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Molecular Diagnosis and Evolutionary Relationship Analysis of Plant Parasitic Tea Garden Nematodes from Different Tea Estates in Sylhet Region of Bangladesh

MAB Siddique
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

TA Asa
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

MMH Sohag
Department of Genetic Engineering and Biotechnology, Jagannath University, Dhaka-1100, Bangladesh.

MSH Chowdhury
Department of Chemistry, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

A Iqbal
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

K Islam
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

Abstract/ Executive Summary:

Nematodes from plant-parasitic sources are ever-present and incidental to plant growth as well as crop production. The damage of tea gardens caused by nematode is often non-specific and easily confused with symptoms. The present study determined the parasitic and non-parasitic nematodes population in different tea gardens of the Sylhet region by their morphological and partial molecular characterization. Out of 13 tea gardens, it was observed that BTRI, Karimpur, Mathiura, and Tarapur tea gardens have the highest number of parasitic and non-parasitic nematodes. After PCR amplification, DNA bands with desired amplicon size were detected by gel electrophoresis. Among thirteen soil samples, nematodes from Malnichara, Karimpur, BTRI, Mathiura, and Finlay had partially confirmed the presence of root-knot nematode (Meloidogyne spp.), root-lesion nematode (Pratylenchus brachyurus), burrowing nematode (Radopholus similis), reniform nematode (Rotylenchulus reniformis) and lance nematode (Hoplolaimus columbus) consequently based on approximately base pair of 1.7, 1.1 and 0.52 kb (different Meloidogyne spp.) 0.52, 0.52, 0.25 and 2.3 kb of specific genes. From evolutionary analysis, it might be said that Meloidogyne species are strongly related with each other making clusters except Meloidogyne natalie where this one is closely related with Hoplolaimus columbus in their evolutionary relationship as remaining others (Rotylenchulus reniformis, Radopholus similis, Pratylenchus brachyurus) are in different clusters in the same clade and this result could be confirmed after sequencing.

Keywords: Evolutionary relationship, Molecular diagnosis, Plant parasitic nematode, Tea garden

Location: Sylhet region.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Tea, Nematodes

Objectives:

The present study determined the parasitic and non-parasitic nematodes population in different tea gardens of the Sylhet region by their morphological and partial molecular characterization.

Methodology:

Tea plant-parasitic nematode containing soil has been collected from different tea gardens of the greater Sylhet region. Considering the depth of 0-20 cm and 20-40 cm, soil samples were collected by using a soil auger. Rhizosphere soil samples were collected from stressed tea bushes. The stressed tea bushes were considered as those that did not have two leaves and a bud, become yellowish, wilting and, stunted growth (Adegbite and Adesiyan 2006). The soil was taken in proper air circulated plastic bag and transported in an insulated box to the laboratory and stored at 4°C until processing on the same day. For the nematode extraction from the soil, a modified Baermann funnel method was used which is easy to isolate the nematodes. In the modified Baermann funnel, the apparatus consists of a plastic box with a mass in it. Two tissue papers (crisscross manner) were placed there. Each soil sample was mixed thoroughly, and 200g of soil was subsampled and was placed over the tissue with distilled water by cover-up and kept still overnight, where nematodes were moved through the tissue and settled at bottom of the box by gravity. The next morning the nematode suspension was collected and identified according to their typical morphological features, considering the presence of Sylhet. The population density was estimated by the formula (number of nematode/ml of suspension) × (total volume of the nematode suspension from 200 g of soil). The counting was done on a nematode counting slide under a dissecting microscope. DNA extraction was done using the standard protocol of Clear®Detections Nematode DNA extraction and purification kit (Product no: EX-N-P-NDEP, Netherlands) which is designed to extract and purify DNA from nematode suspensions and/or multiple cysts. The kit separates DNA from proteins, detergents, and low molecular weight compounds. The purified nematode DNA was used for downstream applications such as PCR. In the present study, 16s rRNA regions from extracted and purified DNA regions were amplified by different nematode-specific primers (Table 1). For the amplification process, PCR mixture has been prepared with an aliquot of 50 µl solution, where 25 µl was the master mixture, 4 µl each for forward and reverse primer, 8 µL for the template, and the remaining are nuclease-free water. All reactions are performed according to the following conditions: initial denaturation has been done for 1 minute with 94°C, then the cycling conditions for the amplification step has been done for 35 cycles and the extension period is for 1 minute at 72°C. Then the preparation was kept for 7 minutes for the final extension and then storage has been done at 4°C. All PCR amplifications were run on the Thermal Cycler (SimpliAmp™ Thermal Cycler, Applied Biosystem®, and the USA). For in silico observation of the expected nematodes species (among them eight sequences were from Meloidogyne spp.) nucleotide sequences were retrieved from NCBI. The sequences were saved into fasta format and were processed through Molecular Evolutionary Genetic Analysis (MEGAX) tools for the evolutionary analysis among the twelve nematodes species. An evolutionary relationship among the twelve species was determined on the comparison of the Polymerase Chain Reaction amplified result of that relevant species. Here, the evolutionary analysis was prepared with the Neighbor-Joining method with a bootstrap replicate value of 1000.

Source of Information: J. Bio-Sci. 29(1): 101-109, 2021 (June)

Article Link (Online Journal): DOI: https://doi.org/10.3329/jbs.v29i0.54826

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Molecular diagnosis is important for the confirmation of plant-parasitic nematode species identification and analysis of their phylogenetic relationships with available sequences in databases. The present study provides the basic information on molecular identification based on PCR band of genomic DNA and a comprehensive framework of evolutionary relationships of plant-parasitic nematodes associated with tea gardens. After DNA sequencing, it will be identified to species level and this study will aid in further characterization of diverse plant-parasitic nematodes in different tea gardens in the future.

Knowledge Management: Journal