Determination of genetic diversity on DNA-A of the begomoviruses infecting pumpkin in Thailand

Name: Wiwat Piyatassee

LCSH: Kasetsart University -- Theses. M.S. (Agricultural Biotechnology) 2020

Classification :.LCCS: SB347

LCSH: Kasetsart University. -- Interdisciplinary Graduate Program -- Theses

LCSH: Pumpkin -- Diseases and pests

LCSH: Pumpkin -- Begomoviruses

LCSH: Begomoviruses

Abstract: Exploring begomovirus-infecting pumpkins in Thailand and study on their genetic diversity based on DNA-A, - the important component for pathogenicity in host plant, was conducted in this research. Pumpkin samples associated with disease symptoms exhibiting yellow mosaic, mosaic, yellow leaf curling and leaf distortion were collected during 2016 to 2018. All samples were preliminary screened with six polyclonal antibodies specific to cucurbit-infecting viruses in enzyme-linked immunosorbent assay and the results showed 16 positive samples with one-three virus infection. Fifty-nine negative samples were further screened for the presence of begomovirus using rolling circle amplification. The templiPhi products obtained from nine samples including KN7, KN44, KN47, KN49, KN51, KN52, CRI136, NMA7 and SNK152. Random digestion with HindIII, BamHI, SmaI, SalI, SacI, PaeI, PstI and KpnI to generate a single cut of each product was performed in which two samples, KN7 and KN44, were completely cut giving a DNA band at approximately 2.7 kb in size. Seven samples, including KN47, KN49, KN51, KN52, CRI136, NMA7 and SNK152, were successfully amplified by PCR method with BEGEN-A degenerate primers designed to be specific with begomoviruses infecting pumpkin in Asia. Nucleotide sequence analysis showed that six samples were closely related to squash leaf curl China virus (SLCCNV) isolate [Thailand] including KN44, KN47, KN49, KN52, CRI136 and SNK152 at 97.95-99.09% identity. Two samples were closely related to SLCCNV isolate [Wax gourd: Nakhon Pathom] at 98.01-98.10% nucleotide identity and KN7 was closely related to squash leaf curl Yunnan virus (SLCuYV) isolate [Thailand] at 98.91% identity. Surprisingly, we obtained the DNA-B from KN44 sample by HindIII restriction enzyme digestion which was closely related to SLCCNV isolate Hn DNA-B at 91.05% nucleotide identity. Phylogenetic reconstruction revealed that eight isolates of SLCCNV were clustered in SLCCNV clade with pairwise identity greater than 98% while SLCuYV isolate KN7 was clustered in SLCuYV clade at 98.9% pairwise identity. Analysis of the recombination events found no recombination evidence in all SLCCNV isolates. Although we found the positive results of the recombination events in SLCuYV isolate KN7 from four methods including RDP, GENECOV, Bootscan and 3Seq, this result was still not accepted under the recombination event criteria. To date, only two begomovirus species infecting pumpkin were reported in Thailand including SLCCNV and SLCuYV. However, the possible threat generated by recombination and reassortment which may cause more diversity and symptom severity should be further studied in the near future. The advantage of our finding in this research provides the useful information for begomovirus detection, disease surveillance and also for the begomovirus-resistance breeding program as well.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Ratchanee Hongprayoon

Role: Thesis Advisor

Created: 2020

Modified: 2025-08-04

Issued: 2025-08-04

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2563/wiwat-piy-all.pdf

CallNumber: SB347 .W58

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Agricultural Biotechnology

Grantor: Kasetsart University

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