Biodegradation of Imidacloprid by the Local Isolate Rhizobium pusense

Imidacloprid is systemic insecticide (1-[(6-chloro-3-pyridinyl) methyl]-N-nitro-2-imidazolidinimine) and the world’s most widely used has significant efficacy against a broad variety of pests and a unique mode of action by using it spreader and irrigation. The persistence of this pesticide in the soil means that it causes environmental damage that must be cleaned up. In this study collected and identified the best bacteria isolate that breakdown imidacloprid from the Plant Protection Director in Baghdad, which has been using neonicotinoid pesticides for years in their own greenhouse for pest control. Using high-performance liquid chromatography HPLC to measuring the residual concentrations of imidacloprid in MSM media at a concentration of 25 mg/L for 21 days to know the best degrading bacterial isolate for imidacloprid. The best bacterial isolate No.43 was able to degrade 50.2% for 21 days of imidacloprid which was identified as Rhizobium pusense according to morphological, cultural and biochemical tests. Moreover, genetic analysis for the 16S rRNA gene and given a new accession number (OP218458.1.) in the GenBank of NCBI. It could be concluded, from this study that the soil previously contaminated for long periods of time with imidacloprid can be treated by degrading the imidacloprid residues in it by isolated bacteria Rhizobium pusense.


Introduction
Pesticides are a significant environmental risk where three million metric tons of pesticides are used globally each year because of their enduring effects on the environment.The organic decomposition of many of these pesticides in the soil is incomplete oni enter the food chain through bioaccumulation and biomagnification, having an effect on both the target and non-target organisms, including people.Since farmers who spray pesticides in greenhouse fields are also affected by them, pesticide residues are viewed as a significant risk factor in society [1,2].One million of the three million individuals who are fatally poisoned by pesticides each year do not even know it, according to the World Health Organization (WHO) [3].Neonicotinoid insecticides are widely used because insect pests are a widespread issue.accounting neonicotinoid around 25% of the global pesticide market [4].These compounds are becoming more and more well-liked as an alternative to pyrethroid and organophosphate due to their unique mechanism of action [5].More and more academics have been focusing on the effects on organisms, the environment and activities using these substances through Studies on aquatic creatures [6], birds [7], bees [8,9] and mammals (including humans) have shown 1259 (2023) 012035 IOP Publishing doi:10.1088/1755-1315/1259/1/012035 2 that exposure to neonicotinoid pesticides may cause acute or chronic toxicity [10,11].As of late.The residual duration after pollution has been removed is an important indication.The amount of time that neonicotinoids stay in the soil varies greatly, from a few hundred to several thousand days [12].Imidacloprid, the first The half-life of one neonicotinoid insecticide is 28-1250 days, while the halflife of clothianidin is 148-6931 days.There hasn't been much investigation into the precise reasons behind the significant inter-annual variation in neonicotinoid residual durations in the environment.The most popular insecticide is imidacloprid, also known as (1-[(6-chloro-3-pyridinyl)methyl] The first neonicotinoid pesticide on the market was N-nitro-2-imidazolidinimine.It is used in more than 100 countries and more than 140 different types of agricultural crops [13].More than two-thirds of the world's imidacloprid is produced in China, with 12,000 tons produced in 2012.2019 China exported 31,595 tons of imidacloprid-related goods in 2017 [6].IMI has been used heavily and widely, but this has led to a number of problems, including environmental contamination, insect resistance, and the extinction of natural adversaries [14].Because of the increased attention being paid to environmental issues, IMI's microbial degradation has been studied in an effort to eliminate IMI residues in ecosystems.It was discovered that IMI can remain in the soil for up to 156 days before it starts to lose its effectiveness [13].The most common method for converting synthetic chemicals into inorganic compounds is biodegradation, and both biotic and abiotic components of soil, such as elements, sunlight, and microbes, aid in this process.Soil microorganisms have been shown to degrade imidacloprid in several studies [15], also found the ability of pseudomonas sp.To biodegradation of imidacloprid and hydrocarbons in soil [16][17][18] and bioremediation of imidacloprid using Azospirillium biofertilizer and Rhizobium biofertilizer [19].This research aimed to isolate the best degrading bacterial species of imidacloprid in communities' soil samples for greenhouses and identify the best.

Collecting Soil Samples
In order to isolate microbes from greenhouse soil, samples were taken from the rhizosphere an area of 50 m 2 in the Plant Protection Directorate that had long been used as Neonicotinoid insecticide in the greenhouse.Soil samples were taken randomly from a depth of 15 to 25 centimeters, bagged in sterile containers, and sent in refrigerated containers to the lab.After the first stages of each experiment were set up, backup samples were placed in the fridge.That's according to the research [20].

Chemicals
The National Center for Pesticide Control's Plant Protection Department provided imidacloprid (purity > 97%).The 99.9% pure HPLC grade solvents were purchased from Merck KGaA in Germany.HPLC-grade water was manufactured by the UK business Romil.Acetonitrile and HPLC grade water were used with a nylon filter (0.45 m).Using aliquots of the working standard solution for HPLC calibration, analytical standards were made.the 1-50 mg/L range.

Culture of Microbe Growth
According to the manufacturer's recommendations, distilled water was used to create the NA (Nutrient Agar) and MSM (Mineral Salt Medium) media, and both were autoclaved for 15 minutes at 121 C to guarantee that the solutions were sterilized (Sigma-Aldrich, USA).Imidacloprid (25 ppm) was administered using a syringe filter, and after the media had cooled, it was placed onto Petri dishes.According to [21] the mineral salt medium (MSM) was employed for isolation and biodegradation testing and contains (g/L) ammonium sulfate (2.0), potassium hydrogen phosphate (0.625), sodium dihydrogen phosphate (0.6), magnesium sulfate (0.2), and calcium chloride.Antarcticite (hexahydrate) (0.15) (pH 7.0).(2018).The medium was cleaned by autoclaving it at 121 degrees for 15 minutes.

Instruments
For the analysis of imidacloprid, an HPLC (model LC-2010 A HT Shimadzu Japan) with a DAD was employed, All data collection and processing were carried out using ChemStation software.Using a 250 4.6 mm Orbit C18 reversed-phase column, each sample was separated.The mobile phase had a 1259 (2023) 012035 IOP Publishing doi:10.1088/1755-1315/1259/1/0120353 binary composition of 60% water and 40% acetonitrile at a flow rate of 1 ml/min and a temperature of 40°C in the oven.The wavelength of the detector was set at 270 nm.The calibration curve was checked each time a sample was examined.was created using outside standards, and a quantification method called linear regression analysis was used.The retention time was used to identify imidacloprid.The Spectrophotometer (Cecil/CE 7200) was used to measure the optical density (OD) of the microorganisms [22].

Isolation of Pesticide-Degrading Bacteria
In a flask (10 2 ml) with 18 ml of sterilized MSM broth, 2.0 g of soil was added.200 cc of MSM broth containing 25 ppm imidacloprid was in a rotary shaker for two hours at a speed of 220 rpm before being transferred to a flask with 5 ml of the combination (pH 7).after a week of incubation at 28 °C in a rotary shaker at 120 rpm.By scattering an aliquot (0.1 ml) of culture MSM agar plate, which contains 25 ppm imidacloprid, and incubating it for 24-72 hours, it was possible to identify bacterial colonies that were resistant to imidacloprid.The colonies on the plates that visually distinguished themselves from the others were then sought out.On NA agar, a few isolated colonies were recultured and incubated for 24-72 hours at 28 °C [23].

Biodegradation of Pesticides in Minimal Salt Medium
All biodegradation studies used 50 ml sterilized falcon tubes.Each tube was given 30 ml of MSM that had 25 mg/L imidacloprid added to it.This MSM functioned as the only source of carbon and nitrogen in a carbon-limited medium.Also used control tubes which contain medium and a pesticide (without any bacteria) to avoid any potential environmental effects such as photodegradation.for imidacloprid.10 7 bacterial cells per ml were cultivated in the falcon tubes for three weeks at 28°C in the dark on a rotating shaker set to 120 rpm.For investigations on pesticide residue, subsamples from each treatment were taken after 1, 7, 15, and 21 days.A one-half milliliter of each subsample was combined with a one-half milliliter of acetonitrile in two milliliters Eppendorf tubes, and the resulting mixture was centrifuged at 12,000 rpm for five minutes to measure the concentration of each treatment.To store the supernatant used Pasteur pipettes were used to transfer it to the amber HPLC vials and kept in a refrigerator.Each sample was injected into the HPLC at a volume of 50 microliters.And also measure the growth bacteria in each falcon tubes at the same time on 550nm optical density (OD) was recorded [22].

Statistic Evaluation
HPLC results demonstrating differences in imidacloprid concentration between treatments, One-way analysis of variance (ANOVA) was conducted.LSD test was used in SPSS 19 to assess mean differences (p 0.05).Controls were not included in the data analysis and were simply used to track the growth of the isolates [24].

Molecular Characterization
Using 16S ribosomal DNA, the bacteria isolate that was most effective and capable of destroying imidacloprid was found.Using the rRNA database at National Center for Biotechnology Information (NCBI), analysis of the sequence, and verification of homogenic data were carried out after bacterial ribosomal RNA amplification.Via used these procedures while extracting DNA.[25].In five ml of LB broth, the bacterial culture was cultivated before one ml of it was transferred to Eppendorf tubes.The supernatant from these tubes was discarded without touching the particle after being centrifuged at 8000 rpm for 3 min.ABC DNA Isolation Kit-recommended extraction of genomic DNA.The amount and integrity of DNA were determined using the spectrophotometer (Nanodrop).

Quantitation of DNA
To guarantee the quality of samples for later use, the extracted DNA concentration was assessed using a Quantus Fluorometer.For every microliter of DNA, Quantifluor Dye was diluted to a volume of 200 µl for each l µl of DNA.DNA concentrations were measured after a 5-minute incubation at room temperature.

Primer Preparation
Provided primers in a lyophilized Quantus Fluorometer by the Promega business.After that, produce a stock solution by dissolving in water free of nuclease to achieve a final concentration of 100 pmol/µl.to organize To obtain 10 pmol/µl in a working solution from primers, add 10 µl of primer stock solution (kept at a freezing temperature of 20 C) to 90 µl of nuclease-free water. of working priming solution.

PCR Reaction Mix Preparation and PCR Thermocycling Conditions
Using the Taq Ready master mix kit and the manufacturer's recommendations, aseptically create the PCR reaction mixture (Promega, USA).Polymerase chain reaction assays were performed at a reaction volume of 50 μl (table 2), and according to the PCR program (table 3).Table 2. according to the manufacturer's guidelines, master mix kit.

Gel Electrophoresis and Sample Preparation
DNA and PCR products were detected using gel electrophoresis, which was observed using ethidium bromide and a UV transilluminator documentation system.Before electrophoresis, 3 microliters of loading dye solution and 7 microliters of DNA extract sample were combined and poured into the wells.In contrast, 10 µL of PCR products were put into each well of the gels for PCR.Size of the PCR detection result, DNA A 100 bp ladder always follows an electrophoretic run.UV Transilluminator documentation system was used to visualize DNA bands [26,27].Once the results were obtained, they were examined using geneious software.

HPLC Calibration
Under experimental conditions, imidacloprid retention times were 4.8 minutes (Fig. 1).To establish the sensitivity of the HPLC, working standard solutions of imidacloprid were prepared at various dilutions (1, 5, 25, and 50 ppm) and used to calibrate the instrument before any sample analyses were injected.
For imidacloprid, the correlation between the quantity of standard solution injected and the resultant peak area was 0.999, suggesting a linear connection.

Isolation and Screening of Pesticide Degrading Bacteria
Different bacterial strains were used to test imidacloprid biodegradation for 21 days.A one-way ANOVA was employed to examine variations between Thirty-seven isolates taken from MSM medium content of the soil samples taken which greenhouses soil in where imidacloprid insecticide has been sprayed for many years was the Plant Protection Department.The One-way analysis of variance (ANOVA) was used to see whether there were any statistically significant differences between the two groups.The daily concentration means (7, 15, and 21) days were shown to be substantially different from one another at 95% confidence interval.was significant for all groups on day 7 (df=4, F=10.202, p 0.001).Days 15 (df=4, F=21.916, p 0.000) and 21 (df=4, F=18.420, p 0.000) were also significant for differences between groups.(Table 4).An ANOVA was combined with a post hoc test to identify the subset of pairs that showed statistically significant differences.Based on the LSD results from days 15 and 21, the isolates were classified into 5 groups on day 21.They were arranged into 8 categories overall.On day 15, there were six distinct bacterial isolates in the first three groups.25 mg/l in MSM medium.Imidacloprid biodegradation in spiking medium showed wide variation with four bacterial strains (Figure 2).Imidacloprid biodegradation was between 12.61% and 39.52% on day 15 of incubation, although it increased afterward to 49.11% and 50.20% of the spiking quantity by day 21 of incubation.Statistically, the analysis found a significant relationship between the growth of isolate and days, also, a significant correlation between bacterial population size and degradation ability.Figure 3 shows the average growth rates of the four biodegradable isolates throughout the course of 28 days of incubation.Isolate No.3 grew at a faster pace than the others, and all of the isolates reached their maximum size by day 7 (No.

Morphological and Biochemical Characterization of Isolate No.43
The best-isolated bacterium for imidacloprid breakdown was found to be No.43 after being analyzed using high-performance liquid chromatography.Gram staining and culture characteristics were used to determine the isolate's identity, which was then confirmed using traditional techniques based on growth.After 24 hours of incubation at 28°C on general medium (Nutrient agar) and a selective medium (MacConkey agar), The cells of bacterial strain No.43 were Gram-negative, rod-shaped.nonendospore-forming, motile, and light pink color in MacConkey agar as shown in (Figure 4).

Molecular Characterization of Pesticide-Degrading Bacteria No.34
The examination of the 16S rRNA gene sequence allowed for the molecular identification of the selected isolates.Using ntBlast and other tools, the isolates' 16S rRNA sequences were compared to similar genomes of other bacteria that were discovered using GenBank.According to the PCR results, the replicated isolate included a 1500 bp band (Fig. 5).The 16S ribosomal RNA gene was sequenced by this band [28].Discovered 16S rRNA segment's BLASTN comparison to existing bacterial sequences in the GenBank database revealed sequence similarity to nitrogenous bases with a percentage of 100% of Rhizobium pusense.And this sequence was published in GenBank (NCBI) under the accession numbers (OP218458.1).

Conclusions
The results of this research demonstrated that imidacloprid might be degraded by certain bacterial species.The chosen soil bacterial isolates were able degrade between 20 and 50 % of imidacloprid by 21 days.However, the bacteria (Rhizobium pusense) introduced in this study was the first locally isolated in Iraq about the degradation of imidacloprid by Rhizobium pusense in MSM media from greenhouse soil samples.These bacterial conversions of imidacloprid open up new avenues for its chemical degradation in soil.

Figure 1 .
Figure 1.Separation of the imidacloprid (9.4 min) 1 ml per minute flow rate on a C18 column.

Figure 2 .
Figure 2. Different bacteria's effects on the levels of imidacloprid (MSM Media).

Table ( 1
) provides a list of the primers used in this research.

Table 1 .
Primers utilized in this Study.

Table 4 .
(1,7,15,21)xcept Isolate No.47, which kept growing until day 28.Isolate No.3 had higher growth recorded by OD after incubation than the other isolates.It was still lower than that recorded by Isolate No.43, which showed much lower efficiency in biodegrading imidacloprid.No.3 isolate was degraded with Only 24.75% of the imidacloprid, and 37.7 % in the No. 47 isolate.Isolate No. 43 outperformed the others in terms of imidacloprid biodegradation, although growth more slowly than the others (Figure3).A one-way ANOVA test of the Significance for Days(1,7,15,21).