Isolation and Identification of bacteria from the agricultural soil samples to tolerate pesticides dimethoate, thiamethoxam and Imidacloprid

In the current study, bacteria from agricultural soil systems that have been polluted with pesticides were isolated, identified, and their ability to tolerate pesticides was examined. Target bacterial species were isolated from Psidium guajava (L) and Abelmoschus esculentus (L) cultivating an agriculture field. From 10 distinct soil samples collected from an agricultural field, 27 bacterial species were extracted, and the capacity of these microorganisms to withstand pesticides was examined. Only three bacterial species (PRB-S1P2, PRB-S1P3, and PRB-S6P1) are capable to grow on Nutrient agar medium with different concentration of pesticides dimethoate, Thiamethoxam and Imidacloprid. Apart from these three, one bacterial species were highly tolerant to all test pesticides. The highest pesticide tolerant bacteria are Pseudomonas nitroreducens was identified through 16s rRNA sequencing and the sequences were submitted to the NCBI with the accession No: ON624333.1. Hence, the bacteria can be subjected to further study of its use in the field of bioremediation.


Introduction
Agriculture is an important part of the economy in many countries. It involves the production of food, fiber, and other goods through the cultivation of crops and the raising of livestock. In many developing countries, agriculture is the primary source of income and employment for a significant portion of the population. However, the agriculture sector also faces numerous challenges, including fluctuating commodity prices, climate change, and increasing competition from cheaper imports. The use of fertilizers and pesticides can be important tools for increasing agricultural productivity and ensuring food security. Overuse of fertilizers can lead to soil degradation and negative environmental impacts, such as water pollution, environmental pollution etc (Bisht and Singh Chauhan 2021)). Similar adverse effects on human health and the environment can result from the incorrect use of pesticides. Pesticides can also have negative impacts on non-target species and can be harmful to humans if not used properly (Bernardes et al 2015).
Chemicals known as pesticides are any substance or combination of substances used to control pests, including insects, weeds, plant disease organisms, and numerous other living organisms like nematodes, arthropods other than insects, and vertebrates that can harm crops and have an impact on food production Any further distribution of this work must maintain attribution to the author-(s) and the title of the work, journal citation and DOI. (WHO 1990, Damalas 2009). Pesticides work by altering the biological processes of the pests they are designed to control. For example, insecticides are designed to kill or control insects, and they work by disrupting the nervous system of the insects or by inhibiting the production of certain enzymes or hormones. Fungicides are designed to control fungi and work by inhibiting the growth and reproduction of the fungi.
The use of pesticides can have negative impacts on the environment and on different forms of life. Pesticides are designed to alter the biological processes of the pests they are designed to control, and they can also have unintended impacts on non-target species, including beneficial insects and other organisms (Pathak et al 2022). The sensitivity of different organisms to pesticides can vary widely, and some species may be more vulnerable to the negative effects of pesticides than others. For example, some species of birds, fish, and other wildlife can be sensitive to certain pesticides, and their populations can be affected by exposure to these chemicals. In addition to their potential impacts on non-target species, pesticides can also have negative impacts on the ecosystems in which they are used (Sharma et al 2019).
There are many different pesticides that are used around the world, and the specific pesticides that are most widely consumed can vary depending on factors such as the types of crops being grown, the pests and diseases that are present in a particular region, and the availability and effectiveness of different pesticides (Tudi et al 2021). According to data from the United Nations Food and Agricultural Organization (FAO), the top ten pesticides by global consumption in 2018 were as follows: Glyphosate (herbicide), Atrazine (herbicide), Acetochlor (herbicide), 2,4-D (herbicide), Metam sodium (fumigant), Paraquat (herbicide), Chlorpyrifos (insecticide), Dicamba (herbicide), Propoxur (insecticide), and Dimethoate (insecticide). It is important to note that these rankings are based on global consumption and do not necessarily reflect the most widely used pesticides in any particular country or region. Additionally, the use of certain pesticides can vary over time as new products are developed and older products are phased out. The use of these pesticides has drawn the scientific community's attention to the search for the possible adverse effects of environment, including aquatic flora and fauna, and humans (Ranasingh 2007, Lokhande et al 2011, Bempah et al 2012. In several countries where fruit and vegetable production is increasing to fulfil the present demand for a balanced diet, consumption of more fruits and vegetables is encouraged. Under such conditions, it will create a severe health risk to the public. In these countries, monitoring foodstuff quality should be followed (Dobrinas et al 2011, Bempah et al 2012, Jardim and Caldas 2012. Pesticides that accumulate in higher soil concentrations mainly involve organochlorine and organophosphorus pesticides (Yang et al 2009, Wang et al 2007, Ghadiri et al 1995, Aiyesanmi and Idowu G 2012. This is because they ate highly persistent and degraded extremely slowly by microorganisms. These pesticides were mainly used to improve crop production. Gradually the adverse effect was known and resulted in a ban in certain countries. But as they are, a persistent trace of such compounds is still found in the soil. They mainly affect the crop and get accumulate in the plant. An animal who used to eat them ultimately accumulated the pesticide within their body, and thus they are involved in the food chain affecting the whole chain (Aktar et al 2009, Yang et al 2009, Weber et al 2010. Dimethoate is an organophosphate insecticide that is used to control a wide variety of pests, including aphids, spider mites, thrips, and whiteflies. It works by inhibiting the activity of the enzyme acetylcholinesterase, which is responsible for breaking down the neurotransmitter acetylcholine (Glinyshkin et al 2021).
Thiamethoxam is a neonicotinoid insecticide that is used to control a wide range of pests, including aphids, thrips, whiteflies, and leafhoppers. It works by binding to the nicotinic acetylcholine receptors in the nervous system of insects, disrupting their ability to transmit nerve impulses and ultimately causing their death (Grant and Lewis 2017).
Imidacloprid is a neonicotinoid insecticide that is used to control a wide range of pests, including aphids, whiteflies, thrips, and leafhoppers. It works by binding to the nicotinic acetylcholine receptors in the nervous system of insects, disrupting their ability to transmit nerve impulses and ultimately causing their death (Simon-Delso et al 2015).
Numerous studies have documented how pesticides affect several soil microorganisms and their metabolic processes, including nitrification, ammonification, respiration, nitrogen fixation, dehydrogenase activity, etc (Meena et al 2020), among others (Agnihotri et al 1981, Rangaswamy and Venkateswarlu 1990, Garg and Tandon 2000, Pandey and Singh 2002, Adiroubane et al 2003. DeLorenzo and Serrano 2003 reported the toxicity of pesticides on marine phytoplankton Dunaliella tertiolecta. In a human or animal organism's body fat, pesticides may be metabolised, eliminated, stored, or bioaccumulated (Pirsaheb et al 2015). The epidermis, gastrointestinal tract, neurological system, respiratory system, reproductive system, and endocrine systems are just a few of the organs that chemical pesticides have been connected to as having negative effects on health (Thakur et al 2014). Furthermore, excessive levels of accidental, intentional, or occupational pesticide exposure can result in hospitalisation and even fatalities (Gunnell et al 2007).
The current study examines the isolation, identification, and pesticide tolerance of bacteria from agricultural soil systems that have been polluted with pesticides.

Materials and methods
2.1. Sample collection Soil samples were collected from Psidium guajava and Abelmoschus esculentus are the crops grown in the Egattur agricultural farms at the range of Latitude: 13°06'32.4'N and Longitude: 79°53'20.8'E, Kanchipuram district, Tamil Nadu, India (figure 1). These crops have been sprayed with biopesticides and chemical pesticides for the last 10 years. Ten different soil samples were taken from rhizosphere area of pesticide contaminated surface and transferred aseptically in the air tight containers to avoid the loss of moisture. The samples were stored in 4°C.

Pesticide stock
Pesticides, dimethoate, Thiamethoxam and Imidacloprid were used to isolate pesticide degrading bacteria from soil sample. The pesticide samples were purchased from Aarathana Agra Agencies located in Ariyalur district, Tamil Nadu. About 4 μl (100 ppm), 8 μl (200 ppm) and 12 μl (300 ppm) of each pesticide was taken from the 30% of stock to obtain the desired ppm. These concentrations were selected to provide a range of doses for the study, covering a spectrum of exposure scenarios and potential degradation rates. All stock solutions were prepared with the help of DDH 2 O.

Isolation of pesticide degrading bacteria
Bacteria were isolated through serial dilution method (Avishai and Charles 2014) using nutrient agar (Hi-Media).

Morphological characterization of isolated bacteria
Morphological characterization of the isolated bacteria was carried out using Gram's Staining method. Each isolate was used for Gram's staining (Bartholomew and Mittwer 1952).

Physical and biochemical methods for bacterial identification
The preliminary study of bacterial isolates were identified through following tests like Oxidase, (29), Indole production test, Methyl red and Voges Proskauer (MR & VP) test ((Norris and Swain, 1971).), Catalase test, Citrate utilization test, Triple Sugar Iron (TSI) Test using standard protocols.

Isolation of genomic DNA from bacteria isolated from soil
The total genetic material (genomic DNA) was isolated from bacteria using the phenol-chloroform procedure (Wright et al 2017). The better pesticide tolerant bacterial genomic DNA was isolated. A PCR reaction was used to amplify the collected DNA. Then, it was kept at 4°C. Using a conventional process, the Agarose gel electrophoresis was utilised to confirm the isolated genomic DNA and amplified product of the 16s rRNA. In order to amplify 16S rRNA from the genomic DNA extracted using bacterial isolate, general forward primer (27F) 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer (1492R) 5'-GGTTACCTTGTTACGACTT-3' (Marchesi et al 1998) were acquired from Eurofins Genomics India Pvt. Ltd., Karnataka, India.

16S rRNA sequencing and analysis
In order to sequence the Polymerase Chain Reaction products, Eurofins Genomics India Pvt. Ltd. of Karnataka, India eluted them from the gel. To find the equivalent with readily available distinct position sequence aliments, 16S rRNA gene sequences were exported into the BLAST programme (Basic Local Alignment Search Tool) available from the National Center for Biotechnology Information's webstore.

Collection of soil sample
In this present study, pesticide contaminated soil sample was collected from Psidium guajava (guava) and Abelmoschus esculentus (lady's finger) agricultural fields in Egattur agricultural farm, Kanchipuram district, Tamil Nadu, India (figure 1).
The ten soil samples were serially diluted and twenty seven microorganisms were found to the nutrient agar plates. By streaking, colonies that were morphologically distinct were selected and moved to fresh agar plates. Based on similar morphological characteristics and growth patterns, three pure colonies were isolated and named it as PRB-S1P2, PRB-S1P3, and PRB-S6P1. The chosen microorganisms were screened for further biochemical analysis and Pesticide tolerant capability.

Isolation of pesticide tolerant bacteria
The colonies were grown on nutrient agar plate amendment with different concentration of pesticides. The plate with three bacterial isolates against three different pesticides revealed the highest growth on sample 1 against dimethoate with 4 ×10 −6 cfu ml −1 in a plate (table 1). 3 ×10 −6 cfu ml −1 on 200 ppm and 1 ×10 −6 cfu ml −1 on 300 ppm respectively. Sample 6 showed the highest colonies against dimethoate of 100 ppm with 3 ×10 −6 cfu ml −1 colonies in a plate (table 2). Sample 1 showed the highest colonies against Thiamethoxam of 100 ppm with 3×10 −6 cfu ml −1 , and each 1 ×10 −6 cfu ml −1 in both 200 and 300 ppm concentration. The number of colonies on the Imidacloprid amadement plate showed 3×10 −6 cfu ml −1 in 100 ppm and 1×10 −6 cfu ml −1 on 200 and 300 ppm (table 1 and figure 2). Sample 6 showed the highest colonies against dimethoate of 100 ppm with 3 ×10 −6 cfu ml −1 colonies (table 2 and figure 3). 1 ×10 −6 cfu ml −1 was present on thiamethoxam amendment plates at the ppm of 100 and 200 and there was no colony on 300 ppm. Likewise, in the imidacloprid plates showed 2 ×10 −6 cfu ml −1 on 100 ppm and 1 ×10 −6 cfu ml −1 on 200 ppm. No colonies were found at the concentration of 300 ppm (tables 1 and 2). Based on similar morphological characteristics and growth patterns, three pure colonies were selected and named it as PRB-S1P2, PRB-S1P3, and PRB-S6P1. The chosen microorganisms were screened for further biochemical analysis (figure 4).

Gram's staining
The gram staining was performed on the strains which were isolated PRB-S1P2, PRB-S1P3, and PRB-S6P1. The stained slides were observed under the light microscopes at oil immersion objective (100X) magnification. All the isolates tested (PRB-S1P2, PRB-S1P3, and PRB-S6P1) were gram-negative, bacillus which absorbs the primary stain, and appears rod-shaped (table 3).

Biochemical tests 3.5.1. Indole production test
The isolates examined in the current investigation lacked the ability to convert tryptophan to indole using 'tryptophanase.' As a result, the surface layer stayed yellow as it had been before the Kovac's reagent was added, rather than developing a deep red colour (table 4).

Methyl red and voges proskauer (MR & VP) test
All the three isolates were screened for MR-VP test. PRB-S6P1 alone changed the colour from yellow to red indicates positive to methyl red test and other two isolates showed negative. S1P3 and S1P2 showed positive reaction for the VP test and there was no colour change on S6P1 strain (table 4).

Citrate utilization test
The isolates PRB-S1P2 and PRB-S1P3 were able to utilise citrate as the only source of carbon and energy during the citrate utilisation test, producing sodium bicarbonate as well as ammonia. As a result, the citrate test's findings indicated that blue colour production had occurred. PRB-S6P1 came out negative (table 4).

Triple sugar iron (TSI) test
PRB-S1P2 and PRB-S6P1 show positive for the TSI test, whereas isolates S1P3100 show negative for the same (table 4).

Catalase test
For the purpose of observing the catalase reaction, cultures underwent a catalase test. 3% hydrogen peroxide was haphazardly sprinkled onto the culture that had been mounted on the slide after 24 h. For the strains of PRB-S1P2, PRB-S6P1, and PRB-S1P3, the catalase test was conducted, and the results were observed to be positive (table 3).

Oxidase test
The oxidase test was done for all the strains (PRB-S1P2, PRB-S1P3, and PRB-S6P1) of and the result was observed to be negative for the oxidase test (table 3). These physical and biochemical results provide information about the biochemical characteristics of the tested bacterial isolates, helping to identify and differentiate them based on their metabolic properties (Xuedong and Yuqing 2015).

Molecular identification of bacteria by 16s rRNA amplification and sequencing
To get high molecular weight and high quality genomic DNA from the isolate PRB-S6P2, genomic DNA extraction was done as previously stated in the methods section. Successful isolation of genomic DNA from the PRB-S1P2 strain. PCR was used to amplify the 16S rRNA from DNA samples of isolated isolates. The 16S rDNA gene fragment was amplified using the universal primers (forward primer 5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse primer 5′-GGTTACCTTGTTACGACT-3′). On a 1.2% agarose gel, the amplified product was confirmed, and a single 1.5 kb-sized fragment was seen (figure 5). The amplified product was sequenced and the sequence was blasted in the NCBI data base (Altschul et al 1990). The result showed that the sequence has 99.59% similarity/identity with the already available sequence of Pseudomonas nitroreducens and 95.52% similarity with Pseudomonas sp. Figure 5. 16S rDNA amplification product of bacterial isolate, PDB-S1P2 in agarose gel. > PDB-S1P2 forward sequence.
Overall, pesticide tolerant bacterial strains were isolated from the soil samples collected from agricultural land. Agar plate containing different concentration (100, 200 and 300 ppm) of pesticides namely, Dimethoate, Thiamethoxam and Imidacloprid were used for screening pesticide tolerant bacteria. Totally 27 isolates were found. Based on the characteristics of bacterial strain on the agar plate, three different isolates were taken to biochemical characterized by using conventional method. Out of three bacterial isolate, isolate, PDB-S1P2 was selected for molecular analysis as this bacterial isolate showed pesticide tolerance in the higher concentration of pesticide. Hence the bacterial isolate, PDB-S1P2 was subjected to molecular analysis such as, 16S rRNA amplification and sequencing of the amplified product (figure 6). The sequence result was blasted in NCBI data base ( figure 7). The blasted result showed that the sequence has 99.59% similarity/identity with the already available sequence of Pseudomonas nitroreducens and 95.52% similarity with Pseudomonas sp.
This result is concordance with the results reported by various studies ( Under sustainable agriculture, only efficient microorganisms can replace artificial fertilisers and pesticides to increase vegetable yield. Because native microorganisms may alter their physiological and genetic makeup in Figure 6. The sequence of 16s RNA of bacterial isolate PDB-S1P2. response to environmental changes, these bacteria can also thrive in pesticide-and environmentally-unfavorable environments.

Conclusion
In the present study, a pesticide-tolerant bacterium was isolated from agricultural fields growing guava and lady's finger. A total of 27 bacterial isolates were obtained. Among these isolates, the bacterial isolate PDB-S1P2 exhibited the highest tolerance to higher pesticide concentrations compared to other isolates. Therefore, PDB-S1P2 was selected for molecular analysis. The blast analysis revealed a 99.59% similarity/identity with the available sequence of Pseudomonas nitroreducens and a 95.52% similarity with Pseudomonas sp. Based on these results, it can be concluded that the isolated soil bacteria exhibiting pesticide tolerance belong to the Pseudomonas species. Since this isolated Pseudomonas species is tolerant to pesticides, it may have the ability to degrade them. Therefore, further studies are necessary to explore the potential use of these bacteria in the field of bioremediation. Additionally, additional research will be required in the future to understand the molecular mechanisms underlying the emergence of pesticide resistance in soil microorganisms.

Acknowledgments
This project was supported by Researchers Supporting Project number (RSP2023R230) King Saud University, Riyadh, Saudi Arabia.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).