Exploration of soil bacterial diversity in organic and conventional agriculture fields in Sri Lanka

Study Extent

This project focused on the organic and conventional agroecosystems of Sri Lanka as the study area. The soil samples were obtained from 25 agricultural fields across the wet zone, intermediate zone, and dry zone, which collectively represented the main agroecological zones of Sri Lanka. Soil sampling was carried out from August 2021 to June 2022. Metagenomic sequencing of the V4 region of the bacterial 16S rDNA gene was employed to explore the soil bacterial diversity within the collected samples, offering a comprehensive insight into the soil bacterial populations across these diverse agroecosystems.

Sampling

Soil samples were collected from four different locations in each of the 25 agricultural fields belonging to the main agroecological zones of Sri Lanka. The soil samples were obtained by pushing a 50ml falcon tube to a depth of 20cm into the soil from the natural surface level. The samples were transported to the laboratory in ice boxes and were stored at -20°C until they were used for the extraction of DNA.

Quality Control

Soil samples were collected from four different locations in each agricultural field and were homogenized to form a composite soil sample prior to the extraction of DNA to obtain a more representative and consistent sample, reducing variability, and offering a more comprehensive view of the soil's overall bacterial diversity. Replicates and controls were maintained during both the DNA extraction and amplification process to eliminate the possible contaminations that could take place. The concentration and purity of DNA were assessed using the Spectradrop microplate reader following both the DNA extraction and amplification steps and prior to high throughput sequencing.

Method steps

1. Soil samples were collected from four different locations in each agricultural field by pushing a 50ml falcon tube to a depth of 20cm into the soil from the natural surface level (Naveed et al., 2016; Wang et al., 2016). The samples were transported to the laboratory in ice boxes. The soil samples from the four different locations in each agricultural field were homogenized to form a composite soil sample, freeze-dried, ball-milled for 30 minutes with 15 metal beads, and passed through a 500μm sieve prior to DNA Extraction. The soil DNA was isolated from 500mg of soil using the HiPurA soil DNA Purification Kit according to the manufacturer’s instructions. DNA extraction was conducted on four 500mg soil samples from each field. The extracted DNA was electrophoresed in 1% agarose gel. The DNA was quantified and its purity was determined using the Spectradrop microplate reader (Yakovleva et al., 2017). The DNA samples that showed a high concentration and purity were pooled and stored at -20 °C until they were used for PCR amplification. The PCR amplification was performed as a two-step program. To amplify the V4 domain of bacterial 16S rDNA for sequencing, PCR amplification was performed using standard forward and reverse primer pair, F515 (GTG CCA GCM GCC GCG GTA A) and R806 (GGA CTA CVS GGG TAT CTA AT) (Zhang et al., 2019). This was the first step of PCR amplification. Next, 3 μL of the PCR product was used as the template for the second step which was performed with barcoded primers. The PCR mixture for both steps contained the Hi-Chrom master mix, forward primer (10X), reverse primer (10X), molecular grade distilled water, and 3μL DNA template in a final volume of 25μL. The amplifications were conducted in a heal force K960 thermal cycler using an initial DNA denaturation step of 94 °C for 1 min, followed by 28 cycles at 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min, and a final elongation at 72 °C for 10 min. The PCR products were electrophoresed in 1% agarose gel. The PCR products from the second step were purified using the HiPurA PCR Product Purification Kit, quantified, and their purity was determined using the Spectradrop microplate reader. Finally, the PCR products were sequenced using high-throughput sequencing technology, and the sequence data were used to access the soil bacterial diversity.

Asia LK