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2INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
COMPARISON OF MICROBIAL POPULATION IN PESTICIDE AND VERMICOMPOST TREATED SOIL
1Shumaila Parveen, 2Aiman Khan, 3Mohd Ikram Ansari
1Research Scholar , Department of Biosciences, Integral University, Lucknow (UP), India 2Student, Department of Biosciences, Integral University, Lucknow (UP), India 3Assistant professor, Integral, University, Lucknow (UP), India https://doi.org/10.5281/zenodo.13842283
Abstract. The application of organic amendments to agricultural soil enhances organic matter, supplies essential nutrients, improves soil structure, boosts water retention, and stimulates microbial activity. This research investigates the reconstruction of soil microbiomes in pesticide-contaminated soils, focusing on sustainable agriculture and environmental health. By evaluating the effects of pesticides on soil microbiomes over time, the study employs in vitro enrichments to explore the complex interactions between agriculture, the environment, and microbiology. We analyzed how pesticide, and organic amendments influenced soil microbial communities. Monthly soil samples were collected from treated pots, including pesticide treatments (Mancozeb), organic (cow manure and vermicompost) and control (no amendments). Our findings reveal that both pesticide and amendment treatments significantly altered soil microbial communities. Additionally, soils treated with pesticides and organics, showed the presence of antibiotic-resistant bacteria, with the absolute abundance of antibiotic resistance genes (ARGs) varying across treatments. Pesticide-treated soils showed the highest ARB levels, particularly ampicillin-resistant bacteria, which accounted for 80% of the population, compared to soils amended with organic treatments. These results provide critical insights into the management of organic amendments in farming systems and the relationships between soil microbial communities and soil function. This study contributes to enhancing agricultural sustainability by reconstructing soil microbiomes affected by pesticide use.
Keywords: soil microbiome, Pesticide impact, Organic amendments, Antibiotic resistance genes (ARGs), Microbial community dynamics.
Introduction
The discovery of antibiotics initially offered a profound sense of relief, with hopes that bacterial infections could be effectively controlled or eradicated [1]. However, as time has passed, bacteria have evolved and developed resistance to these once-powerful drugs. This training is focused on understanding antibiotic resistance in soil environments, an area of growing concern due to the widespread emergence of multidrug-resistant (MDR) bacteria. Antibiotics, a pivotal class of pharmaceuticals introduced prominently with penicillin in 1941, revolutionized medicine by effectively treating bacterial infections and saving millions of lives. They are produced by microorganisms, particularly in soil, where they play a crucial role in controlling the growth of competing microbes [2]. Despite their significant impact, antibiotics are ineffective against viruses and their overuse has led to the rise of antibiotic resistance. Resistance occurs naturally as bacteria adapt to their environments. When exposed to antibiotics, bacteria may undergo genetic changes that render them immune to the medication, ensuring their survival and replication. This adaptive process is a key factor behind the growing challenge of antibiotic resistance. The prevalence of
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
multidrug-resistant bacteria poses a serious threat to global health, as infections that were once easily treatable are becoming increasingly difficult to manage [3].
Soil is a critical environment in this context. It contains a vast and diverse microbial community essential for maintaining soil fertility and agricultural productivity. Soil microorganisms, including bacteria and fungi, contribute to processes such as organic matter decomposition, nutrient cycling, and soil structure maintenance [4]. One gram of healthy soil can harbor millions of bacteria and other microorganisms, all contributing to a balanced and productive ecosystem. The health of soil microbial communities is a key indicator of soil and ecosystem health, and disruptions in these communities can impact agricultural productivity and environmental stability.The use of antibiotics and pesticides in agriculture has raised concerns about their effects on soil health. Pesticides, both organic and synthetic, are employed to manage pests and pathogens but can also have adverse effects on non-target organisms, including beneficial soil microbes [5]. Misuse or overuse of these chemicals can lead to contamination and resistance issues, complicating pest and disease management.Given the current challenges, there is a need to explore sustainable agricultural practices. Integrated Pest and Nutrient Management protocols advocate for the use of eco-friendly alternatives, such as organic farming, biofertilizers, biological control agents, and precision pesticide application [6]. These methods aim to enhance crop production while minimizing negative environmental impacts. Antibiotic resistance mechanisms in bacteria can be classified into several categories: limiting drug uptake, modifying drug targets, inactivating drugs, and active drug efflux. Gram-negative and Gram-positive bacteria exhibit different mechanisms due to structural differences. For instance, Gram-negative bacteria often use all four resistance mechanisms, whereas Gram-positive bacteria may rely less on limiting drug uptake. Understanding these mechanisms is crucial for developing strategies to combat antibiotic resistance. For example, antibiotics like erythromycin and vancomycin, which are commonly used in medicine, can face different resistance challenges. Erythromycin, a macrolide antibiotic, is used to treat a range of infections but faces resistance through various mechanisms. Vancomycin, a last-resort antibiotic, combats severe infections but can be resisted through modifications in cell wall precursors.
This study aims to investigate antibiotic resistance in soil environments, examining the prevalence of resistance, the mechanisms involved, and the impact of agricultural practices. By understanding how soil microbes interact with antibiotics and how resistance develops, we can better address the growing issue of antimicrobial resistance and work towards more sustainable and effective solutions in agriculture and medicine.
Site location and Experimental setup:
The experiment was conducted at an agricultural field near Integral University, Lucknow. Surface soil (0-20 cm) was sieved through a 4 mm mesh, homogenized, and air-dried. A pot experiment using tomato (Lycopersicon esculentum) hybrid was carried out in the Department of Biosciences, Integral University.
Pot experiment design:
The pot experiment used 5 kg of agricultural soil, with treatments including 200g vermicompost and 4g pesticide (Mancozeb) per pot. There were four replicates of two treatments, plus controls. Two-week-old tomato seedlings were transplanted, watered, and maintained under natural light and consistent temperature.
SAMPLE COLLECTION
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Soil samples were collected from the rhizosphere of tomato plants in both treatments. These samples were placed in Ziplock bags, sent to the laboratory, and stored at 4°C for further processing.
Enumeration and isolation of bacteria from soil
Bacteria were isolated using growth pattern differentiation methods, including specimen collection, culturing, and microscopic examination. Techniques involved culture and non-culture methods with solid and liquid media. Plating methods such as streaking and serial dilution were used to separate and identify bacterial species based on colony characteristics and turbidity.
Procedure:
To prepare samples, 10g of soil was suspended in 90ml of 85% saline to create a stock solution. Serial dilution involved transferring 1ml of this stock into the first of four sterilized test tubes, each containing 9ml of saline. This was sequentially transferred to the subsequent test tubes.
Media preparation and plating:
Culturable aerobic heterotrophic bacteria and CFU were assessed by serial dilution and plating on Nutrient Agar. Soil samples (10g) were diluted in saline, plated, and incubated at 37°C for 24 hours. Colonies were counted, and the bacterial population was calculated using the formula:
CFU = (number of colonies x dilution factor) / volume of culture.
Isolation of bacteria from soil:
A soil sample was vortex-mixed (10g in 90ml saline) for 30 minutes, then serially diluted up to 104, plated on nutrient agar, and incubated at 37°C for 24 hours. Ten distinct, fast-growing bacterial isolates were selected based on colony morphology and purified via repeated streaking.
Sub culturing for pure culture preparation:
Streaked Plate Method
The streaking method involves pouring nutrient agar into petri plates and letting it solidify. An inoculating loop, sterilized by flame, is used to streak the inoculum on the media while keeping the plate near the flame. Incubate the plates at 35-37°C for 24-48 hours to isolate pure cultures.Sterilize the inoculating loop in a Bunsen burner until red hot and allow it to cool. Pick an isolated colony and streak it over the first quadrant of the agar plate. Flame and cool the loop, then streak the second quadrant, repeating for three to four quadrants. Incubate at 37°C for 24 hours and observe the colonies.
Antibiotic Sensitivity Test:
Transfer the pure bacterial culture into 5ml of nutrient broth and incubate at 37°C overnight. Adjust the suspension density to match a turbidity standard. Spread 100^l of suspension on nutrient agar plates and let dry. Place antibiotic discs on plates and incubate at 37°C overnight. Measure the zone diameters.
Discussion
The study reveals ampicillin-resistant bacteria in pesticide and vermicompost treatments, with no resistance in the vermicompost treatment. Soil is a major reservoir for antibiotic resistance, influenced by agricultural practices like pesticide use and manure application. Resistance levels vary by location and antibiotic type, highlighting the need for effective management to combat resistance.
Conclusion
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
In conclusion, soil samples from pesticide and vermicompost treatments revealed ampicillin-resistant bacteria in controls and pesticide treatments, but none in the vermicompost treatment. The study highlights that agricultural soils amended with pesticides and vermicompost can harbor antibiotic-resistant bacteria, serving as reservoirs for resistance genes. To combat antimicrobial resistance, it is crucial to implement policies reducing antibiotic use, employ precision agriculture, and explore bioremediation and plant-derived antibiotics. Integrated global strategies and advanced wastewater treatment are essential to address this growing crisis.
REFERENCES
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2. Wang, H., Liu, R., You, M. P., Barbetti, M. J., & Chen, Y. (2021). Pathogen biocontrol using plant growth-promoting bacteria (PGPR): Role of bacterial diversity. Microorganisms, 9(9), 1988.
3. Salam, M. A., Al-Amin, M. Y., Salam, M. T., Pawar, J. S., Akhter, N., Rabaan, A. A., & Alqumber, M. A. (2023, July). Antimicrobial resistance: a growing serious threat for global public health. In Healthcare (Vol. 11, No. 13, p. 1946). MDPI.
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5. Ankit, Saha, L., Kishor, V., & Bauddh, K. (2020). Impacts of synthetic pesticides on soil health and non-targeted flora and fauna. Ecological and practical applications for sustainable agriculture, 65-88.
6. Dutta, P., Bhattacharyya, A., & Kumari, A. (2023). Innovative Integrated Pest Management Paradigm for Sustainable Crop Production with Special Reference to North East India. In Integrated Pest Management in Diverse Cropping Systems (pp. 61-90). Apple Academic Press.
