegradation of polyaromatic hydrocarbons (PAHs) and other petroleum hydrocarbon. Microbial populations have high biodegradation capacity for organic material (Kanat, 2018) [8] . Pyroxasulfone (PYS), a pre-emergence herbicide registered for weed control in maize, wheat and soybean in India. Efficacy of any pesticide depends upon various environmental factors e.g. soil properties, rainfall and its physicochemical properties. The DT50 of pyroxasulfone in soil ranged from a low of 15.4 d to a high of 53.3 d, and loss was more rapid under warm, moist conditions. These results indicate that pyroxasulfone would last long enough to provide residual weed control, but would not persist excessively to injure rotational crops (Mueller, 2017) [16] the information regarding the impact of biotic factors particularly microbial degradation of PYS is not available.

Materials and Methods

Reagents and Chemicals

Technical-grade pyroxsulfone (99.73% pure) was procured from M/S Chromatopak Analytical Instrumentation (India) Pvt. Ltd., Jawahar Nagar, Goregaon West Mumbai-400 062, MS grade Dichloroethane (99.8% pure), Sodium sulphate, Magnesium sulphate, Sodium chloride, MS grdae Acetonitrile, acetone (99.9% pure), Nutrient agar media, Bushnell and Haas agar media.

Screening of Pyroxasulfone Degrading Bacteria by Enrichment Culture 2.2.1 Medium for screening

Bushnell and Haas agar (BHM) media with following composition was used: MgSo4 0.2; CaCl2 0.02; KH2PO4 1.0; FeCl2 0.05; NH4NO3 1.0; Agar 15.0. The medium for screening was composed of BHM media and pyroxasulfone. BHM media was autoclaved and isolated microbes were separately inoculated in BHM broth then supplemented with 10 mg/L pyroxasulfone were incubated at room temperature. Samples were analyzed after 0(after 2 hrs.) 5, 10, and 15 days interval for growth and biodegradation rate. Growth rate was measured at 600 nm on spectrophotometer. Pyroxasulfone was extracted using acetonitrile and analyzed by GC-MS/MS as described in screening procedure.

Screening procedure

The screening procedure of Sharma, 2013 was followed with some modifications. The bacteria was used to previously isolated from pyroxasulfone applied soil at Food Quality Testing Laboratory. All isolated microbes were screened for their efficacy to degrade pyroxasulfone. For determination of degradation potential of isolated microbes, they were separately inoculated in BHM broth supplemented with pyroxasulfone as sole carbon source and were be incubated at room temperature. For screening procedure taken 50 ml BHM media containing pyroxasulfone in separating funnel. Added NaCl and added 15 ml dicholomethane (DCM) in separating funnel and Kept a flask with funnel under the nozzle of separating funnel and added Sodium sulphate or MgSO4 on the cotton plug placed inside the funnel. Mixed the separating funnel properly and released the gas intermittently after shaking allowed the funnel to settle down and extract the DCM layer and also repeated the above step 2, 3 times. Taken 4 ml aliquot in test tube and evaporate it to dryness with TurboVap at 40 ℃. Finally made up the volume to 2 ml using acetonitrile and filtered it in to glass vial and dilute it on 100 μl: 900 μl acetonitrile (1 ml) and quantified on GC-MS/MS.

Screening of Biosurfactant Production using Blue Agar and Blood Agar 2.3.1 Blue agar

The potential of isolation microorganisms to produce biosurfactants, which improve pyroxasulfone's bioavailability was examined. To identify the synthesis of biosurfactants Cetyl Tri Methyl Ammonium Bromide (CTAB) was added to blue agar plates. Culture was spot inoculated and incubated to observe the formation of blue halos around the colonies.

Blood agar

To see hemolysis, cultures were injected in spots on blood agar plates (nutrient agar containing 5% human blood) and allowed to incubate at room temperature. A clear zone surrounding a colony was indicated of hemolytic activity.

Identification of Potential Isolate/s by 16S rRNA

Potential isolate was identified by 16S rRNA sequence homology. Sequencing service was taken from SLS Research Pvt. Ltd, Surat. Sequence homology was searched from available database using nucleotide BLAST tool and isolated organism was identified and gene sequence was submitted for accession number.

Results and Discussion

Isolation of Bacterial Cultures

Twenty six (26) bacterial cultures used in the present study were previously isolated at Food Quality Testing Laboratory. These cultures were for screening of pyroxasulfone degrading isolates to check the degradation of pyroxasulfone.

Screening of Pyroxasulfone Degrading Isolates using GC-MS/MS

The breakdown of pyroxasulfone by different 26 bacterial isolates was studied in BHM broth. Results were shown in Table 1. All of these species were assessed for total pyroxasulfone degradation under similar environmental condition at four interval times (0, 5, 10 and 15 days). The results obtained in the study reveals that there was no any degradation of pyroxasulfone was recorded on 0 day. However pyroxasulfone degradation was recorded in the later sampling intervals (5, 10, 15 DAI) in 26 bacterial strains along with control which was devoid of bacterial inoculation. The maximum degradation of pyroxasulfone was recorded in HP 1 culture on 5th, 10th and 15th DAI which was followed by culture HP 7. The maximum pyroxasulfone degradation HP 1 and HP 7 were the two bacterial species that degraded pyroxasulfone highest in BHM medium as compared to other culture. Although, a consistent pyroxasulfone degradation was also recorded in other cultures along with control on 5 th , 10 th and 15 th DAI. On 15 th DAI, the maximum degradation of pyroxasulfone was recorded in HP 1 culture (67.74%) followed by HP 7 (62.10%), HP 19 (58.90%). Other cultures have also exhibited pyroxasulfone degradation capability which was found to be in the range of 42.05 to 50.82%. However, these cultures were not considered for further study as control treatment exhibited 39.31% degradation on 15 DAI. Where, (Gamma) γ= weak hemolytic activity (Beta) β= produce completely hemolysis with zone around colony.

(Alpha) α= reduction of red color hemoglobin to appear green color colony Among the 26 culture, HP 1 and HP 7 culture recorded highest pyroxasulfone degradation as compared to other. These two cultures viz. HP 1 and HP 7 were identified by 16S rRNA gene sequencing. Based on sequence homology, the cultures HP 1and HP 7 were identified as Streptomyces sp. HP 1 and Staphylococcus sp. HP 7. The sequence of above isolates were submitted to GenBank for accession number and these were assigned the accession number PQ069794 and PQ069798 for HP 1 and HP 7 cultures, respectively. Their phylogenetic tree is given in Photo 3.2.

Conclusion 4.1.1 Screening of Pyroxasulfone Degrading Cultures

In present study, 26 bacterial cultures were screened for their capability to degrade the pyroxasulfone in BHM broth which were previously isolated from farm soil with history of pyroxasulfone application at Food Quality Testing Laboratory of Navsari Agriculture University, Navsari, Gujarat. DCM based liquid extraction method adopted for pyroxasulfone residue analysis from BHM broth and quantified on GC-MS/MS. The BHM broth were spiked with pyroxasulfone at the rate of 10 mg/l and incubated for 15 days with sampling on 0, 5, 10 and 15 th days. The overall pyroxasulfone reduction for each of these species increased gradually throughout the duration of the incubation period although it was maximum for two bacterial species, i.e., Streptomyces sp. HP 1(67.74%) followed by Staphylococcus sp. HP 7 (62.10%) on 15 th day. All isolates showed positive results on blood agar. Similarly, all of the isolates were positive results for biosurfactant synthesis on CTAB blue agar except of HP 13, HP 19, HP21 and HP23.

Identification of Isolates Showing Highest Pyroxasulfone Degradation

The potential isolates, HP 1 and HP 7 which showed highest degradation identified by molecular characterization as Streptomyces sp. HP 1 and Staphylococcus sp. HP 7. Genetic sequences were submitted to GenBank and accession numbers of HP 1 and HP 7 cultures were assigned the accession number PQ069794 and PQ069798 for HP 1 and HP 7 cultures, respectively.

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